| Plant tissue culture is very important for the somatic genetics, and it can be used not only to study on the mechanism of growth, development and differentiation of plant cell, but also to product valuable protein as bioreactor and to improve crop in breeding. In this study, plant regeneration through organogenesis of shoot bud from callus cultures of strawberry was established and the metabolism of sugar, protein and reactive oxygen species (ROS) was investigated in order to understand the mechanism of differentiation of plant tissue and cell in vitro. In addition, the cloning of EGF gene and its transformation to the tobacco and strawberry callus were carried out. The main main results are as follows:1.The establishment of plant regeneration system through organogenesis from callus cultures of strawberryThree types of callus cell lines were established due to the growth regulator added to the MS medium and the culture condition, and they showed different potential for morphogeny after being transferred to the same regeneration medium. The type I callus was induced quickly, and was soft and grayish white, with a smooth, wet-looking surface. The type II callus was light yellow, compact and granular, and grew much more slowly. The type III callus was green, compact and tubercular. The type I callus had lower regeneration capacity and the type III had higher. The callus expressed their totipotency through the direct organogenesis rather than the somatic embryogenesis.The formation of different types of callus were mainly controlled by the growth regulator and light, and there is little relationship between the explants and the type of callus.2. The possible relationship between the metabolism of sugar, protein and ROS(1) Three types of callus had different sugar and protein levels. The type I callus possessed higher soluble sugar and decreased sucrose, in the contrast with II and IIIcallus. The II and III calli showed 6-fold as much as the content of water soluble protein, compared with the I callus. SDS-PAGE suggested that the protein was strongly expressed in the callus that differentiated to form shoot cluster and root. During the period of subculture, the sucrose in calli sharply decrease in 15 week, and lover of the soluble sugar keep constant. The protein level in type II and III callus decreased slowly, while what in the type I callus quickly. The ability of expression of totipotency in calli decreased, especially the callus I lost the ability after 12 week subculture. It seemed that there existed some relationship between the regeneration capacity and level of sucrose and protein.(2) During the differentiation and development of callus, H2O2 productioncoincided with emergence of meristemoid and formation of bud primordium in morphogenic calli, SOD activity increased in the early regeneration culture and decreased thereafter, and catalase activity constantly declined while peroxidase decreased during the 5-d culture and gradually increased thereafter. The inhibition of H2O2 production by DDC decreased the percentage of calli generating shoot bud, and exogenous addition of H2O2 slightly promoted the potential for regeneration of shootbud.In the freshly induced calli, the II and III types of callus had 5-fold and 9-fold higher contents of intracellular H2O2, and 3 -fold and 4-fold higher level of O2-, compared with the I type of callus, respectively. Also, the calli II and III had much higher activities of antioxidant enzymes than the callus I. During the subculture, H2O2 level increased gradually and O2'- rapidly, while the activities of antioxidant enzymes gradually declined. The results indicated that the ROS might play a dual role in the regeneration of strawberry calli. On the one hand, a certain level of ROS may have a positive effect on the regeneration, and on the other hand, high level of ROS is inhibitory for the expression of totipotency in calli.3. The cloning of EGF gene and its transformed to the tobacco and strawberry callusTotal RNA was isolated from m...
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