Agrobacterium -mediated transformation of niger [ Guizotia abyssinica (L. f.) Cass.] using seedling explants

Abstract

A protocol was developed for Agrobacterium-mediated genetic transformation of niger [ Guizotia abyssinica (L.f.) Cass.] using hypocotyl and cotyledon explants. Hypocotyls and cotyledons obtained from 7-day-old seedlings were co-cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm that harbored genes for beta-glucuronidase (GUS), kanamycin, and hygromycin resistance. Following co-cultivation, the hypocotyl and cotyledon explants were cultivated on MS medium containing 1 mg/l 6-benzylaminopurine (BA) for 3 days in darkness. Subsequently, hypocotyl and cotyledon explants were transferred to selective MS medium containing 1 mg/l BA, 10 mg/l hygromycin, 10 mg/l kanamycin, and 500 mg/l cefotaxime. After 6 weeks, hypocotyls and cotyledons produced multiple adventitious shoot buds, and these explants were subcultured to MS medium containing 1 mg/l BA, 30 mg/l hygromycin, and 30 mg/l kanamycin. After a further 3 weeks, the explants (along with developing shoot buds) were subcultured to MS medium containing 1 mg/l BA, 50 mg/l kanamycin, and 50 mg/l hygromycin for further selection. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.1 mg/l alpha-naphthaleneacetic acid, 50 mg/l kanamycin, and 50 mg/l hygromycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southern blot hybridization confirmed the incorporation of the neomycin phosphotransferase II gene into the host genome.