There are two main obstacles to chickpea improvement: 1) the long period of time between the breeders first cross and commercial release of an improved cultivar and 2) a law level of diversity in the cultivated chickpea germplasm. The aim of this study has been to develop tissue culture techniques to enable the in vitro induction and/ or growth and germination of isolated haploid and zygotic embryos of chickpea in order to assist chickpea breeders in overcoming these breeding constraints.
Protocols to enable the production of homozygous breeding material via doubled haploid techniques in chickpea would speed the development and commercial release of improved cultivars. In this study, intact anther and microspore culture techniques were assessed for developing doubled haploid populations. The culture of intact immature anthers yielded callus from all fifteen chickpea genotypes tested. The composition of the MS-based callus induction medium was identified as the most important factor influencing callus induction frequency. A sucrose concentration of 0.075 M was optimum for callus induction and the plant growth regulator 2,4-D was identified as a potent inducer of callusing from intact anthers of chickpea. Upon the withdrawal of the 2,4-D somatic embryogenesis, rhizogenesis and plantlet regeneration was achieved from the callus of a limited number of genotypes. Cauliogenesis was observed from the anther-derived callus of genotype Bumper cultured on callus induction medium containing 2,4-D.
Callusing was also achieved on an MS-based culture medium without the addition of plant growth regulators, however this callus did not produce somatic embryos. The addition of the ethylene inhibitors silver nitrate and silver thiosulphate and the amino acids L-proline and L-serine appeared to enhance the induction of callusing from anthers cultured on medium without plant growth regulators, however the exact role of these compounds remains unknown. Activated charcoal was completely inhibitory to callusing from intact anthers in this study. Although subordinate to the composition of the callus induction medium, the genotype of the donor plant also influenced the success of anther culture attempts. The kabuli genotype Bumper was identified as particularly responsive to callusing, embryogenesis and plantlet regeneration from intact anthers. Donor plant growth conditions, i.e phytotron or glasshouse, were not observed to have a significant effect on callusing response. Cold pretreatment of the buds for at least 48 hours prior to culture was found to improve the callusing response from anthers. Centrifuging the bud, the size of the bud when harvested and the orientation of the anther had no effect on callus production.
The culture of isolated microspores yielded embryos from seven chickpea genotypes. The isolated microspores underwent division to the globular embryo stage when cultured on a membrane supported by solid MS-based medium. The microspores were plated on this membrane in discrete droplets of liquid medium (modified MS medium + 4.5 pM 2,4-D) at a density of 1 x 106 microspores mL-'. Starving the cultured microspores of carbon and/ or applying a 4C pretreatment for a period of 96 or 192 hours enhanced embryogenesis. The effect of either stress on embryogenic response was clearly genotype dependent. Genotype Bumper was more responsive to the carbon starvation treatment whereas genotype Yuma had a requirement for a cold pretreatment period in order to undergo microspore division.
The culture of selfed in-ovule zygotic embryos harvested eight days after pollination from C. arietinum and C. pinnatifidum yielded complete plantlets. A MS-based medium combining the plant growth regulators thidiazuron (1 pM) and zeatin (1 pM) was identified as the most effective for the growth and germination of the embryos to plantlets across both species. C. pinnatifidum was far more responsive to in-ovule embryo culture that the cultivated species. Intercoupled plasma analysis of the composition of selected elements within the endosperm of five annual Cicer species identified clear differences in composition between the five species and the MS nutrient salts. At both 12 and 16 days after pollination, the Cicer endosperm across the five species contained much higher concentrations of the major elements phosphorus and potassium and the minor elements sodium, iron, copper and zinc than the MS medium. The osmotic pressure of the endosperm was shown to decrease as the embryo progressed in age from 8 to 16 days after pollination. The research reported in this thesis indicates that chickpea is amenable to both haploid and zygotic embryo culture. Further research should enable these techniques to be adapted to use in conventional breeding programs
