Initiation of Cell Suspension Cultures and Plant Regeneration from Protoplast of Asparagus

Abstract

Liquid suspension cultures of anther-derived callus of Asparagus officinalis L. were established by shaking mased anther callus in vitamin-fortified MS medium with 2 mg/l 2,4-D and 1,000 mg/l casein hydrolysate at 150 rpm. Subculture was done periodically by inoculating 5 ml of old culture into 20 ml of fresh medium to maintain cell vitality. As the number of subculture generation increased, the growth rate slowed and a lag phase was observed. The prolonged duration of logarithmic phase and the lower maximum cell concentration obtained in subcultures of older generations indicated gradual loss of growth, vigor and vitality with increasing culture age. The highest yield of protoplast from suspensions occurred after one division of cells in subculture. Protoplast division occurred and clusters about 0.1–0.3 mm in diameter formed after one month of culture in Kao's medium. Large size of calli could be seen when these protoplast clusters were plated onto DA medium solidified with 0.6% phytagar. Embryogenic callus and plant regeneration was observed after these protoplast-derived callus were transferred into MS medium with 0.1 mg/l kinetin and 0.3 mg/l NAA.