Producing transgenic Basmati rice with the potato protease inhibitor II (Pinll gene by Agrobacterium- mediated transformation

摘要

The potato protease inhibitor II (Pinll) gene was introduced through Agrobacterium-mediated transformation into three rice cultivars, Basmati 1, Taraori Basmati, and TNG67 (japonica). The Pinll gene cassette with the Pinll wound-inducible promoter wascloned into pCAMBIA 1200 and 1201 vectors. These piasmids were then mobilized mio Agrobacterium strain LBA4404 carrying pSBl plasmid by triparental mating. The resultant strains were used to transform mature-seed scutella calli (3-9 wk old) and/or immature embryo-derived tissues. Rice tissues transformed using Pinll genes cloned in pCAMBIA1201 showed a GUS expression at higher frequencies both immediately after co-cultivation and 4-5 wk after selection. TNG67 and Pusa Basmati 1 tissues were more embryogenic than those of Taraori Basmati and eventually produced hygromycin-resistant calli and plants at higher frequencies. In Taraori Basmati, transformation efficiency was significantly higher when immature embryo-derived tissues were used than with the mature-seed scutella-derived calli. About 90 Hyg~R plants of Pusa Basmati 1 and Taraori Basmati have been obtained. Histochemical analysis showed the expression of thegus gene in 74% of hygromycin-selected transformed plants obtained using pCAMBIA 1201 +Pinli. Polymerase chain reaction analysis revealed the presence of the Pinll gene sequence in 42 of the 51 plants. Of the 27 plants analyzed, 21 were Southern-positive.