Protoplast isolation and culture of wild Kentucky Bluegrass in Gansu

In the present study, the conditions of protoplast isolation and culture were explored using embryogenic callus from wild Kentucky Bluegrass variety Longxi (LX) and Dingxi (DX). The results showed that the optimal conditions for protoplast isolation was enzyme composition including 1.5% Cellulase R10+0.5% Pectolase Y23+1.0% Macerozyme R10+0.3% Driselase with 16 h digestion. The optimal mannitol concentration was 0.6 molL-1 for LX protoplast and 0.5 molL-1 for DX protoplast. The highest yield of protoplasts was 6.59106 numberg-1 for LX and 5.95106 numberg-1 for DX. After purification, DX protoplasts were cultured in KM8P liquid medium. With 3.0105 numbermL-1 plating density and 1.0 mgL-1 2,4D, DX protoplasts had highest cell division frequency of 9.56% and plating efficiency of 4.62%.