Pyramiding of drought adaptive traits and development of doubled haploids in the traits pyramided rice (Oryza sativa L.)

Introgression of traits through routine conventional breeding takes very long time, while doubled haploid technology reduces the breeding time significantly. With an objective of improving drought adaptation of rice with sustained productivity under water limited conditions, epicuticular wax trait was pyramided by crossing wax trait donor line (AC 35310) with TIL 14, a rice line introgressed already with root and WUE traits. With this cross, approximately 400 F-1 seeds were produced and true F-1's was identified through SSR markers. Later, haploid and doubled haploid generation protocol was optimized for the true F-1's, which include standardization of developmental stage of anther explants, pretreatment of anthers, culture conditions (temperature, humidity and light), media for callus induction, shoot regeneration and rooting. Accordingly, early to mid-uninucleate pollen bearing anthers subjected for cold treatment of 8 degrees C for 10 days cultured in N-6 basal media supplemented with growth hormones (2,4-D, auxin, kinetin) was found to be effective in inducing regenerative callus. Shoot regeneration was achieved in N-6 basal media supplemented with kinetin (2 mg/L), NAA (0.25 mg/L), BAP (0.25 mg/L), while, profuse rooting was observed in half strength MS media without any growth hormones. Well-rooted plants were hardened and grown them in pots separately and finally, seeds were harvested from them. Using polymorphic SSR markers, diploids generated from anther wall were identified and discarded while, spontaneously formed doubled haploids were retained and labelled them as DH-0 generation seeds. The ploidy of these spontaneously formed doubled haploids were confirmed again through flow cytometry analysis.