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Development of an efficient high frequency microspore embryo induction and doubled haploid generation system for Indian mustard (Brassica juncea)

Deepak Prem1, Kadambari Gupta1 and Abha Agnihotri2

1Center for Bioresources and Biotechnology, TERI School of Advanced Studies, Darbari Seth Block, IHC complex, Lodhi Road,
New Delhi, 110003, India. Tel.: 91-11-2468 2100 or 2468 2111, Fax: 91-11-2468 2144 or 2468 2145
2
Bioresources and Biotechnology Division, TERI, Darbari Seth Block, IHC complex, Lodhi Road, New Delhi, 110003,
India.Tel: 91-11-2468 2100 or 2468 2111, Fax: 91-11-24682 144 or 2468 2145

Abstract

Indian mustard, Brassica juncea, is an important oilseed crop for the South East Asian region. An efficient doubled haploid generation system still remains a major constrain for crop improvement. Microspores isolated from flower buds containing majority of late uninucleate microspores from two B. juncea genotypes of Indian origin, namely ‘Pusa Bold’ and ‘Varuna’, were cultured in NLN-13 medium containing 10 M silver nitrate. Activated charcoal associated with agarose gel significantly increased microspore embryo induction with an average of 349.5 embryos produced per petri dish containing 2.5 ml of culture suspension at a density of 40,000 cells per ml. The 21-30 days old dicotyledon embryos were germinated on B5 medium containing 2% sucrose (w/v) and 0.1 mg/L Gibberellic acid. A cold treatment of 4C for 10 days in dark, followed by incubation at 25C, resulted in an average of 82.9% embryo germination. The proportion of spontaneous diploids ranged from 0 to 12%. Root dip colchicine treatment was performed and effective chromosome doubling of achieved at 0.34% colchicine for 2 hour duration with an average of more than 60% plants producing doubled sectors. The plants were hardened and grown to maturity with a survival rate of more than 80% under controlled environment growth conditions. The frequency of microspore embryo/ doubled haploid production from the present investigation is about ten times higher than earlier reports and offers a great potential for its use in genetic enhancement of Indian mustard.

Media summary

The poster presents a ten times higher efficient protocol for generation of doubled haploids than earlier reports, thus offering a great potential for genetic enhancement of Indian mustard.

Introduction

Indian mustard or brown condiment mustard is an important oilseed crop for several developing countries in South East Asia. The classical breeding approaches have been successful mainly for enhanced yields and the genetic enhancement of this crop could be greatly accelerated by the use of androgenic haploids through isolated microspore culture. The production of doubled haploids has two chief advantages viz. production of homozygous plants in one step and efficient selection for recessive traits, which highlight its strong potential application as a versatile genetic manipulation tool combinable with both classical breeding and relatively new biotechnologies. Although several studies have been conducted to exploit this approach for genetic enhancement of various Brassica species, production of a large number of haploid embryos in Indian mustard remains a major bottleneck (Prem et al. 2004). The poster presents development of an efficient protocol for generation of large number of microspore derived embryos and their subsequent development into doubled haploid plants.

Material and methods

Plant material

Two B. juncea genotypes of Indian origin namely ‘Pusa Bold’ and ‘Varuna’ were investigated for the purpose. The donor plants were grown in field conditions till bolting and then transferred to a controlled environment growth chamber. Flower buds of sizes 2.5 to 3.5 mm containing majority of late uninucleate microspores were used for isolation of microspores.

Microspore embryo induction and germination

Microspore isolation was carried out as per Coventy et al. (1988) with modifications. NLN medium containing 13% sucrose (w/v) and 10M silver nitrate was used for microspore culture. The effect of addition of activated charcoal with or without association with agarose gel (Gland et al. 1988) was also studied. The produced embryos (21-30 days old) were germinated on B5 medium containing 2% sucrose (w/v) and 0.1 mg/L Gibberellic acid. The effect of a cold pre-treatment prior to incubation at 25 0C was also studied to optimise frequency of embryo germination.

Ploidy determination and production of doubled haploid plants

Root tip cytology (Tsuschiya 1999) was done to ascertain the ploidy status of germinated plantlets. Colchicine treatment for production of doubled haploids was performed by the root dip treatment method wherein, the roots of 25- 30 days old 4-5 leaf stage plantlets were dipped in 0.34 % aqueous colchicine solution for 2 hrs. The colchicine treated plantlets were hardened in glass jars containing commercial grade agropeat and MS medium (Murashige & Skoog, 1962) containing half strength of major salts and devoid of sucrose. The 6- 7 leaf stage hardened plantlets were transferred to the field and were raised to pod setting and maturity.

Results

Microspore embryo induction

The addition of activated charcoal associated with agarose gel significantly increases microspore embryo induction with an average of 349.5 embryos produced per petridish as compared to control media devoid of activated charcoal (Figure 1).

Figure 1. Effect of activated charcoal on induction of microspore embryogenesis in B. juncea

Histogram represents overall mean no. of embryos, irrespective of genotype, produced per petridish containing 2.5 ml of microspore culture suspension at a cell density of 40,000 cells/ ml. The overall mean was worked out from three replicates in an experiment in which approximately 100 buds of each genotype (30-35 buds each replicate) containing microspores at late uninucleate stage were cultured in presence or absence of activated charcoal. Histograms represented by different letters differ significantly according to paired two tailed t- test assuming unequal variance at α= 0.05. Bars represent standard error.

Similar observations have been reported for B. oleracea microspore culture (Dias et al.1999) and for B. napus (Gland et al. 1988) by using activated charcoal as a potent embryogenesis synergist. Addition of activated charcoal to culture media without association with agarose gel, in the present study, was observed to be detrimental for embryo induction. Significant genotypic effect was observed for the microspore embryogenesis from two genotypes with the genotype ‘Varuna’ exhibiting greater microspore re-differentiation ability as compared to Pusa Bold (based on means comparison by paired two tailed t- test at α = 0.05). Such genotype dependent response has been reported for several Brassica species (Lichter 1989). However, the frequency of microspore embryo production reported here is 10 times greater than any published report on B. juncea microspore culture.

Microspore embryo regeneration

A cold treatment of 4C for 10 days in dark significantly increased the normal germination of cultured embryos with more than 80% embryos showing consistently normal germination irrespective of the genotype as compared to those germinated under a 25C control regime (Figure 2). The cold shock treatment presented in this study is similar to that proposed by Coventry et al for B. napus. However, the information on efficiency of microspore embryo regeneration in B. juncea is scanty and in a solitary report Lionneton et al. (2001) have reported a much lower proportion range (0 to 29.2%) for microspore embryo regeneration without the use of a cold treatment.

Figure 2. Effect of cold treatment on microspore embryo germination in B. juncea

Histogram represents overall proportion of microspore embryos that showed normal germination irrespective of donor plant genotype. The proportion was calculated from observations recorded in three replicates from germination of a total of 250- 350 embryos on each individual temperature treatment.. Histograms represented by different letters differ significantly according to paired two tailed t- test assuming unequal variance at α= 0.05. Bars represent standard error

Figure 3. Production of doubled haploids in B. juncea: spontaneous diploids / colchiploids

The proportion of spontaneous diploids was calculated by dividing the number of plants showing haploid chromosome number from the total number of plants observed. A total of 110 microspore embryo derived plants obtained from the two genotypes were used for root tip cytology studies to ascertain the overall proportion depicted above. The overall proportion of survival after colchicine treatment was calculated by dividing the number of healthy green plants by the total number of plants treated. For this purpose, observations were recorded three weeks after the root dip treatment for approximately 200 plantlets irrespective of donor genotype. The chromosome doubling was recorded for approximately 100 plants in terms of development of doubled fertile sectors 8 to 10 weeks after colchicine treatment. The average doubling was calculated by dividing the number of plants showing doubled fertile sectors by the total number of colchicine treated plants. Bars represent standard deviation.

Ploidy status and colchicine treatment

Based on root tip cytology studies, most of the microspore embryo derived plants were observed to have haploid chromosome number and the proportion of spontaneous diploids was observed to range from 0 to 12% for both the genotypes (Figure 3). Similar results for low frequency of spontaneous diploidy have been reported by Lionneton et al. (2001) and Hiramatsu et al. (1995). Approximately, half of the plantlets subjected to colchicine treatment survived (Figure 3). However, the subsequent growth of these plantlets was slow as compared to untreated control plants. The colchicine treated plants were hardened and transplanted to the plastic plots containing sterilized soil and agropeat (2:1). The observations for flower morphology and pollen viability were recorded to ascertain the fertility status of the plants. It was observed that as a result of the colchicine treatment, the plants produced were chimeras that had both fertile and sterile sectors. The plants were segregated as fertile or sterile depending on the presence or absence of fertile sectors respectively and based on this average effective chromosome doubling was observed to range from 57 to 60% plants producing doubled sectors (Figure 3). The plants with fertile sectors were grown to maturity with a survival rate of more than 80% in a controlled environment growth chamber.

Conclusion

The high and consistent microspore embryo regeneration presented here represents a significant increase in the embryo induction and their regeneration into plantlets as compared to any report published as yet for Indian mustard doubled haploid production. Activated charcoal showed a synergist effect substantially increasing frequency of microspore embryogenesis. Apart from induction and germination of microspore embryos, other vital steps namely ploidy determination and colchicine treatment for production of doubled haploid plants have also been standardized with increased efficiency. The protocol presented here is being used at our laboratory for generation of fatty acid profile variability by utilizing haploid induced mutagenesis.

Acknowledgement

The authors gratefully acknowledge the grant of financial support from Indian Council of Agricultural Research, Government of India for this work.

References

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