improved method for electroporation in plant protoplasts: infection of tobacco protoplasts by tobacco mosaic virus particles

• Published in: Plant cell reports 1987-04, Vol.6 (2), p.90-93
• Main Authors: Nishiguchi, M, Sato, T, Motoyoshi, F
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic engineering applications; Genetic technics; Genetics and breeding of economic plants; Methods. Procedures. Technologies; Nicotiana tabacum; Plant breeding: fundamental aspects and methodology; protoplasts; Tobacco mosaic virus; Vectors (cloning, transfer, expression). Insertion sequences and transposons
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/bf00276660

Conditions of electroporation were optimized for introduction of tobacco mosaic virus (TMV) particles into tobacco mesophyll protoplasts (Nicotiana tabacum L. cv. Petit Havana SR1). Compared with conditions for TMV-RNA uptake, a longer electric pulse was necessary at the same voltage to induce TMV particle entry. Up to 80-90% of the protoplasts were infected with TMV particles after exposure to a 10 msec pulse at 200 V (0.67 KV/cm) in a 0.5 M mannitol solution. Protoplast viability was slightly lower than for controls which did not undergo electroporation. The presence of buffer in the mannitol solution reduced the net voltage in the solution which resulted in a significant decrease of the level of infection. These results suggest that the membrane pores resulting from an electrical pulse were wide enough for TMV particles (300 × 18 nm) to enter protoplasts.