| In this experiment,the synchronization regulation and the high-frequecy regenerational technique of somatic embyogenesis from the embryogenic callus derived from transgenic protoplasts(TPEC)was developed by optimized the long-term subcultural methods and conditions of TPEC in litchi;the mechanism of the regeneration of somatic embyogenesis from TPEC was further researched by the determination of some physiological indexes of the activity of POD and amylase,the changes of POD and EST isozyme zymograms,the contents of amylase and total soluble sugar and the microstructure and ultrastructure observation of different somatic embryogenesis by paraffin section and the transmission electron microscopy(TEM);the high-efficiency regenerational conditions of protoplasts from TPEC were optimized from the Q81.535 suspension cells from TPEC cell lines;and the preliminary studies on the propolast fusion with PA-4000 electrofusion instrument were carried out between the Q81.535 suspension cells from TPEC cell lines in litchi and the friable embyogenic calli in longan.The main results were as follows:1.The optimization of the long-term subcultural conditions and the synchronization regulation of TPEC in litchi.The good litchi TPEC were altermatively cultured in the MS medium supplentmented with 1 mg·L-12,4-D and the MS medium supplentmented with 1 mg·L-12,4-D and 50 mg·L(-1) Hygromycin B every 20 days for 2 years,which maintained resistant and ernbryogenetic,and the ability of somatic embryogenesis were higher than that of the control.The synchronization regulation of TPEC cultured in the MS medium supplemented with 1 mg·L-12,4-D and 50g·L-1 sugar was better than that cultured in the MS medium supplement with 0.3 mg·L-12,4-D and 20 g·L-1sugar,through the differentiation of somatic embryos from TPEC cultured in the MS medium supplemented with 5 mg·L-1ZT and 50 g·L-1sugar and 10 g·L(-1- agar,the sizes of embryo tended to uniform,high degree of synchronization,and the frequency of somatic embryogenesis from litchi TPEC was improved immensely.2.Effects of phytohormones,osmoregulation matter,natural appendage on the induction and maturation of somatic embryogenesis from litchi TPEC.Different concentrations of cytokinins(ZT,KT,6-BA)affected the somatic embryogenesis from litchi TPEC differentially,the sequence affecting somatic embryogenesis was ZT＞KT＞6-BA.The results showed that the suitable medium of litchi somatic embryogenesis at high frequency was the MS medium supplemented with 3.0mg.L-1ZT,50 g·L-1sucrose,10 mg·L-1 agar and 100 mg·L-1 inositol which pH value was 5.8.On the medium,the frequency of somatic embryogenesis was up to 100%,the rates of early-stage white cotyledonary embryoids was up to 82%,individual larger,most of which was normal cotyledonary embryos.Coconut milk and longan juice were beneficial to somatic embryo maturation from litchi TPEC,the rates of normal embryos were 48.3%and 45.6%,respectively.However,ABA,BA and PEG were not favorable to the somatic embryo maturation from litchi TPEC,the rates of normal embryos was as low as 17.4%. 3.The researches of the mechanism of the somatic embryogenesis from TPEC in litchi.①The researches of some enzyme activity during the three stages of the long-tem culture,the somatic embryogenesis and the mature.During these stages the POD activity was totally descending trend,but that of spherical embryo was better than of the matural culture in the 15 days;the amylase activity was lower,the double-peak change ofα-amylase was earlier than ofα-amylase;during the different cell lines the activity of POD andβ-amylase in the four TPEC cell lines was higher than CK,but the activity of a-amylasea was lower than CK.The activity of POD andα-amylase was different between the white cotyledon embryo during the early-stage and vitrificational embryos and between the normally mature embryos and earlier mature embryos.②The analysis of POD and EST isozyme zymogram with polyacrylamide gel electrophoresis during the three stages of the long-tem culture,the somatic embryogenesis and the mature.The POD isozyme zymogram of somatic embryos of different TPEC cell lines and different forms and different matural times was different in the isozyme band and the number of the band;EST isozyme was stable,the EST isozyme zymogram in the different cell lines differenate in the isozyme band not in the number of the band,but two new stable isozyme band occurred in the somatic embryogenesis and the mature differenate in the expression levels not in the number of the band.③The changes of the contents of the total sugar and soluble amylase during the three stages of the long-tem culture,the somatic embryogenesis and the mature.The double-peak changes of the total soluble sugar in the somatic embryogenesis and the mature stage occurred in the spherical embryo and the matural culture of the 30 days;The three-peak change of the total soluble amylase occurred in the spherical embryo and the matural culture of the 15 days and 60 days;the contents of the total soluble sugar and amylase was different between the white cotyledonary embryos during the early and vitrified embryos and between the normally mature embryo and earlier mature embryo.④The studies on the microstructural and ultrastructural observation of TPEC somatic embyo by paraffin section and ultrathin section.The microstructure and ultrastructure of somatic embryos in the different stages and different forms were different.The microstructural characteristics of the cells in the early-stage white cotyledonary embryos induced from TPEC cell lines and the normally mature embryo was that the cells were regular forms and contained a lot of cell inclusions,that of the cells in the vitrified embryo and the earlier mature embryo was that the cells were bigger and contained a thinner cell wall,a bigger vacuole,a small cell nuclear,a nucleolus and a littel of cell inclusions.4.The preliminary studies on the propolasts fusion between litehi and longan.The isolation conditions of protoplasts from the Q81.535 suspension cell lines from TPEC cell lines in litchi was that the suspension cells altermatively cultured in the medium without Hygromycin B for 6～8 times in the 5 days were isolated by CPW-13M enzymatic mixture containing 1%cellulose and 1%pectinase under the continuous oscillation culture at 25±2℃in the dark.The yield and viability of the rotoplasts of TPEC reached 10.8×107 protoplasts·g-1and 95.3%,respectively.The high-frequency regeneration of the propolasts with calcium alginate beads suspensionally cultured in the modified KM8P medium supplentmented with 1.0 mg·L-1 2,4-D and 0.2 mg·L-1NAA and 0.5 mg·L-1BA and 0.5 mg·L-1KT and 50 mL·L-1CW and 250 mg·L-1glutamine and0.45 M glucose and 0.1 M sugar in the dark was established.It was better for the propolasts fusion between the Q81.535 suspension cells from TPEC cell lines in litchi and the friable embyogenic calli in longan under the conditions as follows:the propolast density of 5×105 protoplasts·mL-11.0 Mhz AC frequency and 50 V·cm-1AC voltage for 60s,600 V·cm-1DC strength for 10 ms,the pulse for 2-3 times.The rate of the propolasts fusion reached more than 30%.The fusional produces cultured in the KM8P medium supplentmented with 1.0 mg·L-12,4--D and 0.2 mg·L-1NAA and 0.5 mg·L-1BA and 0.5 mg·L-1KT and 50 mL·L-1CW and 250 mg·L-1 glutamine and 0.45 M glucose and 0.1 M sugar formed polycell clusters,and the rate of planting efficiency reached 12.5%.The technique would serve the creation of new germplasm and the new kinds of fruit in litchi and in longan.
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