High subculture frequency, maltose-based and hormone-free medium sustained early development of somatic embryos in maritime pine

Embryonal-suspensor mass (ESM) lines characterized by a spiky morphotype (i.e., developed early embryos escaping from the ESM periphery) were recently shown to produce the best maturation yields in Maritime pine. How to select or preserve such a valuable morphotype during ESM maintenance (prior to maturation treatment) is still unknown. Several maintenance procedures were tested; 2400 ESM from 10 lines were subcultured each 7 or 14 d on maltose- or sucrose-containing medium without plant growth regulator (PGR) or supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine or a low concentration of abscisic acid. Multiple components analyses based on growth rate, macromorphology data, and micromorphology data, collected for 6 mo., allowed for the association of the spiky morphotype with additional traits (defining the phenotype I) such as whitish aspect, high vigor, high growth rate, and complex cellular organization (resulting from high early embryogenic ability). Since a gradual increase in growth rate as well as a decrease in cellular organization were concomitantly observed during the 6 mo. experiment, we concluded that ESM growth and early somatic embryo development were disconnected. In some lines, the progressive loss of early embryogenic ability (aging process) could be decreased using maltose-based and PGR-free medium. For most lines, the aging effect was minimized using a weekly subculture. An improved procedure for ESM maintenance prior to the maturation step is thus proposed.