Preparation of protoplast from Glomerella cingulata

Sustainability is influenced by various aspects especially in the field of agriculture. Agricultural sustainability includes meeting the food resource demands but among the disturbing factors is pathogen infestation. More innovative technologies are needed to overcome with the sustainability of centralized agricultural systems developing technologies which not adversely impact the environment. Environmental sustainability problems with the usage of fertilisers, agricultural machinery, irrigation water, and pesticides are driving this trend. As a result, a new method is required that integrates biological and ecological processes into food production while reducing the usage of harmful agents. Agricultural sustainability indicates an increasing awareness of the benefits of ecological and agronomic natural management, modification, and design, as well as a focus on genotype enhancement using a number of current biological techniques. Among the pathogens in agriculture is Glomerella cingulate, a plant pathogenic fungus that cause anthracnose disease that infects a variety of plants. The fungus secretes cutinase, a degradative and hydrolytic enzyme that degrade cutin in order to traverse the cuticular barrier of the infected plant. In order to do genetic manipulation and study the regulatory for the production of this enzyme, protoplast is needed as a tool in genetics and molecular research and in transformation of DNA. In this study, a protocol for isolation of protoplast from G. cingulata was established including the effects of mycelial age, concentration of enzymes and duration of lytic digestion on the yield of protoplasts. The highest yield of protoplast (7.2x10⁷ protoplasts/ml) was achieved from a 3-day mycelial culture which was treated with 10 mM dithiothreitol and incubated with 4 mg/ml lysing enzyme for 3 hours at 30˚C. The frequencies of regeneration of the isolated protoplast are at 0.4% to 0.6% on a complete medium compared to 0.03% on Potato Dextrose Yeast Extract Agar. The use of protoplast isolation is significant for a variety of genetic modification applications