| 1．Establishment of cryopreservation technique systems of longan and litchi embryogenic cultures by vitrificationIn the experiment, the effects of the factors including the subculturing time, throwing methods and developmental phases on cryopreservation by vetrification of longan embryogenic cultures were investigated. As a result, the cell survival rate after cryopreservation was higher by using longan embryogenic calli after subculturing 12～15 d; the comparison of 3 throwing methods (40℃water bath, room temperature 25℃and tap water)was carried out which indicated that they had the same effect on the survival rate after cryopreservation for longan calli. The survival rate of cryopreservated globular embryoids was higher than that of normal embryogenic calli.By experiment, the effects of low-temperature preculture, throwing methods and illumination condition on cryopreservation of litchi embryogenic calli by vitrification were tested. The results indicated that both 5℃low-temperature preculture for 2 d and using throwing with 40℃warm bath and tap water could all improve the survival rate after cryopreservation by vitrification significantly. In the experiment of recovery growth, we found that if the embryogenic c alli after cryopreservation had been in darkness for 15 d first, and then they were transferred under normal illumination, the rate of recovery growth became faster and the survival rate after cryopreservation was also higher.2．Somatic embryogenesis and plantlets regeneration normally from cryopreserved longan and litchi embryogenic calli by vitrification.Plantlets regenerated normally via somatic embryogenesis after cryopreservation of longan and litchi embryogenic calli. The number of somatic embryos was similar to that of the embryogenic calli before cryopreservation. Through the maturation culture, the somatic embryos could germinate and form plantlets normally. It showed that the ability of somatic embryogenesis and plantlets regeneration didn't changed after cryopreservation of longan and litchi embryogenic calli.3．Test of hereditary stability after cryopreservation of longan and litchi embryogenic calli by vitrificationThe hereditary stability of cryopreserved embryogenic calli from longan and litchi was tested by randomly amplified polymorphic DNAs (RAPD) technique on molecular level. The results of RAPD by 10 primers proved that somaclonal hereditary stability was almost preserved, only appearing little variation. It was founded that there were some differences between the amplified sites of embryogenic cultures before cryopreservation and after cryopreservation in exceptional primers. In the experiment, the variations of cryopreserved longan and litchi embryogenic calli were 3．0～3．5% and 7．48% respectively. The genetic variation of cryopreservation by vitrification was related to environmental stress. 4．Effects of low-temperature preculture on the survival rate and proteins expression for globular embryoids of longanThe study indicated that there were some direct effects on the survival rate and specific proteins expression after cryopreservation of longan globular embryoids. The survival rate of longan globular embryoids after cryopreservation reached the highest (82．58%) when precultured in 5℃low-temperature for 4 d, while the expression of proteins showed 4 novel protein spots appeared, 1 protein spot was up-regulated.The numbers of protein spots novel and up-regulated were 5, which were the most. The survival rate of longan globular embryoids after cryopreservation decreased to 70．25% when precultured in 5°C low-temperature for 4 d, while the expression of proteins showed 4 protein spots disappeared, 153 protein spots were down-regulated. The numbers of protein spots disappeared and down-regulated were 157, which were the most. All these suggested that the proteins novel and up-regulated strengthened the ability of anti-freezing while the proteins disappeared and down-regulated weakened the ability of anti-freezing.The former increased the survival rate after cryopreservation, and the later decreased the survival rate after cryopreservation. It showed that 5℃low-temperature preculture for 4 d was the best preculture method for cryopreservation of longan globular embryoids.5．Identification of novel proteins induced during 5℃low-temperature preculture by biology MS and analysis of their functionsIn the experiment, by using Matrix-Assisted Laser Desorption/Ionization Time of Flight-Mass Spectrometry (MALDI-TOF MS), the peptide mass fingerprinting (PMF) was analyzed in LLT1, LLT2, LLT3 and LLT4 which were the novel protein spots appeared during 5℃low-temperature preculture. The PMF data of these 4 proteins were used in Mascot search. The results indicated that LLT3 was perhaps resistance protein. It was predicted that LLT3 was an AFPs in longan and derived from PI. Moreover, it was predicted that LLT4 was a FDP-ALD in longan which was related to synthesis of compounds. The analysis of the function of proteins predicted that AFPs and proteins concerning synthesis of compounds expressed when 5℃low-temperature precultured for 4 d of longan globular embryoids. |
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