53. Transformation of rice protoplasts by direct uptake of a "reportable chimaeric B. T. alpha-endotoxin protein gene"

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

Transformation of rice by uptake of a selectable marker gene has been achieved. Recently, transgenic rice plants carrying NPTII gene have been obtained by different DNA delivery methods. But no work on transfer of an agriculturally important gene into rice has been reported.

Rice is attacked by several Lepidopteran insects such as Tryporyza incertulas, Chilo suppressalis and Cnaphalocrocis medinalis, causing great damage. B. thuringiensis is considered as an effective agent to control these insects. The glucuronidase (GUS) gene is a very sensitive reporter gene and is widely used for plant transformation work. In this paper, we report a preliminary study on the construction of the B.T. alpha-endotoxin insect-control protein gene into plant expression vector, transformation of this gene into rice protoplasts, and its expression in rice callus.

Plasmid construction: Plasmid pGY61 carries a chimaeric gene consisting of the Nos and CaM 35s promoter, neomycin phosphotransferase II (NPTII) gene from TN5, alpha-endotoxin crystal protein gene from B. thuringiensis Aizaiwai 7-29 linked with the GUS gene, and Nos polyadenylation region. The GUS gene is in the downstream of the B.T. alpha-endotoxin gene.

Protoplast isolation and transformation: Protoplasts were isolated from cell suspension of Oryza sativa variety Taipei 309 line LB4 as previously reported. PEG mediated DNA uptake followed the procedure of Kren et al. (1982).

Gene expression in rice culture: beta-glucuronidase activity was measured by fluorophotometer. A high GUS activity was found in the callus derived from pGY61 treated protoplasts. The activity was 5 times higher than that of the control based on 5 minute reaction (Fig. 1). It demonstrated that the GUS gene has been expressed in the supposedly transformed rice callus, suggesting that the B.T. alpha-endotoxin protein gene has also been transcribed.

Transformation frequency: Under the optimum conditions of PEG treatment, out of 2X10⁶ treated protoplasts, 3,000 microcolonies greater than 1 mm in diameter were obtained on nonselection medium. The plating efficiency was inthe order of 10^-3. Three of the 15 colonies were found to have a high GUS activity, which was 5 times more than that of the control based on 5 minute reaction. The transformation frequency was about 20% based on dividing colonies and 2.0 X 1- ^-4 in terms of the number of protoplasts treated with plasmid. Further evidence of the B.T. a-endotoxin gene expression will be obtained by Southern and Western blotting.

Fig. 1. Assay of B-glucuronidase activity in supposedly transformed rice callus.

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.