| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
The results of this study were summarized as following :
1),to carry out genetic transformation, as a first step, efficient plant regeneration system should be established. In this study, we used various materials which could be collected in any season and any times, and have established plant regeneration systems by the way of somatic embryogenesis from leaflets, seeds and ovaries of sexual and apomicitic guineagrass, and from sexual rice. The somatic embryogenesis on calli of guineagrass was observed and confirmed with eletronic microscope.
2),based on the establishments of plant regeneration systems on guineagrass and rice, several kinds of calli of them were used for gene transformation with 3 kinds of binary vectors containing peta-GUS gene and mediated with agro-bacterium. The calli infected with agro-bacterium were moved onto selection medium containing antibiotics for selection, and survived calli were removed onto regeneration medium for plant regeneration. Complete plants were
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regenerated on hormone free medium in final. The peta-GUS examination was carried out on regenerated plants, indicating the GUS fluorescence be visible. Therefore, the genetic transformation systems have been established in guineagrass and rice.
3),the construction of 3 kinds of binary vectors and apomixis candidate gene ASG-1l were carried out. They were pIG121Hm35S :: ASG1, pSMA35H2 :: ASG1, and pActnos/Hm2 :: ASG1. In them, the antibiotics were contained but the peta-GUS was replaced with ASG1 gene. However, the agro-bacterium of GV3101/PMP90 was used for pSMA35H2 :: ASG1, and the EHA101 was for the others.
4),based on the construction of 3 kinds of binary vectors and ASG1, they were mediated with different agro-bacterium for genetic transformation. As the peta-GUS indicator was replaced, the culture was continued in steps of co-culture with agro-bacterium, selection medium, callus propagation, regeneration medium and hormone free medium for plant regeneration. In final, complete plants were achieved and they are cultured in incubator of P2 level.
5),for the examination and gene expression of ASG1,total DNA were extracted from calli and leaves of guineagrass and rice, respectively. When the primers were made according to the sequences of ASG1, the total DNAs were used as template and amplified with the primers using HITACHI i-chip PCR system. A band with 900bp in size was detected from all the materials provided from guineagrass and rice. This result indicated that apomixis candidate gene ASG1 has been transferred into the regenerated plants. Now, the experiments are in progress, in order to observe whether the reproductive mode of the plants is changed or not. Less
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