Plastid transformation technology is an attractive tool in plant biotechnology. The advantages of transplastomic plants over conventional nuclear transformants are as follows; high-level expression of foreign proteins, absence of gene silencing and position effect, lack of transgene transmission to environment through pollens, etc. Although transplastomic plants are demanded, application of this technology is virtually limited to tobacco. In order to extend transplastomic technology to crops, we have tried to transform wheat plastid. In the present study, two wheat cultivars, Akadaruma and Fielder, were used as the material plants. Two plastid transformation vectors containing either rbcL-psaI or trnV-rps12 / 7 sequence were constructed. Both vectors contain a spectinomycin/streptomycin-resistance gene (aadA) and a GFP gene (gfp) in the middle of chloroplast sequences. The homologous recombination at rbcL-psaI or trnV-rps12/7 should integrate aadA and gfp into chloroplast genome. The calli were obtained from immature embryos, and they were bombarded with the vector DNAs. Two days after the bombardment, explants were transferred to selection medium containing streptomycin. Resistant calli were maintained on the selection medium for 2-4 months, and then transferred to shoot regeneration medium containing antibiotics. The 51 resistant calli were obtained from 7,837 bombarded calli. Further investigations are in progress to regenerate transplastomic wheat.

P-10 N. Ohnishi, E. Himi and Kaz. Noda (Res Inst Biores, Okayama Univ)
DFR (dihydroflavonol 4-reductase) gene structure of common wheat and it ancestor

Wheat (Triticum aestivum L. cv Chinese Spring) has three DFR genes on group 3 chromosomes (TaDFR-A, TaDFR-B, TaDFR-D). The nucleotide sequence of these DFR showed more than 92 % similarity and this amino acid sequence maintained more than 95 % similarity. DFR genes contained three introns: the first intron showed 79.3 % similarity on average, the second intron 69.1 % and the third intron 75.9 %. Similarity between the intron regions was lower than that between cDNAs, especially the second intron showed lower similarity than the other introns. cDNA of T. monococcum and CS DFRs showed 92.8 % similarity on average and Ae. squarrosa and CS was 87.9 % on average. Compared the intron sequences of TaDFR-A and T. mnococcum DFR, the first intron showed 100 % similarity, the second intron 97.8 % and the third intron 98.8 %. While, the introns of TaDFR-B and TaDFR-D showed lower similarity to the introns of T. monococcum DFR. Also the introns of Ae. squarrosa DFR showed higher similarity to those of TaDFR-D than those of TaDFR-A and -B. Especially, the. second intron of Ae. squarrosa DFR was less similar to those of TaDFR-A and -B.

P-11 S.K. Ghimire^1*, H. Tsujimoto² and K. Kato¹ ( ¹Fac Agr, Okayama Univ, ²Fac Agr, Tottori Univ, *dnsl4715@cc.okayama-u.ac.jp)
Analysis of genetic diversity in wheat germplasm introduced from Nepal and Bhutan based on isozyme and RAPD polymorphism

Genetic diversity in wheat accessions collected from various parts of Nepal and Bhutan was analyzed by RAPD and isozyme analysis as well as field observation. Heading date showed large variation, ranging from 18.3 April to 13.5 May. Positive correlation was detected between heading date and the altitude of their collection sites (r=0.424, P < 0.01). Percentage of spring type varied among collection sites, and significant negative correlation with altitude was observed in east sites of Nepal (r=-0.475, P< 0.01). A total of 17 and 53 polymorphic bands were scored by RAPD and isozyme analysis, respectively. Gene diversity in newly introduced accessions was less than that in wheat germplasm introduced from Nepal and Bhutan a few decades ago, indicating rapid genetic erosion. By cluster analysis using isozyme data, a total of 48 populations were grouped into 5 distinct clusters. Most of South Asian countries like India, Nepal and Bhutan were clustered together, indicating their close genetic relationship. Among the Nepalese and Bhutanese wheat, new and old accessions were separated into two different sub-clusters, as also shown by RAPD analysis. Chinese accessions were clearly divided into two groups, and those from Xinjiang, Tibet and Yunnan were grouped with other Himalayan wheat, and independent of those in the Coast and Yangtze River basin of China.