Protoplast Preparation And Preliminary Analysis Of Karyotype Of Ustilago Esculenta

A smut fungus Ustilago esculenta causes enlargement of infected culms of Zizania latifolia, a perennial aquatic grass, and the induced swollen culms are used as a vegetable in China and some southeastern countries. Study on biological characteristics of U. esculenta is helpful in understanding how the fungus plays a part in the formation of the swollen culm.A protocol for analysis of chromosome size and number of the fungus was developed. Fungal protoplasts were prepared following the following method:The fungus was grown for24h. Spores was harvested and then transferred into a Petri dish (100x20mm) containing40ml filter-sterilized protoplasting solution containing10mg/ml Lysing enzymes,10mg/ml Driselase and10mg/ml Cellulase to hydrolyze [volume (ml):weight (g)=20]. The Petri dish was shaken at70rpm at28℃for at least4h.In addition, the chromosome size and number of U. esculenta was estimated by pulsed-field gel electrophoresis (PFGE). The results showed that at least ten chromosome bands were observed with estimated sizes of1822kb,1606kb,873kb,727kb,648kb,564kb,533kb,464kb,438kb, and396kb.