Abstract: The need of MS salts for shoot sprouting and proliferation shows the high salt requirement for the growth of Gymnema sylvestre. Influence of plant growth regulators, IAA, BA, 2,4-D and kinetin on shoot sprouting was investigated. Synergistic effect of Vitamin B2 was studied. To overcome phenolic exudation and the effects of antioxidants, the effect of activated charcoal, citric acid and ascorbic acid were investigated. Incorporation of citric acid (100 mg L-1) to the medium prevented phenolic exudation and increased the production of healthy normal shoots and shoot bud differentiation in Gymnema sylvestre. There was a considerable improvement in rooting as about 53% shoots could be induced to root on 1/2 MS medium within 45 days with a fairly good length and number of roots per shoot. MS medium containing 1 mg L-1 BA+0.5 mg L-1 IAA + 100 mg L-1 Vitamin B2+100 mg L-1 citric acid is best for shoot proliferation and 1/2 strength MS medium with IBA 3 mg L-1 is best for root induction.
INTRODUCTION
Use of Gymnema sylvestre, commonly known as periploca of the woods, has increased recently due to the pharmaceutical potential of gymnemic acids, found in its leaves. The various reports on its multiple uses attracted attention for utilization of the plant for its active principle, gymnemic acid. Gymnemic acid has been reported to effect a natural treatment for diabetes mellitus. At present 90% collection of medicinal plants is from the wild. This will not augur the sustainable use of medicinal plants and also it will not fulfill the needs of the majority of the population. Gymnema sylvestre natural strands are fast disappearing and are threatened with extinction due to its indiscriminate collection, over exploitation of natural resources for commercial purposes and to meet the requirements of the pharmaceutical industry. The attempt for its production and conventional propagation is hampered due to its poor seed viability, low rate of germination and poor rooting ability of vegetative cuttings. Alternative propagation methods would be beneficial in accelerating large scale multiplication, improvement and conservation of the plant.
Due to lack of proper cultivation practices, destruction of plant habitats and the illegal, indiscriminate collection of plants from these habitats, many medicinal plants are severely threatened. Advance biotechnological methods culturing plant cells and tissues should provide new means of conserving and rapid propagation of valuable, rare and endangered medicinal plants.
The present study describes the in vitro propagation, methods of Gymnema sylvestre.
MATERIALS AND METHODS
Plant material: Seeds of Gymnema sylvestre were collected from one single plant grown in the Kodiakarai forest, Nagapattinam district, Tamil Nadu, India, identified by the taxonomist from Botanical Survey of India, Coimbatore, Tamil Nadu.
Seed culture: The seeds were washed in running tap water for 5
min, treated with 2-3 drops of Tween 40 for 10 min and finally the seeds
were washed thrice with sterile distilled water. Seeds were subsequently
surface sterilized with 0.1% HgCl2 for 5 min and washed thrice
with sterile distilled water. The surface sterilized seeds were cultured
on Murasige and Skoog medium supplemented with 2% sucrose and solidified
with 0.8% agar (Himedia) (Ashok et al., 2002). pH of the medium
was maintained at 5.8. Samples were grown at a photoperiod of 16 h light
and 8 h darkness at 25 ± 1°C with the light the light intensity
of 1000 lux provided by cool white fluorescent lamps during the photoperiod.
The resulting seedlings were used as explant source. Seeds and seedlings
of Gymnema sylvestre are shown in Fig. 1.
| Fig. 1: | (a) Seeds and (b) seedlings of Gymnema sylvestre |
In vitro propagation of Gymnema sylvestre
Initiation and multiplication: Auxillary node explants
were collected from a healthy plant. Explants were cultured on MS media
fortified with various concentrations (mg L-1 w/v) of cytokinins
either individually or in combination with auxins (concentration ratio
was specified in the Table 3) were investigated to optimize
salt and hormonal requirements for sprouting and multiple shoot induction.
pH of the media was adjusted to 5.8 with 0.1 N NaOH. All culture media
contained 3% sucrose (w/v) and solidified with 0.8% agar (Bacteriological
grade, Himedia, India (Komalavalli and Rao, 2000), Media was sterilized.
Explants were placed vertically in glass tubes (150x25 mm) containing
20 mL of culture medium and plugged tightly with non-absorbent cotton.
Rooting: Shoots (4-5 cm long) regenerated from different explants were excised and individually transferred to MS medium fortified with various concentrations of auxins (specified in the Table 6) (IAA, IBA and NAA) and onto varying strengths of MS salts (full, 3/4, 1/2, 1/4 and 1/8) for root induction. After 50 days in rooting medium, the rooted micro shoots were removed from the culture medium and the roots were washed in sterile distilled water to remove all traces of agar and transferred to soil.
RESULTS AND DISCUSSION
Influence of medium: Among the three different media tested, MS
medium was found to be the best basal medium for shoot sprouting (6.8
± 1.0%), number (3.61 ± 0.51) and length (2.83 ±
0.04) of shoots with little callus formation followed by B5 and white
medium (Table 1, 2). The shoot buds
which sprouted on white medium showed only limited development even if
they were maintained for longer period in culture as in previous report
(Komalavalli and Rao, 2000). Shoots did not develop on any media in the
absence of cytokinin. In vitro propagation of plants belonging
to Asclepidiaceae have also been shown to have optimum overall growth
in MS medium (Chi Won and John, 1985; Patnaik and Debata, 1996; Komalavalli
and Rao, 1997). Thus the degree of growth and differentiation varied considerably
with the medium constitution (Shekhawat et al., 1993; Das et
al., 1996). The need of MS salts for shoot sprouting and proliferation
shows the high salt requirement for the growth of G. sylvestre.
| Table 1: | Influence of various media supplemented with 0.5 mg L-1 BA on shoot bud induction from axillary node explants of Gymnema sylvestre after 30 days |
| Values are Mean ± SE of 10 replicates | |
| Table 2: | Influence of various media supplemented with 1 mg L-1 BA on shoot bud induction from axillary node explants of Gymnema sylvestre after 30 days |
| Values are Mean ± SE of 10 replicates | |
Influence of plant growth regulators: Axillary shoot sprouting
was initiated at all concentrations of BA alone and in combinations of
BA and IAA, 2, 4-D and kinetin, 2, 4-D and BA, when compared to kinetin
combinations.
| Fig. 2: | Multiple shoot developed from Gymnema sylvestre axillary node explant, (a) Shoot initiated from |
IAA and BA combinations were effective (Table 3). In the present study, combined BA (1 mg L-1) and IAA (0.5 mg L-1) in the culture medium promoted the shoot sprouting frequency and multiple shoot induction whereas Komalavalli and Rao (2000) reported that high concentration of IAA and IBA (above 0.1 mg L-1) induced callus, occasionally root formation occurred at excised ends and prevented multiple shoot induction. In IAA and BA combinations, the shoot number was also increased (Fig. 2). Combinations of 2,4-D and BA was also tested. Shoots were sprouted in all the concentrations. When compared to IAA and BA combinations, the propagation rate was very low. Komalavalli and Rao (2000) reported auxin combined with either BA or kinetin above resulted in low propagation rate. Combination of 2,4-D and BA induced callus formation.
Reddy et al. (1998) reported that kinetin did not improve significantly
the shoot length and the number of proliferating shoots. In the current
report, combined, 2,4-D (1 mg L-1) and kinetin (0.5 mg L-1)
in the culture medium promoted the growth of the shoots. When compared
to kinetin, BA significantly improved the shoot length and the No. of
proliferating shoots. MS medium containing BA was more effective than
kinetin for inducing proliferation of axillary buds as in previous report
(Reddy et al., 1998). Results of BA alone supplemented medium and
combinations BA and IAA, BA and 2 ,4-D, kinetin and 2, 4-D supplemented
medium were analysed. Results were shown in the Table 3.
From the results it was inferred that combinations of IAA (5 mg L-1)
and BA (1 mg L-1) increased the propagation rate, whereas Komalavalli
and Rao (2000) reported that the shoot number increased when auxins (both
NAA and IBA at 0.1 mg L-1) combined with BA and kinetin. Superiority
of BA and kinetin in combination has been found in micropropagation of
other woody perenials (Das et al., 1996; Komalavalli and Rao, 1997).
The shoots formed exhibited phenolic exudation and leaf drop, which inhibited
the conversions of multiple buds into shoots as in earlier report (Komalavalli
and Rao, 2000).
| Table 3: | Influence of plant growth regulators on multiple shoot induction from axillary node of Gymnema sylvestre on MS medium after 30 days |
| Values are mean ± SE of 10 replicates | |
Influence of vitamin B2 (Riboflavin): Synergistic effect
of Vitamin B2 was studied after determining the optimum cytokinin
and auxin levels for shoot sprouting to improve the quality of the shoots.
Different concentrations of Vitamin B2 was added into the medium.
Vitamin B2 100 mg L-1 significantly improved the
shoot sprouting frequency, shoot number and shoot length, prevented yellowing
of leaves and reduced the callus formation at the cut end of the axillary
node explants (Table 4). Thus Vitamin B2
was found to be necessary for the speedy proliferation of axillary buds
and to improve the quality and quantity of Gymnema sylvestre. Komalavalli
and Rao (2000) reported the influence of complex extracts in G. sylvestre
in vitro propagation.
| Table 4: | Influence of Vitamin B2 on multiple shoot induction in 20 day old seedling from axillary node explant of Gymnema sylvestre on MS media fortified with 1 mg L-1 BA+0.5 mg L-1 IAA after 30 days |
| Values are mean ± SE of 10 replicates | |
| Table 5: | Influence of antioxidants on multiple shoot induction from 28 day old seedling axillary node of Gymnema sylvestre on MS medium supplemented with 1 mg L-1 BA+0.5 mg L-1 IAA+100 mg L-1 vitamin B2 after 30 days |
| Values are Mean ± SE of 10 replicates | |
Influence of antioxidants: To overcome phenolic exudation, the
effect of activated charcoal, citric acid, ascorbic acid were investigated.
Incorporation of citric acid (100 mg L-1) to the medium prevented
phenolic exudation, increased the production of healthy normal shoots
and shoot bud differentiation in G. sylvestre (Table
5) as in earlier report (Komalavalli and Rao, 2000). The promotive
effect of citric acid was reported by Shekhawa et al. (1993) in
Prosopris cineraria. Activated charcoal added to the culture medium reduced
the number of shoots per explant, whereas it is reported to have beneficial
effects in Eucalyptus tereticornis (Das and Mitra, 1990) and Sesbania
sesban (Shankar and Mohan Ram, 1999). Reduction in shoot number may
be due to adsorption of essential compounds besides the inhibitory factors
(Weatherhead et al., 1979). Ascorbic acid added into the culture
medium did not give significant results. In marked contrast, ascorbic
acid (100 mg L-1) proved to be better for multiple shoot induction
and to sustain overall growth in the related species of Tylophora
and G. elegans (Komalavalli and Rao, 1997). Thus the culture of
shoots in vitro in the presence of citric acid completely inhibited
the explant browning by controlling the phenolic oxidation and enhanced
multiple shoot induction in G. sylvestre.
| Fig. 3: | Rooting of in vitro derived Gymnema sylvestre plantlets |
| Table 6: | Influence of IBA concentration and MS medium strength on rooting of Gymnema sylvestre derived shoots after 45 days |
| Values are Mean ± SE of 10 replicates | |
Rooting of in vitro desired shoots: The shoots (4-6 cm) were cultured on various strengths of MS basal medium fortified with different concentration of IBA (Table 6). Komalavalli and Rao (2000) reported that IBA was most effective for root induction and survival in the field with minimal callus formation. IBA improved the overall growth of roots and the reduced the time duration of root induction. There was a considerable improvement in rooting as about 53% shoots could be induced to root on half strength MS medium within 45 days with a fairly good length and No. of roots per shoot. Photographs of in vitro derived roots and plantlets are shown in Fig. 3. Thus improvement in overall quality of roots was observed at half strength MS medium as in the earlier report (Komalavalli and Rao, 2000). In contrast, a drastic inhibitory effect on both root formation and elongation was noted at 1/4, 1/8, strength MS medium. No significant difference was observed regarding G. elegans and G. sylvestre (Komalavalli and Rao, 2000). In conclusion, the outlined procedure offers a potential system for improvement, conservation and mass propagation of G. sylvestre from pre-existing meristems of seedling explants. MS medium containing 1 mg L-1 BA+0.5 mg L-1 IAA+100 mg L-1 Riboflavin (Vitamin B2)+100 mg L-1 citric acid is the best for shoot proliferation. MS basal medium supplemented with 3 mg L-1 IBA is the best for root induction. The in vitro propagation of G. sylvestre is not very different from that of G. elegans described earlier and may be applicable for other economically important wood climbers as well. Komalavalli and Rao (2000) reported that MS medium containing 1 mg L-1 BA+0.5 mg L-1 kinetin+ 0.1 mg L-1 NAA+100 mg L-1 malt extract+100 mg L-1 citric acid, the best for shoot proliferation.