Regulation of gene expression during conifer embryogenesis

通过体细胞胚胎发生（SE）的克隆繁殖具有在短得多的时间内将上级材料传递到商业林的潜力，并且与种子生产的有性过程结合使用将有助于保持种子园的正确遗传平衡。目前，在针叶树中成功引入商业胚胎发生的限制被认为是：a）胚胎质量的改善; B）响应基因型的效率和数量; c）从成熟组织诱导SE。为了开发加拿大两种重要的经济树种白色云杉和道格拉斯冷杉的SE，我们一直在研究SE和ZE开发过程中的分子事件。成熟过程中表达的cDNA和基因组序列的分析提供了重要的信息，针叶树胚胎成熟的分子生物学和形成的基础上，继续拟议的研究，将集中在胚胎发育的早期阶段。利用同源胚调控探针（转录因子PmLEC 1和PmCOP 1，在早期道格拉斯冷杉ZE和SE中表达的两个关键基因，（在我实验室分离的））和DD-PCR，研究早期胚胎发育的分子基础，MG、胚柄和其他因子在自花和异花授粉种子中的作用。这些基因的功能分析将通过反向遗传学进行。此外，将检查E和NE培养物在诱导阶段之间的差异，以了解基因产物在SE起始中起什么作用。针叶树E文化的形成是基因型依赖的，诱导频率可以在家庭之间有很大的差异。方法包括基于PCR的SSH cDNA文库与微阵列和/或基于定量RT-PCR的谱分析偶联。基于微阵列的方法将允许同时定量大量转录物，以鉴定SE起始和对PGR的响应期间的协调基因组，所述PGR包括与来自包括道格拉斯冷杉在内的广泛的针叶树基因型的SE频率增加有关的抑制剂处理。本研究具有重要的理论意义和实践意义，对培养高素质人才具有重要的指导意义。

Clonal propagation by somatic embryogenesis (SE) has the potential for the delivery of superior material to the commercial forest in a much shorter time and used in combination with the sexual process for seed production will help maintain the correct genetic balance from seed orchards. Presently constraints to the successful introduction of commercial embryogenesis in conifers are perceived to be: a) improvement of embryo quality; b) efficiency and the number of responding genotypes; c) induction of SE from mature tissues. To develop SE for white spruce and Douglas fir, two economically important species in Canada, we have been examining the molecular events during development of SE and ZE. The analysis of cDNA and genomic sequences expressed during maturation have provided important information on molecular biology of conifer embryo maturation and form the basis of continuing proposed studies that will focus on very early stages of embryogeny. The molecular basis of early embryogeny, role of MG, suspensor, and other factors in self- and cross-pollinated seeds will be studied using the homologous embryo regulatory probes (transcription factor PmLEC1, and PmCOP1, two critical genes expressed during early Douglas fir ZE and SE, (isolated in my lab), and DD-PCR. The functional analysis of these genes will be performed by reverse genetics. In addition, differences between E and NE cultures during the induction phase will be examined to understand what roles the gene products play in initiation of SE. Formation of conifer E culture is genotype dependent and induction frequencies can vary widely among families. Approaches include PCR based SSH cDNA libraries coupled with microarray and/or quantitative RT-PCR based profile analyses. Microarray based methodology will allow the simultaneous quantification of a large number of transcripts to identify coordinated gene groups during SE initiation and response (s) to PGRs including Brassinolide treatment implicated in increased SE frequency from a wide range of genotypes of conifers, including Douglas fir. The research is of fundamental and practical interest and will also be important in training of HQP.