| Summary: | 碩士 === 國立臺灣大學 === 園藝學研究所 === 89 === Chapter I The regulation of plar and apolar grwoth in pre-embryogenic cells of triploid banana Musa AAB cv. Raja.
The extacellular pH in the embryogenic cells suspension of banana Musa AAB cv. Raja auto-regulated in the narrow range 4.0-4.6, and dominated in proliferation growth phase during longterm maintenance culture refreshed with TB5 medium in each 14 days interval. It was showed that the adjusted medium pH at 3.8-5.8. There were changed to 4.15-4.71 after autoclave, and autoregulated to 4.35-4.68 in the second subcutlure generation. In the meantime, all the cell population of different pH treatment remained the same proliferation growth phase active in apolar growth. The change of subculture duration or refresh medium ratio was also without effect on extracellular pH and growth phase. The addition of NaH2PO4 250mg/L was efficiently to buffer the external solution pH at 4.7-5.5, concomitantly induced the pre-embryogenic cells destining to polar growth and forming clustered globules.
It was demonstrated that the growth phase change could be simulated through controlling pH level. Both growth types of proliferation and globularization phase were efficiently directed into multiplication phase via acidic pH3.5-4.0 treatment. Under the acidic condition, the pre-embryogenic cells masses of proliferation phase were activated in releasing homogenous free cells with high embryogenic competenc within 2-3 weeks. In contrast, the globules of globularization phase reproduced less proportion of pre-embryogenic cells and required longer time 5-7 weeks for complete disorganization.
In response to controlling pH4.8-5.3, both aploar growth types of multiplication and proliferation phases were rapidly directed to polar growth of globularization phase, and respectively formed of solid globules and clustered globules.
These results supported the the acid growth model that the acidic condition may interfer the establishment of polar axis in the pre-embryogenic determined cells, and conducted to apolar growth in formation of amophically pre-embryogenic cell masses. The acidic treatment cells were dominated in symmetric cell division, the size ratio between two daughter cells was examined as 1.02±0.02. The lightly acidic condition pH4.8-5.3 may serve as signal for induction of polar axis and resulted in unequal division, the size ratio of two daughter cells was 1.30±0.04 among the cell lines different genomic groups. Therafter, the development fate of apical and basal cells was defined in this research.
Chapter II High efficient synchronization and mass production somatic embryos of triploid banana Musa AAB cv. Raja.
The cell lines in embryogenic suspension culture of banana Musa AAB cv. Raja was dominant in apolar proliferation consisted of pre-embryogenic cell masses (PEMs) and heterogeneous cell population, which displayed highly embryogenic competency assessed on regeneration medium capable to form somatic embryos 9254±1554 with 1/30 ml CPV. But the induced somatic embryos were unsynchronized in growth, and limited in embryo-plantlet conversion rate.
Giving control extracellular pH3.5-4.0 level, the PEMs of proliferation phase were forced to multiplication phase active in releasing pre-embryogenic cells that only reproduced 821 embryoids with 1/30 ml CPV. Subsequently, the pre-embryogenic free cells were treated with controlling pH4.8-5.3 level which were directed to polar growth, and formed uniform globules. It was demonstrated that the 1/30 ml CPV was capable to form 33,429±784 somatic embryos and 89% uniformity on SH3 regeneration medium, it was estimated as high as 106 somatic embryos per ml CPV.
The somatic embryo-plantlet conversion rate was achieved over 90% on MS medium supplemental with NAA 0.1 mg/L medium culture and the trans plant able plantlets were established in less than 30 days. This method could induce synchronization, individualization, normalization of somatic embryogensis, and high plantlet conversion rate. It is benefit for production of massive and qualitative somatic embryos.
The pre-embryogenic cells of multiplication and proliferation phases could be directed to polar growth, and develop to globular body with basal suspensor. Subsequently, exposing the globules in liquid SH3 regeneration which were capable to differentiate morphological bipolarity and become mature somatic embryos. The evidence indicated that the controlling pH directed to polar growth and globularization, and the development of somatic embryogenesis, all the sequences could be conducted under alternative liquid medium condition. The quantities production of somatic embryos in bioreactor would become feasible, but the technique of quality improvement is still required to be studied.
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