Autor: M.R. Boase, M.J. Bendall, T.A. Peters, Gayle B. Marshall
Rok vydání: 2002
Předmět:
Zdroj: Plant Cell, Tissue and Organ Culture. 70:27-39
ISSN: 0167-6857
Popis: A protocol to produce transgenic cyclamen from etiolated hypocotyls of Cyclamen persicum has been developed. Precultured etiolated hypocotyls segments of cv. Sierra Rose were infected with two disarmed Agrobacterium tumefaciens strains, LBA4404 or EHA105 containing the binary vectors pMOG410 or pART 27-10. Both binary vectors contain in their T-DNA region a nos-nptII gene and a 35S-gusA-intron. Transformed cells were selected with 50 mg l−1 kanamycin and shoots were regenerated via adventitious organogenesis in the presence of 1 mg l−1 6-benzylaminopurine (BAP) and 1 mg l−1 indole-3-acetic acid (IAA). One hundred and twelve transgenic shoots produced from three experiments were rooted in the presence of 100 mg l−1 kanamycin and 0.1 mg l−1 α-naphthaleneacetic acid (NAA). Fifty-two of these plantlets (47%) were positive for β-glucuronidase (GUS) enzyme activity, when leaf laminae were assayed histochemically. An enzyme-linked immuno sorbent assay (ELISA) for the nptII protein confirmed expression of the selectable marker gene and validated the kanamycin rooting assay. Northern blot analyses of leaf laminae and petals of seven pMOG410 transformants and of leaf laminae of five pART27-10 transformants showed gusA RNA transcripts only in the transformants. Southern blot analysis of left border integration patterns of seven pMOG410 transformants confirmed the independent nature of the transformants. Petals, scapes, leaf laminae, petioles and corms from 14 independent transformants growing in a containment greenhouse were still GUS positive 33 months after explants were infected. Some seedlings from these transformants were GUS positive showing transgene transmission to sexual offspring and meiotic stability. These results indicate stable, long-term expression of a non-selected transgene can be obtained in transgenic cyclamen.
Databáze: OpenAIRE
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