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

  • Genetic Transformation and Hybridization
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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 β-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 α-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.

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Fig. 1A–E.
Fig. 2A, B.
Fig. 3.

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Abbreviations

BA :

6-Benzylaminopurine

CaMV :

Cauliflower mosaic virus

GUS :

β-Glucuronidase

HPT :

Hygromycin phosphotransferase

NAA :

α-Naphthaleneacetic acid

NOS :

Nopaline synthase

NPTII :

Neomycin phosphotransferase II

PCR :

Polymerase chain reaction

X-Gluc :

5-Bromo-4-chloro-3-indolyl-β-d-glucoronide

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Correspondence to K. Y. Paek.

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Communicated by G.C. Phillips

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Murthy, H.N., Jeong, J.H., Choi, Y.E. et al. Agrobacterium-mediated transformation of niger [Guizotia abyssinica (L. f.) Cass.] using seedling explants. Plant Cell Rep 21, 1183–1187 (2003). https://doi.org/10.1007/s00299-003-0573-1

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  • DOI: https://doi.org/10.1007/s00299-003-0573-1

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