Analysis of embryogenic callus induction and regeneration of indica rice variety of Malaysia

Rice is the main food-crop for more than half of the global population and its demand is increasing due to population growth. Different abiotic and biotic stresses are among the major reasons that lower the yield. Development of new rice varieties through in vitro somatic embryogenesis not only contributes to enhance the yield but also improves the quality of rice. However, exact timing of maintenance of embryogenic competent callus was yet to be established for indica rice, which is considered as a main barrier in genetic modification. Somatic embryogenesis receptor kinase (SERK1) gene is extensively used as an embryogenic marker in many plant species, which is expressed specifically in embryogenic callus. To obtain high callus induction, effect of plant growth regulators (i.e. 2,4-D and NAA), carbon sources (i.e. sucrose, maltose and sorbitol), basal media (i.e. MS, N6 and LS), and pre-heat treatments (i.e. 35°C, 40°C, 45°C and 50°C) with different imbibition periods (3 days, 5 days and 7 days) were investigated for four Malaysian indica rice varieties (i.e. MR220, MR220-CL2, MR232 and Bario). SERK1 gene was quantified by real time PCR in differential stages of callus (14 days, 21 days, 28 days, 35 days and 42 days), different PGR (2,4-D, NAA, NAA+ 2,4-D), and pre-heat treatment. Plant regeneration was also optimised by using different concentrations of plant growth regulators. After regeneration, agronomic studies was carried out between control plant and treatment plant for all varieties. In the present study, highest percentage of callus induction was obtained for MR220 (96%), MR220-CL2 (100%) MR232 (100%) and Bario (95.7%) on MS media with 3 mg/L 2, 4-D and 3% maltose after 21 days of culture of pre-heat treated seed at 45°C for 3 days. The characteristics of embryogenic callus were found to be embryogenic from SEM and histology. Amplification of SERK1 cDNA was referred as detection of the gene of aged 21-days was successfully amplified in all four varieties. The phylogenetic tree analysis showed that SERK1 gene of all varieties were similar to the SERK1 of Oryza sativa Japonica. The Real Time PCR analysis revealed that SERK1 transcript was significantly higher at 21 days old callus on MS media with 2,4-D at 45°C pre- heat treated callus for all four varieties. Further, regeneration was tested for 21 days old callus, where the regeneration frequency were found to be 72%, 89%, 71% and 50% in MR220, MR220-CL2, MR232 and Bario respectively in optimised regeneration media (2mg/L BAP+ 2mg/L Kinetin+0.5mg/L NAA). Regenerated plants grew easily in the glasshouse with 90 –95% survival rate. Agronomic studies did not show any morphological variation but grain weight of in vitro raised plant was significantly higher than control plant in all tested varieties. These findings establish a suitable protocol for in vitro regeneration system to be used in genetic modification studies in indica rice in future