In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls

A high-efficiency two-step culture procedure for direct somatic organogenes is in loblolly pine (Pinus taeda L.) resulting in the formation of multiple shoot structures induced on cotyledons and hypocotyls of mature zygotic em bryos is described. Mature zygotic embryos of eight genotypes of loblolly p ine were used as explants to induce direct somatic organogenesis with this two-step culture method, involving the induction and the differentiation of direct adventitious shoots. After mature zygotic embryos of eight genotype s of loblolly pine were cultured on induction medium containing 2,4-dichlor ophenoxyacetic acid (2,4-D) or alpha -naphthaleneacetic acid (NAA), 6-benzy ladenine (BA), and kinetin for 2-3 weeks, embryos were transferred to diffe rentiation medium. Adventitious shoot regeneration via direct somatic organ ogenesis with the frequency of 8.7-27.8% was obtained from mature zygotic e mbryo cultures of the genotypes tested. The highest mean number of 32.6 adv entitious shoots per mature zygotic embryo was produced from genotype La. T he tissue culture protocol of in vitro shoot regeneration via direct somati c embryogenesis was optimized after examining the periods of the induction culture, chilling treatment, glutamine concentration, and basic medium leve ls. Rooting was achieved on TE medium supplemented with 0.5 mg/l indole-3-b utyric acid (IBA), 0.5 mg/l gibberellic acid (GA(3)), and 1 mg/l 6-benzylad enine (BA), and regenerated plantlets were established in soil. These resul ts suggested that adventitious shoot regeneration via direct somatic organo genesis could be useful for clonal micropropagation of some genotypes of lo blolly pine and for establishing a transformation system of this coniferous species.