Agrobacterium-mediated Transformation Of Rape (Brassica Napus) With MSI-99 Gene And Plant Regeneration

Rape is an important oil plant in world and China. However, diseases increasingly become a main limiting factor on rape production. Introducing resistant genes into rape varieties by transformation technology is a preferred strategy to resolve rape diseases. MSI-99, a broad-spectrum antibacterial peptide, have been successfully introduced into tobacco, banana and tomato, which displayed strong resistance to different kinds of diseases. Transferring MSI-99 gene into rape has not yet been reported. This study begins with establishing highly effective regeneration systems , then introducing MSI-99 gene into two rape varieties via Agrobacterium.The cotyledonary petioles and hypocotyls of two rape varieties , huashuang No.3 and 3905, were used as explants. The optimum regeneration systems for cotyledonary petioles and hypocotyls were obtained by the comparison of 8 different regeneration systems:â‘  The optimum system used for cotyledonary petioles: MS basic medium complemented with 3.0mg/L 6-BA+0.2mg/L NAA+3.4mg/L AgNO3 (huashuang No.3) ; MS basic medium complemented with 5.0mg/L 6-BA+0.2mg/L NAA+3.4 mg/L AgNO3 (3905) ;â‘¡The optimum system used for hypocotyls: MS basic medium complemented with 4.0mg/L 6-BA +2.0mg/L ZT+3.4 mg/L AgNO3+500 mg/ L PVP ;The binary vector pBIN-MSI99 carrying MSI-99 gene was transferred into Agrobacterium LBA4404 by electroporation. Then cotyledonary petioles and hypocotyls explants of huashuang No.3 and 3905 were infected by the Agrobacterium LBA4404/pBIN-MSI99. Adventitious shoots was induced on select and regeneration medium followed by plantlets regeneration. Nine resistance plants were obtained from Kan screen. PCR analysis showed that 7 of 9 resistance plants were positive. Southern bloting analysis confirmed that 3 plants of them showed positive signal. It was indicated that MSI-99 gene was integrated into rape genome.