During the last decade, tremendous developments have been in plant genetic engineering techniques and many important transgenic cereal plants have been successfully produced. Almost all of the new techniques in genetic manipulation depend upon the successful regeneration of plants from in vitro cultures such as callus, suspension cells and protoplasts. In this paper, I briefly describe the contributions of Japanese researchers to the development of in vitro culture and gene transformation of cereals, especially rice, and discuss a number of recent advances in this area of research in Japan. In Japan, rice is the most important crop. On the background of intensive basic researches on rice plants, as early as 1964, after the pioneering work of Furuhashi and Yatazawa (1964), the in vitro culture of rice was established in 1967. After the protoplast culture system was established, the first transgenic rice plants were regenerated from rice protoplasts via the direct gene transformation system in 1988. Recently, the Agrobacterium-mediated transformation system was established in rice plants by a research group at JT (Japan Tobacco Inc.) in 1994. A number of transgenic rice plants with various useful genes introduced, including genes conferring tolerance to environmental stresses, resistance to disease and insects, and improving the quality and yield, have been produced at various laboratories. The environmental risk evaluation of transgenic rice plants has been completed, and virus resistant-rice and low allergenic protein-rice have been developed for commercial use. Among the cereals, wheat has proved to be one of the most difficult species to transform. We established the in vitro culture of wheat in 1978 and succeeded in the production of transgenic wheat plants by using particle bombardment in 1995. The transgenic barley plants have been obtained by both protoplast-mediated and microprojectile-mediated transformation systems. Recently, the Agrobacterium-mediated transformation system has been established in maize. In Japan, the Rice Genome Project has been proceeding intensively and genes beneficial for improving crop plants will be isolated and characterized in rice plants. Isolation of useful genes will allow rapid development of beneficial cultivars in not only rice but also other cereals including wheat, barley and maize.
We previously obtained transgenic rice plants via particle bombardment technology by using the scutellar tissues of mature embryos after a 7-day culture on the callus induction medium. In this experiment, the early events in rice scutellar tissues bombarded with DNA-coating gold particles were observed histologically. After bombardment, the gold particles were uniformly dispersed over the surface of the scutellar epithelium tissue which consisted of the dividing palisade cells. There were some cells containing a single or several particles on the surface of scutellar tissues. Although the particles hardly penetrated into internal cells, the introduction of foreign DNA into only the surface cells of epithelium tissue was sufficient for production of transgenic rice plants, because the single epithelium cell has the competence for transformation and regeneration.
The lipoxygenase (LOX) activity in susceptible and resistant rice leaves after inoculation with the bacterial pathogen Xanthomonas oryzae pv. oryzae was studied. In the compatible combination, the slight suppression of LOX activity was observed from 24 hours to 36 hours after inoculation. In the incompatible combination, LOX activity began to increase 12 hours after inoculation at the latest. This enhancement was observed up to 36 hours after inoculation. The bacteria inoculated onto the compatible rice propagated rapidly, but those inoculated onto the incompatible rice grew poorly. These results suggested the possibility that LOX was related to the bacterial leaf blight resistance of rice. The LOX activity in the susceptible rice leaves inoculated with the extracellular polysaccharide (EPS) mutant was similar to that in the leaves inoculated with the wild strain. This indicated that EPS scarcely affected the induction or suppression of LOX activity in rice leaves. After inoculation of the hrp mutant, the LOX activity in rice leaves was enhanced in the incompatible combination, but was not suppressed in the compatible combination. This indicated the possibility that some metabolic systems or secretion systems involving the hrp gene were related to the suppression of LOX activity in the compatible combination.
Strains of Mesorhizobium huakuii, root nodule bacteria of Astragalus sinicus, were identified from DNA polymorphism amplified by the polymerase chain reaction (PCR) using some known or random primers. Five strains of M. huakuii isolated in Japan and China were examined using SPH (a random primer), nifHDK (a part of the sequence of nif gene) and ERIC (both ends of an repetitive intergenic consensus DNA sequence of enteric bacteria). DNA bands amplified with primers showed distinctly different band patterns between groups of strains isolated in Japan and those isolated in China. Although it was sometimes difficult to identify strains belonging to the same group with only one primer, strains of a group could be identified by comparison of the results obtained with different PCR primers. Strains of M. huakuii isolated randomly from a rice field soil were examined by PCR, and several types of strains were found to survive at the same site. This identification method using PCR was also useful to investigate the infection rate of inoculated strains in a pot cultivation experiment.