Plant regeneration system in recalcitrant rye (Secale cereale L.)

An effective recovery of plants, genetic engineering, somaclonal variation and haploid induction to improve traits of important crops strongly depend on in vitro systems. For plant molecular breeding and gene transformation, tissue culture is powerful tool. Low efficiency of callus initiation, somatic embryo maturation, and plant regeneration is a major problem in rye tissue culture; therefore, rye is recognized as one of the most recalcitrant species for this process. The aim of this study was to develop optimum, stable and reproducible callus formation and regeneration system for rye to overcome this restriction. To establish an efficient protocol, the effects of three types of explants (intact mature embryo, scratched mature embryo and endosperm-supported mature embryo), carbohydrate source (20 g/L of sucrose and 40 g/L of maltose), auxin type [2,4-dichlorophenoxyacetic acid (2, 4-D), 3,6-dichloro-2-methoxybenzoic acid (dicamba) and 4-Amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram)], and auxin concentration (2, 4, 6, 8, 10 and 12 mg/L) on callus formation rate (%), embryogenic callus formation rate (%), responded embryogenic callus rate (%), and regeneration efficiency of rye (Secale cerealeL.) were evaluated. The best explant type for plant regeneration was found to be endosperm-supported mature embryo. Our results revealed that the highest responded embryogenic callus rate (60%) and regeneration efficiency (n = 1.24) were obtained from the MS medium containing maltose and 12 mg/L picloram in endosperm-supported mature embryo. This protocol provides a basis for future studies on genetic transformation of rye.