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The objective of the present study was to develop a comprehensive and reproducible regeneration system for spinach (Spinacia oleracea L. ) from commercially important cultivars and to assess the potential use of spinach for Agrobacterium tumefaciensmediated transformation. Tissue cultures of spinach were initiated from seed material. Axenic shoot cultures of spinach were established on MS-based medium containing 1.0 VM NAA at a temperature of 15°C and under a 16 h photoperiod. These three parameters were found most suitable for the establishment of shoot cultures and the encouragement of axillary shoot growth. Attempts to enhance axillary shoot production of spinach were investigated by the use of a double phase culture system, employing semi-solid and liquid culture media. The application of liquid medium was feasable with a volume of 5 ml for a duration of 7 or 14 d or with a volume of 10 ml for a duration of 7 d, but the multiplication rate of spinach was not increased. Adventitious shoot production was initiated from cultured spinach root explants derived from axenic shoots or hypocotyl explants. Sections from root tips and middle segments exhibited the highest shoot regeneration capacity when cultured on Nitsch and Nitsch (1969) medium supplemented with 20 μM NAA and 5.0 QCM GA3. Histological analysis demonstrated that the regenerating shoots originated directly from the root explants. Adventitious shoots were rooted on MS-based medium containing 1.0 μM NAA and transferred to the glasshouse, where the plants were grown to maturity. Seeds collected from regenerated plants were 95 % viable, producing a homgenous, fertile Rl-generation. Flow cytometric analysis was used to determine ploidy levels of regenerated plants and their progenies and showed that spinach leaf tissue from all generations displayed an even proportion of Go/G1 cells and G2/M cells, which may be characteristic for this species. Transformation studies using in vitro derived spinach explants demonstrated a positive response using two strains of Agrobacterium tumefaciens. The highest transformation rate was achieved with 25 % of explants being GUS-positive, therefore confirming susceptibility of spinach to the binary vector containing both T-DNA border sequences. It was found that best results were obtained with root explants which had been incubated for 8 weeks prior to co-cultivation with Agrobacterium and in vitro material which had been maintained in culture for up to 2 years. This reproducible regeneration system for spinach and the demonstration that spinach is amenable to Agrobacterium-mediated transformation provides the basis for potential commercial application within spinach breeding, aiming to generate an improved crop plant. |
