Agrobacterium-mediated transformation of niger [ Guizotia abyssinica (L. f.) Cass.] using seedling explants.
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.
Murthy
H N
HN
Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, 361-763, Cheongju Chungbuk, Korea.
Jeong
J H
JH
Choi
Y E
YE
Paek
K Y
KY
D019659:Asteraceae / Q000235:genetics* / Q000382:microbiology; D015139:Blotting, Southern; D018014:Gene Transfer Techniques; D005966:Glucuronidase / Q000378:metabolism; D012038:Regeneration; D012231:Rhizobium / Q000235:genetics*; D036226:Seedlings / Q000235:genetics / Q000382:microbiology; D014170:Transformation, Genetic
D005966:Glucuronidase