The Expression Pattern Of Genes Involved In Organogenesis And Somatic Embryogenesis In Arabidopsis

Cell totipotency is one of the characteristics of plant which means that any plant cell has the potency to develope into an intact plantlet. The process that cultured tissues or cell aggregates differentiate into adventitious root, adventitious shoot or flower bud is named in vitro organogenesis, while the process that one somatic cell in vitro develops into a whole plant similar with zygote embryo is named somatic embryogenesis. Up to the present, the mechanism in adventitious shoot regeneration and somatic embryogenesis is still not well understood. Therefore, it is necessary to investigate systematically.In this study, we established a rapid and high frequency system of shoot regeneration. Different from the somatic embryogenesis system, the explants were mature seeds, actually post-geminated seedlings in MS medium containing 2, 4-D. The morphological and histological changes of adventitious shoot regeneration were examined in detail. We analyzed the temporal and spatial expression patterns of genes involved in organogenesis, including miR165a, miR166a and REV. The role of auxin polarity distribution in shoot regeneration was discussed. The results provided information for understanding the molecular mechanism of in vitro organogenesis and shoot regeneration.In addition, unable-differentiated and differentiated callus were obtained from Arabidopsis germinated-seeds, the expression of genes related with somatic embryogenesis was dectected. The results provided important information for further research of somatic embryogenesis.The main results in this study are summarized as follows:1. Establishment of culture system with high frequency of shoot regenerationTo induce in vitro organogenesis from the shoot meristem and produce secondary organs, mature seeds were cultured in liquid medium containing 2, 4-D. After removing auxin, the existing shoot primordium and leaf-like structure developed into adventitious shoot. Longitudinal section of an adventitious shoot showed the regeneration structure was directly connected with the existing vasculature of explantsthrough procambium, while absent of root meristem.2. Expression pattern of several genes involved in organogenesisThe same culture system of shoot regeneration was respectively established with homozygous seeds of miR165a::GUS, miR166a::GUS, REV::GUS, and the expression of miR165a, miR166a, REV in organogenesis in vitro and shoot regeneration were detected by GUS activity.In the presence of auxin, miR165a was not expression in enlaged SAM, then expressed in one and several cells in its inlateral and bilateral. This suggests that miR165a may function in organ inition. Overexpression of miR166a promoted shoot genesis wich futher domenstrated miR165/166 played important roles in shoot regeneration. They also function in adaxial-abaxile polarity cdevelopment of leaf-like structure and adventious shoot presumed from their expression in lateral of these structures. In addition, miR165a was related with secondary leaf organogenesis.REV was expressed both in organ primodium induced by 2, 4-D, and leaf tip of adventitious shoot. Additionally, it seems that REV plays more important role in vascular tissue development for its strong expression in the central primodium cells and leaf vein.3. Auxin polarity distributionDR5::GUS, as a synthetic reporter of auxin-induced transcription, marks an auxin perception maximum in local region. Maximum auxin accumulated at the tip during organ formation induced by auxin, and also at leaf promordium tip of adventitious shoot. On the base of regeneration shoot and its peripheral region also accumulated auxin. Its concentration gradually reduced with adventitious shoot development. Eventually, adventitious roots differentiated from this region. The results indicate that auxin polarity distribution is required for organogenesis in vitro and adventitious shoot development. Adventitious root regeneration also requires local auxin accumulation.4. Expression of genes related with stem cell in Arabidopsis callusThree phenotypes of callus were obtained from Arabidopsis. One type of callus was green-particle and incompact, the second one was yellowish and compact, and the third one was green with tumor-like structure. The green-particle callus cannot differentiate into plant, while the other two have the capability. In order to analyze the molecular mechanism for their different phenotype, WUS expression was analyzed by in situ RNA hybridization in the three types of callus. The result showed that WUS mRNA were detected in somatic embryos in yellowish and compact callus, but absent from green-particle, incompact callus. This suggests WUS may functions in somaticembryogenesis.