Fig. 2. Southern blotting of genomic DNA from rice (probe: aizawai 7-29 gamma-endotoxin gene).
1) Biotechnology Res. Center, Chinese Academy of Agricultural Sciences, Beijing, 100081, China
2) Institute of Crop Breeding and Cultivation, CAAS, Beijing, 100081, China
Lepidopteran insects such as Tryporgza incertulus, Chilo suppressalis and Cnaphalocrocis medinalis have caused severe losses in rice production. Previously,
Table 1. Rice genetic transformation via the pollen-tube pathway
====================================================================
Methods Treatment No.of florets No.of mature frequency of
treated seeds collected seed set(%)
====================================================================
1.Transformation pGYCK 63 272 52 19.1
after cutting ==================================================
top floret TE buffer 118 20 17.0
====================================================================
2.Transformation pGYCK 63 721 354 49.2
after cutting ==================================================
palea TE buffer 102 51 50.0
====================================================================
we succeeded in transferring the Bacillus thuringiensis (B.t.) gamma-endotoxin gene into protoplasts of Taipei 309 and regenerating transgenic plants (Yang et al. 1990). To date this represents the only reported transfer of an agricultural important gene into a commercial rice variety. In this paper we present the results of the transfer of a B.t. gamma-endotoxin gene by the pollen-tube pathway method (Luo and Wu 1988) into a superior Chinese Cultivar, Zhonghua No.11 (developed by the Institute of Crop Breeding and Cultivation, CAAS). This cultivar is resistant to certain diseases, gives relatively high yield, and is widely planted in China.
Because the transformation procedures were carried out in the field, instead of in a greenhouse with constant relative humidity, the pollen-tube pathway procedure was modified. We used two methods to cut the stigma. The first method was to cut away the top one-thirds to half of the floret, and then the stigma. The second method was to cut away most of the palea together with the stigma (just above the ovary) with the whole lemma remained attached.
The results of the genetic transformation experiments are shown in Table 1. The seed set frequency obtained by using the second method to cut the stigma was much higher than that obtained using the first method.
The plantlets were transplanted to normal soil at the three leaf stage. DNA
Fig. 2. Southern blotting of genomic DNA from rice (probe: aizawai 7-29
gamma-endotoxin gene).
1: Negative CK. 2: Undigested #3 plant DNA. 3,4: #3 plant DNA digested with Hind III. 5: Undigested #7 plant DNA. 6,7: #7 plant DNA digested with Hind III. 8: Undigested #8 plant DNA. 9: #8 plant DNA digested with Hind III. 10: Postive CK (4.2 kb gamma-endotoxin gene).
was isolated from leaves and probed with a Hind III restriction fragment from the B.t. aizawai 7-29 gamma-endotoxin gene. Among DNA samples from 387 plants, 11 plants were found to give positive signals by dot blot hybridization (Fig. 1).
Next, DNA ftom the plants #3, #7 and #8 was analyzed by Southern blott- ing. Undigested DNA from these two plants, as well as DNA digested with Hind III, were probed with the Hind III fragment of aizawai 7-29 gamma-endotoxin gene. Fig. 2 showed that positive hybridization signals were found with #3 and #8 plant DNA, either undigested or digested with Hind III, whereas DNA from plant #7 and a control plant did not show any hybridization band. The data suggest that the B.t. toxin gene has been transferred into the cells of the #3 and #8 rice plants.
The expression of beta-glucuronidase activity in the transgenic rice plant #3 was confirmed by histochemical assay (Fig. 3). Since in pGYCK 63, the GUS gene was translationally fused downstream of the B.t. gamma-endotoxin gene, the expression of the GUS gene suggests that the B.t. gamma-endotoxin was also produced in the transgenic rice plant.
This is the first report on transferring a B.t. endotoxin gene into a widely used commercial rice variety.
Fig. 3. Histochemical assay of beta-glucuronidase activity.
Top: Negative CK;
Bottom: Root of #3 plant.
References
H. Yang, S. D. Guo, J. X. Li, X. L. Chen and Y. L. Fan, 1990. Direct uptake of gamma-endotoxin protein gene from Bacillus thuringiensis into rice protoplasts and regeneration of transgenic rice. Genetic Manipulation in Plants 6(1): 13-21.
Z.X. Luo and R. Wu, 1988. A simple method for the transformation of rice via the pollen-tube pathway. Plant Mol. Biol. Reporter 6: 165-174.