Enhancement of Somatic Embryogenesis by a Novel Control Method of Phytohormone

本研究从工程角度对体细胞胚胎发生进行了研究，以期提高再生效率。1)在培养体系中添加核黄素（riboflavin， RF）的影响a)核黄素对合成生长素2,4- d的分解速率与光照强度有很大关系。FAD和EMN对2,4- d的降解也有催化活性，但对2,4- d分解的主要产物光色胺没有催化活性。b)在胡萝卜细胞悬浮培养中加入核黄素提高了细胞和培养基中2,4- d的降低率，也提高了再生效率。c)以疏水烷基链作为生长素分解的连接体，合成了核黄素固定化微球（RF-beads）。d)用RF-beads培养胡萝卜细胞提高了体细胞胚的形成效率，但过量的RF-beads抑制了体细胞胚的再生，这可能是由于在2,4- d降解过程中产生的活性氧对细胞造成了损伤。c)提出了一种具有生长素降解血管的新型培养体系，以实现有效的体细胞胚发生。(2)气相组成分析及气体交换方法研究了胡萝卜细胞培养瓶顶空气体组分对体细胞生成的影响。提出了乙烯气体的抑制作用和二氧化碳气体的抑制作用。b)在水稻愈伤组织悬浮培养顶空中检测到少量的乙醇和乙醛等，可能是胚胎发生的抑制因子。然而，在顶空气体中没有观察到任何促进胚胎发生的物质。

In this research, somatic embryogenesis was investigated for the enhanced regeneration efficiency from the engineering viewpoints.1) Effects of riboflavin (RF) addition in the culture systema) Decomposition rate of 2,4-D, a synthetic auxin, by riboflavin depended greatly on light irradiation intensity. FAD and EMN also showed the catalytic activity for the 2,4-D degradation, but lumichrome, the main product of 2,4-D decomposition, cannot.b) Riboflavin addition into carrot cells suspension culture increased the decrease rate of 2,4-D in the cells and the medium, also enhanced the regeneration efficiency.c) Riboflavin immobilized beads (RF-beads) were synthesized with a hydrophobic alkyl chain as a linker for the auxin decomposition.d) The carrot cell culture with RF-beads increased the efficiency of somatic embryo formation, however the excess amount of the beads inhibited the regeneration probably because the damage to the cells by activated oxygen generated through the 2,4-D degradation.c) A novel culture system with an auxin degradation vessel is proposed for the effective somatic embryogenesis.2) Analysis of gas-phase composition and gas-exchanging methoda) Effects of gas components in flask headspace of carrot cell cultures were investigated for somatic cmryogcnesis. Inhibition of ethylene gas and the suppressive effect of carbon dioxide gas on it were suggested.b) In the headspace of rice callus suspension culture, a small amount of ethanol and acetaldehyde etc. was detected, which could be an inhibitory factor for embryogenesis. However, there could not be observed any substances in the headspace gas that enhance embyogenesis.