Plantlet Regeneration Via Somatic Embryogenesis and Investigations on Agrobacterium tumefaciens Mediated Transformation of Oak (Quercus robur)

Abstract

Immature zygotic embryos at different developmental stages were used as expiant source to induce somatic embryogenesis in Quercus robur throughout several years. Material was collected from several open pollinated trees at different sites. Somatic embryogenic callus could be induced on immature zygotic embryos collected 3 weeks to 18 weeks after anthesis. The highest initiation rate of nearly 30% could be induced approximately 11 weeks after anthesis in the middle of August on 5 μM 2–4D plus 0.5 μM BAP. Embryogenic competence of the callus lines has been maintained by regular subculturing on 1 μM BAP or hormone free media for four years. Maturation media with increased agar concentration (1 %) proved to enhance germination rate significantly. Conversion rate of matured somatic embryos into plantlets could be increased to 80% by a partial desiccation treatment.

Somatic embryogenic callus of oak was shown to be infectable by Agrobacterium tumefaciens. Additional wounding of embryogenic tissue caused significant decreases in regeneration. A cocultivation period of two days seemed to enhance the regeneration capacity of oak somatic embryogenic callus more than in four days cocultivation. Regeneration rate on selection media can be considered as relatively high. In the optimum case 70% regeneration could be observed after two passages. More than 100 lines are presently under cultivation. After several subcultures on kanamycin media samples were tested by PCR with selected primers for the presence of the CAT (Chloramphcnicol acctyltransfcrasc) gene. About 70% of the regenerated embryogenic calli showed positive results, whereas other samples of the kanamycin resistant tissue and also the control exhibited no band of the CAT fragment. PCR-tests with regenerated somatic embryos gave similar results.