D. Report of the Committee on Genetic Engineering (Molecular Biology and Biotechnology of Rice)

Ray Wu, Convener

Progress on the genetic engineering of rice has been advancing at an unusually rapid pace during the last year or two. A large number of rice genes have been identified by cloning and sequencing. For example, a number of abstracts have been published in the proceedings of the Fifth Annual Meeting of Int'l Program on Rice Biotechnology, held in Tucson, Arizona, 1991, sponsored by the Rockefeller Foundation, New York. Many papers were cited in the Rice Genetics Neivsletter, Vol. 8, 1991, in the book Agricultural Biotechnolooy (You and Chen, 1992), and in a review article by Okita (1991). Many of these genes are housekeeping genes and are likely to be expressed in all tissues. Among them, the rice actin 1 gene was shown to be highly expressed in all tissues. Thus, the promoter of this gene is useful for expressing foreign genes in transgenic rice (McElroy et al., 1990; 1991). Several tissue-specific genes have also been identified, and the following promoters from the rice genes are expected to be useful for tissue-specific expression of foreign genes: a genomic clone GOS9 for root (de Pater and Schilperoort, 1992), several alpha-amylase genes including OSamy-c (Kim et al., 1992) and RAmyl-B and other genes (Huang et ai., 1990) for germinating seeds, glutelin genes for immature seeds (Kim and Wu, 1990; Takaiwa ct al., 1991), the PS1 gene for pollen (Zou et al., 1992), and an rbcS for leaf (McElroy, D., T. Hayakawa, K. Shimamoto and R. Wu, manuscript in preparation).

The regulatory sequence of several rice genes have been thoroughly analyzed to increase our understanding of their mode of action; for example, the phytochrome gene (Kay et al., 1989; Dehesh et al., 1990; Quail et al., 1991), and the actin 1 gene (Zhang et al., 1991; Wang et al., 1992).

Importanto? advances have been made on the transformation of rice protoplasts and intact cells, followed by regeneration of fertile plants. Transgenic plants of a dozen japonica and several indica varieties have been obtained (for a review, see Kothari et al., 1992). Reproducible methods for producing fertile transgenic plants include: (1) transformation of protoplasts by polyethylene glycol or electroporation (for a review, see Cao et al., 1991), and (2) transformation of intact suspension cells (Cao et al., 1992), calli (Xu et al., 1992) and immature embryos (Christou et al., 1991) by partcle bombardment (Klein et al., 1987). Introduction of DNA into intact rice cells via the Agrobacterium system followed by plant regeneration has been reported by several groups (Ralneri et al., 1990; Li et al., 1991). However, this method has not yet reached the staoe of reproducibility.

Over the last 20 years or so, very significant advances have been made by conventional breeding in producing a number of agronomically useful rice plants. However, introduction into rice of desirable genes from plants that are not sexually compatible with rice has not been possible. On the other hand, with the advent of genetic engineering and biotechnology, introduction into rice of useful foreign genes from any source, including microorganisms, can be accomplished in principle.

With the availability of useful promoters for high level expression of foreign genes in rice, and with the establishment of transformation/regeneration systems in rice, the time is ripe to initiate work on rice improvements using genetic engineering techniques. Rencently, preliminary experiments have been carried out in several laboratories by transforming rice, using cloned genes, with the aim of producing superior transgenic plants with different novel properties. These experiments include transformation of rice with (a) a Bacillus thuringielisis (B.t.) toxin gene (Xie et al., 1991), and (b) the potato protease inhibitor II gene (pin2) and a cowpea trypsino? inhibitor (CpTi) gene (Xu et al., 1992) with the aim of producing insect-resistant plants; (c) a coat protein gene of rice stripe virus (Yan et al., 1991; Hayakawa et al., 1991) with the aim of producing virus-resistant rice plants. It is likely that within a year, positive results from these and other experiments will be forthcoming.

The next challenge for rice improvement via genetic engineering is to identify and clone additional agronomically useful genes. A rice cystatin gene (Chen et al., 1991) and a rice amylase inhibitor gene were isolated. Once transformed into rice, the plants may become resistant to certain insects, including the rice weevil (Reeck et al., 1991). A rice chitinase gene was cloned (Lamb et al., 1991; Broglie et al., 1991). A barley basic beta-(1-3)-glucanase gene and a 30 kilodalton (kD) ribosome-inactivating protein gene were cloned (Henrik ct al., 1991). Once transformed into rice, the plants may become resistant to fungal diseases, including rice blast disease. In addition, a number of insecticidal proteins have been identified; this includes a number of new B.t. proteins (Bottrell et al., 1991; Riazuddin et al., 1991). Also, several anti-bacterial proteins (Liu et al., 1990; Bulet et al., 1991; Li et al., 1992) and a fungicidal protein were isolated (Liu et al., 1992). Once the protein sequence is determined, the DNA sequence encoding these genes can be cloned.

Finally, investigations have been initiated to identify, and then clone the genes responsible for the following traits: high yield, and tolerance to low temperature, to drought or to salt stresses. These are more difficult tasks because each trait is probably controlled by several genes. However, in principle, the major genes involved in each of these traits can be identified first by RFLP mapping and then by map-based gene cloning (McCouch and Tanksley, 1991). It is almost certain that many different varieties of transgenic rice plants endowed with a number of the above-mentioned useful traits, ano?d other desirable traits, will be grown around the world within the next 20 years.

References

Bottrell, D. G., R. M. Aguda, W. Theunis and C. Demayo, 1991. The IRRI program on Bacillus thuringiensis, in Fifth Annual Meeting of Int'l Program on Rice Biotechnology (in Arizona), Rockefeller Foundation, New York, p. 75.

Broglie, K., I. Chet, M. Holliday, R. Cressman and R. Broglie, 1991. Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani, Science 254: 1194-1197.

Bulet, P., S. Cociancich, J.-L. Dimarcq, J. Lambert, D. Hoffmann and J. A. Hoffmann, 1991. Isolation from a coleopteran insect of a novel inducible antibacterial peptide and of new members of the the insect defensive family, J. Biol. Chem. 236: 24520-24525.

Cao, J., W. Zhang, D. McElroy and R. Wu, 1991. Assessment of rice genetic transformation techniques, in Rice Biotechtioloay (Khush, G. S. and G. H. Toenniessen, eds.), C. A. B. Int'l, London, pp- 175-198.

Cao, J., X. Duan, D. McElroy and R. Wu, 1992. Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells, Plant Science, in press.

Chen, M.-S., B. Johnson, L. Wen, S. Muthukrishnan, K. J. Kramer, T. Morgan and G. R. Reeck, 1992. Rice cystatin: bacterial expression, and insect growth suppressinc, activity of a truncated form of the protein, Protein Expression and Purification 3: 41-49.

Christou, P., T. L. Ford and M. Kofron, 1991. Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos, Bio/Technology 9: 957-962.

Dehesh, K., W. B. Bruce and P. H. Quail, 1990. A trans-acting factor that binds to a GT-motif in a phytochrome gene promoter, Science 250: 1397-1399.

de Pater, B. S. and R. A. Schilperoort, 1992. Structure and expression of a o?root-specific rice gene, Plant Mol. Biol. 18: 161-164.

Hayakawa, T., Y. Zhu, K. Itoh, Y. Kimura, K. Shimamoto and S. Toriyama, 1991. Genetically engineering rice resistant to rice stripe virus, an insect transmitted virus, in Fifth Annual Meeting of Int'l Program on Rice Biotechnology (in Arizona), Rockefeller Foundation, New York, p. 128.

Henrik, T., R. Leah, K. Shriver, J. Logemann and J. Mundy, 1991. Barley seed anti-fungal proteins, in Fifth Annual Meeting of Int'l Program on Rice Biotechnology (in Arizona), Rockefeller Foundation, New York, p. 29.

Hodges, T. K., J. Peng, L. A. Lyznik and D. S. Koetje, 1991. Transformation and regeneration of rice protoplasts, in Rice Biotechnology (Khush, G. S. and G. H. Toenniessen, eds.), C. A. B. Int'l, London, pp. 157-174.

Huang, N., N. Koizumi, S. Reini and R. L. Rodriguez, 1990. Structural organization and differential expression of rice alpha-amylase genes, Nucleic Acids Res. 18: 7007-7014.

Kay, S. A., A. Nagatani, B. Keith, M. Deak, M. Furuya and N.-H. Chua, 1989. Rice phytochrome is biologically active in transgenic tobacco, The Plant Cell 1: 775-782.

Kim, S. Y. and R. Wu, 1990. Multiple protein factors bind to a rice glutelin promoter region, Nucleic Acids Res. 18: 6845-6852.

Kim, J. K., J. Cao and R. Wu, 1992. Regulation and interaction of multiple protein factors with the proximal promoter regions of a rice high pl alpha-amylase gene, Mol. Gen. Genet. 232: 383-393.

Klein, T. M., E. D. Wolf, R. Wu and J. C. Sanford, 1987. High-velocity microprojectiles deliver nucleic acids into living cells, Nature 327: 70-73.

Kothari, S. L., M. R. Davey, P. T. Lynch, R. P. Finch and E. C. Cocking, 1992. Transgenic rice, in Transgenic Plants (Kung, S. D. and R. Wu, eds.), Academic Press, Orlando, FL. Volume 2, pp. 3-20, in press.

Li, B. J., Y. Xu, X. P. Xu, H. Shi, X. Ke and D. Yu, 1991. Few potential techniques for transferring foreign genes into crop po?lants, in Fifth Annual Meeting of Int'l Program on Rice Biotechnology (in Arizona), Rockefeller Foundation, New York, p. 61.

Li, D., X. Chen, W. Zhu, P. Xu, Q. Ge, W. Chen and Y. Zhang, 1992. Study of antagonistic proteins of bacteria to Xanthomonas oryzae pv. oryzae, in Agricultural Biotechnology, China Science and Technology Press, Beijing, China, pp. 703-707.

Liu, H., H. Gu, X. Chen, S. Tang, N. Pan and Z. Chen, 1992. Isolation and partial characterization of an antifungal protein from rice, in Agricultural Biotechnology, China Science and Technology Press, Beijing, China, pp. 6-12.

Liu, J.-Y., N.-S. Pan and Z. L. Chen, 1990. Characterization of an anti-rice bacterial blight polypeptide LCI, Rice Genetics Newsletter 7: 151-154.

McCouch, S. R. and S. D. Tanksley, 1991. Development and use of restriction fragment length polymorphism in rice breeding and genetics, in Rice Biotechnology (Khush, G. S. and G. H. Toenniessen, eds.), C. A. B. Int'l, London, pp. 109-133.

McElroy, D., W. Zhang, J. Cao and R. Wu, 1990. Isolation of an efficient actin promoter for use in rice transformation, Plant Cell 2: 163-171.

McElroy, D., A. D. Blowers, B. Jenes and R. Wu, 1991. Construction of expression vectors based on the rice actin 1 (Act 1) 5' region for use in monocot transformation, Mol. Gen. Genet. 231: 150-160.

Okita, T. W., 1991. The identification and characterization of rice nuclear genes, in Rice Biotechnology (Khush, G. S. and G. H. Toenniessen, eds.), C. A. B. Int'l, London, pp. 199-224.

Quail, P. H., W. B. Bruce, K. Dehesh and J. Dulson, 1991. phyA gene promoter analysis, in Plant Molecular Biology 2 (Herrmann, R. G. and B. Larkins, eds.), Plenum Press, New York, pp. 499-508.

Ralneri, D. M., P. Bottino, M. P. Gordon and E. W. Nester, 1990. Agrobacterium-mediated transformation of rice (Oryza sativa L.), Bio/Technology 8: 33-38.

Reeck, G. R., S. Muthukrishnan and K. J. Kramer, 1991. Cereal inhibitors of insect amylases and sulfhydryl proto?eases, in Fifth Annual Meeting of Int'l Program on Rice Biotechnology (in Arizona), Rockefeller Foundation, New York, p. 30.

Riazuddin, S., E. Khan, S. P. Rubin, S. Karim, R. Makhdoom and A. Sohail, 1991. Entomocidal properties of B.t. isolates from Pakistan and studies on transformation of rice, in Fifth Annual Meeting of Int'l Program on Rice Biotechnology (in Arizona), Rockefeller Foundation, New York, p. 76.

Takaiwa, F., K. Oono, D. Wing and A. Kato, 1991. Sequence of three members and expression of a new major subfamily of glutelin genes from rice, Plant Mol. Biol. 17: 875-885.

Wang, Y., W. Zhang, J. Cao, D. McElroy and R. Wu, 1992. Characterization of cis-acting elements regulating transcription from the promoter of a constitutively active rice actin gene, Mol. Cell Biol- 12: 3399-3406.

Xie, D. X., Y. L. Fan and P. C. Ni, 1992. Transgenic rice plant of a superior Chinese cultivar Zhonghua No. 11 containing the B.t. delta-endotoxin gene in its genome, Science in China Series B 35: 566-569.

Xu, D.-P., T. Wu, J. Cao and R. Wu, 1992. Production and analysis of transgenic rice plants, in Agricultural Biotechnology, China Science and Technology Press, Beijing, China, pp. 91-96.

Yan, Y. T., B. S. Qiu, J. F. Wang, X. M. He, S. Z. Zhao, X. F. Wang and P. Tien, 1991. Expression of rice strip virus coat protein gene in transgenic rice plant, in Fifth Annual Meeting of Int'l Program on Rice Biotechnology (in Arizona), Rockefeller Foundation. New York, p. 96.

You, C. B. and Z. L. Chen (eds.), 1992, In Agricultural Biotechnology, China Science and Technology Press, Beijing, China.

Zhang, W., D. McElroy and R. Wu, 1991. Analysis of rice Act 1 5' region activity in transgenic rice plants, Plant Cell 3: 1155-1165.

Zhu, Q. and C. J. Lamb, 1991. Isolation and characterization of a rice gene encoding a basic chitinase, Mol. Gen. Genet. 226: 289-296.

Zou, J.-T., H. Wang, H.-M. Wu and A. Y. Cheung, 1992. Isolation and characterization of a rice pollen-spo?

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