9w'a-terials and .9w'e-thods -...
TRANSCRIPT
Chapt:er III
.9W'a-terialS and .9W'e-thods
3. 0 ::M.ateria{s anrf nzetfiorfs
The present study "Factors affecting somatic embryogenesis
and plant regeneration in Dendrocalamus asper and
Dendrocalamus tulda (Edible bamboo) sp." was carri ed out
at Department of Biotechnology, Sant Gadge Baba Amravati
University, Amravati. The original species of D. asper and D.
tulda were procured from Central Nursery, Maharashtra
Forest Department, Wadali, Amravati. These plants were
maintained in green house and used as source of explants.
The mother source plants D. asper and D. tulda were a lso
used as a source of explants from Wadali nursery.
3.1. Requirements
3.1.1 Chemicals
The deta il s of different chemicals used and collected from
different agencies to perform experiments are as under.
Sa lts of macro and microelements of analytical grade were
obtained from Loba chemie, Himedia, Sigma, Qualigens, Sd
fine, Merck. Etc.
Vitamins, Amino acids, Sucrose were obtained from Hi media
Laboratories Pvt. Ltd., Bombay.
Myo inositol, Thiamine HCI, Plant growth regulators (Auxin,
e.g. 2,4-D, NAA, IBA, IAA etc. and cytokinin e.g. Kinetin,
SAP) of cell culture tested grade were purchased from M/S
Sig ma Chemicals, Saint Louis, Missouri 63178 USA. and Hi
media Laboratories Pvt. Ltd . , Bombay.
Phytagel was purchased from (Hi media).
Bavisti n (Carbandezim: A systemic Fungicide) were obtained
from M/s BASF India Limited, Bombay.
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Streptomycium Sulphate (a n Antibiotic) was obtained from
M/S Hindusthan Pharmaceuticals, Pune .
Teepol (Detergent l iquid soap- Laboline) was procured from
M/S BDH Glaxo chemicals Limited, Bombay.
HgCl2 NaOCI, an explant sterilan t were used from Hi media. I
3.1.2 Glass wares
All the glasswares used for the experiment during the work were
'Borosilicate Laboratory grade' and were procured from M/s. Borosil
India Limited, Bombay.
3.2 Washing of Glass wares
Glasswares were soaked in 1: 10 Laboline (detergent)
so lu tion in d istilled water for two hours followed by rinsing
with tap water. Then they were soaked in lN HN03 for
overnight. On the next day the glasswares were washed 2-3
times w ith tap water and followed by double distilled water.
After washing they were dried in oven at 110°c. Finally the
glasswares were stored in dust proof cupboards until further
use.
3.3 Sterilization of glass wares
Test tubes and conical flasks were plugged with non
a bsorbent cotton wrapped in muslin cloth. Prior to use all
g lasswares were autoclave d at 1.06 kg/Sq.cm pressure at
approximately 121°c temperature for one hour. Autoclaved
glasswares were then dried in oven at 80-100°C for two
hours. Forceps, sca lpels, blades and scissors were f irst
autoclaved and then during inoculation sterilized by dipping
;n absolute alcohol and holding on flame alternately.
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3.4 Selection of culture media
The basal media deve loped by Murashlge a n d Skoog's ( 1962)
was s t andardized u si ng differen t co n cen tration s and
combllldllOn!:. of growth hormones for In vitro Cdllu::.
Inducti on and regeneration from tissues of D. asper ancJ D.
tulda.
3.S Adjuvant: t:o t:he basal medium
T h e MS rTIGdlum wds sur>pl emcnted with fo llowing ilOjuvant
t or Initiation of growth, est .. 1blis hni<"nt or cu ll1Jr<..·~,
rnctinl<'rh1nce d n d rnultlpllc dtlon of c ulturcs and
rnorphog<' n esls of th e cxp l ant.
• 2, 4 - Dich l orophenoxy acetic iH.ld (Auxin)
• Ndphthi:l l crie acet i c ncid (Auxin)
lndol hutyrlC dCld (Auxin)
3 - .lndo l acellc ac.id (/\uxln)
•
•
• • •
•
6 - Benzyl amino purine (Cyloklnin)
Klnetln (Cy t ok inln )
S u crose (Carbon source)
Thiamine HCI (Vitamin)
Myolnosito l (Vitamin)
Ascorbic Acid (Antiox idant)
3.6 composition of Media
The composit ion of MS basa l medium used for the present
study is given in Table 1. Concentrated so lution (stock
so lutions) conta ining different ingred ient s were prepared in
sterile double distilled water and stored in reagent bottles at
4°c not more than 15 days after preparation.
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Separate stock solutions were prepared for each growth
reg ulators by dissolving it in a minimal quantity of the
appropriate solvents. The stock solutions of auxin ( lmg/ml)
were prepared by adding a few drops of 0.lN NaOH (3ml for
every 10 mg of auxin) to a weighed quantity of auxin an d
t hen making up the required volum e with sterile double
glass distilled water. The stock solutions of cytokinin were
pre pared by adding few drops of 0.1 N HCI (3ml for every 10
m g of cytokinin) to a weighed quantity of cytokinin and then
ma king up the required volume with sterile double glass
distilled water.
3. 7 Preparation of culture media
A more convenient and popular method, were to prepare a
se ries of stock solutions. To prepare Murashige and Skoog's
b asa l medium, four different stock solutions were prepdred.
a) Major salts (20X concentrated)
b) Minor salts (200X concentrated);
c) Iron ( 200X concentrated);
d) Organic nutrients except sucrose (200X concentrated).
:::o r the preparation of stock solutions (a)-(d) each
component were separately dissolved to the last parti cle
and then mixed with the others.
-~e sequence of steps involved in preparing a medium is as
fol lows :
1) Required quantities of Phytagel and sucrose were
weighed and dissolved in double distilled water, about
3/4 volume of the medium, by heating them in water
bath.
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2) Appropriate quantities of the various stock so lu tions,
including growth regulato rs and other special
supp lem e nts w e re added.
3) T h e final volume of the medium was m ade up with
double g lass di s tilled water.
4) Afte r mi x ing w e ll , the pH of t h e m e dium was adjusted
u s ing 0.1 N NaOH and/or 0. 1 N H C I .
5) Tl1 e n t h e m e dium was poured into t h e d es ired
cu ltured vesse l. 15 ml o f the m e dium wa s d is p e nde d In
a 25 X 150 mm c ulture tube, a nd about 50 m l In 150
ml f lask.
6) T ubes a nd co nica l flasks were plugged u sing cotton plugs
wrapp ed w ith muslin cloth and fina lly t ubes w e r e wrapped 0
with a luminum fo ils and autoclaved at 1 2 1 C temperature
a nd 1.06 Kg/Sq.cm pressure for 30 min. After t he steam wc:is
completely re leased, tubes and conica l f lasks were removed.
7) The test tube s la nts w e r e prepared. Th e s l a nt tubes
and flasks were stored in dust free area until further
use .
Table 1 : Stock solution for MS basa l medium
Stock solution I
Constituents
MgS0'4.7H20
K H 2PO.,
KN03
NH4N03
CaCl2.2H20
Stoc k Solution XX
H3l303
MnS0.,.4H,.O
ZnSO.,.lH70
Na2Mo 0 4 .2 112 0
cuso'1.sH7o
CoCl2 .6H;iO
Stock Solution XII
FeS04.7H20
Na2 EDTA.2H20
Stock Solution IV
Inositol
Th iamine HCI
Pyridoxine HCI
Nicotinic Acid
G lycine
Amount (Mg/I)
7400
3400
38000
33000
8800
1240
4460
1/20
50
5
5
5560
7460
20000
100
100
100
400
a . For preparation of stock solutlon III, Dissolved FeS04 . 7H 2 0 and Na:;iEDTA .2H 2 0 separatel y in 450 ml disllllcd wate r by healing and constant stirring. Mixed the two solutions, adjusted the pH to 5.5 and added double distilled water lo makeup the final volume to one liter and stored in umber color bottle b. To prepare one liter of media SO ml of stock solution I, 5 ml each of stock solution II, III, IV were taken.
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Table 2: Disinfectants used for sterili z ing plant material .
Sr . No . Disinfectants Concentration Duration of
( % ) treatme nt
(Min) ,__
1 Benzalkonium Chloride 0.01 - 0.1 5 - 20.
2 Bromine water 1 - 2 2 - 20 - 3 Calcium hypochloride 9 - 10 5 - 30
4 Ethyl alcohol 75 - 95 30 sec
5 Hydrogen p e roxid e 3 - 1 2 5 - 15 - 6 Mercuric chlorid e 0.1 - 1 . 0 2 - 10 -
7 Silver nitrate 1 5 - 3 0
8 Sodium hypoc hlorid e 0 . 5 - 5 5 - 3 0 -
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3.8 Transfer area for aseptic manipulation
All the aseptic manipulations like expla nt preparation,
surface sterilization, inoculation as wel l as their subculturing
operations were carried out i n the lamina r airflow hood . The
working surface wa s c leaned u s in g 0 .5 °10 D e ttol so luti on
( v IV) a nd fin a 11 y wiped with 9 0 °/o a I c oho I. The door of t h e
laminar flow ben c h was c losed and the working a r ea wa s
expos ed to ultra violet ligh t for 3 0 minutes before each
exp e riments.
Tho s urgical Instruments were c lea ned and wrapped In brown
paper /aluminum f o ll an d autoc l aved. Fiiter pap e r of
required s i zes w e r e c u t and kept In th e petriplates and
wrapped in br-own pap e r . All these materi a l s were autoc laved
f o llowing usual procedure a nd s t ore d In dust free area.
Du ring the co u rsc of ex plant prep a rallon, s u rface
s terilization, lnoc vl a llon a nd other aseptic manipulation, all
the s urgica l instruments viz. forceps, sca lpe l, n eed le and
blade were deeped Into Lh e 90°/o a l cohol an d fl a med Lo
s t e rili ze before e very operation. Th e sta ndard s Lerll e
te c hniqu es were fo l lowed as s ugges t e d by Street ( 1977) for
inoc ul ation and s u bcu lLurln g of explants.
3.9 Selection, isolation and preparation of explants
D e ndrocalamus asper and Dendrocalamus tulda plants were
regu larly Irrigated and ma i ntained In the green house. Th e
p lant tissues such as l eaf bid, shoots t i p, nod e seg m e nts and
root segments were se lected from healthy plants. The
iso lated plant tissues were washed with tap water and then
double glass distilled water . The explants were cut in proper
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size in the petriplate and were washed with double glass
distilled water. These exp lants were ready for surface
sterilization .
3.10 Sterilization of Explant
The surface sterilization of explants was performed on laminar
airflow. The leaf, shoot tip, node bud segments and roots were
washed three times with tap water and then with double glass
distilled water. Then the ex plants were ready for surface
sterilization. The different concentrations with different incubation
period of HgCl2 were chosen for the surface sterilization of leaf bid,
shoot tip, node bud segments and root segments.
A. S urface steri lizatio n of Leaf bid, s hoot tip, node bud segm e nts
a nd root segme nts.
The diffe r e nt con centrations of m e r c uric c hloride w ere u sed for the
s urface sleri llz ation of explants, leaf bid, s hoot tip, node bud
segments a nd root segments. The leaf bid, shoot tip, node bud
seg m e nts a nd root segments were s u spended in 0.1 to 1 .0 °/o HgCl2
(W/V) solution for 1 min to 10 min tim e duration. These treated
explants was hed thrice in b eaker containing sterile double dis tilled
water. Afte r washing, these explants w ere blotted by sterile blotting
paper. Then explants inoculated aseptically on MS medium
supplemented with various concentrations of auxin and cytokinin.
The observations were recorded after 3 -4 days, of inoculation in
terms of their survival and occurrence of contamination.
3.11 Incubation room
All the inoculated cultures of D. asper and D. tulda w e re
incubated in culture room. The t e mperature wa s maintai n e d
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0 at 27 + 2 C. The cultures were kept in light for 16 hours
(1000- 4000 lux) and 8 hour dark respective ly.
Out of a l l the four different explants selected and tested for
their growth responses in various combinations of MS media
only new sprout nodal exp lants showed the response.
Therefore it was continued for further study.
3 . 12 Induc tion a nd growth of c a llus
The explant new sprout bud segment were c u ltu red on MS
media fortified with different concentrations of auxin and
c ytoklnln. Ten exp lants and s lants per treatment were taken,
which were replicated thrice. Days required for induction of
ca llus were observed.
Physical appearances of callus were noted after 30 days.
Ca llus growth was measured in terms of fresh weight a nd
dry w e ight after 30 days. The n ew sprout node bud seg ment
showed the better respon se for ca llus induction. Cal l u s was
taken on filter paper and washed 2-3 times with steril e
double glass distil led water, to remove the remaining media
from ca l lus. The ca lli were soaked on butter paper and fresh
weights were d etermi ned after harvesting the tissue on
butter paper. This ca lli were again used for dry weight
determination. For dry weight, ca l lu s ti ssue were dried for 0
overnight in oven at 65 C for 12- 18 hours to remove
complete water. Then the dry weight were taken. The data
was recorded and analyzed statistically for all the
observations.
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3.13 Somatic embryogenesis and multiple shoot
induction
The fresh calli were transferred to different modified MS
media fortified with different concentrations of auxin and
cytokinin either single or in combination, for the induction of
somatic embryos. The different physical and chemical factors
were studied responsible for somatic embryogenesis. The
cu ltures were kept in 16 hours light ( 1000- 4000 lux) and 8 0 0 0
hours dark at the temperature 23 C to 27 C + 2 C. The
embryo formations were observed after incubation of callus.
Note d down the days r eq uired for e mbryo formation as well
as the induction of multiple s hoots. Th e s ubc ulturing of the
multipl e shoots were performed on the same medium . The average number of s hoots were recorded and analyzed statica lly.
Wh e n the height of multipl e s hoots attained 5- 7 cm, t h e shoots were Isolated and transferred on rooting medium . The who le exp e rim e nt was repeated thri ce .
3.14 :Isolation of shoots for root: induction
The Individual shoots were Inoculated on rooting m edia. The
isolate d shoots were washed thoroughly with sterile glass
doubl e distilled wate r and transferred for rooting on full and
half strength MS medium su pplemented with different
concentrations of auxin and cytokinin. The cultures were
kept under 16 hours light (3000-3200 lux) and 8 hours dark.
In individual shoot, root initiation was observed after 10
days of shoot transfer. Each treatment was repeated thrice
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and the observations for average numbers of roots we re
record ed aft er 15 days .
3.15 Hardening of in vitro raised bamboo
T h e in v itro m ature p la ntl e t s w e r e about 7 c m t a ll , whic h
we r e r eady for t h e harde n i ng . Th e p la n t le t s w e r e re m o v e d
f ro m t h e m e d ium a n d r oots w e r e was h e d gen t l y w i t h steri le
dou b le g l a ss dist i ll e d w a l e r to r e move th e excess m e d ia
f r o m t h e r oots. I n v it ro g r o wn r oo t s y stem s a ppea r e d to b e
v e ry d e lica t e, th e r e f o r e ca r e w as t a k e n t o r e t a in a ll th e fibe r
roots Intac t to th e pl a n t l c t s. T h ese p la n t le t s w e r e
tra n s f e rre d into di f f e r e n t harde ning m e dia co n s lllute d s u c h
as 1 ) S o ll : Sa n d, 2 ) Soll : Sa nd : Cow du n g : 3 ) So ll : Sand :
C ow dung : V e rmic ulite.
Tli e tempe r ature, humidity a n d p ti o t o p e rt od we r e mainta in e d
duri n g h a r d e ning th e In vit ro grow n p l an llets . A f te r 1 0 - 15
days , t h e pl a n t tets w e r e tra n s f e rre d Into p o lyth e n e b a g s
with h a rd e n m e dia . T h e n t h e p la n t s we r e tra n s f e rre d t o
green h o u se . Thro ugh o ut th e h a rd e nin g procedure th e
s urv iva l rate o f t h e pla n t was measure d . O b se r vat i o n s o f
s u ccess f u ll y grown p l a n t le t s w e r e t a k e n a f ter 15 a nd 30
days a nd data w e r e s t at is ti c a ll y a n a l y zed.
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3.16 Statistical analysis Ten aliquots of each treatment w ere used for recording
observations. The data obtained was statistically analyzed as per procedure by Panse and Sukhatme (1958) using the formula as follows.
Whe r e
L Xi X =
n
x = mean
S um of ' I' observa tions
=J L (Xi - x )2
S. D. n - I
Whe r e X i observa tions ( I = 1 , 2 , 3 , ...... ... n )
s. D . = S tandard D e vi a tion .
S . D . 5 . E. = \r.;-
Whe re S. E. = Standard Error.
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