Somatic embryogenesis from ovaries, developing ovules and immature zygotic embryos, and improved embryo development of Castanea sativa

Somatic embryogenesis of European chestnut ( Castanea sativa Mill.) was obtained using juvenile tissue cultured on P24 medium with 5 μM 2,4-dichlorophenoxyacetic acid plus 0.5 μM 6-benzylaminopurine (BA) for three weeks and then cultured on 0.89 μM BA. Induction frequency with ovaries ranged from 2.0 to 19.1 % and was observed in tissue collected 2 to 8 weeks postanthesis, ovules used as a starting tissue gained 0.8 to 7.8 %, 3 to 9 weeks postanthesis. Zygotic embryos collected 5 to 10 weeks postanthesis formed 10.5 to 57.1 % somatic embryos, respectively. The culture lines were maintained via secondary embryogenesis on P24 medium with 0.89 μM BA. Development and maturation were stimulated on P24 medium with increased agar concentration (1.1 %). Five plantlets were transferred to substrate and acclimatized successfully in greenhouse.