Agrobacterium mediated biotransformation of Oryza sativa L. involving Aquaporin gene for drought tolerance
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Date
2016
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Abstract
An investigation on “Agrobacterium mediated biotransformation of Oryza sativa L. involving Aquaporin gene for drought tolerance” was carried out in Department of Agriculture Biotechnology, Orissa University of Agriculture and Technology, Bhubaneswar. Rice, Oryza sativa L. (2n = 24) belonging to the family Gramineae and subfamily Oryzoides is the second most important cereal crop and staple food for more than half of the world’s population. Rice is water loving crop requires large quantity of water and for producing one kg rice, about 3000 - 5000 liters of water depending on the different rice cultivation methods. Drought is the first environmental stress responsible for decrease in rice production. The present investigation was carried out for standardization of protocol for in vitro regeneration and genetic transformation of two rice varieties viz., ‘Pratikhya’ ‘and Lalat’. The Agrobacterium strain EHA105 harboring vector pCAMBIA 2301 containing gene of construct HvPIP2,4 driven by promoter 35SCaMV used for the genetic transformation of two rice cultivars ‘Pratikhya’ and ‘Lalat’. MS media fortified with 2, 4-D: 3.0mg/l, Kinetin: 0.5mg/l showed highest callus induction up to 92.67 % in case of var. ‘Pratikhya’ within 4 weeks of culture. The var. ‘Lalat’ showed highest callus regeneration in 2,4-D: 2.0mg/l, Kinetin: 0.5mg/l with 92.67%. The maximum number of shoot buds were obtained in MS media supplemented with BAP: 2.0mg/l, Kinetin: 0.5mg/l, NAA: 0.2mg/l for var. Pratikhya (94.62%) within days 6 weeks of culture. For root induction MS media enriched with NAA (1 – 2 mg/l) + Kinetin (0.2 -05 mg/l)+ BAP (0.2mg/l) reported highest efficiency. Influence of Agrobacterium co-cultivation at different period was investigated and three days period transformation proved best for transformation. The optimum concentration of cefotaxime for washing was standardized as 500 mg/l for controlling the excess growth of Agrobacterium after co-cultivation. In case of cultivar ‘Pratikhya’ 100 mg/l of kanamycin proved lethal dose whereas in cultivar ‘Lalat’ 80 mg/l proved efficient concentration for selection of putative transformats. The putative transformed plants were checked for presence of transgene by using polymerase chain reaction with gene specific primer. Transformation efficiency was recorded 6.52% based on Kanamycin selection and 2.17% PCR based method in var. Pratikhya.