Embryogenic cultures of plants are exposed to various stress factors both in vitro and during cryostorage. In order to safely include the plant material obtained by somatic embryogenesis in combination with cryopreservation for breeding programs, it is necessary to monitor its genetic stability. The aim of the present study was the assessment of somaclonal variation in plant material obtained from embryogenic cultures of Picea abies (L.) Karst. and P. omorika (Pancic) Purk. maintained in vitro or stored in liquid nitrogen by the pregrowth-dehydration method. The analysis of genetic confoimity with using microsatellite markers was performed on cotyledonary somatic embryos (CSE), germinating somatic embryos (GSE) and somatic seedlings (SS), obtained from tissues maintained in vitro or from recovered embryogenic tissues (ETc) and CSE obtained after cryopreservation. The analysis revealed changes in the DNA of somatic embryogenesis-derived plant material of both Picea spp. They were found in plant material from 8 out of 10 tested embryogenic lines of P abies and in 10 out of 19 embryogenic lines of P. omorika after in vitro culture. Changes were also detected in plant material obtained after cryopreservation. Somaclonal variation was observed in ETc and CSE of P omorika and at ETv stage of P abies. However, most of the changes were induced at the stage of somatic embryogenesis initiation. These results confirm the need for monitoring the genetic stability of plants obtained by somatic embryogenesis and after cryopreservation for both spruce species.
The Crassulaceae family mainly comprises herbaceous leaf succulents, some of which are used as ornamentals. The development of the embryo suspensor in Sedum reflexum L. was investigated using cytochemical methods, light and electron microscopy. The full development and functioning of the suspensor occurs during the late globular and heart-stage embryos. The suspensor consists of a large basal cell and a single row of 6-10 chalazal cells. The basal cell produces a branched haustorium which invades ovular tissues. The walls of the haustorium and the micropylar part of the basal cell form the wall ingrowths that are typical for transfer cells. The dense cytoplasm filling the basal cell is rich in profiles of endoplasmic reticulum, active dictyosomes, mitochondria, plastids, microtubules, bundles of microfilaments, microbodies and lipid droplets. The present work reveals that the suspensor structure in S. reflexum markedly differs from that found in other representatives of Crassulaceae. Ultrastructural analysis and cytochemical tests (including proteins, insoluble polysaccharides and lipids) indicate that in S. reflexum the embryo suspensor is involved mainly in absorption and transport of metabolites from the ovular tissues to the developing embryo proper via the chalazal suspensor cells.
We investigated direct and indirect formation of somatic embryogenesis in Brassica oleracea var. botrytis (cauliflower), a very important vegetable crop worldwide. Direct somatic embryogenesis, which is rather rare, was achieved in culture of 2-week-old hypocotyl explants of Brassica oleracea var. botrytis on MS medium supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5; 1.0; and 1.5 mg/l kinetin. Initial induction of embryogenic callus was achieved on MS supplemented with very low concentrations of 2,4-D (0.05 mg/l and 0.1 mg/l). Indirect somatic embryogenesis from leaf sections was obtained on MS supplemented with 0.05 or 0.1 mg/l 2,4-D. We examined various stages of somatic embryos (globular, heart, torpedo, cotyledonary). More embryos per explant were produced through the indirect pathway (23-25) than through the direct pathway (14-19). The number of embryos produced was high. There is a potential for recurrent, repeated or secondary somatic embryogenesis, possibly an unlimited source for mass propagation and ideal for synthetic seed production in this species. Plant regeneration was achieved on half-strength MS medium without any hormones.