INTRODUCTION
From ancient times plants and plant parts are being used as medicines to treat
various diseases or other health problems. Medicinal plants constitute an important
resource, used by the indigenous medicinal system which is about 3000 years
old. The demand for plant based raw materials used predominantly in the pharmaceutical
and cosmetic industries have grown enormously during the recent years. About
seventy percent of these plant based raw materials for the pharmaceutical and
cosmetic industries come from the subtropical and tropical parts of the world
(Singh and Chowdhery, 2002).
Recently World Health Organization (WHO) has compiled a list of 20,000 medicinal
plants used in different countries. However, approximately 10,000 plants are
used for phyto-therapy in Indian system of medicine. According to Biological
Conservation Letter (Villa-Lobos, 1994), more than 7,000
species of plants found in various ecosystems are said to be medicinal in the
country. The global market for medicinal plant materials and herbal medicines
is estimated to be worth several billion dollars a year. International export
trade in medicinal plants is dominated by China which exports 1,21,900 tonnes
of materials a year and India exports 32,600 tonnes annually (Rajasekharan
and Ganeshan, 2002). So, India is one of the world’s richest sources
of medicinal and aromatic plants. The position of India is tenth among plant
rich countries of the world and fourth among the Asian countries.
Medicinal plants possess unlimited and untapped wealth of chemical compounds
with high drug potential, which make these plants useful as sources of biomedicines.
About 80% of the population in India and other developing countries depend on
traditional medicine mostly from plant origin. In India 2,500 species belonging
to more than 1000 genera, are regularly used in production of Ayurvedic, Unani,
Siddha and Tribal medicines (Rajasekharan and Ganeshan,
2002). The Central Council of Research on Ayurveda and Siddha Medicine has
drawn a list of 2430 commonly used medicinal plants having greater demand for
manufacture of galenicals, mixtures, compound formations and patent medicines
(Gupta, 1998). About 75% of these plants are collected
from tropical and 25% are from temperate forests. For the preparation of indigenous
system of medicine, 30% of materials are roots, 14% bark, 16% whole plants,
5% flowers, 10% fruits, 6% leaves, 7% seeds, 2% wood, 4% rhizomes and 6% stems
are used (Singh and Chowdhery, 2002).
Now days, demand for plant-based medicine in both developed and developing
countries like India is increasing because of the growing recognition that those
herbal medicines are non-narcotic. The rising demand in the organized manufacturing
sector has ruthlessly exploited the wild growing plant population those have
bulk use (Nayar and Sastry, 1987,1998).
Unfortunately, it is observed that, besides other factors like urbanization,
industrialization, development projects, etc., many such useful plants run the
risk of extinction, due to over-exploitation. It was reported that out of 2,800
plants, used in traditional medicines practiced in different parts of our country,
the drug potential of only 5% of these plants have been studied chemically or
pharmaceutically (Sabnis and Daniel, 1990). Of great concern
is the fact that before their medicinal values are fully known, many plants
may be lost from the local floras. The urgency and seriousness of the problem
has rightly deserved and drawn worldwide attention.
Legumes have a long history of use in agriculture. The family includes herbs,
shrubs, trees and vines distributed throughout the world, especially the tropical
rain forest. Along with uses in agriculture, human food and animal forage, timber
and dye legumes are having a greatest value of medicines and can be utilized
and explored for its medicinal value. Worldwide, there are a total of 790 genera
and 17,600 species of the legumes, of these, there are 163 genera and 1,252
species that are used as sources of medicinal plants. Among the sources of Oriental
herbal medicines, the Leguminosae is the fourth largest family in terms of numbers
of medicinal genera and species that are used, following the Gramineae (grasses,
grains), Compositae (daisies, dandelions) and Orchidacea (orchids). Very few
number of medicinal legumes are encouraged for cultivation, still there is large
number of plants (wild and endemic) are now unexplored and unattended (Hu,
1980; Wee and Keng, 1992; Ling,
1995).
Desmodium gangeticum (L.) DC (Syn. Hedyserum gangeticum) of Fabaceae
is known as Salaparni or Prishniparni in Hindi and Sanskrit is a medicinal legume.
It has multiple uses in the Ayurveda system. Three pterocarpenoids namely gangetin,
gangetinin and desmodin were isolated from hexane extract of roots of D.
gangeticum (Purushothaman et al., 1971).
During routine pharmacological screening gangetin exhibited significant anti-inflammatory
and anti-fertility activity in albino rats (Ghosh et
al., 1983; Pillai et al., 1981). The
plant is commonly used as bitter tonic, digestive, anti-catarrhal, anti-emetic,
in inflammatory conditions of the chest and others (Nayar
et al., 1956). The plant also shows anthelmintic, aphrodisiac, astringent,
diuretic properties. Roots are chewed daily for the cure of typhoid and pneumonia.
The plant parts form part of the compositions of popular Ayurvedic medicines
such as Dasamula Kwatha (M/s Zandu Pharmaceuticals), Dashmularishta (Dabur,
Himalayan Drugs).
In vitro propagation system proved to be highly useful for high rate
multiplication than the clonal propagation by conventional means (Hunter,
1986). Now, methods for micropropagation of high alkaloid producing lines
were established and routinely followed (Schoner and Reinhard,
1986) as in other medicinal plants like Atropa (Staba
and Chung, 1981) and Dioscorea (Heble and Staba,
1980). It was found that Desmodium gangeticum grows mainly in South
East Asia and Northern Australia, is a rare species, mostly found in Sal forest,
shows annual seed set (in the month of March-May) and low rate of multiplication
in the natural condition. Due to its high medicinal value D. gangeticum,
there is an urgency to develop appropriate technique for mass propagation of
this valuable species and to domesticate it for future use. In the present study
a protocol for regeneration and mass propagation of D. gangeticum has
been established using nodal explants.
MATERIALS AND METHODS
Plant material and surface sterilization: Healthy plants of Desmodium
gangeticum (L) DC were collected, grown in the nursery beds of the experimental
gardens. For the purpose of micropropagation young shoots were harvested from
3-6 months green house grown plants. These shoots were defoliated. The first
2-3 nodes from the apical region and 1-2 nodes from the basal region of these
shoots/branches were discarded. The stem node segments (0.8-1.2 cm) were cut
with the help of a clean razor/blade/scalpel so that each contained a dormant
axillary bud. These nodal explants were washed under running tap water for another
5-10 min. The explants were surface-sterilized in batches of 15-20 in an aqueous
solution of 0.1% (w/v) mercuric chloride (HgCl2) for about 7-10 min.
Following surface sterilization all the explants were rinsed 3-4 times in sterile
distilled water and inoculated on to the surface of sterilized nutrient agar
media.
Culture medium: Murashige and Skoog (1962) basal
medium was supplemented with various concentrations of Benzyladenine (BA) and
Kinetin (Kn) with 2% (w/v) sucrose and gelled with 0.8% (w/v) agar (Bacteriological
Grade, Himedia, Mumbai, India). The pH of the medium was adjusted to 5.8 by
addition of 0.1 N KOH/NaOH or 0.1 N HCl.). Three to five explants were transferred
onto each culture flask containing 30 mL of medium. For each treatment at least
15 replicates were maintained.
All the cultures were incubated in a growth room with a 16 h photoperiod (cool,
white fluorescent light (30 μmol m sec-1) and the temperature
maintained at 25±2°C, with 50-80% relative humidity.
Proliferated microshoots were separated and those measuring 2-3 cm and above
were individually planted onto the basal MS medium with or without the supplementation
of auxins (0.25-1 mg L-1) like IAA (indole-3-acetic acid; Sigma,
USA) or IBA (indole-3-butyric acid, Sigma, USA) for rooting.
Plantlets with healthy root systems were washed free of the agar gelled medium
(especially the root portions) under running tap water, dipped for 4-5 min in
an antifungal solution (0.1% w/v `Bavistin', Bayer India) and transplanted in
small (5 cm diameter) plastic tea cups, earthen pots or poly-bags containing
autoclaved sand:soil:compost/farmyard manure (1:1:1). The pots were watered
with 5 mL of ¼ strength MS major and minor salts on alternate days for
the first week and hardened under a polyethylene tent in the greenhouse for
at least one week. Relative humidity was 85% at 28-30°C under daylight conditions
(during May-July). After hardening for 7-10 days the plants were transferred
to bigger pots (25 cm diam.) containing a non-sterile sand: soil: compost mixture
(1:3:1) and shifted to field conditions.
Scoring of data: All the data observed, collected and documented on
axillary bud multiplication, organogenesis and somatic embryogenesis were statistically
analyzed by mean, standard error and some of the results were compared by DMRT
(Duncan, 1955).
Results are presented in Mean±SE of three independent experiments each
with 20 replicates in order to find out the accuracy of results.
RESULTS
Response of nodal explants of Desmodium gangeticum: The response
of nodal explants of Desmodium gangeticum on different culture medium
has been studied through establishments of culture and bud break response of
nodal explants with axillary bud. Data were recorded on multiplication and proliferation
of shoot buds, shoot growth, quality of shoots, rooting and plantlets establishment.
Culture establishment and bud break response through nodal explants with
axillary bud multiplication and proliferation of shoots: The response of
Desmodium gangeticum nodal explants cultured on different shoot multiplication
media during the first initiation passage over a period of three weeks has been
presented in Table 1. The data in Table 1
include the percentage of explant response, days required to bud break response,
number of shoots formed per responsive explant and the shoot growth in terms
of shoot length and number of nodes formed per such developed shoot.
Culture medium devoid of growth regulators (control) failed to stimulate the
bud break response in the cultured explants even when the cultures were maintained
beyond the normal observation period of three to four weeks. Higher levels of
the cytokinin (0.25-1.5 mg L-1 of Kn or BA) elicited bud break response
in varying percentage of the cultured explants. Depending upon the concentrations
of Kn or BA tested, the proportion of explants showing bud break varied from
41±0.577 to 100% and the number of days required for such a response
also varied from 4-10 days (Table 1).
| Table 1: |
Shoot formation on stem node explants of Desmodium gangeticum
cultured on semi solid in the first passage after second week |
 |
| Data (Mean±SE) of three independent experiments each
with 20 replicates, NR: Not responded, Means followed by the same letter
within the column are not significantly different at p<0.05 as tested
by the multiple range test of Duncan (1955) *Callus
formation Shoots measuring <0.5 cm not taken into account for calculation
of shoot length, Highest and least response are presented in bold |
|
| Fig. 1(a-h): |
In vitro morphogenesis of Desmodium gangeticum
and their derived plants, (a) Axillary shoot bud from nodal explant after
1 week, (b-c) Nodal explant with well developed shoots, (d) Nodal explant
with multiple well developed shoots, (e) Nodal explant with multiple shoots
after second passage, (f) Excised shoots showing well developed roots, (g)
Fully developed plantlets after 6 week of hardening on sand:soil (1:1) on
poly bag and (h) Fully developed plantlets after 12 weeks of hardening on
sand:soil (1:1) on pots |
The best response was obtained when the basal medium was supplemented with
0.5 mg L-1 of BA (Fig. 1a). In this medium, an
average of 5.00±0.577 days required for bud break and 1.56±0.181
mean number of shoots having 2.9±0.057 cm shoots length and 5.50±0.288
number of nodes were produced (Fig. 1b). The least response
obtained in Kn 0.1 mg L-1. The response was 41±0.577% and
9.33±0.66 number of days required for bud break (Table
1). The explants from the control set when transferred any time within the
second week of incubation on to fresh medium containing either Kn or BA also
exhibited bud break response.
Shoot multiplication: The potential for shoot multiplication in Desmodium
gangeticum appeared to be very strong in the presence of a cytokinin like
Kn or BA in the culture medium. In all cultures where Kn or BA supplement was
used at least one shoot emerged per axillary bud soon after the bud break (Fig.
1b, c). During the third week more number of shoot buds
(about 1-5 buds) appeared around the main shoot on some explant depending on
the concentration of the cytokinin used. Normally, when the cytokinin used was
Kn, the shoots formed in the cultures were healthy with thicker shoots and well-formed
leaves (Fig. 1d). On the other hand, shoots formed in medium
containing BA were thinner with smaller leaves. Although the mean number of
shoots obtained per explant with BA treatment was slightly higher than that
with Kn there was not much difference in the overall shoot growth i.e., both
in terms of mean shoot length and the mean number of nodes per shoot (Table
1). Treatment wise both the cytokinins BA and Kn appeared to be most effective
when used singly at 0.5 mg L-1 concentration. At higher concentrations
of BA and Kn the explants occasionally formed small mass of callus.
After the primary culture period of three weeks the shoots attained a length
of about 1.5 to 3 cm. At this stage the main shoot was aseptically excised/removed
and used for rooting experiments. The explants following removal of the shoots
were recultured on fresh media supplemented with either 0.5 mg L-1
of Kn or BA and maintained for about 4 weeks for the second passage. During
the second passage the shoot forming potentiality of these residual explants
appeared to be much stronger than that of the fresh explants during the primary
culture passage. The response of these explants has been presented in Table
2. Explants recultured on simple basal medium devoid of cytokinin produced
3-5 shoot buds within the first week.
| Table 2: |
Shoot formation on stem node explants of Desmodium gangeticum
in the second passage after excision of shoots |
 |
| Data (Mean±SE) of three independent experiments each
with 20 replicates, Means followed by the same letter within the column
are not significantly different at p<0.05 as tested by the multiple range
test of Duncan (1955), *Callus formation. Shoots
measuring <0.5 cm not taken into account for calculation of shoot length |
The shoot multiplication rate remarkably improved as soon as the basal medium
was supplemented with 0.5 mg L-1 of Kn or BA. Thus after a 4-week
period of second passage, with Kn, the shoot multiplication rate increased up
to about 5.76±0.088 shoots per explant (Fig. 1e) and
to as high as 33.8±0.057 shoots per explant with BA (Table
2). Unlike in the cultures with Kn treatment, invariably there was the formation
of callus at the cut shoot base prior to the formation of multiple shoot buds
in all cultures with BA treatment. In general, after culture period of 5 weeks
the shoot multiplication as well as the shoot growth was more pronounced with
BA treatment than Kn treatment.
Shoot growth and quality: The overall growth response of the shoots
regenerated on various media during both the primary passage and the second
passage was recorded in terms of mean shoot length and mean number of nodes
per shoot (Table 1, 2).
Shoot growth on nutrient media supplemented with lower levels of cytokinins
(0.1 mg L-1 of BA or Kn) was visibly poor during the primary passage,
where the mean shoot length did not exceed 1.5 to 2 cm after three weeks of
observation period. These shoots had only 1-3 nodes on an average. Growth of
the regenerated shoots was optimal at a concentration of 0.5 mg L-1
of BA (2.9±0.057 cm length with 5.50±0.288 nodes) followed by
that of 0.5 mg L-1 of Kn (1.72±0.005 cm length with 4.66±0.881
nodes) in the nutrient medium (Table 1). The appearance of
the shoots was also more or less normal.
During the second passage shoots produced on Kn (0.5 mg L-1) media
were had a somewhat stunted appearance with shorter internodes (2.38±0.004
length with 3.22±0.005 nodes) and thinner leaves but when these were
sub cultured on media with lower Kn level (0.25 mg L-1) the shoots
became normal after about a 2 weeks growth period. On the other hand, shoots
produced on BA (0.5 mg L-1) supplemented media were taller and had
longer internodes (3.85±0.071 length with 3.97±0.005 number of
nodes). But such shoots had a somewhat hyper hydrated or vitrified look.
| Table 3: |
Root formation of Desmodium gangeticum affected by
various concentrations of IAA and IBA |
 |
| Data (Mean±SE) of three independent experiments each
with 20 replicates, NR: Not responded, Means followed by the same letter
within the column are not significantly different at p<0.05 as tested
by the multiple range test of Duncan (1955) |
However, when such shoots were transferred to media devoid of cytokinin or
with lower levels of BA or Kn (0.1-0.5 mg L-1) the normal growth
resumed and the appearance of the shoots improved gradually. Irrespective of
the concentration of the cytokinin used in the medium there was a considerable
degree of leaf fall from the developing shoots. However, in spite of the leaf
fall these shoots did not die and behaved normal during the rooting experiment.
Rooting and plantlets establishment: When regenerated shoots attained
a length of about 3 cm or more, they were excised and planted on semi solid
basal medium alone or supplemented with varying concentrations (0.25, 0.5, 1.0
or 1.5 mg L-1) of either IAA or IBA for induction of rooting. The
rooting responses of shoots on different media which included rooting percentage,
days required for root initiation, mean number of roots per shoot and mean root
growth over a period of three weeks have been presented in Table
3. There was no rooting in case of shoots planted on basal media devoid
of growth regulator (control). Similarly, at lower levels of IAA (0.25 and 0.5
mg L-1) or IBA (0.25 mg L-1) treatments also there were
hardly any rooting in the cultured shoots during the 3 weeks observation period.
In all cultures with higher levels of IAA (1.0 and 1.5 mg L-1) or
IBA (0.5, 1.0 and 1.5 mg L-1) treatments, root primordia emerged
from the shoot base starting from day 6 to 16 after shoot inoculation (Fig.
1f) and soon after that the root growth was rapid. IBA was more effective
than IAA in induction of rooting as days required to rooting was only 6-8 days
as against the 12-16 days required for similar response in case of the latter.
Further, the IBA treatments also recorded better percentage of rooting than
the IAA treatments (Table 3). IAA always produced fewer (only
2-3) roots than that (4-7 roots) obtained with IBA. Occasionally, there was
leaf drop during rooting but such leaf drop was not a problem for shoot survival
because such shoots developed new leaves sooner or later when transplanted in
potted soil. During the rooting experiments in all the cultures supplemented
with the auxins IAA or IBA there was considerable thickening of the shoot base
prior to root initiation. Depending on the concentrations of auxin in the medium
there was also in some cultures simultaneous callusing and rooting at the thickened
shoot base.
For acclimatization, rooted plantlets were removed from the culture medium,
washed free from the agar medium, treated with some antifungal solution and
transferred to small poly-cups containing vermiculite and then to sterile potting
medium contained in poly-bags or earthen pots following the procedure as described
at appropriate section under ‘material and methods’. The potting mix
consisted of garden soil, farmyard manure (compost) and sand in the ratio of
1:1:1(v: v: v). The pots with the tender plantlets were kept under small polythene
tents in a green house for hardening purpose. After about 7-10 days of hardening
the polythene tents were removed. Soon the plants developed new leaves and started
to grow. The micropropagated plants were maintained in the green house for about
two weeks and subsequently transferred to the open for normal growth (Fig.
1g, h).
DISCUSSION
The dependence of cultured explants on bud break response and shoot multiplication
was extensively discussed (George. and Sherrington, 1984).
This has also been recently reported in the case of micropropagation of many
medicinal plants like Hemidesmus indicus (Patnaik
and Debata, 1996), Gmelina arborea (Thirunavoukkarasu
and Debata, 1998) and Plumbago zeylanica (Sahoo
and Debata, 1998). In many other plants like Saussurea lappa by Arora
and Bhojwani (1989) while multiple shoots originated from leaf axils in
stem node explants in the growth hormone supplemented nutrient media, the stem
portion below the node often formed callus. Such situation did not hinder the
overall shoot multiplication rate in this species. Premature leaf fall is not
an uncommon phenomenon as it is often encountered in in vitro micropropagation
experiments of many species like Sassafras randaience (Wang
and Hu, 1984). In these studies the problems of leaf fall were easily overcome
by supplementation of certain growth substances.
As observed by Kokate (1995), a large number of medicinal
plants that generate multiple shoots in culture of axillary and shoot tip meristems
have opened up new avenues for production of pharmaceuticals with preservation
and propagation of elite genotypes. Protocol for micro propagation through shoot
bud cultures of a number of medicinal plants like Calotropis procera
(Das et al., 2005) and Houttuynia cordata
(Handique and Bora, 1999) etc., were developed.
In most of the cultures by supplementation of BA in the medium produced more
number of shoots produced which agrees with the result obtained by Singh
and Tiwari, 2010 in Clitoria ternatea The shoots obtained from the
multiplication medium were rooted on full strength of MS with supplementation
of IBA also reported by Dvin et al. (2011).
Starting with a single stem node explant 3-5 plants could be obtained after
three weeks of primary culture. Following excision of these shoots from the
explant and reculturing on fresh media for four weeks one could get about 35-40
shoots. Each of these shoots offering 3-4 nodes for the next culture cycle,
about 105-160 shoots can be obtained after another 2 months. Thus starting with
one explant one would expect to obtain such number of plants through only two
culture cycles involving shoot multiplication, rooting and hardening in about
8-10 weeks.
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