Improved protocol for efficacious in vitro androgenesis and development of doubled haploids in temperate japonica rice

Abstract

DH (Doubled haploid) is the immortal mapping population and an outcome of single meiotic cycle, contributed from male partner. An improved procedure was developed for high frequency androgenesis in japonica genotypes, K-332 and GS-88 and their F1s. A total of 207 fertile, green, di-haploid plants were generated from K-332 × GS-88 hybrids using the improved anther culture protocol. The investigation was carried out to evaluate callus induction potential and regeneration response for the genotypes and the derived F1s on N6 media and modified N6 media (N6M). Whereas, N6 failed to induce callusing, agarose solidified N6M media supplemented with 4% maltose, growth regulators; NAA (2 mg/l), 2, 4-D (0.5 mg/l), Kinetin (0.5 mg/l), and silver nitrate induced high calli percentage of 27.6% in F1s, 9.5% and 6.7% in GS-88 and K-332 respectively. Murashige and Skoog (MS) media supplemented with 3% sucrose, and the hormonal combination BAP (2 mg/l), Kinetin (1 mg/l) and NAA (1 mg/l) induced high green shoot regeneration rates (0-60.0%). The effect of cold pre-treatment at 4°C and the stage of anther collection and their interaction was studied. The effect of cold pre-treatment (CP) of collected boots at 4°C (for CP2: 2, CP4: 4, CP6: 6 and CP8: 8 days) at different stages of panicle emergence (BES4-6: 4-6, BES7-10: 7-10, BES11-13: 11-13, BES>13: more than 13 inches was worked out in relation to the effect on response of calli induction, albino regeneration, green plant regeneration and number of shoots/green calli. CP referred to the number of days for which the collected boots were incubated before they were inoculated. BES was the length (inches) between flag leaf and penultimate leaf at the time of boot collection. We concluded that CP6 and BES7-10 showed better response to callus proliferation and regeneration of plantlets across genotypes. The appropriate pre-treatment, stage of anther collection and favourable media composition resulted in high calli induction and green plant regeneration rates in recalcitrant japonica genotypes. The modified N6 media resulted into efficient callus induction and is expected to be useful for studies which aim at rapid generation of mapping populations for genetic studies.

Fig 1. Confirmation of hybridity of F₁ individuals with polymorphic SSR marker RM72.

M: 100 bp DNA ladder (Genetix Biotech Asia, New Delhi, India), C: Blank.

Fig 2. Calli induction and proliferation of F₁ derived pollen.

a: Callus initiation, b: Initial growth, c: Proliferation of white nodular calli from anthers inoculated on N6_M media.

Fig 3. Green plant regeneration from F₁ derived pollen on N6_M media.

a: Emergence of green spots in the pollen derived calli inoculated on regeneration media b. Development of shoots from green calli.

Fig 4. Green plant regeneration from the pollen derived calli on regeneration media.

a: Development of roots (combination B), b: Development of shoots (combination C), c: Development of roots and shoots (combination D); d: Sub-culture of individual plantlets.

Fig 5. Variation for grain type in doubled haploid progenies derived from K-332 × GS-88.