An improvement of tomato protoplast culture for rapid plant regeneration

Abstract

Tomato mesophyll protoplasts were cultured in TM2 medium containing 5.7 μM α-naphthaleneacetic acid and 2.4 μM benzyladenine and were incubated either in stationary culture or on an orbital shaker at 25–30 strokes per min, in combination with interval addition of fresh medium. The effects of stationary and shaking conditions on the growth of the colonies and their subsequent shoot organogenesis were significantly different. The cultures maintained in stationary condition without adding fresh medium accumulated a thin membranous layer on the medium surface and whitish substance in the medium that seemed to precede cell browning and premature colony death. Mild shaking conditions along with the reduction of colony density by one half by dividing the contents of one dish into two dishes, after adding 2 ml of fresh medium on the 4th day and further addition of fresh medium (0.5 ml) on the 8th day of plating, provided optimal conditions for colony growth and suppressed thin layer and whitish substance accumulation. Ten-day-old colonies raised through this protocol regenerated shoots rapidly (within 19–20 days after initial plating) after transfer to regeneration medium (MS medium with 2.8 μM zeatin riboside, 0.06–0.1 μM gibberellic acid, 4% sucrose and 1% type VII agarose) directly bypassing the callus phase.