Sello Simone, Moscatiello Roberto, La Rocca Nicoletta, Baldan Barbara, Navazio Lorella
Department of Biology, University of PadovaPadova, Italy.
Botanical Garden, University of PadovaPadova, Italy.
Front Plant Sci. 2017 Aug 18;8:1444. doi: 10.3389/fpls.2017.01444. eCollection 2017.
Photosynthetic cell suspension cultures are a useful experimental system to analyze a variety of physiological processes, bypassing the structural complexity of the plant organism . Nevertheless, cell cultures containing functional chloroplasts are quite difficult to obtain, and this process is usually laborious and time-consuming. In this work a novel and rapid method to set up photosynthetic cell suspension cultures from the model plant was developed. The direct germination of seeds on a sucrose-containing agarized culture medium supplemented with 0.25 μg/ml 6-benzylaminopurine and 0.5 μg/ml 2,4-dichlorophenoxyacetic acid caused the straightforward formation of green calli at the level of seedling hypocotyls. The subsequent transfer of these calli in liquid culture medium containing the same concentrations of phytohormones and gradually decreasing sucrose levels allowed for the establishment of chloroplast-containing cell suspension cultures, containing functional chloroplasts, in a much faster way than previously described procedures. Pulse amplitude modulation analyses, measurements of oxygen evolution and electron transport rate, together with confocal and electron microscopy observations, confirmed the photosynthetic efficiency of these cell suspension cultures. The described procedure lends itself as a simple and effective way to obtain a convenient tool for a wide array of structural and functional studies on chloroplasts.
光合细胞悬浮培养物是一种有用的实验系统，可用于分析各种生理过程，绕过植物有机体的结构复杂性。然而，获得含有功能性叶绿体的细胞培养物相当困难，而且这个过程通常既费力又耗时。在这项工作中，开发了一种从模式植物建立光合细胞悬浮培养物的新颖快速方法。在含有0.25μg/ml 6-苄基腺嘌呤和0.5μg/ml 2,4-二氯苯氧乙酸的含蔗糖琼脂培养基上直接萌发种子，导致在幼苗下胚轴水平直接形成绿色愈伤组织。随后将这些愈伤组织转移到含有相同浓度植物激素且蔗糖水平逐渐降低的液体培养基中，从而能够以比先前描述的方法快得多的方式建立含有功能性叶绿体的含叶绿体细胞悬浮培养物。脉冲幅度调制分析、氧气释放和电子传递速率的测量，以及共聚焦和电子显微镜观察，证实了这些细胞悬浮培养物的光合效率。所描述的方法本身是一种简单有效的方法，可用于获得一个方便的工具，用于对叶绿体进行广泛的结构和功能研究。
