Somatic embryogenesis of hybrid Pinus elliottii var. elliottii × P. caribaea var. hondurensis and ploidy assessment of somatic plants

A protocol is described for somatic embryogenesis (SE) Pinus elliottii × P. caribaea, an interspecific pine hybrid of major economic importance. Different embryogenesis conditions were tested, from time of initiation to plant regeneration. Immature megagametophytes containing zygotic embryos of five open pollinated (OP) mother trees were used for SE initiation. An established rate of 10.2% was achieved for explants inoculated on modified Litvay’s medium (mLV), supplemented with BAP (4.5 µM) and 2,4-D (9 µM). Initiation of SE was influenced by the mother-tree genotype however, modification of culture conditions enabled establishment of embryogenic cell lines (ECLs) from all OP seed families. Fifty-two percent of somatic embryos developed to the cotyledonary stage on mLV, supplemented with 40 µM ABA and 6% w/v sucrose. Cotyledonary stage somatic embryos converted to plants at 45–86%. Four months after acclimatization in a greenhouse, plant survival rates ranged between 78 and 94%. Flow cytometry analysis of ploidy, did not show any variation in ECLs and somatic seedlings.