Obtaining Transgenic-callus of Moringa oleifera PKM-1 with Insect-resistant Gene (sck+Cry1-Ac) via Agrobacterium-mediated Transformation

PurposeThe Lepidopteran pest, Noorda blitealis, has become increasingly harmful in the production of Moringa oleifera. In order to improve the insect resistance of M. oleifera, the present study was performed to introduce insect-resistant genes into M. oleifera by Agrobacterium tumefaciens-mediated transformation method, so as to obtain transformants.

MethodsThe marker gene nptⅡ was screened by using the modified cowpea trypsin inhibitor-derived gene signal-CpTI-KDEL (sck) and Cry1-Ac gene from Bacillus thuringiensis, which used by endoplasmic reticulum localization. The leaves and stems of M. oleifera PKM-1 were subject to genetic transformation mediated by EHA105. Resistant plants were obtained by kanamycin-resistant selection. Meanwhile, PCR and NPTⅡ ImmunoStrip detection were used to confirm the integration and expression of the nptⅡ gene.

ResultThe leaves and stems of M. oleifera could be directly transformed through mediation using A. tumefaciens without pre-culture, the optimal transformation condition was: the OD₆₀₀ value of the fermentation broth of genetically engineered bacteria was 0.2, with two minutes infection time and two days appropriate co-culture time. The addition of acetosyringone or not had no obvious effect on the resistant callus rate, and the use of 200 mg/L Timentin could effectively inhibit the growth of A. tumefaciens. PCR detection of kanamycin-resistant callus showed that the marker gene nptⅡ had been successfully integrated into the genome of M. oleifera PKM-1; the NPTⅡ ImmunoStrip test showed that NPTⅡ protein was expressed.

ConclusionsM. oleifera PKM-1 kanamycin-resistant callus is obtained in this study, which provides reference material for the molecular breeding of M. oleifera.