Plant Regeneration from Protoplast-Derived Somatic Embryos of Asparagus officinalis L.

Abstract

Summary A method for regenerating plants from embryogenic calli derived from protoplasts of asparagus is described. The protoplasts were isolated from calli of a selected crown of the genotype G-203 of Asparagus officinalis L. Optimum protoplast yield was obtained from calli of 8 to 10 d of age after subculture following enzymatic digestion. Of the isolated protoplasts 80 % to 90 % were viable. The highest plating efficiency (12.45 %) was obtained when cells were cultured at a density of 1 × 10 5 protoplasts/mL in 1/2 strength of Murashige and Skoog (MS) medium with 1 mg/L NAA, 0.5 mg/L zeatin, 0.5mg/L folic acid, 0.05 mg/L biotin, 1 g/L glutamine, 0.2 g/L casein hydrolysate, 0.6 M glucose, 0.1 M mannitol, and 0.1 % (w/v) Gelrite. A remarkable decrease in plating efficiency was recorded in the presence of both spermine and spermidine in the culture medium. Colonies were visible after 30 d and grew consistently into slimy and friable embryogenic calli after transferring them onto MS medium containing 1 mg/L 2,4-D, 3 % (w/v) sucrose and 0.2 % (w/v) Gelrite. Somatic embryos were induced from such calli when cultured on 1/2 MS medium with only 1.% (w/v) sucrose devoid of any growth hormones. After transplanting on 1/2 MS medium with 1 % (w/v) sucrose and 1 mg/L GA3, 40–45 % of embryos germinated normally and grew into plantlets within 30 d. These plantlets from protoplast-derived somatic embryos were diploid revealing 2n = 20 chromosomes.