Effect of zeatin on in vitro embryogenesis and plant regeneration from anther culture of hexaploid triticale (x Triticosecale Wittmack)

To achieve results sooner, cereal crop selection programs usually combine conventional methods, such as selection of parents and large-scale cross-breeding with haploid technology, a methodology which allows obtaining homozygous lines from the F1 hybrids. Methods of androgenesis (anther culture and isolated microspore culture techniques) have gained widespread use for selection of wheat and triticale. Currently, the main issue for the androgenesis in Triticale is the low efficiency of green plant regeneration. The present work, for the first time ever, utilizes cytokinin zeatin as an exogenic phytohormone in the induction medium, and determines its concentration optimal for improving embryo formation and green plant regeneration from the triticale anther culture. The aim of this research is to increase efficiency of the triticale anther culture, and study the effects of adding cytokinin zeatin to the nutrient medium on embryogenesis induction and regeneration. Two lines of spring triticale, YaTKh-327-11 and Zernokormovoye 5 (facultative), and two lines of winter triticale, T-968 and T-45, were used. Donor plants for the haploid technology were grown in the irrigated field of Kazakh Research Institute of Agriculture and Plant Growing LLP (Kazakhstan, Almaty Region). Cut spikes were subjected to low temperature (4 °C for 14 days), and then the anthers, after they were isolated, to high temperature (32 °C for 3 days). The spikes were sterilized with 0.1 % solution of mercuric chloride. Modified mW14 medium was used as the basic nutrient medium for embryogenesis induction. Five variants of nutrient medium were studied, with concentration of phytohormone zeatin gradually increasing in each subsequent variant (0.2 mg/l, 0.4 mg/l, 0.6 mg/l, 0.8 mg/l, 1.0 mg/l), and medium without zeatin served as control. The study conducted on 4 genotypes of triticale has shown that addition of zeatin to the nutrient mediums in concentrations of 0.2-0.8 mg/l increased the rate of androgenic structure formation by 42.3-65.2 %. Maximal effect on the androgenic structure formation was achieved at 0.4 mg/l concentration of zeatin, with 112 androgenic structures (AS) per 100 anthers on average compared to 67.8 AS per 100 anthers in control group. In the embryogenesis inducing nutrient mediums with 0.4-0.6 mg/l zeatin concentrations the rate of embryogenesis was 16.9-24.1 % higher compared to the control, with embryos having bipolar structure, and producing stem and roots during the regeneration, which indicates positive effect of zeatin on differentiation and organogenesis of the dividing microspore cells. All the variants in the experiment showed a significant increase in the rate of regeneration compared to the control with no zeatin added. In embryos transplanted from the medium containing 0.6 mg/l zeatin the rate of green plant regeneration was the highest reaching 6.3 pcs/100 anthers. It has been established that addition of zeatin and the effect of genotype were the statistically significant factors for androgenic structure formation and regeneration. Efficiency of spontaneous chromosome doubling in triticale amounted to 26.5 %, which has allowed producing 97 double haploid lines from the promising lines of triticale without colchicination.