Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Abstract

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11 μM NAA and 4.44 μM BA or 26.85 μM NAA and 13.32 μM BA. The callus proliferation was more efficient on medium supplemented with 26.85 μM NAA and 13.32 μM BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11 μM NAA and 4.44 μM BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89 μM GA3 in combination with 0.54 μM NAA, 11.42 μM IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42 μM IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10 weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.