Objective Conditions for simultaneous transformation of multiple genes in Indica rice, Minghui 86, were studied to facilitate the development of high yield and resistant breeds.
Method Two multi-gene vectors designated as P5 and P8 that harbored the high-yield RRM2, drought-tolerant HS1, herbicide-resistant EPSPS, and insect-resistant Bt as well as the apoptosis-inhibiting iap and gene that promotes cell regeneration p35 were transformed to the receptor Minghui 86 using agrobacterium-based transformation methodology. The receptor, callus culture, agrobacterium concentration, infection time, co-culture, and screening concentrations of G418 and glyphosate were evaluated for the selection.
Result Under same culture conditions, the recovery rate of Minghui 86 immature embryos was significantly higher with a better callus quality than that of the mature embryos. The optimal transformation conditions were determinated to be a bacterial concentration of OD600=0.4-0.6, an infection time of 15-20 m, co-culture for 2-3 d with the addition of sterile filter paper in medium, and the screening concentration of G418 at 150 mg·L-1 and of glyphosate at 800 mg·L-1. The PCR detection confirmed that GUS in the polygenic vector P5 was successfully transferred into Indica cv. Minghui 86.
Conclusion The optimized culture conditions afforded significantly improved callus and resistance induction rates in Minghui 86.
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