Agrobacterium-mediated transformation of indica rice cultivars using binary and superbinary vectors

The Agrobacterium-mediated transformation system was extended to two indica cultivars: a\udwidely cultivated breeding line IR-64 and an elite basmati cultivar Karnal Local. Root tips and shoot tips\udof seedlings, and scutellar-calli derived from mature seeds showed high-efficiency Agrobacterium tumefaciens\udinfection and stable transformation. In addition to the superbinary vector pTOK233 in\udAgrobacterium strain LBA4404, almost equally high levels of transformation were achieved with a relatively\udmuch smaller (13.1 kb) binary vector (pCAMBIA1301) in a supervirulent host strain AGL1. In both\udcases, as well as in both cultivars, while 60–90% of the infected explants produced calli resistant to the\udselectable agent hygromycin, 59–75% of such calli tested positive for GUS. A high level (400 μM) of acetosyringone\udin the preinduction medium for Agrobacterium and a higher level (500 μM) in the cocultivation\udmedium was necessary for an enhancement in transformation frequency of the binary vector to levels\udcomparable to a superbinary. Hygromycin-resistant calli could be produced from all the explants used.\udTransformants could be regenerated for both cultivars using the superbinary and binary vector, but only\udfor calli of scutellar origin. In addition to the molecular confirmation of hpt and gus gene transfer and\udtranscription, absence of gene sequences outside the transferred DNA (T-DNA) region confirmed\udabsence of any long T-DNA transfer