Efficient Plant Regeneration from Suspension-cultured Cells of Tall Bearded Iris

Abstract

A protocol was developed for efficient plant regeneration of Iris germanica L. `Skating Party' from suspension cultures. Suspension cultures were maintained in Murashige and Skoog (MS) basal medium (pH 5.9) supplemented with 290 mg·L –1 proline, 50 g·L –1 sucrose, 5.0 μ m 2,4-D, and 0.5 μ m Kin. Suspension-cultured cells were transferred to a shoot induction medium (MS basal medium supplemented with 10 mg·L –1 pantothenic acid, 4.5 mg·L –1 nicotinic acid, 1.9 mg·L –1 thiamine, 250 mg·L –1 casein hydrolysate, 250 mg·L –1 proline, 50 g·L –1 sucrose, 2.0 g·L –1 Phytagel, 0.5 μ m NAA, and 12.5 μ m Kin). Cell clusters that proliferated on this medium differentiated and developed shoots and plantlets in about 5 weeks. Regeneration apparently occurred via both somatic embryogenesis and shoot organogenesis. A series of experiments was conducted to optimize conditions during suspension culture to maximize subsequent plant regeneration. Parameters included 2,4-D and Kin concentrations, the subculture interval, and the size of cell clusters. The highest regeneration rate was achieved with cell clusters ≤280 μm in diameter, derived from suspension cultures grown for 6 weeks without subculturing in liquid medium containing 5 μ m 2,4-D and 0.5 μ m Kin. Up to 4000 plantlets with normal vegetative growth and morphology could be generated from 1 g of suspension-cultured cells in about 3–4 months. Chemical names used: 2,4-dichlorophenoxyacetic acid (2,4-D); kinetin (Kin); 1-naphthaleneacetic acid (NAA).