Plant Regeneration from Mesophyll and Cell Suspension Protoplasts of Sweet Potato, Ipomoea batatas (L.) Lam.

Abstract

Mesophyll protoplasts of sweet potato, variety Chugoku No.25, were isolated from shoot tip cultures grown in vitro. After the surface of the leaves was scratched with sterilized carbo-rundum, they were plasmolyzed for I hour in 0.3 M sorbitol and 0.05 M C_aCl₂. The enzyme solution for isolation contained 0.1%Pectolyase Y-23, 2 % Cellulase Onozuka RS, 5 mM C_aCl₂ and 0.5 M mannitol in a modified K3 medium (Kao and Michayluk 1981). The crude protoplast suspension was filtered through a nylon mesh and protoplasts were isolated by discontinuous density gradient centrifugation. The initial medium used was a modified KM8p medium (Kao and Michayluk 1975) sup-plemented with 0.1 mg/l zeatin (ZEA), 0.1 mg/1 2, 4-dichlorophenoxyacetic acid (2, 4-D). 0.5 M mannitol and 10 g/l sucrose. The cell colonies formed were plated on a mQdified MS medium (Murashige and Skoog 1962) for callus forma-tion containing 20 g/l sucrose, 2 g/l Gellan Gum, 0.5 mg/l 2, 4-D and I mg/1 abscisic acid (ABA) with or without 0.5 mg/l kinetin (KlN) or 0.5 mg/l ZEA. Furthermore, the calli were transferred to a MS medium for regeneration supplemented with 30 g/l sucrose, 2 g/l Gellan Gum and 1 mg/l KlN or 0.5 mg/l ZEA. Compact calli were induced on the medium supplemented with ZEA, and regenerated plants were obtained. The effects of silver nitrate (AgNO₃) and drying on plant regeneration were investigated. AgNO₃ was not effective for plant regeneration, but the drying treatment for 3 days prior to transfer to the medium for regeneration was effective. Cell suspension protoplasts were isolated from the embryogenic calli initiated from the shoot apical domes in the same manner as used for mesophyll protoplast isolation. The initial culture medium and the callus proliferation medium were identical to those used for mesophyll protoplast. Drying treatuent was also effective to enhance the frequency of adventitious bud formation, and regenerated plants were obtained.