|Keywords (Extracted from title, table of contents and abstract of thesis)
rice (oryza sativa), basmati, (basmati 370, super basmati,basmati pak, basmati 385, jhona, ks282, rp15, rp18, rp20 and taipei 309, basmati 370, super basmati, irri6 and br11, transgenic rice plants
Rice, after cotton, is second most important cash crop of Pakistan. Pakistani Basmati rice holds a great commercial value and it is distinguished by its long grain and aroma. Insect attack is one of the vital cause of its production loss in every year. Inspite of enormous efforts made to develop insect resistant Basmati varieties no results are still visible to combat insect attack. Under the present investigations, efforts have been made to develop an alternative measure of pest resistance by genetic manipulation of Basmati rice. For this purpose twelve rice varieties (Basmati 370, Super Basmati, Basmati Pak, Basmati 385, Jhona, KS282, RP15, RP18, RP20 and Taipei 309) were screened out through an elaborate study in tissue culture response and regeneration potentiality. In the study of comparative tissue culture response the varieties Basmati 370, Super Basmati, IRRI6 and BR11 produced higher percentage of embryogenic calli proving themselves as suitable varieties for regeneration and genetic manipulation. In vitro tissue culture experiments five explants namely scutellum, immature embryo, leaf base, mesocotyl and root were selected as the target tissue explants. scutellum proved to be the most suitable explant. for callus formation in all rice varieties. The mesocotyl explant showed least response in callus induction and was not further used in regeneration experiments. Considering their better tissue culture performance and economic importance the varieties Basmati 370 and IRRI6 were selected for regeneration study.
In a comparative study Basmati 370 proved to be the best variety giving 91 % regeneration frequency from somatic embryogenesis. Four explants (scutellum, immature embryo, leaf base and root) were used as the target tissue explants for regeneration. Scutellum gave highest regeneration (91 %) followed by immature embryo (74%). The embryogenic calli were subjected to nine regeneration media supplemented with different phytohormones. The MS medium containing sorbitol 3% sucrose 3%, NAA 1 mg/L, BAP 2mg/L, casamino acids 0.2% and higher concentration (0.4%) of phytagel was found to be the best medium for regeneration in tested rice varieties. In meristem culture the highest regeneration frequency was observed in meristem isolated from four day old seedlings. Variable range of regeneration response was observed in different rice varieties. The varieties IRRI6 and Basmati 370 gave the highest percentage of regeneration Le. 55% and 50% respectively. Basmati 370 was selected as the target variety for transformation studies because of its best in vitro tissue culture response and economic importance in the country.
To accomplish an efficient transformation in Basmati 370 a number of methods namely, PEG mediated transformation of protoplasts, electroporation of intact tissue, pollen tube path way and agrobacterium mediated transformation were used. None of them was found suitable for stable transformation of this local rice variety. Attempts were taken to use biolistic gun for bombarding the target gene into the Basmati rice. As a new approach, genetic transformation with home made biolistic gun needed for optimization of the tool and efficient demonstration of gene transfer technology. Conditions of biolistic bombardment were optimized by using GUS reporter gene. Conditions for stable transformation were established by bombarding hygromycin (hph) resistant gene. Biolistic method was found suitable for genetic transformation of indica rice. Pesticidal gene CyIA(c) was selected as a target gene for transformation. The target gene coated on tungsten particles was bombarded into leaf base and meristem of mature embryo of Basmati 370. Transformation efficiency at the rate of 0.45 and 0.39% was obtained respectively from leaf base and mature embryo meristem by using biolistic bombardment. A number of sixty two putative transformants were produced by bombarding 10,000 mature embryo and 3000 meristem. A number of forty putative transformed plants were successfully transferred into soil and their toxic level was evaluated by insect feeding assay. Out of forty putative transformants nine plants were finally screened out for molecular analysis on the basis of 33% and above insect mortality.
The results of dot blot and southern blot analysis confirmed the presence and integration of CryIA(c) gene into the genome of three putative transformants. One of these plant produced viable seeds. Molecular analysis (PCR, dot blot and southern blot) of T1 progeny plants confirmed the presence, stable integration and inheritance of CryIA(c) gene in T1 generation. Expression of the integrated gene was confirmed by western blot analysis. Incorporation of pest resistance in Basmati 370 was finally confirmed by insect bioassay.