january - april 2003 volume 37 number 1 - ku...

Kasetsart Journal : N

atural Science January - A

pril 2003 Volum

e 37 Num

ber 1

January - April 2003

Volume 37 Number 1

The Kasetsart Journal

Advisor : Napavarn Noparatnaraporn

Rangsit Suwanketnikom

Editor-in-Chief : Ed Sarobol

Associate Editors : Wanchai Chanprasert, Natural Science

Suparp Chatraphorn, Social Science

Editorial Board : Natural Sciences Social Sciences

Amara Thongpan Suwanna Thuvachote

Pornsri Chairatanayuth Pongpan Trimongkholkul

Onanong Naivikul Nongnuch Sriussadaporn

Praparat Hormchan Patana Sukprasert

Korchoke Chantawarangul

Aree Thunyakijjanukij

Overseas Members

G. Baker (Mississippi State University, USA.)

A. Bruce Bishop (Utah State University, USA.)

John Hampton (Lincoln University, New Zealand)

Helen H. Keenan (University of Stathclyde, Scotland)

Chitochi Miki (Tokyo Institute of Technology, Japan)

Eiji Nawata (Kyoto University, Japan)

Manager : Orawan Wongwanich

Assistant Managers : Surai Suwannarat

Business Office : Kasetsart University Research and Development Institute (KURDI)

Kasetsart University, Chatuchak, Bangkok 10900.

The Kasetsart Journal is a publication of Kasetsart University intended to make available the results

of technical work in the natural and the social sciences. Articles are contributed by Kasetsart University faculty

members as well as by those from other institutions. The Kasetsart Journal : Natural Sciences edition is issued

four times per year in March, June, September and December while The Kasetsart Journal : Social Sciences

edition is issued twice a year in June and December.

Exchange publications should be addressed to

The Librarian,

Main Library,

Kasetsart University,

Bangkok 10900, Thailand.

KASETSART JOURNALNATURAL SCIENCE

The publication of Kasetsart University

VOLUME 37 January - April 2003 NUMBER 1

Screening Methods for High Yield Corn Inbreds in Honeycomb Design and Performances

of Their Hybrid Combinations

.............................................. Krisda Samphantharak and Tanapong Ouanklin 1

Development of Male and Female Parents of F1 Hybrid in Chinese Cabbage

................. Ekapote Payakhapaab, Tragool Tunsuwan, Chokchai Chimonkon,

................................................... Dumdern Karadee and Maneechat Nikonpun 5

Soybean Yield and Nutrient Composition as Affected by Soil and Foliar Fertilizations

........................ Chin Theng Phiv, Chawalit Hongprayoon, Peerasak Srinives,

.................................................... Arunsiri Kumlung and Yongyuth Osotsapar 14

Preliminary Test of Polyploidy Induction in Cotton (Gossypium arboreum)

Using Colchicine Treatment

.... Arunee Wongpiyasatid, Praparat Hormchan and Ngamchuen Rattanadilok 27

Cloning and Nucleotide Sequence of Four tRNA Genes in Mitochondrial Genome

of Thai Walking Catfish, Clarias macrocephalus Günther

......................................................... Pradit Sangthong and Amnuay Jondeung 33

Relation of Paralumbar Nerves and Conus Medullaris to the Vertebrae of Swamp Buffaloes

................................. Narong Chungsamarnyart, Worawut Rerkamnuaychoke

..................................................................................... and Nati Nilnophakoon 41

Isolation of Anti-malarial Active Compound from Yanang (Tiliacora triandra Diels)

............................................................. Chalerm Saiin and Sutthatip Markmee 47

Synergistic Effects of Sesame Oil with Cypermethrin on the Survival and Detoxification

Enzyme Activity of Plutella xylostella L. Larvae

......... Suraphon Visetson, John Milne, Manthana Milne and Pintip Kanasutar 52

Development of Fish Strip from Hybrid Clarias Catfish Surimi Fortified with Konjac Flour

........ Benjawan Chotpradit, Mayuree Chaiyawat and Nongnuch Raksakulthai 60

Antiaflatoxigenic Effect of Lactic Acid Bacteria Isolated From Some Thai Fermented Foods

............ Siriporn Stonsaovapak, Ladda Wattanasiritham and Aree Shuvisitkul 65

Using of Extrusion Process for Preparation of Instant Cereal Beverage Powders based

on Corn and Soybean

..... Chulaluck Charunuch, Pracha Boonyasirikool and Chowladda Tiengpook 72

The Optimum use of Salinity, Nitrate and Pond Depth and b-Carotene Production

of Dunaliella salina

....... Orapin Bhumibhamon, Udom Sittiphuprasert, Naiyana Boontaveeyuwat

........................................................................................ and Jantana Praiboon 84

Quantity and Distribution of Plant Nutrients on Eutrophication in Bang Pra Reservoir,

Chonburi Province

........ Ratcha Chaichana, Chumlong Arunlertaree, Boonsong Srichareondham

.................................................................................. and Narong Veeravaitaya 90

Fisheries in the Mun River: A One-Year Trial of Opening the Sluice Gates of the

Pak Mun Dam, Thailand

.Tuantong Jutagate, Chaiwut Krudpan, Praneet Ngamsnae, Kanjana Payooha

...................................................................................... and Thanatip Lamkom 101

Synthesis of Barium Titanate as an Electroceramic Raw Materials

................................................................................... Nuchnapa Tangboriboon 117

Kasetsart J. (Nat. Sci.) 37 : 1 - 4 (2003)

Screening Methods for High Yield Corn Inbreds in HoneycombDesign and Performances of Their Hybrid Combinations

Krisda Samphantharak and Tanapong Ouanklin

ABSTRACT

Plant selection method is changing accordingly with emerging new concepts of selections. One

of the most widely discussed concept is plant selection under nil competition environment in honeycomb

designs to avoid plant to plant competition, minimize soil heterogeneity, promote highest expression of

genetic potential, enhance differentiation among lines and facilitate line selection. This study designed

to compare moving circle selection and prediction criterion, PC = X ( ) /Xs X Sp- 2 with conventional

visual grid selection (selection 1 plant out of each 19 plants in the same row) in honeycomb design.

Grouped replicated R-49 honeycomb design and 40 replicated plants was used to screen 49 S7 inbreds

under nil competition environment. As a results, moving circle selection identified highest number of

diverse and good combine lines followed by PC and visual grid selection when tested in conventional

plant spacing, 0.75 ¥ 0.25 m. Top-7 hybrids were derived from top–5 inbreds of moving circle selection

while only 3 and 1 hybrids in the top-7 were derived from top-5 inbreds of PC and visual selection,

respectively. The results suggested that moving circle selection was the most effective method of

selection under this experimental conditions. However, considering time and cost efficiency, visual grid

selection is more practical for the identification of potential inbreds.

Key words: honeycomb, prediction criterion, moving circle

INTRODUCTION

Genetic and environmental interaction(GxE) is one of the most decisive factors for thesuccess or failure of plant selection. There are twokinds of environment, the one that can be controlledand the one that can not be controlled. Eventhough,plant densities are controllable environment butthere are different views for the optimum plantdensities for the effective line screening. It is acommonsense that plant screening should be doneunder the conditions that plants will be grown.However, conditions in farmers’ fields are variedwidely and the optimum conditions are impossibleto ascertain. To solve the problem, multilocation

yield trials are needed but it is very costly andpractically will carry out only for the mostpromising lines on the final screening. In addition,yield per unit area can be improved by increasingplant densities or increasing yield per plant withthe same densities. Troyer and Rosenbrook (1983)and Russell (1991) suggested that selection shouldbe done under higher plant densities as means toimprove grain yield of maize. Selection under highplant densities also increase heritabilities and gainsfor many traits (Eagles and Lothrop, 1994).Indirectly, selection under higher plant densitiesshould verify progenies that can tolerate morelimited moisture supplies, effectively use availablenutrients, effective in partitioning of available

Department of Agronomy, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.

Received date : 20/01/03 Accepted date : 26/03/03

2 Kasetsart J. (Nat. Sci.) 37 (1)

photosynthates and survive greater pressures forsusceptibility to diseases and pests (Hallauer, 1990).On different poin of views, Fasoula and Fasoula(2000) suggested a selection under nil competionenvironment in honeycomb designs by movingcircle selection for effective control of soilheterogeneity and full expression of genotypes.Moreover, potential yield per plant ( X ), toleranceto stress (predicted by standardized entry mean,X /Sp) and response to input (predicted by thestandardized selection differential, ( ) /Xs X Sp- ),were proposed for analysis of yield components orprediction criterion (PC) = X ( ) /Xs X Sp- 2 . Thisstudy was conducted to evaluate the effectivenessof each selection method; moving circle, PC andvisual selections in honeycomb design in order toidentify useful inbreds and hybrid combinations.

MATERIALS AND METHODS

Forty-nine S7 inbreds from different sourcesof germplasm; Pioneer3012, Pioneer3013,Pacific328, Pacific700, CPDK888, CPDK999,G5445A, SW3853, Cargill919, Pop28 (HS), Ki32and Ki42 were planted in grouped replicated R-49honeycomb design with 40 replications. Plantspacing was equilateral triangle of side 0.86 m.,three seeds per hill and thined to 1 plant per hill at14th day after planting. Three selection methods;visual grid selection (1 out of 19 plants in the samerow), moving circle selection (1 out of 19 plants inthe circle) and prediction criterion, PC =X ( ) /Xs X Sp- 2 as proposed by Fasoula andFasoula (1997b) were applied in the sameexperimental plot. Selections were based onprediction criterion values and selectionfrequencies of each inbred by the other two selectionmethods. Top-5 inbred lines from each selectionmethod were selected.

The remnant seeds of selected inbreds wereseparately planted in non-replicated honeycombdesign, 0.86 m. spacing among plants and 3 plantsfrom each line were selected and bulked, separately.They were crossed in diallel series and the derived

hybrids and 4 checks were planted in randomizedcomplete block design with conventional spacing(0.75 ¥ 0.25 m.), 4 row plot of 5 meter rows and 2replications. Yields and desired agronomic traitswere recorded.

RESULTS AND DISCUSSION

Nil competition environment of each cropis depended upon plant types and root systems ofeach crop. For maize, Onenanyoli and Fasoulas(1989) used plant to plant space of 1.25 m. to avoidcompetition among plants. As a matter ofconvenience, the present study used plant to plantspace of 0.86 m. which fitted to the conventional0.75 m. row spacing being used at Suwam Farm.Under the present study plant to plant space of 0.86m. seemed to be adequate for corn inbreds becausewide gap among plants and full expression ofplants were observed.

Five selected inbreds out of 49 inbreds byeach selection method were presented in Table 1.From total of 15 selected inbreds (3 selectionmethods), only 8 inbreds were different. Theremaining 7 inbreds; 3 selected inbreds (Agron13,Argon26 and Agron27) from PC and 2 selectedinbreds (Agron26, Nei9201) from visual selectionwere overlapped with selected inbreds from movingcircle method. The other 2 selected inbreds(Agron4, Agron6) from visual selection wereoverlapped with selected inbreds from PC method.Considering the 5 selected inbreds of each selectionmethod and their original sources presented inTable 1, selected inbreds from PC methodcomprised of 3 inbreds originated fromPioneer3013 (Agron4, 6 and 26) 1 inbred fromG5445A (Agron27) and 1 inbred from SW3853(Agron13). The visual selection method rendered3 inbreds from Pioneer3013 (Agron4, 6 and 26) 1inbred each from Cargill 919 (Agron21) and Pop28(Nei9201). The moving circle method rendered amore diverse inbreds; 2 inbreds from SW3853(Agron12 and 13) and 1 inbred each fromPioneer3013 (Agron26), G5445A (Agron27) and

Kasetsart J. (Nat. Sci.) 37 (1) 3

Table 1 Selected top-5 of 49 S7 inbreds by each of 3 selection methods planted in grouped replicated

R-49 honeycomb design with equilateral triangular side of 0.86 m. and 40 replications.

Prediction criterion1 Moving circle selection Visual grid selection

Entry2 PC value Entry2 Frequency Entry2 Frequency

Agron27 3.11 Agron26 17 Agron6 11



Agron6 2.53 Nei9201 14 Agron21 6

Agron26 2.52 Agron13 11 Nei9201 6

1 PC = X Xs X Sp( ) /- 2

2 Original sources of inbreds in Table 1:

Pioneer3013 = Agron4, Agron6 and Agron26

G5445A = Agron27

SW3853 = Agron12 and Agron13

Cargill 919 = Agron21

Pop28(HS) = Nei9201

pop28 (Nei9201). Table 2 showed yields and otheragronomic traits of top-10 hybrids out of 28 hybridsfrom diallel cross of 8 selected inbred lines fromthe 3 selection methods. The high effeciency ofmoving circle selection was obviously displayed.Top –7 hybrids were derived from selected inbredsof moving circle selection and there were only 3and 1 hybrids in top-7 hybrids which had inbredsin common with inbreds from PC and visualselections, respectively. The Agron6 x Agron12hybrid ranked 8th comprised of inbreds from visualand moving circle selection. The Agron6 x Nei9201and Agron4 x Agron27 hybrids ranked 9 th and10 th derived from crossing of inbreds from visualselection and PC, respectively. The top5 hybridswere comparable to checks (hybrids derived fromearly generation testing for combining abilityprogram previously conducted) but statistically,better than the commercial hybrid, Pioneer3013.Therefore, selection for inbred per se under nilcompetition environment or for their combiningabilities were equally effective for the identificationof inbreds of which could render hybrids withsimilar yield levels, eventhough, they were different

inbreds. However, high yield inbreds hadadvantages on seed production and maintaining ofinbred lines. Using selection frequency of movingcircle selection and visual grid selection to identifystable lines in replicated honeycomb designs shouldbe an effective selection method for high yield andstable inbreds without any complicate calculationas compared to PC method. However, if moreselected inbreds (10 inbreds) from each selectionmethod were saved, all 3 methods were equallyeffective to identify potential inbred lines.Considering time and cost efficiency, visual gridselection with selection frequency of each lineshould be the most effective method for theidentification of potential inbreds. For populationimprovement, selection for inbred per se under nilcompetition environment in honeycomb designsfollowed by hybrid yield trials under high densitiesshould be a good combination to get high yieldinbreds and hybrids which can be grown underwide ranges of plant densities (Tokatlidis et al.,2001).


LITERATURE CITED

Eagles, H.A. and J.E. Lothrop. 1994. Highlandmaize from central Mexico-its origin,characteristics, and use in breeding programs.Crop Sci. 34 : 11-19

Fasoula, D.A. and V.A. Fasoula. 1997a.Competitive ability and plant breeding. PlantBreed. Rev. 14 : 89 -138.

Fasoula, D.A. and V.A. Fasoula. 1997b. Geneaction and plant breeding. Plant Breed. Rev.15 : 315-374.

Fasoula, V.A. and D.A. Fasoula. 2000. Honeycombbreeding : Principles and application. PlantBreed. Rev. 18 : 177-250.

Fasoulas, A.C. and V.A. Fasoula. 1995.

Honeycomb selection designs. Plant Breed.Rev. 13 : 87-139.

Hallauer, A.R. 1990. Methods used in developingmaize inbreds. Maydica 35: 1-6.

Onenanyoli, A.H.A. and A.C. Faoulas.1989. Yieldresponse to honeycomb selection in maize.Euphytica 40 : 43-48.

Russell, W.A. 1991. Genetic improvement of maizeyields. Adv. Agron. 46 : 245-298.

Tokatlidis, I.S., M. Koutsika-Sotiriou and A.C.Fasoulas.2001. The development of density-indepdent hybrids in maize. Maydica 46 : 21-25.

Troyer, A.F. and R.W. Rosenbrook.1983. Utilityof higher plant densities for corn performancetesting. Crop Sci. 23 : 863-867.

Table 2 Means of agronomic traits and grain yields of top-10 hybrids (S7x S7) from diallel cross of 8

selected inbreds from 3 selection methods planted at Suwan Farm in conventional row spacing

0.75 ¥ 0.25 m.

Hybrid Grain yield Days to Days to Ear Plant Shelling 100

at 15% tasseling silking height height (%) grain

moisture -50% -50% (cm) (cm) weight

(kg/ha) (g)

Agron12 X Agron27 6275a 52i 50j 133gh 195fg 85f-j 23d-f

Agron26 X Nei9201 6018a-c 51j 50k 155fg 211c 86c-f 25a

Agron13 X Agron26 5837b-d 52f 50j 117d-f 202d 86cd 24cd

Agron27 X Nei9201 5743b-e 50i 50k 118de 202d 84k-m 24bc

Agron26X Agron27 5712b-e 52i 51h 109jk 199de 83k-m 25ab

Agron12 X Agron26 5481d-g 53f 53f 117ef 194hi 88ab 22gh

Agron13X Agron27 5462e-g 51k 50j 111ij 186kl 83mm 21ik

Agron6X Agron12 5343f-h 53f 52g 119cd 197e-g 85f-j 23ef

Agron6X Nei9201 5331f-h 52i 52g 106lm 197e-g 82mm 23c-e

Agron4 X Agron27 5168g-i 53g 54d 109jk 173n 85c-g 18pq

Checks:

Agron14 X Agron29* 6043ab 50i-m 51i 102op 188jk 84g-k 23f

Agron20 X Agron29* 5712b-e 52e-i 51i 92u 185kl 86c 20k-m

Agron30 X Agron32* 5656c-f 52e-i 48l 101p 174n 84h-l 17q-s

Pioneer 3013 5343f-h 48m 54d 97qr 179m 83k-n 20jk

Mean 4574 52 52 103 184 84 20

CV (%) 17 1.36 1.35 4.67 3.57 3.05 7.64

* Top hybrids of selected inbreds (S7) from early generation testing (topcross) program previously conducted.


Development of Male and Female Parents of F1 Hybrid inChinese Cabbage

Ekapote Payakhapaab, Tragool Tunsuwan, Chokchai Chimonkon,Dumdern Karadee and Maneechat Nikonpun

ABSTRACT

Male and female parents were improved to produce good hybrid progenies of Chinese cabbage,

a highly self-incompatible vegetable crop. Hybrid seed production using bees was also studied. Inbred

seeds of 4 Chinese cabbage lines were produced using bud pollination which is a conventional technique

for inbred lines. Results showed that line 40-9 gave the highest seed weight of 0.165 g/plant. Using seed

set analysis technique to check self-incompatibility, lines 23-3-4, 27, 27-3-7, and 40-9 were found to be

self-incompatible among the 9 Chinese cabbage lines evaluated. However, when fluorescent microscope

technique was used, the self-incompatible lines were 23-3-4, 27, 27-3-7, 40-9, and 142. Four inbred lines

with self-incompatibility were selected to produce seeds. When line 40-9 was used as female parent in

reciprocal crosses by bee pollination, the progenies gave the highest seed yield, an indication of maternal

effect. Crosses 40-9 x 142-5, 40-9 x 27-3-7 and 40-9 x 23-3-4 gave high seed weight of 4.8, 3.9 and 2.7

kg/rai, respectively. Comparison of 11 Chinese cabbage hybrids with 3 commercial varieties showed that

hybrid 142-5 x 40-9 gave the highest head yield with 6,170 kg/rai, 36.3% higher than commercial

varieties. Other crosses such as hybrids 23 x 27, 23 x 142, 27-3-7 x 23-”.-4, 27-3-7 x 142-5 and 40-9 x

23-3-4 had good horticultural characteristics. These results indicate that improvement of parental lines

is necessary to enhance good F1 hybrid.

Key words : Chiness cabbage, hybrid


INTRODUCTION

Chinese cabbage is not only a popular

vegetable throughout the country but also an

economical crop for export. Chinese cabbage grown

in Thailand are both open-pollinated and F1 hybrid

varieties. They are all imported from abroad so

seed price is very high. Popular Chinese cabbage

grown by farmers are hybrid varieties from Japan,

Taiwan and Korea.

The Vegetable Seed Production Thailand

project was supported by the International

Development Research Centre, Canada through

the Faculty of Agriculture, Chiang Mai University.

The project aimed to develop varieties of some

Brassica crops including Chinese cabbage and

their seed production. Chinese cabbage varieties

were collected from local markets and other

countries. Good F1 hybrid varieties were developed

from these varieties (Tunsuwan et al., 1997). Seed

production of open-pollinated Chinese cabbage

was tested in few locations under highland condition

and the results were satisfactory. Chimonkon et al.

(1997) developed self-incompatibility

Department of Horticulture, Faculty of Agriculture, Chiang Mai University Chiang Mai 50200, Thailand.


characteristic of Chinese cabbage for F1 hybrid

seed production. This character can reduce labor

cost because emasculation of female parent is not

needed. Therefore, the price of F1 hybrid seeds is

reduced. Very often, the hybrid seeds can be

collected from both parents.

Self-incompatibility is a special

characteristic of some Brassica crops which

promotes cross-pollination and prevents self-

pollination of these crops. This characteristic is

controlled by multiple allelic genes at a single

locus. Pollen tube germination is inhibited by

stigma if they carry the same self-incompatible

genes (Nasrallah and Nasrallah, 1993; Nasrallah

et al., 1994; Isogai et al., 1987). The is a sporophytic

reaction in which 2n chromosome of S locus of

both parents controls the reaction (Gaude et al.,

1993; Pastuglia et al., 1997 and Nasrallah and

Nasrallah, 1993). Chinese cabbage also carries

self-incompatible genes of S locus. It is classified

into a sporophytic reaction (Opena et al., 1988).

The self-incompatible genes were used to develop

F1 hybrid Chinese cabbage for high yield and good

quality. Additionally, bee pollination was studied

for F1 hybrid seed production.


1. Hand pollination of an unopened flower budfor inbred seed production

The seeds of Chinese cabbage Inbred lines

23-3-4, 27-3-7, 40-9 and 142-5 were germinated

for two days and then vernalized at 4-5∞C for 15

days before they were planted in the field in winter

of 1999 at Chiang Mai University. The

inflorescence was covered with paper bag before

blooming. When some flowers of the inflorescence

started to open, the paper bag was removed. The

young unopened flowers were forced to open by

forceps. The pollen from opened flowers of the

same inflorescence was used to pollinate the young

flowers. All other opened flowers were removed.

The inflorescence was covered with the same

paper bag. The seeds were collected at maturity.

2. Testing for self-incompatibility levelsTwo methods of testing for self-

incompatibility levels of Chinese cabbage were

used: seed set analysis and fluorescent microscope.

In seed set analysis method, unopened and opened

flowers of the same inflorescence were pollinated

with pollen from the same plant (Shinohara, 1981

; Opena et al., 1988). Ten plants with healthy

inflorescence from each inbred lines were selected.

About 3-4 healthy inflorescences per plant were

covered with paper bags. The open flowers were

removed before bagging. Two to three days after

bagging, both unopened and opened flowers were

emasculated and pollinated with pollen from the

same inflorescence of the same plant. The unopened

and the opened flowers were marked with string

and the inflorescence was covered with the same

paper bag. When the seeds matured, the number of

pods that set and number of seeds per pod were

counted.

Fluorescent microscope method was used

to observe pollen tubes in styles of female flowers

(Kho and Baer, 1968). Unopened and opened

flowers were taken from a plant and pollinated

with pollen from the same plant. The opened

flowers were emasculated and cross-pollinated

with pollen from different varieties.

Three types of flowers were collected: the

self-pollinated of the unopened flower, the self-

pollinated of the opened flower, and the cross-

pollinated of the opened flower. The flowers were

put on a slide in a petri dish containing potassium

dichromate (K2CrO4) solution underneath the slide.

The potassium dichromate kept the atmosphere in

the petri dish at 98%. The flowers were left in the

petri dish for 24 hrs. The styles of the flowers were

dissected and placed in 1 N sodium hydroxide.

The sample was boiled at 60∞C for 30 minutes. The

styles were washed with distilled water, stained

with aniline blue solution (0.2% diluted in 2%

potassium phosphate), and kept for 24 hrs. in a


refrigerator. Then they were squashed with glycerin

on a slide and observed under fluorescent

microscope. The number of pollen tubes in each

style was counted.

3. F1 hybrid seed production by beesFour inbred lines of Chinese cabbage, 23-

3-4, 27-3-7, 40-9 and 142-5, were vernalized at 4-

5∞C for 15 days. They were planted in a field at a

distance of 30 cm between plants and 50 cm

between rows. Six seedlings were planted for each

line with 3 plants per row. Plot size was 2 x 2 m

with two plots per crossing. Each plot was planted

with 2 inbred lines, side by side. Twelve possible

crosses were made including reciprocals. A bee

hive was placed in each cross making sure that the

number of bees in each net was enough for

pollination. A salan net was used to cover the plots

to protect against insects and to keep honey bees

inside the net. The mature seeds were harvested

when pods dried.

4. Varietal evalution of F1 hybrid Chinesecabbage and control varieties

Randomized complete block design with 3

replications was used for this experiment. The F1

hybrid varieties were tested against 3 control

varieties such as Chang, Bomb 159 and Tapa 23.

Each treatment was planted in a 2 x 2 meters plot.

Eight plants were planted per plot at a spacing of

40 x 50 cm. Guard rows were planted around each

replication.

RESULTS

1. Hand pollination of an unopened flower budfor inbred seeds production

Inbred lines of Chinese cabbage were

different in seed set (Table 1). Line 40-9 gave the

highest seed weight of 0.165 g/plant, followed by

27-3-7 and 27 with 0.153 and 0.123 g/plant,

respectively. It was observed that unopened flowers

in the middle of an inflorescence produced the

highest seed number while unopened flowers at

the top and bottom of the inflorescence produced

few seeds or did not set seed at all. It was also

observed that the flowers which gave good seed

set were big but the yellow color of petals did not

yet show up.

2. Testing for self-incompatibility levelsTesting for self-incompatibility levels by

seed set analysis methodThe self-incompatibility levels of the plant

is indicated by the number of seeds set in the

opened flowers. Some inbred lines such as lines

27-3-7 and 40-9 showed strong self-

incompatibility. When the range of 0-25% of seed

setting was used to indicate strong self-

incompatibility levels, the strongest self-

incompatible line was 27-3-7 which showed 22.01

% seed set. It was followed by 40-9 and 23-3-4

with 24.72 and 29.24 % seed set, respectively.

When 25.5% of setting was used to indicate weak

self-incompatibility, the weak self-incompatible

Table 1 Seed weight from self pollinated Chinese cabbage.

Line Seed weight/plant (g)

40-9 0.165

27-3-7 0.153

27 0.123

23 0.076

142-5 0.047

23-3-4 0.032


Table 2 Levels of self-incompatibility of Chinese cabbage lines, tested by seed set analysis.

Number of pod Number of seed

Line Unopened Opened Unopened Opened Seed set1 Conclusion2

flower flower flower flower (%)

23 15 15 210 80 38.10 WSI

23-3-4 15 15 236 69 29.24 WSI

27 15 15 193 96 49.74 WSI

27-3-7 15 15 231 51 22.01 SI

40-9 15 15 267 66 24.72 SI

142 15 15 253 88 34.78 WSI

142-5 15 15 245 79 32.24 WSI

1 0-25%-self-incompatibility, 26-50% weak self-incompatibility, 51-75% weak self compatibility and 76-100% self-compatibility.2 SI = self-incompatibility

WSI = week self-incompatibility

lines were 23, 27, 142, and 142-5 (Table 2 and

Figure 1).

Testing of self-incompatibility levels byfluorescent microscope technique

Chinese cabbage inbred lines were tested

for their self-incompatibility levels by fluorescent

microscope technique. Pollen tubes in styles of

unopened and opened flowers were observed using

fluorescent microscope after they were self-

pollinated. The inbred lines showed weak self-

incompatibility, weak and levels (Table 3). The

weak self-incompatible lines, 23-3-4, 27, 27-3-7,

40-9, and 142, showed low percentage of pollen

tubes in styles. Inbred lines 23, 40, and 142-5

showed some pollen tubes in their styles. They

were classified in both weak self-incompatible

and weak self-compatible groups. The self-

compatible line was 23-3-1 which showed 80% of

pollen tubes in opened flowers when self-pollinated

(Figure 2).

Comparing the two methods of evaluating

self-incompatibility levels, the results were

somewhat different. Seed set analysis method

showed strong self-incompatibility levels in lines

27-3-7, 40-9, and 23-3-4. However, the fluorescent

microscope technique showed weak self-

incompatibility levels in lines 23-3-4, 27, 27-3-7,

40-9, and 142. Some pollen tubes were found in

these lines under the fluorescent microscope. This

method of evaluation might be more sensitive than

the seed set analysis method. The pollen tubes

which were found in these lines might not be able

to convey to seed setting.

3. Bee pollination for F1 hybrid seed productionFour Chinese cabbage inbred lines, 23-3-4,

27-3-7, 40-9, and 142-5, were reciprocally crossed

with the use of bees. Seed yield of the crosses is

shown in Table 4. Seed weight ranged from 0.03 to

4.8 kg/rai. Cross 40-9 x 142-5 gave the highest

seed yield, 4.8 kg/rai. It was followed by crosses

40-9 x 27-3-7 and 40-9 x 23-3-4 which yielded 3.9

and 2.9 kg/rai, respectively. Seed yield as such

was rather low for seed production. There might

be some problems in bee pollination. Probably, the

size of the salan net (2.5 x 2.5 m) may be too small

for bee activities. The bees tend to stay in the hive

or are held on the salan. Maternal effects were

detected in line 40-9 as a mother plant compared to

the other mother lines (Table 4).


Table 3 Levels of self-incompatibility of Chinese cabbage lines, tested by fluorescent microscope

technique.

Control Self pollinated Cross pollinated

Line Unopened Opened Unopened Opened Unopened Opened Conclusion

flower (%) flower (%) flower (%) flower (%) flower (%) flower (%)

23 - - 801 50 80 100 WSI2,WSC

23-3-1 - - 100 80 100 80 SC

23-3-4 - - 100 30 80 100 WSI

27 - - 50 30 30 100 WSI

27-3-7 - - 80 30 100 100 WSI

40 - - 80 50 100 50 WSI,WSC

40-9 - - 100 30 80 100 WSI

142 - - 30 30 100 80 WSI

142-5 - - 80 50 100 100 WSI,WSC

1 Number of pollen tube in style in percentage, 0-25%-self-incompatibility, 26-50% weak self-incompatibility, 51-75% weak

self-compatibility and 76-100% self-compatibility.2 WSI = weak self-incompatibility

SC = self-compatibility

WSC = weak self-compatibility

4. Varietal evaluation of F1 hybrid Chinesecabbage and control varieties

Evaluation of 11 F1 hybrid varieties of

Chinese cabbage obtained from the crosses against

3 control varieties showed that most F1 hybrid

varieties gave higher fresh head yield than the

control ones (Table 5).

Head fresh weight of F1 hybrid varieties

ranged from 3,665 to 6,170 kg/rai. Cross 142-5 x

40-9 gave the highest head fresh weight of 6,170

kg/rai followed by 23 x 142 and 27-3-7 x 142-5

which yielded 5,551 and 5,150 kg/rai, respectively,

These yields were significantly different from all

control varieties which gave head fresh yield

ranging from 3,625 to 4,527 kg/rai. Considering

head weight before trimming, the F1 hybrid

Figure 1 Self-compatible and self-incompatible

inflorescence of Chinese cabbage.Figure 2 Pollen tube in a style of female in

Chinese cabbage flower.


Table 4 Seed weight of F1 hybrid Chinese cabbage.

Cross Seed weight Cross Seed weight

(kg/rai) (kg/rai)

40-9 x 142-5 4.826 27-3-7 x 23-3-4 0.197

40-9 x 27-3-7 3.941 142-5 x 27-3-7 0.144

40-9 x 23-3-4 2.714 23-3-4 x 40-9 0.128

142-5 x 40-9 1.642 23-3-4 x 142-5 0.085

23-3-4 x 27-3-7 1.403 27-3-7 x 142-5 0.051

27-3-7 x 40-9 0.773 142-5 x 23-3-4 0.030

Table 5 Fresh weight before and after trimming, trimming percentage, and solidity of Chinese cabbage

head of F1 hybrid and control varieties.

Cross and Head yield Head weight Head weight Trimming Solidity of head HSI

variety (Kg/rai) before trimming after trimming (%) (g/cm3)

Cross142-5 x 40-9 6,170.0 a3 1,390.0 b 964.0 a 30.65 0.5719 a 1.228 e

23 x 142 5,551.0 b 1,329.0 b 867.3 b 34.74 0.5507 a 1.308 d

27-3-7 x 142-5 5,150.0 b 1,484.0 a 804.7 b 45.77 0.4566 c 1.311 d

27 x 23 4,911.0 c 1,248.0 b 767.3 c 38.52 0.4110 d 1.279 d

27-3-7 x 40-9 4,851.0 c 1,333.0 b 758.0 c 43.14 0.4916 b 1.356 c

40-9 x 142-5 4,736.0 c 1,181.0 c 740.0 c 37.34 0.5857 a 1.299 d

23 x 27 4,646.0 c 1,299.0 b 726.0 c 44.11 0.4499 c 1.432 b

40-9 x 27-3-7 4,414.0 d 1,107.0 c 689.7 d 37.70 0.4418 c 1.227 e

142 x 23 4,186.0 d 1,112.0 c 654.0 d 41.19 0.4511 c 1.289 d

40-9 x 23-3-4 3,785.0 e 974.0 d 591.3 e 39.29 0.5020 b 1.569 a

27-3-7 x 23-3-4 3,665.0 e 1,173.0 c 572.7 e 51.18 0.4243 d 1.513 a

VarietyChang 4,527.0 d 1,486.0 a 707.3 d 52.40 0.5269 a 1.404 b

Tapa 23 3,917.0 e 1,152.0 e 612.0 e 46.88 0.3997 d 1.343 c

Bomb 159 3,625.0 e 1,473.0 a 566.3 e 61.55 0.3931 d 1.427 b

C.V. (%) 7.65 6.38 7.65 - 6.58 3.35

LSD.05 588.33 135.78 91.926 - 0.053 0.076

1. Solidity = MHW/(0.524d12d2)

MHW = mean head weight

d1 = head width

d2 = head length

2. Head shape index; HSI = head width/head length

3. Means follow by the same letters indicate no differences at P = .05 by least significant difference.


varieties gave head weight ranging from 970 to

1,484 g (Table 5). The range was more or less

similar to fresh head weight of the control varieties

which ranged from 1,152 to 1,486 g. However,

when the outer leaves were trimmed off, most of

the F1 hybrid varieties showed higher head weight

than the control (Table 5, Figure 3 and 4). Head

weight of the F1 hybrid varieties after trimming

ranged from 572.7 to 964 g/head, while the control

varieties showed a range of 566.3 to 707.3 g/head.

The head weights of these varieties were

significantly lower than most of the F1 hybrid

varieties.

The control varieties showed higher

percentage of trimming than most (BE SPECIFIC)

of the F1 hybrid varieties (Table 5). Variety Bomb

159 gave the highest trimming percentage of

61.55%. A good control variety such as Chang

showed 52.40%. It was followed by variety Tapa

23 with 46.88%. The F1 hybrid varieties showed a

range of 30.65 to 51.18%. Therefore, the marketable

yield of most of the F1 hybrid varieties should be

higher than the control. Head solidity of the control

variety, Chang, was not significantly different

from high yielding F1 hybrid varieties, 142-5 x 40-

9 and 23 x 142. However, varieties Bomb 159 and

Tapa 23 were less solid than all of the F1 hybrid

varieties. High solidity of Chinese cabbage is

more perferable than low solidity.

Another important characteristic of Chinese

cabbage is head length. Long head is more

preferable than short one, therefore, high head

shape index is preferred.

Cylindrical head shape was observed in the

F1 hybrids 40-9 x 23-3-4 and 27-3-7 x 23-3-4 with

head shape index of 1.569 and 1.513, respectively,

which were significantly different from the control

varieties. However, there were many F1 hybrid

Figure 3 Head of F1 hybrid Chinese cabbage

142-5 x 40-9 (50) and control varieties

(1-Chang, 2-Bomb 159 and 3-Tepa 23).

Figure 4 Head of F1 hybrid Chinese cabbage 27-

3-7 x 23-3-4 (44) and control varieties

(1-Chang, 2-Bomb 159 and 3-Tapa 23).


Table 6 Horticultural characteristics of F1 hybrid Chinese cabbage.

Cross- Solidity of Thickness Head Color of Color of

Cross section of head of petiole shape outer leaves inner leaves

petiole

23 x 27 Flat Firm Thick Obovate Pale green Yellow

23 x 142 Flat Firm Semi Obovate Light green Yellow

27 x 23 Flat Firm Semi Obovate Pale green Yellow

27-3-7 x 23-3-4 Flat Firm Thick Obovate Pale green Yellow

27-3-7 x 40-9 Semiround intermediate Thick Obovate Light green Yellow

27-3-7 x 142-5 Flat Firm Thick Obovate Pale green Yellow

40-9 x 23-3-4 Flat Firm Thick Ovate Light green Yellow

40-9 x 27-3-7 Flat Firm Thick Obovate Pale green Yellow

40-9 x 142-5 Flat Firm Thick Obovate Pale green Yellow

142 x 23 Flat Firm Thick Obovate Pale green Yellow

142-5 x 27-3-7 Flat Firm Thick Obovate Light green Yellow

varieties which their head shape index ranged

from 1.227 to 1.311. Their heads were rather

round which is undesirable in the market.

Other horticultural characteristics of most

of F1 hybrid varieties were more or less the same

(Table 6). They had very tight heads, oval shape,

thick petioles, pale and light green color of outer

leaves, and yellow color of inner leaves.

The F1 hybrid varieties had certain levels

of disease resistance. Even though the hybrids

were not screened for disease resistance, soft rot is

a common disease for Chinese cabbage, The disease

was not observed on the F1 hybrid and the control

varieties.

DISCUSSION

Improvement of F1 hybrid varieties of

Chinese cabbage requires good male and female

parental lines with high levels of self-

incompatibility. Comparison ot self-

incompatibility test by using seed set analysis and

fluorescent microscope technique showed that the

former method was better than the later. Results

showed that lines 23, 23-3-4, 27-3-4, 40-9, and

142-5 were self-incompatible and may have good

potential for F1 hybrid seed production. Among

the lines tested for seed production using bud

pollination technique, line 40-9 gave the highest

seed weight of 0.165g/plant.

Production of F1 hybrid seeds of the crosses

showed that self-incompatibility could be utilized.

Results showed that there was maternal effect.

When line 40-9 was used as a female parent, the

crosses gave the highest seed weight.

Varietal evaluation of F1 hybrid Chinese

cabbage showed that F1 hybrid 142-5 x 40-9 gave

higher head weight than standard varieties.

However, the head shape was rather round. There

were other good potential F1 hybrids such as 23 x

27, 23 x 142, 27-3-7 x 23-3-4, 27-3-7 x 142-5 and

40-9 x 23-3-4.

CONCLUSION

Inbred lines 23, 23-3-4, 27-3-7, 40-9 and

142-5 were suitable to be used as parental lines due

to the high levels of self-incompatibility which is


good for F1 hybrid seed production. All the lines

gave high seed yield when used as a female parent.

Among these lines, 40-9 showed maternal effect.

It also gave high seed yield when inbred seeds

were produced by self-pollination of the unopened

flowers.

Bee pollination of F1 hybrid seed production

was not satisfied (SATISFACTORY?) because

seed yield was quite low. Production of F1 hybrid

seed by bee pollination is useful due to low cost in

inbred and hybrid seed production. However,

whether this technique is expropriated

(APPROPRIATE?) or not needs to be evaluated

further.

Most F1 hybrid varieties gave high head

yield but their heads were rather round which is not

desirable. Good potential F1 hybrid varieties were

142-5 x 40.9, 23 x 27, and 23 x 142.

LITERATURE CITED

Briggs, F.N. and P.F. Knowles. 1967. Introduc-tion to Plant Breeding. Reinhold Publishing

Corporation: A Subsidiary of Chapman-

Reinhold, INC., 426 p.

Chimonkon, C., M. Nikornpun and T. Tunsuwan.

1997. Varietal trial of Chinese cabbge,pp.

120-126. In National Vegetable Meeting,

15th, Bangkok,

Gaude, T., A. Friry, P.Heizmann, C. Mariac, M.

Rougier, I. Fobis and C. Dumas. 1993.

Expression of a self incompatibility gene in a

self compatible line of B.oleracea. Plant Cell5 : 75-86.

Isogai, A., S. Takayama, C. Tsukamoto, Y. Ueda,

H. Shozawa, K. Hinata, K. Okazaki and A.

Suzuki. 1987. S-locus-specific glycoproteins

associated with self-incompatibility in

Brassica campestris. Plant Cell Physiol.28(7) :1279-1291.

Kho, Y.O. and J. Baer. 1968. Observing pollen

tubes by means of fluorescence. Euphytica17 : 298-302.

Nasrallah, J.B. and M.E. Nasrallah. 1993. Pollen-

stigma signaling in the sporophytic self

incompatibility response. Plant Cell 5 : 1325-

1335.

Nasrallah, J.B., J.C. Stein, M.K. Kandasamy and

M.E.Nasrallah. 1994. Signaling the arrest of

pollen tube development in self-incompatible

plants. Science. 266 : 1505-1508.

Opena, P.T., C.G. Kuo and J.Y.Yoon. 1988.

Breeding and Seed Produciton of ChineseCabbage in the Tropic and Subtropics.

Technical Bulletin No.17. AVRDC. Taiwan.

P. 92.

Pastuglia, M., V. Ruffio-Chable, V.Delorme,

T.Gaude, C. Dumas and J.M. Cock. 1997. A

function S locus anther gene is not required

for the self incompatibility response in

B.oleracea. Plant Cell 9 : 2065-2076.

Shinohara, S. 1981. Principles of Vegetable SeedProduciton. Tsukuba International

Agriculture Training Center, Textbook V.C.

No.26. 225 p.

Tunsuwan, T., C. chaimongkul and M. Nikornpun.

1997. Hybrid Chinese cabbage seed

production project, pp. 105-119. In NationalVegetable Meeting, 15th, Bangkok.


Soybean Yield and Nutrient Compositionas Affected by Soil and Foliar Fertilizations

Chin Theng Phiv1, Chawalit Hongprayoon1,Peerasak Srinives2, Arunsiri Kumlung1 and Yongyuth Osotsapar1

ABSTRACT

Soil fertilizer application and foliar fertilization offer a possible means of increasing soybean

[Glycine max (L.) Merr.] yield in Thailand but little is known of appropriate foliar fertilizer use to

supplement soil fertilization. Field experiment was conducted twice to determine the effects of soil N P

K fertilization together with foliar fertilizers containing macronutrients and micronutrients on growth,

yield and nutrient composition of soybean (Sukhothai 1 and KUSL 20004 cultivars). The treatments were

arranged in 3 ¥ 3 factorial experiment in randomized complete block. Three methods of soil fertilization

were control (S0), 18 kg N ha-1 at 7 days after seeding (DAS) (S1), and 18 kg N ha-1 at 7 DAS + 18 – 18

– 18 kg N – P2O5 – K2O ha-1 at 30 DAS (S2). Foliar fertilizer contained both macronutrients and

micronutrients. The three methods of foliar fertilization were control (F0), 3 applications at 34, 42 and

49 DAS (F1) and 6 applications at 20, 27, 34, 42, 49 and 56 DAS (F2). Throughout the studies, soil and

foliar fertilizations did not significantly affect growth, yield and yield components of soybean. The

concentration of N P K Fe and Zn in shoot at 68 DAS and N P K and Ca in leaves at 89 DAS were not

consistently affected by soil and foliar fertilizations. The remarkable effects of soil fertilizers on the

concentrations of Ca Mn and Cu in shoot, and Mg Fe Mn and Cu in leaves were observed. Foliar

fertilizations increased Fe and Cu concentrations in leaves. The nutrient concentrations of soybean shoot

and leaves under this investigation were in sufficient ranges which were agreeable with soil test results.

This finding indicated that the soil can provide sufficient nutrients for soybean growth and yield under

this condition. Therefore, soil and foliar fertilization will not be economically feasible.

Key words: soybean, soil fertilizer, foliar fertilizer, macronutrients, micronutrients

1 Department Soil Science, Faculty of Agriculture, Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140 , Thailand.2 Department of Agronomy, Faculty of Agriculture, Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140 , Thailand.


INTRODUCTION

Soybean [Glycine max (L.) Merr.] has

efficient symbiotic nitrogen fixation that can

provide over 80% of the nitrogen in the crop at

maturity, however in most cases fixation accounts

for 25 – 75% of total plant nitrogen (Deibert et al.,

1979). One metric ton of soybean grain removes

approximately 59 kg N, 60 kg P, 19 kg K and

different amount of other macronutrients and

micronutrients (Fageria et al., 1997). To maintain

soil fertility, at least nutrients (other than nitrogen)

removed in the grain should be returned into soil

by applying the fertilizers. Soybeans grown in

Thailand were reportedly response to N P and K

fertilization. The recommendation rates for soybean

producers are 18 kg N, 36 kg P2O5 and 18 kg K2O

ha-1 for clay loam and silty loam soils. However,

only 36 kg P2O5 ha-1 was applied at planting for

soybean grown in clayey texture soil (Division of

Soil Science, 1999). The suitability of these

fertilizer rates to different soybean cultivars grown

in different soil types need further clarification.

Foliar fertilization of soybean with a liquid

N–P–K–S fertilizer during pod filling period (R5

to R6) has received considerable attention in various

parts of the United States since 1975 (Garcia and

Hanway, 1976). During seed growth period,

soybean plants translocate large quantities of N, P,

K and S from leaves to developing pods and seeds

resulting in decreasing photosynthesis and

ultimately premature senescence (Sinclair and de

Wit, 1975). Soybean yield was increased by foliar

boron application due to increasing final number

of branches and pods on branches (Schon and

Blevins, 1990). Foliar spray containing Ca and B

improved pod development and pod retention of

soybean in field condition (Weaver et al., 1985).

Furthermore, increasing soybean yield from foliar

B + Mg treatment was resulted from an increased

number of pods on the main stem and branches

(Reinbott and Blevins, 1995).

Some information is available on the

effectiveness of foliar applied fertilizers for

leguminous crops in Thailand. Foliar applications

of B and Fe can ameliorate the deficiency problems

of these elements on blackgram, peanut and

mungbean. Two foliar applications of borax, at a

very low rate of 50 gha-1, during flower

development and pod set, were as effective in

correcting boron deficiency in backgram as a high

rate applied to soil (Rerkasem, 1989). Five foliar

applications of 0.5% FeSO4 solution at 10, 20, 30,

40 and 50 days after emergence was the most

effective way to alleviate iron chlorosis, and

substantially improved yield of peanut (Ratanarat

et al., 1990). Mungbean plants given a foliar spray

with a nutrient solution contained 0.5% Fe, Zn and

Mn recovered from the chlorosis and produced

greater number of pods (Oonkasem and

Thavarasook, 1988). Foliar fertilization of a

solution of 5 gL-1 ferrous sulfate was effective in

correcting chlorosis that was induced by iron

deficiency, and it enhanced both growth and yield

of susceptible mungbean cultivars (Ohwaki et al.,

1997).

At present, foliar fertilization together with

soil application have been practiced in many areas

of Thailand but limited information was available

(Pongsakul and Ratanarat, 1999). Suanmalee et al.

(1990) reported that only soil N P K fertilization or

soil fertilization in combination with foliar

applications of N P K fertilizers significantly

increased soybean yield in Pak Chong soil series

(clayey, kaolinitic, Oxic Paleustults) and Wang

Saphung soil series (fine – clayey, mixed Ultic

Haplustalfs). However, N P K foliar fertilization

without soil fertilizer application did not improve

soybean yield. It is obvious that research on the

effects of foliar fertilizers containing both

macronutrients and micronutrients on soybean

yield in this country is almost non. More studies

are, therefore, needed to clarify the roles of foliar

fertilization on soybean yield improvement. The

objective of this study was to determine the effects

of soil N P K fertilization together with foliar

fertilizers containing macronutrients and

micronutrients which applied during vegetative

and reproductive stages on growth, yield and

nutrient composition of soybean.


Field experiment was conducted on

Kamphaeng Saen soil series (fine-silty, mixed,

Typic Haplustalfs) at the Asian Regional Center

(ARC) of the Asian Vegetable Research and

Development Center (AVRDC), Kamphaeng Saen

Campus, Kasetsart University during November

2001 – March 2002 and was repeated during May

– August 2002. Before land preparation of each

cropping, a composite soil sample was collected



from the depth of 0 to 20 cm and tested for pH,

electrical conductivity (EC), organic matter (OM),

P, K, Ca, Mg, Fe, Mn, Zn and Cu. Briefly, pH was

analyzed in a 1 : 1 soil/water ratio, EC of saturated

extract by EC meter, organic matter by Walkley

and Black method, P by the Bray P–2 method, K,

Ca and Mg by ammonium acetate method (Jackson,

1973). Soil Fe, Mn, Zn and Cu were detemined by

atomic absorption spectrophotometry on the DTPA

extract (Linsay and Norvel, 1978). The soil test

values for the first and second trials are shown in

Table 1.

Before planting of soybean, land was

ploughed once and harrowed twice. Each plot

measured 5 m in length and 3 m in width. The

planted spacing between hills was 25 cm and

between rows was 60 cm. Soybean cultivars

‘Sukhothai 1’ and ‘KUSL 20004’ were used in the

first and second trials, respectively. Soybean seed

was inoculated with rhizobium bacteria before

seeding. About 3-4 seeds were dropped in each

hill. Upon establishment, plants were thinned to 2

plants per hill. To minimize moisture stress, furrow

irrigation was applied throughout the growing

season. Weeds were controlled chemically and

hand weeded as necessary.

The experimental design was randomized

complete block with four replications. The fertilizer

treatments were factorially arranged in 3¥3 soil

application and foliar fertilization methods. Soil

fertilizer application methods were control (So),

18 kg N ha-1 at 7 days after seeding or DAS or

growth stage Vc (S1) and 18 kg N ha-1 at 7 DAS

+ 18-18-18 kg N- P2O5-K2O ha-1 at 30 DAS or

growth stage R1 (S2). Ammonium sulfate (21–0–0)

and compound fertilizer (15–15–15) were used in

soil fertilizer treatments. The foliar fertilization

methods were control (water sprayed plots, F0),

foliar fertilizer application 3 times at 34, 42 and, 49

DAS or growth stages R2, R3 and R4 (F1) and

application 6 times at 20, 27, 34, 42, 49 and 56

DAS or growth stages V4, V6, R2, R3, R4 and R5

(F2). The concentration of each element in solution

applied at early growth stages was lower than

those used at later stages. The detail of soil and

foliar fertilizer applications were given in Table 2.

The sources of nutrients for foliar fertilizer mixture

were urea (46–0–0), ammonium sulfate (21–0–0),

potassium nitrate (13–0–46), magnesium sulfate

(10% Mg and 14% S), Fe EDTA (13.2% Fe), Zn

EDTA (14% Zn), Mn EDTA (13% Mn), Cu EDTA

(14% Cu) and calcium-boron-molybnenum

solution (6% Ca, 2% B and 0.2% Mo) under the

commercial trade name B PlusTM*.

Alkyl aryl polyethoxylate and sodium

alkylsulfonate alkylate 60% adjuvant was used at

the rate of 0.1 mlL-1 . All foliar fertilizer solutions

were applied with backpack hand sprayer in the

early morning (8 – 10 am). The nozzle size was

adjusted to deliver an appropriate rate and uniform

distribution. Foliar fertilizer mixtures were sprayed

uniformly across each plot.

Table 1 Soil test values for the first and second fertilizer trials conducted at ARC, Kasetsart University,

Kamphaeng Saen.

Trial pH EC OM P K Ca Mg Fe Mn Zn Cu

(dSm-1) (%) (mg kg-1)

1 6.5 0.8 1.4 238.4 131.6 1721.5 246.6 30.8 1.3 36.2 1.1

2 6.8 1.1 1.9 130.1 143.1 1921.6 232.7 24.9 1.0 52.3 1.2

* Use of trade name does not imply endorsement of the product name nor criticism of similar ones not named.


To study plant growth parameters, 5 plants

were collected from two sampling rows at 46, 54,

69 and 89 DAS or growth stages R4, R5, R6 and

R7 of the first trial and 65 and 79 DAS or growth

stages R6 and R7 of the second trial. The plants

were cut at ground level and determined for node

number, branch number and plant height. After

growth measurement, plant samples were air-dried

at 70∞C for 3 days to measure dry weight. Whole

plant samples were taken at 68 DAS or growth

stage R6. They were washed thoroughly with tap

water containing mild detergent, rinsed three times

with deionized water, dried and ground for chemical

analysis (Schon and Blevins, 1990).

Ten plants were randomly collected from

sampling rows at maturity and counted for number

of pods per plant and number of pods on branches.

One hundred pods were randomized and counted

for the total number of seeds to obtain average

number of seeds per pod.

The soybean was harvested at maturity

when 95% of total pods turned brown (R8). Grain

yield was measured from a 5 m length of the center

two rows. Plant number per plot was recorded

before threshing. Grain yield was weighed and a

grain sample was collected from each plot to

determine grain moisture. The grain yield was

adjusted to 13% moisture. Subsamples from each

plot were obtained to detemine 100 - seed weight.

Samples of trifoliate leaves (including

petioles) consisting of 25 uppermost fully expanded

leaves were randomly collected one week before

harvesting from each plot to analyze for the nutrient

concentration of leaves in the last growth stage.

Leaf samples were washed in 0.1 N HCl for

approximately 30 seconds, rinsed in deionized

water for 30 seconds to remove of residual foliar

fertilizer (Moraghan, 1991). Leaf samples were

dried in a hot air oven at 70∞C and ground to pass

a 2-mm screen. Plant samples were digested with

perchloric/nitric/sulfuric tertiary acid. Total N was

determined by auto analyzer and P by vanado-

molybdate colorimetry. The concentrations of K,

Ca, Mg, Fe, Mn, Zn and Cu were determined by

atomic absorption spectrophotometry (Walsh,

1971).

Data were analyzed with analysis of

variance (ANOVA) using IRRISTAT package.

The treatment means were separated using

Duncan’s multiple range test.

Table 2 Soil fertilizer rates, foliar fertilizer concentrations and application schedule of the fertilizer

trials.

Treat- Dates and rates of soil and foliar fertilizer application

ment 7DAS* 20DAS 27DAS 30DAS 34DAS 42DAS 49DAS 56DAS

(kg N ha-1) (2L/plot) (2L/plot) (kg N-P2O5-K2O ha-1) (3L/plot) (4L/plot) (4L/plot) (4L/plot)

S0 - - - - - - - -

S1 18 - - - - - - -

S2 18 - - 18–18–18 - - - -

F0 - - - - - - - -

F1 - - - - P3 P3 P3 -

F2 - P1** P2 - P3 P3 P3 P3

* DAS = Days after seeding;** P1, P2 and P3 = Concentration (mg kg-1) of elements in the solution as follows :

P1 = 250 N, 50 P, 100 K, 25 S, 15 Mg, 30 Ca, 15 Fe, 10 Mn, 7.5 Cu, 7.5 Zn, 10 B, 1 Mo

P2 = 500 N, 100 P, 200 K, 50 S, 30 Mg, 45 Ca, 30 Fe, 20 Mn, 15 Cu, 15 Zn, 20 B, 2 Mo

P3 = 750 N, 150 P, 300 K, 75 S, 45 Mg, 60 Ca, 45 Fe, 30 Mn, 22.5 Cu, 22.5 Zn, 30 B, 3 Mo



1. Plant growthThe effects of soil and foliar fertilizer

applications on plant height, number of nodes per

plant, number of branches per plant and dry weight

of shoot are shown in Tables 3 and 4. Soil and

foliar fertilizer treatments did not influence plant

height at 46 days after seeding (DAS) of the first

trial and 79 DAS of the second trial (Table 3).

However, soil fertilizer treatments decreased plant

height at 69 DAS of the first trial but increased that

of the second trial. The heavy rainfall at that plant

age in the first trial may contribute to this variation.

Faliar treatments did not show any influence on

soybean height. The results corroborated the work

of Edmisten et al. (1994) which indicated that

foliar treatments of complete fertilizers at 10 – 14

days intervals starting at 2 – 3 leaf stage had no

effect on height of cotton. There was an interaction

between soil and foliar treatments on plant height

at 79 DAS of the second trial. This interaction

indicated that one application of soil fertilizer (S1)

increased plant height when foliar fertilizer was

not used.

Soil fertilizer and foliar fertilizer

applications did not result in a significant increase

in number of nodes per plant (Table 3). The result

revealed that the effect of fertilizer treatments on

plant height may be due to the increase in length of

the internodes.

There was no response of shoot dry weight

to soil fertilizer treatments. Foliar fertilizer

application, however, significantly increased dry

weight at 89 DAS of the first trial (Table 4). The

interaction between soil and fertilizer treatments

remarkably affected dry weight at 54 DAS of the

first trial. This interaction indicated that one

application of soil fertilizer (S1) in plots without

foliar fertilizer (F0) tended to increase dry weight

of soybean. In contrast to the result of this

experiment, Haq and Mallarino (2000) indicated

that foliar fertilization with N P K at early season

seldom influenced dry weight of soybean at the R2

Table 3 Plant height (cm) and number of nodes per plant of soybean after soil and foliar fertilizations.

Fertilizer Height (1st trial) Height (2nd trial) Node no. (1st trial) Node no. (2nd trial)

treatment 46 DAS 69 DAS 65 DAS 79 DAS 46 DAS 69 DAS 65 DAS

SoilS0 60.4 91.9 b 85.6 a 86.7 11.3 14.7 16.3

S1 58.9 87.7 a 90.9 b 89.6 11.2 15.3 16.1

S2 56.4 87.9 a 91.0 b 88.2 11.2 14.6 16.5

F- test ns * ** ns ns ns ns

FoliarFo 56.7 88.1 87.9 87.5 a 11.3 14.9 16.8

F1 59.7 90.6 90.4 89.3 ab 11.0 15.0 16.5

F2 59.3 88.2 89.4 87.5 a 11.3 14.9 16.5

F-test ns ns ns ns ns ns ns

S x F ns ns ns * ns ns ns

CV (%) 9.4 4.9 4.5 4.2 5.4 5.0 5.4

ns = not significant different ,* significant different at P £ .05, ** significant different at P £ .01

Means in each column followed by the same letter are not different by DMRT at P £ .05


growth stage.

It was noted that foliar treatment did not

significantly increase number of branches per plant

in the cultivar ‘Sukhothai 1’ (Table 5). Therefore,

the promotion of six split foliar fertilizer

applications (F2) on plant dry weight was likely

due to the effects of treatments on increasing dry

weight of leaves, branches or stems. Similar results

were obtained by Poole et al. (1983).

Soybean is relatively sensitive to

phytotoxicity due to foliar fertilizer treatments

(Weaver et al., 1985). The ammonium

polyphosphate mixed solution damaged the foliage

more than potassium polyphosphate mixed solution

or urea alone. Repeated applications of foliar

fertilizer increase leaf injury (Parker and Boswell,

1980). Visual leaf injury evaluation in this

experiment was made 3 days after each spray.

Only less than 5% of leaf area was affected by F1

treatment, a little more leaf burn developed on

mature leaves sprayed with F2 solution. Although

leaf burn was not serious and the plants recovered

within two weeks without an adverse effect on

plant growth, this may cause negative effect on

foliar treatments in general.

The number of branches per plant of

‘KUSL 20004’ soybean from the second trial was

shown in Table 6. Soil and foliar fertilizer

treatments did not cause a considerable increase in

number of branches per plant at 89 DAS of the first

trial (Table 5), and 65 DAS of the second trial

(Table 6).

Soybean canopy was relatively dense from

35 DAS. Foliar application tended to increase

humidity in leaf canopy which was favorable for

the growth of pathogens and pest control by

chemicals was not effective under this condition.

2. Soybean yield and yield componentsSoybean yield of the first trail is shown in

Table 5. The yield of the second trial is not presented

due to the damage of plants by white flies during

the final stage of seed filling which seriously

affected seed yield but not some yield components

(Table 6).

Soil and foliar fertilizer applications had

Table 4 Dry weight (kg ha-1) at different stages of soybean growth after soil and foliar fertilizations.

Fertilizer First trial Second trial

treatment 54 DAS 69 DAS 89 DAS 65 DAS 79 DAS

SoilS0 544 1995 2515 2562 2608

S1 586 2101 2733 2494 2547

S2 536 2087 2523 2419 2679

F – test ns ns ns ns ns

FoliarFo 539 2039 2572a 2395 2673

F1 565 2048 2399a 2633 2603

F2 560 2096 2800b 2444 2559

F – test ns ns * ns ns

S x F ** ns ns ns ns

CV (%) 11.5 14.3 13.9 12.8 11.9

ns = not significant different, *significant different at P £ .05, ** significant different at P £ .01



Table 5 Yield and yield components of ‘Sukhothai 1’ soybean in the first trial.

Fertilizer Branches/plant Pods/plant Seeds/plant Weight of Grain yield

treatment 89 DAS 46 DAS 89 DAS 89 DAS 100 seeds (g) (kg ha-1)

SoilSo 3.1 8.8 47.6 140.8 14.7 2254

S1 2.9 9.2 51.2 142.5 14.3 2225

S2 2.6 9.4 53.2 139.3 14.0 2238

F – test ns ns ns ns ns ns

FoliarFo 2.7 9.1 52.5 139.2 14.8 2229

F1 3.0 8.1 51.0 140.8 14.0 2281

F2 2.9 10.2 49.2 140.7 14.2 2208

F – test ns ns ns ns ns ns

S x F ns ns ns ns ns ns

CV (%) 29.8 22.9 14.8 4.70 8.2 16.8

ns = not significant different

Table 6 Yield and yield components of ‘KUSL 20004’ soybean in the second trial.

Fertilizer Branches/plant Pods on main stem Pods on branches Seeds/pod Seeds/plant

treatment 65 DAS 65DAS 79 DAS 65 DAS 79 DAS

SoilS0 3.6 39.9 39.5 20.0 19.4 2.4 141.7

S1 3.4 40.9 40.1 20.6 19.5 2.5 140.5

S2 3.4 40.8 40.6 24.3 19.4 2.3 141.1

F – test ns ns ns ns ns ns ns

FoliarF0 3.6 40.3 40.0 21.1 19.9 2.4 141.0

F1 3.3 40.9 40.5 20.0 19.0 2.5 139.7

F2 3.6 42.6 39.7 23.8 19.4 2.4 143.6

F – test ns ns ns ns ns ns ns

S x F ns ns ns ns ns * ns

CV (%) 15.2 7.8 6.5 24.8 22.3 9.4 3.8

ns = not significant different, *significant different at P £ .05

no significant effects on seed yield of soybean in

the first trial. Even, split application of foliar

fertilizer (F1 and F2) did not increase seed yield

over the control (Table 5). Further increasing to

six - split application gave no increase in seed

yield. On the contrary, Suanmalee et al. (1990)

reported that soil fertilization significantly

increased yield of soybean grown in Pak Chong


and Wang Saphung soil series. Supplementing

with six applications of foliar fertilizers in 10 days

interval did not further increase grain yield in

either locations. The effective response of soybean

yield to soil fertilizer treatments in both soils was

mainly due to low level of available P and K.

The effects of fertilizer treatments on yield

components of both trials are shown in Table 5 and

6. Total pods per plant at 46 and 89 DAS of the first

trail was not increased by increasing soil and foliar

fertilizer applications. Average number of pods

per main stem and pods on branches per plant were

also slightly affected by soil and folair fertilizer

applications. One application of soil fertilizer (S1)

or three split applications of foliar fertilizers (F1)

slightly increased the average number of seeds per

plant but not on seed size of the first trial (Table 5).

Both soil and foliar fertilizer treatments did not

affect branch number, pod number per main stem,

pod number on branches, number of seeds per pod,

and number of seeds per plant of the second trial

(Table 6). Our results are in contrary to the finding

of Parker and Boswell (1980) which indicated

yield reduction of soybean in NPKS foliar treated

plots and presented the positive correlation between

leaf injury and yield depression. Little leaf injury

produced from foliar fertilizer treatments could

possibly explain a lack of positive yield response

or a small yield decrease. It did not result in a

significant yield reduction (Haq and Mallarino,

2000). In order to avoid leaf burning and to improve

the chance of positive yield response, Poole et al.

(1983) suggested that foliar fertilization should be

conducted before 0800 or after 1700 hours which

is not practical to general field conditions. Although

our sprayed solution contained both boron and

magnesium but the result of foliar application was

not in agreement with the works of Schon and

Blevins (1990), and Reinbott and Blevins (1995)

which suggested that foliar treatments with both

nutrients promoted higher soybean yield, mainly

due to increase in number of branches per plant,

number of pods on branches, and seed size.

3. Shoot and leaf nutrient concentrationsSoil and foliar fertilization treatments did

not show significant effect on N P K and Mg in

shoot sampled at 68 DAS (Table 7). However, the

effect of soil fertilization on shoot Ca content was

inconsistent. Calcium concentration in shoot from

soil fertilizer treated plots of the first trial was

relatively lower than control. Little influence of

soil treatments on Ca in shoot was noted in the

second trial. The data in Table 5 showed slight

trend towards increasing shoot dry weight with

soil fertilization. The relatively larger biomass

production in these treatments may contribute to

the dilution of Ca in shoot.

Concentrations of Fe Zn Mn and Cu in

soybean shoot were shown in Table 8. Shoot Fe

and Zn were not affected by soil fertilizer treatments

but two split applications of soil fertilizers (S2)

remarkably increased Mn and Cu in soybean shoot

of the first trial and Cu in shoot of the second trial.

The content of Zn Mn and Cu was not affected by

foliar treatments of both trials. Shoot Fe declined

in foliar treated plant of the first trial but tended to

increase in the second trial. This indicated the

inconsistent effect of foliar fertilization on Fe

content in soybean shoot.

Concentration of macronutrients in leaves

sampled at 89 DAS are shown in Table 9. Soil and

foliar fertilizer applications did not significantly

increase the concentration of N P K and Ca in

leaves, but one application of soil fertilizer

(S1)remarkably increased leaf Mg content.

Macronutrient concentration in leaves from all

treatments are in sufficient range. Our data are in

contrary with the finding of Boote et al. (1980) that

foliar application of N P K and S during pod filling

stages increased the concentration of all the

elements in soybean leaves without significantly

improved seed yield. Generally, soybean plant

requires high amount of N for seed production.

The major part of N is accumulated in seed during

pod filling stages. As much as 75% of the total N

is found in the seeds at harvest time (Vasilas et al.,


Table 7 N, P, K,Ca and Mg concentrations (% dry weight) in shoot at 68 DAS of both trials.


treatment N P K Ca Mg N P K Ca Mg

SoilSo 2.80 0.22 2.27 1.58a 1.31 2.18 0.21 2.39 1.17 0.98

S1 2.79 0.23 2.29 1.44b 1.23 2.42 0.24 2.59 1.19 1.02

S2 2.73 0.24 2.33 1.48b 1.24 2.23 0.23 2.48 1.18 1.06

F – test ns ns ns * ns ns ns ns ns ns

FoliarFo 2.73 0.22 2.16 1.51 1.25 2.18 0.22 2.39 1.12 0.99

F1 2.78 0.24 2.37 1.53 1.31 2.27 0.23 2.57 1.21 1.04

F2 2.82 0.23 2.36 1.48 1.28 2.38 0.23 2.49 1.20 1.01

F – test ns ns ns ns ns ns ns ns ns ns

S x F ns ns ns * ns ns ns ns ns ns

CV (%) 11.5 9.1 9.3 7.4 6.3 13.8 9.2 10.0 13.1 8.1

ns = not significant different, *significant different at P £ .05


Table 8 Fe, Zn, Mn and Cu concentrations (mg kg-1) in shoot at 68 DAS of both trials.


treatment Fe Zn Mn Cu Fe Zn Mn Cu

SoilS0 150.8 18.4 67.7 b 9.4 a 77.4 15.3 27.6 7.0 a

S1 172.7 23.5 59.6 a 10.5 a 99.9 19.3 32.0 11.6 b

S2 175.3 25.7 72.4 b 16.6 b 100.2 19.9 31.4 12.4 b

F – test ns ns * ** ns ns ns **

FoliarF0 195.0 b 23.6 70.1 12.6 90.4 18.0 27.6 10.6

F1 147.7 a 21.3 63.4 12.7 90.3 18.5 33.0 9.3

F2 165.7 a 22.7 66.2 11.2 96.7 18.0 30.3 10.7

F – test * ns ns ns ns ns ns ns

S x F ns ns ** ** ns ns ** ns

CV (%) 28.0 38.4 16.1 22.6 30.2 32.2 18.0 27.0

ns = not significant different, * significant different at P £ .05, ** significant different at P £ .01



1995). The results of our experiments indicated

that N concentration in soybean leaves collected

one week before harvesting was not affected by

either soil or foliar fertilizer treatments. A well

established soybean-rhizobium symbiosis from

inoculation may be effective in providing enough

N to plants.

It is important to note that the soil used in

this experiment was previously grown to soybean

and amended with duck manure and chemical

fertilizers. The roots and stover were incorporated

to the soil after harvest. The C:N ratio of soybean

roots and stover was favorable for fast

mineralization and would provide some available

N to soybean in the succeeding crop (Goss et al.,

2002). The residual N benefit of soybean stover to

succeeding crop was 13.16% of their total N or

equal to 12.7 kg N ha-1 (Yataputanon et al., 2002).

Nitrogen gained from symbiotic fixation together

with the mineralized N from soil organic matter

may have contributed to sufficiency of this element

in untreated control.

The soil and foliar fertilizer applications

did not affect the concentration of P K and Ca in

leaves but one application of soil fertilizer (S1)

significantly increased Mg content in leaves as

compared to control (Table 9). However, there

was no marked influence of foliar fertilization on

Mg concentration in leaves. The result of leaf

analysis at 89 DAS also revealed that soil and

foliar fertilization did not change nutrient

composition especially N P K and Ca as compared

to control. The values of N P K Ca and Mg were in

sufficient ranges for soybean at this growth stage.

These were consistent with the adequacy of

available P, extractable K Ca and Mg in Kamphaeng

Saen soil series. It is obvious that these nutrients

were not the limiting factors under the studied

conditions.

The concentration of Fe Zn Mn and Cu in

leaves sampled at 89 DAS are shown in Table 9.

Soil fertilizer application did not affect the

Table 9 N, P, K, Ca, Mg, Fe, Mn, Zn and Cu concentrations in leaves of ‘Sukhothai’ 1at 89 DAS of the

first trial.

Fertilizer N P K Ca Mg Fe Zn Mn Cu

treatment (%) (mg kg-1)

SoilS0 2.51 0.26 1.98 2.05 0.80 b 297 a 27.0 75.9 a 14.6 a

S1 2.45 0.27 2.03 2.16 1.03 a 219 b 29.7 97.8 b 15.6 b

S2 2.45 0.28 2.08 1.97 0.96 ab 215 b 28.9 99.8 b 15.9 b

F – test ns ns ns ns ** ** ns * *

FoliarF0 2.43 0.27 2.03 2.11 0.98 172 a 26.6 a 82.7 a 14.8 a

F1 2.37 0.28 2.04 2.05 0.95 220 b 25.9 a 91.0 b 15.0 a

F2 2.47 0.27 2.03 2.01 0.89 237 b 33.6 b 99.8 c 15.9 b

F – test ns ns ns ns ns ** * * *

S x F ns ns ns * ns ns ns ns ns

CV (%) 18.1 8.7 7.7 10.3 14.8 26 23.4 16.7 12.3

ns = not significant different, * significant different at P £ .05, ** significant different at P £ .01

Means in each column followed by the same letter are not different by DMRTat P £ .05


concentration of Zn but one application of soil

fertilizer (S1) remarkably increased Fe Mn and Cu

concentration in leaves. In addition, three

applications of foliar fertilizer (F1) significantly

increased Fe and Mn concentration while six

applications (F2) markedly increase concentration

of Zn and Cu in leaves. Our finding is in agreement

with the work of Bednarz et al. (1999) in cotton.

However, none of the four elements were deficient

in untreated plots and the increases of Zn and Cu

were beyond the plant required concentration

(Reuter ond Robinson, 1997).

Application of foliar fertilizers containing

macronutrients and micronutrients at reproductive

stage have been shown to increase soybean seed

yield in some studies (Schon and Blevins, 1990;

Smith et al., 2000). However, the other studies

showed that foliar fertilization of soybean either

did not influence or decrease yield (Parker and

Boswell, 1980; Freeborn et al., 2001). Generally,

nitrogen limitation of this crop during early to mid

pod filling stages is due to rapidly decreasing of

N2-fixation by Bradyrhizobium spp. (Haper, 1987).

Furthermore, root activity also decreases during

that growth period and nutrient uptake is not

sufficient to meet the seed demand for nutrients

(Garcia and Hanway, 1976). However, the contrary

result from the study on 15N-labeled urea absorption

and translocation of soybean indicated that changes

in the rate of nutrient absorption by root during

pod-fill were minimal and unlikely to be a major

factor determining the effectiveness of foliar

fertilization (Vasilas et al., 1978). Early season

foliar fertilization for soybean was also studied if

foliar application could increase P and K supplied

to young plants (Haq and Mallarino, 2000).

However, they reported that foliar fertilization of

soybean with various nutrient mixtures resulted in

very small and infrequent yield increase. Addition

of a mixture of micronutrients to the N P K S

fertilizer did not result in additional yield response

(Mallarino et al., 2001). These findings are in

agreement with the result of our experiment which

indicated that three-split foliar fertilization at

reproductive stage (F1) or two-split applications

at early season together with four-split applications

at seed filling stage (F2) did not increase yield of

irrigated soybean in this fertile soil. In addition,

positive response to foliar fertilization tended to

occur when soil or weather condition reduced

plant growth and nutrient availability (Haq and

Mallarino, 2000). It is obvious from the result of

soil test before planting, shoot analysis at 68 DAS

and leaf analysis at 89 DAS that there was no

limited nutrient for soybean under this studied

condition. Soil and foliar fertilizations in this

fertile soil, therefore, will not offset the application

costs.

CONCLUSIONS

Three methods of soil fertilization and three

methods of foliar fertilization did not significantly

affect growth, yield and yield components of

soybean. The concentrations of nutrients in shoot

at 68 DAS and leaves at 89 DAS were not

consistently affected by soil and foliar fertilizations.

The nutrient concentrations of soybean shoot and

leaves were in sufficient ranges. This finding

indicated that soil can provide sufficient nutrients

for soybean growth and yield under this condition.

LITERATURE CITED

Bednarz, C.W., N.W. Hopper and M.G. Hickey.

1999. Effects of foliar fertilization of Texas

Southern High Plains cotton : Leaf phosphorus,

potassium, zinc, iron, manganese, boron,

calcium and yield distribution. J. Plant Nutri.22 : 863-875.

Boote, K.J., R.N. Gallaher, W.K. Robertson, K.

Hinson and L.C. Hammond. 1980. Effects of

foliar fertilization on photosynthesis, leaf

nutrition, and yield of soybeans. Agron. J. 72

: 271-275.

Deibert, E.J., M.D. Jeriego and R.A. Olson. 1979.


Utilization of 15N fertilizer by nodulating and

nonnodulating soybean isolines. Agron. J. 71

: 717-723.

Division of Soil Science. 1999. FertilizerRecommentation for Field Crops.Department of Agriculture, Bangkok, 60 p.

(In Thai).

Edmisten, K.L., C.W. Wood and C.H. Burmester.

1994. Effects of early-season foliar

fertilization on cotton growth, yield and

nutrient concentration. J. Plant Nutr. 17 :

683-692.

Fageria, N.K., V.C. Ballger and C.L. Jones. 1997.

Growth and Mineral Nutrition of FieldCrops. 2nd ed. Marcel Dekker Inc., New

York. 624 p.

Freeborn, J.R., D.L. Holshouser, M.M. Alley,

N.L. Powell and D.M. Orcutt. 2001. Soybean

yield response at reproductive stage to soil-

applied nitrogen and foliar-applied boron.

Agron. J. 93 : 1200-1209.

Garcia, L.R. and J.J. Hanway. 1976. Foliar

fertilization of soybeans during the seed-filling

period. Agron. J. 68 : 653-657.

Goss, M.L., A. de Varennes, P.S. Smith and J.A.

Ferguson. 2002. N2 fixation by soybean grown

with different levels of mineral nitrogen, and

the fertilizer replacement value for a following

crop. Can. J. Soil Sci. 82 : 139-145.

Haq, M.U. and A. P. Mallarino. 2000. Soybean

yield and nutrient composition as affected by

early season foliar fertilization. Agron. J. 92

: 16-24.

Harper, J.E. 1987. Nitrogen metab olism, pp. 497-

533. In J.R. Wilcox (ed.). Soybean :Improvements, Production, and Uses. 2nd

ed. Agron. Mono. 16. ASA, CSSA, and SSSA,

Madison, WI.

Jackson, M.L. 1973. Soil Chemical Analysis.Prentice-Hall, New Delhi. 498 p.

Lauer, D.A. 1982. Foliar fertilization of dry beans

with Zn and NPKS. Agron. J. 74 : 339-344.

Linsay, W.L. and W.A. Norvel. 1978. Development

of a DTPA soil test for zinc, iron, manganese

and copper. Soil Sci. Soc. Am. J. 42 : 421-

428.

Mallarino, A.P., M.U. Haq, D. Wittry and M.

Bermudez. 2001. Variation in soybean

response to early season foliar fertilization

among and within fields. Agron. J. 93 : 1220-

1226.

Moraghan, J.T. 1991. Removal of endogenous

iron, manganese, and zinc during plant

washing. Commun. Soil Sci. Plant Anal. 22

: 323-330.

Ohwaki, Y., Y. Kraohaw, S. Chotechuan, Y. Egawa

and K. Sugahara. 1997. Differences in

responses to iron deficiency among various

cultivars of mungbean (Vigna radiata (L.)

Wilczek). Plant and Soil 192 : 107-114.

Oonkasem, B. and C. Thavarasook. 1988.

Micronutrient deficiency of mungbean in

Takhli soil, pp. 172-185. In Proceedings of the3rd Mungbean Conference. Kanchanaburi,

November 21-23, 1988. (In Thai).

Parker, M.B. and F.C. Boswell. 1980. Foliar injury,

nutrient intake, and yield of soybean as

infuenced by foliar fertilization. Agron. J. 72

: 110-113.

Pongsakul, P. and S. Ratanarat. 1999. On overview

of foliar fertilization for rice and field crops in

Thailand, pp. 199-220. In A. Suriyapan, E.

Hansakdi, P. Parkpian, R. Simmons and C.

Traynor, (eds.). Proceedings of the 2nd

International Workshop on FoliarFertilization. April 4-10, 1999. Bangkok.

Poole, W.D., G.W. Randal and G.E. Ham. 1983.

Foliar fertilization of soybeans. I. Effects of

fertilizer sources, rates and frequency of

application. Agron J. 75 : 195-200.

Ratanarat, S., W. Masangsan, P. Vadeesirisak, C.

Ditsantia and V. Phanuvas. 1990. Effects of

iron foliar spray and specific Rhizobium strains

on yield components of three peanut cultivars,

pp. 120-128. In Proceedings of 8th NationalGroundnut Research Meeting. 3-5 May


1989. Roi-Et, Thailand (In Thai).

Reinbott, T.M. and D.G. Blevins. 1995. Response

of soybean to foliar-applied boron and

magnesium and soil applied boron. J. PlantNutr. 18 : 179-200.

Rerkasem, B. 1989. Boron deficiency in food

legumes in Northern Thailand. J. Soil Fert.16 : 130-152. (In Thai with English abstract).

Reuter, D.J. and J.B. Robinson. 1997. PlantAnalysis : An Interpretation Manual.CSIRO, Victoria, Australia. 572 p.

Schon, M.K. and D.G. Blevins. 1990. Foliar boron

applications increase the final number of

branches and pods on branches of field grown

soybeans. Plant Physiol. 92 : 602-607.

Sinclair, T.R. and C.T. de Wit. 1975. Photosynthate

and nitrogen requirements for seed production

by various crops. Science 189 : 565-567.

Smith, G., W. Weibold, T.L. Niblack, P. Scharf

and D.G. Blevins. 2000. Yield components of

soybean plants infected with soybean cyst

nemathode and sprayed with foliar

applications of boron and magnesium. J. PlantNutr. 23 : 827-834.

Suanmalee, N., N. Tiaranan, R. Deemark and Y.

Soravisuitra. 1990. Foliar fertilization forsoybean production on Pak Chong and

Wang Sapung soil series. Annual Report,

Field Crops and Fertilizer Research Group,

Soil Science Division, Department of

Agriculture, Thailand. (In Thai).

Vasilas, B.L., J.O. Legg and D.C. Wolf. 1978.

Foliar fertilization of soybeans : absorption

and translocation of 15N – labeled urea. Agron.J. 70 : 787-791.

Vasilas, B.L., R.L. Nelson, J.J. Fuhrmann and

T.A. Evans. 1995. Relationship of nitrogen

utilization patterns with soybean yield and

seed filling period. Crop Sci. 35 : 809-813.

Walsh, L.M. 1971. Instrumental Methods forAnalysis of Soil and Plant Tissue. ASA,

CSSA and SSSA, Madison, WI. 221 p.

Weaver, M.L., H. Timm, H. Ng, D.W. Burke and

M.J. Silbernagel. 1985. Pod retention and

seed yield of beans in response to chemical

foliar applications. HortScience 20 : 429-

431.

Yataputanon, J., P. Chaivanakupt, S. Wunprasert

and T. Arayangkul. 2002. N-fixation of

soybean and residual effect from N-fixation

of soybean to rice yield in rice-soybean

cropping system using N-15 technique. J.Soil and Fert. 24 : 1-21. (In Thai with English

abstract).


Preliminary Test of Polyploidy Induction in Cotton(Gossypium arboreum) Using Colchicine Treatment

Arunee Wongpiyasatid1, Praparat Hormchan2 and Ngamchuen Rattanadilok3

ABSTRACT

Two local varieties of Gossypium arboreum, PM2 and PM3 were treated with colchicine solution

for polyploidy induction. Two colchicine solutions, colchicine solution # 1 (0.1% colchicine) was

derived from powder sold by Sigma and colchicine solution # 2 (0.5% colchicine) from drug tablets for

gout treatment. Three experiments were undertaken which were, Experiment 1: apical meristem

dropping with colchicine solution # 1; Experiment 2: seed treatment with colchicine solution # 2.

Experiment 3 : apical meristem dropping with colchicine # 2. All were compared with the untreated

(water treatment) controls. The results showed % germination of PM2 and PM3 after seed treatment to

be lower than those of the controls. The same were found with their heights. Eleven, nine and three

presumably polyploidy plants of PM2 in Experiment 1, PM3 in Experiment 1 and PM3 in Experiment 2

respectively were found to have stomata sizes of 24.9%, 34.9% and 31.4% increased and stomata

frequencies of 24.2%, 46.5% and 45.9% decreased compared to those of the controls respectively.

Key words: cotton, Gossypium arboreum, colchicine, polyploidy

INTRODUCTION

Cotton originally cultivated in Thailand

were native cotton, Gossypium arboreum origin

and the so-called ‘Indian cotton’. Earlier attempt

to improve cotton production was by introducing

the ‘Cambodian cotton’ from Cambodia to Thailand

in the early 1950’s. The so-called ‘Cambodian

cotton’ was in fact the American Upland cotton,

Gossypium hirsutum introduced to Cambodia

earlier (Na Pompeth, 1994). G. arboreum consists

of both white and brown cotton which produces

short, sparse seed hairs that are not spinnable. It is

diploid and possess 13 pairs of chromosome

(2n=26) while G. hirsutum is allotetraploids with

26 pairs of chromosome (4n=52) (Fryxell, 1969)

American cotton, G. hirsutum is very likely

an alloploid derived from hybridization between

Gossypium thuberi and G. arboreum (http://www.

biology. ualberta.ca/courses hp/gen275/problem-

set-key-2-0.1 htm) followed by chromosome

doubling resulting in well-developed seeds.

However, hybridization between different species

may not be so successful due to different

chromosome numbers or sizes. Polyploidy,

therefore, could be artificially induced by some

treatments, such as colchicine. In the late 1930’s,

it was discovered that colchicine inhibited the

formation of spindle fibers and effectively arrested

mitosis at the anaphase stage. At this point, the

chromosomes have multiplied but cell division

have not yet been taken place resulting in polyploidy

1 Department of Applied Radiation and Isotopes, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.

2 Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.

3. Department of Agronomy, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.



cells. In later year, a number of other mitotic

inhibitors including oryzalin, trifluralin,

amiprophos-methyl and N2O gas have been

identified and used as doubling agents (Bouvier et

al., 1994; van Tuyl et al, 1992; Taylor et al, 1976).

Colchicine treatment is the classical method

to induce doubling of chromosome number. The

techniques used for chromosome doubling of barley

haploid with colchicine have been reported by

Jensen (1974), Subrahmanyam and Kasha (1975)

and Thiebaut and Kasha (1977). Yan (2001)

demonstrated that ploidy of waxflowers could be

doubled using colchicine. The tetraploid plantlets

were found to have large leaves with fewer large

stomata than the diploid, an indicator of increased

ploidy level. Most species of Fuchsia are either

diploid or tetraploid. Addink (2002) reported that

a crossing between a diploid and tetraploid of

Fushia often resulted in a triploid which was

mostly sterile and could not be used for further

breeding. Doubling the chromosome number with

colchicine treatment from the diploid parent and

the triploid could solve breeding problem in Fushia.

Hybrid HAR was obtained from crossing of G.

hirsutum and G. arboreum and G. raimondie using

colchicine (DOA, 1984). Stephens (1947) also

reported the morphology of cotton hybrids through

colchicine treatment.

Chromosome counting which is time

consuming and laborous is not suitable for detection

mixoploidy in tissues with lower proportion of

dividing cells such as leaves (Uhlik, 1981) Recently

stomata size, leaf index value, stomata frequency,

pollen grain diameter and other changes in plant

morphology were found to be useful indicators in

the primary screening for new ploidy levels.

Evan(1955) and Speckman et al (1965) stated that

stomata length was the accurate indicator of

polyploidy level in many plants. Wright (1976)

also showed that stomatal measurement was a

quick way to determine whether or most of the

leaves on a branch were polyploidy. The purpose

of this study is to induce polyploidization in G.

arboreum by colchicine treatment using plant

attributes as primary indicators.


Two local varieties of G. arboreum, PM2

(light brown cotton) and PM3 (white cotton) were

used for polyploid induction through colchicine

treatment. PM2 and PM3 were grown in pots, 30

pots/variety/treatment. Three seeds per pots were

grown to obtain three plants. Twenty pot plants

were treated with colchicine while 10 pot plants

with just water.

Colchicine solutions were derived from

two sources

1) Sigma sold colchicine powder was

diluted in water into 0.1 % colchicine solution

(colchicine solution # 1)

2) Colchicine tablets (drug for gout

treatment) were dissolved in water to obtain 0.5%

colchicine solution (colchicine solution # 2)

Three experiments were undertaken as

followed

Experiment 1A few drops of colchicine solution # 1 were

applied on the apical meristem of seedlings between

the first pair of true leaves. To aid in absorption,

cotton balls were placed on the treated spots. The

treatments were repeated during 8:00-9:00 am for

3 consecutive days. The controls were treated with

water.

Experiment 2This experiment was achieved by soaking

seeds in colchicine solution # 2. The bottles with

seed treatment were periodically shaken. After 24

hours, the treated seeds were washed in the flowing

water for 3 hours, and then planted in the pots. The

controls were treated with water.

Experiment 3The similar procedure was followed as in


Experiment 1 except colchicine solution # 2 (with

no cotton balls covered the apex) was used instead

of colchicine solution # 1.

All plants, both treated and untreated (the

controls) in all experiments were allowed to grow

normally. Seed germination, plant height, stomata

size, and stomata frequency per 1 mm of leaf area

were checked.

Stomata measurementSimilar sizes of treated and untreated leaves

from 3-4 months old plants were sampled, 5 leaves/

plant. For stomata sizes, lower epidermis from

both left and right sides of each leaf sample were

peeled off, placed on glass slides covered with

cover glasses and measured under 40X

stereomicroscope. Stomata lengths were measured

employing ocular micrometer, 10 measurements/

leaf (5 from left and 5 from right sides of the mid

vein). It was replicated 5 times. The values obtained

were computed into micrometer (mm) using stage

micrometer.

To obtain stomata frequency, the

measurements were undertaken under 40X

stereomicroscope through TV monitor. Surface

cells of 5 leaves/plant, treated and the control,

were used, 10 measurements/plant The

measurements were undertaken from both left and

right sides of each leaf.


Changes in the morphological

characteristics such as plant height, stomata size

and stomata frequency were important indicators

for the detection of ploidy levels in M1 generation

of cotton varieties. Table 1 presents % germination

of PM2 and PM3 after seed treatment (Experiment

2) with colchicine solution # 2 to be lower than

those of the controls. The results half agreed with

explanation from Addink (2002) who stated that

colchicine with too high concentration could inhibit

the development of living part resulting in mortality

of organism. Although most treated seeds survived

to produce healthy plants, only a few could become

polyploidy. This might be due to the fact that even

though colchicine solution # 2 had high contration

of colchicine (0.5%), the starch suspension from

tablets could interfere with the absorbtion of

colchicine through the seed coats. However, the

Table 1 Averaging percent germinations and height of G. arboreum the local varieties, PM2 and PM3,

after colchicine treatments compared to the (untreated) control.

Germination (%) Plant Height (cm)

Experiment Treatment PM2 PM3 1st measurement/1 2nd measurement/2

PM2 PM3 PM2 PM3

1 Control - - 12.50 13.15 62.40 66.90

Treated - - 9.70 11.15 60.50 68.70

2 Control 95 100 14.40 13.05 71.10 89.60

Treated 78 72 11.90 11.95 66.40 94.80

3 Control - - 11.75 10.60 60.40 62.90

Treated - - 10.10 11.50 60.50 60.90

1 Averaged heights of PM2 and PM3 plants from the first measurement on Days 75, 60 and 45 after planting for Experiment 1,

2 and 3 respectively.2 Averaged heights of PM2 and PM3 plants from the second measurement on Days 130, 120 and 100 after planting for Experiment

1, 2 and 3 respectively.


24 – hour period of seed soaking still allowed

sufficient time for some seeds to receive high

doses resulting in no germination while some

obtained low or no dose resulting in

polyploidization or normal germination

respectively.

The averaging heights of treated plants of

PM2 and PM3 in all experiments were also found

to be lower than those of the untreated except that

of PM3 treated plant in Experiment 3 on the first

measurement. This exception might be similarly

explained to the result of % germination that the

starch suspension from tablets in colchicine

solution could prevent absorbtion of colchicine

doses through meristem droppings, hence, no

apparent height different from the control to be

seen. For seedling treatment, colchicine solution #

1 revealed more effectively than 0.5 % from drug

tablets with different effects. Stebbin (1950) stated

that the decrease growth rate of polyploids was

caused by the reduced ratio of cell division. The

supply of the cells with auxin, a phyto-hormone

was interrupted, the respiratory intensity was

reduced and the activity of many enzymes was

disminished. The results on plant height on first

measurement also agreed with Kerr (2001) and

Wright (1976) who stated that the induced 4n

seemed to grow more slowly and growth

abnormality were the first indication of successful

colchicine treatment.

However, the inhibition continued for 5-6

weeks after which the surviving plants of both

varieties in all experiments resumed normal growth

and development as appeared on the second

measurements. This was confirmed by Yan (2001)

who treated shoot tips of waxflower by immersion

in 0.05 % colchicine and found them to exhibit a

deformation of the new growth but it was only

transient, later growth returned back to normal.

In Table 2 the stomata size of selected

plants are found to range from 26.25-34.65, 32.35-

37.40 and 30.15-33.85 mm while those of the

control are 22.50-27.25, 24.65-27.75 and 22.05-

26.65 mm for PM2 in Experiment 1, PM3 in

Experiment 1 and PM3 in Experiment 2

respectively.

Compared to the control plants, the treated

plants of both cotton varieties had epidermis with

Table 2 Anatomical differences in leaves between the controls and the treated cotton plants of the two

cotton varieties, PM2 and PM3.

Ranges of Stomata

Experiment Variety Treatment Stomata Length Length (±S.D) Frequency (±S.D) 1

(mm) (mm)

1 PM2 Control 22.50-27.25 25.20±1.25 427.3±19.30

Treated 26.25-34.65 30.40±3.01 324.0±89.60

Difference - 20.90% increase 24.20% decrease

1 PM3 Control 24.65-27.75 26.00±1.00 451.50±33.02

Treated 32.35-37.40 35.00±1.54 241.70±15.47


2 PM3 Control 22.05-26.65 24.70±1.00 451.50±33.02

Treated 30.15-33.85 32.40±1.95 244.44±43.00


1 = number of stomata per 1 mm2


larger stomata sizes but lower stomata frequency

(Table 2). It was found that the leaves of plants of

PM2 in Experiment1 and PM3 in Experiment 1 and

2 had 24.2, 46.5 and 45.9 % fewer stomata that

were 20.9, 34.9 and 31.4 % longer in length than

those of the untreated plants respectively. The

differences were observed by microscopy as

described in the procedure.

The results agreed with those of Uhlik

(1981) who reported that the polyploid plants

usually had gigantic characteristics such as thicker,

wider and greener leaves with greater stomata size

and larger flowers. Stomata size, frequency of

stomata including pollen grain diameter were

favorable used as preliminary indicator of plants

with polyploidy levels. Tan and Dunn (1973) also

studied the correlation of the above characters

with ploidy levels of Bromus inermis and found

the possitive correlations of stomata length and

pollen grain diameter while negative correlation

of stomata frequency with ploidy levels. That

meant stomata length and pollen grain diameter

increased with the increasing of polyploidy levels.

The opposite was noticed in stomata frequency.

The results agreed with Yan (2001) who reported

that the leaves of the tetraploid plantlets of

waxflower had 54% fewer stomata that were 15%

shorter in length than those of the diploid plantlets.

Collins (1933, 1960) and Kerns and Collins (1947)

studied the diploid, triploid hybrids and diploid

and autotetraploid plants of pineapple variety

‘Smooth Cayenne’ and found that the sizes of cell,

trichomes and stomata also increased with the

ploidy levels while the stomata frequency was

reduced. Sax and Sax (1973) also found greater

somata frequency in diploid than tetraploid leaves

of equal area of Tradescantia camaliculata.

In Experiment 1, chimera was observed

from 5 out of 11 PM2 polyploidy plants which

indicated that as colchicine rarely acted on cells in

a growing point, artificial induction usually resulted

in a mixture called mixoploid or chimera (Wright,

1976). Similarly reported by Addink (2000) and

Kehr (2001), they explained colchicine treatments

to plant constituting of many cells to have chimera

appearances. The results showed that the induced

polyploidy plants still composed of plants with

differing number of chromosomes.

Primary screening of morphological

characteristics revealed 22 plants [11 plants of

PM2 (Experiment 1), 8 plants of PM3 (Experiment

1) and 3 plants of PM3 (Experiment 2)] to be

presumably polyploids with higher ploidy levels.

The 0.1% solution of colchicine derived from

colchicine powder sold by Sigma seemed to give

better results than the 0.5 % solution from the drug

tablets. The rate of polyploidy occurrence from

seedling in Experiment 1 appeared to be higher

than seed treatment in Experiment 2. To confirm

polyploidization, the selected plants will be

furtherly analyzed and chromosome number will

be determined.

CONCLUSION

The preliminary investigation had

demonstrated that

1. 0.1% colchicine solution derived from

powder sold by Sigma gave better results than

0.5% colchicine solution derived from drug tablets.

2. The presumably polyploidy plants of

PM2 and PM3 had slower growth at the beginning

and later resumed normal growth, larger stomata

and less stomata frequency compared to those of

the controls.

ACKNOWLEDGEMENTS

This research work was financially

supported by Kasetsart University Research and

Development Institute (KURDI).

LITERATURE CITED

Addink, W. 2002. Colchicine : Use in plant

breeding work to induce mutations


(polyploidy). file://A:\Colchicine.htm.

Bouvier, L., F.R. Fillon and Y. Lespinasse. 1994.

Oryzalin as an efficient agent for chromosome

doubling of haploid apple shoots in vitro.

Plant Breeding 113 : 343 – 346.

Collins, J.L. 1933. Morphological and cytological

characters of triploid pineapples. Cytologia 4: 248-256.

Collins, J.L. 1960. The Pineapple. Interscience

Publishers, N.Y. 295 pp.

DOA. 1987. Cotton. Academic Document 9 : 213

(in Thai).

Evans, A.M. 1955. The production and

identification of polyploids in red clover, white

clover and lucerne. New Phytol 514 : 149 –

162.

Genetic 275. 2002. Problem Assigment # 2Answer Key. http://www.biology.ualberta.

ca/courses.hp/gen275/problem-set-key-

2.01.htm.

Kerr, A. 2001. Tetraploidy conversion : An easyand effective method of colchicinetreatment. http://members.tripod.com/

~h_syriacus/tetraploidy.htm.

Kern. K.R. and J.L. Collins. 1947. Chimera in the

pineapple : colchicine induced tetraploids and

deiploid-tetraploids in the cayenne variety. J.of Heredity 38 : 322-330.

Jensen, C.J. 1974. Chromosome doubling

techniques in haploids, pp. 153-190. In K.L.

Kasha (ed.). Haploids in higher plants-Advances and potential. University of

Guelph, Canada.

Na Pompeth, B. 1994. Technical aspects of research

in Thailand with emphasis on integrated pest

management approach, pp. 37-50. In CottonSectors in Continental (itak) Southeast Asia.Regional Conference Proceedings, Vientiane,

Lao PDR, Oct 26-28, 1994. French Ministry

of Foreign Affairs/ CCL/CIRAD-DORAS

Project.

Sax, K. and H.J. Sax. 1973. Stomata size and

distribution in diploid and polyploid plants. J.Arnold Arboreum 18 : 164-172.

Speckmann, G.J., J. Post, Jr. and H. Dijkstra. 1965.

The length of stomata as an indicator for

polyploidy in rye-grasses. Euphytica 14 :

225-230.

Stebbins, G.L. 1984. Polyploidy and the

distribution of the arctic-alpine flora : new

evidence and a new approach. BotanicaHelvetica 94 : 1-13.

Stephens, S.G. 1947. Cytogenetics of Gossypium

and the problem of the origin of New World

cotton, pp. 431-442. In Advances in GeneticI. New York Academic Press.

Subrahmanyam, N.C. and K.J. Kasha. 1975.

Chromosome doubling of barley haploids by

introus oxide and colchicine treatments. Can.J. Genet. Cytol. 17 : 573-583.

Tan, Geok-Yong and G.M. Dunn. 1973.

Relationship of stomatal length and frequency

and pollen-grain diameter to ploidy level in

Bromus inermis Leyss. Crops Sci. 13 : 332-

334.

Taylor, N.L., M.K. Anderson, K.H. Wuesenberry

and C. Watson. 1976. Doubling the

chromosome number of trifolium species

using nitrous oxide. Crop Sci. 16 : 516-518.

van Tuyl, J.M., B. Meijer and M.P. van Dien.

1992. The use of oryzalin as an alternative for

colchicine in in-vitro chromosome doubling

of Lilium and Nerine. Acta Hort. 325 : 625-

629.

Uhlik, J. 1981. Kompendium pro postgradualni

studium genetiky a slechteni. Praha VSZ :105 – 195.

Wright, J.W. 1976. Introduction to ForestGenetics. Academic Press, New York.

Yan, G. 2001. Chromosome doubling of waxflower

plantlets regenerated in vitro, pp. 11-20. In

The Proceeding Biology of Waxflower. Areport for the Rural Industries Research and

Development Cooperation.


Cloning and Nucleotide Sequence of Four tRNA Genesin Mitochondrial Genome of Thai Walking Catfish,

Clarias macrocephalus Günther

Pradit Sangthong and Amnuay Jondeung

ABSTRACT

The Hind III digested fragment of Thai walking catfish mitochondrial DNA was cloned and its

nucleotide sequence was determined. This fragment, consisting of 668 bp, was composed of partial

WANCY region and COI gene. The partial WANCY region was consisted of partial tRNATrp gene,

complete tRNAAla, tRNAAsn, tRNACys, and tRNATyr genes, and origin of light strand replication (OL).

These genes and non-coding sequence (OL) were the same in their organization as those found in other

vertebrates. The partial nucleotide sequence of COI stared with GTG and its inferred amino acids were

highly conserved as previously described in other fishes. Interestingly, the intergenic spacer between

tRNACys and tRNATyr was ten nucleotides in length, which might be unique among Clariid species.

Key words: nucleotide sequence, tRNA genes, Thai walking catfish, Clarias macrocephalus

INTRODUCTION

Thai walking catfish, Clarias

macrocephalus Günther, is one of five Clariid

species found in Thailand and lives in freshwater

swamps, marshes and canals throughout the country

and Southeast Asia (Smith, 1945). Thai fish farmers

have cultured Thai walking catfish for so long by

collecting catfish fry from natural waters such as

paddy fields, swamps and canals (Sidthimunka,

1971). After Tongsanga et al. (1962) had

successfully induced spawning in C.

macrocephalus, the fingerling production was done

by artificial insemination. Since then, the Thai

walking catfish has become one of the most popular

farming species in Thailand. In 1987, the African

catfish, Clarias gariepinus,was introduced to

Thailand and successfully hybridized with C.

macrocephalus (Lawonyawut et al.,1992). The

successful interspecific hybridiazation has resulted

in changing the farming species into the hybrid

ones. This change also has impact on Thai

walking catfish, which is now becoming shortened

and threatened. Therefore, it is very inevitable to

set up the sustainable conservation and

management programs. In making decision

regarding the conservation on the genetic resources

of this species, an understanding of the amount and

distribution of its genetic variation is necessary

(Allendorf and Ryman, 1987). The molecular

genetic information, such as protein polymorphism,

mitochondrial or nuclear DNA variation will be

facilitated in order to understand the genetic

variability of this species.

The vertebrate mitochondrial genome is a

small duplex, covalently closed circular DNA and

varies in size ranging from 15-20 kb. All animal

mitochondrial genomes contain 37 genes, 2 for

Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.



ribosomal RNAs, 13 for proteins and 22 for tRNAs

and one major non-coding region (Boore, 1999).

Animal mitochondrial DNA (mtDNA) is haploid

and maternally inherited without recombination.

MtDNA changes at a rate as much as 5-10 times

higher than that of nuclear DNA. This means that

genes in mtDNA have higher variability than

single-copy genes in nucleus. Therefore,

mitochondrial DNA analyses have been widely

used to study the population structure and gene

flow, hybridization and phylogenetic studies

(Moritz et al., 1987) Although the complete

mitochondrial DNA sequences of many fish species

have been reported but none of Clarias species

(Boore, 1999).The objective of this study was to

sequence a cloned fragment of Thai walking

mtDNA in order to obtain a sequence for uses in

the further studies. In this report, the first partial

nucleotide sequence of WANCY region and of

COI gene in mitochondrial genome of Thai walking

catfish was presented.


Mitochondrial DNA isolation.The mtDNA was isolated by a slightly

modified method of White and Densmore III(1992).

The fresh eggs of single individual of Thai walking

catfish (Clarias macrocephalus Günther) was

homogenized for 1 stroke in 15 ml TEK (50 mM

Tris-HCl pH 7.5, 10 mM EDTA, 1.5% KCl) buffer.

The homogenate was poured into a 50 ml

polypropylene tube and underlayered with 15 ml

of 15% sucrose in TEK buffer. The homogenate

was then centrifuged at 5,500xg for 10 min. The

supernatant was transferred to the new

polypropylene tube and centrifuged at 8,000xg for

30 min. The pellet was suspended in 5-10 ml cold

EST (100 mM EDTA, 150 mM NaCl, 10 mM Tris-

HCl pH 8.0) buffer for 2 cycles and centrifuged at

8,000xg for 15 min. The mitochondrial pellet was

resuspended in 500 ml EST and transferred to a

microfuge tube. The 18% SDS solution was added

to the pellet suspension until final concentration

was 2%. All previous steps were carried out at 4∞C. The suspension was allowed to stand at room

temperature for 10 min. The 250 ml 5M NaCl was

added into the microcentrifuge tube and centrifuged

at 8,000 g for 15 min. The supernatant was

successively extracted by phenol-chloroform-

isoamyl alcohol (25:24:1) and chloroform isoamyl

alcohol (24:1), respectively. The mtDNA was

precipitated with two volumes of ice-cold absolute

ethanol overnight and centrifuged at 12,000xg for

10 min at 4∞C. After washing with 70% ethanol,

the mtDNA was vacuum dried, resuspended in 30

ml TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH

8.0) buffer and stored at –20∞C until use.

Cloning and sequencing.Isolated mtDNA was completely cleaved

with Hind III and yielded 5 fragments; 8.8, 3.2,

3.0, 1.1 and 0.6 kb. All Hind III digested fragments,

covering entire genome of mtDNA, were randomly

ligated to pUC 18 with T4 DNA ligase and

transformed into E. coli JM 109 as described by

Sambrook et al. (1989). Transformed colonies

were screened for recombinant plasmid by using

X-gal color system. Recombinant plasmids were

characterized by cleaving with Hind III.The

inserted fragments were compared to each fragment

of intact mtDNA molecules digested with the

same restriction enzyme. It was found that there

were only two clones, pMmt H500 and pMmt

H1000, which contained 0.6kb- and 1.1kb-inserted

fragments respectively. The inserted fragment in

pMmt H1000 clone was too large to be analysed

only with a few reactions. Thus the result will be

presented in the next report.

The recombinant, pMmt H500, was sent to

Bioservice Unit (BSU) of The National Center for

Genetic Engineering and Biotechnology for DNA

sequencing. Dye terminator labeling method was

applied for sequencing on an automatic sequencer

(377 DNA sequencer, PE Biosystem). Nucleotide

sequence data were analyzed by the basic BLAST


program available on www.ncbi.nlm.nih.gov/. The

tRNA genes were aligned with CLUSTAL W

program (Thompson et al., 1994) available on

http://dot.imgen.bcm.tmc.edu:9331/. The obtained

mtDNA sequence of Thai walking catfish was

deposited at GenBank data libraries under the

accession number AF 322219.


The nucleotide sequence of the L-strand of

the Thai walking catfish mtDNA obtained from

the clone pMmt H500 is shown in Figure 1. The

total length of the partial mitochondrial was 668

bp. The overall base composition of this fragment

of L-strand was 39.04%A, 25.75%T, 26.65%C

and 18.56%G. An alignment of this fragment with

mitochondrial sequences of other organisms

deposited in GenBank database revealed that it

contained a partial cluster of 5 tRNA genes, origin

of L-strand replication (OL), partial sequence of

COI and some intergenic spacers.

A partial cluster of 5 tRNA genes was

composed of a partial sequence of tRNATrp gene

and 4 complete genes; tRNAAla, tRNAAsn,

tRNACys and tRNA Tyr. The four tRNA genes

were identified by their sequence homologies to

other vertebrate tRNAs; their specific anticodons

and their potential to fold into cloverleaf secondary

structure showed mismatch and variable atypical

base-pairing in their stem regions (Figure 2). These

tRNAs ranged in size from 69-73 nucleotides. All

proposed cloverleaf structures contained 7-8 bp in

the amino acid arm, 5-6 bp in the TYC stem, 5 bp

in the anticodon stem and 4-5 bp in the DHU stem.

These structures showed variabilities from their

counterparts found mtDNA of bichir (Noack et al.,

1996) and Japanese sardine(Inoue et al., 2000)

which were found consistently 7 bp in the amino

acid stem, 5 bp in the TYC stem, 5 bp in the

AAGCTTTAAGTAGGAGTGAAAATCTCCTAATCTCTGCCATAAGACTTGCAGGACTCTATCCCACATCTTCTGAATGCAACTCAGAC

ACTTTAATTAAGCTAAAGCCTTACTAGATGAGAAGGCCTCGATCCTACAAACTCCTAGTTAACAGCTAGGCGCTCAAACCAACGAG

CATTCATCTACTTTCCCCGCCGCCTAAGCATAAAGGCGGGGAAAGCCCCGGCGGGGGTTTAACCTGCATCTTTAGATTTGCAATCT

AACATGTTATACACCACAAGGCTTCAATATTTACTGATAGGAAAAGGACTCAAACCTTTGTACATGGAGCTACAATCCACCGCCTA

M T I T R W F F S T N H K D I G T L Y L V F GACCCTCGGCCATCCTACCTGTGACGATCACACGCTGATTTTTCTCAACCAACCATAAAGACATTGGCACCCTTTATCTAGTATTTG

A W A G M V G T A L S L L I R A E L A Q P G A L L G D DGTGCCTGAGCCGGAATAGTCGGCACAGCCCTGAGCCTACTAATCCGAGCAGAACTGGCACAGCCTGGGGCTCTTCTAGGAGATGAC

Q I Y N V I V T A H A F V M I F F M V M P I M I G G F G NCAGATCTATAATGTTATTGTCACCGCCCACGCCTTCGTAATAATCTTCTTTATAGTAATACCAATTATGATTGGAGGCTTCGGAAA

W L V P L M I G A P D M A F P R M N N M SCTGACTTGTGCCCCTAATAATTGGTGCCCCCGATATAGCATTCCCACGAATAAATAACATAAGCTT

OL

tRNATrp (partial sequence)

COI (partial sequence)

tRNAAla

tRNACys

tRNATyr

tRNAAsn

Figure 1 The partial L-strand nucleotide sequence of Thai walking catfish mitochondrial genome. The

direction of transcription is denoted by arrows. Beginning and end of each gene are indicated

by a vertical bar (|). The non-coding sequences, origin of L-strand replication (OL) and

intergenic spacer, are overlined with thick and dotted lines respectively. The deduced amino

acid sequence for partial COI is shown above the nucleotide sequence (one-letter abbreviation

is placed above the first nucleotide of each codon). The Hind III retriction sites are underlined.


anticodon stem, and 3-4 bp in the DHU stem.

These animal mt-tRNAs are usually smaller and

lower G+C content and have more nonstandard

base pairs in their stem regions than those of their

nuclear counterparts (Brown, 1985). The order of

gene arrangement of these 4 tRNAs reported here

had similar organization of tRNA genes called

WANCY region, a coding region of 5

mitochondrial tRNAs (tryptophan, alanine,

asparagine, OL, cysteine and tyrosine)(Seutin et

al.,1994), which was reported in mitochondrial

genome of other fishes(Tzeng et al., 1992; Zardoya

and Meyer, 1996 ; and Inoue et al., 2000).

The origin of light strand replication (OL)

in a non-coding DNA sequence of approximately

30 nucleotides is located in the WANCY

TAGATGA

ATCTACT

G

AT

G

G

A

AG

CTG

C T T CCG TAGG

TC

TTGAA

CCTAG

GGATC

TC GCGGAGCT

GT

T T

TG

GTA

TT

tRNAAsn

5í�3í�

TG

G GAA

TTA

A

TG TGACA T

TT

ACGAA

TTAGA

AATCT

GCCTTG

AA

CGGGGC

T

TT

C

T

AC

ATA

CG C CCGCAGG

tRNACys

5û�3û�

TG

G TAG

C

TT

ATGGC

GTGGA

TACCT

T

T

AG

TTG

T T T CCAAAGG

GTAGGA

GTATCCT

CA

G G

TGCC GA

GG CATG

tRNATyr

5û�3û�

T T CGAAG T

AAGGCTT

TTCTGAA

A

TACGT C TCG TGG AG

TCTGA

AGACT

TA

T A

A

G

TG

CGTAT

GAA

T

AG

ATG

ë�

ë�

ë�

ë�

ë�

ë�

ë�

ë� ë�

ë�

tRNAAla

3û�5û� Aminoacyl arm

DHU armTYC arm

Variable arm

Anticodon arm

Figure 2 The putative secondary structure of four Thai walking catfish mitochondrial tRNAs; tRNAAla,

tRNAAsn, tRNACys and tRNATyr, based on sequence of Figure 1.


region(Seutin et al.,1994). In Thai walking catfish

mtDNA fragment, the OL found between the

tRNAAsn and tRNACys and is 35 nucleotides in

length. The OL sequence had capability to form a

stable stem-loop structure with 11 bp in the stem,

13 nucleotides in the loop (Figure 3). The four

nucleotides of the OL stem were parts of tRNACys

gene. It was slightly different from that of lungfish

mtDNA which about half of OL stem shared with

tRNACys gene (Zardoya and Meyer, 1996). The

Thai walking catfish OL also had a conserved

motif, 5’-GCCGG-3’, at base with tRNACys gene.

This conserved motif of OL has been also reported

in Atlantic cod, loach, bichir and human (Noack et

al., 1996; Tzeng et al., 1992 and Anderson et al.,

1981). The most interesting character of Thai

walking catfish OL is a A-T rich sequence in the

loop, which has been reported in bichir (Noack et

al., 1996), African clawed frog (Roe et al.,1985)

and human (Anderson et al., 1981). The role of the

thymine rich sequence in loop region in the human

is shown to be involved in synthesis of RNA

primer for light-strand replication process (Wong

and Clayton, 1985).

The partial sequence of COI, as shown in

Figure 1, is 305 nucleotides in length. The derived

amino acid sequence of a part of COI gene, coding

for cytochrome c oxidase subunit I, is shown in

5û�GGGG GCC T

G CG CG C

G CA TA TA T

C

AG CC

AT

G

T

CCA

C

T

CT

CG

3û� ATGAG

Loach12

(Crossostoma lacustre)

AGA3û� 5û�GCGG TGC

CC

AT

C

C

CC

TG

C

G

G CC GG CG CG CG CA T

T AG CA TA T

A T

C

Atlantic cod15

(Gadus morhua)

G CG CC GG CG CG CA T

A TA TG CG C

TC

TT

A

T

AT

TG

T

G

5û�GGGC CCG GT3û� CCACBichir10

(Polypterus ornatipinnis)

G CG CC GG CG CG CG C

G CA TA TA T

GAT3û� GGGGCCGC5û�

TC

G

AT

T

G TG

T

T

C

Thai walking catfish

(Clarias macrocephalus)

A

G CG CC GG CG CG CA T

G CA TA TG C

GAT3û� GGGG GCC 5û�

TC

GC

C

T

TT

GC

T

T

Human16

(Homo sapiens)

Primer�initiation

T

Figure 3 The stem-loop structures for the origin of light strand replication (OL) of Thai walking catfish

mtDNA and other vertebrates. The underlined sequence represents conserved motif.


Figure 4. The COI gene starts with the initiation

codon of GTG. Normally, this triplet codon

specifies valine within open reading frame. GTG

as initiation codon has been previously reported in

many fish species such as freshwater loach (Tzeng

et al., 1992), lungfish (Zardoya and Meyer, 1996),

bichir (Noack et al., 1996), deep sea fish (Miya

and Nishida, 1999) and Japanese sardine (Inoue et

al.,2000), but not in Atlantic cod (Johansen et

al.,1990), which use GUG as the initiation codon

for the COI gene. The inferred amino acid sequence

of partial COI is also compared to those sequences

of cod, loach and trout (Figure 4). It was found that

the homologies of TW catfish COI compared to

that of other fish ranges from 96 to 97%. This

shows that COI polypeptide sequence is highly

conserved as previously described in Atlantic cod,

loach and trout (Johansen et al., 1990; Tzeng et

al., 1992; Zardoya et al. 1995)

Interestingly, the intergenic spacer between

tRNACys and tRNATyr is 10 nucleotides in length.

It has been reported that the intergenic spacers in

many fish species are absent or small number of

nucleotides (Miya and Nishida, 1999; Inoue et al.,

2000) Long spacer has been found between

tRNAThr and tRNAPro genes of 8 cod fish species

which varies in size ranging form 25 to 99 bp and

their variation can be determined both at

intraspecific and interspecific levels(Bakke et al.,

1999) Thus, the intergenic spacer found here might

be a unique feature of the Clariid species.

CONCLUSION

The mtDNA of Thai walking catfish was

isolated from freshly ripen eggs, cleaved with

Hind III and cloned into pUC18. A recombinant

clone, pMmt 500 contaning about 0.6kb-inserted

fragment, was screened by using X-gal system and

sent for sequencing of its inserted fragment. The

inserted mtDNA fragment was found to be 668 bp

in length and composed of a partial WANCY

region and a partial COI gene. The sequence was

analyzed and discussed comparatively with

mitochondrial sequences of other organisms.

LITERATURE CITED

Allendorf, F.W. and N. Ryman. 1987. Genetic

management of hatchery stocks, pp. 141-159.

In N. Ryman and F. Utter (eds.). PopulationGenetics & Fishery Management.University

of Washington Press. Seattle.

Anderson, S., A.T. Bankier, B.G.Barrell, M.H.L.

de Bruijn, A.R. Coulson, J. Drouin, I.C.

Eperon, D.P. Nierlich, B.A. Roe, F. Sanger,

Figure 4 The inferred amino acid sequence of partial COI gene of Thai walikng catfish (Mac) and those

of other fishes: Atlantic cod (Cod), Loach (Loa), and Trout (Tro). A dot indicates amino acid

similarity.

10 20 30 40 50 60Mac MTITRWFFSTNHKDIGTLYLVFGAWAGMVGTALSLLIRAELAQPGALLGDDQIYNVIVTA 60Cod .A.......................................S.................. 60Loa .A.......................................N.................. 60Tro .A.......................................S.................. 60

70 80 90 100Mac HAFVMIFFMVMPIMIGGFGNWLVPLMIGAPDMAFPRMNNMS 101Cod ............L.........I.................. 101Loa .............L................H.......... 101Tro ......................I.................. 101


P.H. Schreier, A.J. Smith, R. Staden and I.G.

Young. 1981. Sequence and organization of

the human mitochondrial genome. Nature290 : 457-465.

Bakke, I., G.F. Shields and S. Johansen. 1999.

Sequence characterization of a unique

intergenic spacer in Gadiformes mitochondrial

DNA. Mar Biotechnol. 1 : 411-415.

Boore, J.L. 1999. Survey and summary: Animal

mitochondrial genomes. Nucleic Acid Res.27 : 1767-1780.

Brown, W.M. 1985. The mitochondrial genome

of animals, pp.95-130. In R.J. MacIntyre (ed.).

Molecular Evolutionary Genetics. Plenum

Press, New York.

Inoue, J.G., M. Miya, K. Tsukamoto and M.

Nishida. 2000. Complete mitochondrial DNA

sequence of Japanese sardine, Sardinops

melanostictus. Fisheries Sci. 66 : 924-932.

Johansen, S., P.H. Guddal and T. Johansen. 1990.

Organization of the mitochondrial genome of

Atlantic cod, Gadus morhua. Nucleic AcidsRes. 18 : 411-419.

Miya, M. and M. Nishida. 1999. Organization of

the mitochondrial genome of a deep-sea fish,

Gonostoma gracile (Teleostei: Stomiiformes):

first example of transfer RNA gene

rearrangements in bony fishes. Mar.Biotechnol. 1: 416-426.

Moritz, C., T.E. Dowling, W.M. Brown. 1987.

Evolution of animal mitochondrial DNA:

Relevance for population biology and

systematics. Ann. Rev. Ecol. Syst. 18 : 269-

292.

Lawonyawut, K., B.J. McAndrew, D.J. Penman

and P. Sodsuk. 1992. Electrophoretic and

morphological studies of the hybrid catfish,

pla duk bigoui (female Clarias macrocephalus

Gunther X male C. gariepinus Burchell), pp.

137-139. In Proceedings of InternationalWorkshop on Genetics in Aquaculture andFisheries Management, University of

Sterling, 31st August –4th September 1992.

Noack, K., R. Zardoya and A. Meyer. 1996. The

complete mitochondrial DNA sequence of

the bichir (Polypterus ornatipinnis), a basal

ray-finned fish: ancient establishment of the

consensus gene order. Genetics 144 : 1165-

1180.

Roe, B.A., D.P. Ma, R.K. Wilson and J.F.H. Wong.

1985. The complete nucleotide sequence of

the Xenopus laevis mitochondrial genome. J.Biol. Chem. 260 : 9759-9774.

Sambrook, J., E.F. Fritsch and T. Maniatis. 1989.Molecular Cloning: A Laboratory Manual.2nd ed.,Cold Spring Harbor Laboratory Press,

Cold Spring Harbor, NY.545 p.

Seutin, G., B.F. Lang, D.P. Mindell and R. Morais.

1994. Evolution of the WANCY region in

amniote mitochondrial DNA. Mol. Biol. Evol.11 : 329-340.

Sidthimunka, A. 1971. Production of catfish

(Clarias spp.) and its problems in Thailand.

Thai Fish. Gaz. 24(4) : 497-506.

Smith, H.M. 1945. The Fresh-water Fishes ofSiam, or Thailand. United States Government

Printing Office, Washington. 622p.

Thompson, J.D., D.G. Higgins and T.J. Gibson.

1994. CLUSTAL W: improving the sensitivity

of progressive multiple sequence alignment

through sequence weighting, position-specific

gap penalties and weight matrix choice.

Nucleic Acids Res. 22 : 4673-4680.

Tongsanga, S., A. Sidthimunka and D. Menasveta.

1962. Induced spawning in catfish (Clarias

macrocephalus Gunther) by pituitary hormone

injection, pp. 205-213. In Indo-PacificFisheries Council Proceedings, 10th Session,

IPFC Secretariat, FAO Regional Office for

Asia and Far East, Bangkok.

Tzeng, C.S., C.F. Hui, S.C. Shen and P.C. Huang.


the Crossostoma lacustre mitochondrial

genome: conservation among vertebrate.

Nucleic Acids Res. 20 : 4853-4858.

White, P.S. and L.D. Densmore III. 1992.


Mitochondrial DNA isolation, pp. 29-58. In

A.R.Hoelzel (ed.). Molecular GeneticAnalysis of Population: A PracticalApproach. Oxford University Press, New

York.

Wong, T.W. and D.A. Clayton. 1985. In vitro

replication of human mitochondrial DNA:

accurate initiation at the origin of light-strand

synthesis. Cell 42 : 951-958.

Zardoya. R. and A. Meyer. 1996. The complete

nucleotide sequence of the mitochondrial

genome of the lungfish (Protopterus dolloi)

supports its phylogenetic position as a close

relative of land vertebrate. Genetics 142 :

1249-1263.

Zardoya, R., A. Garrido-Pertierra and J.M. Bautista.


the mitochondrial DNA genome of the rainbow

trout, Oncorhynchus mykiss. J. Mol. Evol. 41

: 942-951.


Relation of Paralumbar Nerves and Conus Medullaris tothe Vertebrae of Swamp Buffaloes

Narong Chungsamarnyart, Worawut Rerkamnuaychoke and Nati Nilnophakoon

ABSTRACT

The last thoracic (T13) and the first three lumbar spinal nerves (L1-L3) of the 20 adult swamp

buffaloes specimens were dissected and observed the relation of their crossing to the tip of transverse

processes of first five lumbar vertebrae (TL1-TL5). Their dorsal and ventral branches crossed obliquely

caudolaterally on the dorsal and ventral surface of the lumbar transverse processes, respectively. The T13

crossed the anterior border of TL1 tip (20 specimens). Most of L1 (8 specimens and two of the right side)

crossed the posterior border of TL2 tip. The L1 of 6 specimens crossed the anterior border of TL3 tip.

Some variations of L1 crossed the anterior border of TL2 tip and the posterior border of TL3 tip. The L2

crossed the anterior (10 specimens and one of the right side) and the posterior (7 specimens) border of

the TL4 tip, and a few specimens variably crossed the posterior border of TL3 tip. Most of ventral branch

of L3 were lining caudolaterally under psoas muscles. The ventral branch of L3 in some specimens (4

specimens and two of only right and left side) have a branch on the dorsal surface of the psoas muscles

and crossed the posterior border of TL5 tip. The conus medullaris of spinal cord of all specimens were

taper to filum terminale at the caudal part of the first sacral vertebra.

This study showed the variation in the course of the lumbar spinal nerves. It might be recommended

that the effective paravertebral anesthesia nerve blocks in swamp buffaloes will be injected to the tip of

the first five lumbar transverse processes, and infiltrated to anterior and posterior of them for the last

thoracic and the first three lumbar spinal nerves blocks, respectively. The epidural anesthesia in swamp

buffaloes will be safe for the spinal cord by injection at sacrococcygeal foramen since the spinal cord is

taper to filum terminale in the first sacral vertebra.

Key words: paralumbar nerves, spinal cord end, swamp buffalo

Department of Veterinary Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand.


INTRODUCTION

The local anesthesia of spinal lumbar nerves

or epidural anesthesia are the safety anesthesia in

large animals for ruminotomy, caesarian sections

and exploratory laparotomy. The lumbar

paravertebral nerves block in cow has been in

clinical practice (Cakala, 1961, de Lahunta and

Habel, 1986). The anesthesia injection points are

located by palpation and injection at the tip of

lumbar transverse processes which the dorsal and

ventral branches of the last thoracic spinal (T13)

and the first three lumbar spinal nerves (L1, L2 and

L3) are upper and under lining on lumbar transverse

processes as follows; the last thoracic spinal nerve

crossed the anterior border of the first lumbar


transverse process Tip (TL1), the first lumbar

spinal nerve (L1) crossed the posterior border of

the second lumbar transverse process tip (TL2),

the second lumbar spinal nerve (L2) crossed the

posterior border of the fourth lumbar transverse

process tip (TL4), and the third lumbar spinal

nerve (L3) are running straight caudally along the

bodies of vertebrae, under psoas muscles.

Therefore, the relationship between lumbar spinal

nerves and the lumbar transverse processes are

important to locate the injection points for lumbar

paravertebral nerve blocks, but those injection

points in the swamp buffalo has not yet elucidated.

The end of spinal cord (conus medullaris)

in relation to the spinal vertebrae is important to

locate the safety point of epidural anesthesia

injection. The safety injection points should be

posterior to the end of spinal cord and anterior to

the end of dura mater. The end of spinal cord and

the end of dura mater of the horse, ox, dog and cat

have been reported and applied in clinical epidural

anesthesia (Seiferle, 1951; de Lahunta and Habel,

1986). The end of spinal cord of the horse, cow,

dog and cat terminate at the first or second sacral

vertebrae, the first sacral vertebra, the sixth lumbar

vertebra and the sixth lumbar to the third sacral

vertebrae, respectively. While the end of dura

mater of horse, cow, dog and cat locate at the third

sacral vertebra, fourth sacral vertebra, first sacral

and first coccygeal vertebra, respectively.

However, the relation between the end of

spinal cord and spinal vertebrae of the swamp

buffalo has not yet reported. Thus this study try to

elucidate the relation of the end of spinal cord to

the body of sacral vertebrae and the lumbar spinal

nerves to the lumbar transverse processes of

vertebrae.


The posterior part of vertebrae of 20 adult

swamp buffaloes were collected from slaughter

house, including the last two thoracic with ribs, the

pelvis and coccygeal vertebrae. Thirteen buffalo

specimens were 3-4.5 years old. Six specimens

were 8-10 years old and 1 year old was one

specimen. The dorsal and ventral branches of 13th

thoracic, 1st, 2nd and 3rd lumbar spinal nerves were

dissected and observed of their crossing to the tip

of lumbar transverse processes (TL). Then the

specimens were fixed with 10% formalin for

dissecting the end of spinal cord and observed the

relation of the cord with the sacral vertebrae.

RESULTS

The dorsal and ventral branches of the 13th

thoracic, 1st, 2nd and 3rd lumbar spinal nerves

crossed obliquely caudolaterally on the dorsal and

ventral surface of the succeeded lumbar transverse

processes, respectively. The dorsal branches

perforated the longissimus dorsi muscle to

subcutaneous layer at the top of transverse

processes tip of succeeded vertebrae. (Figure 1a)

The ventral branch of the 13th thoracic

spinal nerve (VT13) of all 20 specimens crossed

the anterior border of transverse process tip of the

first lumbar vertebra (TL1 ). (Figure 1a, 1b, and

2a)

Most of specimens (8 specimens and two

of the right side) had ventral branches of the 1st

lumbar spinal nerve (VL1) crossed the posterior

border of transverse process tip of the 2nd lumbar

vertebra (TL2), while the VL1 of 6 specimens

crossed the anterior border of transverse process

tip of the 3rd lumbar vertebra (TL3 ) (Figure 1a, 1b,

and 2a). There were 4 specimens and one of the left

side which VL1 crossed the anterior border of

transverse process tip of the second lumbar vertebra

(TL2). It was one of the left side of specimen

which VL1 crossed posterior border of transverse

process tip of the third lumbar vertebra (TL3).

The ventral branch of the 2nd lumbar spinal

nerve (VL2) of 10 specimens and one of the right

side crossed the anterior border of the 4th transverse

process tip (TL4). The other 7 specimens had VL2


Figure 1a and 1b. The dorsal and ventral views of lumbar region of swamp buffalo specimen No. 16

(B16) show the course of last thoracic (T13) and first three lumbar spinal nerves (L1-L3). The

dorsal (DT13) and ventral (VT13) branches of T13 cross the anterior tip of first lumbar

transverse process (TL1). The dorsal (DL1) and ventral (VL1) branches of L1 cross the anterior

part of TL3 tip. The dorsal (DL2) and ventral (VL2) branches of L2 cross the posterior part of

TL4 tip. The dorsal (DL3) and the right ventral (DVL3, dorsally psoas muscle branch)

branches of L3 cross the posterior part of TL5 tip. The dorsal (DL4 and DL5) and ventral (VL4

and VL5) branches of L4 and L5 run caudolaterally on the dorsal and ventral surface of pelvic

girdle, respectively.


Figure 2b. The dorsal view of opened sacral region of buffalo specimen No. 20 (B20) shows the filum

terminale of spinal cord (arrow) at the caudal part of the first sacral vertebra (S1), and the end

of dura mater at the third sacral vertebra (S3). S2; the second sacral vertebra.

Figure 2a. The ventral view oflumbar region ofswamp buffalo speci-men No. 20 (B20)shows the course of theventral branches of thelast thoracic (VT13)and the first three lum-bar spinal nerves (VL1-VL3). The VT13crosses the anterior partof the first lumbar trans-verse process (TL1) tip.The VL1 crosses theanterior part of TL3 tip.The VL2 crosses theposterior part of TL4tip. The dorsally psoasmuscle branch of leftVL3 crosses the poste-rior part of TL5 tip.


crossing the posterior border of the TL4 tip (Figure

1a, 1b, and 2a). The VL2 of a few specimens (2

specimens and one of the left side) showed variably

crossed the posterior border of TL3 tip.

The ventral branch of the 3rd lumbar spinal

nerve (VL3) of the most specimens (14 specimens

and two of only left and right sides) were lining

caudolaterally under psoas muscles to the inguinal

region. The VL3 in some specimens (4 specimens

and one of the left side) have a branch on the dorsal

surface of the psoas muscles (DVL3) which it

crosses the posterior border of transverse process

tip of the fifth lumbar vertebra (TL5) (Figure 1b,

right side and 2a, left side).

The ventral branch of the 4th,5th and 6th

lumbar spinal nerves were lining caudally along

the body of vertebrae to the dorsolateral wall of

pelvic cavity (Figure 1b). These nerves lied far

from the median plane about 3.5-4.5 cm.

The conus medullaris of spinal cord of all

specimens were tapered to filum terminale at the

caudal part of the first sacral vertebra (Figure 2b).

The end of dura mater of all specimens were

located caudal part of the third sacral vertebrae

(Figure 2b).

DISCUSSION

The location of ventral branch of 13th

thoracic spinal nerve of swamp buffalo was at the

same point as of the cattle (Cakala, 1961, de

Lahunta and Habel, 1986) since the ventral branch

of buffalo also crossed the anterior border of

transverse process tip of the first lumbar vertebra.

The ventral branches of the 1st, 2nd, 3rd

lumbar spinal nerves of swamp buffaloes were

greater variable distribution than those of the cattle

(Cakala, 1961, de Lahunta and Habel, 1986). It

might be the cause of the small number of the

specimens (only 20 specimens) and the transverse

process tips of swamp buffaloes are more lateral or

far from the emerging point (intervertebral

foramen) of each lumbar spinal nerves. Thus the

distribution of nerves are more variable lining.

However, the transverse process tips are the easiest

clinical palpation for locating anesthesia injection

points in buffaloes. As the results, it might be

recommended that the local anesthesia infiltration

points of 1st lumbar spinal nerve (L1) in buffaloes

are at the tips of both second and third transverse

process of lumbar vertebra (TL2 and TL3), but the

infiltration anesthesia point of L1 in the cattle is

only the posterior border of TL2 tip (Cakala,

1961). However, the tip of TL2 of buffaloes was

the main infiltration point for L1, since the

percentage of ventral branches of L1 (VL1) crossed

posterior border of TL2 tip were 45% (8 specimens

and two of the right side in 20 specimens) and the

crossing of the anterior border of TL2 tip were

20% (4 specimens and one of the left side in 20

specimens). The VL1 of the others specimens

(30% or 6 specimens in 20 specimens) and 5%

(one specimen) crossed the anterior border and

posterior border of TL3 tip, respectively.

The ventral branch of L2 (VL2) crossed the

anterior border and posterior border of TL4 52.5%

(10 specimens and one of the right side) and 35%

(7 specimens), respectively. It suggested that the

main anesthesia infiltration point for the 2nd lumbar

spinal nerve (L2) in the buffalo might be the tip of

the 4th transverse process tip (TL4). It was similar

to the recommended point in cattle (Cakala, 1961),

but in buffaloes might be need the minor anesthesia

infiltration point for L2 at the tip of TL3 since the

VL2 crossed the posterior border of TL3 12.5% (2

specimens and one of the left side). This infiltration

point will also anesthetize the more caudal lining

of L1 as mentioned above.

The ventral branch of the 3rd lumbar spinal

nerve (VL3) of the most specimens (75% or 14

specimens and two of only left and right sides)

were similar to cattle by lining caudal and slightly

lateral under psoas muscles to the inguinal region

(de Lahunta and Habel, 1986). Some specimens

(or 4 specimens and two of only right and left

sides) having a branch of VL3 on the dorsal


surface of the psoas muscles and crossed the

posterior border of transverse process tip of the

fifth lumbar vertebra (TL5). Therefore, the

infiltration anesthesia of the udder will be injection

3.5-4.5 cm from the median plane to the L3 at

emergence point. This injection point was a little

shorter than the original method of paravertebral

anesthesia in cattle, 5 cm from the median plane

(Farquharson, 1940). However, the width of

vertebral bodies are variable on the body size of

animals.

The ventral branch of the forth lumbar

spinal nerve (VL4) for the udder might also be

anesthetize by injection at emergence point from

intervertebral foramen since it straight caudally

along the bodies of vertebrae to the dorsolateral

wall of pelvic cavity.

The conus medullaris of spinal cord and

the end of dura mater of all specimens were similar

with the cattle (Seiferle, 1951) because they

terminated at the caudal part of the first and third

sacral vertebrae, respectively. This might be

recommended that the lumbosacral epidural

anesthesia can be done in adult buffaloes as similar

procedure as in adult cows (de Lahunta and Habel,

1986).

CONCLUSION

The branch of the lumbar spinal nerves in

swamp buffaloes were greater distribution than in

the cattle. It is recommended to inject at the tip of

the first five lumbar transverse processes and

infiltration to both anterior and posterior part of

them for local anesthesia the last thoracic and first

three lumbar spinal nerves. The epidural anesthesia

in swamp buffaloes will be applied at

sacrococcygeal foramen as similar as in cattle

because of the conus medullaris of spinal cord is in

the first sacral vertebra, and the end of dura mater

is located caudal part of the third sacral vertebra.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the

Kasetsart University Research and Development

Institute for the research fund.

LITERATURE CITE

Cakala, S. 1961. A technic for the paravertebral

lumbar block in cattle. Cornell Vet. 51 : 64-

67.

De Lahunta A.D. and R.E. Habels. 1986. AppliedVeterinary Anatomy. W.B. Saunders Com.

Philadelphia.

Farquharson, J. 1940. Paravertebral lumbar

anesthesia in the bovine species. JAVMA 97

: 54-57.

Seiferle, E. 1951. Zur Ruckenmarks-topographie

von Pferd und Rind. Abstr. JAVMA 118 :

379-383.


Isolation of Anti-malarial Active Compound from Yanang(Tiliacora triandra Diels)

Chalerm Saiin and Sutthatip Markmee

ABSTRACT

Malaria remains to be one of the serious problems in tropical countries because of the increase in

antimalarial drug resistance. This investigation was to study the extraction of bioactive compounds with

anti-malarial activity from Yanang (Tiliacora triandra Diels) root. The dried root was extracted with

chloroform : methanol : ammonium hydroxide mixture (50:50:1). Further isolation and purification of the

crude extract using column chromatography and crystallization techniques provided two pure alkaloid

compounds: tiliacorinine (I) and tiliacorine (II) with 0.0082 and 0.0029 percent yield, respectively.

Structures of I and II were confirmed by spectroscopy techniques and compared with reference data.

Key words: anti-malarial, Yanang, Tiliacora triandra Diels, tiliacorinine, tiliacorine

INTRODUCTION

Today malaria is found throughout the

tropical and sub-tropical regions of the world and

causes more than 300 million acute illnesses and at

least one million deaths annually. Moreover, the

increase of drug resistance of Plasmodium

falciparum remains to be serious problems (WHO,

1998). Thailand is a resource of medicinal plants

and many of them have claimed to be used as

antimalarials. Hence, utilization of these plants

has been considered.

Tiliacora triandra Diels, one of the

medicinal plants, known in Thai as Yanang, belongs

to Menispermaceae family. Its root has been widely

used as antipyretic agent for all kinds of fever and

also prescribed in the preparation of antimalarial

in folk medicine (Fumio et al., 1990; Norman and

Nuntavan, 1992). Tiliacorinine (I) and tiliacorine

(II) which are bisbenzylisoquinoline alkaloids have

been found and isolated by using a combination

of chromatography and counter-current distribution

in Tiliacora species, Tiliacora racemosa Colebr

(Anjaneyulu et al., 1969) and their in vitro

antimalarial activity against Plasmodium

falciparum have been studied (Thaweephol et al.,

1987). The isolation of tiliacorinine and tiliacorine

from T. triandra roots by column of ion exchange

resin; Amberlite IRA 400 has been reported

(Thaweephol et al., 1974). Further preparative

thin layer chromatography have more frequently

used for purification of these two compounds

(Pichaet and Boondate, 1981; Thaweephol et al.,

1987). In this report we describe a simple column

chromatographic method without further

purification of the diastereomeric alkaloids,

tiliacorinine and tiliacorine from T. triandra roots.

Our method differs from those reported earlier

with regard to stationary phase material, eluting

solvent composition, and application.

Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences, Naresuan University,

Phitsanulok 65000, Thailand.




ChemicalsSilica gel 60 F254 size 0.040-0.0063 mm

for column chromatography, thin layer

chromatography (TLC) aluminium sheets 20 ¥ 20

cm coated with silica gel 60 F254 and kieselguhr

were obtained from Merck (Damstadt, Germany).

Chloroform, methanol and ethyl acetate were

commercial grade and were obtained from Rattana

Trading Co. (Thailand). Analytical grade petroleum

ether and heptane were obtained from SNP Co.

(Thailand). Analytical grade ammonia was

obtained from Carlo Erba (Thailand). Analytical

grade ether was purchased from BDH Co.

(Thailand). Mayer’s reagent was prepared

according to the reference method of USP 23.

Extraction of alkaloids from roots of T. triandraThe air-dried ground root of T. triandra

was bought from Manora O-soad store,

Phitsanulok, Thailand, dried in hot air oven at 70∞C for 3 hours, milled to fine powder and then dried

with the same procedure. The dried powder (2 kg)

was macerated in chloroform : methanol : ammonia

(50:50:1) at room temperature for 24 hours and

filtered. The filtrate was evaporated under reduced

pressure until dry. The residue from the filtration

was further macerated following the same

procedure until the portion of the filtrate gave a

negative result with Mayer’s test (USP 23). All

evaporated extracts were combined and dissolved

in small amount of chloroform. The chloroform

layer was extracted in 10% sulfuric acid several

times until the chloroform layer gave the negative

result with Mayer’s test. The combined aqueous

layer was basified with dilute ammonia to

precipitate water insoluble alkaloids. The mixed

alkaloids were collected and dried in vacuum

desiccator.

Isolation of tiliacorinine and tiliacorineThe mixed alkaloids (9.974 g) were

dissolved in chloroform : methanol (1:1), and then

filtered. The precipitate was discarded and the

filtrate was evaporated under reduced pressure.

The separation of active alkaloids was performed

using column chromatography on a silica gel 60

F254 (4 ¥ 40 cm) eluted with petroleum ether,

heptane and finally with chloroform : methanol :

ethyl acetate (5:1:3), with 30 ml fractions being

collected as follows: fractions 1-34 (A), 35-41 (B),

42-52 (C), 53-69 (D), 70-81 (E), and 82-145 (F).

The collected fractions were monitored using thin

layer chromatography on silica gel GF254 having

chloroform : methanol (9:1) as the solvent system.

Collection B and D were evaporated and then

crystallized in ether to give small pale yellow

needles of tiliacorinine (165 mg) and tiliacorine

(58 mg), respectively. Melting point measurement

(Buchi 535, Japan), ultraviolet-visible (UV-Vis)

spectrophotometry (Lamda20, Perkin Elmer,

Tiliacorinine (I) Tiliacorine (II)

N

O

O

N

CH3

CH3

OCH3 OH

H

OCH3

H1234

1’2’3’

4’ 4’3’2’

1’43 2 1

N

O

O

N

CH3

CH3

OCH3 OH

OCH3

H

H

RS


Germany), infrared (IR) spectrophotometry (KBr

pellet technique, Spectra 2000, Perkin Elmer,

Germany), and proton nuclear magnetic resonance

(1H-NMR) spectrometry (Jeol JMN-A500

spectrometer, using tetramethylsilane as the

internal standard) were used to identify the

structures. Visualization of TLC was observed

under ultraviolet light or by spraying with Mayer’s

reagent.


The extraction of 2 kg grounded root of T.

triandra gave 9.974 g total alkaloids. Further

isolation provided two pure alkaloid compounds

from collection B (165 mg, 0.0082%) and collection

D (58 mg, 0.0029%). The UV spectra of compounds

from collection B and D were identical at lmax 290

nm. The IR spectra of these two compounds are

shown in Table 1.

The 1H-NMR spectra of collection B and D

were in accordance with those of tiliacorinine and

tiliacorine as reported by Guha et al. (1976). The1H-NMR spectrum in CD3Cl of collection B

(Figure 1) showed the 2¢-NCH3 resonance at lower

field (d2.65, s, 3H) and the 2-NCH3 at higher field

(d2.32, s, 3H). The signals of two OCH3 appeared

at 3.87 (s, 3H) and 3.99 (s, 3H). 1H-NMR spectrum

in CD3Cl of collection D (Figure 2) showed the 2¢-NCH3 resonance at lower field (d2.64, s, 3H) and

the 2-NCH3 gave rise to a signal at higher field

(d2.34, s, 3H). The signals of two OCH3 appeared

at 3.87 (s, 3H) and 3.93 (s, 3H). Two of these

compounds, from collection B and D also had six

protons representing the two sets of methylene

bridge protons (d2.82, m, 3H, and d3.00, m, 3H),

the two methine neighbors situated on the two

heterocyclic rings and nine aromatic protons.

The UV-Vis, IR and 1H-NMR spectra of

compound from collection B were slightly

distinguishable from that of collection D. These

results confirmed that the compounds of both

samples were either tiliacorinine or tiliacorine.

However, comparing the melting points of these

compounds (Table 2) to those reported by

Thaweephol et al. (1987), it could be concluded

that collection B (Rf 0.73) was tiliacorinine and

collection D (Rf 0.57) was tiliacorine.

CONCLUSION

Tiliacorinine and tiliacorine, diastereomeric

compounds from the roots of T. triandra were

completely separated by using simple column

chromatography and crystallization techniques.

This method has never been used with this plant

before. The result provided high percentage yield

of tiliacorinine and tiliacorine that could be used

for structure modification and determine the

antimalarial activity relationship. The designing

and modifying of tiliacorine and tiliacorinine

structures are in progress to increase antimalarial

activity for further study.

Table 1 IR spectra bands of compounds from collection B and D.

The bands Frequency (cm-1)

Collection B Collection D

O-H stretching 3,394 3,402

C-H stretching of aromatic moieties 1,587 and 1,502 1,587 and 1,498

C-O stretching 1,277-1,122 1,273-1,118


Figure 2 The 1H-NMR spectrum of collection D in CD3Cl.

Figure 1 The 1H-NMR spectrum of collection B in CD3Cl.


Table 2 Melting point of tiliacorinine and tiliacorine.

Compounds Melting point (∞C)

Thaweephol et al. Our result

Tiliacorinine 162-175 167-170 a

Tiliacorine 264-267 262-264 b

a compound crystallized from collection Bb compound crystallized from collection D

ACKNOWLEDGEMENTS

This work was supported by a grant from

the Faculty of Pharmaceutical Sciences, Naresuan

University, Thailand. We wish to thank Ms.

Kanyarat Sompu and Ms. Wilasinee Kuangaw for

their technical assistance.

LITERATURE CITED

Anjaneyulu B., T.R. Govindachari, S.S. Sathe, N.

Viswanathan, K.W. Gophinath, and B.R. Pai.

1969. Alkaloids of Tiliacora racemosa Colebr.

Tetrahedron 25 : 3091-3105.

Fumio I., D. Supanee, K. Naoko, F. Yuichi, A.

Masaki, R. Nijsiri, and M. Isamu. 1990.

Chemical and biological studies on some Thai

medicinal plants. J Sci Soc. 16 : 25-31.

Guha K. P., P.C. Das , B.Mukherjee, R. Mukherjee,

G.P. Juneau, and N.S. Bhacca. 1976. Structure

of tiliamosine: A new diphenyl

bisbenzylisoquinoline alkaloid from Tiliacora

racemosa. Tetrahedron Letters 47 : 4241-

4244.

Norman R.F. and B. Nuntavan. 1992. ThaiMedicinal Plants Recommended forPrimary Health Care System. Bangkok:

Mahidol University. 402 p.

Pichaet W. and P. Boondate. 1981. Alkaloids of

Tiliacora triandra. Aust. J Chem. 34 : 2001-

4.

Thaweephol D., K. Panida and N. Kazumitsu.

1974. Isolation of active principle from

Yanang. Journal of Department ofMedicinal Science of Thailand 16 : 75-81.

Thaweephol D. and C.Pranee. 1987. Isolation of in

vitro antimalarial priciples from Tiliacora

triandra Diels. Journal of Department ofMedicinal Science of Thailand 29 : 33-38.

United States Pharmacopeial Convention, 1995.

The United States Pharmacopeia, TheNational Formulary : USP 23, NF18 1995,

Rockville, MD. 2391 p.

World Health Organization, 1998. The WorldHealth Report 1998-Life in the 21stCentury: a Vision for All. Geneva. 241 p.


Synergistic Effects of Sesame Oil with Cypermethrin on theSurvival and Detoxification Enzyme Activity of

Plutella xylostella L. Larvae

Suraphon Visetson1, John Milne2, Manthana Milne3 and Pintip Kanasutar1

ABSTRACT

Two types of insect-toxicity tests, (1) contact and (2) no-choice leaf dipping test, were conducted

using the insecticide cypermethrin without piperonyl butoxide (PB), cypermethrin with PB and cypermethrin

with sesame oil against the 2nd –3rd instar larvae of Plutella xylostella L. Sesame oil showed good

synergism with cypermethrin yielding synergistic ratios (SR) that ranged from 1.54 – 6.33 in the contact

method and 2.04-5.88 in the no-choice leaf dipping method and were comparable to using PB, which

showed SR’s of 6.33 and 6.71, respectively. Both PB and sesame oil mixed together with cypermethrin

inhibited monooxygenase activity by approximately two-third but induced glutathione–S-transferase ca.

2-3 folds in both methods. The synergists had no effect on esterase activity (CF ca. 1.2).

Residues of cypermethrin in the larvae increased by 1.29 – 2.57 folds in the treatments with added

sesame oil compared to a 2.86 fold increase when PB was added, using the contact method. The no choice

leaf dipping method revealed that cypermethrin residue levels increased by 2.82 – 6.91 fold with added

sesame oil and 8.27 fold with added PB. This indicated that both PB and sesame oil played the same role

in the inhibition of an enzyme, possibly monooxygenase. Field trials with Chinese kale showed the same

trends that were evident in the laboratory work. The addition of sesame oil to the insecticide reduced the

larval population by 70-80 percent while the addition of PB reduced the larval population by up to 88

percent in the kale crop. Monooxygenase activities of insect larvae collected in the field from kale sprayed

with cypermethrin plus synergist (sesame oil or PB) were lower than those from kale treated with

insecticide alone. The results in terms of synergism and changes in enzyme metabolism were discussed.

Key words: synergistic effects, sesame oil, cypermethrin, detoxification enzyme, Plutella xylostella L.


1 Department of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.2 Department of Biology, Faculty of Science, Mahidol University, Bangkok 10100, Thailand.3 Department of Agriculture, Bangkok 10900, Thailand.

INTRODUCTION

Although insecticides create manyproblems such as pollution, insecticide resistantinsects, disruption of biodiversity, residues inagricultural products, and most of all, high cost ofproduction, insecticides have been proven to beone of the most effective methods in insect control.

In Thailand, of all vegetable insect pests, thediamondback moth (Plutella xylostella Linn.) isone of the most serious in vegetable. It is resistantto many insecticides in most parts of Thailandwhere insecticides are frequently used. Fortunately,Thailand is one of the countries with the mostdiverse plants in Southeast Asia. Many Thai farmersutilize plant products for insect control. Some

plants contain secondary plant substances such asazadirachtin from neem seed kernels that inhibithormone production in lepidopterous larvae(Schmutterer, 1990). Others, like rotenone fromderris (Derris elliptica Benth) (Visetson and Milne,2001) and selinnadien from tubers of nutgrass(Cyperus rotundus L.) (Visetson et al., 2001) havebeen effectively used in the control of thediamondback moth larvae. However, other uses ofplant products in insect control, e.g., as synergistswith insecticides have never been investigated.Sesame is widely grown in the northeastern part ofThailand. Thai farmers usually plant sesame cropsafter the rice has been harvested. Plants use left-over moisture in the soil from the rice crop andgrow very fast. Sesame oil has many uses: as acosmetic for skin protection, in medicine as anadjuvant for many drug emulsions for ulcer, incooking as a Thai food additive. Furthermore, thechemical structure of the oil is similar to that of theinsecticide synergist, piperonyl butoxide, whichhas played a large role in reducing population ofpyrethroid resistance insects (Collins, 1990).

This research was conducted to investigatethe synergistic effects of sesame oil and piperonylbutoxide with cypermethrin on the survival anddetoxification enzyme activity of P. xylostellalarvae. The inhibition by cypermethrin ofmonooxygenase, esterase and glutathione-S-transferase activity after addition of these synergistswould show how these synergists interact withdetoxification mechanisms. These results couldbe beneficial to Thai farmers as well as tobusinessmen who are looking for ways to reducethe cost of insecticides by the addition of analternative synergist.


Insect larvae and plant samplesDiamondback moth larvae were collected

from a vegetable producing area in Kanchanaburiprovince, 150 kms west of Bangkok. Larvae werereared under laboratory conditions (23 ± 2∞C)

with methods modified from Visetson et al. (2001).F2 –generation was used for all experiments.Sesame pods collected from Nongpai district(Petchaboon province, 230 kms north of Bangkok)were air-dried. Sesame seeds from the pods wereground and screw- pressed to obtain esame oilwhich was collected into a bottle and stored at-20∞C until used in tests.

Efficacy testsThe toxicities of various concentrations

(0.01- 0.7% w/v) of cypermethrin with or without10% piperonyl butoxide (PB) and varied 0.5-10%sesame oil on 2nd – 3rd instar larvae of Plutellaxylostella L. were determined using a no choiceleaf dipping method and a contact method. Fivereplicates comprising 20 larvae in each replicatewere used in each test. A 5% emulsifier, triton X-100, was mixed into each test solution before trialscommenced. The no –choice leaf dipping methodused a leaf circle disk of Chinese kale with adiameter of 5 cm which was given to the larvae asfood. The contact method was done by allowing20 larvae to move freely in a petri-dish previouslysprayed with test solution. A completelyrandomized design with 5 replicates was used.Mortality was evaluated after 24 hours of exposure.All experiments were run at 23 ± 2∞C. In the caseof control mortality, Abbott’s formula (Matsumura,1975) was employed. LC50 values were calculatedfrom regression equations with cypermethrin usedas control groups.

Detoxification enzyme assaysThe surviving larvae from each treatment

were used in in vitro assays to optimize enzymeactivity of esterase, glutathione-S-transferase andmonooxygenase activities following the methodof Visetson and Milne (2001) by usingparanitrophenyl acetate, dichloronitrobenzene andaldrin as a substrate for the three enzymes. Proteinmeasurement was done according to the method ofLowry et al. (1951) and Bovine Serum Albuminwas used to quantify all enzyme activity.



The coefficient of determination (r2) wasdetermined for both insect larval mortality andenzyme activity. Synergist ratios (SR) andcorrection factors (CF) were quantified to measurethe effectiveness of synergists and changes inenzyme levels, respectively.

Determination of residuesUsing the contact and leaf dipping methods

with the LC50 level, larvae were exposed to variousconcentration of cypermethrin with or without PBor sesame oil for 24 hours and then transferred topetri-dish. Live larvae (approximately 0.5 g) wereassayed for cypermethrin by a method modifiedfrom Visetson (1991) using a disposable pasturepipette column. The column was previously packedwith 1 g aluminium oxide, followed by 0.5 g silicicacid. Live larvae (0.5 g) were crushed with 10 mgtrisodium citrate and 5 mg disodium hydrogenorthophosphate and the mixture was then loadedinto the column. The column was eluted with 15ml of 4% acetone in hexane. The eluent wasconcentrated to 0.5 ml and injected into a GLC/(Varian, USA) equipped with a 63Ni electroncapture detector and a 20 m capillary column,packed with 5% SE-30 on GAS-Chrom Q 80-100mesh. The conditions for chromatography were:injector temp. 220∞C, column 190∞C, detector285∞C, carrier gas flow (N2) 45ml/min. Theseconditions gave 99.8% recovery of cypermethrin.

Field experimentsField trials using Chinese kale were

separately undertaken to confirm laboratory results.The experiment used a randomized complete blockdesign with 5 replicates. Plot size was 6 x 2 squaremeters. Spraying at the rate of 80 litre/rai was doneonce a week, beginning on 10-day old Chinesekale. Seven sprays at 7 days interval were appliedduring the experiment. Larval numbers wereregularly checked at fixed points before and afterspraying for 24 hours. Duncan multiple range test(DMRT) was employed for means comparisonswith p<0.05 according to Finney (1964).

RESULTS AND DISCUSSIONS

Efficacy testsLC50 values for cypermethrin in both types

of test methods, (1) contact and (2) no choice leafdipping methods, against 2nd – 3rd instar larvae ofthe Plutella xylostella L were 0.57 and 0.47% w/v, respectively (Table 1). The addition of 10% PBresulted in synergist ratios (SR) of 6.33 and 6.71folds for first and second methods, respectivelywhereas addition of 10% sesame oil gave the SR of5.7 and 4.27 folds, respectively. In addition, sesameoil plus PB added to cypermethrin did not increaseSR values. The results indicated that both PB andsesame oil played the same role in increasingcypermethrin efficacy. These results were similarto those of Visetson and Milne (2001) who foundthat addition of PB and diethyl maleate to rotenoneincreased rotenone efficiency. A larger SR wasobtained when 20% sesame oil was added but atthis point it was not very economical in terms ofthe cost. The correlation between concentrationand mortality in most experiments indicated r2 of0.87 – 0.99 except that the addition of 0.5%sesame oil showed r2 of 0.69 only in the contactmethod. Hence, this low concentration of sesameoil added might not be sufficient to deplete thedetoxification mechanisms in the larvae. This resultwas similar to that of Visetson (1991) who workedwith PB added to cyfluthrin in the control ofTribolium castaneum. Furthermore, the resultsshowing higher insecticide efficiency after additionof PB confirmed Collins (1990)’s works whoshowed that PB may inhibit one of detoxificationenzymes possibly monooxygenase in insects.

Detoxification enzyme assaysCypermethrin alone gave little increased

monooxygenase activity by ca. 1.12 folds and giveno change in esterase and glutathione-S-transferaseactivity in both test methods (Table 2). Elevatedmonooxygenase levels after application ofpyrethroids have been reported by a number ofworkers (Rose, 1985; Hung and Sun, 1989;


Table 1 LC 50 values of cypermethrin (0.01 – 0.7% w/v) with or without piperonyl butoxide (PB) and

sesame oil against 2nd – 3rd instar larvae of Plutella xylostella L. using no choice leaf dipping

method and contact method.

Application methods1

Treatment (% w/v) Contact Leaf dipping

LC50 (%w/v) r2 Regression eq.4 LC50 (%w/v) r2 Regression eq.

[ SR] [SR]

None3 0.57 ± 0.02c2 0.98 Y = 22.64 + 48.0X 0.47 ± 0.05b 0.97 Y = 33.92 + 34.22X

10% PB 0.09 ± 0.01a 0.99 Y = 46.24 + 41.80X 0.07 ± 0.02a 0.99 Y = 46.98 + 43.16X

[ 6.33] [ 6.71]

0.5% oil 0.37 ± 0.23bc 0.69 Y = 36.93 + 35.32X 0.22 ± 0.25b 0.76 Y = 41.80 + 37.26X

[1.54 ] [2.14 ]

1.0% oil 0.21 ± 0.22b 0.87 Y = 39.89 + 48.10X 0.23 ± 0.12b 0.84 Y = 39.62 + 45.12X

[2.71 ] [2.04 ]

10.0% oil 0.10 ± 0.06a 0.91 Y = 45.72 + 42.80X 0.11 ± 0.09a 0.97 Y = 45.36 + 42.16X

[5.70 ] [4.27]

10% oil + 10% PB 0.11 ± 0.03a 0.97 Y = 43.81 + 56.21X 0.12 ± 0.09a 0.93 Y = 44.04 + 49.64X

[5.18] [3.92]

20.0% oil 0.09 ± 0.03a 0.89 Y = 46.24 + 41.80X 0.08 ± 0.09a 0.99 Y = 46.54 + 43.16X

[6.33] [5.88 ]

1 means followed by different letters within the same column are significantly different at P < 0.052 means ± SD, 5 replicates, 20 individual /replicate, 24 hours check per batch from F2-generation for all experiments.3 ”None” means no PB or sesame oil was added to the various concentration of cypermethrin. SR = (LC50 none)/ (LC50 with PB

or sesame oil) while r2 was a correlation determination between concentration and mortality.4 Y = dependent variable (% mortality), X = independent variable (dose of cypermethrin with or without PB or sesame oil)

Visetson, 1991). Monooxygenase activity wasinhibited after PB was added giving a CF of 1.72fold but giving no change of esterase andglutathione-S-transferase activity in the leafdipping method. These results were similar tothose of Collins (1990) and Visetson (1991) whoworked with Tribolium castaneum. On the otherhand, the CFs for esterase and glutathione-S-transferase were slightly elevated in the contactmethod. These might be due to slight cross-synergisms of PB with esterase and glutathione-S-tranferase found by Prabharker et al. (1988).Although the statistical analysis showed nosignificant different, the addition of 10% sesameoil to cypermethrin trended to give CF of ca. 1.6for monooxygenase activity indicating inhibitionwhile no CF change was obtained when more oilwas administered. This was an indication that

sesame oil was a synergist with cypermethrin thatplayed more or less the same role as PB inmonooxygenase inhibition as proposed by Raffaand Priester (1985). The mixed oil - PB treatmentdid not show differences monooxygenase activity.CF from either PB or oil only treatments is alsoindicate that both sesame oil and PB gave the samemonooxygenase inhibition level.

Determination of cypermethrin residuesThe residues of cypermethrin in the larvae

from contact and leaf dipping method were 0.07and 0.11 ppm, respectively (Table 3). The additionof 10% PB and 20% sesame oil increasedcypermethrin in the larvae showing “residueincreases” (RI) of 2.86 and 2.57 folds in thecontact method and 8.27 and 6.91 folds with theleaf dipping method, respectively. The increased


cypermethrin in the larvae after addition of sesameoil or PB indicated that might be due tomonooxygenase inhibition when the synergistswere added. Both synergists might block an activesite or bind with apoenzymes of monooxygenasemaking it inactive resulting in increasedcypermethrin levels. Furthurmore, the oil plus PBtreatment showed little difference in larvalcypermethrin levels from those of either the PB orthe sesame oil treatment alone. This was alsoanother indication of monooxygenase inhibitionshowing RI of 3.0 and 6.55 in the contact and leafdipping methods, respectively. Higher levels of

accumulated insecticides in insects in terms ofinsecticide metabolism have been reported beforeby a number of workers using labeled insecticideswith synergists: trans-permethrin by De Vries andGeorghiou, (1981), diazinon by Forgash et al.(1962), DDT and dieldrin by Palpp and Hoyer,(1968).

Field experimentsCypermethrin alone reduced larval numbers

in the field by given reduced number (RN) of 1.67.Greater RN (ca. 8.34) was obtained forcypermethrin with 10% PB and for cypermethrin

Table 2 Detoxification enzyme activity of 2nd – 3rd instar larvae of Plutella xylostella L. after addition

of PB or various amounts of sesame oil to cypermethrin at LC50 from Table 1.

Types of detoxification enzyme activity 1,

Treatment (% w/v) Monooxygenase2 Esterase Glutathione-S-transferase

(product produced/min/mg protein)3

[CF]4

Contact Leaf dipping Contact Leaf dipping Contact Leaf dipping

Control 4,412 ± 187.14ab 18.16 ± 3.23b 1.12 ± 0.46a

- - -

None5 5,120 ± 231b 5,461 ± 568b 14.24 ± 0.31ab 15.76 ± 1.17ab 1.04 ± 0.10a 2.07 ± 0.33a

- - -

10%PB 3,671 ± 926ab 3,182 ± 170a 12.14 ± 0.32ab 20.11 ± 1.56ab 0.54 ± 0.16a 2.31 ± 1.12a

[1.39] [1.72] [1.17] [0.78] [1.93] [0.89]

0.5% oil 4,003 ± 243ab 4,678 ± 164ab 12.12 ± 0.96ab 14.79 ± 1.07a 2.43 ± 1.15ab 3.89 ± 1.68 a

[1.28] [1.17] [1.17] [1.07] [0.43] [0.53]

1.0% oil 3,650 ± 331ab 3,783 ± 892ab 10.45 ± 0.47a 17.37 ± 3.54ab 2.17 ± 1.32ab 3.23 ± 1.15a

[1.41] [1.44] [1.36] [0.91] [0.48] [0.64]

10.0% oil 3,200 ± 786ab 3,312 ± 760ab 12.87 ± 0.67ab 19.12 ± 1.23b 3.53 ± 0.11b 3.15 ± 1.12a

[1.60] [1.65] [1.11] [0.82] [0.29] [0.66]

10% oil + 10% PB 3,450 ± 461ab 3,498 ± 312ab 12.88 ± 0.43ab 18.97 ± 3.37ab 3.09 ± 1.11b 3.86 ± 1.09a

[1.48] [1.56] [1.11] [0.83] [0.34] [0.54]

20.0% oil 3,100 ± 879a 3,301 ± 639a 13.54 ± 0.36ab 19.43 ± 7.12ab 3.19 ± 0.12b 3.11 ± 1.14a

[1.65] [1.65] [1.05] [0.81] [0.33] [0.67]

1 means followed by different letters within the same column are significantly different at P < 0.05, DMRT2 means ± SD, 5 replicates, n= 10-15 of 2nd – 3rd instar lavae were employed, 24 hour check per batch from F2-generation for

all experiments.3 enzyme assays were followed Visetson and Milne (2001), the unit of monooxygenase, esterase and glutathion-S-transferase

are picM aldrin epoxidation/min/mg protein, nM paranitrophenol produced/min/mg protein and nM DCNB conjugated

product/min/mg protein.4 CF is a correlation factor = (enzyme activity of none)/ (enzyme activity of treatment).5 “None” means no PB or sesame oil was added to the concentration of cypermethrin while control means spraying with water

onto the larvae.


Table 3 Residues (ppm) of cypermethrin found in larvae of Plutella xylostella L. in the two assay

methods (contact and leaf dipping) after exposure to cypermethrin at LC50 level with and

without PB or sesame oil added.

Residue (ppm) of cypermethrin

Treatment (% w/v) Contact Leaf dipping3

(RI)4 (RI)

None1 0.07 ± 0.01a2 - 0.11 ± 0.06a -

10% PB 0.20 ± 0.11b [2.86] 0.91 ± 0.37b [8.27]

0.5 % oil 0.09 ± 0.06ab [1.29] 0.31 ± 0.12ab [2.82]

1.0% oil 0.09 ± 0.08ab [1.29] 0.54 ± 0.11ab [4.91]

10.0% oil 0.11 ± 0.06ab [1.57] 0.62 ± 0.09b [5.64]

10% oil + 10% PB 0.21 ± 0.07b [3.0] 0.72 ± 0.08b [6.55]

20.0% oil 0.18 ± 0.07b [2.57] 0.76 ± 0.08b [6.91]

1 “None” means no synergist was added to the cypermethrin.2 means followed by different letters within the same column are significantly different at P = 0.05, DMRT3 means ± SD, 5 replicates with 100 individual of 2nd – 3rd instar lavae of F2 generation per replicate were employed, exposure

for 24 hours for all experiments.4 RI was residue increase derived from (residues found in oil and PB or sesame oil added)/ (residue found in “none”).

with 10% sesame oil, RN was 5.74. In other words,the addition of sesame oil to the insecticide reducedthe larval population by 70-80 % while the additionof PB reduced the larval population by up to 88 %in the kale crop. These results showed higher RNvalues than those found by Visetson and Milne(2001) who, using derris extracts with PB, showedthat the addition of PB to cypermethrin reducedthe larvae in chinese kale by up to 50%. Thedifference might be due to a variety of ecologicaleffects. Season has often been reported to causemajor variation in terms of insect infestation incrops. This research was done in the rainy seasonwhen more larvae were found in the area while thework of Visetson and Milne (2001) was done insummer when there were less larvae were found.No further increase in RN was detected when amixture of PB and sesame oil or a higher percentageof sesame oil was added (Table 4). Thedetoxification enzyme activity of larvae from thefield experiment indicated more or less the sametrend as that in laboratory experiments exceptmonooxygenase activity. The monooxygenase

activity varying from 1.1- 1.5 folds was obtainedwhile esterases and glutathione-S-transferaseactivities showed no significant differences from“none”. The field results indicated that 10% sesameoil added to cypermethrin exhibited the same levelof monooxygenase inhibition as the addition of10% PB. This result confirmed the experiments ofYu (1986), Brattsten (1988) and Yang et al. (2001)who pointed out that plant allelochemicals couldeither reduce or inhibit detoxification mechanismsin terms of enzyme systems and hence increased ordecreased insect susceptibility to insecticides. Thiswas similar to the result of Rose (1985) who foundthat mid guts of polyphagous lepidopterous larvaeshowed stronger aldrin epoxidase activity than didthose of monophagous lepidopterous larvae. So,this research also gave strong evidence that sesameoil could stop monooxygenase function indiamondback moth larvae and that this oil could beused as a synergist in place of PB. However, theresults of enzyme activity of purified enzyme bothinduced and inhibited forms after sesame oil wasapplied with other insectcides would reveal the


Table 4 Means numbers ± SD enzyme activities of 2nd – 3rd instar Plutella xylostella L. larvae, found

on the leaves of Chinese kale after application of cypermethrin at LC50 (from Table 1) with or

without PB or sesame oil.

Enzyme activity

Treatment (% w/v) Number of larvae/10 plants Monooxygenase Esterase Glutathione-S-transferase

(product produced/min/mg protein)2,3

[RN]4,5 [CF]

Control 35.13 ± 2.24c1 4,412 ± 187a 18.16 ± 9.23a 1.12 ± 0.12a

- - - -

None 21.21 ± 8.23b 5,822 ± 100b 22.14 ± 0.05ab 2.22 ± 0.36ab

[1.67] - - -

10% PB 4.21 ± 0.89a 3,825 ± 254a 24.67 ± 3.25b 3.12 ± 1.76b

[8.34] [1.52] [0.89] [0.71]

0.5 % oil 10.12 ± 3.41ab 5,273 ± 385b 24.23 ± 1.56b 3.46 ± 1.11b

[3.47] [1.10] [0.91] [0.64]

1.0 % oil 5.61 ± 2.32a 4,982 ± 146a 15.24 ± 3.89a 3.33 ± 1.54b

[6.26] [1.17] [1.45] [0.67]

10.0 % oil 6.12 ± 3.13a 4,481 ± 119a 21.11 ± 1.12ab 3.12 ± 1.11b

[5.74] [1.29] [1.05] [0.71]

10% oil + 10% PB 5.32 ± 1.67a 3,898 ± 540a 22.33 ± 2.67ab 3.23 ± 1.11b

[6.60] [1.49] [0.99] [0.69]

20.0 % oil 6.24 ± 2.41a 4,211 ± 931a 21.44 ± 1.11ab 3.98 ± 1.25b

[5.63] [1.38] [1.03] [0.56]

1 means followed by different letters within the same column are significantly different at P = 0.05, DMRT2 None means no PB or oil was added to the cypermethrin while control means spraying with water onto the kale.3 enzyme assays were followed Visetson and Milne (2001), the unit of monooxygenase, esterase and glutathion-S-transferase

are picM aldrin epoxidation/min/mg protein, nM paranitrophenol produced/min/mg protein and nM DCNB conjugated

product/min/mg protein.4 RN was reduced number which was derived from the division of larvae found in untreated control and number found in treated

one.5 3 replicates with 100 individual of 2nd – 3rd instar lavae were employed, exposure for 24 hours for all experiments.

extent to which sesame oil could be used as asynergist. If sesame oil acts synergistically with allother insecticides, the farmers can reduce the useof insecticides and increase their effectiveness byaddition of sesame oil in formulations.

CONCLUSIONS

Sesame oil showed good synergism withcypermethrin. Both PB and sesame oil mixedtogether with cypermethrin inhibitedmonooxygenase activity. Residues of cypermethrinin the larvae increased by 1.29 – 2.57 folds in thetreatments with sesame oil added. This indicated

that both PB and sesame oil played the same rolein inhibition, possibly monooxygenase. Field trialsinvolving larvae population in Chinese kale gavesimilar results as in the laboratory. The addition ofsesame oil to the insecticide spray reduced thelarval population by 70-80 percent while theaddition of PB reduced the larval population in thekale by up to 88 percent.

ACKNOWLEDGMENTS

This research was supported by theKasetsart University Research and DevelopmentInstitute (KURDI), Kasetsart University, Thailand.


LITERATURE CITED

Brattsten, L.B. 1988. Potential role of plantallelochemicals in the development ofinsecticide resistance, pp 313-348. In P.Barbosa and D. Letourneau (eds.). Novelaspects of insect plant interactions. Wiley,New York.

Collins, P.J. 1990. A new resistance to pyrethroidsin Tribolium castaneum (Herbst). Pestic. Sci.28 : 101-115.

De Vries, D.H. and G. P. Georghiou. 1981.Decreased nerve sensitivity and decreasedcuticular penetration as mechanisms ofresistance to pyrethroids in a (IR)-trans-permethrin-selected strain of the house fly.Pestic. Biochem. Physiol. 15 : 234-241.

Finney, D.J. 1964. Statistical Methods inBiological Assay. 2nd ed. Charles Griffin andCompany Limited, London. 238p.

Forgash, A.J., B.J. Cook, and R.C. Riley. 1962.Mechanisms of resistance in diazinon-selectedmulti-resistant Musca domestica. J. Econ.Ent. 55 : 544-551.

Hung, C.F. and C.N. Sun. 1989. Microsomalmonooxygenase in diamondback moth larvaeresistant to fenvalerate and piperonyl butoxide.Pestic. Biochem. Physiol. 33 : 168-175.

Lowry O.H., N.J. Rosebough, A.L. Parr and R.J.Randall. 1951. Protein measurement with thefolin phenol reagent. J. Biol. Chem. 193 :256-275.

Matsumura, F. 1975. Botanical insecticides, pp.94-99. In Matsumura (ed.). Toxicology ofInsecticides. Plenum, New York.

Palpp, F.W.Jr. and R.F. Hoyer. 1968. Insecticideresistance in the house fly: decreased rate ofabsorption as the mechanism of action of agene that acts as an intensifier of resistance. J.Econ. Ent. 61 : 1298-1303.

Prabharker, N., D.J. Coudriet, and N.C. Toscano.1988. Effect of synergists on organophosphateand permethrin resistance in sweetpotato

whitefly (Homoptera: Aleyrodidae). J. Econ.Ent. 81 : 34-39.

Raffa, K.F. and T.M. Priester. 1985. Synergists asresearch tools and control agents in agriculture.J. Agric. Ent. 2 : 27-45.

Rose, H.A. 1985. The relationship between feedingspecialization and host plants to aldrinepoxidase activities of midgut homogenasesin larval Lepidoptera. Ecol. Ent. 10 : 455-467.

Schmutterer, H. 1990. Properties and potential ofnatural pesticides from the neem tree,Azadirachta indica. Annu. Rev. Entomol.35 : 271-297.

Visetson, S. 1991. Insecticide resistancemechanisms in the rust-red flour beetle(Tribolium castaneum. Herbst). PhD-thesis,The University of Sydney, NSW, Australia.

Visetson, S. and M. Milne. 2001. Effects of rootextract from derris (Derris elliptica Benth) onmortality and detoxification enzyme levels inthe diamondback moth larvae (Plutellaxylostella Linn.). Kasetsart J. (Nat. Sci.) 35: 157-163.

Visetson, S., M. Milne and J. Milne. 2001. Toxicityof 4,11-selinnadien-3-one from nutsedge(Cyperus rotundus L.) tuber extracts todiamondback moth larvae (Plutella xylostellaL.), detoxification mechanisms and toxicityto non target species. Kasetsart J. (Nat. Sci.)35 : 284- 292.

Yang, X, D.C. Margolies., K.Y. Zhu and L.L.Buschman. 2001. Host plant-induced changesin detoxification enzymes and susceptibilityto pesticides in the twospotted spider mite(Acari: Tetranychidae). J. Econ. Ent. 94 :381-387.

Yu, S.J. 1986. Consequences of induced foreigncompound-metabolizing enzymes in insects,pp 153-174. In L. B. Brattsten and S. Ahmad(eds.). Molecular aspects of insect-plantassociations. Plenum, New York.


Development of Fish Strip from Hybrid Clarias Catfish SurimiFortified with Konjac Flour

Benjawan Chotpradit, Mayuree Chaiyawat and Nongnuch Raksakulthai

ABSTRACT

The hybrid catfish surimi was prepared and used as raw material for fish strip. Fish strip was

processed by mixing hybrid catfish surimi, flour, salt, sugar, soy sauce and pepper for 10 minutes before

rolling into a thin sheet, cooked at 80∞C for 50 minutes in a hot air oven, then baked in a microwave oven

at a high power level for 35 seconds to expand and dry before cutting into a strip. To increase fibre of fish

strip, 1% of konjac flour could be added with moisture content of the mixture adjusted to 70% and 0.1-

0.2% sodium bicarbonate added. Dietary fibre of product from this process could be increased for 1.65

folds. The developed fish strip was more accepted in taste, texture and overall likeliness than the market

product.

Key words: fish strip, hybrid Clarias catfish, surimi, fibre, konjac flour

Department of Fishery Product, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand.


INTRODUCTION

In Thailand, at the present time, snacks

become more influence to people who live in rural

areas especially children. Many kinds of snacks

have been produced; however, most of those snacks

are made of starch and sugar, therefore, their

nutritive values are rather low. Fish can be added

to increase the protein content of snacks, e.g., in

fish strip that is one of the popular snacks with a

market share of about 10% (Boonyasirikul, 1998).

In general, fish strip is made from marine fish or

surimi. Freshwater fish has not yet been used as

raw material. Since hybrid Clarias catfish (Clarias

macrocephalus x C. gariepinus) is the second

highest species raised, the price is sometimes low

tremendously. Therefore new value added product

development may create market demand and a

way to assist the farmers. Surimi is stabilized

myofibrillar proteins obtained from mechanically

deboned fish flesh that is washed with water and

blended with cryoprotectants (Park and Morrissey,

2000). Washing process help to removes

compounds such as sarcoplasmic protein, inorganic

salts, low-molecular weight substances, lipids,

and blood components (Mireles DeWitt and

Morrissey, 2002). The objectives of this study are

to investigate the feasibility of using hybrid catfish

surimi as raw material for fish strip and increasing

dietary fibre in the product by fortification with

konjac flour.



1. Preparation of hybrid catfish surimi

3. Determination of appropriate dryingtemperature

An appropriate drying temperature was

determined by varying drying temperatures at 60,

70, 80, 90, and 100∞C for 50 minutes. The prepared

fish sheath was dried in a hot air oven at the above

temperature treatments before cooking in a

microwave oven at high level energy for 35 seconds

then cut into a strip of 2 mm width and 15 cm

length.

Fish strip was sensory evaluated for

appearance, color, flavor, taste, texture and overall

acceptability by 15 panelists using hedonic scoring

(1 - 9, 1 = dislike extremely, 9 = like extremely) to

determine the appropriate drying temperature. The

commercial sample with the ingredients of fish

meat, tapioca flour, sugar, salt and soy sauce given

full scores of 9 in all quality attributes was used to

compare with the experimental samples.

Experimental design was RCBD. Analysis of

variance and DMRT were employed. TA-HD

Texture Analyzer was used in measuring tensile

strength (g) of the products. Linear expansion was

calculated as:

Expansion (fold) =

Thickness of

Thickness of

fish strip after cooking in microwave oven (mm)

fish strip before cooking in microwave oven (mm)

4. Fortification of konjac flourKonjac flour of 0, 0.5, 1.0, 1.5 and 2.0% (w/

w) of the total mixture were prepared by suspending

small amount of water and added into the fish

mixture. The moisture of the mixture was adjusted

to 70% before thin sheath making as in 2. Sensory

evaluation and physical properties were determined

as in 3. Dietary fibre was analyzed according to

AOAC (1995).

5. Effect of sodium bicarbonate additionSodium bicarbonate at 0, 0.1, 0.2 and 0.3

% (w/w) of fish mixture with 1% (result of 4)

konjac flour was added and processed as in 2.

Sensory evaluation and physical properties were

Fresh hybrid catfish

Heading, Gutting and Filleting

Washing

Deboning

Leaching 3 times (20 min each)

1st, minced meat: iced water + 0.2%salt = 1:5 by wt

2nd, minced meat: iced water + 0.2%salt = 1:5 by wt

3rd, minced meat: iced water + 0.3%salt = 1:5 by wt

Pressing

Mixing with 3% sugar

Hybrid catfish surimi

Figure 1 Preparation of hybrid catfish surimi.

Source: Kongpun (1996)

2. Processing of fish stripIngredients for making fish strip comprised

74% hybrid catfish surimi, 10% corn starch, 2.5%

wheat flour, 2.5% tapioca flour, 6% sugar, 2.5%

soy sauce, 1.5% salt and 1% pepper.

Catfish meat was prepared into surimi

according to Kongpun (1996). All ingredients

were Kenwood mixed for 10 minutes and rolled on

plastic film into a thin sheath of 1.0-1.5 mm thick.

The fish sheath was dried in a hot air oven before

cooking in a microwave oven at high level of

energy for 35 seconds to reach 14-16% moisture

content then cut into a strip of 2 mm width and 15

cm length.


determined as in 3.


Optimum conditions were considered from

texture, linear expansion (fold), tensile strength

(g) and acceptability. Sensory evaluation scores

and physical characteristics of fish strip dried at

different temperatures are shown in Table 1. It was

found that drying temperature significantly affected

physical properties of fish strip. Sample dried at

80∞C got the highest sensory evaluation score for

texture and linear expansion after cooking was

also the highest but the tensile strength was the

lowest among all treatments, although it was higher

than that of the commercial sample.

Sensory evaluation scores and physical

characteristics of fish strip fortified with different

quantity of konjac flour are shown in Table 2. It

was found that the linear expansion of fish strip

decreased when concentration of konjac flour

increased. It could be concluded that the maximum

Table 1 Sensory evaluation scores and physical characteristics of fish strip dried at different temperatures.

Attribute Sensory evaluation scores at different drying temperature (∞C)1/

60 70 80 90 100 Com. sample

Appearance 6.73±0.90b 6.83±0.84b 6.73±0.88b 7.00±0.91b 6.73±0.88b 9.00±0.00a

Color 6.73±1.03bc 6.53±0.83bcd 7.03±1.31b 6.47±0.99cd 6.03±0.85d 9.00±0.00a

Flavor 6.80±1.08b 7.13±0.74b 7.00±0.84b 7.00±0.92b 6.73±1.03b 9.00±0.00a

Taste 6.27±1.33c 6.20±1.32c 7.13±0.99b 7.15±0.99b 6.27±1.03c 9.00±0.00a

Texture 6.77±0.65c 6.73±0.96c 7.37±0.69b 6.93±0.59bc 6.57±0.49c 9.00±0.00a

Acceptability 7.27±0.67bc 7.07±0.70c 7.53±0.64b 7.30±0.59bc 7.27±0.64bc 9.00±0.00a

Expansion (fold) 1.28±0.11b 1.38±0.08a 1.33±0.10a 1.16±0.11c 1.03±0.14d Not determined

Tensile strength (g) 332.3±42.7bc 325.5±36.8bc 312.2±23.2b 336.4±32.8bc 349.2±35.2c 276.8±11.3a

1/ Values in the same row followed by different letters are significantly different (P£0.05)

Table 2 Sensory evaluation scores and physical characteristics of fish strip fortified with different

concentrations of konjac flour.

Attribute Sensory evaluation scores of fish strip with different concentrations

of konjac flour 1/

0 (%) 0.5 (%) 1.0 (%) 1.5 (%) 2.0 (%)

Appearance 7.60±0.71a 7.40±0.54a 7.13±0.74ab 6.70±0.59b 5.93±0.80c

Color 7.23±0.90a 7.00±0.75a 7.43±0.90a 6.27±0.80b 6.03±0.85b

Flavor 7.47±0.83a 7.37±0.61ab 7.07±0.96abc 6.87±0.91bc 6.73±0.70c

Taste 7.60±0.74a 6.93±0.70a 7.10±0.85ab 6.60±0.51bc 6.10±0.95c

Texture 7.33±0.97a 7.20±0.80ab 7.10±0.54ab 6.53±1.35bc 5.93±1.10c

Acceptability 7.57±0.88a 7.23±0.56a 7.07±0.70a 6.37±0.67b 5.80±0.77c

Expansion (fold) 1.70±0.30a 1.62±0.09a 1.51±0.11ab 1.31±0.17c 0.87±0.13d

Tensile strength (g) 257.7±51.0a 269.4±32.7ab 282.9±26.9ab 301.2±46.1bc 334.8±34.2c



Table 3 Sensory evaluation scores and physical characteristics of fish strip prepared with different

concentrations of sodium bicarbonate.

Attribute Sensory evaluation scores of fish strip with different concentrations

of NaHCO31/

0 (%) 0.1 (%) 0.2 (%) 0.3 (%) Com. sample

Appearance 7.00±1.25cd 7.80±0.56b 7.37±0.48bc 6.57±0.59d 9.00±0.00a

Color 6.17±1.13d 7.20±0.56b 6.77±0.68bc 6.43±0.98cd 9.00±0.00a

Flavor 6.60±1.18b 7.20±0.62b 6.80±1.00b 6.67±1.11b 9.00±0.00a

Taste 6.87±0.91c 7.57±0.62b 7.27±0.53bc 7.00±1.19c 9.00±0.00a

Texture 6.27±1.28c 7.47±0.91b 7.00±0.38b 6.23±0.70c 9.00±0.00a

Acceptability 6.83±0.99b 7.37±0.58b 7.10±0.47b 6.20±1.58c 9.00±0.00a

Expansion (fold) 1.24±0.15c 1.58±0.11b 1.71±0.11b 2.08±0.57a Not determined

Tensile strength (g) 324.1±28.9c 276.0±32.4b 277.7±42.0b 221.1±38.2a 271.0±19.4b


concentration of konjac flour used was 1.0 %.

Increasing a concentration of konjac flour lowered

sensory evaluation scores of the products. However,

sensory evaluation scores of samples with 1 %

konjac flour were not significantly different from

the samples with 0 or 0.5 % konjac flour (P>0.05).

Dietary fibre of the sample with 1 % konjac flour

was 0.76% (AOAC, 1995) increased by 1.65 folds

from sample with out konjac flour.

Sensory evaluation scores and physical

characteristics of fish strip prepared with different

concentrations of sodium bicarbonate compared

with commercial sample are shown in Table 3. It

was found that the higher the concentration of

sodium bicarbonate used the increase in the

expansion of fish strip was obtained. However,

from the sensory evaluation scores on texture and

overall acceptability, it was concluded that

appropriate concentrations of sodium bicarbonate

were 0.1 – 0.2 %.

Fish strip prepared at the most appropriate

conditions of konjac flour and sodium bicarbonate

was compared with commercial sample. The results

were shown in Table 4. The sensory evaluation

scores for all attributes except flavor of the prepared

sample were higher than that of the commercial

sample. The score for flavor of the prepared sample

was lower, although not significant, due to the

specific flavor of hybrid catfish. However, the

scores for taste and overall acceptability were

significantly higher (P£0.05).

Table 4 Sensory evaluation scores of prepared fish strip and commercial sample.

Attribute Appearance Color Flavor Taste1/ Texture Overall1/

acceptability

Prepared sample 6.90±0.81 6.80±0.94 6.33±0.79 7.40±0.66a 7.10±0.91 7.17±0.75 a

Com. sample 6.70±0.75 6.83±1.25 6.80±0.70 6.60±0.95b 6.80±1.19 6.33±0.96 b

1/ Values in the same column followed by different letters are significantly different (P< 0.05)


CONCLUSION

1. Sample dried at 80∞C for 50 minutes

then cooked in a microwave oven at high for 35

seconds contained 14-16 % moisture content.

2. The addition of konjac flour should not

be higher than 1%.

3. Sodium bicarbonate increased the linear

expansion of the sample and softened its texture.

Therefore addition of 0.1 - 0.2% sodium

bicarbonate in sample with 1% konjac flour was

recommended.

4. Prepared fish strip has higher sensory

evaluation score than the commercial sample in

taste and overall acceptability (P£0.05) but

appearance, color, flavor and texture were not

significantly different.

ACKNOWLEDGEMENTS

This work was partially supported by thesis

and Dissertation Support Fund, Graduate School,

Kasetsart University.

LITERATURECITED

AOAC. 1995. Official Methods of Analysis. 16th

ed. Association of Official Analytical

Chemists. Arlington, Virginia.

Boonyasirikul, P. 1998. Production of Snacks at

the Institute of Food Research and Product

Development. Kasetsart University, Bangkok,

pp. 47-52. In 30 years, the Institute of FoodRescarch and Product Development,Kasetsart University. Kasetsart University,

Bangkok.

Kongpun, O. 1996. Yield and quality of surimi

from hybrid Clarias catfish and effect of

food additives on gel forming ability.

Fisheries Gazette 49 (1) : 48-54.

Mireles DeWitt, C.A. and M.T., Morrissey. 2002.

Pilot plant recovery of catheptic proteases

from surimi wash water. BioresourceTechnology 82 : 295-301.

Park, J.W. and M.T., Morrissey. 2000.

Manufacturing of surimi from light muscle

fish, pp 23-59. In J.W. Park (ed.). Surimi andSurimi Seafood. New York.


Antiaflatoxigenic Effect of Lactic Acid Bacteria IsolatedFrom Some Thai Fermented Foods

Siriporn Stonsaovapak1, Ladda Wattanasiritham1

and Aree Shuvisitkul2

ABSTRACT

Eighty – seven isolates of lactic acid bacteria (LAB) were isolated from some Thai fermented

foods. They comprised Lactobacillus, Pediococcus, Leuconostoc, Lactococcus, Streptococcus, and

Enterococcus. Each of these isolates was tested for its antifungal activity against Aspergillus flavus. Of

eighty – seven LAB isolates, the two isolates with high antifungal activity were chosen for further

identification. They were identified as Lactobacillus plantarum and L. brevis. The effects of LAB

supernates on growth and aflatoxin production in culture of A. flavus grown in malt extract broth for 14

days at 25∞C were also studied. Both of them showed inhibitory ability on growth and aflatoxin

production in cultures.

Key words: lactic acid bacteria, Thai fermented foods, antiaflatoxigenic

1 Institute of Food Research and Product Development, Kasetsart University, Bangkok 10900, Thailand.

2. Department of Science Service, Ministry of Science, Technology and Environmental, Bangkok 10400, Thailand.


INTRODUCTION

Aflatoxins are secondary metabolites

produced by Aspergillus flavus, A. flavus subsp.

parasiticus, and A. nomius in various foods and

agricultural commodities. Aflatoxins have been

shown to be toxigenic, carcinogenic, mutagenic,

and teratogenic to different species of animals

(Campbell and Stoloff, 1974). Aflatoxin B1 is the

most potent hepatocarcinogen in many animal

species (Chu, 1977). Aflatoxins have been reported

to be produced in cereal grains, peanuts, tree nuts,

figs, seeds and fermented products including cheese

and fermented meats such as salami, sausage, and

country cured hams (Gourama and Bullerman,

1995). Therefore, the presence of aflatoxins in

foods presents a potential hazard to human health.

Lactic acid bacteria (LAB) are gram

positive, non – sporulating microaerophilic bacteria

whose main fermentation product from

carbohydrates is lactate. They comprise both

cocci and rods. LAB are commonly found in foods

including fermented meat, vegetables, fruits,

beverages and dairy products (De Vuyst and

Vandamme, 1994). They are also of paramount

importance in food technology because of their

contribution to flavour and aroma development

and to spoilage retardation. The inhibition of

growth of food spoiling bacteria can be due to one

or more of the antibacterial substances produced

by LAB namely organic acids, hydrogen peroxide

and proteinaceous substances with a bactericidal

or bacteriostatic mode of action, such as

bacteriocins (Vandenbergh, 1993). The

antibacterial effects of LAB and their metabolites

are well documented and have been extensively


investigated, but more research on the antifungal

effects is needed (Klaenhammer, 1988;

Stonsaovapak et al. , 1994).

The present study was undertaken to

demonstrate the inhibition of aflatoxitgenic fungi

by LAB isolated from some Thai fermented foods,

in order to reduce the health hazard of aflatoxins.

The inhibition of toxigenic molds by LAB could

be of great public health significance.


Mold cultureThe organism used in this study was

Aspergillus flavus, obtained from the Department

of Agriculture, Ministry of Agriculture and

Cooperatives, Bangkok, Thailand. It was

maintained on slants of potato dextrose agar (PDA

; Merck, Darmstadt) at 4∞C until further use.

Preparation of spore suspensionCulture of the fungi was grown on PDA

slants for 7 to 10 days at 25∞C until well sporulated.

The spores were harvested by adding 10 ml of

sterile water and aseptically dislodging the spores

with a sterile inoculating loop. Spore suspensions

were aseptically filtered through sterile cheesecloth

to remove mycelial debris. The spore suspensions

were further adjusted with sterile water to give a

final spore concentration of approximately 104

spores / ml. The spore concentration was

determined on PDA plates using standard pour

plate technique at 25∞C for 2 to 3 days. The PDA

used in this study was not acidified, and the pH of

this medium after sterilization was 5.6 ± 0.2

Bacterial strains and culture conditionsLAB used in this study were isolated from

locally available Thai fermented foods including

fish and shellfish, pork, vegetables, and rice (Table

1). 25 g of each sample was added to 225 ml of

sterile phosphate buffer pH 7.0 and thoroughly

homogenized for 1 minute in a stomacher. 0.1 ml

of the appropriate dilutions were spread onto the

surface of MRS plates (MRS medium; Merck,

Darmstadt) and incubated for 3 days at 30∞C.

Colonies of LAB were randomly selected from

MRS plates. They were propagated and maintained

in MRS broth.

Preparation of culture supernatantsFor preparation of culture supernatants, the

randomly selected isolates LAB were grown in

MRS broth at 30∞C for 18 h without shaking. Cells

were removed by centrifugation at 8,000 rpm for

10 minutes, followed by filtration of the culture

supernatants through a 0.45 mm pore size filter

(Millipore).

Table 1 Thai fermented foods used in the experiment.

Vegetable Fish and shellfish Pork Rice

Pak-Sian-Dong Pla-Som Nham Khao-Mak

Naw-Mai-Dong Pla-Paeng-Daeng Sai-Krok-Prieo

Pak-Kard-Dong Pla-Ra

Tua-Ghog-Dong Nam-Bu-Du

Sa-Tau-Dong Kung-Som

Hom-Dong Tai-Pla

Kung-Jom

Hoi-Dong


Screening of antifungal activity by LABApproximately 104 spores / ml of the A.

flavus (indicator organisms) were used for testing

the antagonistic activity. A. flavus was spread with

a swab on PDA plates, 5 ml of LAB culture

supernantant was spotted on freshly prepared lawn

of A. flavus (Mayr – Harting et al., 1972). After

incubated plates for 3 – 5 days at 25∞C, the plates

were checked for inhibition zones. The culture

supernatant which were qualified as positive were

neutralized to pH 7 with 0.1 N NaOH and were

tested again for antifungal activity. LAB that had

high antifungal activity were further identified

and were used in inhibition test.

Identification of LABLAB were identified by gram staining,

catalase test, cell morphology, growth at selected

temperatures, thermal resistance, and carbohydrate

fermentation pattern (Weiss, 1992).

Inhibition test of fungal growth and aflatoxinproduction

Tests for inhibition of fungal growth and

aflatoxin production were assayed in flask cultures.

One millilitre of the final spore suspension

containing about 104 spores / ml of A. flavus was

inoculated in 125 ml conical flasks containing 10

ml of malt extract broth (Merck, Darmstadt).

Different concentrations of selected LAB supernate

were added to the flask. The flask cultures were

then incubated for 14 days at 25∞C. After 14 days

of incubation, the survival of mold growth were

determined on PDA plates. Duplicate samples

were taken for each assay, and experiments were

replicated three times to reduce variability

Determination of approximate aflatoxin

content in the cultures were carried out according

to the method reported by Bullerman et al., (1977).

Flasks were samples aseptically by removing

duplicate portions of 0.1 ml of broth for aflatoxin

determination. The culture filtrate was extracted

with 5 ml of chloroform by liquid / liquid extraction.

Total aflatoxin content was then determined by

measuring UV absorption at 362 nm using a

spectrophotometer and calculating total aflatoxin

content using the molar extinction coefficient of

21,800 reported for aflatoxin B (Asao et al., 1963).

The approximate aflatoxin content was calculated

from three replicate experiments.


Eighty – seven isolates of lactic acid bacteria

were isolated from locally available Thai fermented

foods including vegetables, fish and shellfish,

pork and rice. The genus of all LAB isolates were

identified according to the method described by

Weiss (1992). The results showed that LAB

isolated from Thai fermented foods were

Lactobacillus (73 isolates), Pediococcus (6

isolates), Leuconostoc (2 isolates), Lactococcus

(2 isolates), Streptococcus (2 isolates), and

Enterococcus (2 isolates) (Table 2).

The results showed that many kinds of

LAB can be found in Thai fermented foods. It was

also shown that Lactobacillus was the predominant

genus among LAB isolated in this study. This

finding agreed with those of Charernjiratrakul and

Rodpradit (1997), who studied on the isolation and

identification of lactic acid bacteria from Thai

fermented foods.

Each of these isolates were tested for

antifungal activity against A. flavus by using spot-

on-lawn assay. The result showed that of the 87

LAB isolates studied, 47 isolates did not show any

inhibitory ability against A. flavus. Twenty – three

isolates had low inhibitory ability producing

inhibition zones of less than 6 mm. Ten isolates

were considered to have low inhibitory ability too,

by producing 6-10 mm inhibition zones. Five

isolates were considered to have moderate

inhibitory ability producing 10-18 mm inhibition

zones. Two isolates had high inhibitory ability

producing inhibition zones of more than 20 mm

(Table 3).


Table 2 The genus of lactic acid bacteria isolated from Thai fermented foods.

Genus Number of LAB

Isolates %

Lactobacillus 73 83.90

Pediococcus 6 6.90

Leuconostoc 2 2.30

Lactococcus 2 2.30

Streptococcus 2 2.30

Enterococcus 2 2.30

Table 3 Antifungal activity of lactic acid bacteria

isolated from Thai fermented foods

against A. flavus.

Number of isolates A. flavus

Inhibition zone

47 -

23 +

10 ++

5 +++

2 ++++

- = no inhibition zone

+ = inhibition zone of less than 6 mm

++ = inhibition zone of 6 – 10 mm

+++ = inhibition zone of 10 – 18 mm

++++ = inhibition zone of more than 20 mm

When the pH of the culture supernatants of

the two isolates which gave high inhibitory were

adjusted to 7 with 0.1 N NaOH before testing their

antifungal activity, the supernatants remained their

ability to inhibit the growth of A. flavus. It indicated

that fungal inhibition was not due to the lactic acid

produced.

Antifungal activities of LAB had been

reported by some investigators (Batish et al., 1991),

and the inhibitory compound was polypeptide.

Two isolates of LAB with high inhibitory

ability were morphologically, physiologically, and

biochemically characterized and identified

according to the method described by Weiss (1992).

The result showed that L1 isolate identified as

Lactobacillus plantarum and L2 isolate was L.

brevis (Table 4).

The effects of L1 and L2 supernates on

growth and aflatoxin production in culture of A.

flavus grown in malt extract broth for 14 days at

25∞C were presented in Table 5. No growth or

aflatoxin could be detected in the culture to which

10% of L1 supernate had been added, whereas

slightly growth and trace of aflatoxin production

were obtained when 10% of L2 supernate was

added to the cultures.

It has also been reported previously that

Lactobacillus cell-free supernatant inhibited the

production of aflatoxin (Karunaratne et al., 1990).

It was shown that the aflatoxin inhibition was

probably due to an inhibitory metabolite other

than hydrogen peroxide and low pH. The aflatoxin

reduction was due to low-molecular-weight

inhibitory compounds. Partial purification and

characterization of the inhibitor showed that it was

a heat-stable compound.

From the available literature and also this

study on the effect of LAB on mold growth and

mycotoxin production, it would appear that LAB

have the potential as biological control agents in

foods to prevent mold growth. The antifungal

biopreservatives have the potential to constitute

suitable food preservatives that are safe, effective,

and acceptable to consumers, regulatory agencies,


Table 4 Morphological, physialogical, and biochemical characteristics of the 2 isolates of lactic acid

bacteria having high inhibitory ability.

Characteristics Lactic acid bacteria isolates

L1 L2

Morphology Rod Rod

Gram stain + +

Catalase test - -

Growth at 15∞C + +

Gas production - +

Hydrolysis of arginine - +

Fermentation of

Arabinose - +

Cellubiose + -

Esculin + -

Galactose + +

Gluconate + +

Glycerol - -

Inulin - -

Lactose + +

Maltose + +

Mannitol + -

Melezitose + -

Melibiose + +

Raffinose + -

Rhamnose - -

Ribose + +

Salicin + -

Sorbitol + -

Sucrose + -

Trehalose + -

Xylose - +

L. plantarum L. brevis

and the food industries. Many reports showed that

the inhibition of mycotoxins by LAB was due to

factors other than acidity, and there is a strong

indication that some inhibitory compounds are

protein in nature. It is recommended that in future

studies more research is needed to purify and

identify inhibitory compounds.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the

Kasetsart University Research and Development

Institute for provided funding this study.


LITERATURE CITED

Asao, T., G. Buchi, M.M. Abdel – Kadir, S.B.

Chang, E.I. Wick and G.N. Wogan. 1963.

Aflatoxin B and G. J. Am. Chem. Soc. 85 :

1706 – 1707.

Batish, V.K., L. Ram, and S. Grover. 1991.

Interaction of Streptocccus lactis subsp.

diacetylactis DRC – 1 with Aspergillus

parasiticus and A. fumigatus in milk. Cult.Dairy Prod. J. 26 : 13 – 14.

Bullerman, L.B. , F.Y. Lieu, and S.A. Seier.

1977. Inhibition of growth and aflatoxin

production by cinnamon and clove oils,

cinnamic aldehyde and eugenol. J. Food Sci.42 : 1107 – 1109.

Campbell, T.C. , and L. Stoloff. 1974. Implication

of mycotoxins for human health. J. Agric.Food Chem. 22 : 1006 – 1015.

Charernjiratrakul, W. and A. Rodpradit. 1997.

Isolation, screening and identification of lactic

acid bacteria from Thai fermented foods.

Songklanakarin J. Sci Technol. 19 : 181 –

188.

Chu, F.S. 1977. Mold of action of mycotoxins and

related compounds. Adv. Appl. Microbiol.40 : 352 – 357.

De Vuyst, L. , and E.J. Vandamme. 1994. Lactic

acid bacteria and bacteriocins : their practical

importance, pp.1 – 11. In L. De Vuyst and

E.J. Vandamme (eds.). Bacteriocins ofLactic Acid Bacteria. Blackie Academic &

Professional, Glasgow.

Gourama, H., and L.B. Bullerman. 1995.

Antimycotic and antiaflatoxigenic effect of

lactic acid bacteria : A review. J. Food Prot.58 : 1275-1280.

Karunaratne, A., E. Wezenberg and L.B.

Bullerman. 1990. Inhibition of mold growth

and aflatoxin production by Lactobacillus

spp. J. Food Prot. 53 : 230 – 236.

Klaenhammer, T.R. 1988. Bacteriocins of lactic

acid bacteria. Biochimie. 70 : 337 – 349.

Mayr – Harting, A., A.J. Hedges and R.C.W.

Table 5 Growth and aflatoxin production of A. flavus grown in malt extract broth containing inoculum

level of 104 spores / ml and inhibitor (selected LAB supernate) after 14 days of incubation at

25∞C.

Inhibitor Concentration A. flavus

(%) Log CFU/g Aflatoxin (mg/ml)

Control 6.80 ± 0.02a 2.75 ± 0.10a

L1 supernate 1 4.02 ± 0.03 1.84 ± 0.04

(L. plantarum) 5 2.28 ± 0.01 0.08 ± 0.02

10 NGb NDc

L2 supernate 1 5.00 ± 0.03 2.02 ± 0.06

(L. brevis) 5 3.28 ± 0.08 1.25 ± 0.04

10 1.20 ± 0.01 1.08 ± 0.01

a Values are mean ± SD, n = 3b NG, no growthc ND, not detected


Berkeley. 1972. Methods for studying

bacteriocins, pp. 315-422. In J.R. Noris and

D.W. Ribbons (eds.). Methods inMicrobiology, vol. 7 A. Academic Press,

Inc., New York.

Stonsaovapak, S., J. Kaneko, and K. Izaki. 1994.

Characterization of bacteriocin produced by

Pediococcus acidilactici isolated from

fermented pork in Thailand. Kasetsart J.(Nat. Sci.) 28 : 310 – 313.

Vandenbergh, P.A. 1993. Lactic acid bacteria,

their metabolic products and interference with

microbial growth. FEMS Microbiol. Rev.12 : 221 – 237.

Weiss, N. 1992. The Genera Pediococcus and

Aerococcus, pp. 1502-1507. In A. Balows,

H.G. Truper, M. Dworkin, W. Harder and

K.H. Schlesifer (eds.). The Prokaryotes, volII Springer Verlag New York Inc., New York.


Using of Extrusion Process for Preparation of Instant CerealBeverage Powders based on Corn and Soybean

Chulaluck Charunuch, Pracha Boonyasirikooland Chowladda Tiengpook

ABSTRACT

Preparation of the instant cereal beverage powders based on corn and soybean from extrusion

process has been studied. To examine the effect of particle size of corn grit (13, 23 and 33 mesh) and the

composition between corn grit and isolated soy protein (84:10, 74:20 and 64:30) on the properties of

product and evaluate nutritional value of acceptable product. The results showed that the differences in

particle size of corn grit were not significant effect (p > 0.05) on the chemical composition (moisture and

protein content) and most of the physical properties of product but the differences in the composition

between corn grit and isolated soy protein were significant effect (p £ 0.05) on the protein content and

the physical properties of product (bulk density, reconstitution index, water absorption index, water

solubility index and viscosity). The highest acceptable product consists of particle size of corn grit

equaled to 13 mesh and the composition between corn grit and isolated soy protein equaled to 84:10 which

reconstituted well, good soluble and moderate viscosity had adequate protein content and appropriate

pattern of essential amino acid for good nutritive consuming.

Key words: instant cereal beverage powders,extrusion process,corn,isolated soy protein


Institute of Food Research and Product Development, Kasetsart University, Bangkok 10900, Thailand.

INTRODUCTION

In the present Thai social condition is

competitive, the way of life is urgent and urban

citizens are faced with chronic traffic problems.

Hence their need for food which can be easily

prepared and convenient for consumption is ever

increasing. In the aspect of food industry, more

technologies for food product research and

development are needed satisfy needs of

consumers. Apart from being convenient to prepare

and easy for consumption, these food products

should also have sufficiently high nutrition, to

result in good quality of life for the consumers.

Especially for school age children who are often

faced with traffic problems, causing them not to

have time for breakfast before going to school.

And not having breakfast reduces the sugar level in

the blood, resulting in learning, and working

effiency in working age people are also reduced.

With this reason, instant beverage powders are

another choice for consumers who need

convenience and quickness in food preparation for

family members. They are also health food for

consumers in all gender and age groups.

In the current bad economic condition,

agro - industry promotion is vital for the country’s

economic development. Because the agricultural

sector is the source of principle income for the

country, and be the economic sector creating large

number of employment. Therefore, according to

the 8th National Economic and social Development

Plan (Year 1997-2001), the government policies

stress increased value of agricultural goods

according to market demands, and to promote

agricultural goods processing. Because of this

reason, the research work stresses development of

food products from corn and soybean, which are

important agricultural raw materials in Thailand,

whose prices are low and production quantities are

sufficient for domestic consumption. They are

processed into instant beverage powders which

can be prepared in short length of time by dissolving

in water. The products are suitable for those who

have no time for food preparation. Moreover, they

are also developed into high nutrition protein

drinks for those who are health conscious. In the

aspect of instant food processing from cereals

which can dissolve well in water, the traditional

process is to make the food hot, cooked and dried

by drum drier. Flaked product is obtained, and the

product is then grounded and sifted through a

mesh with required size. After extrusion, cooking

technology was introduced in the food industry,

diverse production processes and various instant

food products from cereals were developed (Hauck,

1980), including instant beverage powders. This is

because extrusion system (Harper, 1981) has the

ability to make cereals gelatinize and form

expanded products with the property of good water

absorptibility. Moreover, it is also beneficial in the

aspects of its high productivity, energy efficient

and production step reduction.

Concerning foreign research works related

to instant beverage powder production, most of

raw materials used are in the form of liquid or high

viscosity liquid. Therefore they tend to be made

hot and dried by using spray drier (Holsinger et al.,

1974; Guy and Vetterl 1975; King, 1985). But if

the main raw materials used were cereals and in

characteristics of powders, the process used tend

to be one of the two systems (Anderson et al.,

1971) which one is the drum drying system, making

them hot and then dry by using roller machine, and

the another is extrusion cooking system, making

them hot and reduce moisture within the extruder,

such as in the research work “Instant Beverage

Mixes” (Bookwalter et al., 1971) which used cereals

as raw materials, and they are made hot and

cooked by using single screw extruder. The product

obtained is then grounded and flavored and

improved nutritive value by mixing with other

food materials such as milk powder. This product

can be dissolved in hot water and ready for

consumed. The research works in Thailand are

rather few and often use drum drying system in

production, such as the research work “

Development of instant high fiber processed food”

(Tangkanakul et al., 2000) which boiled and

steamed agricultural raw materials mix, after which

water was added and grounded until fine. The food

was then dried by using double drum drier, and

made into powder which was also consumed as

instant beverage powder. And from the result of

survey on instant cereal beverage powders available

in Thailand, it was found that most of the products

use drum drier production system. Imported

products such as the brand names “GOLD

ROAST”, “SUPER” and “VITAMAX” are

imported from Singapore, while other products

manufactured locally are “NESVITA”, “INNA”,

“MONIEGOLD” and “GOOD TIME”. With

“NESVITA”, which is a rather popular brand

manufactured by Nestle Foods (Thailand) Co.,

Ltd., being produced by using cereal mix and

made hot, cooked and dried by using double drum

drier. After which it is grounded to the required

size, obtaining the instant cereal powders (Cereal

Base Type 02) which can be flavored with other

ingredients and be consumed as instant beverage

powders. Moreover, the Cereal Base Type 02 is

also sold to other manufacturers for flavored and

sold as instant cereal beverage powders with brand

names “MONIEGOLD” and “GOOD TIME”.

For this reason the research work used

extrusion technology to produce instant cereal



beverage powders, in order to develop domestic

production and make them more diverse. This can

also help to reduce import quantity of this type of

products and is also greatly beneficial for solving

the country’s economic problems.


Preparation of raw materialsCorn grit (13 and 33 mesh) were supplied

by Thai Maize Products Ltd. Corn grit (23 mesh),

getting from corn grit (13 mesh) which was

grounded and sieved for screening the required

size. Isolated soy protein (Profam 974), full fat soy

flour and flavorings were supplied by Heinz Win

Chance Ltd., the Royal Project and Givaudan

Roure Ltd. respectively. After preparation, raw

materials were examined by proximate analysis

(A.O.A.C., 1990) and particle size distribution.

Experimental designTo study the production of instant cereal

beverage powders from extrusion process, the 3¥3 factorial in randomized complete block design

was employed with two independent variables at

three levels of variation. The independent variables

were size of corn grit (13, 23 and 33 mesh) and the

composition between corn grit and isolated soy

protein (84 : 10, 74 : 20 and 64 : 30). Dependent

variables were moisture and protein content, bulk

density, reconstitution index, water absorption

index, water solubility index and viscosity.

Experimental data were analyzed by using the

Statistical Analysis System (SAS) and a second

order polynomial equation was fitted to each

response variable.

Extrusion processFor each test run of experimental design (9

experimental units/1 replicate; 3 replicates), the

weighed raw materials (the composition between

corn grit and isolated soy protein equaled to 84 :

10, 74 : 20 or 64 : 30, full fat soy flour 4%,

vegetable oil 1% and mixture of vitamins and

minerals 1%) at each size of corn grit were

thoroughly mixed by a mixer before fed into a

laboratory co – rotating twin – screw extruder

(Hermann Berstorff Laboratory Co-rotating Twin

Screw Extruder ZE25¥33D). This extruder

comprises of 7 parts of barrel ended with a 24.5

mm thick die plate with one circular die hole

(diameter 3.0 mm). The barrel length – to – diameter

ratio (L/D) of the extruder was 870 : 25. The

mixture of raw materials were fed into the extruder

with a volumetric twin screw feeder (K- Tron

soder AG5702, type 20, Switzerland) and water

was pumped to the ingredients to achieve required

moisture content. Temperature of barrel 1-7 and 9

was 30, 35, 65, 135, 155, 175, 130 and 125∞Crespectively. The other operating condition were

adjusted at screw speed 350 rpm, feed rate 319-

375 g/min, water rate 19-26 g/min, feed moisture

15-17% and melting temperature 152-155∞C. After

extrusion, the extruded samples were dried in the

electric oven at 80∞C for 10 min and grounded by

Fitz Mill (mesh size 0.6 mm) to obtain instant

cereal powders. Finally, the instant cereal powders

(50%) was mixed with sugar (25%), skim milk

powder (10%), creamer (14.4%), malt flavor

(0.2%) and milk cream flavor (0.4%) to produce

the instant cereal beverage powders.

Chemical and physical properties examinationThe final products, instant cereal beverage

powders were examined chemical and physical

properties as below.

Moisture and protein content (A.O.A.C.

1990).

Bulk density (Akpapunum and Markakis,

1981). The loose bulk density of product was

determined by transferring 50 g product into a 250

ml graduated glass cylinder and measuring the

volume of the products off the scale. The packed

bulk density was determined in a similar way, but

the volume was measured after tapping the cylinder

until the products settled (about 2 min). Both of the


loose and packed bulk density were calculated as:

Bulk density (g/ml) = mass of sample

Volume occupied by sample

Reconstitution index (Ihekoronye and

Oladunjoye, 1988). The reconstitution index was

determined by mixing 7.5 g of products with 50

ml. of warm water (50∞C) for 90 sec and measuring

the sediment formed in a graduated cylinder, 10

min after the mixing.

Water absorption index and Watersolubility index (Anderson et al., 1969 and

Damardjati and Luh, 1987). A 2.5 g of the ground

sample was suspended in 30 ml of water in a 50 ml

tared centrifuge tube. The sample was stirred

intermittenly over a 30 min period and centrifuged

at 3000¥g for 10 min. The supernatant was poured

carefully into a tared evaporating dish. The

remaining gel was weighed and the WAI was

calculated as follows:

Water absorption index (WAI) =

Weight of gel - Weight of ground dry sample

Weight of ground dry sample

The supernatant liquid from the WAI study

was vacuum dried at 70∞C until constant weight

was reached. The amount of dried solid (%)

recovered from evaporating the supernatant was

expressed as water solubility index.

Viscosity. The viscosity of dispersions

containing 7.5 g of product in 50 ml of water was

measured by Brookfield Digital Viscometer, model

RVDV-III (Operating conditions:- U-L Adaptor,

ULA Spindle, 16 ml sample, 25∞C and 10 rpm).

Sensory evaluationThe final products, instant cereal beverage

powders at each size of corn grit and each


protein were conducted with trained panels (18) in

balanced incomplete block experimental design

(t=9, k=4, r=8, b=18, l=3) who have experienced

with food product development by using 9-point

hedonic scale (1-extremely dislike to 9-extremely

like) to determine the preference in color, odor,

flavor, texture and overall acceptant of products.

Nutrition labeling and protein qualityThe nutritive value of the most appropriate

instant cereal beverage powder was evaluated in

the forms of nutrition labeling and pattern of

essential amino acids. The nutrition labeling based

on the Announcement of the Public Health Ministry

No.182, 1998. Additionally, the protein quality

was assessed by comparing essential amino acids

of this product with standard pattern of essential

amino acids set by joint FAO/WHO committee.


Chemical composition and particle size of rawmaterials using for instant cereal powders fromextrusion process

The principal raw materials using for

production of instant cereal powders from extrusion

process in this research work was corn grit. This is

because apart from being an important agricultural

raw material in Thailand with cheap price and

sufficient production quantity for domestic

consumption, corn also has properties suitable for

extrusion process. Because it can expand well and

gives good corn flavor retained after extrusion

(Moore, 1993). The raw material used together

with corn was soybean which improve extruded

product for higher nutritive value in case of protein

content and pattern of essential amino acids than

product made from only one type of cereal.

Moreover, the heat from extrusion process also

reduced trypsin inhibitor which is a toxic substance

in soybean not required by the body (Konstance et

al., 1998). Types of soybean using in this research

were in the form of isolated soy protein and full fat

soy flour. Usage of isolated soy protein gave

benefits in the aspects of increasing protein quantity

(Table 1), without the disadvantage of bean smell

in the product. While apart from being raw material

with high nutritional value concerning protein and


Table 1 Chemical composition and particle size of raw materials.

Chemical composition (%) Average

Raw materials Moisture Fat Protein Ash Dietary 1/ particle size

fiber (mesh)

Corn grit (Large) 11.95 1.43 6.26 0.40 3.89 13

Corn grit (Medium) 11.67 1.30 6.46 0.45 2.42 23

Corn grit (Small) 11.80 1.64 6.36 0.54 2.88 33

Isolated soy protein 3.46 3.02 86.75 4.94 4.60 > 100 2/

Full fat soy flour 2.65 22.10 40.14 5.06 17.20 > 100 3/

Source : 1/ Food and Nutrition Technical Services, Institute of Nutrition, Mahidol University.

2/ Protein Specialties Division, Archer Daniels Midland Company, USA.

3/ Sahaviriya Pure Science Co., Ltd.

fat, full fat soy flour is also source of dietary fiber

(Table 1). Furthermore, usage of oil from full fat

soy flour together with vegetable oil which added

to the raw materials about 1-2 percent, was also

beneficial in the aspect of food material lubrication,

helping the food product to expand well and

consistently, and have good texture

(Boonyasirikool and Charunuch, 1999)

The effect of particle size of corn grit and thecomposition between corn grit and isolated soyprotein on the product qualities

Due to the interactions between particle

size of corn grit (13, 23 and 33 mesh) and the


protein (84:10, 74:20 and 64:30) did not have

significant effect (P>0.05) on the moisture content

and the physical properties of product (bulk density,

reconstitution index, water absorption index, water

solubility index and viscosity) but only had

significant effect (P£0.05) on the protein content

as shown in Table 2, so it should be considered

additionally on the effect of main factors (particle

size of com grit or the composition between corn

grit and isolated soy protein) as shown in Table 3

and 4. It was found that particle size of com grit did

not have significant effect (P>0.05) on the chemical

composition (moisture and protein content) and

most of the physical properties of product, but the

differences in the composition between corn grit

and isolated soy protein had significant effect

(P£0.05) on the protein content and the physical

properties of product , except the moisture content.

Isolated soy protein has increased protein content

which caused the product less soluble and

reconstitute not well, reduced viscosity also.

Because of this, raw material which consists of

starch molecules such as corn can expand and

absorb water well, while raw material which consist

of protein molecules such as isolated soy protein

has less expand after extrusion and the property of

dissolving into homogeneous solution with

difficulty (Moore, 1993). Thus, the composition

between corn grit and isolated soy protein was an

important factor for producing high quality of

instant cereal beverage powders.

Because the studied factors (particle size of

corn grit and the composition between corn grit

and isolated soy protein) were quantitative factors,

hence response surface could be studied to show

trends of response when levels of studied

quantitative factors changed as shown in Table 5.

Moreover, the effect of particle size of corn

grit and the composition between corn grit and

isolated soy protein caused the product difference

significantly (P£0.05) on sensory evaluations as


Tab

le 2

Eff

ect o

f int

erac

tions

bet

wee

n pa

rtic

le s

ize

of c

orn

grit

and

the

com

posi

tion

betw

een

corn

gri

t and

isol

ated

soy

pro

tein

on

som

e ch

emic

al a

nd

phys

ical

pro

pert

ies

of in

stan

t cer

eal b

ever

age

pow

ders

.

Tre

atm

ent

Bul

k de

nsity

(g/

ml)

Rec

onst

itutio

n in

dex

(ml)

Wat

erW

ater

Size

of

corn

Cor

n gr

it :

Moi

stur

ePr

otei

nL

oose

bul

kPa

cked

Sedi

men

tC

lear

abso

rptio

nso

lubi

lity

Vis

cosi

ty

grit

(mes

h)Is

olat

ed s

oy(%

)(%

db)

dens

itybu

lk d

ensi

tyin

dex

inde

x(C

ps)

prot

ein

(%)

1384

:10

3.33±0

.39A

12:9

8±0.

30D

0.44±0

.01A

0.52±0

.04A

53.0

0±0.

.25A

1.00±0

.25A

1.95±0

.05A

55.9

7±2.

78A

168.

33±9

.95A

1374

:20

3.15±0

.42A

17.6

1±0.

32C

0.40±0

.06A

0.47±0

.08A

52.0

8±0.

14A

1.92±0

.14A

1.40±0

.18A

56.7

9±2.

20A

94.9

7±9.

12A

1364

:30

3.27±0

.24A

20.4

5±0.

77B

0.42±0

.03A

0.49±0

.06A

49.5

8±1.

59A

4.42±1

.59A

1.54±0

.26A

53.7

9±2.

65A

48.4

3±8.

73A

2384

:10

3.19±0

.37A

12.6

5±0.

31D

0.43±0

.03A

0.52±0

.06A

52.9

2±0.

14A

1.08±0

.14A

2.00±0

.02A

56.1

3±2.

67A

175.

13±1

9.92

A

2374

:20

3.11±0

.49A

17.2

6±0.

69C

0.40±0

.04A

0.48±0

.06A

51.7

5±0.

25A

2.25±0

.25A

1.39±0

.12A

56.4

0±1.

03A

88.0

7±20

.90A

2364

:30

3.34±0

.14A

21.4

5±0.

27A

0.40±0

.04A

0.48±0

.06A

48.1

7±2.

36A

5.83±2

.36A

1.68

v0.1

2A52

.59±

0.37

A30

.97±

6.85

A

3384

:10

3.17±0

.22A

12.6

0±0.

26D

0.44±0

.02A

0.54±0

.05A

52.5

8±0.

29A

1.42±0

.29A

1.86±0

.04A

57.7

5±0.

81A

140.

57±1

5.40

A

3374

:20

3.14±0

.02A

17.2

3±0.

20C

0.42±0

.05A

0.52±0

.08A

50.8

3±0.

76A

3.17±0

.76A

1.39±0

.18A

56.1

4±0.

91A

87.0

7±28

.53A

3364

:30

2.82

±0.2

4A21

.45±

0.48

A0.

41±0

.05A

0.50

±0.0

9A48

.08±

2.27

A5.

92±2

.27A

1.66

± 0.2

8A53

.98±

1.69

A28

.60±

5.47

A

In a

col

umn,

mea

ns w

ith th

e sa

me

lette

r ar

e no

t sig

nifi

cant

ly d

iffe

rent

at 0

.05

sign

ific

ance

leve

l.

78 Kasetsart J. (Nat. Sci.) 37 (1)T

able

3E

ffec

t of

mai

n fa

ctor

(si

ze o

f co

rn g

rit)

on

som

e ch

emic

al a

nd p

hysi

cal p

rope

rtie

s of

inst

ant c

erea

l bev

erag

e po

wde

rs.

Size

of

Bul

k de

nsity

(g/

ml)

Rec

onst

itutio

n in

dex

(ml)

Wat

erW

ater

corn

gri

tM

oist

ure

Prot

ein

Loo

se b

ulk

Pack

edSe

dim

ent

Cle

arab

sorp

tion

solu

bilit

yV

isco

sity

(mes

h)(%

)(%

db)

dens

itybu

lk d

ensi

tyin

dex

inde

x(C

ps)

(%)

133.

25±0

.32A

17.0

1±3.

29A

0.42

±0.0

4A0.

49±0

.06B

51.5

6±1.

73A

2.44

±1.7

3A1.

63±0

.03A

55.5

1±2.

59A

103.

91±5

2.96

A

233.

21±0

.33A

17.1

2±3.

84A

0.41

±0.0

3A0.

49±0

.05B

50.9

4±2.

45A

3.05

±2.4

5A1.

69±0

.28A

55.0

4±2.

34A

98.0

6±64

.60A

B

333.

04±0

.24A

17.0

9±3.

85A

0.42

±0.0

4A0.

52±0

.07A

50.5

0±2.

30A

3.50

±2.3

0A1.

64±0

.26A

55.9

6±1.

94A

85.4

1±51

.21B

In a

col

umn,

mea

ns w

ith th

e sa

me

lette

r ar

e no

t sig

nifi

cant

ly d

iffe

rent

at 0

.05

sign

ific

ance

leve

l.

Tab

le 4

Eff

ect

of m

ain

fact

or (

com

posi

tion

betw

een

corn

gri

t an

d is

olat

ed s

oy p

rote

in)

on s

ome

chem

ical

and

phy

sica

l pr

oper

ties

of i

nsta

nt c

erea

l

beve

rage

pow

ders

.

Cor

n gr

it :

Bul

k de

nsity

(g/

ml)

Rec

onst

itutio

n in

dex

(ml)

Wat

erW

ater

Isol

ated

soy

Moi

stur

ePr

otei

nL

oose

bul

kPa

cked

Sedi

men

tC

lear

abso

rptio

nso

lubi

lity

Vis

cosi

ty

prot

ein

(%)

(% d

b)de

nsity

bulk

den

sity

inde

xin

dex

(Cps

)

(%)

84:1

03.

23±0

.30A

12.7

4±0.

31C

0.44±0

.02A

0.52±0

.04A

52.8

3±0.

28A

1.17

±0.2

8C1.

94±0

.07A

56.6

2±2.

15A

161.

34±2

0.85

A

74:2

03.

13±0

.32A

17.3

6±0.

43B

0.41

±0.0

4B0.

49±0

.07B

51.5

6±0.

69B

2.44

±0.6

9B1.

39±0

.14C

56.4

4±1.

33A

90.0

3±18

.64B

64:3

03.

14±0

.31A

21.1

2±0.

69A

0.41

±0.0

3B0.

49±0

.06B

48.6

1±1.

96C

5.39±1

.96A

1.63±0

.21B

53.4

5±1.

71B

36.0

0±11

.24C

In a

col

umn,

mea

ns w

ith th

e sa

me

lette

r ar

e no

t sig

nifi

cant

ly d

iffe

rent

at 0

.05

sign

ific

ance

leve

l.


shown in Table 6. That is difference in products,

when the composition between corn grit and

isolated soy protein changed, could be seen more

clearly when compared to changing of the particle

size of corn grit. Product with the composition

between corn grit : isolated soy protein equaled to

84:10 had more trend of higher liking scores of

product concerning color, odor, flavor, texture and

overall acceptance, when compared to product

with the composition between corn grit : isolated

soy protein equaled to 74:20 and 64:30, for all

values of particle size of corn grit (13, 23 and 33

mesh). Because of increasing protein quantity

such as the result in Table 4, it caused the product

to have the property of dissolving into

homogeneous solution with difficulty or poor

reconstitution which brought the organoleptic

properties of product get worse.

Nutritive value of instant cereal beveragepowders in the forms of nutrition labeling andpattern of essential amino acids

Choose the representative product of instant

cereal beverage powders from extrusion process

for nutritional evaluation by using size of corn grit

equaled to 13 mesh and composition between corn

grit and isolated soy protein equaled to 84:10 due

to the results of the effect of main factors on Table

4 which stated that the product using composition

between corn grit and isolated soy protein equaled

to 84:10 had more trend for good reconstitution

with less separation and sensory evaluation on

Table 6 which showed that the product using size

of corn grit equaled to 13 mesh and composition

between corn grit and isolated soy protein equaled

to 84:10 had higher score of preference in color,

odor, flavor, texture and overall acceptance. From

nutritional evaluation of this products in the forms

of nutrition labeling (Announcement of the Pulbic

Health Ministry, No.182, 1998), it was found that

this product has good nutrition condition for health

(The Committee, 1989) because it consists of

higher protein quantity (4g per one serving) when

compared to other available products. And the

quantity of protein is equaled to 8 percent of Thai

RDI which is nearly to protein quantity in food

Table 5 Response surface function of some chemical composition and physical properties of products

when particle size of corn grit and the amount of isolated soy protein changed.

Chemical composition and Response surface function

physical properties

Moisture Y = 3.0213+0.0352A-0.0086B-0.0007A2-0.0007AB+0.0005B2

Protein Y = 8.4514-0.0348A+0.5124B-0.0007A2+0.0035AB-0.0043B2

Bulk density

- Loose bulk density Y = 0.5225-0.0041A-0.0065B+0.0001A2-0.00004AB+0.0002B2

- Packed bulk density Y = 0.6076-0.0037A-0.0085B+0.0001A2-0.00005AB+0.0002B2

Reconstitution index

- Sediment Y = 52.7978-0.0369A+0.1845B+0.0008A2-0.0027AB-0.0083B2

- Clear Y = 1.2022+0.0369A-0.1845B-0.0008A2+0.0027AB+0.0083B2

Water absorption index Y = 3.2531+0.0147A-0.1842B-0.0006A2+0.0006AB+0.0039B2

Water solubility index Y = 54.8528-0.2184A+0.4963B+0.0070A2-0.0040AB-0.0141B2

Viscosity Y = 264.6380+0.2398A-10.1789B-0.0339A2+0.0198AB+0.0864B2

When A = particle size of corn grit

B = amount of isolated soy protein


claimed as source of protein (according to Nutrition

claim, appendix to announcement of the Public

Health Ministry No.182, 1998, which stated that

food claimed as source of protein must consist of

10-19 percent of protein quantity required for Thai

RDI). Furthermore, the product was considered to

have suitable protein quantity, because nutritional

regulation stated that approximately 12 percent of

total energy should be received from protein

(Whitney and Hamilton, 1981). In the aspects of

quantities of fat, saturated fat, and sodium, they

are in “low” of nutrition claim standard, and there

was no cholesterol. This product also had Vitamin

A, B1, B2, Ca and Fe in the quantities of 20, 4, 8,

8 and 4 percent of Thai Recommended Daily

Intakes respectively. Concerning with dietary fiber,

it was found that there was rather little (0.4 g per

100 g of product) which is stated to zero value in

the form of nutrition labeling. Hence for further

product development, more dietary fiber

reinforcement raw materials should be added.

Moreover, the protein quality was assessed

by comparing essential amino acids of this product

with standard pattern of essential amino acids set

by joint FAO / WHO committee, as shown in

Table 8. It was found that this product has good

quality of protein because of nearly every type of

essential amino acids be according to the standard

set by FAO/WHO, except threonine, which is

deficient in small amount and showed the chemical

score equaled to 97.5 percent.

CONCLUSION

Production of instant cereal beverage

powders based on corn and soybean by using

extrusion process showed that the differences in

particle size of corn grit (13, 23 and 33 mesh) did

not have significant effect (P>0.05) on the chemical

composition (moisture and protein content) and

most of the physical properties of products but the

differences in the composition between corn grit

and isolated soy protein have significant effect

(P£0.05) on the protein content and the physical

properties of product (bulk density, reconstitution

index, water absorption index, water solubility

index and viscosity). The suitable product should

have the composition between corn grit and isolated

soy protein equaled to 84:10 because it can

reconstitute well, good soluble and moderate

Table 6 Sensory evaluation of instant beverage powders based on corn and soybean from extrusion

process.

Treatment Organoleptic properties

Size of Corn grit : Overall

corn grit Isolated soy Color Odor Flavor Texture acceptance

protein

13 84:10 7.42±0.49A 7.14±0.63A 7.19±0.78A 7.26±0.39A 7.39±0.43A

13 74:20 7.32±0.34AB 6.76±1.06AB 6.67±0.80B 6.42±0.88B 6.64±0.79B

13 64:30 7.12±0.21AC 6.67±0.55AC 6.47±0.44BC 6.59±0.68BC 6.50±0.35B

23 84:10 7.40±0.35A 7.11±0.64A 7.12±0.65AD 6.90±0.46ACD 7.02±0.51AB

23 74:20 7.19±0.53AD 6.96±0.68A 6.72±0.94BD 6.76±0.52BDE 6.74±0.84BC

23 64:30 6.69±0.49E 6.20±0.58C 6.04±0.53CE 6.07±0.84B 6.01±0.59D

33 84:10 7.34±0.54AF 6.92±0.59A 6.80±0.57AB 7.01±0.53ACE 6.91±0.40B

33 74:20 6.92±0.59BCDEF 6.78±0.44AD 6.62±0.46B 7.06±0.30AE 6.80±0.39B

33 64:30 6.89±0.54CDE 6.31±0.58BCD 6.40±0.50BE 6.11±0.48B 5.90±0.40CD

In a column, means with the same letter are not significantly different at 0.05 significance level.


Tab

le 7

Nut

ritio

n la

belin

g of

inst

ant b

ever

age

pow

ders

bas

ed o

n co

rn a

nd s

oybe

an f

rom

ext

rusi

on p

roce

ss c

ompa

red

with

mar

keta

ble

prod

ucts

.

Nut

riti

on f

acts

A1B

11/M

arke

tabl

e Pr

oduc

ts

NE

SVIT

AG

OO

D T

IME

P.P.

CO

RN

MIL

K

Serv

ing

size

: 1

pack

age

(30

g)Se

rvin

g si

ze :

1 pa

ckag

e (3

0 g)

Serv

ing

size

: 1

pack

age

(30

g)Se

rvin

g si

ze :

1 pa

ckag

e (3

0 g)

Serv

ing

per

pack

age

: 1Se

rvin

g pe

r pa

ckag

e : 1

Serv

ing

per

pack

age

: 1Se

rvin

g pe

r pa

ckag

e : 1

Am

ount

per

ser

ving

Am

ount

per

ser

ving

Am

ount

per

ser

ving

Am

ount

per

ser

ving

Cal

orie

s 12

0 C

alor

ies

from

Fat

20

Cal

orie

s 12

0 C

alor

ies

from

Fat

20

Cal

orie

s 13

0 C

alor

ies

from

Fat

16

Cal

orie

s 14

0 C

alor

ies

from

Fat

40

% D

aily

Val

ue*

% D

aily

Val

ue*

% D

aily

Val

ue*

% D

aily

Val

ue*

Tot

al F

at 2

g3

%T

otal

Fat

2 g

3 %

Tot

al F

at 2

g3

%T

otal

Fat

4.5

g7

%Sa

tura

ted

Fat 0

.5 g

3 %

Satu

rate

d Fa

t 1.5

g7

%Sa

tura

ted

Fat 0

g0

%Sa

tura

ted

Fat 4

g20

%C

hole

ster

ol 0

mg

0 %

Cho

lest

erol

0 m

g0

%C

hole

ster

ol 0

mg

0 %

Cho

lest

erol

Les

s th

an 5

mg

1 %

Prot

ein

4 g

Prot

ein

2 g

Prot

ein

3 g

Prot

ein

2 g

Tot

al C

arbo

hydr

ate

23 g

8 %

Tot

al C

arbo

hydr

ate

23 g

8 %

Tot

al C

arbo

hydr

ate

20 g

7 %

Tot

al C

arbo

hydr

ate

22 g

7 %

Die

tary

Fib

er 0

g0

%D

ieta

ry F

iber

Les

s th

an 1

g4

%D

ieta

ry F

iber

3 g

12 %

Die

tary

Fib

er 1

g4

%Su

gar

11 g

Suga

r 13

gSu

gar

5 g

Suga

r 9

gSo

dium

60

mg

3 %

Sodi

um 1

50 m

g6

%So

dium

120

mg

3 %

Sodi

um 1

30 m

g5

%%

Dai

ly V

alue

*%

Dai

ly V

alue

*%

Dai

ly V

alue

*%

Dai

ly V

alue

*V

itam

in A

20 %

• V

itam

in B

14

%V

itam

in A

0 %

• V

itam

in B

12

%V

itam

in A

0 %

• V

itam

in B

112

%V

itam

in A

Les

s th

an 2

% •

Vita

min

B1 4

%V

itam

in B

28

%•

Cal

cium

8 %

Vita

min

B2

4 %

• C

alci

um20

%V

itam

in B

212

%•

Vita

min

C0

%V

itam

in B

2 4

% •

Cal

cium

Les

s tha

n 4

%Ir

on4

%Ir

on 2

0 %

• Fo

lic A

cid

20 %

Cal

cium

20 %

• Ir

on4

%Ir

on L

ess

than

2 %

*Per

cent

Dai

ly V

alue

s ar

e ba

sed

on a

200

0 ca

lori

e di

et.

You

r da

ily v

alue

s m

ay b

e hi

gher

or

low

er d

epen

ding

on

your

cal

orie

nee

ds.

Tot

al F

atL

ess

than

65g

Satu

rate

d Fa

tL

ess

than

20g

Cho

lest

erol

Les

s th

an30

0m

gT

otal

Car

bohy

drat

e30

0g

Die

tary

Fib

er25

gSo

dium

Les

s th

an2,

400

mg

Cal

orie

s pe

r gr

am:

Fat

= 9

;C

arbo

hydr

ate

= 4

;Pr

otei

n =

4

1/In

stan

t bev

erag

e po

wde

rs o

pera

te a

t par

ticle

siz

e of

cor

n gr

it eq

uale

d to

13

mes

h an

d th

e co

mpo

sitio

n be

twee

n co

rn g

rit a

nd is

olat

ed s

oy p

rote

in e

qual

ed to

84:

10


viscosity. Moreover, the acceptable score is the

highest also. Even though the protein content is

less than other products but it is still more than

marketable products, and it has adequate protein

content and appropriate pattern of essential amino

acids for good nutritive consuming, Therefore,

this research work is beneficial in the aspects of

application of extrusion process for diversity of

products in food industry and promote Thai

agricultural raw materials by giving them value

added.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the

financial support of the Kasetsart University

Research and Development Institute (KURDI).

LITERATURE CITED

Akpapunum, M.A. and P.Markakis. 1981.

Physicochemical and nutritional aspects of

cowpea flour. J. Food. Sci. 46 : 972-973.

A.O.A.C. 1990. Official Methods of Analysis.

Vol. II, 15th ed., Association of Official

Analytical Chemists, Inc., Arlington, Virginia.

1298 P.

Anderson, R.A., H.F. Conway, V.F. Pfeifer and

E.L. Griffin. 1969. Gelatinization of corn

grits by roll-and extrusion-cooking. CerealScience Today 14 : 4-12.

Anderson, R.A., V.F. Pfeifer, G.N. Bookwalter,

and E.L. Griffin. 1971. Instant CSM food

blends for world-wide feeding. Cereal ScienceToday 16 : 5 – 11.

Bookwalter, G.N., H.F. Conway, and E.L. Griffin.

1971. Instant beverage mixes, United States

Patent.

Boonyasirikool, P. and C. Charunuch. 1999.

Development of broken rice-based ready-to-

eat breakfast cereal by extrusion process.

Kasetsart J. (Nat. Sci.) 33 : 415-429.

Damardjati, D.S. and B.S. Luh. 1987.

Physicochemical properties of extrusion-

cooked rice breakfast cereals, pp. 251-263. In

Trends in Food Processing I : MembraneFiltration Technology and ThermalProcessing and Quality of Foods.

Table 8 Comparison of essential amino acid contents between instant beverage powders (A1B1) and

FAO/WHO standard (1972) in milligrams per gram of protein.

Essential amino acids A1B11/ FAO/WHO2/

Isoleucine 43 40

Leucine 104 70

Lysine 57 55

Methionine + Cystine 55 35

Phenylalanine + Tyrosine 78 60

Threonine 39 (97.5)3/ 40

Tryptophane 15 10

Valine 50 50

Source : 1/ Division of Nutrition, Department of Health, Ministry of Public Health,2/ Food Composition Table for use in East Asia (FAO, 1972)3/ Limiting amino acid with chemical score in parenthesis

amino acid content in foodChemical score = ¥ 100

amino acid content in FAO/WHO standard


Proceedings of the 7th World Congress of

Food Science and Technology. October 1987.

Singapore.

Guy, E.J. and H.E. Vetterl. 1975. A high quality

protein, vitamin, and mineral fortified

chocolate flavored powder for beverage use.

J. Dairy Sci. 58 : 432-435.

Harper, J.M. 1981. Extrusion of Foods. Vol. I,

CRC Press Inc., Boca Raton, Florida. 212 p.

Hauck, B.W. 1980. Marketing opportunities for

extrusion cooked products. Cereal FoodsWorld 25 : 594-595.

Holsinger, V.H., C.S. Sutton, L.F. Edmondson,

P.R. Crowley, B.L. Berntson, and M.J.

Pallansch. 1974. Production and properties of

a nutritious beverage base from soy products

and cheese whey, pp. 16-17. In Proceedingsof International Congress of Food Scienceand Technology. Philadelphia, Pennsylvania,

USA.

Ihekoronye, A.I. and M.G. Oladunjoye. 1988.

Formulation and Physicochemical properties

of high-protein food beverage powders based

on protein concentrate from the Nigerian “red

skin” groundnut. Trop. Sci. 28 : 219-237.

King, V. 1985. Studies on the production of banana

juice powder using spray drying. J. ChineseAgr. Chem. Soc. 23:62-72.

Konstance, R.P., C.I. Onwulata, P.W. Smith, D.

Lu, M.H. Tunick, E.D. Strange, and V.H.

Holsinger. 1998. Nutrient-based corn and soy

producls by twin-screw extrusion. J. FoodSci. 63 : 864-868.

Moore, G. 1993. Raw materials and in extrusion.

Ingredients Extra 2 : 2-5.

Tangkanakul, P., N. Vatanasuchart, M.

Pongpipatpong, and P. Tangtrakool. 2000.

Development of Instant high fiber processed

food. Kasetsart J. (Nat. Sci.) 34 : 117-124.

The committee on recommended daily dietary

allowance for Thais. 1989. RecommendedDaily Dietary Allowance for Healthy Thais.Department of Health, Ministry of Public

Health. 161 p.

Whitney, E.N. and E.M.N. Hamilton. 1981.

Understanding Nutrition. 2nd ed., West

Publishing Co., St. Paul, Minnesota. 629 p.


The Optimum use of Salinity, Nitrate and Pond Depth forbbbbb-Carotene Production of Dunaliella salina

Orapin Bhumibhamon1, Udom Sittiphuprasert2, Naiyana Boontaveeyuwat1

and Jantana Praiboon1

ABSTRACT

Dunaliella salina, halotorelant green algae was collected from the East Coast of Thailand. It has

a massive accumulation of b-carotene when grown under defined growth conditions such as high light

intensity, high salt concentration and nitrate deficiency. The present study investigated the optimization

of salinity, nitrate and pond depth for cell growth and b-carotene production of the alga in question.

Cultivation was done in three stages. These were indoor cell growth cultivation, outdoor cell growth

cultivation and outdoor b-carotene production. The optimum salinity for cell growth of indoor and

outdoor cultivation (5 l) was 9% NaCl, which has specific growth rate (m) of 0.579 (d-1) and 0.981 (d-1).

The optimum salinity for outdoors b-carotene production (5 l) when use 40% inoculum (2.16 ¥ 106 cell

ml-1) was 12% NaCl. This produced b-carotene content of 51.73 mg ml-1. In addition, the concentrations

of medium used were 100%, 75% and 50% to decrease nitrate concentration in the starter ponds for

increasing b-carotene production in the next step. The results showed that 50% medium gave minimum

nitrate concentration of 4.5 mg l-1. For the effect of pond depth, the light expose of the alga were used

9, 11, 13 and 18 cm the ratios of carotenoid to chlorophyll were 7.48, 6.25, 5.54 and 3.35, respectively.

Therefore the suitable pond depth for b-carotene production from D. salina was 9 to 11 cm.

Key words: b-carotene production, Dunaliella salina, pond depth, salinity, nitrate


1 Department of Biotechnology, Faculty of Agro-industry, Kasetsart University, Bangkok 10900, Thailand.2 Sriracha Fishery Research Station, Faculty of Fisheries, Kasetsart University, ChonBuri 20130, Thailand.

INTRODUCTION

Dunaliella is a unicellular green algae

capable of growing in a wide range of salt

concentrations from 0.2% to saturation (around

35%). It produces and accumulates large amounts

of b-carotene when cultivated under high light

intensity, high salt concentration and nitrate

deficiency conditions. More than 10% of the dry

weight of D. salina is b-carotene (Ben-Amotz and

Avron, 1983). b-carotene has important nutritional

characteristics, as it is the most effective precursor

of vitaminA. Moreover, b-carotene is also used as

food and cosmetics coloring agent; as pro-vitaminA

in animal feed; in medical treatment of disease

such as erythropoietic protoporphyria (EPP) (Ben-

Amotz and Avron, 1990); and as a potent

antioxidant which reduced the incidence of cancer

in humans (Ziegler, 1989). The aim of the present

study was to optimized the culture conditions for

D. salina b-carotene production.


StainD. salina DS1197 was collected from the

Eastcoast of Thailand. It was isolated and cultured

in Borowitzka’s medium (Borowitzka,1988).

Optimum salinity for indoor cell growthThe algae were cultured in 150 ml

transparent plastic cone tubes placed in a water

bath. The inoculum was approximately 0.5 ¥ 106

cell ml-1. Cells were grown in a modified

Borowitzka’s medium having 0.5 g l-1 of KNO3 at

30∞C. Light intensity was supplied to the culture

tube surface at 8 klx on daylight for 16 hrs. A

mixture of CO2 and air (2% CO2) was provided at

12 ml min-1. Salinity of 6%, 9% and 12% NaCl

were used for cell growth.

Optimum salinity for outdoor cell growthThe cells from indoor cell growth

(maximum cell density) were centrifuged at 2,000

rpm for 10 min. The living cell pellets were

transfered to culture medium to make up a starting

cell of about 0.5 ¥ 106 cell ml-1. The cells were

grown in a 5 l plastic tray with controlled shaking

of 12 rpm in the open air outdoor. Pure CO2 was

supplied to the culture at the rate of 72 ml min-1 for

8 hr daylight. As for indoor culture, cells were

grown in a medium of 9% salinity.

Optimum salinity for outdoor bbbbb-caroteneproduction

Cell culture of outdoor cell growth was

used to inoculate 40% of the culture volume (2.16

¥ 106 cell ml-1) for further study of b-carotene

production. The outdoor culture was carried out in

5 l plastic trays at 100% outdoor light exposure.

Pure CO2 was supplied at the rate of 72 ml min-1

for 8 hr daylight. The cultures were cultivated in a

Borowitzka’s medium without KNO3 at different

salinity of 9%, 12%, 15% and 18% NaCl.

Optimum nitrate concentration for outdoorcell growth

The cells from indoor cell growth

cultivation (maximum cell density) were transfered

to culture medium to make up a starting cell of

about 2 ¥ 105 cell ml-1. The cultures were grown

in 300 l of outdoor raceway pond under 100%

outdoors light exposure. Culture agitation was

provided by a paddle wheel. Three different

Borowitzka’s media concentration of 100%, 75%

and 50% were supplied to brine (9% NaCl) to

reduce nitrate concentration in order to increase b-

carotene production in the next step.

Optimum pond depth for outdoor bbbbb-caroteneproduction

The cells from outdoor cell growth

(optimized nitrate concentration) were transferred

to culture medium (without KNO3) to make up

starting cell of about 2 ¥ 105 cell ml-1. The cells

were grown in an outdoor raceway pond under

100% outdoors light exposure. CO2 was bubbled

into the pond to maintain the culture pH of 8.0.

There were 2 separated sets of experiment.

Experiment 1: The pond depths of 9 cm

(250 l working volume) and 18 cm (500 l working

volume).

Experiment 2: The pond depths of 11 cm

(300 l working volume) and 13 cm (350 l working

volume).

Pigment analysisChlorophyll and carotenoid were extracted

from the alga pellet using 90% acetone and assayed

as described by Borowitzka (1991).

bbbbb-carotene analysisThe filtrate of 5-10 ml of culture through

whatman GF/C filter was wrapped in an aluminum

foil and frozen at - 20∞C until analysis. The

extraction was done in the dim light 10-15 ml cold

90% acetone with gentle grinding. The supernatant

was collected by centrifuging at 3,000 rpm for 15

min. b-catrotene in supernatant was detected by

HPLC as described by Borowitzka (1991).




1. Effect of salinity for indoor cell growthThe results showed that higher salinity

medium affected the intracellular mechanism by

inhibit cell division of D. salina more than lower

salinity medium (Figure 1). The specific growth

rates (m) of 6%, 9% and 12% NaCl were 0.463,

0.597 and 0.511 d-1, respectively. Thus, the optimal

salinity for indoor cell growth of D. salina were

9% and 12% NaCl same in the previous report

found by Borowitzka (1988).

2. Effect of salinity on outdoor cell growth (5liter)

The problem of outdoor culture was

protozoan contamination when growth under low

salinity that effected cell growth and yield.

Although the 9% NaCl were optimal for cell

growth of the indoor culture, it also good for

outdoor cell growth. The cultures at 9% and 12%

NaCl were investigated for cell growth and

protozoan contamination. The results were similar

to those of the indoor cultures at the same salinity.

The specific growth rate of culture at 9% NaCl was

higher than that of 12% NaCl. Specific growth

rates of the cultures at 9% and 12% NaCl were 0.98

and 0.51 d-1, respectively (Figure 2). Protozoan

contamination was not found in either culture. The

salinity of 9% NaCl was therefore considered

optimal for outdoor cell growth culture of D.

salina DS1197.

3. Effect of salinity on outdoor b-caroteneproduction (5 liter)

The culture was cultivated by using

inoculum size of 40% of the culture. The inoculum

culture was 2.16 ¥ 106 cell ml-1. The culture was

investigated for b-carotene production at different

salinity of 9%, 12%, 15% and 18% NaCl for 15

days. The highest b-carotene of the culture at 12%

NaCl was 51.73 mg ml-1 (Table 1).

4. Effect of nitrate concentration on outdoorcell growth (300 liter)

The source and concentration of nitrogen

can provoke important change in the growth an

biochemical composition of microalgal.

Manipulation of nitrogen concentration of the

culture medium was found to be a simple technique

to effect significant differences in the protein,

carbohydrate, lipid and pigment content of

Dunaliella (Uriarte et. al., 1993). D. salina can

accumulate highest b-carotene when growth under

nitrate deficiency. Thus, the medium concentration

of 100%, 75% and 50% were used to reduce nitrate

Figure 1 Effect of salinity on cell growth in

indoor cultivation of D. salina.

Figure 2 Effect of salinity on outdoor cell growth

of D. salina.

Cultivation period (day)

0 2 4 6 8 10

Cel

l gro

wth

(Ct/C

0)

0

2

4

6

8

10

12

14

16

9% NaCl12% NaCl


concentration were 36, 12 and 4.5 mg.l-1

respectively (Figure 3) and increase b-carotene

production. There were non-significant differences

in the cell concentration is shown in Figure 4. The

medium concentration selected for further culture

in the present study was 50% of Borowitzka’s

medium due to gave minimum nitrate

concentration.

5. Effect of pond depth on bbbbb-carotene productionPond depth is an one factor for b-carotene

production which there has relationship with light

quality and intensity absorbed by this alga. Thus,

different pond depths were use to search for

optimum b-carotene production. The results were

as follows:

Experiments 1: The cultivation was carried

out during August 1999. Pond depths of 9 and 18

Table 1 b-carotene production of outdoor cultivation in different salinity.

Salinity

9 % NaCl 12 % NaCl 15 % NaCl 18 % NaCl

Cultivation period (d) 15 15 15 15

Initial cell ¥ 106 cell ml-1(C0) 2.16 2.16 2.16 2.16

Final cell ¥ 106 cell ml-1(Ct) 1.79 1.66 1.38 1.12

Initial b-carotene (mg ml-1) 9.48 9.01 7.58 6.32

Final b-carotene (mg ml-1) 48.71 51.73 43.33 38.04

cm were used gave carotenoid to chlorophyll ratios

to be 7.48 and 3.35, respectively (Figure 5).

Experiment 2: The cultures were carried

out during September 1999. Pond depths of 11 and

13 cm were used gave carotenoid to chlorophyll

ratios of 6.25 and 5.54, respectively (Figure 6).

According to the result of these experiments,

the pond depth suitable for further culture of b-

carotene production was 9 to 11 cm due to gave

highest carotenoid to chlorophyll ratios.

Large scale outdoor mass culture where the

system is more complex and other factors interact

to affect growth and carogenesis. These factors

included perdition by protozoa, variable mixing of

the culture and the interactions of these factors

with nutrient supply, salinity, temperature and

light. In addition, cell density and pond depth as an

important factor for b-carotene production due to

Cultivation peroid (day)1 2 3 4 5 6 7 8

Nitr

ate

conc

entr

atio

n (m

g.l-1

)

0

20

40

60

80

100

120

100% of medium75% of medium50% of medium


1 2 3 4 5 6 7 8

Cel

l con

cent

ratio

n (¥

10-5

cel

l.ml-1

)

0

5

10

15

20

25

30

100% of medium75% of medium50% of medium

Figure 3 Effect of medium concentration on ni-

trate concentration.

Figure 4 Effect of medium concentration on cell

concentration.


relationship with light quality and intensity

absorbed by this alga. The optimum cell density is

not fix, but is influenced by the type of algae

culture, culture depth (pond depth), turbulence

and environmental conditions (temperature a

available irradiant). At cell density below the

optimum, productivity decreases, because the

biomass present cannot absorb all the available

light energy. Above optimum cell density

productivity decrease, because a portion of the

culture is in the dark and biomass is lost due to

respiration (Grobbelaar, 1995). When b-carotene

production in plastic tray (lab scale) and in raceway

pond (pilot scale) were compared. The results

(Table 2) show that b-carotene production of lab

scale was higher than that of pilot scale because of

the difference in cell density and pond depth. The

lab scale had higher cell density and less pond

depth than pilot scale. Thus, light energy absorbed

by this alga would increase to give maximum b-

carotene accumulation and production.

CONCLUSION

The optimal conditions for cell growth and

b-carotene production of D. salina DS1197 as

follows: The optimum salinity of indoor and

outdoor cell growth was 9% NaCl while the

optimum salinity for outdoor b-carotene production

was 12% NaCl. The minimum nitrate concentration

Table 2 Comparison of b-carotene production lab scale (plastic tray, 5 l) and pilot scale (raceway pond,

250-300 l).

Laboratory scale Pilot scale

(5 L) 9 cm (250 l) 11 cm (300 l)

Cultivation period (d) 15 14 15

Initial cell (cell ml-1) 2.16 ¥ 106 2.03 ¥ 105 1.95 ¥ 105

Final cell (cell ml-1) 1.66 ¥ 106 3.70 ¥ 105 5.12 ¥ 105

Initial beta-carotene (mg ml-1) 9.01 1.36 0.95

Final beta-carotene (mg ml-1) 51.73 3.65 3.84

beta-carotene (pg cell-1) 31.10 9.80 7.29

Cultivation peroid (day)

0 2 4 6 8 10 12 14 16

Car

oten

oid

to c

hlor

ophy

ll ra

tio

0

1

2

3

4

5

6

7

8

9 cm18 cm


0 2 4 6 8 10 12 14 16

Car

oten

oid

to c

hlor

ophy

ll ra

tio

0

1

2

3

4

5

6

7

11 cm13 cm

Figure 5 Effect of pond depth, 9 and 18 cm. on

carotenoid to chlorophyll ratios.

Figure 6 Effect of pond depth, 11 and 13 cm. on

carotenoid to chlorophyll ratios.


was 4.5 mg l-1 when growth under 50%

Borowitzka’s medium and optimum of raceway

pond depth for b-carotene production was 9 to 11

cm.

ACKNOWLEDGEMENTS

The research was supported by KURDI,

Kasetsart University under National Research

Council, Bangkok, Thailand.

LITERATURE CITED

Ben-Amotz,A. and M. Avron. 1983. On the factor

which determine massive b-carotene

Accumulation in the halotolerant alga

Dunaliella bradawil. Plant Physiol. 72 : 593-

597.

Ben-Amotz,A. and M. Avron. 1990. The

biotechnology of cultivation the halotolerant

alga Dunaliella. Trends Biotechnology 8 :

121-126.

Borowitzka,M.A. 1988. Modified Johnson’sMedium for Dunaliella spp., Cited by

S.Powtongsook. Cultivation of MicroalgaeDunaliella salina for Beta-caroteneProduction M.Sc. Thesis, Chulalongkorn

University, Bangkok.

Borowitzka, M.A. 1991. Standard methods for

total carotenoid assay suitable for Dunaliella

salina, pp. 243-248. In A. Vonshak and M.A.

Borowitzka (eds.). Laboratory Manual:Research Seminar and Workshop on MassCulture of Microalgae. Silpakorn University,

Thailand, Nov. 1991.

Grobbelaar, J.U. 1995. Influence of areal density

on b-carotene production by Dunaliella salina.

J.Appl.Phycol. 7 : 69-73.

Uriarte, I., A., Farias, A.J.S. Hawkins and B.L.

Bayne. 1993. Cell characteristics and

biochemical composition of Dunaliella

primolecta Butcher condition at different

concentration of dissolved nitrogen.

J.Appl.Phycol. 5 : 447-453.

Ziegler, R.G. 1989. A review of epidemiological

evidence that carotenoids reduce the risk of

cancer. J.Nutrition 119 : 116-122.


Quantity and Distribution of Plant Nutrients on Eutrophicationin Bang Pra Reservoir, Chonburi Province

Ratcha Chaichana1, Chumlong Arunlertaree11, Boonsong Srichareondham2

and Narong Veeravaitaya3

ABSTRACT

The main objectives of this study were to study the quantity and variation of plant nutrients in water

bodies supplied to Bang Pra reservoir including the characteristics of distribution and variation of plant

nutrients and phytoplankton in Bang Pra reservoir. This study was conducted during March 2001 –

February 2002.

In the inflowing brooks, the quantity of nitrite and total ammonia were significantly different

(p<0.01) both in each brook and in each month. It was demonstrated that the quantity of nitrate and

orthophosphate were significantly different (p<0.01) in each month but not different in each brook

(p>0.05).

In Bang Pra reservoir, the quantity of nitrite in each water sample was different (p<0.05) and the

quantity of total ammonia was significantly different (p<0.01). When considering both quantity of nitrite

and total ammonia in each month, it was shown that they were significantly different (p<0.01). Statistical

analysis indicated the significant difference of othophosphate quantity (p<0.01) in each month but not in

each water sample station (p>0.05). Given these conditions, Bang Pra reservoir can be classified as a

eutrophic lake.

High quantity of plant nutrients in Bang Pra reservoir was mainly found at water sample stations

of water receiving areas located at the mouth of brooks.

The biological analysis revealed that phytoplankton, which was mostly found in Bang Pra

reservoir was Aulacoseira, in Division Chromophyta. Moreover, in June, November 2001, and January

2002, Aulacoseira was found in such large quantity compared to other months that these periods could

be regarded as periods of phytoplankton bloom.

Key words: eutrophication, plant nutrients, phytoplankton, reservoir

1 Faculty of Environment and Resources Studies, Mahidol University, Nakhon Pathom 73170, Thailand.2 Inland Fishery Resources Development and Research Institute, Department of Fisheries, Bangkok 10900, Thailand.3 Department of Fishery Biology, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand.


INTRODUCTION

Bang Pra reservoir is greatly affected by

unsustainable developments such as the excessive

utilization of chemical fertilizers by farmers and

inadequate soil and water conservation in

agricultural areas that directly cause ecological

transformation, especially from eutrophication.

Eutrophication occurred in 1984 and exterminated

a number of fish from the decomposition of

exceeding algae blooms. Setkit et al. (1987)

conducted a research on the types and nutritive


importance of plant nutrients in Bang Pra reservoir.

His study emphasized on the selection of

appropriate species of fish released in the reservoir

in order to eradicate excessive algae and aquatic

plant blooms, which propounded the problem of

exaggerated productivity and distribution of algae

in the reservoir. This evidence apparently showed

that Bang Pra reservoir was encountering with the

substantial amount of phytoplankton. Furthermore,

Chookajorn et al. (1991), reported to the Royal

Irrigation Department, responsible for the reservoir,

that in the dry season the excessive algae blooms

had killed a number of fishes.

Accordingly, the importance of the

eutrophication problem induced to this study, which

was conducted based on the study on quantity and

distribution of plant nutrients on eutrophication in

Bang Pra reservoir in the Province of Chonburi.

This research studied ecological characteristics of

Bang Pra reservoir that leads to the eutrophication

phenomena. This study justified the characteristics

of plant nutrients and aquatic plant dispersions in

the reservoir, including the environment that

appeals to eutrophication.


The conditions of water sample collection

were considered according to these followings.

1. A study on quantity and variation of

plant nutrients in each area and season was

investigated from five inflowing brooks supplying

water into Bang Pra reservoir; the brook number

one (A1 : Sukreap brook), the brook number two

(A2 : Ruam brook), the brook number three (A3 :

Tha Sai brook), the brook number four (A4 : Kru

brook), and the brook number five (A5 : Nong Kor

– Bang Pra brook). There were five water sample

stations from five inflowing brooks and water

sample composed of an equal mixture of surface

was taken from each station.

2. A study on distribution and variation of

plant nutrients in each area and season was

conducted by collecting water samples in Bang

Pra reservoir. The conditions of water sample

collection were considered from;

- Four samples were collected from the

water receiving area (the water sample station

number one (B1), two (B2), three (B3) and four

(B4)).

- One sample was collected from the

reservoir with eutrophication problem (the water

sample station number five (B5)).

- One sample was collected in front of

the dam (the water sample station number six

(B6)).


center of the reservoir (the water sample station

number seven (B7)).


pumping station of the Royal Irrigation Department

that supplies raw water for consumption (the water

sample station number eight (B8)).

Water samples from five inflowing brooks

and from reservoir were collected throughout the

year from March 2001 – February 2002.

The thermal stratification in Bang Pra

reservoir was also examined according to area and

season. There were eight study stations that were

the same areas as the study on distribution of plant

nutrients and phytoplankton in Bang Pra reservoir.

All data collected in situ were depths, temperature

and dissolved oxygen. Measurements were made

at a series of depth intervals at every one-meter

throughout water column at all stations.

Biological data examined phytoplankton

taxa and their quantitative distribution in Bang Pra

reservoir were collected at the same selected

stations as water quality (B1, B2, B3, B4, B5, B6,

B7, B7, and B8). At each station, ten liters of

sample was equally taken from the surface of

water (approximately one metre deep) using 37-

micrometer mesh size of phytoplankton net every

month from March 2001- February 2002. The ten

ml. of phytoplankton sample taken from each

station was immediately preserved with five % of


formalin solution.

Water samples collected from brooks were

analyzed for their physical and chemical properties

whereas water samples collected from Bang Pra

reservoir were investigated physical, chemical

and biological properties as shown in Table 1.

Analytical procedures used for quantitative

determination of the chemical properties were

based on standard methods for the examination of

water and wastewater (APHA, AWWA and WPCP,

1980)

The quantitative statistical determination

was investigated by cluster analysis and

multidimensional scaling (MDS) ordination (Clark

and Warwick, 1994 and Ludwig and Renold,

1988).

RESULTS

Water quality of supplying brooks and BangPra reservoir

The results of the variation of plant nutrients

in the five brooks demonstrated that the maximum

average quantity of nitrate (3.16±4.01 mg/l) was

found in the brook number two while the maximum

average quantity of nitrite (1.20±1.63 mg/l), total

ammonia (2.14±1.51 mg/l) and orthophosphate

combined (1.15±2.82 mg/l) was found in the brook

number three.

When considering the variation of plant

nutrients of the five brooks by month, it revealed

that the maximum average quantity of nitrate

(6.03±3.30 mg/l) reached its peak in December

whereas the maximum average quantity of nitrite

(1.80±2.97 mg/l) reached its peak in July. For the

maximum average quantity of total ammonia

Table 1 Parameters and analytical methods of samples.

Parameters Analytical methods

Physical

Depth* (m) Plummet

Temperature* (∞C) Thermometer

Dissolved Oxygen* (mg/l) DO meter

Turbidity (NTU) Nephelometric method

Transparency* (m) Secchi disc

pH* pH meter

Conductivity* (ms/cm) Conductivity meter

Chemical

Nitrate (mg/l) Cadmium reduction column method

Nitrite (mg/l) Griess-Ilosvay diazotization

Total ammonia (mg/l) Nesslerization

Orthophosphate (mg/l) Ascorbic acid method

Biological

Chlorophyll a (mg/l) Fluorescent techniques

Genus composition and abundance Sedqewick rafter counting chamber

of phytoplankton

* Remark; Field study.


Figure 1 The monitoring stations of field data collection in the five brooks located around Bang Pra

reservoir and the monitoring stations of field data collection in Bang Pra reservoir.

Water sample station

B1

B8

B6

B2

B7B3

B4B5

A1

A2

A3

A5

A4

(3.93±0.72 mg/l) and orthophosphate (4.52±3.79

mg/l), the results showed that both of them reached

their peaks in October.

The study on the variation of plant nutrients

in Bang Pra reservoir showed that nitrate at every

water sample station was undetectable. However,

both nitrite (0.68±0.53 mg/l) and total ammonia

(1.38±1.25 mg/l) were found at the water sample

station number one whereas orthophosphate

(0.22±0.47 mg/l) was found at the water sample

station number three.

When considering the variation of plant

nutrients in Bang Pra reservoir for each month, it

revealed that nitrite (2.05±0.59 mg/l), total

ammonia (2.82±1.02 mg/l) and orthophosphate

(0.80±0.41 mg/l) reached their peaks in September,

November and October, respectively.

The biological study in Bang Pra reservoir

demonstrated that there were seven divisions of

phytoplankton in total: Cyanophyta, Chlorophyta,


Figure 2 The average quantity of plant nutrients in the five brooks (A) and in Bang Pra reservoir (B).

-1

0

1

2

3

4

5

6

7

Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb

mg/

l

NO3 NO2

NH3 PO4

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0


mg/

l

NO2

NH3

PO4

Figure 3 The characteristic of Aulacoseira.

(http://keisou.hp.infoseek.co.jp/chuusin/

Aulacose/aulsei.html)

Chromophyta, Chrysophyta, Bacillariophyta,

Euglenophyta, Pyrrophyta and four unidentified

genera. Both identifiable and unidentifiable genera

totaled to 75 genera. The most prevalent

phytoplankton found in Bang Pra reservoir was in

genus Aulacoseira, Division Chromophyta. In

addition, it was discovered that 84.47% of

phytoplankton found in Bang Pra reservoir was

Aulacoseira.

DISCUSSION

Water quality and seasonal distribution of plantnutrients in the inflowing brooks and Bang Prareservoir

The consequences of the variation of plant

nutrients; nitrate, nitrite, total ammonia and

orthophosphate in the brooks carrying water into

Bang Pra reservoir, showed that the quantity of

plant nutrients in the hot season (March to May)

was low compared with those in the rainy (June to

October) and cold (November to February) season.

During the rainy season, plant nutrients in the five

brooks would first increase in June and incresae a

second time in October due to the high rainfall in

the beginning and the end of the rainy season that

vastly carried plant nutrients into the five brooks.

During the cold season, plant nutrients in each

brook would decrease but remained higher than

that during the hot season. Therefore, it could be

concluded that the rain was a significant factor of

washing and carrying plant nutrients remaining in

the surrounding areas into the five brooks.

However, it could not be clearly concluded

why each brook had different amount of plant

nutrients. After surveying the surrounding areas of

the five brooks located around Bang Pra reservoir,

since most areas are agricultural areas having


similar characteristics. Consequently, it might be

likely that different agricultural activities such as

kinds and quantity of fertilizers used in agricultural

area might cause plant nutrients remaining in

agricultural area to be carried into the five brooks

in different quantities.

Another assumption considering different

quantities of plant nutrients in each brook might be

that agriculturists have inappropriate water and

soil conservation plan causing different remaining

plant nutrients in the agricultural area.

The water analyses results at Bang Pra

reservoir showed that the physical and chemical

quality of water met the level of water quality

standard of Thailand (Water quality division, 1991)

and water quality for fishery purposes

(Duangsawasdi and Somsiri, 1985). Despite the

change in seasons from hot to rainy season and

from rainy to cold season, water quality in Bang

Pra reservoir remained in acceptable standard but

slightly changed.

During the hot season, nitrate in the reservoir

was very low and undetectable. This was because

nitrate could not be washed from the surrounding

areas into Bang Pra reservoir due to lack of rain.

Moreover, nitrate found in each brook was rather

low as well. Considering the variation of nitrite,

total ammonia and orthophosphate in various

seasons, it showed that those plant nutrients were

lowest during the hot season whereas they had

large amount in the beginning and the end of the

rainy season. This was because plant nutrients

found in the five brooks carrying water into Bang

Pra reservoir during those periods were high too.

During the cold season, plant nutrients began to

decrease and with little change in scattering in

every water sample station.

Although each water sample station had

similar trend in changing and distributing of plant

nutrient, it still had different amount of plant

nutrients due to its location. For instance, water

sample stations that were the representatives of

water receiving areas mostly had larger amount of

plant nutrients than other stations located far from

that area.

Apart from that, this study also revealed

that phosphorus was the limiting factor in the

water quality of Bang Pra reservoir. This was

because when considering the N:P ratio referring

to the criteria of Sven-Olof and Walter (1989), it

showed that the N:P ratio of Bang Pra reservoir,

which was equal to 9.477 was higher than the

criteria of Seven-Olof and Walter (1989) who

stated that if a ratio of N:P exceeding seven,

phosphorus would be a limiting factor.

Furthermore, when using chlorophyll a in

order to compare with Helmut (1991) for classifying

the fertility of water in Bang Pra reservoir, it

revealed that a range of chlorophyll a in Bang Pra

reservoir was between 0.6062 – 1.8146 mg/l,

which was higher than that in a eutrophic lake,

0.01 – 0.50 mg/l (Helmut, 1991).

The change of phytoplankton in each water

sample station was not different at 0.5 limit.

Nevertheless, phytoplankton found each month

were different at a significant level of 95 %.

Phytoplankton in Bang Pra reservoir were scattered

in the similar quantity in almost every water sample

station whereas they had exponentially different

quantity in each month. Phytoplankton were low

during the hot season whereas they were relatively

high during the rainy and cold season. This was

because plant nutrients in Bang Pra reservoir during

the hot season were lower than those during the

rainy and cold season.

In June or the rainy season, phytoplankton

swiftly increased in every water sample station

and its total amount equaled to 784,260,000 units

per cubic metre. The main genus of phytoplankton

found was Aulacoseira, Division Chromophyta.

This increase of phytoplankton was a phenomenon

of phytoplankton bloom. This was due to the large

amount of rainfall in June that carried the remaining

plant nutrients in the agricultural area into Bang

Pra reservoir. Moreover, plant nutrients

substantially supplied from the five brooks were


also a vital factor that increased plant nutrients in

Bang Pra reservoir during this period.

After June, phytoplankton in every water

sample station began to decrease until October.

However, in November, quantity of phytoplankton

suddenly increased to the maximum, equaling

1,786,430,000 units per cubic metre. This showed

that there was phytoplankton bloom again. This

study also revealed that phytoplankton at the water

sample station number two, four and seven had

considerably increased, equaling to 3,444,700,000,

325,095,000 and 256,655,000 units per cubic metre,

respectively. The main genus of blooming

phytoplankton, Aulacoseira was most commonly

found. The significant cause of the blooming of

this genus was that during the end of the rainy

season, Bang Pra sub-watershed received the most

rainfall, thereby causing large amount of plant

nutrients carried into the brooks and subsequently

into the reservoir. When water in the five brooks

containing a large amount of plant nutrients flowed

into the reservoir, it undoubtedly caused a swift

growth of phytoplankton.

During the cold season, phytoplankton

decreased again in December. This was because

some parts of plant nutrients in Bang Pra reservoir

began to sink into the bottom of the reservoir while

other parts were used by living organisms.

However, in January, there was a tremendous

increase in the amount of phytoplankton equaling

1,252,185,000 units per cubic metre. There were

largest amount of phytoplankton, equaling

189,135,000 units per cubic metre in the water

sample station number two. The main genus of

phytoplankton found was still Aulacoseira, which

was the same genus appearing in June and

November, when the blooming occurred.

Aulacoseira bloom occurred in January because

some parts of plant nutrients in Bang Pra reservoir

still remained from the rainy season. This enabled

phytoplankton to use these plant nutrients for their

growth. Moreover, in January, total ammonia in

Bang Pra reservoir in every water sample station

increased again because total ammonia found in

the five brooks during the mentioned period

increased simultaneously. This might possibly

bring about phytoplankton bloom in Bang Pra

reservoir in January.

Another potential assumption that might

lead to phytoplankton bloom in Bang Pra reservoir

in January was a phenomenon of thermal

stratification. This study discovered that in

December, there was a trend of thermal

stratification in the reservoir. This caused the

Figure 4 The average quantity of phytoplankton in the Bang Pra reservoir.

0.00E+00

5.00E+07

1.00E+08

1.50E+08

2.00E+08

2.50E+08


unit/

cubi

cmet

re


lower part of the water mass to be unmixable with

the upper part of water mass. Accordingly, the

remaining plant nutrients in the bottom of reservoir

could not be carried to mix on the surface of the

reservoir. This caused little amount of plant

nutrients in the surface of the reservoir in December

when compared with those in January. In January,

there was no thermal stratification in Bang Pra

reservoir. Consequently, plant nutrients existing

in the bottom of the reservoir could be carried to

mix with the upper part of the water mass. This

caused the surface of the reservoir to have large

amount of plant nutrients again in January and

Figure 5 The dendrogram for hierarchical clustering of phytoplankton found in Bang Pra reservoir

(above) and the MDS ordination of phytoplankton, which were separated as two groups, stress

value = 0.01 (below).

Jan

Jun

No

v

Oct

Dec

Sep

Feb Ju

l

Au

g

May

Mar

Apr

100

80

60

40

Sim

ilar

ity

MarAprMay

Jun

JulAugSep

OctNov

Dec

Jan

Feb

Stress: .01

phytoplankton bloom occurred once again.

The study on the variation of phytoplankton

was consistent with the determination of cluster

analysis and multidimensional scaling (MDS)

ordination. Cluster analysis (above) and MDS

(below) showed that the groups of phytoplankton

found in June, November and January were highly

diversified and the most well distributed when

compared with other phytoplankton groups.

Therefore, the diversity and distribution of

phytoplankton in each month could be divided

into two groups as shown into Figure 5; the first

group consisted of June, November and January


whereas the second group comprised March, April,

May, July, August, October, December and

February.

When considering the quantity and

dominant species of phytoplankton found in the

reservoirs in Thailand (Chantsavang et al., 1989,

Dumrongtripob and Janesirisak, 1996, Kasisuwan

and Sukkasem, 1994, Mapairoj and

Traichaiyaporn, 1996, Pitaktansakul, 2000, Somsiri

et al., 1995 and Sukollapun and Chabjinda, 1997),

it showed that the amount of phytoplankton were

normally somewhat low when compared with

those found in Bang Pra reservoir, except at the

Lumtakong reservoir, Nakhon Ratchasima

province (Kakkaeo et al., 2002) where it contained

large quantity of phytoplankton similar to Bang

Pra reservoir. The Lumtakong reservoir is known

as a eutrophic reservoir. In addition, there were

other reports concerning a phenomenon of

phytoplankton bloom. For instance, the study of

Kakkaeo et al. (2002) at the Lumtakong reservoir

found that in December 1998, there was a rapid

increase in quantity of blue green algae, equaling

2,200,000,000 units per cubic metre. This amount

approximated to quantity of phytoplankton that

bloomed mostly in Bang Pra reservoir in November,

equaling 1,786,430,000 units per cubic metre.

After studying dominant species of

phytoplankton found in various reservoirs in

Thailand (Chantsavang et al., 1989,

Dumrongtripob and Janesirisak, 1996, Kasisuwan

and Sukkasem, 1994, Mapairoj and

Traichaiyaporn, 1996, Pitaktansakul, 2000, Somsiri

et al., 1995 and Sukollapun and Chabjinda, 1997),

it was found that phytoplankton, which was the

dominant species was mostly in Division

Cyanophyta, genus Oscillatoria, Anabaena and

Microcystis; these are dramatic bloom species or

dominant species found in a eutrophic lake. This

was consistent with the study of Sven-Olof and

Walter (1989) and Maitland (1978) who stated

that phytoplankton, in Division Cyanophyta, genus

Oscillatoria, Aphanizomenon and Microcystis or

Diatoms, genus Melosira, Fragilaria,

Stephanodiscus and Asterionella, are the main

groups of phytoplankton found in a eutrophic lake.

However, Aulacoseira, the dominant genus of

phytoplankton in Bang Pra reservoir, has never

been found or reported to bloom in any eutrophic

lake before.

CONCLUSION

The seasonal change played an important

role in the amount and variation of plant nutrients

in the five brooks.

The quantity and change of plant nutrients

in Bang Pra reservoir mainly depended on plant

nutrients carried from the five brooks that supply

water to Bang Pra reservoir. The trophic state of

Bang Pra reservoir was a eutrophic lake and the

distribution of plant nutrients in this reservoir was

mostly found at the inflowing stations to the

reservoir than other water sample stations located

far away from the water receiving areas.

The biological consequences showed that

Aulacoseira was the dominant genus found in

Bang Pra reservoir, particularly with the high

peaks in June, November 2001 and January 2002.

ACKNOWLEDGEMENTS

This study has been completed with the

help and encouragement of my supervisor,

Assistant Professor Dr. Chumlong Arunlertaree,

Faculty of Environment and Resources Studies,

Mahidol University, Mr. Boonsong

Srichareondham of the Inland Fishery Resources

Development and Research Institute, Department

of Fisheries and Mr. Narong Veeravaitaya,

Department of Fishery Biology, Faculty of

Fisheries, Kasetsart University. I also wish to

thank Mr. Chirdsak Vongkamolchoon, the director

of Chonburi Inland Fishery Development and

Research Center. Thanks to the Department of

Environmental Science, Faculty of Science,


Kasetsart University and the Postgraduate

Education, Training and Research Program in

Environmental Science, Technology and

Management, Mahidol University for providing

me with a grant to study and conduct my thesis.

LITERATURE CITED

APHA, AWWA and WPCP. 1980. StandardMethod for the Examination of Water andWaste Water. 15th edition. American Public

Health Publisher, New York. 1134 p.

Chantsavang, B., T. Chookajorn, S. Duagsawasdi

and P. Sordsuk. 1989. Hydrobiological andFishery Resource Survey in Kang KrachanReservoir Phetchaburi Province. Technical

Paper Number. 108. National Inland Fisheries

Institute, Department of Fisheries, Bangkok.

36 p. (in Thai)

Chookajorn, T., B. Chansavang, S. Tharnsuthus

and P. Kaewjaroon. 1991. Fishery ResourceSurvey in Bang Pra Reservoir, Chon BuriProvince. Technical Paper Number. 120.

National Inland Fisheries Institute,

Department of Fisheries, Bangkok. 22 p. (in

Thai)

Clark, K.R. and R.M. Warwick. 1994. Change inMarine Community: an Approach toStatistical Analysis and Interpretation.Plymouth Marine Laboratory, Plymouth.

144 p.

Dumrongtripob, J. and S. Janesirisak. 1996.

Hydrobiological and Fishery ResourceSurvey in Siridthon Reservoir in 1993.Technical Paper Number 17. Ubonratchathani

Freshwater Fisheries Development Center,

Ubonratchathani. 67 p. (in Thai)

Duangsawasdi, M. and C. Somsiri. 1985. WaterProperties and Water Analytical Methodsfor Fishery Research. National Inland

Fisheries Institute, Department of Fisheries,

Bangkok. 115 p. (in Thai)

Helmut, K. 1991. Control of Eutrophication in

Inland Waters. Redwood Press, Wiltshire.

337 p.

Kakkaeo, M., P. Kaeojaruen, M. Aeimsub, N.

Promkrouy and W. Somchan. 2002.

Abundance, Density and Distribution ofFishery Resources in LumtakongReservoir, Nakhon Ratchasima Province.Technical Paper Number. 212. National Inland



Kasisuwan, S. and R. Sukkasem. 1994.

Hydrobiological and Fishery ResourceSurveys in Khlongkhla Reservoir, SongkhlaProvince. Technical Paper Number.3. Pattani

Inland Fisheries Development Center, Pattani.

31 p. (in Thai)

Ludwig, J.A. and J.F. Renold. 1988. StatisticalEcology; a Primer on Methods andComputing. John Wiley & Sons, New York.

337 p.

Maitland, PS. 1978. Biology of Freshwater.Blackie and Son Ltd., London. 243 p.

Mapairoj, P. and S. Traichaiyaporn. 1996. UsingPhytoplankton as Bio-Indicator in StandingWaters, Chiang Mai. Department of Biology,

Faculty of Science, Chiang Mai University,

Chiang Mai. 85 p. (in Thai)

Somsiri, C., S. Suravit and C. Mesuk. 1995. WaterQuality and Biodiversity of Plankton inRajjaprabha Reservoir, Surat ThaniProvince. Technical Paper Number. 173.

National Inland Fisheries Institute,

Department of Fisheries, Bangkok. 87 p. (in

Thai)

Pitaktansakul, R. 2000. Diversity of FreshwaterAlgae in Eutrophic Waters and OptimumConditions for the Growth of Mycrocystisaeruginosa Kutzing. MS. Thesis. Kasetsart

University, Bangkok. (in Thai)

Setkit, S., P. Sitasit and S. Manusmongkol. 1987.

Species and Nutritive Values of SomeNatural Food Plants in Bang Pra Tank.Technical Paper Number. 69. National Inland




Sukollapun, S. and K. Chabjinda. 1997.

Hydrobiological and Fishery ResourceSurveys in Maekuang Reservoir, Chiang-Mai Province. Technical Paper Number.1.

Inland Fisheries Division, Bangkok. 32 p. (in

Thai)

Sven-Olof, R. and R. Walter.1989. The Control

of Eutrophication of Lakes and Reservoirs.vol.1. The Parthenon Publishing Group, New

Jersey. 314 p.

Water Quality Division. 1991. Water QualityStandard of Thailand. Department of

Environmental Quality Standard. The Bureau

of National Environmental Committee.

Bangkok 135 p. (in Thai)


Fisheries in the Mun River: A One-Year Trial of Openingthe Sluice Gates of the Pak Mun Dam, Thailand

Tuantong Jutagate, Chaiwut Krudpan, Praneet Ngamsnae, Kanjana Payoohaand Thanatip Lamkom

ABSTRACT

The changes in fisheries during the trial on the opening of sluice-gates of Pak Mun Dam (July 2001

to June 2002) were investigated. There were 184 fish species from 44 families found, with some species

recognized to extirpate from the Mun River. Shannon’s diversity index ranged from 1.92 to 3.14. Catch

per unit of fishing effort ranged from 0.38 to 1.70 and 0.61 to 2.71 kg set-1 night-1 in down- and up-stream,

respectively. Fish upstream migrations during the rainy season were observed. Incomes from fishing

activities were 16,366 Thai Baht yr-1 per household. Traditional activities, especially related to fisheries,

returned and income from fishing also increased during the study period.

Key words: Pak Mun Dam, fishes and fisheries


Fisheries Programme, Faculty of Agriculture, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand.

INTRODUCTION

Fisheries in the Mun River, the largest

Mekong River tributary in Thailand, have been an

issue of interest since the construction of Pak Mun

Dam in 1990. The dam is about 22 m high (with the

operating head of 17 m) and 300 m wide. The

length of the reservoir outflow from the dam to the

Mekong River is 4.5 km and at the full storage

level, water flowing into the dam is backed up 35-

km upstream (Roberts, 2001). The dam was

inaugurated to impound in 1994. After the 1994

impoundment, there were major concerns on the

potamodromous behavior of many antipodal

species, the transformation from rheophilic- to the

limnophilic- environment, as well as changes and

lost in fishing activities (Roberts, 1994).

The Mun River is the longest river in the

north-east of Thailand, 641 km from its origin to

the Mekong confluence, with a 117,000-km2

catchment area (Duangswasdi and Chookajorn,

1991). Schouten et al. (2000) reported 265 fish

species recorded in the Mun watershed before

1994 and about 10 were introduced species. Fish

are the cheapest source of animal protein and

source of income for the local people, who live in

the vicinity to inland water bodies in Asia (De

Silva, 2001) and the migratory fishes of the Mekong

River Basin support many important community-

based artisanal fisheries (Roberts, 1993; Ahmed et

al., 1996). Warren and Mattson (2000) stated that

most of Mekong fishes were migratory species,

albeit to different degrees, for reproductive, trophic

or dispersal purposes. Poulsen and Jørgensen

(2000) also reported that many Mekong indigenous

species have to move to the Mekong tributaries

such as the Mun River in Thailand for their

reproductive purpose. However, since the

construction of the Pak Mun Dam, it is likely to be

a bio-geographic barrier and has a major impact


for the upstream movement of migratory fishes

from the Mekong River to the Mun River (Roberts,

2001). Schouten et al. (2000) reported that after

the completion of the Pak Mun Dam, fishes and

fisheries was haphazard and performance of the

fish ladder, pool and weir type, had never been

properly evaluated.

In June 2001, The Royal Thai Government

tried to solve these controversies and ordered the

Electric Generation Authority of Thailand (EGAT)

to open all the sluice gates to monitor the changes

during the one-year trial (from July 2001 to June

2002) and to investigate the methods for

rehabilitation of fishes and fisheries. The objective

of the study was get the information of changes in

fish species and fisheries in the Mun River for a

compromised management.


Sampling sitesThe sampling sites were divided into two

main areas, a downstream site (from Mun

confluence to dam) and an upstream site (from

dam to Pibun Mungsaharn District), by using the

dam as the barrier. Many rapids, slightly sharp

curves and narrower width were found in the

downstream site, while the bottom was flatter and

wider at the upstream site. The downstream and

upstream sites were subdivided into 11 and 17

stations, respectively (Figure 1).

Data collection and analysisEach station had its own data collector. The

study period was from July 2001 to June 2002

since the Thai Government had allowed opening

of sluice gates for only one year. Fishers were free

to decide when they went to fish as well as what

fishing gear(s) should be used in their given areas

while the sampling dates were set by the project.

Two field-staff members meet with the data

collectors fortnightly, to check on data collected

and brought the samples back. Fish samples were

packed in 10% formalin and were taken to the

Faculty of Agriculture, Ubon Ratchathani

University (70 km from the Pak Mun Dam).

Samples were weighed, measured and

taxonomically classified.

Figure 1 Map of the sampling area and stations.

Note Sampling stations


Species diversity was calculated by using

Shannon’s diversity index (H’) and the relative

evenness (J’);

H’ = Â Pi ln Pi ----------------------------- (1)

and

J’ = H’/Hmax ------------------------------ (2)

where, Pi was the relative abundance, i.e.

the number of individuals for each species divided

by the total number of individuals for all species

(S) in each sample and Hmax was the natural

logarithm of S (Begon et al., 1990).

Non-parametric chi-square (c2) was used

for comparing catch per unit of fishing effort

(CPUE; kg set-1 night-1) between the main sampling

sites. Analysis of variance (ANOVA) and the

least-significant different (LSD) were used as tools

for analyzing the difference among CPUE in each

month, which was transformed to ln(1+CPUE)

(Green, 1979), by equation

Yij = m + ai + Œij ------------------- (1)

where, Yij was ln(1+CPUE) of the ith month

in the jth station, m was the grand mean ln(1+CPUE),

ai was the month effect and Œij was the error

term. The SYSSTAT program (Wilkinson, 1989)

was used as a tool in statistical analysis.

Information of incomes from fishing

activities, from 1990 to 1999, was derived from

the data, collected by the Interior Ministry staff for

development planning. In this study, the 2002

income was calculated from the payment, paid to

the data collectors. Changes in their fisheries and

fishery-related activities were from group

discussions with representatives, local fishers, in

the study area.

RESULTS

Fish species compositionDuring the study period, there were 184

fish species from 44 families found. Among these,

34 species were Mekong endemic species, 11 were

introduced species and 48 were species claimed by

the fishers that they were not seen in the study

areas for a long time, therefore were defined as

uncommon species (Table 1). Most catches were

from five families viz. Cyprinidae, Pangasidae,

Bagridae, Siluridae and Cobitidae. Also included

in the catches were two common stocking fishes

namely carp, Cyprinus carpio (Linn.) and Nile

tilapia, Oreochromis niloticus (Linn.) as well as a

stocking shellfish, giant freshwater shrimp,

Macrobrachium rosenbergii (De man). After the

opening of the sluice gates, H’ values continuously

increased until November, when the water receded,

and then tended to decline slightly. The lowest H’

value was in July and the peak was in November,

and the index ranged from 1.92 to 3.14 (Table 2).

Species, that probably extirpated from the

Mun River before construction of the dam

(Schouten et al., 2000; Roberts, 2001) were also

caught viz. mad barb, Leptobarbus hoeveni

(Bleeker), giant mottled eel, Anguilla marmorata

(Quoy & Gaimard), Laotian shad, Tenualosa

thibaudeaui (Durand), Mekong giant catfish,

Pangasinodon gigas (Chevey) and sheathfish,

Wallagonia leeri (Bleeker). Moreover, many

species, which were not in the list of reportedly

utilized fishes of the Pak Mun fish ladder

(Pholprasith et al., 1997), were also found in the

upstream area.

FisheriesCatch per unit of fishing effort (CPUE)

ranged from 0.38 to 1.70 and 0.61 to 2.71 kg

set-1 night-1 in down- and up-stream, respectively

(Table 3). From the c2-test, there was difference in

CPUE between the two main sampling areas (P-

value = 0.039) and CPUE tended to increase after

the gate-opening, especially in the upstream area

(Figure 2). However, a similar decline was observed

in CPUE from February to April and an increase,

again in June, in both down- and up-stream areas.

From ANOVA, there was no statistical difference

in terms of monthly ln (1+CPUE) in the down- and

up-stream areas, where P-values are 0.087 and

0.558, respectively. However, from LSD-test, ln


(1+CPUE) in downstream in February and June

were significant different from each other (P-

value < 0.05) (Table 3). When categorized into

groups, Pangasiisd, Bagriids, Siluriids and some

Cyprinids, a homogenous group in the wet season

showed statistical difference (P-value < 0.05) to

the other months (Jutagate, accepted). The black

shark minnow, Morulius chrysophekadion

(Bleeker) and Pangasiids largely contributed in all

catches in terms of weight and number, respectively

(Figure 3).

For the changes in the fishing-gear use by

discussing with the local fishers and observing,

decreasing of the water level in the upstream area

allowed the resumption of trap fisheries, especially

in the rapid areas. On the other hand, during the

normal condition of sluice gate closing, well

operated fishing gear such as small lift net

(“Dahng” in Thai), used to fish Thai river sprat,

Clupeichthys aesarnensis (Wongratana) and small

shrimps, could not be employed during the trial

period, since the amount of target species and

shrimps decreased. No such changes were observed

in the use of longline and gillnet but the fishers

mentioned that the yields were higher when the

gates were opened than during the closing of sluice

gate period.

Socio-economics aspectsThe income from fishing activities alone

showed a remarkable change in the past ten years

(1992-2002) from 4,336 (±783) Thai Baht yr-1 per

household in 1992 to 16,366 (±116) Thai Baht

yr-1 per household in 2002. During this period, the

trend of increasing income continued from 1990 to

1996 and then declined from 1999 to date (Figure

4). Peak income was in 1996, two years after

impoundment.

In terms of fishery-related traditional

activities, the interviewees, from the group of

fishers, claimed that the benefits gained from this

trial period were that when they helped one another

to fish, they had strengthened their relationships

among neighbors and locals on joint use of fishing

gears techniques and fishing maneuvers were

transfered to the younger generation. Furthermore,

household members, who went to work in other

cities came back home to fish again.

DISCUSSION

Fish species diversity in the Mun River,

especially in Ubon Ratchathani Province area has

fluctuated considerably from 115 (Tantong and

Siripan, 1969), 75 (Team Consulting Engineers,

1982), 68 (Duangsawasdi and Chookajorn, 1991),

70 (Duangsawasdi and Duangsawasdi, 1992), 152

(Schouten et al., 2000) and, recently, 59 species in

the reservoir area (Jutagate et al., 2001). The

differences in number may have been from

misidentified fishes and misuse of sampling

techniques. Unfortunately, specimens from

previous studies were not maintained for further

re-checking. However, in this study, all specimens

were collected and maintained at the Ubon

Ratchathani University Museum of Fisheries

(UBUMF).

The high Shannon diversity (H’) index in

November, which was the period of the start of the

dry season, meant that the fishers could use many

types of gear. Moreover, this was also the time that

the adult fishes and new recruits migrated back to

the main stream (Poulsen and Jørgensen, 2000).

Continuous increasing of CPUE at the upstream

area indicated the migration of the downstream

fishes from the Mekong River. Homogeneous

grouping of CPUE data refers to the occurrence of

the migration (Warren, 1999). It is believed that

the three main purposes of migration, in this river

system, are reproductive, trophic and dispersal

(Warren et al., 1998). In Mekong River system,

indigenous fish species present more or less

migratory habits in both longitudinal and lateral,

where a raising of the water level and changes in

water color and turbidity are among the main

triggers to migrate upstream and then move back


to the main stream when the water level recedes

(Poulsen and Jorgensen, 2000). The remarkably

high CPUEs in June and February were good

indicators, respectively, of the upstream and

downstream migration. Dry season upstream

migrations of some species were also observed by

Warren et al. (1998).

Contributions of P. gigas, Bagrid catfish,

Hemibagrus nemurus (Val. in Cuv. & Val.) and M.

rosenbergii implied the success of fish stocking

program of the EGAT cooperating with Department

of Fisheries (DOF). One of P. gigas was found

with a labeled tag in its first dorsal fin base and the

two latter market-species were stocked annually.

Meanwhile, Java barb, Barbodes gonionotus

(Bleeker) and minnow Labiobarbus leptocheilus

(Val. in Cuv. & Val.), which were stocked from

1999-2000 at a high rate (Head of Pak Mun Fishery

Conservation Unit, pers. com.), were never found

in large numbers in any catches. It could imply that

the rapid areas were not the suitable niches for

these two species.

Tongkum (1991) reported that the income

from fisheries of the main two impacted districts,

Khong Jiem and Pibun Mungsaharn, was 16,893

Thai Baht yr-1 per household. Peak income from

fishery occupation was in 1996, due to the increase

in fish production after impoundment, and this

was always lapper in newly dammed reservoirs

elsewhere in Thailand (for example, Ubolratana

and Sirinthorn), before income dropped and then

stabilized (Fishery Extension Division, 1995). The

slight increase in income from 1990 to 1994 was

due to the increase of fish prices from the changes

of the value of Thai Baht. The fluctuation in 1999

and 2002 was probably caused by the closing and

opening the sluice gates.

In regard to any optimized fishery

management which can be measured, Charles

(2001) stated that there were three main paradigms

in fishery management viz., conservation,

rationalization and social/community. A proper

management scheme should balance these three

paradigms. However, with the management of the

Pak Mun Dam, fishes and fisheries were on one

side and the electric generation was on another

side. In terms of conservation and social/

community paradigms, the fish migratory issue

must get priority. Meanwhile, the power supply

was amongst the most important in the

rationalization paradigm.

From the fisheries viewpoint, since the fish

ladder does not perform well and is not suitable for

many species found in the Mun River (Schouten et

al., 2000), the fishes should have a chance to

migrate upstream to complete their life cycle.

Allowing the opening of all sluice gates at a

particular time, especially during the rainy season,

this should be considered by the authorized

organizations and the Thai Government. However,

the dilemma occured because from July until 15th

September was still in the closed fishing season

but from the observation during the study period,

the fishers fished heavily during this period.

Therefore, the Pak Mun Fishery Conservation

Unit should concentrate on patrolling and stopping

any forbidden fishing gear uses during this time to

let the fishes have a chance to move upstream.

CONCLUSIONS

1. During the study period, the number of

fish species and income from fisheries increased

considerably compared to the previous closed

sluice gate period.

2. Most fish species likely migrated

upstream in the beginning of the rainy season and

migrated down stream after the water receded in

beginning of the dry season.

3. If any compromised management is

implemented to meet the requirements of both

fisheries and power supply, in terms of fisheries,

the period of opening the sluice gates, which

covers the rainy season, should be considered.


Table 1 Species found during the opening the Pak Mun Dam sluice gate’s trial.

No. Scientific name Common name A B C D

Family Dasyatidae (stingrays)

1 Himantura polylepis (Bleeker) Freshwater giant stingray *

Family Notopteridae (featherbacks)

2 Notopterus notopterus (Pallas) Bronze featherback

3 Chitala blanci (Aubenton) Royal featherback *

4 Chitala lopis (Bleeker) Giant featherback *

5 Chitala ormata (Gray) Clown featherback

Family Anguillidae (true eels)

6 Anguilla marmorata Quoy & Guimard Giant mottled eel *

Family Clupeidae (herrings)

7 Clupeichthys aesarnensis Wongratana Thai river sprat * *

8 Tenualosa thibaudeaui (Durand) Mekong shad *

Family Engraulidae (anchovies)

9 Setipinna melanochir (Bleeker) Freshwater anchovy

Family Cyprinidae (carps or barbs)

10 Barilius koratensis Smith Korat barilius

11 Cyprinus capio Linnaeus Common carp * *

12 Hypophthalmichthys molitrix Valenciennes Bighead carp *

13 Paralaubuca typus Bleeker Silver knife barb *

14 Paralaubuca barroni (Fowler) Barron knife barb

15 Luciosoma bleekeri (Steindachner) Bleeker Apollo shark

16 Macrochirichthys macrochirus (Valenciennes) Laotain sword barb *

17 Parachela maculicauda (Smith) Scissortail Asian hatchet

18 Parachela oxygastroides (Bleeker) Giant Asian hatchet

19 Raiamas guttatus (Day) Trout barb *

20 Amblypharyngodon chulabhornae Chao Fah rasbora

Vidthayanon & Kottelat

21 Esomus metallicus Ahl Striped flying barb *

22 Rasbora sumatrana (Bleeker) Sidestripe rasbora

23 Rasbora myersi Brittan Silver rasbora

24 Rasbora rubrodorsalis Donoso-Buchner & Red fin rasbora

Schmidt

25 Rasbora borapetensis Smith Red tail rasbora

26 Rasbora spilocerca Rainboth & Kottelat Dwarf scissortail rasbora

27 Rasbora trilineata Steindachner Scissortail rasbora

28 Leptobarbus hoeveni (Bleeker) Mad barb

29 Probarbus jullieni Sauvage Seven-line barb *

30 Thynnichthys thynnoides (Bleeker) Tiny scale barb *

31 Amblyrhynichthys truncatus (Bleeker) Bigeye barb *


Table 1 (cont.) Species found during the opening the Pak Mun Dam sluice gate’s trial.


32 Cosmocheilus harmandi Sauvage Bala shark

33 Cyclocheilichthys heteronema (Bleeker) Branched barbel *

sensory barb

34 Cyclocheilichthys apogon (Val. in Cuv. & Beardless sensory barb *

Val.)

35 Cyclocheilichthys armatus (Val. in Cuv. & Silver sensory barb

Val.)

36 Cyclocheilichthys repasson (Bleeker) Pointed nose sensory *

barb

37 Cyclocheilichthys enoplos Bleeker Giant sensory barb *

38 Cyclocheilichthys furcatus Sontirat Mekong sensory barb * *

39 Mystacoleucus marginatus (Val. in Cuv. & Yellowtail hook barb *

Val.)

40 Mystacoleucus atridorsalis Fowler Spotted black tip hook * *

barb

41 Mystacoleucus ectypus Kottelat Black tip hook barb *

42 Puntioplites proctozysron (Bleeker) Cubic barb *

43 Puntioplites falcifer Smith Longfin cubic barb *

44 Sikukia gudgeri (Smith) Gudger barb * *

45 Barbodes altus (Gunther) Golden barb *

46 Barbodes gonionotus (Bleeker) Java barb *

47 Barbodes schwanenfeldi (Bleeker) Tinfoil barb *

48 Hypsibarbus malcomi (Smith) Malcom barb

49 Hypsibarbus suvatti Rainboth Suvatti barb

50 Hypsibarbus wetmorei (Smith) Wetmore barb

51 Hypsibarbus lagleri Rainboth Lagler barb * *

52 Discherodontus ashmeadi (Fowler) Red tail barb * *

53 Poropuntius deauratus (Val. in Cuv. & Val.) Yellow tail brook * *

Barb

54 Scaphognathops bandanensis Boonyaratpalin Bandan sharp-mouth * *

& Sirungroj barb

55 Scaphonathops stejnergeri Smith Stejnerger sharp- mouth * * *

barb

56 Hampala dispar Smith Spotted wolf barb * *

57 Hampala macrolepidota (Kuhl & van Hasselt Banded wolf barb *

in van Hasselt)

58 Puntius chola Hamilton Chola common barb

59 Puntius aurotaeniatus Tirant Samet common barb

60 Puntius binotatus (Val. in Cuv. & Val.) Spotted common barb




61 Puntius orphoides (Val. In Cuv. & Val.) Red cheek common barb *

62 Puntius partipentazona Fowler Siamese tiger barb

63 Bangana behri Fowler Mekong dolphin labeo *

64 Henicorhynchus siamensis (Sauvage) Common Siamese barb

65 Henicorhynchus lineatus (Smith) Striped Siamese barb * *

66 Henicorhynchus lobatus (Smith) Pointed nose Siamese

barb

67 Henicorhynchus ornatipinnis Roberts Red fin Siamese barb * *

68 Cirrhinus microlepis Sauvage Giant violet Siamese * *

barb

69 Cirrhinus chinensis Gunther Mud carp *

70 Cirrhinus migrala (Hamilton) Mrigal *

71 Morulius chrysophekadion (Bleeker) Crow Labeo *

72 Labeo dyocheilus (McClelland) Thick lip labeo *

73 Labeo rohita (Hamilton) Rohu *

74 Labiobarbus leptocheilus (Val. in Cuv. & Striped longfin labeo *

Val.)

75 Lobocheilus melanotaenia (Fowler) Bluegill labeo *

76 Osteochilus hasselti (Val. in Cuv. & Val.) Red spotted robust labeo *

77 Osteochilus melanopleura (Bleeker) Black eye robust labeo

78 Osteochilus lini Fowler Lin robust labeo *

79 Osteochilus microcephalus (Val. in Cuv. & Black striped robust *

Val.) labeo

80 Osteochilus waandersi (Bleeker) Black striped robust

labeo

81 Crossocheilus oblongus Kuhl & van Hasselt Oblong algae eater * *

in van Hasselt

82 Crossocheilus reticulates (Fowler) Reticulated algae eater

83 Crossocheilus atrilimes Kottelat Mekong algae eater *

84 Epalzeorhynchos munense (Smith) Mun red tail shark * *

85 Epalzeorhynchos frenatus (Fowler) Red fin shark * *

86 Mekongina erythrospila Fowler Mekong Labeo * *

Family Characidae

87 Myletes bidens (Spinx & Agassiz) Pacu *

Family Gyrinocheilidae (algae eater)

88 Gyrinocheilus pennocki Fowler Spotted algae eater * *

Family Cobitidae (loaches)

89 Acanthopsis sp.1 (large spotted) Horse face loach *

90 Acanthopsis sp.2 (small spotted) Horse face loach *




91 Acanthopsoides sp.1 Pygmy Horse face loach

92 Botia helodes Sauvage Tiger botia *

93 Botia eos Taki Sun botia * *

94 Botia lecontei Fowler Silver botia

95 Botia morleti Tirant Skunk botia *

96 Botia modesta Bleeker Redtail botia *

97 Lepidocephalichthys hasselti (Val. in Cuv. & Hasselt sand loach

Val.)

98 Lepidocephalichthys sp.1 Sand loach

Family Balitoridae (river loaches)

99 Homaloptera smithi Hora Smith climbing loach *

100 Nemacheilus pallidus Kottelat Spotted brook loach *

Family Bagridae (Bagrid catfishes)

101 Pseudomystus siamensis (Fowler) Bubble bee mystus

102 Mystus atrifasciatus Fowler Mekong striped mystus *

103 Mystus mysticaetus Roberts Southeast Asia striped

mystus

104 Mystus singaringan (Bleeker) Long adipose mystus

105 Mystus bocourti (Bleeker) High fin mystus

106 Hemibagrus sp.1 White bagrus *

107 Hemibagrus nemurus (Val. in Cuv. & Val.) Yellow bagrus *

108 Henmibagrus filamentus (Fang & Chaux) Filamentous bagrus

109 Hemibagrus wyckii (Bleeker) Black bagrus

110 Hemibagrus wyckioides (Chaux & Fang) Red fin bagrus

111 Bagriichthys obscurus Ng Shortfin blunt-nose

catfish

112 Bagriichthys macracanthus (Bleeker) High fin blunt-nose

Family Pangasiidae (river catfishes) catfish

113 Pangasianodon gigas Chevey Mekong giant catfish * *

114 Pangasianodon hypophthalmus (Sauvage) Iridescent shark *

catfish

115 Helicophagus waandersi Bleeker Rat mouth shark

catfish

116 Pangasius pleurotaenia (Sauvage) Big eye shark catfish *

117 Pangasius conchophilus Roberts & Snail eating shark

Vidthayanon catfish

118 Pangasius bocourti Sauvage Bocourt shark catfish *

119 Pangasius macronema Bleeker Long barbel shark

catfish




120 Pangasius larnaudii Boucourt Black eye shark

catfish

121 Pangasius sanitwongsei Smith Sanitwong shark catfish *

Family Schilbeidae (Schilbeid catfishes)

122 Laides longibarbis (Bleeker) Flatted barbel shark

catfish

Family Siluridae (sheathfish)

123 Belodontichthys truncatus (Bleeker) Dracula sheathfish

124 Hemisilurus mekongensis Bornbursh & Mekong sheathfish * *

Lundberg

125 Micronema apogon (Bleeker) Bronze sheathfish

126 Micronema bleekeri (Gunther) Blue sheathfish

127 Micronema micronema (Bleeker) Gray sheathfish *

128 Kryptopterus cheveyi Durand Chevey sheathfish

129 Kryptopterus palembangensis Bleeker Giant glass catfish *

130 Kryptopterus cryptopterus (Bleeker) Riverine sheathfish *

131 Ompok siluroides (Lecepede) Black ear sheathfish

132 Wallago attu (Schneider) Crocodile sheathfish

133 Wallagonia leeri Bleeker Black sheathfish *

Family Sisoridae (Sisorid catfishes)

134 Bagarius yarrelli Sykes Crocodile catfish

135 Glyptothorax lampris Fowler Torrent catfish

Family Clariidae (walking catfishes)

136 Clarias batrachus (Linnaeus) Walking catfish

137 Clarias macrocephalus Gunther Broad-head walking

catfish

138 Clarias sp.1 (hybrid) Hybrid walking catfish *

Family Loricariidae (sucker armer cat)

139 Hypostomus plecostomus (Linnaeus) Lorica sucker catfish *

Family Mochokidae

140 Synodontis eupterus Boulenger Longfin upside-down *

catfish

Family Salangidae

141 Neosalanx sp.1 Mekong salang * *

Family Sundasalangidae (Noodlefish)

142 Sundasalanx mekongensis Kottelat & Ng Mekong noodlefish *

Family Poecilidae

143 Poecilia reticulata (Peters) Guppy *




Family Oryziidae (rice fish)

144 Oryzias minutilus Smith Pygmea rice fish

145 Oryzias sinensis Chen, Uwa & Chu Chinese rice fish *

Family Hemirhamphidae (halfbeaks)

146 Dermogenys siamensis Fowler Siamese half beak

Family Belonidae (needlefishes)

147 Xenentodon canciloides (Bleeker) Long beak tropical gar *

Family Syngnathidae (pipefishes)

148 Doryichthys contiguus Kottelat Pygmy pipefish * *

Family Indostomidae

149 Indostomus spinosus Brite & Kottelat Spiny tropical * *

Family Synbranchidae (swamp eels) stickleback

150 Monopterus albus (Zieuw) Swamp eel

Family Mastacembelidae (spiny eels)

151 Macrognathus semiocellatus Roberts Ocellated spiny eel

152 Macroghathus siamensis (Gunther) Peacock spiny eel

153 Mastacembelus favus Hora Tire track spiny eel

154 Mastacembelus armatus (Lacepede) Zig-zag spiny eel

Family Chaudhuriidae (dwarf swamp eels)

155 Chaudhuria caudata Annandale Burmese spineless eel *

Family Ambassidae (glassfishes)

156 Parambassis siamensis (Fowler) Siamese glassfish *

Centropamidae

157 Lates calcarifer (Bloch) Barramundi *

Family Scienidae (drums)

158 Bosemania microlepis (Bleeker) Smallscale croaker *

Family Coiidae (Tiger fish)

159 Coius undecimradiatus Roberts & Kottelat Narrow-band tigerfish *

Family Toxotidae (archerfishes)

160 Toxotes chatareus (Hamilton) Largescale archerfish

Family Cichlidae (cichlids)

161 Oreocromis niloticus (Linnaeus) Nile tilapia *

Family Eleotridae (sleepers)

162 Oxyeleotris marmorata Bleeker Marbled sleeper goby

Family Gobiidae (gobies)

163 Branchygobius mekongensis Larson & Mekong Bubble bee * *

Vidthayanon goby

164 Glossogobius giuris (Hamilton) Gangetic tank goby * *




165 Tridentiger ocellatus (Fowler) Mekong ocellate goby *

166 Gobiopterus chuno (Hamilton) Glass goby *

167 Uniden. goby Deep water goby *

Family Nandidae (leaffishes)

168 Nandus oxyrynchus Ng, Vidthayanon & Ng Leaf tigerfish *

169 Pristolepis fasciatus (Bleeker) Compressed perch *

Family Anabantidae (climbing perch)

170 Anabas testudineus (Bloch) Climbing perch *

Family Belontiidae (gouramies)

171 Betta smaragdina Ladiges Mekong fighting fish *

172 Trichogaster pectoralis (Regan) Snakeskin gourami

173 Trichogaster trichopterus (Pallas) Three spot gourami

174 Trichopsis schalleri Ladiges Colorful croaking

gourami

175 Trichopsis vittatus (Cuv. in Cuv. & Val.) Croaking gourami

Family Osphronemidae (giant gouramies)

176 Osphronemus goramy Lacepede Giant gourami * *

Family Channidae (snakeheads)

177 Channa limbata (Cuvier) Red tail snakehead

178 Channa lucius (Cuv. in Cuv. & Val.) Marble snakehead

179 Channa striata (Bloch) Green tail snakehead *

180 Channa micropeltes (Cuv. in Cuv. & Val.) Giant snakehead *

Family Soleidae (soles)

181 Brachirus harmandi (Sauvage) Ovate sole

Family Tetraodontidae (puffers)

182 Chonerhinus nefastus Roberts Green bottle puffer

183 Tetraodon leiurus Bleeker Spotted green puffer

184 Tetraodon cambodgensis Chabanaud Ocellated puffer

Note: A: Species, which reportedly utilize fish-ladder (Pholprasith et al., 1997)

B: Mekong endemic species

C: Uncommon species

D: Introduced species


Table 2 Shanon’s diversity indice and relative evenness during the study periods.

Month Shanon’s index ± SE Relative evenness ± SE

July 2001 1.92 ± 0.65 0.55 ± 0.20

August 2001 2.92 ± 0.18 0.81 ± 0.50

September 2001 2.12 ± 0.57 0.58 ± 0.17

October 2001 3.10 ± 0.18 0.81 ± 0.04

November 2001 3.14 ± 0.13 0.82 ± 0.02

December 2001 2.70 ± 0.32 0.76 ± 0.08

January 2002 2.35 ± 0.33 0.70 ± 0.09

February 2002 2.45 ± 0.27 0.73 ± 0.07

Martch 2002 2.16 ± 0.33 0.66 ± 0.09

April 2002 2.49 ± 0.23 0.71 ± 0.07

May 2002 2.44 ± 0.37 0.66 ± 0.09

June 2002 2.50 ± 0.23 0.74 ± 0.06

Table 3 Average CPUE at Pak Mun Reservoir from July 2001 to June 2002.

Month CPUE ± SD (kg set-1 night-1) ln (1+CPUE)

Downstream Upstream Downstream Upstream

July 0.43 ± 0.10 1.40 ± 0.95 0.358ab 0.875ab

August 0.38 ± 0.17 1.71 ± 0.94 0.322b 0.998ab

September 0.64 ± 0.35 0.61 ± 0.18 0.495ab 0.476a

October 0.59 ± 0.17 1.17 ± 0.27 0.464ab 0.775ab

November 0.85 ± 0.28 1.67 ± 0.34 0.615ab 0.982ab

December 0.69 ± 0.28 1.94 ± 0.70 0.525ab 1.078ab

January 0.50 ± 0.17 2.62 ± 0.97 0.405ab 1.287ab

February 1.62 ± 0.69 2.71 ± 1.06 0.963c 1.312b

Martch 0.98 ± 0.39 2.28 ± 0.77 0.683ab 1.188b

April 0.79 ± 0.14 1.54 ± 0.45 0.584ab 0.932ab

May 0.73 ± 0.22 2.04 ± 0.45 0.548ab 1.112ab

June 1.70 ± 0.16 2.19 ± 0.77 0.993c 1.160b

Note: the same letter, in each column, mean that there is no significant different (µ = 0.05)


Downstream

0.00

0.50

1.00

1.50

2.00

2.50

J A S O N D J F M A M J

Month

CPU

E (

kg s

et-1

nig

ht-1

)

Upstream

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

J A S O N D J F M A M J

Month

CPU

E (

kg s

et-1

nig

ht-1

)

Figure 2 Temporal changes in CPUE ± SD (kg set-1) during the study periods.

Figure 3 Contribution of catches during the study periods.


ACKNOWLEDGEMENTS

This research was funded by the Royal

Thai Government. We gratefully acknowledged

all in situ data collectors and the fishers who were

involved in this study. We thank our field staff,

Sangkom Yaowachai and Ekapot Puekpan for

their brilliant work and a special thank to Dr.

Michael D. Hare for the English editing of the

manuscript.

LITERATURE CITED

Ahmed, M., T.S. Tana and N. Thuok. 1996.

Sustaining the gifts of the Mekong: fisheries

in Cambodia. Watershed 1 : 33-38.

Begon, M., J.L. Harper and C.R. Townsend. 1990.

Ecology: Individuals, Populations andCommunities: 2nd ed. Blackwell Sciet. Publ.,

London. 945 p.

Charles, A. T. 2001. Sustained Fishery Systems.Blackwell Sci. Inc., London. 527 p.

De Silva, S.S. 2001. Reservoir fisheries: broad

strategies for enhancing yields, pp. 7-15. In

S.S. De Silva (ed.). Reservoir and Culture-based Fisheries: Biology and Management.Canberra: ACIAR.

Duangswasdi, S. and T. Chookajorn. 1991.

Fisheries Characteristic, Species andDistribution of Fishes in the Mun River.Tech. Pap. No. 125, National Inland Fisheries

Institute, Department of Fisheries. 12 p. (In

Thai)

Duangswasdi, M. and S. Duangswasdi. 1992.

Fishery Resources and Fisheries Activitiesin Mun River. Tech. Pap. No. 136. National

Inland Fisheries Institute, Department of

Fisheries. 53 p. (In Thai)

Fishery Extension Division 1995. Damming andFishery Resources. Royal Thai Department

of Fisheries, Bangkok. 41 p. with annex. (In

Thai)

Green, R.H. 1979. Sampling Design andStatistical Methods for EnvironmentalBiologists. A Wiley-Interscience Publication,

New York. 257 p.

Jutagate, T., C. Krudphan, P. Ngamsnae, T.

Lamkom and K. Payooha. Catch per unit of

fishing effort as the evidence in fish migratory

in the Mekong tributary in Thailand. J. Fish.Mgmnt. and Ecol.

Jutagate, T., T. Lamkom, K. Satapornwanit, W.

Naiwinit and C. Petchuay. 2001. Fish species

diversity and ichthyomass in Pak Mun

Figure 4 Changes in income ± SD (Thai Baht yr-1) per household from fisheries at the study area from

1990-2002.

0

5000

10000

15000

20000

1988 1990 1992 1994 1996 1998 2000 2002 2004

Year

Inco

me

(Tha

i Bah

t yr-

1 )

3,693 – 606

4,336 – 9365,737 – 1,545

14,248 – 2,914

8,718 – 1,621

16,367 – 402


Reservoir, five years after impoundment.

Asian Fish. Sci. 14 : 417-425.

Pholprasith, S., P. Sihapitukgiat, B.

Sricharoendham and K. Su-aroon. 1997.

Fishes Passing through the Pak Mun FishLadder and Some Factors AffectingMigration. National Inland Fisheries Institute

Tech. Pap. No.193. Bangkok, Department of

Fisheries. 119 p. (In Thai)

Poulsen, A.F. and J.V. Jorgensen 2000. FishMigration and Spawning Habit in theMekong Mainstream: A Survey UsingLocal Knowledge (Basin-Wide). Mekong

River Commission, Vientiane. 134 p.

Roberts, T. R. 1993. Artisanal fisheries and fish

ecology below the great water falls of the

Mekong River in Southern Laos. Nat. Hist.Bull. of Siam Soc. 41 : 31-62.

Roberts, T.R. 1994. Just another dammed river?

Negative impacts of Pak Mun Dam on fishes

of the Mekong Basin. Nat. Hist. Bull. ofSiam Soc. 41 : 105-133.

Roberts, T.R. 2001. On the river of no returns:

Thailand ‘s Pak Mun Dam and its fish ladder.

Nat. Hist. Bull. of Siam Soc. 49 :189-230.

Schouten, R., P. Sripatraprasit, S. Amornsakchai

and C. Vidthayanon. 2000. Fish, andFisheries Up- and Downstream of the PakMun Dam. World Commission on Dams,

Pak Mun Dam case study. 51 p.

Tantong, A. and N. Siripan. 1969. A survey on

fishes and fishing gears in the Mun River,

Ubon Ratchathani, pp. 10-35. In AnnualReport of Ubon Ratchathani FreshwaterFisheries Station, Department of Fisheries.

(In Thai)

Team Consulting Engineers 1982. Environmentaland Ecological Investigation of Pak MunProject. Vol. 2: Main Report, Part IV:Fishes and Fisheries. Electric Generation

Authority of Thailand, Bangkok. 47 p.

Tongkum, T. 1991. A Study on the FisheryResources and Socio-economics Status inthe Lower Mun River. Electric Generation

Authority of Thailand, Bangkok. 27 p. (In

Thai)

Warren, T.J. 1999. A Monitoring Study to Assessthe Localized Impacts Created by the NamTheun-Hinboun Hydro-Scheme onFisheries and Fish Populations. Final Report.

The Theun-Hinboun power company.

Vientiane, Lao PDR. 68 p.

Warren, T.J., Chapman, G.C. and Sinhanouvong,

D. 1998. The upstream dry-season migration

of some important fish species in the lower

Mekong River of Southern Lao PDR. AsianFish. Sci. 11 : 239-251.

Warren, T.J. and Mattson, N.S. 2000. Fish passes

and migrations: Can fish passes mitigate the

impacts of water related development on fish

migrations in the Mekong Basin? Mekong

Fish: Catch and Culture 6(2) : 1-4.

Wilkinson, L. 1989. SYSSTAT: the System forStatistics. Evanston Inc., IL. 882 p.


Synthesis of Barium Titanate as an Electroceramic Raw Materials

Nuchnapa Tangboriboon

ABSTRACT

Nowadays, most electronic and electrical parts of equipment in Thailand such as substrates,

magnetics, capacitors, ferroelectric, piezoelectric, RAMs, DRAMs, FERAMs, loudspeakers and devices

in ultrasonic cleaning are imported. Barium titanate powder is one of electroceramic raw materials. It is

used to produce parts of electroceramic products. In this project, barium titanate was produced by

hydrothermal synthesis and thermal treatment process. The mol ratios of titanium dioxide to barium

hydroxide in the thermal treatment process were 0.5:1, 1:1, and 1:0.5 at 700, 1000 and 1200∞C ,for 3 and

5 hours. Results of all experiments were analysed by x-ray diffraction (XRD), scanning electron

microscope (SEM), particle size distribution and liquid pycnometer technique. A good condition for

barium titanate synthesis is from the hydrothermal reaction at the weight ratio of 5:20, 90∞C , and 72

hours. The obtained powders possessed high purity, perovskite structure and a density of 6.2520 g/cm3.

In addition, an average particle size at the accumulated particle size of 50 percent (d50) is less than 1

micron. For the thermal treatment process, the appropriate condition is 0.5 mol titanium dioxide to 1 mol

barium hydroxide at 700∞C for 3 hours. An average particle size at d50 is larger than 5 microns and the

density is 4.7100 g/cm3 because its structure is composed of another composition.

Key words: barium titanate, perovskite structure, hydrothermal synthesis, thermal treatment process

INTRODUCTION

Panne et al.(1992) reported that Barium

titanate (BaTiO3) is an extensively studied and

widely utilized perovskite-type electroceramics.

There are two important crystalline phases of

barium titanate being used in the microelectronics

industry as reported in William (2002). The

tetragonal phase of barium titanate is used in

a broad array of electronic devices due to its

ferroelectric properties, while the cubic form,

although not ferroelectric, has a high dielectric

constant that makes it suitable as capacitors. The

main objective in barium titanate synthesis is to

create smaller, more uniform particles to allow for

finer ceramic layers to be used in multilayer

capacitors, piezoelectric, capacitor and actuator

(and, thus, achieve device miniaturization) without

the loss of dielectric properties as reported in

Kingery et al.(1992). Controlling the phase,

composition homogeneity, particle size, density

and mono-dispersity, microstructure, and the cost

of particle production are important concerns in

developing techniques for synthesizing barium

titanate. In this research, two methods of barium

titanate synthesis are studied. One method is

thermal treatment process and the other is

hydrothermal process. The hydrothermal process

is based on Michael (2000).

Department of Material Engineering, Faculty of Engineering, Kasetsart University, Bangkok 10900, Thailand.




The synthesis of barium titanate as an

electroceramic raw materials consisted of two

methods. The principle of mol ratio mixing of two

components are based on Ree and Dippel .(1992).,

as follows :

Method 1 Thermal treatment process (Figure 2)

1. Mix titanium hydroxide and barium

hydroxide at the mol ratios of 0.5:1, 1:1, and 1:0.5.

2. Pour the mixture from the first step into

a ceramic crucible and heat in a furnace at

temperatures of 700,1000 and 1200∞C for 3 and 5

hrs.

3. Characterize the powder obtained from

the second step by XRD, SEM, particle size

analyzer, and liquid pycnometer technique.

Figure 1 Perovskite structures in Ferroelectric materials.

Figure 2 Flow chart of barium titanate synthesis by the thermal treatment process.

+

mol ratios of TiO2 : Ba(OH)2 0.5:1, 1:1, 1:0.5

Ba(OH)2TiO2

A ceramic crucible

Heat at temperatures of 700,1000, and 1200∞C for 3 and 5 hrs.

Barium titanate powder


Method 2 Hydrothermal synthesis (Figure 3)

1. Mix 20 g. of barium hydroxide with 5 g.

of titanium hydroxide(Ba:Ti equals to 1:1 mol

ratio) in a Teflon bottle.

2. Add 30 ml. of distillate water into the

mixture in the Teflon bottle, cover and shake

vigorously.

3. Heat the mixture from the second step

in an oven at 90∞C for 24 and 72 hrs to activate

the reaction.

4. Wash the precipitate from step 2 with

100 ml. of 1 molar of formic acid and filter.

5. Dry the filtered cake at 90∞C for 24 hrs.

6. Characterize the powder as received by

XRD, SEM, particle size analyzer, and liquid

pycnometer technique.


The suitable condition for barium titanate

synthesis by thermal treatment process is 0.5 mol

of titanium dioxide and 1 mol of barium hydroxide

at 700∞C for 3 hrs. The powder obtained has a

density of 4.7100 g/cm3 which is less than

theoretical density value because of Al4Ti2SiO12

mixed in perovskite phase.

Hydrothermal synthesis for barium titanate

powder is a condition of 5 g. of titanium dioxide

Figure 3 Flow chart of barium titanate synthesis by the hydrothermal synthesis.

weight ratio 20 g : 5 g (1:1 mol ratio of Ba:Ti)

Temperature 90∞C for 24 and 72 hrs

Ba(OH)2TiO2

Add 30 ml. of water

in a Teflon bottle

Dilute with 100 ml. of 1M. formic acid

Filter

Dry the filtered cake at 90∞C

Barium titanate powder


mixed with 20 g. of barium hydroxide (mol ratio

1:1), dried at 90∞C for 72 hrs. Barium titanate

powder has characteristic close to the theory. The

powder has less aggregation and better defined

microspherical shape (solid sphere with smooth

surface) than that of thermal treatment process.

Figure 4 shows crystalline phase compositions,

most of them are perovskite structure, as Hung and

Riman.(1998). The average particle size is smaller

than 1 micron, as shown in Figure 5. The

microstructure of barium titanate powder, as-

prepared by SEM is shows in Figure 6. In this

method, 100 ml. of 1 M. of formic acid was added

to reduce the barium carbonate impurity level in

the barium titanate suspension, the procedure was

slightly modified from the work of Eckert et

al.(1996). Formic acid was added immediately

during the suspension was still hot. The suspension

was allowed to stand for 15 min at room

temperature; then it was centrifuged and washed

with deionized water twice, and was removed by

pipette. The remaining white paste was air dried to

form a cake, and dried in an oven overnight. The

other hydrothermal synthesis condition is to use 5

g. of titanium dioxide to 20 g. of barium hydroxide,

dry at 90∞C for 24 hrs., but the crystalline phase

compositions are composed of the perovskite,

cubic and tetragonal. The density was reduced to

3.1191 g/cm3

CONCLUSION

Hydrothermal synthesis is a suitable method

to make barium titanate from 5 g. of titanium

dioxide of 20 g. of barium hydroxide (Ti:Ba equals

1:1) at 90∞C for 72 hrs. Barium titanate powder

obtained is filtered with formic acid during

hydrothermal process to purify. The powder

obtained has monodispersed-nanocrystalline

structure, high density, no aggregation and high

purity.

ACKNOWLEDGEMENTS

The author is grateful to the Faculty of

Engineering, Kasetsart University for the grant

support of this research and L.M.S Instrument

Co.ltd. for analysing the particle size distribution.

Figure 4 Crystalline phase compositions.

(a) X-ray diffraction of barium hydroxide.

(b) X-ray diffraction of titanium dioxide.

(c) X-ray diffraction of the mixture of titanium

dioxide and barium hydroxide at a ratio of

5:20 at 90∞C for 72 hrs.


LITERATURE CITED

Eckert J.O.Jr., C.C. Hung-Houston, B.L. Gersten,M.M. Lencka, and R.E. Riman.1996. Kineticsand mechanisms of hydrothermal synthesisof barium titanate. J. Am. Ceram. Soc. 79 :2929-2939.

Hung C.C. and R E. Riman. 1998. X-rayspectroscopy investigation of hydrother maland commercial barium titanate powders. J.Am. Ceram. Soc. 81(6) :1589-1599.

Kingery. W.D., H. K Bowenz. and Uhlann. 1992.Introduction to Ceramics. John Wiley andSons., Inc., New York. 1032 p.

Michael Z.C. Hu. 2000. Wet-chemical synthesisof monodispersed barium titanate particleshydrothermal conversion of TiO2 micro-spheres to nanocrystalline BaTiO3 PowderTechnology 110 : 2-14.

Panne A. D., D J. Eichorst. and L F.Francis.1992.Molecular Precursors for the ChemicalProcessing of Advanced ElectricalCeramics. John Wiley and Sons, Inc., NewYork. 14 p.

Ree W.S.,Jr. and K A. Dippel. 1992. New Group2 Organometallic Precursors to Metal Oxides.,John Wiley and Sons, Inc., New York. 6 p.

William D.C.,Jr. 2002. Introduction of MaterialsScience and Engineering. John Wiley andSons, Inc., New York. 871p.

Percent cumulative of particle size

particle size (micron)

Figure 5 Particle size distribution of barium titanate (hydrothermal synthesis at 90∞C for 72hrs.) by

particle size analyzer.

Figure 6 Particle size distribution of barium

titanate (hydrothermal synthesis at

90∞C, 72 hrs.) by SEM manification

5,000 and 10,000.

NOTICE TO CONTRIBUTORS

The Kasetsart Journal (Natural Science) is published by Kasetsart University Research and

Development Institute (KURDI) under authorization of Kasetsart University to serve the interest of

scientists and researchers. Research articles must be a report of the original work in any aspects of natural

sciences. The Journal emphasizes and promotes the highest standards of ethics in the conduct of reporting

research. Appropriateness is judged by the scientific approach to study the problems related to natural

sciences. The governing operational factor is the general soundness and integrity of science being reported

as judged by peer reviewers and/or the scientific editors. The Journal is published quarterly and is issued

in March, June, September and December.

GENERAL EDITORIAL POLICIES

Authorship criteria and author responsibilitiesKasetsart Journal is proud of its high quality research journals and is dedicated to maintaining

its high level of professionalism. However, because of reports of fraud and plagiarism in the scientific

literature, we remind authors of their obligations when submitting manuscripts for publication.

Authorship criteria. Authorship is restricted to those who (1) have contributed substantially to one or

more of the following aspects of the work (conception, planning, execution, writing, interpretation, and

statistical analysis) and (2) are willing to assume public responsibility for the validity of the work.

Exclusivity of work. The corresponding author must verify, on behalf of all authors (if more than one),

that neither this manuscript nor one with substantially similar content has been published, accepted for

publication, or is being considered for publication elsewhere.

CopyrightCopyright to published manuscripts becomes the sole property of the KURDI. In instances where

the work cannot be copyrighted (works authored solely by government employees as part of their

employment duties), this requirement is waived.

Reproduction of all or a portion of a Kasetsart Journal article by anyone, excluding authors, is

prohibited, unless permission is received from KURDI. Authors have the right to reproduce extracts from

the paper with proper acknowledgment and retain the right to any patentable subject material that might be

contained in the article.

DisclaimerOpinions expressed in articles published in this journal are those of the author(s) and do not

necessarily represent opinions of the KURDI. KURDI does not guarantee the appropriateness for any

purpose, of any method, product, process, or device described or identified in an article. Trade names, when

used, are only for identification and do not constitute endorsement by KURDI.

Criteria for manuscript acceptanceManuscript acceptability is based primarily on appropriateness and importance of the topic;

clarity of objectives; originality; appropriateness of the experimental design, methods and statistical

analysis; depth of the investigation; substance of the results; thoroughness with which the results are

discussed ; and appropriateness of the conclusions.

ReprintsFollowing acceptance of a paper and prior to publication, the author will be given appropriate

number of reprints free of charge.

MANUSCRIPT REQUIREMENTS

Unless otherwise stipulated, the style and format of manuscripts submitted to Kasetsart Journal

(Natural Science) should follow the “Scientific Style and Format: The CBE Manual for Authors, Editors

and Publishers” 1994, 6th ed. (Cambridge University Press, New York). For convenience, refer to articles

in the latest issue of the journal or for more details, contact the Kasetsart Journal editorial office.

Use the English language (American spelling and usage) and the SI system (Système International

d’Unitès, often referred to as “International Units”) for measurements and units.

Manuscripts preparationManuscripts on original research should include the following elements.

Title page, as p. 1. Include:

• Full title (be concise)

• Name(s) of author(s) and author affiliation(s) with complete address(es)

• Contact information for the corresponding author, including full name, complete mailing address,

telephone, fax, and electronic mailing address.

• Previous address of author(s) if research was conducted at a place different from current affiliation.

Abstract, as p. 2. Include:

• An abstract not exceeding 200 words; all acronyms and abbreviations defined; no references cited.

State what, where and how it was done, major results.

• Five key words.

Introduction. In two pages or less, review pertinent work, cite key references, explain importance of

the research, and state objectives of your work.

Materials and Methods. Provide sufficient detail so work can be repeated. Describe new methods in

detail; accepted methods briefly with references. Use subheadings as needed for clarity.

Use of trade names. Trade names are to be avoided in defining products whenever possible. If

naming a product trade name cannot be avoided, the trade names of other like products also should be

mentioned, and first use should be accompanied by the superscript symbol TM followed in parentheses by

the owner’s name. If a product trade name is used, it is imperative that the product be described in sufficient

details so the nature of the product will be understood by professionally trained readers. Do not use trade

names in titles.

Use of abbreviations and acronyms. At first text use, define in parentheses. Do not use

abbreviations and acronyms in titles.

Statistical analysis. If variation within a treatment (coefficient of variation, the standard

deviation divided by the mean) is small (less than 10%) and difference among treatment means is large

(greater than 3 standard deviations), it is not necessary to conduct a statistical analysis. If the data do not

meet these criteria, appropriate statistical analysis must be conducted and reported.

Results. Present results concisely using figures and tables as needed. Do not present the same

information in figures and tables.

Discussion. Discuss principles and relationship, point out exception. Show agreement with published

research work. The significance of the work or conductions should be presented in the end of discussion.

Conclusion. State conclusions (not a summary) briefly.

Literature Cited. List only those references cited in the text. Required format of references is described

below.

Acknowledgments. List sources of financial or material support and the names of individuals whose

contributions were significant but not deserving of authorship. Acknowledgment of an employer’s

permission to publish will not be printed.

Appendix. This section is rarely needed in a research paper but can be added if deemed necessary (e.g.,

complicated calculations, detailed nomenclature).

Tables. Number each table with Arabic numerals. Place a descriptive caption at the top of each table.

Print one table per page. Columns and their headings are usually (but not always) used to display the

dependent variable(s) being presented in the table. Foot-notes should be identified by lower case letters

appearing as superscripts in the body of the table and preceding the footnote below the table.

Figures (graphs, charts, line drawings, photographs). Supply one illustration per page with the figure

number indicated under the illustration. Figure captions should be double-spaced and listed consecutively

on page(s) separate from figures: use Arabic numerals. Include one original set of illustrations with an

original manuscript, marked, “ORIGINAL.” Illustrations for other copies of the manuscript may be

photocopies, provided they are clear. Exceptions are photomicrographs, gel electrophoretic patterns, etc.;

furnish four glossy photographs for each of these.

Authors are responsible for obtaining permission to reproduce previously copyrighted illustrations.

Proof or certification of permission to reproduce is required.

Lettering, data lines, and symbols must be sufficiently large so as to be clearly visible when the figure

is reduced to a size commonly used in the journal. When a color presentation is deemed necessary, please

note this in the cover letter of the submission.

LITERATURE CITED FORMAT

Manuscripts should follow the name-year reference format of the Council of Science Editors

(formerly Council of Biology Editors). Cite only necessary publications. Primary rather than secondary

references should be cited, when possible. It is acceptable to cite work that is “in press” (i.e., accepted but

not yet published) with the pertinent year and volume number of the reference. Work that is “submitted”

but not yet accepted should not be cited.

In text. Cite publications in text with author name and year. Three or more authors use “et al.”

In parenthetical citations, separate author and year with a comma. Use suffixes a, b and c to separate

publications in same year by the same author. Semi-colons separate citations of different authors. Cite two

or more publications of different authors in chronological sequence, from earliest to latest. For example:

• The starch granules are normally elongated in the milk stage (Brown, 1956).

• Smith et al. (1994) reported growth on vinasse.

• …and work (Dawson and Briggs, 1984,1987) has shown that . . .

• …and work (Dawson, 1984; Briggs, 1999) has shown that . . .

In Literature Cited section. List only those literature cited in the text. References should be

listed alphabetically by the first author’s last name. Single author precedes same author with co-authors.

Type references flush left as separate paragraphs. Do not indent manually. Write the names of book and

journal in bold letters. Let the text wrap with first line hanging indented. Use the following format.

• Journal articles: Author(s). Year. Article title. Journal title, volume number: inclusive pages.

Example: Citation in text: (Smith et al., 1999)

Smith J.B., L.B. Jones and K.R. Rackly. 1999. Maillard browning in apples. J. Food Sci. 64 : 512-518.

• Books: Author(s) or editor(s). Year. Title. Publisher name. Place of publication. Number of pages.

Example: Citation in text: (Spally and Morgan, 1989)

Spally M.R. and S.S. Morgan. 1989. Methods of Food Analysis. 2nd ed. Elsevier. New York. 682 p.

• Chapter: Author(s) of the chapter. Year. Title of the chapter, pages of the chapter. In author(s) or editor(s).

Title of the book. Publisher. Place of publication.

Example: Citation in text: (Rich and Ellis, 1998)

Rich R.Q. and M.T. Ellis 1998. Lipid oxidation in fish muscle, pp. 832-855. In J.J. Moody and W. Lasky,

(eds.). Lipid Oxidation in Food. 6th ed. Pergamon. New York.

For journal abbreviations and other examples of reference formats please refer to articles in a

2000 issue of this journal or contact the editorial office at KURDI.

EDITORIAL REVIEW AND PROCESSING

Peer Review. All submitted manuscripts are screened by the Scientific Editor for importance,

substance, appropriateness for the journal, general scientific quality, and amount of new information

provided. Those failing to meet current standards are rejected without further review. Those meetings these

initial standards are sent to expert referees for peer review. Referees identities are not disclosed to the author.

Author identities are also not disclosed to the referees. Referee comments are reviewed by an Associate

Editor and he/she, often after allowing the author to make changes in response to the referee’s comments,

advises the Scientific Editor to either accept or reject the manuscript. The Scientific Editor informs the

author of the final decision. The review process ordinarily is completed within 3 months. If the process is

delayed beyond that point, authors will be notified.

Rejected manuscripts. Rejected manuscripts may, in some instances, be appropriate for

publication in other journals. Rejected manuscripts including original illustrations and photographs will be

returned to authors.

Accepted manuscripts. The author(s) will be asked to review a copyedited pageproof. The

author(s) is responsible for all statements appearing in the galley proofs. The author will be informed of the

estimated date of publication.

Inquiries regarding status of the manuscript. Direct inquiries to: Orawan Wongwanich,

Manager, Kasetsart University Research and Development Institute (KURDI), Kasetsart University, 50

Pahonyothin Road, Chatuchak, Bangkok 10900, Tel. 66-2-579-0032, 579-5548, 561-1474, Fax. 66-2-

561-1474, E-mail: [email protected].

SUBMISSION MANUSCRIPT

Submit the following items.

Cover letter. Identify the corresponding author and provide his/her full name, address, numbers

for telephone and fax, and e-mail address.

Manuscript. Double-space all components of the manuscript except tables. In 12 point Times

or Times New Roman.Type on one side of A4 paper. Use one inch margins. Number all pages.

Send an original manuscript (with original figures, marked “ORIGINAL”) and 3 photocopies.

Hard copies are required. Staple each manuscript copy, including the original manuscript in the upper left

corner. Include no paper clips or binders.

Disk. Include an IBM-formatted, 3-l/2" disk, containing the manuscript in Microsoft Word

(version 97 preferred).

MAIL MANUSCRIPT TO:

Orawan Wongwanich, Manager,

Kasetsart University Research and Development Institute (KURDI)

Kasetsart University,

50 Pahonyothin Road, Chatuchak,

Bangkok 10900, Thailand

Tel. 66-2-579-0032, 579-5548, 561-1474

Fax. 66-2-561-1474

E-mail: [email protected]

PRE-SUBMISSION CHECKLIST

(see “Instructions for Authors” for additional information.)

Cover letter & form❑ Full contact information for the corresponding author (full name, address, phone,

fax, e-mail).

Manuscript❑ Original manuscript (with original figures, marked ORIGINAL)

❑ Three photocopies

❑ Page numbering

❑ Each manuscript stapled in upper left corner (no paper clips or binders)

❑ Abstract of less than 200 words

❑ 5 key words

❑ Figure captions listed consecutively on a page separate from figures

DISK❑ 3-l/2 in IBM-formatted disk with full manuscript Microsoft Word (version 97 preferred) file.

(submission of the title page and abstract by electronic means is an acceptable alternative)

Kasetsart Journal

Application for Membership

Name : _________________________________________________________________________

Title : _________________________________________________________________________

Home Address : __________________________________________________________________

Tel : ___________________________________ Fax : ______________________________

Office Address : __________________________________________________________________

Tel : ___________________________________ Fax : ______________________________

E-mail Address : __________________________________________________________________

Type of application : � New Membership

� Membership Extension

Membership type : � Regular (non › government) Membership

� Student Membership

Submission information :

Student membership (Natural Science)

� Application and 60 bahts dues payment

� Letter of Recommendation from major advisor or the copy of student ID

Student membership (Social Science)


� Letter of Recommendation from major advisor or the copy of student ID

Regular membership (Natural Science)


Regular membership (Social Science)


Send application materials to

Manager of KU Journal

KURDI Kasetsart University

50 Paholyothin, Bangkhen Bangkok 10900

Payment information :

� Cash

� Send money order made payable to KURDI , Kasetsart Post Office

Preferred address for mail

� Home

� Office

Do Not Write In This Space

For Office Use Only

Membership Number ______________________

Receipt Number __________________________ Receipt Date ________________________

___________________________ Finance Officer

( __________________________ )

Date ___________________________

Letter of Recommendation

This is to certify that _______________________________________________________

is currently the ____________________________ (first - , second -, third -, fourth › year, graduate)

student majoring _________________________ , Faculty of _____________________________

, ______________________________University.

( __________________________________)

Academic Title ______________________

Major Advisor

Date _______________________________

january - april 2003 volume 37 number 1 - ku...

Documents