| Dwarf culture can be realized by using dwarf rootstocks which has become a inevitable tendency of apple culture development,so domestic and foreign scholars have paid much attention to dwarf rootstocks because of their some characters with early fruit,good quality,high yield and efficient.It is important to improve dwarf rootstocks by using regeneration technology in order to overcome their shortage.The apple dwarf rootstocks P59 and SH40 were explored as the experimental materials. The key factors which influenced regeneration,such as kinds and levels of hormone, selection and treatment of explants, culture conditions, polarity and maturity of leaves,were investigated in order to establish the high efficient regeneration system and make the base for transformation.Meanwhile the changes of endogenous polyamine and hormones contents were studied in order to provide theoretical basis for regeneration of adventitious shoots.The main results were as follows:1, The different places and concentrations of agar affected the growth status of tube-shoots significantly.When the Qingdao agar was used,we observed that the tube-shoots were strong,the growth of stems and leaves was normal,and the leaves were older.When the Guangdong agar was used,the growth of some stems and leaves was abnormal,the leaves were young,and the phenomenon of vitrification was serious. With the concentration of Guangdong agar rising,the phenomenon of vitrification was lightened.When the compound of Guangdong and Qingdao agar was used and the concentration of Guangdong agar was below 5.0g/L,the tube-shoots were strong,the growth of stems and leaves were normal,the leaves were young,and the phenomenon of vitrification disappeared.Considering the indexes of shoot height, internode length, the number of top leaves and growth status of tube-shoots synthetically, the growth status of tube-shoots was best with the compound of Guangdong agar 4.0g/L and Qingdao agar 2.0g/L.2, The different plant growth regulators had significant effects on leaf regeneration.It was shown that the regeneration rate rose obviously by using cytokinin BA and TDZ. The regeneration rate of TDZ was higher much times than that of BA ,so TDZ had the best regeneration efficiency.The optimum concentrations of BA and TDZ were 2.0 mg/L, 8.0mg/L respectively,and the regeneration rate were 85.62%,74.52% respectively.The auxins NAA was necessary for induction of adventitious shoots.The leaves in vitro were not able to regenerate without NAA.When some concentration of NAA was added to the medium,the leaves had the regenerative capacity.The optimum concentration of NAA was O.lmg/L.3^ Dark incubation was necessary for leaf regeneration.lt was investigated that direct exposure of leaves to light failed to regenerate adventitious shoots,but the regeneration rate rose obviously after incubation in the dark. If dark time was too long.it was disadvantage to induction of adventitious shoots,the best dark time was 20 days.4> The culture time of tube-shoots had some effects on regeneration.If the culture time was too short,the leaves were small and immature so as to regeneration rate declined.If the culture time was too long,the regeneration rate declined also due to leaves being older.The best culture time was 25~30d.5^ The different parts of the leaf had the different regenerative capacity.We observed that the basal leaf segments had the best regenetrative capacity,and the regenetrative capacity of middle segments was better than that of top segments.6> The effects of operation mode on leaf regeneration were significant.Incision could obviously prmote leaf regeneration,and the regeneration rate of leaves with abaxial surface uppermost was superior to that of leaves with abxial surgace uppermost.7-. Different additives had the different effects on regeneration.CH was able to increase the regeneration rate at the range of some concentration of cytokinin.AgNO3 was disadvantage to leaf regeneration,so the regeneration rate declined after using AgN03.8n Endogenous polyamine contents were closely associated with induction of adventitious shoots.The polyamine contents had the higher level during the early stage of leaf regeneration,and the polyamine contents in dark incubation was higher than those in light incubation obviously,while the polyamine content had the lower level during the late stage of leaf regeneration,and there was nearly no difference of polyamine contents between in dark and light incubation.Meanwhile,Put and Spm contents were reached the peak on the 10th day of culture,and both Spd and total polyamine contents and the ratios of Spd/PuU Spd/Spm and Spd/Put+Spm+Spd were reached the peaks on the 5th day of culture.The above results indicated that the high endogenous polyamine levels were closely associated with the induction of adventitious shoots.9> The endogenous hormone contents and balance were closely associated with induction of adventitious shoots.The hormone contents had the higher level during the early stage of induction of adventitious shoots,and the homone contents in dark incubation was higher than those in light incubation obviously.During the induction course of adventitious shoots in dark incubation,both ZR and ABA contents and the ratios of ABA/IAA and ZR/IAA were increased at the early stage,and then gradually declined,whileIAA and GA3 contents were declined.The above results indicated that the induction of adventitious shoots need high ZR and ABA contents levels and some concentration of IAA and GA3 contents,and the higher ratios of ABA/1AA and ZR/IAA were closely associated with induction of adventitious shoots. |
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