Enhancing genetic modification in recalcitrant plants: An investigation in chili (Capsicum annuum) through the optimized tape sandwich protoplast isolation and polyethylene glycol-mediated transfection.
IF 1.4 4区 生物学Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
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引用次数: 0
Abstract
Chili presents challenges for Agrobacterium-mediated transfection due to its highly recalcitrant nature. One way to overcome this challenge is by using PEG-mediated transfection of protoplasts, which enhances the likelihood of successfully introducing transgenes into the cells. The tape sandwich method for isolating chili leaf protoplasts was optimized by adjusting enzyme concentrations and incubation duration, resulting in a high yield of 1.3×106 cells ml-1 per 0.1 g of leaves. The efficiency of transfecting GFP-encoding plasmids and Cas9 protein using PEG molecules of different sizes was also examined. The highest plasmid transfection efficiency was achieved with 5 µg of plasmid in 50 µl-1, with an average efficiency of 48.71%. For Cas9 protein transfection, the most effective treatment involved using 1000 µg of protein in 100 µl-1, mediated by 40% PEG 4000, resulting in an average efficiency of 2.94% due to protein aggregation. Nevertheless, this optimized protocol reduces the time required for chili protoplast isolation and enhances plasmid transfection efficiency by nearly 50%.
期刊介绍:
Plant Biotechnology is an international, open-access, and online journal, published every three months by the Japanese Society for Plant Biotechnology. The journal, first published in 1984 as the predecessor journal, “Plant Tissue Culture Letters” and became its present form in 1997 when the society name was renamed to Japanese Society for Plant Cell and Molecular Biology, publishes findings in the areas from basic- to application research of plant biotechnology. The aim of Plant Biotechnology is to publish original and high-impact papers, in the most rapid turnaround time for reviewing, on the plant biotechnology including tissue culture, production of specialized metabolites, transgenic technology, and genome editing technology, and also on the related research fields including molecular biology, cell biology, genetics, plant breeding, plant physiology and biochemistry, metabolic engineering, synthetic biology, and bioinformatics.