Repetitive Somatic Embryogenesis of Ocotea catharinensis Mez. (Lauraceae): Effect of Somatic Embryo Developmental Stage and Dehydration

Abstract

Repetitive embryogenesis of Ocotea catharinensis from globular/early cotyledonary somatic embryos was successfully supported by WPM supplemented with 22.7 g l⁻¹ sorbitol, 20 g l⁻¹ sucrose, 400 mg l⁻¹ glutamine and 2 g l⁻¹ Phytagel. The best medium to induce repetitive embryogenesis in cotyledonary somatic embryos was half strength WPM supplemented with 20 g l⁻¹ sucrose, 400 mg l⁻¹ glutamine, 1.5 g l⁻¹ activated charcoal and 2 g l⁻¹ Phytagel. The mature somatic embryos gradually air dehydrated showed repetitive embryogenesis after subculture on half strength B5 medium supplemented with 20 g l⁻ sucrose, 20 g l⁻¹ Phytagel, 1.5 g l⁻¹ activated charcoal, 115.6 µM gibberellic acid and 214.8 µM naphthaleneacetic acid. The early cotyledonary, cotyledonary and mature somatic embryos tolerated respectively 95, 86 and 54% fresh weight losses without losing their repetitive embryogenesis potential. Cotyledonary and mature somatic embryos gradually air dehydrated in sealed Petri dishes showed 40–41% repetitive embryogenesis respectively after 20 days and 12 weeks desiccation storage. Repetitive embryogenesis in cotyledonary somatic embryos was significantly stimulated by chemical dehydration with 0.5 M sorbitol and 56% repetitive embryogenesis was achieved even after exposure to 2 M sorbitol for 24 h. The cotyledonary somatic embryos when alginate-encapsulated showed 47% repetitive embryogenesis even after chemical dehydration in 1.5 M sorbitol for 4 days followed by 1 h air dehydration, but failed to survive to the same dehydration conditions without encapsulation. The optimized repetitive embryogenesis and desiccation protocols offer the possibility to use in vitro techniques for continuous reliable somatic embryo production and short term germplasm storage.