Effect of ABA, the auxin antagonist PCIB and partial desiccation on stone pine somatic embryo maturation

Plant regeneration by somatic embryogenesis (SE) was achieved in Pinus pinea L., a forest species of economic importance for its edible seeds, but improvements in the SE protocol are needed to make this technology feasible for breeding programs. In the present study, different maturation treatments on medium with high concentration of sucrose and gelling agent were tested. The effects of abscisic acid (ABA) concentration and culture procedure, the presence of the auxin antagonist 2-(4-chlorophenoxy)2-methylpropionic acid (PCIB), and the partial desiccation of embryonal masses before maturation on the reduction of proliferation and promotion of maturation in six embryogenic lines were evaluated. Increasing ABA concentration neither reduced proliferation nor improved maturation. The highest number of mature embryos was produced with 121 mu M ABA in line 1F11 or 161 mu M ABA in line 2F47. The culture procedure did not affect growth rate, but monthly subcultures onto maturation medium increased the normal embryo production 13-fold by compared with no subculturing. PCIB decreased proliferation only when it was included during the 12 weeks of the maturation period, and did not improve somatic embryo production. Partial desiccation of embryonal masses between 5 to 26% water loss did not reduce proliferation but enhanced maturation by 1.7 to 4.7-fold compared with the control, depending on the embryogenic line. Up to 256 normal cotyledonary embryos per gram fresh weight from the best line and culture condition were obtained. Somatic embryos germinated and converted to plants at over 70%. Although improvements in maturation are provided, problems such as growth arrest of somatic seedlings and low rates of acclimatization still remain to be solved before SE can be used for large scale plant production in stone pine.