Plant Regeneration from Cell Suspension Culture of Potato(Solanum tuberosum L.)
Ali Akbar Ehsanpour
In the present study, for callus production leaf and
stem segments of potato cultivar White Desiree were cultured on MS medium
supplemented with 2,4-D, NAA and Kinetin (callus production medium). Calli
then were transferred in the same liquid medium for cell suspension production.
In the next step cell suspensions were transferred back to the callus
production medium. Finally, calli derived from cell suspension were cultured
on 6 different shoot initiation media (S1-S6). However, on S6 medium with
combination of GA3 and BAP more than 80% of the calli produced shoot buds
and shoots. Fully grown shoots then were rooted and produced whole plants.
Chromosome and morphological analysis showed no somaclonal variation among
Potato is an important commercial
crop world wide. The crop is damaged by many pests and diseases making
it an important candidate for genetic manipulation. Creation of novel
germplasm through techniques of tissue culture and gene transfer holds
great potential for improving the quality, resistance to diseases and
agronomic characters of potato (Jayaree et al., 2001). Moreover,
a narrow genetic background resulted from a long time hybridization within
the species makes barriers for potato breeders to rapid breeding program
to meet the market demands for new varieties.
In a long history of conventional breeding, it has been
proved to be difficult to increase the frequency of desired genes and
to improve the selection accuracy and efficiency for target traits since
the tetrasomic inheritance of the cultivated potato (Solanum tuberosum
L.). In recent 20 year, including plant tissue culture and molecular cloning,
plant biotechnology has approached an efficient and rapid way for creating
new varieties and their reproduction (Dai et al., 2000).
Plant cell culture is a desired system for selection of
mutants with a simpler protocol than protoplast culture and a higher mutation
rate than involving intact plant, so, it is important in the era of biotechnological
breeding, particularly in mutants resistant to environmental stress. Until
middle of 1990s, there were about 1700 varieties of 154 plant species
reported to be bred through mutation strategy (Xu, 1998) and most of which
were come from the induction combining together of cell and tissue culture.
Using cell culture and artificial induction, potato mutants resistant
to Phytophthora infestance and Fusarium oxysparium were
produced (Li and Zeng, 1990). However, it is rather difficult in potatoes
to gain mutants than in other crops, which due partly to its less-optimized
cell suspension culture techniques and its high ploidy level that usually
accompanied with a low mutation rate. Therefore, it is necessary in addition
to further improve the techniques for cell suspension culture and selection
of single mutant cells (Qi et el., 1996).
Qi et al. (1996) established the cell suspension
culture line from the plantlet leaves of cv. Chunshu 1 and obtained vigor
cells. The plants were regenerated from a further research (Qi et al.,
2000). Feng (1990) constructed cell suspension lines of cvs. Gannongshu
1 and Russet Burbank using leaves and nodes as explants. With the same
varieties, Zhang and Dai (2000) researched on the effects of status of
callus, periods of subculture of the callus and different kinds of medium
on the quality of suspended cells which were originally from leaves and
tubers. More recently, Wang and Zhang (2002) looked at the suitable explants
for cell suspension and the results showed that cotyledon, hypocotyls
and stem tips seemed to be desired explants with a loose structure and
rapid growth of the callus and vigor cells isolated. For this, in vitro
regeneration system of plants via organogenesis or embryogenesis is a
prerequisite. Efficient plant regeneration from a range of explants tissues,
including leaf, stem and tuber, for several potato genotypes has been
reported by Hulme et al. (1992).
The ability of shoot regeneration and organogenesis under
in vitro condition may vary among species, cultivars (clones) and
especially the donor tissue. For example, plants have been regenerated
from isolated, protoplasts of potato cultivar Delaware (Ehsanpoue and
Jones, 2001). The results indicate that the organogenesis and plant regeneration
in potato is highly dependent on the genotype, origin of the explants,
growth regulators added to the culture medium and culture conditions.
Potato cultivar White Desiree is widely grown locally in Isfahan and so
far, no report has been published on plant regeneration using cell suspension
culture for this cultivar. The present studies describe optimum plant
regeneration system from cell suspension culture of potato cultivar White
Desiree. This system can be used fro selection of desirable cell line
and subsequent plant regeneration.
In this study a commercial potato tubers
cultivar White Desiree were supplied kindly by Potato Biotechnology Research
Center in University of Isfahan. Healthy young plants were grown in the
glasshouse under normal day light for 2-3 months. As explant, young internodes
with at least one auxiliary bud were harvested from fully grown plants.
Explants were then washed with tap water and transferred to 70% ethanol
for 30 sec and then were surface sterilized with sodium hypochlorite (5%v/v)
containing two drops of Tween 80 for 20 min followed by 3-4 washes with
sterile distilled water. Explants were then cultured on MS medium (Murashige
and Skoog, 1962) supplemented with sucrose (30 g L-1), agar
(8 g L-1). For callus induction, leaf and stem segments of
in vitro grown plants were transferred to callus proliferation, medium
described by Ehsanpour and Amini (2003). Cultures were maintained in the
culture room with 16/8 light-dark photoperiod at 25±2°C. The experiments
were carried out with 10 replications and 4 explants in each replication.
For cell suspension production, calli then were transferred to the liquid
callus production medium. The pH of media was adjusted to 5.8 and then
were autoclaved for 20 min at 121°C. In the next step, after two subcultures,
cell suspensions were transferred to the plant regeneration medium supplemented
with different growth regulators according to Table
Chromosome counting of regenerated plants derived from cell suspension
cultures were propagated on hormone free MS medium. Root-tip squash was
carried out as described by Karp (1991) from regenerated plants.
RESULTS AND DISCUSSION
stem segments of potato cultivar White Desiree were produced friable callus
on callus production medium (S7). However, leaf segments produced more
callus than stem segments. Cell suspension was also produced from callus
on the same liquid S7 medium. Growth curve of cell suspension showed that
a lag phase with approx 2-3 days, then cells moved to exponential phase.
The interval time between subcultures of cell suspension was 10-11 days.
Well grown cell suspension after 2 subcultures were then transferred to
regenerated media (S1-S6). On S1 and S2 medium after 3 weeks no shoot
bud regeneration was observed and calli became brown. When calli were
cultured on S3 medium, after 2 weeks they turned green but after 6-7 weeks
no shoot bud differentiation was observed. On S4 and S5 medium we observed
similar results as S3 but the calli showed higher growth rate (data are
not shown). However, in these media no regeneration was observed even
after 8 weeks. When calli were transferred on S6 medium they grow better
than other media and 80% of them gradually turned dark green and produced
granular callus after 8 weeks post culture in this medium on the surface
of the calli shoot buds were initiated. Figure
1 shows different stage of plant regeneration from cell suspension.
When regenerated plants were analyzed for chromosome counting, none of
them showed chromosome variation. They have also showed normal morphology
as parent plants. After acclimatization some of the regenerated plants
were transferred to the pot and they grow very well.
Cell suspension cultures are rapidly dividing homogenous suspensions
of cells grown in liquid nutrient media. Cell suspensions are used for
generating large amounts of cells for quantitative or qualitative analysis
of growth responses and metabolism of novel chemicals, as well as for
studies of cell cycle and plant regeneration system under standard conditions.
In addition, cell suspensions serve as an ideal material for the isolation
of protoplasts used intransient gene expression assays and Agrobacterium-mediated
transformation (Raffata et al., 1988). The establishment of suspension
cultures of Arabidopsis thaliana cells derived from leaf and hypocotyls
calli has been reported earlier (Karp, 1991).
of plant regeneration
from cell suspension of potato cultivar
White Desiree in S6 medium. A: Cell suspension, (B) Callus proliferation
derived from cell suspension, (C) Shoot bud initiation, (D)
Plant regeneration, (E) Chromosome analysis from regenerated
plants and (F) Fully grown regenerated plant in the pot
When carrying out a plant cell culture, there are three
important things that must be considered. First the plant part of interest
must be isolated from the intact plant. Next the appropriate environment
to promote optimal growth must be discovered and applied. This may vary
depending on the cells of interest. Finally, these procedures must be
carried out in a sterile environment to prevent growth of microorganisms.
These conditions have already been applied for callus and cell suspension
culture of several different plants. For example callus and cell suspension
has successfully been produced in Medicago sativa L. using combination
of three hormones (Kinetin, NAA, 2,4-D) (Amini and Ehsanpour, 2004). In
the same medium (S7) combination of plant growth regulators associated
with 1 g L-1 yeast extract promoted callus formation from cell
suspension of potato cultivar White Desiree. Lindeque et al. (1991)
have also used similar combination of plant growth regulators for callus
formation of potato cultivar BP1. It seems S7 medium can be recommended
for callus and cell suspension culture of potato cultivar White Desiree.
Anjum and Hakoom (2004) and Anjum (2001) reported that production of cell
suspension from explants is highly genotype dependent. They have also
reported that combination of plant hormones in the medium is very important
too. In the present investigation, when calli derived from cell suspension
of potato White Desiree was transferred to S1 to S5 medium despite of
presence of auxin and cytokinin in the culture medium no differentiation
of shoot bud was observed. In S2, S3, S4 and S5 medium only calli became
green. This observation indicating that chloroplast differentiation occurred
on the surface of the cells in the presence of cytokinin in the medium.
It might be as a result of the interaction of these hormones or interaction
of endogenous with exogenous hormones of the cells. In these media no
shoot buds were initiated. However, when callus-derived cell suspension
was cultured on S6 medium containing BAP and GA3 at the beginning, it
turned green and gradually produced green granular callus and finally
shoot buds were initiated on the surface of the calli. In this medium
it seems the combination and possibly synergism between GA3 and BAP promoted
shoot bud differentiation. Regenerated plants did not show any chromosome
variation or morphological changes. Patricia et al. (2004) detected
some somaclonal variation in callus culture of potato cultivars. However,
the culture condition for plant regeneration from cell suspension of potato
cultivar White Desiree used in this study was optimized. This procedure
can be used for regeneration of plant from selected of desirable cell
lines in the future.
Authors thank the Graduate Council of University
of Tarbiat Moalem and University of Isfahn for their supports.
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