愈伤组织向基质纤维生长形成愈伤组织/纤维结合体的诱导及机理

The genetic and metabolism of callus is more stable than that of suspension cells, and the key is how to realize continuous large-scale cultivation. So we invented curtain-like anchoring cultivation method and its bioreactor. To achieve continuous large-scale culture, agar gel and other gel were used to stick and fix the callus to the bed of bioreactor. It was found that only when the bed surface fluff fibers embedded the neogenesis callus and formed a callus/fiber combination structure, the callus will not come off and overgrow the bed surface after a large number of proliferations. Then the conversion from aseptic culture to open type none asepsis condition culture could be completed online and the purpose of continuous harvesting callus online could be obtained. Therefore, In this project, in order to reveal the the inducing and biological mechanism for the growth and differentiation of the callus to the bed fluff fiber and the formation of the callus/fiber structure, the correlation will be studied that the formation and differentiation of callus "transportation system" to the bed fluff fiber, adhesion tightness, proliferation rate of the new proliferation cell, with the kind, thickness, length, density, submicrostructure, chemical composition of the bed fluff fiber, composition of nutrient solution,etc, through histoanatomy observation and analysis of the callus/fiber structure by microscopic and submicroscopic observation, physicochemical analysis. The results will provide the basis for screening and optimization the kind of the bed fiber of the curtain-like anchoring culture bioreactor.

团队在进行南方红豆杉细胞培养生产紫杉醇研究时发现，愈伤组织紫杉醇产量高，且遗传与代谢较悬浮细胞稳定，关键需解决连续规模化培养问题，为此课题组研究发明了帘状锚固培养法及其生物反应器，试图将愈伤组织用琼脂等凝胶粘固接种到反应器悬垂的帘状培养床上实现连续规模化培养。结果发现，当床面绒毛纤维嵌入新生愈伤组织形成愈伤组织/纤维结合体时，愈伤组织大量增殖后才不会脱落并长满床面，才可在线实现无菌培养到开放式带菌培养的转换，达到可在线连续收获愈伤组织的目的。因此，本项目拟采用显微、亚显微观测、理化分析等，通过愈伤组织/纤维结合体的组织解剖学观察分析，弄清愈伤组织“运输系统”分化形成、新增殖细胞向床面绒毛纤维生长分化及其附着紧密度、增殖速率等与床面绒毛纤维种类、粗细、长短、密度、亚显微结构、化学组成及培养液组成等的相关性，揭示愈伤组织细胞向床面绒毛纤维生长分化形成愈伤组织/纤维结合体的诱导机制与生物学机理，为所发明帘状锚固培养生物反应器培养床纤维种类的筛选与优化提供基础

为了实现南方红豆杉愈伤组织细胞规模化培养生产紫杉醇，研究发明了帘状锚固培养生物反应器及培养法，针对前期研究中发现的科学问题申请了“愈伤组织向基质纤维生长形成愈伤组织/纤维结合体的诱导及机理”课题，现采用光学显微观测、高效液相色谱、原子吸收光谱检测及模拟反应器传质流程自主创制的“纤维渗透浸润传质模拟装置”等，对14种具代表性植物的韧皮、木材、竹材、秸秆共16种材料自制成的纤维及其渗透浸润传递 MS 培养液的速率进行了研究。结果发现16种纤维的光学显微形态学尺寸、结构均存在很大差异。传递过程包括培养液沿纤维细胞表面渗透浸润（初始阶段）和毛细微液流渗透浸润传递（平稳阶段）两个阶段。初始阶段在小构树茎皮与水稻秸秆纤维集束中的渗透浸润速度最快，均为4.00 cm·min-1，是最低马尾松木材纤维0.55 cm·min-1的近8倍。集束中纤维排列方向有序组与无序组间的差距在0.02～1.06 cm·min-1（幅度为0.43%～30.29%）之间。平稳阶段单位质量水稻与小麦秸秆纤维集束单位时间传递培养液的体积速率明显高于其他种类，分别为192.53 mL·h-1·g-1和176.31 mL·h-1·g-1，是青皮竹、慈竹纤维（81.91 mL·h-1·g-1，82.32 mL·h-1·g-1）的两倍多，差距幅度急剧下降，纤维排列方向有序组与无序组间的差距增至11.56～68.90 mL·h-1·g-1（幅度为5.72%～51.96%）之间。两个阶段传递速率与纤维细胞微观形态学尺寸、结构间的对应性分析结果显示，无对应相关性，表明微观形态学结构与尺寸不是影响植物纤维传递速率的主导因素。不同种类植物纤维集束渗透浸润传递MS 培养液的速率存在很大差异，影响差异的主导因素不是纤维细胞微观尺度的物理因素，而是化学因素，且存在吸收和吸附其中化合物和离子成分效应。纤维排列方向的有序性会影响纤维集束的传递速率。我们的研究结果为研究液体在纤维集束中渗透浸润传递提供了一个平台，这对利用纤维集束进行毛细质量传递及相关技术的发展具有重要意义。