Abstract:
Rice is one of the most important and staple food for more than 50% of the world population.
Demand for rice is increasing all over the world. Despite the demand rice productivity is
affected by both the biotic and abiotic factors. Among the biotic factors, losses due to insect
pests are substantial. Rice leaffolder (Cnaphalocrosis medinalis) is one of the important pests
of rice in Asia which causes yield losses of 1 million tons of rice per year resulting in several
billion dollars per year.
An efficient and reproducible Agrobaclerium mediated transformation and regeneration
procedure for local rice has not been established so far. We here report high frequency of
Agrobaclerium tumefaciens mediated transformation of embryogenic calli of mature rice
seeds, and in vitro regeneration of rice for three varieties (Bg 350, Bg 94-1 and Bg 352).
Embryogenic calli were transformed with Agrobacterium tumefaciens strain LBA 4404
harboring binary plasmid pCAMBIA 1301 containing GUS and hpt II driven by CaMV 35 S
promoter and NOS terminator as marker genes. Cocultivation was for 2 days followed by
callus induction (without hygromycin) for 2 weeks. Afterwards selection of transformants was
carried out for 4 - 5 weeks with 30 mg/L hygromycin.
Exclusion of selection with hygromycin for 2 weeks (resting period) followed by selection for
several weeks had enhanced the active proliferation of transformed calli and regeneration
during selection. During 5th week of selection greening was observed and regenerating calli
were transferred to shoot regeneration medium followed by rooting and acclimatized under
contained environment in a green house. Transgene integration was confirmed through PCR
analysis and transformation'efficiencies of 28% for Bg 350, 17% for Bg 94-1 and 16% for Bg
352 were observed.
Developed and optimized transformation procedure for local rice was utilized for the
introduction of Cry genes of Bacillus thuringiensis into a local rice variety. Embryogenic calli
of sub species indica variety Bg 94 -1 were transformed with Agrobacterium tumefaciens
strain GV 3101 carrying binary plasmid pCAMBIA 1305.1 with Cry 1C and Cry 2A
transgene constructs. These genes were driven by CaMV 35S d promoter and NOS terminator
together with GUS Plus and hpt II as marker genes. Transformation, regeneration and
recovery of putatively transformed rice with Cry genes were carried out according to the
optimized procedure for local rice.
The integration of the respective transgenes in the rice genome was observed through PCR
analysis for Cry 1C and Cry 2A, sequencing of the genomic DNA of transformants and
histochemical GUS analysis. Transformation efficiencies of 32% and 6% were observed for
Cry 2A and Cry 1C. Insect Bioassays with rice leaffolder larvae in To transgenic plants
revealed high toxicity to rice leaffolder larvae where mortalities of 89% and 83% were
observed for Cry 2A and Cry 1C transgenic plants.
The gene transfer technology developed by this project after appropriate biosafety measures
could be adopted by Sri Lankan rice scientists in their efforts in enhancing insect resistance
breeding in local rice varieties. Adoption of these Bt rice varieties would lead to reduction of
the insecticide use in rice crop production.