J. NOBRE1*, M.E. CANNELL1, M.R. DAVEY2 AND P.A. LAZZERI1
1. Department of Biochemistry and Physiology, IACR-Rothamsted,
Harpenden, Herts AL5 2JQ, UK;
2. Department of Life Science, Faculty of Science, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
* Present address: Direcção Regional de Agricultura do Algarve,
Braciais, Patacão, 8000 Faro, Portugal.
Introduction
Protoplast-mediated transformation was the first method of direct gene transfer used in plants and the first transgenic cereal plants were produced via direct DNA uptake into protoplasts around ten years ago (e.g. Toriyama et al. 1988). However, transgenic barley plants have been regenerated only recently, using particle bombardment (Wan & Lemaux 1994, Hagio et al. 1995) and via direct gene transfer into protoplasts from embryogenic cell suspensions (Funatsuki et al. 1995, Salmenkallio-Marttila et al. 1995).
Protoplast transformation methods do not call for highly specialised equipment, they allow the production of large numbers of independent transformants and the selection of transformants is efficient. However, totipotent protoplasts are usually obtained from cell suspensions, which are difficult to establish, accumulate genetic aberrations over time and rapidly lose their regeneration capability (Lazzeri & Shewry 1993). To avoid these constraints, we have recently developed a new approach using protoplasts isolated directly from immature scutella, and have shown that these protoplasts can regenerate fertile plants (Nobre et al. 1996a).
Building on this development, a system for the regeneration of transgenic barley plants via PEG-mediated transformation of scutellum protoplasts is reported in the present paper.
Materials and Methods
Immature embryos of the Australian malting cultivar Clipper were conditioned in a high osmoticum medium (Nobre et al. 1996b) and scutellum protoplasts were isolated as described previously (Nobre et al. 1996a). Two plasmid constructs were used in co-transformations: pAct1-DGus, containing the uidA gene driven by the actin 1-D promoter and pCaIneo which contains the neo fragment of the npt II gene under the control of the CaMV35S promoter and the Adh1 intron from maize. Transformation was performed basically according to a procedure described previously for barley cell suspension protoplasts (Lazzeri et al. 1991). PEG-treated protoplasts were embedded in Na-alginate (1.0 x 106 protoplasts / ml) and were co-cultivated with the feeder cell line HTA 42A (tritordeum cell suspension) in L8D1 protoplast medium as described previously for scutellum protoplast culture (Nobre et al. 1996). For the selection of microcolonies, feeder cells were removed after 14 days of culture, and protoplast culture medium was replaced by a standard cell suspension medium containing 15 mg l-1 G418. Culture medium was replaced every 7 days, with a regime of 4-5 one-week passages of selection, until calluses reached 2-3 mm diameter (4-6 weeks). Antibiotic-resistant colonies were subcultured to callus induction and plant regeneration media (Nobre et al. 1996a), in the absence of the antibiotic. Plants regenerated from colonies surviving selection were screened for NPT activity by enzymatic NPTII-ELISA assays and transgene integration was assessed by Southern analysis.
Results and Discussion
To date, 3 transgenic plants have been regenerated from 17 antibiotic-resistant callus lines, recovered from the transformation of 0.9 X 106 protoplasts. The transformants show normal morphology and have now set seed. Tests for NPT activity by ELISA were positive and Southern analysis has confirmed transgene integration.
A high ratio of albino plants and chromosomal aberrations among regenerants were reported to occur from the transformation of protoplasts isolated from barley cell suspensions (Funatsuki et al. 1995, Salmenkallio-Marttila et al. 1995). Although or initial experiments have produced only three plants to date, they demonstrate the feasibility of this approach for barley transformation, and our previous results showing that plants regenerated from scutellum protoplasts show little somaclonal variation and have high fertility suggest that this system may offer a new alternative to the standard techniques for the regeneration of fertile transgenic barley plants.
Acknowledgements: IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.
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