Studies On Optimization System For MRNA Differential Expression In Process Of Adventitious Root Formation, Rooting And Transplantation In Apple Tissue-cultured Seedling

Apple rootstocks M₂₆shoots propagated in vitro were used for the establishment ofthe rooting culture system which agar replaced by vermiculite after a brief inductionpretreatment, apple rootstocks SH₂₈and SH₂₉in vitro were used for the effect ofenvironmental factor on the growth of the plantlets after transplanted, and M₂₆shoots invitro were used for study of establishment of gene differential screening of initiation anddevelopment of adventitious roots. The results were as follows:1. The optimization PCR system for mRNA differential display in tissue-cultured seedling contains: annealing temperature45℃，cDNA （diluted for10times）2μL，Taq polymerase2.0U，random primer2.5μmol·L^-1，anchor primer2.5μmol·L^-1, dNTPs125μmol L^-124primer combinations （8random primers and3anchor primers）were used in PCR ampli-fication for the test materials under the optimization PCRsystem. The results showed:11primer combinations can amplify bands, and nine ofthese primer combinations have better production of amplification, the main band is clear, DNA banding patterns have significant differences.41differential genes were amplified by the nine primer combinations. Among them,38genes were specially expressed,2genes’ expressions increased while1genes’ expressions decreased after the induction.2. M₂₆shoots in vitro which were cultured in agar medium containing0.6mg·L^-1IBAfor7～9days were interposed in vermiculite medium. The shoots’ rooting rates wereexceeded94%and were significant higher than CK of agar medium. And the number oflateral roots were significant higher than cultured in agar medium containing0.6mg·L^-1IBA for11～13days. The rooting rates of the shoots which cultured in vermiculitemedium containing20g·L^-1sucrose were95.18%. But the number of rooting, number oflateral roots and length of roots in shoots were significant higher than which not containingsucrose. Water content of vermiculite medium impacted the rooting of the shootssignificantly. When water content was from50%to90%, the rooting rates of shoots were exceeded96%and significant higher than CK of agar medium. But number of rooting wasnot remarkable difference. When water content was50%or60%, the shoots had thehighest number of lateral roots which was6.69or6.46and significant higher than othertreatments. The shoots cultured in agar medium had the longest length of roots by2.89cm.The rooting rates increased significantly by100%when M₂₆ shoots in vitro which werecultured in agar medium containing0.6mg·L^-1IBA for7～9days were interposed invermiculite medium containing20g·L^-1sucrose and60%water content after dipped by100mg·L^-1IBA, the number of rooting, number of lateral roots or length of roots wereincreased substantially, and the root quality had significant improvement.3. It was studied that the combination of induction of adventitious root anddomestication used vermiculite medium system established formerly. The results showedthat the rooting rates, the number of rooting or the length of roots were no differencebetween in or out Test-tube rooting of Test-tube Seedlings used vermiculite mediumsystem. But the numbers of lateral root cultured out were significant higher than in it.4. Plantlets cultured in the culture chamber for8d and moved to the greenhouse toexercise for17d is the best combination; cultured for8d in the culture chamber andexercised for11d、17d and25d in the greenhouse,17d is also the best time to exercise. Theplant height, leaf number, leaf area and single leaf area of the transplanting tissue culturebetween the two groups of treatment are significantly improved. The survival rates of theplantlets were significant higher when using the mix of turfy soil and vermiculite at ratio of1:1or1:2, and could reach88.24%or90.77%respectively, compared to the using of theratio of2:1. Meanwhile, the plantlets had more growth amount when transplanted in thesubstrate mixed with turfy soil and vermiculite at ratio of1:1. The turfy soil pre-treatedwith autoclave had an effect on the increasing of the leaf area of the plantlet and the singleleaf area, and contributed to more healthy plantlets. The supplement of0.1%Bacillussubtilis in the substrate had no significant effect on the plantlet growth. The watering of thenutrient solutions could accelerate the plantlet growth, which could be seen40days afterthe application, and the formula of MS showed the best in general.