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E.C. Cocking and M.R. Davey
Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
The soil bacterium Agrobacterium tumefaciens, with its associated plasmid, is used as a vector for introducing DNA into the genomes of dicotyledonous plants, but it has not proved suitable for cereals. This failure to achieve Agrobacterium-induced transformation in cereals has led to increased interest in assessing other transformation methods, especially the direct uptake of DNA.
In 1986 polyethylene glycol (PEG) had been used to induce the uptake of chimaeric plasmid into rice protoplasts (variety 5924) and colonies of rice callus had been selected resistant to kanamycin. However no plant regeneration was achieved from these transformed colonies (Uchimiya et al. 1986).
When reproducible, efficient plant regeneration from rice protoplasts became possible utilising the Japonica variety Taipei 309 (Abdullah et al. 1986), a systematic study was undertaken utilizing this Taipei 309 rice protoplast system to obtain transformation of protoplasts, and, coupled with this, reproducible regeneration of transgenic rice plants.
We observed that the resistance of untreated rice protoplasts to kanamycin was influenced by the culture period. Their division was inhibited by 50μg/ml of kanamycin when they were exposed to the antibiotic after 7 days of culture. Fourteen-day old protoplasts were inhibited by 100μg/ml of kanamycin, but exposure to kanamycin after 4 weeks did not prevent colony formation even at 200μg/ml. Consequently, transformed cells were selected by incorporating 100 μg/ml of kanamycin into the liquid medium bathing the agarose sectors after 14 days of culture. The possible toxicity of PEG to rice protoplasts led us to investigate the use of electroporation as an alternative for DNA delivery.
Protoplasts were isolated from established cell suspensions of Oryza sativa Taipei 309 line LB1 (Abdullah et al. 1986). Plasmid, pCaMVNEO carrying a chimaeric gene consisting of the CaMV35S promoter, the NPTII gene from Tn5 and the nos polyadenylation region, was isolated from E. coli. For PEG-mediated DNA uptake, 1 ml containing 1.4X106 rice protoplasts was mixed with 20μg of sheared calf thymus DNA and 5μg of unrestricted pCaMVNEO followed by dropwise addition of 40% PEG 6000. For electroporation 1 ml containing 4.25 X 106 rice protoplasts was mixed with 50μg of sheared calf thymus DNA and 10μg pCaMVNEO and given 3 pulses, at 10 sec. intervals, of 500 to 2500V with 20 to 50 nF capacitance. The rice protoplasts were transformed to kanamycin resistance following uptake of pCaMVNEO induced by electroporation, PEG and PEG combined with electroporation. Protoplast-derived colonies selected on medium containing 100 μg/ml of kanamycin expressed NPTII activity, and contained DNA that hybridized to a 1.0 Kb BamHI fragment of pCaMVNEO carrying the NPTII gene. Expression of the transformation frequency in relative terms (number of kanamycin resistant colonies compared to the number of colonies on kanamycin free medium) gave frequencies of 26%, 8.5% and 2.9% following electroporation, PEG and PEG with electroporation, respectively (Yang et al. 1988).
Having established that electroporation was the superior procedure for the transformation of Taipei 309 rice protoplasts when utilizing pCaMVNEO chimaeric plasmid, we then attempted to obtain transgenic rice plants by regeneration of plants from transformed callus since we knew that untreated protoplast-derived callus of Taipei 309 was capable of plant regeneration on N6 medium (Abdullah et al. 1986). In these experiments we investigated the regeneration capability of kanamycin resistant colonies which developed following exposure of heat- shocked protoplast-derived cells to the regeneration medium containing kanamycin, only 2 plantlets were regenerated from 400 resistant colonies and these plantlets failed to survive in culture. In contrast, about 30% of the kanamycin resistant protoplast-derived colonies maintained in the dark at 27°C on N6 medium without kanamycin produced one or more somatic embryos within 2 to 3 weeks. Of 400 kanamycin resistant colonies transferred to N6 medium without kanamycin, 6 colonies produced 12 green plants. Genomic DNA isolated from these regenerated plants hybridized to the internal 1.0 Kb BamHI fragment of pCaMVNEO containing the NPTII gene; confirming the presence of foreign DNA in the regenerated rice plants. Interestingly only 2 of the six regenerated plants derived from individual kanamycin resistant colonies gave a positive enzyme assay for NPTII activity (Zhang et al. 1988).
This ability to produce transgenic rice plants from protoplasts now provides the opportunity to assess the feasibility of introducing non-selectable genes into rice plants by co-transformation.
References
Abdullah, R., E.C. Cocking and J.A. Thompson, 1986. Efficient plant regeneration from rice protoplasts through somatic embryogenesis. Bio/Technology 4: 1087-1090.
Cocking, E.C. and M.R. Davey, 1987. Gene transfer in cereals. Science 236: 1259-1262.
Uchimiya, H., T. Fushima, H. Hashimoto, H. Harada, K. Syono and Y. Sugawara, 1986. Expression of a foreign gene in callus derived from DNA-treated protoplasts of rice (Oryza sativa L.). Mol. Gen. Genet. 204: 204-207.
Yang, H., H.M. Zhang, M.R. Davey, B.J. Mulligan and E.C. Cocking, 1988. Production of kanamycin resistant rice tissues following DNA uptake into protoplasts. Plant Cell Reports (in press).
Zhang, H.M., H. Yang, E.L. Rech, T.J. Golds, A.S. Davis, B.J. Mulligan, E.C. Cocking and M.R. Davey, 1988. Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts. Plant Cell Reports (in press).
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