Study On The Mechanisms Of Somatic Embryogenesis In Arabidopsis

The zygotic embryo-like structure can be induced from plant organs, tissues and cells. The initiation of this structure, called somatic embryo, is different from that of zygotic embryo. Somatic embryogenesis is an important way to generate whole plants. The developmental process of somatic embryo resembles that of zygotic one, probably due to conserved cellular and molecular mechanism between two processes. Thus, somatic embryogenesis has been recognized as a potential model for studying the plant cell totipotency and the developmental events in plant embryogenesis. Here, we focus on studying the mechanisms of the somatic embryo (SE) induction in Arabidopsis. The main results are as follows:1. Somatic embryogenesisTo induce the SEs, the discs-like embryonic calli were induced in the presence of auxin. By removal of auxin, the somatic proembryos occurred on the edges of the embryonic calli inside. Then, cotyledon primordia were observed on the somatic proembryos. Eventually, mature somatic embryo is composed of cotyledons, hypocotyl and radicel.2. Expression patterns of several genes involved in embryogenesisSeveral genes including LEC1, LEC2, FUS3, PEI1 and ABI3, are involved in zygotic embryo development. The expressions of these genes during somatic embryogenesis were determined by semi-quantitive RT-PCR. The results showed that LEC1, LEC2, FUS3, PEI1 and ABI3 genes appeared to be up-regulated at 2 days after the embryonic calli transferred to the auxin-free liquid medium. Furthermore, localization of LEC2 transcripts was analyzed by pLEC2:: beta-glucuronidase (GUS) expression. The LEC2 gene expression was detected in somatic proembryos. Then, the signals were detected in the cotyledon primordia and mature cotyledons of SEs, indicating that expression patterns of the genes involved in zygotic embryogenesis are regulated during SE induction.3. Expression patterns of CLV3 and WUSTo study the initiation of the stem cells for somatic embryogenesis, we examined the expression patterns of CLV3 and WUS which were reported to express in the shoot apical meristem (SAM) during embryogenesis. The results showed that localization of the WUS transcripts was identified in a group of undifferentiated cells below layers (L) 4~6, which were called organizing center cells at the early stage of somatic embryogenesis. Then, the CLV3 signals were accumulated in L 1~3 of proembryos. When the cotyledons emerged, the CLV3 and WUS transcripts were detected in the shoot meristem of somatic embryos. Further study showed that the transgenic tissues with antisense WUS cDNA could not generate embryos. Moreover, transcripts of several genes involved in embryogenesis could not be detectable in these cultured tissues. Thus, the results indicate that the organizing center is induced earlier than stem cells during SE induction, and WUS expression is essential for theSE formation.4. Auxin polar transport and distribution Auxin is essential for somatic embryogenesis. We found that auxin polar distribution occurred during the SE induction process. If the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) were added in liquid medium containing 2,4-D, auxin polar distribution and expression pattern of WUS were destroyed in the calli; and after 14-day culture, the SEs were failed to be induced. Auxin polar distribution might be involved in PIN-dependent transport. Polar localization of PIN1 was observed in the cells that would form proembryos. The co-expression analysis showed that the WUS expression domain partially overlapped the PIN1 expression one at the early stage of SE induction. Later, polar localization of PIN1 flew to the region in which cotyledon premordia would be produced, and whereas WUS was constitutively expressed in the cells of organizing center in the shoot meristem. Thus, the results suggest that auxin polar transport and distribution play important roles during somatic embryogenesis.5. Effect of different levels of auxin It was found that appropriate level of auxin was critical for induction of SEs. At other levels of auxin, SE could not be induced, and WUS expression pattern was altered. These results indicate that reorganization of auxin polar distribution and formation of organizing center requires the appropriate level of auxin.In this study, the results suggest that auxin polar transport and formation of organizing center cells are essential for induction of SEs. And WUS may function through promoting expressions of genes involved in embryogenesis during somatic embryogenesis.