Somatic embryogenesis and plant regeneration from immature cotyledons of European chestnut (Castanea sativa Mill.)

Abstract

Here, we established a protocol for induction of somatic embryogenesis and plant regeneration from immature cotyledons of open-pollinated seeds of European chestnut (Castanea sativa Mill.) cultivars ‘Osmanoglu’ and ‘Sariaslama’. Basal media, Murashige and Skoog medium (MS), Driver and Kuniyuki Walnut medium (DKW), and Woody Plant Medium (WPM) supplemented with l-glutamine or casein hydrolysate, with or without silver nitrate, agar or gelrite, and various plant growth regulator (PGR) combinations were tested in initial cultures for induction of somatic embryos. The effects of initial cultures on the percentage of somatic embryos and average number of embryos per cotyledon explant, subcultured monthly, were determined at the end of 4 mo. Interactions were observed among the different treatments for ‘Osmanoglu’ cultivar, with the highest rates of somatic embryogenesis (4.7–9.7%) being obtained in MS, DKW, or WPM basal media supplemented with (1) 6-benzyladenine (BA; 1 mg/L) + kinetin (KIN; 2 mg/L) + indole-3-butyric acid (IBA; 0.01 mg/L); (2) BA (1 mg/L) + 1-phenyl-3-(1,2,3-thiadiazol-5-yl; TDZ 0.1 mg/L) + IBA (0.01 mg/L), and (3) KIN (2 mg/L) + TDZ (0.1 mg/L) + IBA (0.01 mg/L) PGR combinations plus l-glutamine or casein hydrolysate, with or without silver nitrate, and with either gelrite or agar. The highest percentages (12.0% and 11.2%) of somatic embryogenesis for ‘Sariaslama’ were obtained in DKW supplemented with PGR combinations of (1) BA (1 mg/L) + KIN (2 mg/L) + IBA (0.01 mg/L), (2) BA (1 mg/L) + TDZ (0.1 mg/L) + IBA (0.01 mg/L), respectively. The average number of somatic embryos ranged between 0 and 0.65 per explant for ‘Osmanoglu’ and between 0 and 0.49 per ‘Sariaslama’ explant. For germination of somatic embryos, root, shoot, and plantlet regeneration, different treatments included desiccation, cold and gibberellic acid (GA3), and BA alone or in combination with auxins (IBA or α-naphthaleneacetic acid, NAA; 0.1 mg/L). The highest rate of somatic embryos regeneration (27.5%) occurred using MS basal media with half-strength microelements containing 0.1 mg/L BA + 0.1 mg/L NAA, after treatments of desiccation, or desiccation plus cold or GA3 (3 mg/L).