Sivanandhan Ganeshan, Bae Solhee, Sung Chaemin, Choi Su-Ryun, Lee Geung-Joo, Lim Yong-Pyo
Molecular Genetics and Genomics Laboratory, Department of Horticulture, College of Agriculture and Life Science, Chungnam National University, Daejeon 34134, Korea.
Department of Horticulture, College of Agriculture and Life Science, Chungnam National University, Daejeon 34134, Korea.
Plants (Basel). 2021 Nov 30;10(12):2636. doi: 10.3390/plants10122636.
Chinese cabbage is an important dietary source of numerous phytochemicals, including glucosinolates and anthocyanins. The selection and development of elite Chinese cabbage cultivars with favorable traits is hindered by a long breeding cycle, a complex genome structure, and the lack of an efficient plant transformation protocol. Thus, a protoplast transfection-based transformation method may be useful for cell-based breeding and functional studies involving Chinese cabbage plants. In this study, we established an effective method for isolating Chinese cabbage protoplasts, which were then transfected with the pCAMBIA1303 binary vector according to an optimized PEG-based method. More specifically, protoplasts were isolated following a 4 h incubation in a solution comprising 1.5% (/) cellulase, 0.25% (/) macerozyme, 0.25% (/) pectinase, 0.5 M mannitol, 15 mM CaCl, 25 mM KCl, 0.1% BSA, and 20 mM MES buffer, pH 5.7. This method generated 7.1 × 10 protoplasts, 78% of which were viable. The reporter gene in pCAMBIA1303 was used to determine the transfection efficiency. The Chinese cabbage protoplast transfection rate was highest (68%) when protoplasts were transfected with the 40 μg binary vector for 30 min in a solution containing 40% PEG. The presence of and in the protoplasts was confirmed by PCR. The methods developed in this study would be useful for DNA-free genome editing as well as functional and molecular investigations of Chinese cabbage.
大白菜是多种植物化学物质的重要膳食来源，包括硫代葡萄糖苷和花青素。由于育种周期长、基因组结构复杂以及缺乏高效的植物转化方案，具有优良性状的大白菜优良品种的选育和开发受到阻碍。因此，基于原生质体转染的转化方法可能有助于大白菜植株的细胞育种和功能研究。在本研究中，我们建立了一种有效的大白菜原生质体分离方法，然后根据优化的基于聚乙二醇（PEG）的方法用pCAMBIA1303双元载体对其进行转染。更具体地说，将大白菜在含有1.5%（/）纤维素酶、0.25%（/）离析酶、0.25%（/）果胶酶、0.5 M甘露醇、15 mM氯化钙、25 mM氯化钾、0.1%牛血清白蛋白和20 mM MES缓冲液（pH 5.7）的溶液中孵育4小时后分离原生质体。该方法产生了7.1×10个原生质体，其中78%是活的。用pCAMBIA1303中的报告基因来测定转染效率。当原生质体在含有40%PEG的溶液中用40μg双元载体转染30分钟时，大白菜原生质体转染率最高（68%）。通过聚合酶链反应（PCR）证实了原生质体中基因的存在。本研究中开发的方法将有助于无DNA基因组编辑以及大白菜的功能和分子研究。
