Somatic embryogenesis and Plantlet Regeneration under Salinity Stress Conditions in Mature Caryopsis Culture of Indian Red Rice (Oryza sativa L.) and Assessment of Genetic Fidelity in Regenerants

The main objective of this study was to establish the stable and efficient protocol for somatic embryogenesis and plantlet regeneration under NaCl-salt stress conditions in traditional Indian red rice (cv. Poongar) using mature caryopsis as explant and also to assess the genetic fidelity in regenerated plantlets. High frequency (98.88±1.33%) of callogenesis was recorded in MS (Murashige and Skoog, 1962) medium fortified with 10µM of 2,4-D (2,4-Dichlorophenoxyacetic acid) while maximum percentage (95.0±3.9%) of somatic embryogenesis and also maximum number (47.3±3.9) of somatic embryo per callus was recorded in presence of 5µM of 2,4-D alone. Furthermore, maximum frequency (85.33±2.02%) of germination of somatic embryos into plantlets was found in MS medium supplemented with 2.0mg/L of IAA (Indole-3-acetic acid), 3.0mg/L of BAP (6-Benzylaminopurine) and 1.0mg/L of Kn (Kinetin). During the salinity stress treatments, various concentrations (10mM, 25mM, 50mM and 100mM) of NaCl were used in this study in order to induce somatic embryogenesis and plantlets regeneration. Significantly, 100mM of NaCl shows the strong inhibitions for callus induction (26.67±0.63%) and somatic embryogenesis (31.3±1.8%),, moreover, weight of the callus was also found to get decreased (94±0.5mg) than control (156±4.3mg) treatment. Interestingly, the highest concentration 150mM of NaCl was proved to be completely inhibitory and <1% of callogenesis was recorded. Moreover, due to the inhibitory response of NaCl, number of somatic embryos per callus was also decreased to 12.7±0.9 whereas the low concentration (10mM) of NaCl proves to be the little inhibitory and thus maximum frequency (91.1±2.33% and 67.5±2.33%) for both callogenesis and embryogenesis respectively. To produce salinity tolerant plantlet, the somatic embryos were transferred to regeneration medium supplemented with respective concentrations of NaCl. The high concentration (100mM) of NaCl causes very low percentage (20.7±0.6%) of regeneration of somatic embryo whereas the very low concentration of NaCl (10mM) supports the high frequency (74.52±1.2%) of plantlet regeneration. These salinity tolerant regenerated plantlets were later aacclimatized into autoclaved soil mixed with vermiculite at the ratio of 3:1. Furthermore, RAPD / ISSR-PCR profiles were analyzed to confirm genetic homogeneity between in vitro regenerated control plantlet and salinity tolerant plantlet. Among the four primers tested, all the primers could show clear polymorphic DNA bands. Hence, the stress tolerant plant and control plantlets are probably not homogenic in nature.