The Plant Tech. Core Laboratory of Agricultural Biotechnology Research Center(ABRC) was established in April 2007 in view of the broad and rapid development of the field of agricultural biology research, and the integration of the center's manpower in the research of transient transfection and morphological anatomy of model plants. The main object of the laboratory is to provide services to the members of the Academia Sinica. At this stage and in the future, the Core will continue to cooperate with the research directions of the ABRC to assist the members at the Center in research and publication. The current regular services include provision of Arabidopsis plant material, rice and Arabidopsis transient transfection, or other model plants (applied for on an individual project basis) transient transfection, plant tissue sectioning, and the construction of CRISPR /SpCas9 target sites vectors. In addition to developing model plant-related technology platforms, the Core actively establishes agronomic crop protoplast isolation and regeneration system, and improvement of transient transfection systems.
1. Transient transfection: E. coli with a vector to be transfected into protoplasts directly provided by the applicant. (Applicable to subcellular localization, BiFC protein-protein interaction, GUS assay, luciferase assay, transcriptome level analysis, CRISPR editing efficiency assessment). The Core provides Arabidopsis and rice protoplasts as transfection platforms. After transfection with PEG 4000-Ca2+, protoplasts are returned to applicants for subsequent experiments.
a. Arabidopsis transfection: Under the constraints of the laboratory space and instruments, we modified the method which established by Dr. Jen Sheen to remove the epidermal cells from the leaves to break down the cell wall with enzymes, which can be isolated 106 protoplasts from the rosette leaves of three mature Arabidopsis plants, which solved the limitation on plant materials and reduced the time consumed by experiments (Wu et al., 2009).
b. Rice transfection: The Core uses 10 to 14-day-old of rice stem and sheath for protoplast isolation and transfection. Transient transfection rice protoplasts can provide a variety of monocotyledonous plant gene expression evaluation (Lin et al., 2018).
2. Tissue section: Academia Sinica has two core laboratories for electron microscopy and one pathology laboratory. They are good at using scanning electron microscopy and transmission electron microscopy to observe the gene expression of animal systems. Although, some members of ABRC have experience in paraffin sectioning, this method sometimes limits choice of samples or cannot meet the needs of the experiment, and is not suitable for some plant tissues, especially root sections. In order to obtain a better solution, our laboratory established a resin sectioning and low-melting-point agarose sectioning system, and provides PAS, toluidine blue, fast green and safranin staining, which can be observed with a fluorescence microscope to increase detection sensitivity and solve image unvisualized problems.