Plant Regeneration from Mesophyll Protoplasts of Goodenia scaevolina F. Muell.

Abstract

To establish a protoplast culture system of Goodenia scaevolina F. Muell., cotyledonary mesophyll protoplasts were isolated and cultured. Because seeds of G. scaevolina are difficult to germinate, the smoke medium was employed to stimulate their germination to obtain cotyledons. After the pre-treatment of cotyledons in a liquid medium, consisting 1/2 strength of MS supplemented with 0.6 M sucrose at 25°C in the dark for 1-2 hr, they were transferred to a solution containing 1 % Cellulase Onozuka RS, 0.1 % Pectolyase Y-23, 0.6 M mannitol and protoplast washing salts and kept at 25°C in the dark for 15 hr. Protoplasts were cultured in 1/2 MS liquid medium supplemented with 1 μM 2, 4-dichlorophenoxyacetic acid, 0.1 μM, 6-benzylaminopurine (BAP) and 0, 6 M sucrose. Using the sucrose for an osmoticum and a carbon source enabled protoplasts to grow up to callus in the initial medium without any additional treatment. When the calli grew to 1-2 mm in diameter, they were transferred onto 1/2 MS solid medium, supplemented with 0.1 μM indole-3-butyric acid (IBA), and 10 μM BAP for plant regeneration. After rooting on MS medium supplemented with 2 μM IBA, the regenerated plantlets were acclimatized and subsequently flowered.