Optimization of In vitro Androgenic Protocol for Development of Haploids and Doubled Haploids in Indica Rice Hybrid, KRH 4

摘要

In vitro androgenesis is an attractive biotechnological technique that provides rapid transformation of heterozygous hybrids into homozygous lines and thus, significantly reducing the duration of breeding cycle. Due to difficulty in generating embryogenic cal1i, low plant regeneration and frequent albino plant regeneration, the prospective benefits of doubled haploid technology have not been exploited fully in indica rice hybrids. The present study, therefore aims at optimizing a previously standardized in vitro androgenic protocol to improve callus and shoot regeneration efficiency in an indica rice hybrid, KRH 4. Accordingly, the most potential anthers for androgenesis were obtained from boots with flag leaf to penultimate leaf distance of 10-12 cm. In particular, anthers at 4/6th position (ratio of length of stamen to the glume) within a spikelet were shown to have the highest callus induction efficiency. Further, callusing was advanced by 18 days and callus induction was improved by 13 per centwhen sucrose was replaced with maltose a carbon source to the previously standardized callus induction media for KRH 4 rice hybrid. Finally, among the optimized callus induction media, T media containing N_6 basal media supplemented with 2,4-D (2 mg/L),NAA (1 mg/L), kinetin (0.5 mg/L), maltose (3) and clerigel (0.2) and maintained at a pH of 5.8, had the highest callus induction efficiency and also produced more embryonic calli. Similarly, shoot regeneration efficiency of embryonic calli (2 mm) wasenhanced by inoculating embryonic calli in R, regeneration media containing N_6 basal media supplemented with kinetin (3 mg/L), NAA (0.1 mg/L), BAP (1 mg/L), sucrose (3) and clerigel (0.2) maintained at a pH of 5.8 and kept under absolute dark for 48 hrs followed by transferring them to BOD incubator maintaining 16-h light/8-h dark regime at 27 ± 2℃.