Effects of different concentration of 2,4-D on somatic embryogenesis using thin cell layers (TCL) culture of alfalfa hypocotyls in cv Karysary and Rangerland

This study was carried out for evaluating morphogenesis responses of hypocotyl thin cell layers (TCL) of two alfalfas cultivars (Medicago sativa L. cv. Karysary and Rangelander) and also the effect of different 2,4-D concentrations on somatic embryogenesis was investigated. Sterilized seeds were cultured on SH medium without hormone. After a week, TCL (0.1 mm) obtained from hypocotyl segments were transferred to SH medium containing 2,4-D (1.5 mg/l). Produced calli were transferred to embryogenesis medium (SH medium containing four 2,4-Dconcentrations: 0, 5, 10, 15 mg/l) and one level of kin (1 mg/l). Then calli were transferred to regeneration medium (BOi2Y without hormone) and finally to the ½MS medium without hormone for plantlets growth. Callus induction was observed in all cultures of hypocotyl TCLs. After callus induction, the growth of the calli was performed suitability independent of the cultivar type. In regeneration medium, the somatic embryos appeared initially direct from TCL or indirect from callus as green dots that enlarged as the embryo develops through the globular, heart, torpedo and cotyledonary stages. The analysis of variance results showed that among different concentrations of 2,4-D there were significant differences in percentage of somatic embryogenesis (PSE) and number of somatic embryos per callus (NSC) (p<0.01). So that, maximum PSE and NSC occurred with 10 mg/l 2,4-D and minimum PSE and (NSC) without 2,4-D. PSE and NE (number embryogenesis) in Rangelander cultivar were significantly more than Karysary cultivar. Different concentrations of 2,4-D and cultivar × 2,4-D interaction had significant effect on plant regeneration (p<0.05). Plant regeneration was observed in SH medium of with and without 2,4-D. However, maximum plant regeneration was obtained in 5 and 10 mg/l of 2,4-D. Therefore, 2,4-D had the highest role on somatic embryogenesis from hypocotyl TCL.