MSA - Cell and molecular biology/Physiology & Genetics
Thomas, Elizabeth , Cubeta, Marc .
The world after Novozyme 234: a method for preparing protoplasts of Rhizoctonia solani .
Studies that require the generation of fungal protoplasts for various applications have relied on the use of the extracellular enzymes cellulase, hemicellulase, Glucanase GV-L, pectinase, Rhozym HP 150, and Novozyme 234. Of the various enzymes that have been deployed to generate protoplasts from Rhizoctonia solani, the use of Novozyme 234 has met with the greatest success. However, due to the limited availability of this enzyme, there has been an ongoing search in the Rhizoctonia community for a suitable replacement for generating protoplasts. In this study, a method was developed to generate protoplasts from mycelium of R. solani using an enzyme cocktail made up of cellulase, lysing enzymes from Trichoderma harzianum, and driselase. The procedure involved growing R. solani in potato dextrose broth supplemented with yeast extract for 65 hr, followed by harvesting and washing the mycelium in 1 M sorbitol. After enzyme digestion, protoplasts were collected, rinsed, and resuspended in STC (sorbitol, Tris-Cl, CaCl2) buffer. The mean number of protoplasts released ranged from 0.19 X 107 ml -1 to 0.37 X 107 ml -1 depending on time of digestion. The size of protoplasts ranged from 2.5 to 15 µM with varying numbers of nuclei per protoplast. Increased time of digestion resulted in larger protoplasts with more vacuoles. This method will serve as a useful tool for generating protoplasts for future transformation and protoplast fusion experiments.
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1 - North Carolina State University, Department of Plant Pathology, Raleigh, NC , 27695
Presentation Type: Oral Paper:Papers for Topics
Location: White Pine/Cliff Lodge - Level C
Date: Monday, July 27th, 2009
Time: 11:30 AM