Agrobacterium tumefaciens-mediated transformation of greenhouse-grown Brassica rapa ssp. oleifera

• Published in: Plant cell reports 1999-05, Vol.18 (9), p.773-777
• Main Authors: Kuvshinov, V, Koivu, K, Kanerva, A, Pehu, E
• Format: Article
• Language: English
• Subjects: 2,4-D; Agrobacterium; Agrobacterium tumefaciens; Bacteriology; benzyladenine; beta-glucuronidase; biochemical techniques; Biological and medical sciences; Biotechnology; Brassica rapa; Brassica rapa oleifera; Brassica rapa subsp. oleifera; Chlorophenoxyacetic acids; cultivars; Cultivation; culture media; Eukaryotic cell cultures; explants; Fundamental and applied biological sciences. Psychology; Genetic engineering; genetic markers; Genetic technics; genetic transformation; greenhouse production; Greenhouses; histochemistry; hygromycin B; In vitro propagation: entire plant regeneration from tissues and cell cultures; in vitro selection; inflorescences; internodes; kanamycin; Methods. Procedures. Technologies; naphthaleneacetic acid; Plant cells and fungal cells; regrowth; reporter genes; Shoots; Silver; silver nitrate; stems; strain differences; Transgenic animals and transgenic plants; Transgenic plants
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s002990050659

Greenhouse-grown plants of turnip rape Brassica rapa ssp. oleifera (syn. B. campestris) cv. Valtti and Sisu were transformed by Agrobacterium tumefaciens infection. Of the three A. tumefaciens strains tested (C58C1, EHA105 and LBA4404), LBA4404 gave the best results. Segments excised from one to two upper internodes of an inflorescence-carrying stem served as explants for the Agrobacterium infection. Cultivation of the explants horizontally during the first 3 days of co-cultivation with A. tumefaciens following immediate selection of transformed tissue of the stem segments placed vertically basal side down were critical. Use of silver nitrate (5-10 mg/l) in the culture medium and Micropore (3 M) paper tape for sealing plates was also beneficial. Transgenic shoots were recovered using either hygromycin or kanamycin (20-25 mg/l) selection. Hygromycin was preferable, as the proportion of 'escapes' was 90% under kanamycin and 10% under hygromycin selection. Regeneration was achieved by culturing the explants for 3-6 days on 0.5 mg/l of 2,4-di-chlorophenoxyacetic acid and 1-2 weeks on 2-3 mg/l of 6-benzyl aminopurine with/without 0.05 mg/l alpha-naphthaleneacetic acid. Recovered shoots were then cultured on hormone-free MS medium. This culture program gave 60-80% shoot regeneration. Regenerants were tested by histological beta-glucuronidase staining and southern blotting. The recovery rate of transgenic shoots was 4-9% of the number of explants used in the experiments.