【作者】 朱蠡庆；

【导师】 王伯初；

【作者基本信息】 重庆大学 ， 生物医学工程， 2005， 硕士

【摘要】 植物不仅是地球生态系统的主体,而且是人类的一个巨大的可再生自然资源库。高等植物作为多细胞生物体由一个单细胞逐渐发育为成熟个体的过程,包括了细胞和组织的形态发生,以及特定组织细胞的分化,其间许多奇妙而有趣的现象一直吸引广大研究者的注意。植物的生物力学研究把细胞内和细胞间的相互作用以及细胞与外界环境间的相互作用的生物物理学模型与整体联系起来,并将在生态环境的改善和作物产量的提高等方面发挥积极作用。在农业工程和植物组织工程中,如何促进植物组织生长是大家关注的重点,而其中力学因素对植物单细胞生长、发育、分化的影响是一个很有潜力的切入点。作一项为基础性研究,本课题首先面临以下三个问题:植物组织感知外界应力刺激的方式是什么?植物细胞的生长、分裂、分化与植物细胞力学信号的关系是什么?植物组织信号传导过程中的力学信号机理是什么?解答这些问题,就要建立正确的模型并预测植物细胞对力学信号的反应,同时建立一个有效的实验平台以获取可以量化的实验参数,同时还要具备大量获得植物单细胞的手段,这两项基础性的工作正是本研究的主要内容。本研究主要应用生物力学、机械设计、电路设计、单片机、电机学、植物生理学等学科的基本理论和研究方法,研制出一套能进行反馈控制连续加载的系统。该系统可以对培养细胞的琼脂施加一个恒定的微小压力,同时保持了细胞的活性。系统的关键是能够实现很高的精度,可以对分离出的植物单细胞施加非常轻微的载荷,其范围在50mN～300mN 之间。微应力施加实验装置的主控模块是以16 位单片机SPCE061A 为核心,整个电路多采用芯片,应力大小可调节和显示。应力变化的信号通过应变片和单通道动态应变仪来检测和放大,并由步进电机实现应力的施加。该装置具有操作、控制简便,维护方便,成本不高,节省人力,具有一定的自动化程度等优点。该装置的研制为细胞力学试验装置和实验手段的改进做出了有益探索,其设计原理及工作原理不仅可应用于植物细胞力学的研究,还可以应用于生物力学研究的其它领域。本研究需要将植物单细胞包埋于琼脂糖培养基中进行培养并使其分裂,原生质体分离是获取植物单细胞的主要方法,本研究选用加拿大狭叶紫锥菊愈伤组织为材料,研究影响其原生质体得率和活力的因素,最终掌握有效获取大量原生质体的方法。通过设计一个正交试验以及其它相关的研究,结果表明采用纤维素酶浓度为2.0%,果胶酶浓度为1.0%,半纤维素酶浓度为0.5%,甘露醇浓度为0.7mol·L-1 的酶液配方酶解时间为8h 时,选用浅黄绿色、质地均匀且呈疏松颗粒状的紫锥菊愈伤组织易游离出原生质体;原生质体的产量可达每克鲜重含更多还原

【Abstract】 Plants play a significant role in ecosystem. Furthermore, it is an important reproducible source for human being. The ontogeny of every high plant as multicellular organism involves the following developmental processes: from a single cell to a mature individual, plant cells will undergo morphogenesis and differentiation. Nowadays, researchers pay more and more attention to these attractive phenomena. Plant biomechanics is an approach aiming at studying the interaction of the biophysics model and the environments, which are related to intro-cellular condition and extra-cellular environment. How to promote plant tissue growth is an attractive aspect in agriculture engineering and plant tissue engineering. The influence of mechanics factors on development and differentiation of single plant cell has been a potential method. In our research, three questions were proposed, how does plant tissue recognize environmental stress stimulate? Which effect do mechanics signals have on development and differentiation of plant cells? What signal transduction mechanism is? The correct model should be founded to predict the responses of pant cell signal transduction. Comprised the two key works, an effective experiment platform should be established to collect process data, and an efficient approach must be mastered to get enough plant single cells. To research the controlled biomechanics, the instrument of loading mechanical stress was developed. This sophisticated research instrument related to a device applying controllec mechanical forces to test specimens, particularly biological specimens, such as living tissue growing in cultures and single cell embeded in agarose. This study mainly intended to arrange for accurate manipulation of a specimen, and accurate measure of mechanical forces that it applied to, which proceeded in the light of basic theories, research means of biomechanics, mechanical design, circuitry, SCM, electromotor and plant physiology, etc. The instrument was a microprocessor-controlled mechanical loading system, specifically engineered for plant cell in sterile culture. The core of the control module of the instrument was SCM SPCE061A, and some chips in the circuit were used in it. The arrangement provided load between 50mN-300mN. A mass of plant single cells are needed in plant cell mechanics investigation. In this paper, Echinacea augustifolia from Canada was selected as material for protoplast isolation. The callus of Echinacea augustifolia were used for isolation and a orthogonal experiment was designed to research the factors influencing the process of protoplasts preparation, the result indicated that it was easy to isolate protoplast from buff-green Callus of Echinacea augustifolia which looked like granule with Symmetrical character. It was showed that the best enzyme solution concentration was composed of Cellulase 2.0% (w/v), Pectinase 1.0% (w/v), Hemicellulase 0.5% (w/v), 0.7mol·L-1 mannitol, and 50.0×104/gFW protoplasts were obtained after 8 hours later. Finally, we summarized the integrated approach including callus induction, callus culture, isolation, purification, identification and calculation of protoplasts.更多还原