Indian Journal of Biotechnology

Vol 7, October 2008, pp 531-535

Response of some Iranian wheat genotypes to anther culture system

Behnam Naserian Khiabani*, Cirus Vedadi, Esfandiar Rahmani and Mir Ahmad Mosavi Shalmani

School of Agricultural, Medical and Industrial Research

Nuclear Science and Technology Research Institute, Mahmood Abad Road, Rajaee Shahr, Karaj, Iran

Received 1 May 2006; revised 27 June 2008; accepted 20 July 2008

The response of five Iranian wheat cultivars and four segregating F3 wheat lines was investigated in anther culture

system for haploid plantlet regeneration. Anthers were plated onto P4 induction medium and cold and gamma irradiation

were also applied to study their effect on initiation of calli and regeneration of plantlets. Significant differences were found

between genotypes. Most of the segregating lines showed high response to anther culture than cultivars. The high calli and

plantlets production in segregating lines may be attributed to heterosis effect. Gamma irradiation in wheat anther culture

does not promote anther culture response. The results showed significant difference between genotypes and gamma

irradiation. The line of F32005 with 4 Gy dose (without cold pretreatment) produced the highest amount of calli (52%) per

100 anthers inoculated, whereas Red-Bofgi genotype (with cold treatment and 4 Gy dose) produced the lowest percentage of

calli (0.25%). The results indicated that both the androgenic response and regeneration ability were greatly genotype

dependent. It could be concluded that genotypic response to anther culture is vital.

Keywords: Anther culture, cold treatment, low dose gamma ray, wheat

Introduction

An efficient doubled-haploid production technology,

inducing homozygosity, can greatly reduce the time

and cost of cultivar

development1-3

. In breeding

programmes of wheat cultivars, in vitro anther culture

technique has been utilized to obtain haploid plants

from hybrid plants in the F1 generation. This technique

speeds up the process of development of new cultivars

by several years, in addition to simplifying and making

the selection process more efficient4,5

. Previously, low

efficiency of doubled-haploid production has limited

exploitation of this method for crop improvement.

Androgenesis, a process by which pseudo embryos

(embryoids)

capable to germinate into plants are

produced from microspores (pollen embryogenesis), is

of significant interest for developmental and genetic

research as well as for plant breeding and

biotechnology. This process produces genetically true-

breed, doubled-haploid (DH) plants. By producing DH

progeny, more number of possible gene combinations

for inherited traits is manageable6.

DH plant production of wheat have been reported

by microspore and/or anther culture (androgenesis),

ovule culture (gynogenesis), Hordeum bulbosum L. or

maize (Zea mays L.) pollination methods (alien

species

chromosome elimination), and an alien

cytoplasm system. Microspore and anther culture

methods have the potential to produce more than a

thousand haploid plants per cultured anther

7. All other

methods are limited to one haploid plant per floret and

they are effective only for a narrow range of

responsive genotypes, while the other genotypes

remain recalcitrant. Thus, more effective methods are

needed for inducing androgenesis in large populations

of microspores for a wide range of genotypes.

However, the basic requirement for DH wheat is to be

independent of donor genotype to a considerable

extent to make it feasible for conventional breeding

system. With regard to haploid wheat production, calli

production, embryogenesis and plantlet regeneration

from embroygenic callus are important steps. The

production of DH lines of cereals from anther culture

is limited by relatively low callus/embryoid induction

frequency, genotype dependent response and poor

regeneration; a large number of regenerated plants are

albino. To coordinate different factors involved in

each step a generalized medium has been presented

by some researchers8.

Induction of androgenesis in anther culture may be

affected by various factors including genotype

dependence, which cause low induction efficiency.

_____________________

*Author for correspondence:

E-mail: bnkhiabani@yahoo.com

INDIAN J BIOTECHNOL, OCTOBER 2008

532

Most advances toward improving anther-microspore

culture methods have been focused primarily on the

concept of using "stress" treatments to induce

androgenesis from the preprogrammed gametophytic

to the sporophytic pathway 9-16

. In most experiment,

pretreatment of cold was used for this purpose. Cold

pretreatment inhibited disorderly mitotic division,

thereby increased the frequency of microspores with

the two equivalent cells; such cells have high ability

to embryogenesis17,18

. Gabriela et al18

have shown in

rice that cold pretreatment was essential for the

induction of callus from anthers of the parental lines

and the F1 hybrids. These effects were genotype

dependent. Auxins were essential for the induction of

callus, and the type and concentration of auxins as

well as the type of carbon source also affected the

induction18

. Further, Ling et al19

demonstrated that

low dose of gamma ray (upto 7 Gy) can improve

anther culture response.

In view of the above, present study was conducted

to determine the influence of several genotype, cold

pretreatment and low dose gamma ray on the

androgenic plant regeneration from wheat anther

culture.

Materials and Methods The experiment was carried out with 9 Iranian wheat

genotypes containing 5 cultivars and 4 F3 lines (Table

1) in the Tissue Culture Laboratory of Nuclear

Agriculture Division. Seeds of each genotype were

planted in four 0.5 m2 plots as replications. Spike of

primary and secondary tillers were harvested from

donor plants shortly before heading, when most of

microspores were at mid to late uninucleate stage. One

or two anthers from each spike were examined

microscopically to verify microspore developmental

stage before culture (Fig. 1). Cold pretreatment of

wheat spikes kept in 12 cm Petridishes were given at

4ºC for 1 wk in a dark room before the anther culture.

Radiation pretreatment of wheat spikes was executed at

2 and 4 Gy. Cold and radiation pretreatments were

conducted separately as well as in combination. Spikes

were surface sterilized with 5% (v/v) NaOCl and then

washed 3 times in distilled water. About 150-200

anthers were isolated in each 8 cm Petridishes and then

were sealed with parafilm. Cultures were placed in

dark at 30ºC and 4 Petridishes were used for each

treatment. Modified P4 induction medium containing

200 mg L-1 glutamine, 5 mg L

-1 Kn, 2 mg L

-1 2,4-D

with 10% (w/v) ficol was used for callus induction.

Modified regeneration medium 190-2, containing

0.5 mg L-1

Kn and 0.5 mg L-1

NAA, was used for calli

regeneration into plantlets (Table 2). Calli reaching

Fig. 1—Microspore at mid (A) and late (B) uninucleate stage

Table 1—Plant materials used and their source

Cultivars/Lines Source

Tajan Spring wheat from north area of Iran

Atila Spring wheat from north area of Iran

Mahoti Spring wheat from central area of Iran

White Bofgi Spring wheat from central area of Iran

Red Bofgi Spring wheat from central area of Iran

F3 2208 Progeny of hybrid: 1-66-22/Tajan

F3 2005 Progeny of hybrid: MV17 /Mahooti

F3 2044 Progeny of hybrid: Rsh/Atilla

F3 2o97 Progeny of hybrid: Tbs/Falat/Attila

Table 2—Composition of CHB (induction) and 190-2

(regeneration) media

Medium composition P4

(mg L-1)

190-2

(mg L-1)

KNO3 1150 1000

Ca(NO3)2,4H2O 100 100

(NH4)2SO4 100 200

KH2PO4 200 300

CaCl2.2H2O - -

MgSO4.7H2O 125 200

ZnSO4.7H2O - 3

MnSO4.4H2O - 8

H3BO3 - 3

KI - 0.5

KCl 35 -

CuSO4.H2O - 0.025

CoCl2 - -

Thiamin HCl - 1

Pyridoxine HCl - 0.5

Nicotinic acid - 0.5

Calcium pantothenate - -

Ascorbic acid - -

Glysine - 2

Mayo inositol - 100

Glutamine - -

Sucrose 90000 30000

Ficol 100000 -

FeSO4.7H2O 32 27.8

Na2EDTA 32 37.3

Agar - 7000

pH 5.6-5.8 5.6-5.8

KHIABANI et al: HAPLOID PLANTLET REGENERATION IN WHEAT GENOTYPES

533

1-2 mm in size was transferred to regeneration

medium to produce platelets. Analysis of variance and

mean comparison were done by MSTATC, and

SPSS11.0 softwares.

Results

Inoculated anthers produced calli within 30 to 35 d

(Fig. 2). After transferring calli to regeneration

medium, green and albino plantlets were generated

within 10 to 14 d (Fig. 3). Then plantlets were

transferred to test tubes containing MS medium (Fig.

4). Number of calli per 100 anthers and number of

plantelets per 100 anthers were analyzed in each

treatment. Table 3 shows summary of analysis

variance.

Analysis of variance showed highly significant

difference between genotypes, cold treatments, and

interaction effects (p=0.0001) on the capacity of

androgensis. However, gamma irradiation did not

shown any significant effect.

Duncan's multiple range test showed that the

genotypes have significant difference according to

their androgenic ability (Table 4). F3 2044 and F3 2005

produced the highest percentage of calli and plantlet

as compared to other genotypes. However, Atila and

White Bofgi produced the lowest percentage of calli

and plantlets. Thus, genotypic effect on induction of

calli and plantlet regeneration was significant.

Cold treatment was found ineffective on

androgenic ability of genotypes. The anthers

incubated at 4ºC for 1 wk produced calli and plantlet

less than the control. The genotypes not treated with

cold produced 20.8% calli and 16.4% plantlets,

whereas 12% and 8.8% were the corresponding

figures for the genotypes under cold treatment.

Interaction effects showed significant differences

between genotypes. All the genotypes tested in the

present study showed low response to the cold

pretreatment.

Significant interaction effects between cold

pretreatment and gamma irradiation are presented in

Table 5. The data showed that combination of cold

pretreatment and gamma irradiation have negative

effect on androgenesis ability of genotypes as per the

Duncan's multiple range tests at 5% level. With 4 Gy

dose (without cold pretreatment), the line of F3 2005

produced the highest amount of calli (52%) per 100

anthers, whereas Red-Bofgi genotype (with cold

treatment and 4 Gy dose) produced the lowest

percentage of calli (0.25%).

Discussion The present results indicate that genotype is an

important factor in haploid regeneration and confirms

pervious finding20-22

. These strong genotypic

differences are yet to be overcome for breeding

purposes in most of the cereals. In wheat, anther

culture ability can be divided into three independently

inherited components: callus induction, plant

regeneration and green plant formation, and it usually

governed by more than one gene23,24

. In the present

study, genotype seems to be one of the major

Fig. 2—Anthers in induction medium produced calli within 30 to 35 d

Fig. 3—Calli regeneration after transfering onto 190-2 medium

Fig. 4—Green haploid plantlets transferred into MS medium in

test tubes

INDIAN J BIOTECHNOL, OCTOBER 2008

534

determinants of callus and embryoid production, since

wide range of variations in callus initiation (4.83-

31.54%) and haploid plantlet formation (2-24.13%)

were observed among the genotypes studied. Both

genetic and environmental factors affect the callus

formation and plant regeneration in cereal anther

culture and significant interactions are often observed

among these factors. In this respect, cold pretreatment

and low dose of gamma irradiation were used and

results showed that gamma irradiation was ineffective

in wheat anther culture system. The gamma irradiation

did not show stimulatory effect on embryoid

production and also on frequency of plant regeneration.

The present findings were in contrast to the report of

Ling et al 19

, which may be because stimulation effect

of low dose gamma ray on wheat anther culture

response is genotype dependent.

The use of cold pretreatment on wheat anthers (prior

to culture) has been shown by others25,26

to raise the

induction frequency of pollen callus and to slightly

improve the production of green plantlets. The present

results showed that cold pretreatment appeared to be

ineffective (when compared to direct culture). The cold

pretreatments had no influence in the propensity of a

particular genotype to produce predominantly calli.

They also did not affect on the rate of plant

regeneration. Therefore, these traits may be influenced

largely by the genotype of anther donor. The results

further indicate that cold treatment is not an essential

key point of androgenic induction in genotypes studies.

This corresponds to the reports of Karimzadeh et al25

and Marsolais et al26

, and in contrast to the findings of

Benito-Moreno et al27

.

The present results indicate that both the androgenic

response and regeneration ability were greatly

genotype dependent and their regulation was

genetically independent. This is because androgenic

ability and plant regeneration differ in the same

genotype, and their response to different kind of

treatment is not the same. These are in agreement with

previously reported studies25,28-32

.

In the present study, most of the segregating lines

showed high response to anther culture than cultivars,

it seems that heterosis effect is the reason of this high

calli and plantlets production in segregating lines. On

the basis of similar response of F3 progenies and their

parents to haploid plant production, it can be concluded

that androgenic traits are highly heritable. This

corresponds to the findings of others20,33-37

.

References 1 Hu H & Yang H Y, Haploids of higher plants in vitro (China

Academic Publishers, Beijing/Springer-Verlag, Berlin) 1986.

2 Hu H, In vitro induced haploids in wheat, in In vitro haploid

production in higher plants, edited by M S Jain, S K Sopory

and R E Veilleux (Kluwer Academic Publishers, Dordrecht,

the Netherlands) 1997.

3 Liu W, Zheng M Y & Konzak C F, Improving green plant

production via isolated microspore culture in bread wheat

(Triticum aestivum L.), Plant Cell Rep, 20 (2002) 821-824.

4 Camergo C E O, Ramos L C S, Ferreira Filho A W P, Felico J

C, Pettnelli Jr A et al, Diaplóides de trigo: Produção de grãos,

Table 3—Analysis of variance for callus induction and plantlet

regeneration

Calli/100 anthers Plantlets/100 anthers Source of

variation df

MS F value MS F value

Genotype 8 2149.198 49.6163٭٭14.5854 1240.208 ٭٭

Cold pretreatment 1 4204.671 97.0689٭٭36.6098 3112.963 ٭٭

Gamma

irradiation

2 103.292 2.3846ns 258.931 3.0451 ns

Genotype×Cold 8 202.807 4.6820٭٭3.0036 255.400 ٭٭

Genotype×Gamma 16 408.771 9.4369٭٭4.2770 363.675 ٭٭

Cold×Gamma 2 2586.338 59.7081٭٭25.0867 2133.144 ٭٭

Genotype×Gamm

a×Cold

ns 1.5686 133.378 ٭٭ 2.5323 109.692 16

Error 162 43.316 - 85.031 - P<0.001; ns not significant ٭٭

Table 4—Comparison of genotype for induction of calli and

plantlets regeneration by Duncan's multiple range tests.

Anthers isolated from

genotype

Mean of calli/

100 anthers

Mean of

plantlets/100 anthers

F3 2044 31.54A 24.13A

F3 2005 29.17A 21.13AB

F3 2o97 21.00B 10.21C

Red Bofgi 18.58B 18.58B

F3 2208 12.96C 10.42C

Mahoti 12.79C 11.33C

Tajan 10.08CD 10.00C

Atila 6.417DE 5.500CD

White Bofgi 4.833E 2.458D

Means followed by the same latter are not significantly difference

(p=0.05)

Table 5—Comparison of interaction between cold pretreatment

and gamma irradiation for induction of calli and regeneration of

plantlets (per 100 anthers)

Pretreatment Treatment

Gamma

irradiation

(Gy)

Mean of

calli/100

anthers

Mean of

plantlet/100

anthers

Control 0 19.11AB 17.31A

Control 2 21.47B 18.50A

Control 4 26.44A 18.47A

Cold 0 14.44C 12.33B

Cold 2 8.583D 4.944C

Cold 4 8.194D 4.278C

Means followed by the same latter are not significantly difference

(p=0.05)

KHIABANI et al: HAPLOID PLANTLET REGENERATION IN WHEAT GENOTYPES

535

características agronômicas e tolerância à toxicidade de

alumínio, Bragantia, 58 (1999) 235-246.

5 Ramos L C S, Camrgo C E O, Ferreira Filho A W P, Yokoo E

Y, Castro J L et al, Melhoramento do trigo: Avaliação de

linhagens diaplóides obtidas via cultura de anteras, Sci Agricola,

57 (2000) 177-183.

6 Konzak C F, Sunderman D W, Polle E A & McCuiston W L,

Spring wheat plant design for conservation tillage crop

management systems, in STEEP—Conservation concepts and

accomplishment, edited by L Elliott (Washington State

University Publications, Pullman, WA) 1987, 247-273.

7 Devaux P, Comparison of anther culture and the Hordeum

bulbosum method for the production of doubled haploids in

winter barley, Plant Breed, 100 (1988) 181-187.

8 Hu T C, Ziaudin A, Simion E, Kasha K G, Isolated microspore

culture of wheat (Tritium aestivum L.) in a defined medium I.

Effects of pretreatment, isolation methods and hormones, In

Vitro Cell Dev Biol, 31 (1995) 79-83.

9 Touraev A, Indrianto A, Wratschko I, Vicente O & Heberle-

Bors E, Efficient microspore embryogenesis in wheat (Triticum

aestivum L.) induced by starvation at high temperature, Sex

Plant Reprod, 9 (1996) 209-15.

10 Touraev A, Vicente O & Heberle-Bors E, Initiation of

microspore embryogenesis by stress, Trends Plant Sci, 2 (1997)

297-302.

11 Hu T & Kasha K, A cytological study of pretreatments used to

improve isolated microspore cultures of wheat (Triticum

aestivum L.) cv. Chris, Genome, 42 (1999) 432-441.

12 Zhou H & Konzak C F, Influence of genetic and environmental

factors on anther culture response of wheat, J Appl Genet, 38

(1997) 393-406.

13 Simonson R L, Baenziger P S & Gustafson V D, Wheat anther

culture as affected by various cultural changes and supplements,

J Appl Genet, 38 (1997) 381-392.

14 Reynolds T L, Pollen embryogenesis, Plant Mol Biol, 33 (1997)

1-10.

15 Zheng M Y & Konzak C F, Effect of 2-4-dichlorophenoxyacetic

acid on callus induction and plant regeneration in anther culture

of wheat (Triticum aestivum L.), Plant Cell Rep, 19 (1999) 69-

73.

16 Ohnoutková L, Novotny J, Mullerova E, Vagera J & Kucera L,

Is a cold pretreatment really needed for induction of in vitro

androgenesis in barley and wheat?, in Proc COST Action 824 on

‘Biotechnological approaches for utilisation of gametic cells’,

held on 1-5 July 2000 (Bled, Slovenia) 2000, 33-37.

17 Henry H & De-Buyser J, Wheat anther culture, in Biotechnology

in agriculture and forestry, vol B, edited by Y P S Bajaj

(Springer-Verleg, Berlin) 1990, 285-350.

18 Gabriela T T, Uriel M A, Guadalupe S M, DeJesús S A , Blanca

M B et al, The effects of cold-pretreatment, auxins and carbon

source on anther culture of rice, Plant Cell Tissue Organ Cult,

71 (2002) 41-46.

19 Ling D X, Luckett D J & Darvey N L, Low dose gamma

irradiation promotes wheat anther culture reponse, Aust J Bot, 39

(1991) 467-474.

20 Moieni A, Lokos-Toth K & Sarafi A, Evidence for genetic

control and media effect on haploid regeneration in anther

culture of hexaploid wheat (Triticum aestivum L.), Plant Breed,

116 (1997) 502-504.

21 Moieni A & Sarafi A, Haploid regeneration by anther culture

and its relationship to agronomic traits in the parent and progeny

pure lines of a composite cross of hexaploid wheat, Cereal Res

Commun, 26 (1998) 127-135.

22 Tersi M, Xynias I N, Gouli-Vavdinoudi E & Roupakias D G,

Anther culture response of F1 durum×bread wheat hybrids after

colchicine application, Plant Breed, 125 (2006). 457-460.

23 Lazar M D, Baenziger P S & Schaeffer G W, Combining

abilities and heritability of callus formation and plantlet

regeneration in wheat (Triticum aestivum L.) anther cultures,

Theor Appl Genet, 68 (1984) 131-134.

24 Szakacs E, Kovacs G, Pauk J & Barnabas B, Substitution

analysis of callus induction and plant regeneration from anther

culture in wheat (Triticum aestivum L.), Plant Cell Rep, 7 (1988)

127-129.

25 Karimzadeh G, Kovaes G & Barnabas B, Effect of cold

treatment and different culture medium on the androgenic

capacity two winter wheat genotypes, Cereal Res Commun, 23

(1995) 223-227.

26 Marsolais A, Seguin-Swart A G & Kasha K J, The influence of

anther cold pretreatments and donor plant genotypes on in vitro

androgenesis in wheat (Triticum aestivum L.), Plant Cell Tissue

Organ Cult, 3 (1984) 69-79.

27 Benito-Moreno R M, Macke F, Hauser M T, Alwen A &

Heberle-Bors E, Sporophytes and male gametophytes from in

vitro cultured tobacco pollen, in Sexual reproduction in higher

plants, edited by M Cresti, P Gori & E Pacini (Springer-Verlag

Berlin) 1988, 137-142.

28 Ouyang J W, Zhou S M & Jia S E, The response of anther

culture temperature in Triticum aestivum, Theor Appl Genet, 66

(1983) 101-109.

29 Deaton W R, Metz S G, Armstrong T A & Mascia P N, Genetic

analysis of anther culture response of three spring wheat crosses,

Theor Appl Genet, 74 (1987) 334-338.

30 De Buyser J, Hachemi-Rachedi S, Lemmee M L, Sejourne S,

Marwtte J I et al, Aneuploid analysis of anther culture response

in wheat, Plant Breed,. 109 (1992) 339-342.

31 Henry Y & De-Buyser J, Effect of the IB/IR translocation on

anther culture ability in wheat (Triticum aestivum L.), Plant Cell

Rep, 4 (1985) 307-310.

32 Szarejko I, Anther culture for doubled haploid production in

barley (Hordeum vulgare L.), in Doubled haploid production in

crop plant, edited by M Maluszynski, K J Kasha, B P Forster &

I Szarejko (Kluwer Academic Publishers, Dordrecht, the

Netherland) 2003, 35-42.

33 Moieni A & Sarrafi A, Genetic analysis for haploid-regeneration

response of hexaploid wheat anther cultures, Plant Breed, 114

(1995) 247-249.

34 Ghaemi M & Sarrafi A, The effect of "D" genome from

synthetic wheat lines in anther culture response, Plant Breed,

112 (1994) 76-79.

35 Ekiz H & Konzak C F, Preliminary diallel analysis of anther

culture response in wheat (Triticum aestivum L.), Plant Breed,

113 (1994) 47-52.

36 Agache S, Bachelier J, DeBuyser J, Henry Y & Snape J W,

Genetic analysis of anther culture response in wheat using

aneuploid, chromosome substitution and translocation lines,

Theor Appl Genet, 77 (1989) 7-11.

37 Zamani I, Gouli-Vavdinoudi E, Kovacs G, Xynias I, Roupakias

D et al, Effect of parental genotypes and colchicine treatment on

the androgenic response of wheat F1 hybrids, Plant Breed, 122

(2003) 314-317.