China Lycoris Axillary Buds Occurred Away From The Tissue Culture Process Development Learning Mechanism

Lycoris, mainly distribute in China, is a peculiar genus in monocots. It is diversity in flowerpattern as well as colors and also abundant in galanthamin and starch, which made Lycoris itselfgreat economic value.In view of morphogenesis, endogenous hormones, isozymes and gene, the developmentprocess of axillary bud regeneration was specifically studied in this research. And the mainresults are as follows:1. A complete definite-bud regeneration in vitro culture system with stable induction rateand proliferation rate was established through the induction, proliferation and rooting mediums.Extrogenous-cytokinin played an important role in the initiation of axillary bud and itssubsequent development. The morphogenesis process was described by serial sections andscanning electron microscope (SEM) in details.2. Genetic variability of micropropagated plantlets, also called somaclonal variation, wasassessed by two different PCR-based markers, ISSRs (inter-simple sequence repeats) and SRAPs(sequence-related amplified polymorphisms) markers. Both markers showed extremely lowmutation rates, which suggested that the established system is appropriate for clonal propagationand germplasm of Lycoris chinensis preservation.3. The mechanism of in vitro regeneration was sketchily discussed by3isozymes atprotein level in this paper. The results showed that there is a correlation between structuralchanges and protein activity and was proved by POX, AMY and ATP isozymes.4. CKs,IAA,GAs and ABA were token as judgement indexes to analyze the function ofendogenous-hormones. The results showed that these endo-hormones played different roles in thecourse of organogenesis development. Endo-cytokinin and auxin interacted together to changethe physiological status. They promoted "axillary bud primordium" transiting from "dormancystate" to "development state", organ initiation and morphogenesis. The interactions among theseendogenous-hormones affected the expression of downstream organogenesis genes and proteins.5. A proper RNA extraction method was established for analyzing the axillary buddevelopment mechanism at gene level. Efficiency of this method was testified by3kinds ofmaterials of5species of Lycoris.6. Complete-length cDNAs encoding KNOX was isolated from the bulb of L.chinensis byRACE. Bioinformatics analysis showed that the cloned LcKNOX1shared a high similarity withperviously reported monocotyledon KNOX protein.7. The expression profiles of LcKNOX1at11sampling times were analyzed byquantitative real time PCR. The results showed that there was a significant linear positivecorrelation between the level of CKs and the expression of LcKNOX1. That is to say, theconcentration of endo-cytokinin was directly associated with the elevation of expression ofLcKNOX1. Meanwhile, based on the results of qPCR and endogenous hormones, preliminary suggestions of reverse guidance for improving the in vitro culture process was proposed.