Liquid Selection System for Basmati Bar Transformants
Published on RICE BIOTRCHNOLOGY QUARTERLY
VOLUME 28, OCTOBER 1996
National Research Centre on Plant Biotechnology
Indian Agricultural Research Institute
Using pAHC25, a plasmid containing gus and bar gene coding regions driven by the maize ubiquitin promoter, first exon and first intron (Ubi I), 5 day precultured mature embryos of basmati rice (Oryza saliva L.) cultivar Kamal Local were subjected to microprojectile bombardment. 1510 embryos were processed through a solid media selection system with 4mg/I phosphinothricin (PPT) supplemented modified MS medium ( 35mMKNO3 and 5mM (NH4)2S04 instead of 19mM KNO3 and 21 mM NH4NO3 of MS basal medium) (Khanna and Raina in press). 110 I embryos were processed through a liquid selection system using modified MS medium supplemented with 30g/I sorbitol, lg/I caesin acid hydrolysate, 2g/l proline, 500mg/I glutamine, 12.5 mM phosphate buffer (pH 5.8), 0.5 mg/1 kinetin and 0.5 mg/I NAA.
After reducing the total nitrogen content of the media to half of its original level ( 17.5 mM KNO, and 2.5 mM (NH,)2S0,) at the end of three weeks, somatic embryogenesis could be induced in the liquid selection medium, in otherwise necrotic looking calli. 20-50 somatic embryoids could be seen budding out from each primary calli. Most of them died within a week due to selection pressure.
The resistant ones grew in size, detached themselves from the parent cal Ius, and produced adventitious embryos. These microcalli were regenerated on MS regeneration medium and 14 putative transformants were recovered using this approach. 12 of them rooted in PPT supplemented 1/2 MS medium. PCR and Southern analysis confirmed IO of these to be transformants for bar gene. Cotransformation frequency of gus was more than 50%. The solid selection procedure failed to give any transformants and while most of the calli died during primary selection, a few proliferating structures recovered from the primary calli died during secondary selection.
The liquid selection system was found to be more efficient as it allowed living cells to pop ou't from amidst the dead cells and because the cells were continously bathed in PPT and the medium was changed every week, the number of escapes was low.!