Morphogenesis Of Spring Durum Wheat In Mature Embryo Cultures
Abstract
O. V. Bychkova, L. P. Khlebova, D. V. Ereschenko
Mature wheat embryo is a convenient type of explants because of its unlimited availability at any time of the year. But the regenerative capacity of the calli derived from mature embryos is low due to the peculiarities of their hormonal status. A high-performance protocol for culturing these explants is necessary to develop to use them in various areas of applied plant biotechnology. Induction and maintenance of a high rate for unorganized growth in plant cell cultures take place on a nutrient medium with high levels of an exogenous auxin, but the presence of a cytokinin is required to induce differentiation processes. We have carried out a study of the various morphogenetic processes in mature embryo cultures of three spring durum wheat genotypes, depending on the time of their cultivation on the callus induction medium. Mature embryos were cultured in the dark at 26 ± 1 °Con Murashige & Skoog (MS) medium containing MS basal salts and vitamins supplemented with 0.7% agar, 3% sucrose, as well as 2 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) (callus induction medium). For morphogenesis induction a part of calli was transferred every five days to a differentiating medium of the same composition of salts and vitamins supplemented with 0.5 mg L-1 2,4-D and 0.5 mg L-1 kinetin. Cell cultures were grown in the light at 22 – 24 °C with a 16-hour photoperiod. Six variants of time intervals for callus proliferation on the induction medium have been studied (5, 10, 15, 20, 25, 30 days). A variant of cell culturegrowing without transferring to the differentiating medium was examined too. Frequencies of callus induction, morphogenesis induction and regeneration capacity (relatively morphogenetic calli) were calculated. We found active callus induction was visible on the 5th – 7th day after placing explants on the MS inducing medium. The greatest level of callusogenesis (92.3%) was discovered under incubating cultures on the original medium for 30 days. After the short-term cultivation of explants on the initiating medium (for five days) new calli on the differentiating medium were not initiated. In this variant, proliferation of the before induced cell clusters was taking place. This resulted in a low frequency of callus formation (44.3%). Development of the primary callus on the inducing medium for 20 – 30 days helped to keep the competence in somatic tissues of mature embryos and generated the largest number of morphogenetic structures of different qualities. The way of morphogenesis depended on the time interval for cell culture growing on the initial medium. Rhizogenesis decreased by 25% after increasing the incubation period to 15 days. This was followed by active nodular structure formation in calli and plant regeneration. For Oasis variety and 12S2-24 line the most effective variant for the realization of regenerative capacity of morphogenetic calli was to incubate cultures on the induction medium for 15 – 20 days and then to transfer them to the differentiating medium. For Pamyati Yanchenko variety the best variant was to grow calli on the induction medium for 25 days. We have shown the significant effects of a genotype and cultivation conditions at different developmental stages of mature embryo cultures from durum wheat. The specificity of a variety began to manifest after 5 – 10 days staying on the induction medium.
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