The genus Withania is an important member of the family Solanaceae.
Twenty three species of the genus Withania have been reported (Negi
et al., 2006). Plants of Withania genus are distributed in
the east of the Mediterranean region and South Asia (Negi
et al., 2006; Atta-ur-Rahman et al.,
1999). Withania coagulans is commercially important because of the
ability possessed by its berries to coagulate milk (Negi
et al., 2006; Hemalatha et al., 2008).
This plant is well known in the indigenous system of medicine for the treatment
of ulcers, rheumatism, dropsy, consumption and sensile debility (Atta-ur-Rahman
et al., 1999). It has received much attention in recent years due
to the presence of a large number of steroidal alkaloids and lactones known
as withanolides (Hemalatha et al., 2008).
Biotechnological tools are important for multiplication and genetic enhancement
of the medicinal plants by adopting techniques such as selection, multiplying,
in vitro regeneration, genetic transformations and conserve the critical
genotypes of medicinal plants (Tripathi and Tripathi, 2003;
Rout et al., 2000). The production of withanone
and withanolide A from Withania somnifera by undifferentiated callus
cultures and cell suspensions induced using the natural variations of cells
(different explants) and Plant Growth Regulators (PGRs) is noteworthy (Sharada
et al., 2007). Tissue cultures having different morphology analyzed
for withanolide production showed that the inherent biosynthetic capability
of the donor plant was retained in cultures and they produced withanolides in
vitro (Sharada et al., 2007). In vitro
culture is more advantageous over a single shoot formation for rapid clonal
multiplication as well as for its conservation (Saritha
and Naidu, 2007). Protocols for in vitro regeneration in W. somnifera
were developed by Rani and Grover (1999). However,
according to present evidence, there is no report about W. coagulans
Therefore, the present study was undertaken in order to examine the potential of different explants in responding to in vitro conditions with the possibility of developing a protocol for the in vitro culture of W. coagulans.
MATERIALS AND METHODS
Production of plantlets as the explant source: Mature fruits of W.
coagulans were collected from the medicinal plant garden of Agriculture
and Natural Resources of Saravan College, Saravan, Iran, in the summer 2008.
Each fruit (berry) contains many seeds. After removing the pulp, endocarp was
manually broken to release the intact seeds. Seeds were thoroughly washed in
running tap water for 30 min to remove any adherent particles and were rinsed
in 70% (v/v) ethyl alcohol for 30 sec followed by 2% (v/v) sodium hypochlorite
for 10 min. Seeds were then washed under sterile conditions with sterile distilled
water and imbibed overnight (for 16 h) in sterile distilled water at 25±2°C.
Two seeds were inoculated in each glass culture tube (2.5x15 cm) containing
20 mL of MS (Murashige and Skoog, 1962) basal medium with
2% (w/v) sucrose and 0.8% (w/v) agar (Merck) without growth regulators. The
pH of the medium was adjusted to 5.8 before autoclaving for 10 min at 121°C.
The cultures were incubated at 25±2°C, with 24 h light (30 μmol/m2/sec)
provided by cool white fluorescent tubes in growth chamber (Kulkarni
et al., 2000). Leaf and internode segments excised from 1 month old
aseptic seedlings were used as explants.
Callus initiation: The leaf explants (0.5x0.5 cm) and internode (0.5 cm long) from W. coagulans plantlet inoculated on agar-solidified MS medium. In the first experiment, leaf segments were cultured on MS medium supplemented with 2, 4-dichlorophenoxyacetic acid (2 ,4-D, 2-4 mg L-1) with combination of 6-benzyladenine (BA, 0.5-1 mg L-1) or kinetin (Kin, 0.5-1 mg L-1). For the callus induction from internode segments, explants were cultured on MS medium in the presence of 2, 4-D (2-4 mg L-1) in association with BA (0.25-0.5 mg L-1). The pH of the medium was adjusted to 5.8 before autoclaving at 121°C for 20 min. All chemicals were purchased from Merck. Cultures were maintained at 25±2°C in a 16/8 h light/dark cycle with a light intensity of 40 μmol/m2/sec provided by cool white fluorescent. Callus was subcultured after 15 days on the original callus-inducing medium. The frequency of callus induction and callus dry weight was determined 8 weeks after culture initiation.
Shoot induction and rooting: For shoot induction, calli were cultured on MS medium containing 2 mg L-1 BA with 0.5 mg L-1 IBA and kept under the same conditions employed in callus initiation for 8 weeks. After 8 weeks, the number of shoots per treatment was recorded. To induce roots, elongated shoots were excised and transferred into culture tubes (23x150 mm) containing 20 mL of half strength MS medium supplemented with IBA or Kin (1-2 mg L-1) either alone. A set containing MS medium without growth regulators served as control. Data were recorded after 4 weeks of culture. The rooted plantlets were washed with tap water to remove rooting medium debris. The young plants were transplanted into boxes containing a soil and sand mixture (2:1) autoclaved at 120°C for 20 min under non sterile conditions and gradually exposed to ambient humidity. After 10 days, the acclimated plants were transferred to a greenhouse, maintained under partial shade and irrigated daily. The percentage of survival was recorded at 2 weeks intervals.
Statistical analysis: All experiments had five replicates per treatment and each experiment was repeated twice. Observations based on the percentage of culture response with regard to leaf and internodes induction, dry weight of callus, number of shoots and roots per explant and roots length were recorded. Results were subjected to Analysis of Variance (ANOVA) and mean values were separated according to Duncans multiple range test at p = 0.05. The statistical program used was MSTATC. The results are expressed as the Mean±SD of experiments.
Callus initiation: Callus initiation appeared for both explants after
14-16 days and callus was creamish-white, light green, compact or friable (Fig.
1A, B). Table 1 shows callus induction
for leaves and internode segments. Leaf explant produced 100% callus induction
in all media. Callus was also creamish and friable (Fig. 1A).
Highest callus growth in terms of dry weight (76±5.34 mg) was observed
in MS medium fortified with 2 mg L-1 2, 4-D + 0.5 mg L-1
Kin. Concentrations of BA (1 mg L-1) enhanced the growth response
in leaf explants. Kin at 1 mg L-1 showed a lower growth response
In second experiment, Internode explants showed an initial swelling at the
cut end in all medium after the second week. The percentage culture response,
callus color and texture varied according to the type of medium used (Table
1). Internode explants produced significantly very low percentage of callus
formation (25-42%) (Table 1). The comparison of callusing
potential of different explants showed that leaf explant is the best one, as
its callusing capacity was 100%, but highest dry weight (86±3.68 mg)
occurred in callus derived from internode explant in the presence of 4 mg L-1
2, 4-D with combination of 0.25 mg L-1 BA. Moreover, though leaf
segment was best in callusing potential, but all callus be brown after subculture
even by using 15 days intervals.
||Callus induction and plantlet regeneration from leaf and internode
segment explant of W. coagulans. (A) Induction of callus from leaf,
(B) internode segment drived callus, (C) induction of multiple shoot from
internode derived callus in MS+2 mg L-1 BA with 0.5 mg L-1
IBA, (D) shoot elongation, (E) rooting of regenerated shoot and (F) establishment
induction from leaves and internode segments of W. coagulans on
MS medium supplemented with various growth regulators
|B: No response. LG: Light green, CW: Creamish-white, C: Compact,
F: Friable. Values represent Means±SD of 5 explants per treatment
in five repeated experiments. Means followed by the same letter(s) are not
significantly different by the DMRT at 5% probability level
of various concentrations of IBA and Kin on rooting after 6 weeks of incubation
in half strength MS medium
|Values represent Means±SD. S: Swelling, R: Rooted,
C: callusing, -: No response
Shoot induction and rooting: Leaf explants failed to respond morphogenetically
to a growth regulator and non shoot regeneration yield. Shoot induction was
obtained just from callus induced from internodes explant 6 weeks after transferring
the callus to MS medium containing 2 mg L-1 BA with 0.5 mg L-1
IBA (Fig. 1C), but shoot regeneration yield was unsatisfactory.
The regeneration frequency varied from 18 to 33% and, by day 45, on average
of 3-5 shoots were regenerated per explant up to two subcultures (Fig.
1D), average length of shoots per culture was 3.5 cm.
Regenerated shoots were rooted best (100%) on half strength MS medium containing
1-2 mg L-1 IBA (Fig. 1E). Medium with 2 mg L-1
IBA produced highest number of roots (23 roots/plant) with an average root length
of 6.5±0.41 cm (Table 2). Rooted plantlets transferred
to soil and sand mixture (2:1) autoclaved at 120°C for 20 min (Fig.
1F), showed 90% survival when transferred to outdoor.
Withania coagulans is normally propagated through seeds, but these methods
are not efficient in producing large numbers of planting stock due to poor germination
rate with low viability of seeds. Biotechnological interventions for in vitro
regeneration, mass micro-propagation and gene transfer methods in medicinal
species have been practiced with success especially in the last decade (Tripathi
and Tripathi, 2003; Rout et al., 2000). The
induction of callus growth and subsequent differentiation and organogenesis
is accomplished by the differential application of growth regulators and the
control of conditions in the culture medium (Tripathi and
Tripathi, 2003). Protocols for in vitro regeneration in W. somnifera
were developed (Rani and Grover, 1999) but the present
study describes for the first time callus induction and plant regeneration from
In vitro response of W. coagulans was varying depending on the
explants. The comparison of callusing potential of different explants showed
that leaf explant is the best one, as its callusing capacity was 100%, but no
shoots could be regenerated from this source of callus and callus were turned
brown after three subcultures. These results corroborate with the findings of
Rani and Grover (1999) on W. somnifera. They
reported callus induction from the leaves, hypocotyls, roots and cotyledonary
leaf segments of W. somnifera. Maximum callusing (100%) was obtained
from root and cotyledonary leaf segments grown on the medium supplemented with
a combination of 2 mg L-1 2, 4-D and 0.2 mg L-1 kinetin.
In the present investigation, it was observed that average of 3-5 shoots were
regenerated per explant up to two subcultures. A similar finding of was reported
in W. somnifera (Siddique et al., 2004;
Sivanesan and Murugesan, 2008). Present results suggest
that the ability to form multiple shoots is dependent on explant source. Rani
and Grover (1999) reported Maximum shoot multiplication on the medium containing
2 mg L-1 BA, also reported that Regenerated shoots rooted best on
MS medium containing IBA (2 mg L-1) alone and IBA (2 mg L-1)
with IAA (2 mg L-1) in W. somnifera. For rooting, the various
levels of IBA and Kin were tested. Result showed that 2 mg L-1 IBA
proved to be most effective, the maximum number of rootlets with 6.5±0.41
root length (cm) obtained with 2 mg L-1 IBA (Table
2). It seems that Cytokinens (Kin) have no effect on rooting in W. coagulans
regenerated microshoots. These results corroborate with the findings of Sivanesan
and Murugesan, (2008) on W. somnifera. A successful tissue culture
method of propagation must result in re-establishment in soil of a high frequency
of tissue culture derived plant (Sivanesan and Murugesan,
2008). In this study, regenerated plantlet of W. coagulans showed
75% of survival rate when transferred to outdoor. Rani and
Grover (1999) and Sivanesan and Murugesan (2008)
obtained 83 and 87% survival rate in the W. somnifera, respectively.
The present study describes here in vitro culture of W. coagulans
for first time. In summary, present experiment show that Use of internodes for
micro propagation was beneficial than leaf explants. It will also be of use
in conservation and genetic transformation studies aimed at improving the plant.
However, the yield of shoot regeneration was unsatisfactory (18-33%). Regenerated
shoots were rooted best (100%) on half strength MS medium containing IBA (2
mg L-1) and produced 23 roots with an average root length of 6.5±0.41
cm. Since, the success rate of vegetative propagation in Withania sp.
is very low (Kulkarni et al., 2000), It is hoped
that a standard protocol to induce multiple shoots in culture may achieve.
The authors sincerely acknowledge the deputy of Research of Sistan and Baluchestan University, Zahedan, Iran, for its financial assistance under project No. 86016-87/4/13. Thanks are also due to Majid Jafari, Department of Medicinal and Aromatic Plant, College of Agriculture and Natural Resources of Saravan, Saravan, Iran, for his help and cooperation.