The present study was undertaken at the Centre for Bioresources and Biotechnology, TERI School of advanced studies during 2001 -2005 for the genetic improvement of Indian mustard (B. juncea). The broad objectives of the study were; developing an efficient protocol for DH production using anther/ microspore culture, using the DH protocol in conjugation with chemical mutagenesis for generation of mutant B. juncea germplasm and evaluation of the generated variability for agro-morphological and biochemical traits. Three Indian varieties of B. juncea namely Pusa Bold, Varuna and BIO-902 were used in the present study to achieve these objectives.
The factors that affect haploid totipotency in anther and microspore cultures were studied. For anther culture these included optimum explant (bud or anther), cold pre-treatment or high temperature incubation, varying sucrose concentrations, various combinations of growth regulators in culture medium, culture of anthers from various bud sizes and different donor plant growth conditions. The isolated microspore culture experiments focused on the influence of donor plant growth environment, microspore development stage, culture media composition and incubation conditions on microspore embryogenesis. The conditions to obtain efficient regeneration of the microspore embryos were also standardized.
Pollen embryos were produced consistently from anthers obtained from 2.1 to 3.5 mm buds of late sown donor plants on NLN medium containing 13% sucrose with or without NAA (0.2 mg/L) and BA (0.02 mg/L). High temperature incubation of cultured anthers at 32 ± 1 °C for three days was essential for triggering pollen totipotency. The culture of bud/ anthers on media containing different sucrose and growth regulator concentrations was antagonistic for pollen embryogenesis. The plants regenerated from the pollen embryos were predominantly spontaneous diploids.
The correlation analysis between the bud size and microspore development stage revealed that the bud size is an accurate marker for donor plants grown under controlled conditions, however, the same did not hold true for the field grown plants. The buds containing late uninucleate microspores collected from plants grown under normal field conditions up to bolting stage and then transferred to controlled environment were observed to be most responsive to microspore embryogenesis. Genotype dependence for microspore totipotency was observed and a significant effect of genotype wise bud size selection was also established.
NLN medium containing 13% sucrose was found to be most suitable for induction of embryogenesis. The fortification of this medium with AC (activated charcoal added directly to culture medium), PVP, colchicine or growth regulators (BA and NAA) was observed to be antagonistic for microspore embryogenesis, while addition of silver nitrate (10.0 μM) had a significant synergistic effect. A post culture high temperature incubation of microspores at 32.5 ± 1 0C for 10 to 15 d was found most suitable for triggering microspore embryogenesis. The addition of AC in association with agarose gel to the culture medium (NLN medium containing 13% (w/v) sucrose and 10.0 μM silver nitrate), along with efficient selection of buds having maximum number of late uninucleate microspores, resulted in high frequency microspore embryogenesis with more than 250 embryos produced per Petri dish on an average for the three genotypes.
The germination response for embryos produced in presence or absence of AC was similar. Air drying of microspore derived embryos was found essential for their germination. The incubation of the microspore derived embryos at 4 ± 1 oC for 10 d in dark resulted in normal germination of more than 80% of the cultured embryos. The majority of plants produced from these embryos were predominantly haploids. Among different diploidization treatments, the treatment of three to four leaf growth stage plants with 0.34 % colchicines for 2 to 3 hours resulted in more than 70% survival and 60% doubling frequency in treated plantlets.
The response of B. juncea zygotic embryos to chemical mutagenesis was studied with an aim to derive a tangible relationship between mutagen concentration, exposure duration and induced lethality in terms of germination of treated zygotic embryos. Subsequently, the results were extrapolated to generate mutant B. juncea germplasm. Three strategies were tested for exploiting haploid mutagenesis namely, mutagenesis of isolated microspores, microspore derived embryos and donor plants used for microspore culture.
While EtBr induced severe lethality at minimum concentration and exposure, variation for zygotic embryo germination was observed after treatment with ENU and EMS for varying concentrations and exposure durations. The exposure duration had a non-linear relationship with percent survival, while a linear decrease in the same resulted with increasing concentrations of mutagens. A linearisable non-linear multiple regression model was developed to predict survival of zygotic embryos after chemical mutagenesis.
The exposure of isolated microspores to EMS and ENU caused a sharp decline in embryogenic response with increasing mutagen concentration. Distinct genotypic effect influenced lethality was observed in the mutagenized microspores. Out of the various concentrations tested, maximum induction of microspore embryos was observed from 5.0 μM for either of the two mutagens. EMS was a more effective mutagen for isolated microspore mutagenesis as compared to ENU since irrespective of the concentration used, EMS mutated microspores produced embryos in about three times higher frequency as compared to those treated with ENU.
The lethality of induced mutations at microspore level was further expressed at all growth stages as the microspore embryos developed to DH plants. The genotype and mutagen concentration were important factors that influenced lethality of induced mutations for haploid embryo mutagenesis. The lethality of the mutagen treatment was mostly pronounced at embryo germination level. This was also operative at survival of plantlets after colchicine treatment. EMS mutants showed a higher colchicine treatment survival as compared to the ENU mutants. The mutants from high mutagen concentration, (10.0 and 20.0 μM), exhibited greater post colchicine exposure lethality as compared to those treated at lower concentration (1.0 to 5.0 μM).
Microspore embryos were produced from EMS mutant donor plants while the ENU mutant donor plants did not produce any embryos. Embryogenesis response of the microspores cultured from mutant donor plants was reduced as compared to normal donor plants. The lethality of induced mutation at this level was mainly functional at the plantlet development level after normal embryo germination. A comparison of the three haploid mutagenesis strategies revealed that chemical mutagenesis at the haploid embryo level resulted in a greater number of mutants as compared to mutagenesis at isolated microspores or donor plant levels.
The germplasm developed from anther culture and the haploid mutagenesis strategies was evaluated for agro-morphological and biochemical traits. Further the stability of the desirable mutations was tested in the next filial generation. Calibrations were developed for non-destructive estimation of oil, moisture and meal protein contents using NIRS for biochemical evaluation. The present results demonstrate the high precision of NIRS for estimation of seed biochemical components for the three Brassica species, B. juncea, B.rapa and B. napus, irrespective of harvest season and seed coat colour.
The anther culture derived DH lines exhibited variations in several agromorphological traits, while their biochemical profile was similar. Evaluation of within line variability in the anther culture derived generation established that the DH progenies were homozygous for a majority of agro morphological traits.
Both EMS and ENU resulted in generation of considerable variability for agro morphological traits. Amongst the morphological traits of the DH mutants, appresed pod phenotype and pronounced variation in leaf size was recorded. The pooled leaf length comparison of mutants and non- mutant DH plants suggested that both large and small leaf phenotype appeared in highest frequency for the ENU mutants followed by EMS, and in comparatively low frequencies among the non- mutant DH populations. Significant variations were observed for most of the important agronomic traits and the total number of pods per plant was observed to be most variable among the mutant progenies.
An overall comparison established that low concentrations of EMS (1.0 to 2.5 μM) and relatively higher concentrations of ENU (2.5 to 5.0 μM) promoted higher frequency of positive mutants with promising yield potential. Among the biochemical parameters studied, the most pronounced effect of chemical mutagenesis was manifested in variation for total glucosinolate content and FA profile. Desirable phenotypes with reduced glucosinolate content from high (more than 100 μM/ g meal) to moderate (less than 60 μM/ g meal) were recovered from 2.5 to 5.0 μM EMS mutagenesis. Positive mutations for FA profile in the form of non- detectable erucic acid, and moderately high oleic acid (more than 45%), were obtained from 2.5 μM EMS and 5.0 μM ENU mutagenesis.
Among the three genotypes used for haploid mutagenesis Pusa Bold produced majority of the positive mutants for agronomic and biochemical characteristics followed by Varuna and BIO-902. Majority of the positive mutants for agronomic and biochemical profile were produced from haploid embryo and isolated microspore level mutagenesis. Most of the desirable mutations were fixed in the next generation, however, some unexpected variations in the agronomic and biochemical traits were observed which need further investigations.
The salient outcomes of the present study are summarized below:
1. Establishment of an efficient DH generation protocol using microspore embryogenesis in B. juncea that resulted in about four time higher frequency of microspore embryo induction and regeneration than previously published reports thus making the DH technology worth applied research in Indian mustard.
2. The present study for the first time established an empirical relationship for biological damage in B. juncea caused by varying concentration and exposure duration of two important alkylating agents EMS and ENU. The equations developed will be of value in academic and applied research.
3. This is the first report of haploid mutagenesis in B. juncea. The simultaneous use of the three haploid mutagenesis approaches and the detailed comparison of lethality induced by mutagen exposure in single cells or in multicellular haploid tissue, as presented in this study, provide valuable insight for its use in genetic enhancement in B. juncea.
4. The non-destructive NIRS calibrations developed from a heterogeneous sample set of three Brassica species for simultaneous determination of oil, moisture and protein content are of immense value in efficient germplasm screening for Brassica quality improvement programs.
5. The DH approach conjugated with chemical mutagenesis resulted in a wide spectrum of useful variations. These include agronomically and biochemically desired genotypes for dwarf plant height, higher number of pods per plant, long pod architecture, increased yield in community, reduced erucic acid in the seed oil and reduced glucosinolate content in seed meal. These mutants form novel germplasm sources for desired traits and can be utilized for genetic enhancement and value addition in oilseed brassicas.