53. Transgenic rice plants produced from protoplasts following direct uptake of Bacillus thuringiensis gamma-endotoxin protein gene

Lab. of Molecular Biology, Biotechnology Research Centre, Chinese Academy of Agricultural Sciences, 30 Baishiqiao Road, Beijing 100081, China

Transformed rice calli have been produced in our lab by direct uptake of a reportable chimeric B. T. delta-endotoxin protein gene (Yang et al. 1988). We have found regeneration of the transformed callus and the expression of the gene transferred into rice plants.

Construction and deletion of delta-endotoxin protein gene: The gene was cut from recombinant plasmid pUCF33. The size of the delta-endotoxin protein gene was 3.8 kb. This gene fragment was inserted into plant-expression vector pBI121.2 to construct a recombinant plasmid pGY61. According to Fishhoff et al. (1987), the deleted gamma-endotoxin protein gene was expressed in plant tissues better than was the complete gene. So, we cut a 1.4 kb fragment from 3' end of the gene and inserted it into the vector pBI121.2 to make another recombinant fragment pGYCK63. The characteristics of these two plasmids were that both carried a chimeric gene consisting of CaMV35s promotor, delta-endotoxin protein gene fused with beta-glucuronidase (GUS) gene and polyadenylation region of the Nos gene. The delta-endotoxin protein gene and GUS gene were fused genes sharing one promotor and terminator. The GUS gene was on down-stream of the d-endotoxin protein gene. The difference between the two plasmids was that pGY61 contained a complete delta-endotoxin protein gene with a 3.8 kb size while pGYCK63 contained the deleted gene fragment with a 2.4 kb size.

Protoplast isolation, transformation and regeneration: Rice protoplasts were isolated from cell suspension of variety Taipei 309 following the method of Thompson et al. (1986). Plasmids pGY61 and pGY63 were transformed into the protoplasts by polyethylene glycol (PEG) method outlined by Krens et al. (1982). The protoplasts were embedded in KPR agarose medium and cultured at 28 deg.C in dark. The first division was observed on the third to fifth day, and the second division was on the 7th to 10th day after isolation. When microcolonies grew up to 1 mm in diameter, they were transferred into differentiation medium. One month later when the shoots and roots were differentiated from the protoplast- derived callus, the plants were transferred to a light. As the seedlings grew up to 10 cm high, they were transplanted into ordinary soil. Up to now, about 40 green plants have been regenerated from the plasmid-treated protoplasts.

Confirmation of transgenic rice plants: Southern analysis was made in the protoplast-regenerated plants. The 3.0 kb KpnI fragment of gamma-endotoxin gene labelled with ³²P was used as the probe, and was hybridized with transgenic plant DNA digested with HindIII. Positive hybridization bands were observed in extracts at least from some transgenic rice plants (Fig. 1), indicating that the B.T. delta-endotoxin gene was integrated in the rice genome. Furthermore, high GUS activities were detected from the extracts of protoplast-regenerated rice plants (Fig. 2), suggesting that the delta-endotoxin gene was expressed in the rice plants. Confirmation by Western blotting is in progress.

Lane 1. 3.0 kb B.T. gamma-endotoxin protein gene fragment cut from plasmid pUCF33 with KpnI.

Lane 5. DNA of a rice plant regenerated from protoplasts not treated with plasmids.

Fig. 2. Beta-glucuronidase (GUS) activity in extract of protoplast-regenerated rice plants.

Abscissa: time (hours). Ordinate: 4-methyl-umbelliferone (4-MU) in u moles. Rice: Regenerated from plasmid-treated protoplasts. Control: Rice plants regenerated from protoplasts not treated with plasmid.

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Krens, F. A., L. Molendijk, G. J. Wullens and R. A. Schilperoort, 1982. In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296: 72-74.

Thompson, J. A., R. Abdullah and E. C. Cocking, 1986. Protoplast culture of rice (Oryza sativa L.) using media solidified with agarose. Plant Sci. 47: 123-133.

Yang, H., S. D. Guo, J. X. Li, X. J. Chen and Y. L. Fan, 1988. Transformation of rice protoplasts by direct uptake of a "reportable chimaeric B. T. gamma-endotoxin protein gene". RGN 5: 141-142.