Studies On Regulation Of Somatic Embryogenesis And Related Anatomy And Proteomics In Camellia Nitidissima Chi

In this experiment the repetitive in vitro regenerating system originated from the adult stem-tips was used as the material resource for the studies on the regulation of somatic embryogenesis and the related developmental anatomy in Camellia nitidissima Chi.(yellow camellia; besides, 2-Dimensional Electrophoresis was applied to analyze protein expression of different developmental phases during somatic embryogenesis. The abnormal developing pathway in Camellia nitidissima Chi., through which somatic embryos formed multiple cotyledons and flower-like shape, (named as MCFS pathway) was a special phenomenon in plant development, which had not been reported. The studies on the phenomenon was conducive to fathom the mechanism of some specific processes of plant in vitro morphogenesis and important to consummate the theory of the developmental biology. The obtained results showed as follows:1．The effects of different combinations of plant growth regulators on the callus induction were compared. The results indicated that the pieces cut from somatic cotyledonary embryo derived from the repetitive somatic embryogenesis system established in our laboratory were cultured on the medium adding 2mg/L 2,4-D and 1mg/L KT and subcultured every 7 days, the calli formed after 3～4 times, and then somatic embryogenesis occurred on the differentiation medium without growth regulators, and many somatic embryos with multiple cotyledons and flower-like shape (named as MCFS embryo) were observed..2．The repetitive MCFS embryogenesis system was established. Almost the same type of MCFS embryos clould be obtained on MS medium adding 0．22mol/L mannitol and 0．12 mol/L sucrose after augmenting osmotic pressure of the medium step by step and selecting typical materials every generation. After being transferred onto the 1/2 MS medium adding 0．2mol/L NAA and 2mol/L BA, the abnormal somatic embryos turned magenta or even became brown; finally, the browned body generated nearly synchronized and identical secondary MCFS embryos on the surface. Going back to the first step with the secondary MCFS embryos could repeat the process.3．The anatomic observations on the materials of different developmental phases in the MCFS pathway and normal materials by paraffin sections were compared. The materials from MCFS embryos had unique morphologic features, and the transforming order from cotyledon to leaf was from leaf margins of both sides to main vein. The MCFS embryo was distinct from lee mutant of Arabidopsis by comparison.4．The 2-DE analyses of the expression of the total proteins of the materials in the different phases during the MCFS pathway and normal developmental pathway, including globular embryos, cotyledon embryos, mature embryos, plantlets, and the calli induced by adding 2mg/L 2,4-D and 1mg/L KT, was carried out..During the normal developmental pathway, from the globular embryo phase to the cotyledon embryo phase, there were 4 protein spots disappeared or weakened obviously, i.e., CnPGl(63．9KD), CnPG2(29．9kD), CnPG3(30．6kD) and CnPG4(29．1 kD), among which CnPG2(29．9kD), CnPG3(30．6kD) and CnPG4(29．1kD) weakened obviously since the cotyledon embryo phase; and there were 9 new protein spots in the cotyledon embryo phase, i.e., CnPCl(73．3kD), CnPC2(70．5kD), CnPC3(35．4kD), CnPC4(44．1 kD), CnPC5(28．4kD), CnPC6 (20．1kD), CnPC7(19．1kD), CnPC8( 17．4kD), CnPC9(19．0kD), among which CnPC1 (73．3kD) and CnPC2(70．5kD) weakened gradually later and completely disappeared in the plantlet phase. From the cotyledon embryo phase to the mature embryo phase, there were 25 protein spots disappeared or weakened obviously, i.e.,CnPC10(58．4kD), CnPC4(44．1 kD), CnPC11(48．1kD), CnPC12 (45．0kD), CnPC13(40．0kD), CnPC14(19．0kD), CnPC 15(42．1 kD), CnPC6(20．1 kD), CnPG4 (29．1kD), CnPC7(19．1kD), CnPC 16(23．0kD), CnPC17(18．8kD), CnPC9(19．0kD), CnPC8 (17．4kD), CnPC 18(21．3kD), CnPC 19(35．1kD), CnPC20(29．2kD), CnPC21(30．4kD), CnPC22 (20．7kD), CnPC23(19．0kD), CnPC24(18．9kD), CnPC25(16．0kD), CnPC26(32．6kD), CnPC27 (32．3kD), CnPC28(28．4kD). among which CnPC4(44．1 kD), CnPC6(20．1 kD), CnPC7(19．1kD), CnPC8(17．4kD), CnPC9(l9．0kD) appeared in the cotyledonary embryo phase only and CnPG4 (29．1kD) completely disappeared in the cotyledonary embryo phase. There were 3 new protein spots in the mature embryo phase, i.e., CnPM1(21．0kD), CnPM2(18．5kD), and CnPM4(15．3kD). CnPM3(16．3kD) strengthened obviously. From the mature embryo phase to the plantlet phase, there were 16 protein spots disappeared, i.e., CnPM5(72．5kD), CnPM6(73．1kD), CnPM7(60．6kD), CnPM8(71．1kD), CnPG2(29．9kD), CnPG3(30．6kD), CnPM9(27．6kD), CnPM 1(21．0kD), CnPM10 (21．5kD), CnPM 11(21．0kD), CnPM12(20．0kD), CnPM3(16．3kD), CnPM13(15．7kD), CnPM14 (14．1kD), CnPM15(13．1kD), CnPM16(12．2kD), among which CnPM 1(21．0kD) only appeared in the mature embryo phase, CnPG2(29．9kD) and CnPG3(30．6kD) weakened since the globular embryo phase, then disappeared in the mature embryo phase. There were 5 new protein spots in the plantlet phase, i.e., CnPP1(55．7kD), CnPP3(57．2kD), CnPP4(55．3kD), CnPP5(55．3kD), CnPP6 (47．2kD). CnPP2(57．5kD) existed in all the phases of the normal developmental pathway, and dramatically increased in expression level in the plantlet phase.The discrepancy of protein expression in MCFS pathway was as follows:①CnPG5(31．4kD) had already appeared in the globular embryo phase of the normal developmental pathway, but had not appeared until the cotyledonary embryo phase of MCFS pathway.②CnPC4(44．1kD) and CnPC5(28．4kD) had not appeared until the cotyledonary embryo phase of the normal developmental pathway, but had already appeared in the globular embryo phase of MCFS pathway.③CnPG4(29．1kD) weakened since the globular embryo phase and disappeared before the mature embryo phase during the normal developmental pathway. In contrast, during MCFS pathway, its expression amount was obviously less in the globular embryo phase than that of the normal developmental pathway, and the protein spot disappeared in the cotyledonary embryo phase.④From the cotyledonary embryo phase to the mature embryo phase during MCFS pathway, CnPP2(57．5kD) and CnPM17(46．4kD) disappeared, which had not yet happened during the normal developmental pathway.⑤CnPM 1(21．0kD) had never appeared during the MCFS pathway.⑥CnPD(14．9kD) which existed from the globular embryo phase to the mature embryo phase was exclusive for the MCFS pathway, but it never appeared in any phase during the normal developmental pathway.In the plantlet phase, there were 5 more protein spots during the normal developmental pathway than that during the MCFS pathway; besides, in the plantlet phase during the MCFS pathway, CnPC7(19．1kD) still existed.CnPD(14．9kD) and CnPC5(28．4kD) were also observed in the callus phase by comparing with the globular embryo phase during the MCFS pathway.