Induction And Molecular Mechanism Of Plant Somatic Embryogenesis By Leafy Cotyledon (LEC) Gene

Leafy Cotyledon (LEC) genes including LEC1and LEC2are key genes in plant embryo development. During embryo morphogenesis phase, LEC genes are required to specify suspensor cell fate and cotyledon identity. In embryo maturation stage, LEC genes are required for the acquisition of desiccation tolerance and the expression of many maturation-specific genes. They are the key regulators in plant somatic embryogenesis which can induce vegetative-to-embryonic transition.Somatic embryogenesis is the process that somatic cells, under inductive conditions in vitro, generate embryonic cells, which undergo a series of morphological and biochemical changes resulting in the formation of somatic embryos. Somatic embryogenesis has been recognized as a model of the plant cell totipotency. Many crops have low regeneration rate, which has became the limiting factor in plant genetic engineering. Increasing the regeneration rate, for example, the rate of somatic embryogenesis, and building an efficient plant regenerate system is the prerequisite of improving crops by genetic engineering, especially soybean, cotton and peanut that are difficult for in vitro regeneration.LEC genes can induce somatic embryogenesis in Arabidopsis. And the transgenic plants can’t complete the whole life cycle if constitutive express LEC genes. In this article, we constructed an inducible expression vector which contain35S:AtLEC:GR gene and transformed them into tobacco (Nicotiana tabacum) aiming to establish a flexible system to use the LEC genes. DEX (dexamethasone) was used as the inducer.Overexpression of AtLEC gene induced vegetative-to-embryonic transition in tobacco and obtained plenty of somatic embryos, which proved the function of LEC gene in somatic embryogenesis. Using tobacco we built an efficient plant regenerate system which could be used for cotton and other crop improvement through gene engineering.The main results of this study are listed:1. Construction of plant inducible expression vector and tobacco transformation: Two inducible expression vectors pART-AtLEC2:GR and pCAMBIA2300-AtLECI:GR were constructed. Agrobacterium-mediated transform method was used for tobacco transformation. After co-culturing in dark, antibiotic screening and root generation we obtained16LEC2transgenic lines and19LEC1transgenic lines. Finally, we had7and5homozygous transgenic lines respectively.2. Induction of embryonic callus and somatic embryogenesis:Transgenic seeds germinated in MS supplemented with dexamethasone (DEX). LEC1transgenic tobacco seedlings grown in DEX medium showed embryonic morphology. The cotyledons were fleshy and failed to expand. However, these seedlings could not produce callus and embryo-like structure. LEC2transgenic tobacco seedlings grown in DEX containing medium produced plenty of embryonic callus on the shoot apical meristem region. Subsequently, somatic embryo-like structures developed from these embryonic callus and somatic embryos. Different DEX concentrations were used in the above experiments. The results indicated that5μM,10μM were the best concentration for transgenic tobacco regeneration. Each embryonic callus could produce plenty of regeneration blastemas and they could develop into normal plants.3. The effect of LEC2expression on endogenous hormone:We determined the level of endogenous hormone in LEC2transgenic and WT tobacco by HPLC. The results showed that IAA in LEC2transgenic tobacco embryonic callus induced for20d by DEX and regenerated callus cultured on MSo for20d were2.87and5.27times higher than that in callus from WT tobacco on the MS media with hormone respectively; zeatin were1.25and6.21times higher than wild typed callus respectively.4. Gene expression profiling of LEC transgenic plants and WT plant:We analyzed the gene expression pattern in LEC1transgenic and WT tobacco seedlings which were treated with30μM DEX for12d by DGE (Digital Gene Expression Profiling). The results showed that the expression level of embryogenesis related genes, such as MADS-box9, SERK1and several seed storage protein genes(LEA protein, oleosin, caleosin,7S,11S globulins and vicilins) was up regulated markedly. The auxin response and transport related genes such as ARF3, ARF8, ARF5and PIN1were up regulated. The expression of ACO, a gene that involved in ethylene signal transduction and have negative effect on somatic embryogenesis, was down regulated.We also analyzed the gene expression pattern in LEC2transgenic seedlings which were treated with30μM DEX for20d by DGE. The results showed that the expression level of MADS-box9, SERK1, LEC1-Like and seed storage protein genes was up regulated markedly. The expression of ARF8, ARF5, P1N1and PIN2was increased in the transgenic seedlings after DEX treatment. The expression of IAA13gene which has negative effect on auxin signal transduction was down regulated. The expression of LEC2gene inhibited the expression of GA3ox2which encoding a key enzyme in GA biosynthesis. The results suggested that LEC2gene might induce somatic embryogenesis through regulation of embryo-related genes and biosynthesis of plant hormone.5. Gene expression confirmation using RT-PCR:Primers were designed according to the sequences of other plant species. Tobacco cDNA was used as template for PCR amplification. The results of semi-quantitative RT-PCR analysis showed that embryogenesis related genes AGL15, WOX2and ABI3expressed in embryonic callus of transgenic tobacco treated with DEX. Seed storage protein gene, SUS2, expressed in both embryonic callus of transgenic tobacco treated with DEX and callus from WT tobacco on the MS media with hormone.