D. Report of the Committee on Genetic Engineering

Within the last two years or so, methods for plant regeneration from rice protoplasts were reported by six groups (Fujimura et al. 1985; Abdullar et al. 1986; Lei et al. 1986; Yamada et al. 1986; Cocking and Davey 1987; Kyozuka et al. 1987). Very recently, transgenic rice plants were produced using two different approaches. In one method, a plasmid containing a neomycin phosphotransferase (NPTII) gene was introduced into the rice floret via the pollen tube pathway. Four transgenic plants were obtained and were shown by hybridization analysis to contain integrated copies of the NPTII gene. These plants also showed functional NPTII enzyme activity (Luo and Wu 1988). In the second approach, protoplasts were directly transformed with reporter-gene containing plasmids in three different laboratories. Toriyama et al. (1988) introduced a plasmid containing a NPTII gene into rice protoplasts by electroporation. The calli were screened by the antibiotic G418 to select the transformed samples. Five transgenic plants were regenerated and were shown to contain the NPTII gene and enzyme activity. Zhang and Wu (1988) incubated a plasmid containing a Beta-glucuronidase (GUS) gene with rice protoplasts in the presence of polyethylene glycol (PEG). Approximately 380 plants were regenerated without any selection pressure. Out of these, 61 showed GUS activity assayed at the calli stage and a number of plants also gave positive GUS enzyme assays. DNA hybridization analysis of 100 plants, including the 61 which showed positive enzyme activity in the calli, gave 86 positives. Thus, they have regenerated a large number of transgenic plants. Cocking and Davey (this issue of RGN) introduced a plasmid containing a NPTII gene into rice protoplasts by electroporation, which yielded a higher transofrmation frequency than PEG; 400 kanamycin resistant colonies were selected. Of these, six colonies produced 12 green plants. However, only two of the six regenerated plants gave a positive enzyme assay for NPTII activity.

Recently, a RFLP genetic map for rice (Oryza sativa) was constructed using randomly selected single-copy DNA clones; 135 markers were mapped on the 12 rice chromosomes. This molecular map covers 1389 cM of the rice genome and exceeds the current classical maps by more than 20%. The map was generated using an F\2\ segregating population (50 plants) from a cross between an Indica and a Javanica rice variety. DNA isolated from primary trisomics were used to assign linkage groups to each of the 12 rice chromosomes (McCouch et al 1988).

An ABA and salt inducible gene (RAB21) was identified by differential screening of an IR36 cDNA library. Although its function is currently unknown, the gene has been characterized by DNA sequence analysis, transcript mapping, and analysis of its in vivo and in vitro synthesized protein product. RAB21 mRNA accumulates very rapidly after induction in suspension cell culture and does not require protein synthesis, indicating that performed proteins may regulate its response to ABA and salt (Mundy and Chua 1988).

Several clones including a-amylase genes were isolated from an IR26 genomic library and could be divided into four different groups based on their restriction map (Ou-lee et al. 1988). One of the genes, OSamy-a, was analyzed in detail. By carrying out a DNA mobility-shift assay, a protein "factor" in the extract of rice cells (mainly aleurone and scutellum) was found to bind specifically to a 500 bp fragment (HS500) in the upstream region of the OSamy-a gene. The presence of this "factor" is dependent on treatment of rice tissues with gibberellin (GA). The approximate position of the "factor"-DNA interaction was defined by an exonuclease III digestion experiment. A region was identified which includes a 22 bp sequence containing an imperfect tandem repeat: CTTTTT ATCTCTTTTAAATGAG. This sequence may be the binding site of the "factor", an interaction which may be responsible for stimulating the synthesis of a-amylase mRNA in response to GA induction (Ou-lee et al. 1988).

Richard et al. (1986) found that ADH activity can be induced in rice embryos during anaerobiosis. Furthermore, ADH was induced by the synthetic auxin, 2, 4- dichlorophenoxyacetic acid (2,4-D), in calli derived from roots of 4-day-old rice seedlings. Kadowaki et al. (1988) reported that the ADH activity in rice roots was induced under anaerobic conditions. Xie and Wu (unpublished observations) found that either anaerobiosis or 2,4-D induces ADH activity in rice endosperm, 5-day-old seedlings, roots and, unexpectedly, etiolated leaves and green mature leaves.

Glutelin is the major seed storage protein of rice. The cDNA coding for a glutelin precursor (57 kDa) was cloned and sequenced. The gene codes for a 37 amino acid signal peptide sequence at the NH\2\ terminus, which is followed by a 269 amino acid acidic subunit (32 kDa) and a 193 amino acid basic subunit (20 kDa) and a 193 amino acid basic subunit (20 kDa) (Takaiwa et al. 1986). A similar cDNA clone coding for a glutelin precursor was isolated by another group and sequenced. The clone also codes for a precursor polypeptide which undergoes post-translational processing. The sequence of the precursor shares 32% of the amino acid positions with those of the glycinin A\1a\B\1b\ precursor (Higuchi and Fukazawa 1987). A genomic clone encoding the glutelin sequence was identified. The gene contains three short introns and codes for a prepro- glutelin protein of 499 amino acids, identical to that deduced from the type II glutelin cDNA. In the 5'-flanking region, a sequence similar to a consensus sequence for the storage protein genes in legumes (the legumin box) was detected (Takaiwa et al. 1987).

Genetic engineering research in rice is supported by grants from the Rockefeller Foundation.

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