Transformation Studies of Rice Oryza Sativa

Abstract

Under the objective (a) of the proposal, twelve varieties of Oryza sativa were screened for the evaluation of regeneration ability. Those varieties included RP18, BR11, Taipei 309, Basmati Pak, super Basmati, Jhona, IRR16, ks282, Bas 370 and Bas 385. All these varieties were subjected to callus induction. Leaf bases, mesocotyl, scutellum and root explants were cultured on various media formulation. Variety Bas 370 and Super Bas gave best response in all the explants used for callus induction while IRR16, production lowest response in callus formation in all the tissue explants studied.. Out of twelve varieties, two, Bas370 Pak, Super Basmati, IRR16, BR11 and RP15 produced plants without callus formation. Under the objective (b) of the proposal, experiments were carried out of established regeneration conditions of protoplasts of Oryza sativa Bas 370 and IRR16. Calli were used to isolate protoplasts enzymatically with a mixture containing cellulose 10g/1, pectolyase 1.0 g/1 and MES 5Mm in CPW13M salts. The protoplasts were purified by centrifugation at a speed of 80xg for 15 minutes. Isolated protoplasts were either embedded in KPR medium containing sea plaque agarose or cultured in liquid medium in the feeder layer plates. Cultures were kept in the dark at 25+20C and were fed with fresh medium at 12- 15 days interval using reduced osmoticum. The dividing protoplasts were observed after three days. Condition of transformation of rice protoplasts and explants were optimized by transient expression of foreign genes in these tissues. In the local varieties of rice, isolated protoplasts fom leaf bases of aseptically grown seedling were used. Chemically induced DNA uptake procedure was used to transfer GUS gene in the isolated protoplasts. A GUS substrate MUG (4-methyl-umbelliferyl -D- glucuronide) was used and GUS activity was determined by examining the microtiter plates under UV. Fluorescence was observed in the extract of protoplasts after 48 hours. When leaf bases of rice seedling were electroporated with DNA to transformed as well as non-transformed leaf base of rice seedlings. Condition were also established for transient as stable expression of genes into leaf bases and mature embryos of rice using home-made particle acceleration gun. Beta-glucoronidase gene was introduce into leaf bases for transient expression. Leaf bases isolated from 5 days old etiolated seedlings of rice were kept on petri plates containing MS medium solidified with 1% agar and bombarded. About 70% of leaf bases showed transient expression. A selectable marker gene, hph which confer hygromycin resistance in transgenic tissues was used for stable transformation. Mature embryos excised aseptically and were kept on MS medium and bombarded with DNA- coated tungsten particles. After 5 days these embryos were transferred to selection medium containing hygromycin at concentration of 50ug/ml. The green fast growing plants were selected for southern blotting to confirm the presence of introduced marker gene. Southern blots gave positive indication of presence of presence of marker gene in rice genome. Under the optimized condition of stable transformation of marker gene, the co-transformation of Bt gene, Cry IA(C) has been achieve. Mature embryos were bombarded with tungsten coated particles. DNA from plasmid PROB5 and ubi-Cry IA(C) was mixed in 1:3 ratio and were used to coat tungsten particles. Leaf bases (2mm) were excised from the resultant plants and cultured on medium containing hygromycin (30ug/ml). Transformed plants were identified by their ability to grow on selection medium while the non-transformed plants became necrotic and subsequently died. Putative transformation were multiplied in vitro and then transferred in 50:50mixture of peat moss and clay. Under the objective(c) the regenerated plants of IRR16 were transferred to soil through an intermediate stage of semi-controlled condition in the greenhouse. These plants bore viable seeds. The transgenic plants were grown at 28oC day temperature and 25oC night temperature eith 14 hours photoperiod of 2,500 lux. The photoperiod was gradually decreased to 10 hours day and 14 hours night to get fertile spikelets. Viable seeds were obtained and germinated into R1 plants.