Factors Affecting Isolation of Protoplasts from Leaves of Grape (Vitis Vinifera)
A method of isolating grape mesophyll protoplasts was investigated to facilitate the use of genetic engineering techniques to improve this economically important woody plant species. Grape leaves were macerated under aseptic conditions using Cellulysin and Macerase in combination with a variety of different factors known to promote the isolation of protoplasts from herbaceous plants. The effects of several factors influencing protoplast isolation could be evaluated quickly by using leaf disks 1 cm in diameter and known volumes of maceration and wash media. The best yields of mesophyll protoplasts were obtained using medium-sized leaves of well-fertilized grapevines kept in the dark for 24 hr prior to maceration in 1% Cellulysin, 0.5% Macerase, 0.7 M mannitol, 5 ppm 2,4-dichlorophenoxyacetic acid, 0.1 ppm benzylaminopurine, and one-tenth strength Murashige and Skoog medium, then incubated at 22°C in the light (1,000 lux) for 24 hr. Over 5,000 protoplasts per cm2 of leaf were produced using these conditions. This method of screening factors affecting protoplast isolation would be applicable to other species as well.