Agrobacterium tumefaciens-mediated transformation of Indian mulberry, Morus indica cv. K2: a time-phased screening strategy

Abstract

An efficient and reproducible protocol for the production of transgenic plants was developed for Morus indica cv. K2 by Agrobacterium tumefaciens-mediated transformation. The hypocotyls, cotyledon, leaf and leaf callus explants precultured for 5 days on regeneration medium were co-cultivated with a bacterial suspension at 10⁹ cells/ml for 3 days in the dark. Infectivity of A. tumefaciens strain LBA4404 was more than that of strains GV2260 and A281, and among the various plasmids tried, pBI121 and pBI101:Act1 transformed nearly 100% of the explants followed closely by p35SGUSINT. About 90-100% of the explants tested positive in the β -glucuronidase (GUS) histochemical assay performed after 3 days of co-cultivation. This high level of transient expression, however, decreased to 20-25% after 15 days. Gus activity was most stable in the callus explants, which emerged as the explant of choice for transformation. The transformed explants were selected on 50-75 mg/l kanamycin for 1 month, and 25-50% of the explants developed adventitious buds. On the basis of kanamycin-resistant shoots produced from the total number of explants inoculated, the transformation efficiency was 44%. After 1 month, 40% of these shoots displayed high gus activity as assessed by the GUS fluorometric assay. On a selection-free root induction medium, 80% of the shoots developed roots and 90% of the potted plantlets acclimatized to the growth room conditions. The 3-month-old regenerates showed gus and nptII(neomycin phosphotransferase II) gene activity as assayed by the GUS fluorometric assay and nptII enzyme assay, followed by PCR polymerase chain reaction (54.5%) analysis after 6-months. Transgene integration into the nuclear genome of 1-year-old regenerates was confirmed in 10 of the 18 transformants tested by Southern analysis. The transformation efficiency as defined by the number of transgenic plants produced from the total number of explants co-cultivated was 6%.