|Budget Amount *help
¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 2000 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1999 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1998 : ¥1,500,000 (Direct Cost : ¥1,500,000)
The results of this study were summarized as the following.
1), To clarify the mechanisms of aposporous initial cell (AIC) appearance and apomictic embryo sac formation, several apomictic and obligately sexual varieties of bahiagrass were studied cytological and quantitatively. After megasporogenesis, the megaspore usually degenerated. AIC appeared and was divided into 4-nucleate embryo sac. The numbers of AIC increased as the ovary grew before anthesis. At anthesis, the sac in micropylar end (first sac) was dominantly formed. After anthesis, the first sac divided dominantly. In final, it occupied the whole space of the ovule. And the embryos of the other sacs coexisted with the first sac.
2), The nucellar ultrastructure was analyzed during the meiocytic stage and during aposporous embryo sac formation in apomictic guineagrass with TEM.Degeneration of dyed stage of MMC was observed in this study as the first time. The chalazal nucellar cells are meristemic until the tetrad stage. Later,
they can turn into AIC.Their increased cell size, large nucleus, and the presence of many vesicles can recognize the AIC.
3) AIC appearance stage-specific gene, ASG-2, has been isolated and characterized from guineagrass using a novel differential screening strategy according to Chen et al.(1997). Sequence analysis indicated that the ASG-2 cDNA (1199 bp) codes for a protein of 173 amino acids. The amino acid sequence alternatively is related to the sequences of RD22, seed-specific and drought-induced gene of Arabidoposis thaliana, and USP, unknown seed protein precursor of Vicia faba.
4) Megasporogenesis (A1), AIC appearance (A2-1) and AIC-derived embryo sac formation (A2-2) of ovary were observed with in situ hybridization. The ASG probes were not detected in A1. As the ovary grew into A2-1, AIC cell gave the strong signals. In the A2-2, the signals were only detected in embryo sacs, especially in the reproductive cells. The sexual young buds of guineagrass showed no signal in any stages tested.
5) Culture conditions for somatic embryogenesis and plant regeneration from leaflet tissues of guineagrass are established. Embryogenic calli and somatic embryogenesis were initiated from yellowish calli derived from leaflets. The embryogenic calli were confirmed using SEM.Now, making transgenic plant is in progress. Less