Somatic embryogenesis in rice (Oryza sativa L.)

Abstract

Rice is the staple food for more than half of the world’s population, but over time the yields have plateaued and there are higher incidences of pest and diseases. This problem can be tackled by combining biotechnological tools with crop improvement methods. Genetic modifications can be used to improve yield, tolerance to pest and disease and even incorporate climate resilience in the crop, which is the need of the hour for the rapidly growing population. For the application of genetic transformation techniques, standardised in vitro regeneration protocols are necessary and among the many techniques, somatic embryogenesis is a promising technique as it produces large number of plantlets. Somatic embryogenesis is a type of asexual reproduction whereby somatic or gametic cells are induced to form somatic embryos under favorable in vitro conditions by exploiting the cellular totipotency of plant cells to form entire plantlets. Thus, this study was conducted to standardise the protocol for somatic embryogenesis and regeneration in two rice varieties, Jyothi (PTB 39) and Nagina 22 (N22). Jyothi is a very popular, red and long bold grained rice variety cultivated predominantly in the Kole and Kuttanad regions of Kerala. N22 is a deep rooted aus type Indian rice variety, well known for its high tolerance to drought and heat. The mature seeds and leaves of these varieties were used as explants in this research programme. Experiment 1 involved studies to standardise the medium for callus induction and somatic embryogenesis. In the preliminary study, the effect of medium (MS and N6), carbon source (sucrose and maltose) and 2,4-D at different levels (0, 1, 2, and 3 µM) were observed, and the treatment combination MS + 2,4-D (3 µM) with maltose performed the best among the sixteen treatments in both varieties. The effect of medium with 2,4-D and BAP on callus induction from leaf explants was studied and none of the varieties responded to the treatments. When the effect of 2,4-D and kinetin on somatic embryogenesis was investigated, the somatic embryogenesis per cent was higher in the treatment combination MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) in Jyothi (80%) and the treatments MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) in N22 (85.71%). Moreover, both varieties exhibited some rhizogenesis from the callus, which reduced the capacity of the calli for somatic embryogenesis as well as regeneration. During this study, the seeds germinated regardless of treatment and the germination per cent varied from 75 to 97.22 per cent in Jyothi while it ranged from 58.33 to 100 per cent in N22. Regarding the callus induction frequency (CIF), 2,4-D (3 µM) in MS medium containing maltose exhibited the highest CIF in both the varieties (N22 with 100 per cent CIF and Jyothi with 69.44 per cent CIF). The callus was developed from the scutellar region of the seeds. Callus induction required six to seven days in both the varieties. It was observed that three week old calli started forming somatic embryos when sub cultured on MS medium supplemented with 2,4-D (0 and 0.4 µM) and Kinetin (0, 0.4, 1, 1.5 and 2 µM), and incubated in dark. Embryogeneic calli per cent was highest in treatment MS + 2,4-D (0.4 µM) + Kinetin (0.4 µM) in Jyothi (87.50%) and in treatments MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) in N22 (85.71%). Non-embryogenic calli formation was the lowest in the treatment MS +2,4-D (0.4 µM) + Kinetin (0.4 µM) for Jyothi (12.50%), while MS +2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) for N22 (14.29 %). For the development of somatic embryos into plantlets, the embryogenic calli were sub cultured on medium with NAA and BAP and incubated in 16 hours light and 8 hours dark conditions. Embryoid formation was observed in both varieties within two weeks, but not all the embryoids developed into plantlets due to degeneration. In Experiment 2 for regeneration studies, N22 formed two plantlets from somatic embryos in the treatment combinations MS + NAA (0.2 µM) + BAP (0.8 µM) and MS + NAA (1 µM) + BAP (3.5 µM) whereas, Jyothi required the addition of maltose (20 and 30 g/l) and sorbitol (25 and 30 g/l) in the medium for embryoid development. Seventeen embryoids formed in Jyothi, but only three developed into plantlets in the treatment MS + NAA (1 µM) + BAP (3.5 µM) with 30 g/l each of maltose and sorbitol. Jyothi plantlets needed an average of 15.67 days for shoot initiation and 29.67 days for root initiation. In N22, the average number of days for shoot initiation and root initiation were 8.50 and 18.50 respectively. All the plants obtained from this study were green and healthy, with the characteristics of normal rice plants formed from seeds. However, these plantlets required additional time for proper root development in basal MS medium or MS medium with IBA and NAA, prior to planting out into pots. Experiment 3 entailed hardening and pot culture. The plantlets of Jyothi and N22 were first transferred to distilled water for a few days to let the leaves to unfurl and then planted in sterilised potting mixture. The plants were then kept under 16 hour photoperiod for 10 days. These plants were further hardened in a polytunnel and later grown in a rain shelter using standard agronomic practices. N22 and Jyothi plants grew with healthy leaves and formed panicles within two months but N22 exhibited poor tillering.