Microprojectile based particle bombardment in development of transgenic indica rice involving AmSOD gene to impart tolerance to salinity

Abstract

Genetic transformation of Pusa Basmati 1, a high yielding scented indica rice variety has been successfully achieved using AmSOD gene housed in a binary vector pSFSOD1 under the transcription control of ubiquitin promoter and nos terminator through microprojectile bombardment for increased tolerance towards salinity stress. Initially the protocol was optimized with binary vector pCAMBIA1305.2 harbouring GUS Plus™ reporter gene involving embryogenic calli developed from mature embryos of three rice varieties viz., Pusa Basmati 1, Taraori Basmati and one new plant type-super rice variety- IR70485–15–3-2 using PDS He 2000. The reporter gene showed significant GUS expression, which indicates maximum transformation efficiency at transient level under optimized parameters such as helium pressure-1100 psi, internal vacuum pressure-28″, flight distance-9 cm, microcarrier-1 μM diameter, particle volume-15 μl and microcarrier launch assembly at 6 cm for efficient bombardment of the embryogenic calli. The bombarded calli were transferred to MS medium supplemented with mannitol (0.2 M) and sorbitol (0.2 M) for 4 h, found to be most appropriate osmoticum in bombardment shock recovery and their proliferation. This best optimized protocol was used to dispense AmSOD gene for development of transgenic lines of the most in vitro culture responsive variety Pusa Basmati 1 to impart tolerance to salinity. Putative transgenic plants were developed from the bombarded calli, which were acclimatized, hardened and grown to maturity and analyzed by using molecular, physiological and biochemical assays to determine the introgression of the transgene into the variety. Further, inheritance study showed typical Mendelian ratio for monogenic dominant transgene in the segregating T2 generation. Among progenies tested, PBT 3–2, PBT 6–4 and PBT 8–9 were found to be appreciably tolerant to salinity stress with high yield at different growth stages, which prospects amply for more production and profitability to attain farm prosperity by large-scale cultivation of transgenic Pusa Basmati 1 on saline soils.