In vitro culture and transformation studies of spinach (Spinacia oleracea L.).
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
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.