Download Chapter - V Micro Propogation of Gymnema sylveste R.Br.

Chapter - V
Micro Propogation of
Gymnema sylveste R.Br.
Chapter-V
Introduction
Gymnema sylvestre R.Br. is a valuable medicinal plant
belonging to the family Asclepiadaceae, distributed over most parts of
India and Africa. The plant is popularly known as ‘gurmar5 (destroyer
of sugar) for its distinctive property of temporarily impairing the taste
of sweetness.
Morphology and distribution
Gymnema sylvestre is large, more or less pubescent, woody
climber found in the Deccan Peninsula, extending to parts of northern
and western India. It is occassionaly cultivated as a medicinal plant.
Leaves are opposite usually elliptic or ovate (1.25-2.0 in. x 0.5-12.5 in.).
Flowers are small, yellow in umbellate cymes, follicles terete,
lanceolate, upto 3 in. in length.
The plant is stomachic, stimulant,
laxative and diuretic. The medicinally active parts of the plant are the
leaves and the roots. The complex mixture of the active principle
namely gymnemic acid were isolated from Gymnema leaves (Yoshikawa
et al, 1989; Yoshikawa et al, 1991; Maeda, 1989).
In ancient times, the Ayurvedic physicians observed that
chewing a few leaves of Gymnema sylvestre suppressed the taste of
sugar. It is well recognised in traditional medicine as a remedy for
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Chapter-V
diabetes mellitus, stomachic and diurea, (Sastri, 1956). Later the
clinical tests showed that regular use over a period of 3-4 months
helped to reduce glucosuria, the appearance of carbohydrates in urine.
Recent clinical trials conducted in India have shown that an extract of
Gymnema sylvestre is useful for controlling Blood Sugar. Studies of
early 1930s showed that the leaves of Gymnema cause hypoglycemia
which is explained on the assumption that the drug indirectly
stimulates the insulin secretion of the pancreas, since it has no direct
effect on the carbohydrate metabolism.
The recent pharmacological and clinical studies have shown
that Grymnema sylvestre acts on two sites.
a)
First, the taste buds in the oral cavity.
b)
Second, the absorptive surface of the intestines.
The structure of those taste buds which detect sugar in the mouth
is similar to the structure of the tissue that absorbs sugar in the intestine.
The important active ingredient of Gymnema sylvestre is an organic
acid called gymnemic acid (Fig.l), which is made up of molecules
whose atom arrangement is similar to that of glucose molecules. These
molecules fill the receptor locations on the taste buds for a period of one
or two hours, thereby preventing the taste buds from being activated by
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Chapter-V
any sugar molecules present in the food. It has been also noted that
Gymnema sylvestre takes away the bitter taste of bitter substances.
However, it has no effect on pungent, salty, astringent or acidic tastes.
Gymnema sylvestre significantly reduces the metabolic effects
of sugar by preventing the intestines from absorbing the sugar
molecules during the process of digestion. Because of the change in the
absorption of sugar, there is a consequent change in the blood sugar
level.
1
2.
3.
4
R2
R.
tigloyl
2-methylbutloyl
2-methylbutloyl
tigloyl
R
acetyl
acetyl
H
H
5
6
glcUA
H
Fig.l Gymnemagenin
Chemical constituents
Gymnema shows the presence of Gymnemic acid (GA), (+)
quercitol, lupeol, P-anyrin, stigmasterol etc. The active principle is
Gymnemic acid (Fig.l). GA I, II, III and IV are anti-sweet components
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Chapter- V
of the leaves Gymnema sylvestre (Yoshikawa et al, 1989). They all
contain a glucuronic acid moiety, the gymnemagenin aglycone esterified
at position C-21 and C-28. A second series of GA-V, VI and VII has
been also reported. GA VII is 3-O-glucuronide of gymnemagenin and
GA V is the O-glucuronyl - 22, 22- bis - O - tigloyl substitution pattern.
GA VIII - IX are also ester saponin, have an oxoglycoside moiety
attached to the glucuronic acid residue (Tsuda et al, 1989).
Propagation
The plant has been propogated by cuttings and to some extent
by seed germination. In cutting propagation, the success rate is
marginal. Constraints in seed propagation is the very shot span of
seed viability. Therefore, exploration of methods for its propagation
through tissue culture offers an effective alternative method for rapid
multiplication and conservation of this useful plant.
Recently the plant has been recognised by natural products
industry in North America and Europe. Number of commercial, over
the counter herbal products are now available that contain varying
amount of Gymnema (Liberti, 1993). In India, the fresh leaves are
available for Rs.60/kg. Hence it is felt that there is a great need for
cultivation of this important medicinal plant.
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F i g. 2: Gymn ema sylvestre
(a)
(b)
Mature plant which served as a source of explant growing at the
Botanical garden of MS University of Baroda.
Flowering branch
♦ s*
Chapter- V
Materials and methods
Plant material
Gymncma sylvestre plants, growing in the campus of M.S.
University of Baroda (Fig.2) served as the initial source of explants.
Tender twigs were collected from the mature mother plants. The shoots
were cut into pieces containing 2-3 nodes and washed thoroughly under
running tap water (30 min). The explants were given mild detergent
(teepol, 0.5%) wash for 2 minutes followed by thorough rinsing of
water to remove the traces of the detergent. Later on, the shoot pieces
were subjected to pre-treatment (2 hours) in a solution containing
bavistine (1%; w/v) and chloromphenicol (0.5%; w/v.)
The pretreated explants were surface disinfected with HgCl2
(0.1%; w/v) and repeatedly rinsed with sterile distilled water. These
explants were then cut into 1-1.5 cm long nodal segments. The single
node explants were then inoculated aseptically on MS Medium
supplemented with various combinations of auxin and cytokinin. For
multiplication, shoots generated from single node explants were
excised and sub-cultured as in vitro explants to MS medium
supplemented with various concentrations of BA and IAA/NAA.
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Chapter-V
In vitro raised shoots were subjected to induction of roots. Rooted
shoots in plastic pots (containing only vermiculate) were covered with
polyethylene bags were kept in the culture room for 3-4 weeks. Such in
vitro derived plantlets were gradually exposed to the culture room
conditions by gradually making holes in the polyethylene bags for
acclimatization. After thorough hardening, the plantlets were transplanted
to earthern pots containing a mixture of garden soil and compost (1:1;
w/v) and transferred to the garden. The plants were frequently subjected
to mist irrigation with spraying bottle for 1-3 weeks.
Tissue culture raised as well as seed germinated plants transferred
to the field were compared for their morphology and growth parameter.
Results and discussion
With the indiscriminate removal of natural flora, one third of the
biodiversity will be lost by the year 2030 and this does not exclude
medicinal plants (Nayar, 1997). Therefore, there is an urgent need for in
situ and ex situ conservation of germ-plasm. In recent years medicinal
plants like Plantago ovata, Catharathus roseus, Gloriosa superba,
Gymnema sylvestre etc. have been introduced as inter crops in
plantations. However, the time has come for the production of quality
planting material and value adition by appropriate technology. There is
growing optimism that micropropagation may provide uniform planting
material (George, 1996).
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Chapter-V
In the present study micropropagation of medicinally important
plant Gymnema sylvestre has been achieved by using nodal explants in
MS medium supplemented with varying concentration of BAP. In the
preliminary experiments there was severe problem of contamination
and senescence of the explants. The problems of contamination was
mitigated by the treatment of the explants with pretreatment solution
prior to inoculation. The pretreatment solution contained fungicide,
bavistin (1%; w/v) and antibiotic, chloramphenicol (0.5%; w/v).
Around 80% of the explants remained contamination free (Table-1).
Table-1: Control of contamination with different pretreatment
solutions in Gymnema sylvestre
Concentration of Bavistin
(%;w/v)
Contamination
(%)Mean±SE
0.00
0.25
0.50
1.00
1.50
100 + 3.0
90 ±4.1
60 + 3.5
20 ±4.1
20 ± 4.2
The effect of season on the rate of bud sprouting and
contamination were also studied. The studies proved that the maximum
contamination occurred during the period of July-August. The maximum
bud sprouting obtained in the months of March-June (Table-2).
Explants cultured in the MS basal medium did not show any response.
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Chapter- V
Within a week’s time the explants senescenced. This showed the
requirement of growth regulators in the medium. Emergence of shoot in
the
nodal
explant
cultured
in
basal
medium
with
subsequent
s
development
of shoots in medium supplemented with PGR’s is observed
in Woodfordia fructicosa (Krishnan and Seeni, 1994). Ocimum sanctum
(Pattnaik and Chand, 1996) and Tanacetum parthenium (Stojakowska
and Kiesiel, 1997). There are various reports on eytokinin induced shoot
initiation in vitro (Sen and Sharma, 1991; Purohit and Dave, 1995; Jain
and Nessler, 1996; Patil and Jayanthi, 1997).
Table-2 : Seasonal influence on contamination and sprouting of
nodal buds of Gymnema sylvestre
Contamination
(%)Mean±SE
Bud sprouting
(%)Mean±SE
January
53.20 ± 0.90
47.70 ± 0.20
February
45.30 ±2.10
52.40 ± 0.40
March
40.05+ 1.10
60.10 + 0.70
April
39.05 ± 1.40
58.40 ± 0.40
May
38.20 ± 0.70
61.30 ±0.40
June
3131+ 0.90
62.30± 0.50
July
72.70 ± 1.00
31.80 ± 0.69
August
71.55 ± 0.60
30.70 ±0.80
September
69.02 ± 1.20
30.30 ± 1.40
October
66.75 ± 0.90
31.80 ± 1.10
November
63.70 ± 1.12
35.20 ± 1.00
December
56.30 ± 2.44
48.80 ± 1.50
Month of collection
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Fig.3: Establishment of nodal exaplnts of Gymnema
(a)
(b)
(c)
Explants inoculated on MS medium supplemented with BA (4.4
pM)
Bud sprouting
In vitro nodal explants showing elongation growth of axillary
buds
Chapter-V
The addition of cytokinin promotes precocious axillary shoot
development (Hussey, 1976; Bhojwanf4nd Razdah, 1983). Although
BA and KN are equally effective in axillary bud proliferation, BA has
been considered to be more active (Rahman and Blake, 1988, Misra,
1996). Bud sprouting was initiated with various growth regulators
(Table-3).
Table-3: Effect of various growth regulators on bud sprouting in
Gymnema sylvestre
Bud sprouting
(%)Mean+SE
Concentration (pM)
BA
: NAA
4.44
4.44
: 0.00
: 0.53
BA
: IAA
4.44
: 0.57
KIN
: NAA
4.65
4.65
: 0.00
: 0.53
KIN
: IAA
4.65
: 0.57
80+1.2
0.00
:
60+1.5
80 + 2.5
70 + 2.7
50 + 1.8
40 + 2.3
50+1.7
0.00
MS medium with 4.4 pM BA showed the maximum percentage of bud
sprouting (Fig.3). Kinetin also showed bud initiation but not as induced
by BA. The nodal explants on BA (4.4 pM) in combination with NAA
(0.53 pM) showed better response than those on BA with IAA.
89
Fig.4: Induction of multiple shoots in Gymnema
(a)
(b)
Multiple shoots formation
Further growth of multiple shoots
Chapter- V
The in vitro raised nodal explants proved to be more beneficial for shoot
proliferations. The micropropagated plants show greater capacity for
morphogenesis than the cuttings excised from the field grown plants
(Jha and Sen, 1992; George, 1996). Since MS medium supplemented
with BA (4.4 pM) alone was giving good response, different
concentrations of BA were studied for the multiple shoot formation
(Table-4).
Table-4: Effect of various concentrations of BA on the number and
length of shoots formed per explants in Gymnema sylvestre
Concentration
(pM)
2.22
4.44
8.88
Number of
shoots
Mean±SE
Shoot length (cm)
Mean± SE
Number of
nodes
Mean± SE
2.60 ± 0.30
5.50 ±0.90
4.60 ± 0.70
1.40 ±0.40
3.40 ± 1.00
2.00 ±0.60
1.40 ±0.40
3.40 ± 1.00
1.80 ±0.70
MS medium supplemented with 4.4 pM BA gave the best response
(Fig.4). Reddy et al (1998) reported incorporation of NAA (1.6 pM)
with high concentration of BA (22 pM) in the medium. But in the
present study only BA (4.4 pM) gave good response. Explant
inoculated at higher concentration of BA alone or in combination with
NAA produced clumps of highly-reduced shoots with smaller leaves
(Reddy et al, 1998). In Hemidesmus indicus. Patnaik and Debata
(1996) reported such abnormal shoots in low concentration of BA.
90
Chapter-V
Reddy (1968) reported callus formation at the basal portion of the
node. But in this study there was no callusing found. Purohit and
Dave (1996) reported the response of KN at different concentrations
on proliferation of shoots. The present investigation showed no
response on MS medium with kinetin. When the regenerated shoot
attained a length of more than 3 inch, they were excised and planted
on half strength MS basal medium as well as MS medium with
various auxins like NAA, IAA and IBA (Table-5).
Table-5 : Effect of auxins on in vitro root formation from
regenerated shoots of Gymnema sylvestre
Concentration
(pM)
Root induction
(%)Mean±SE
No of roots
(%)Mean±SE
5.37
46.60 ± 9.40
4.00 ± 0.80
5.71
13.30 ± 5.40
1.33 ± 0.40
4.92
6.60 ± 7.70
0.33 ± 0.30
66 60 ± 9.40
6.66 ± 1.69
NAA
IAA
IBA
Ms Basal
('/2 Strength)
One of the major obstacles to micropropagation of plants is
rooting and acclimatization of plantlets (Torres, 1989). The cultures
which are inoculated on auxin free half strengths MS basal medium,
showed root initiations. However, rooting was slow and the
percentage was very less. With the addition of auxin callus formation
91
Fig.5: Germination of Gymnema seeds
(a)
(b)
Germination in vermiculate
In vitro germination seed on MS basal medium
Fig 6: Induction of rooting on shoot cuttings
(a)
(b)
Pulse treated (IBA) shoot cuttings transferred for rooting
Shoot cutting treated with IBA (2 4 j,iM) for 30 seconds showing
shoot growth
Chapter-V
was there which did not favour the root formation.
Thus ex vitro
rooting is preferred in commercial laboratories (Alderson et al, 1988).
Gymnema is conventionally propagated by cuttings and seeds.
The seeds were germinated in the field to see the frequency of seed
germination. The percentage rate was very low as 33% (Fig.Sa) the
cotyledonary leaves were very sensitive and the mortality rate was
more after germination. Rate of in vitro germination of seeds also
showed very low percentage as 40% (Fig.5b). This could be due to the
seeds loosing its viability very fast.
Pulse treatment with IBA (2.45 mM and 4.9 mM) (Fig.ba) was
given to the cuttings from the mature plant. The cuttings were
planted, in the sterile soil in the plastic cups. The cuttings which
were given treatment of 2.45 mM for 30 seconds achieved sprouting
and rooting after 6-7 days. But the percentage rate of growth was
very low (Fig.6b). This indicates the low percentage of propagation
through cuttings. In general the plant is propagated by cuttings and
seeds. But in our experiments the seed germination rate and the
propagation rate through cuttings showed the constrains in the
conventional propagation methods.
So the tissue culture method
offers an effective alternative method for rapid multiplication.
92
Fig.7: Induction of callus from leaf explants of Gymnema
(a)
(b)
Callus formation on MS medium supplemented with BA (11 pM)
and 2,4-D (2.26 pM)
Further proliferation of callus on same medium
Chapter-V
The Gymnema plants are known for its sugar masking property
exhibited by its leaves.
The leaves were inoculated for the callus
culture. The explants on culture medium supplemented with various
combinations of BA and 2, 4-D showed swelling, curling and
followed by the induction of callus at the cut ends (Fig.7a). The callus
induction was noted within 4-5 days on explants inoculated on media
supplemented with 11 pM BA and 2.26 pM 2, 4-D (Table-6).
Table-6 : Effect of various combinations of 2,4-D and BA on callus
formation from the leaves of Gymnema sylvestre
BA
2,4-D\^
00
2.2
6.6
8.8
11.00
00
+
+
+
-
++
2.26
6.78
+
+
+
H—1—h
+
4444
9.04
11.3
+
4
++
4
+
-
-
+
+
+
++
+
4 4*
+ + + All the four leaflets showed response
+ + + Three leaflets responded
++
Only two leaflets responded
+
Only one leaflets responded
No response
+
When different levels of BA were used in combination with 2.26 pM
2,4-D a maximum of 70% of callus induction was observed in 11 pM
BA. The callus induced on this combination was friable and fast
growing (Fig.7b). The callus was maintained further in the same
medium (Table-7).
93
Fig.8: Callus induction from immature seeds
(a)
(b)
Callus (close-up) on MS medium supplemented with BA (II
pM) & 2,4-D (2.26 pM)
Further proliferation
Chapter-V
Table-7 : Effect of various concentrations of growth regulators
on subsequent sub culturing of the callus.
Degree of response
Plant growth
regulators (pM)
BA
2,4-D
11.1
0.00
0.00
2.26
11.1
2.26
PI
P2
+ +
+
+
++
4-
+++
+++
H"
"h
4" + 4" 4*
P3
Callus induction was studied in the immature seeds. The immature
pods were* dissected and the seeds were inoculated in MS medium
supplemented with various combinations of BA, NAA and 2, 4-D
(Table-8). The explants showed swelling and enlargement on the medium
supplemented with combinations of BA (11 pM) and 2,4-D (2.26 pM)
thin layer of callus at the edges of explants was observed (Fig.8).
Table-8: Effect of various growth regulators on the callus induction
on immature seed explants
Concentration
(pM)
BA
11.1
0.00
0.00
Degree of response
NAA
2.68
2,4-D
2.26
2,4-D
2.26
4.5
6.7
+
4* 4-
++++
+++
4*
+ + + +, + + +,++,+ indicates degree of response and - as no response
94
Chapter- V
The unorganised callus of Saponaria vaccaria Lin were revised
from seeds on MS medium supplemented with various concentrations of
IAA and 2, 4-D (Singhal et al5 1994). Callus induction was observed in
Aristolochia leaves when it was inoculated on the medium containing
combination of kinetin (0.45-23 pM) NAA (0.46-9.2 pM) and IAA (0.55.7 pM) (Remeshree et al, 1994).
Initiation of callus was observed in Calotropis gigantea Lin on
MS media supplemented with KN (0.5 mg/1) and 2, 4-D (1 mg/1). In
our studies simple MS medium supplemented with 11.1 pM of BA
and 2.26 pM of 2, 4-D gave fast growing and profuse callusing than
the medium supplemented with NAA. Even though the callus growth
was very fast, it failed to give regeneration of shoots and roots from
the callus.
In brief, the major problems of severe contamination and
browning was minimized by the pretreatment of explants with the
solution containing fungicide (bavistin, 1%; w/v) and antibiotic
(chloramphenicol, 0.5%; w/v). The effect of seasonal variations on
bud sprouting was studied and the records proved the maximum bud
sprout during March to June. The shoot initiation and multiplication
were studied with respect to various auxins and cytokinins. Maximum
95
Fig.9: Regenerated plants of Gymnema
(a)
(b)
In vitro grown plantlet transferred to sterile soil for hardening
In vitro grown plant showing good growth in the field
Fig 10: Micropropagated plants of Gymnema
(a)
(b)
(c)
In vitro grown plantlets showing uniform growth plantlets
Seed generated plants
The seed grown plantlets showing morphological differences
Chap ter-V
multiplication obtained in MS medium supplemented with 4.4 pM
BA.
Eventhough the percentage was very low, the root initiation
observed in half strength basal media. The callus initiation obtained in
MS medium containing BA (11.1 pM) and 2, 4-D (2.6 pM).
The in vitro raised shoots were removed from the medium
carefully.
Plantlets were transfer to the plastic pots (containing
vermiculate) which were covered with polythene bags and kept in
culture room for 2-3 weeks.
Such in vitro derived plantlets were
gradually exposed to the culture room conditions by making holes in
the polythene bags for acclimatization. After thorough hardening the
plantlets were transferred to eathern pots containing mixture of garden
soil and compost (1:1) transferred to the garden.
The plans were
subjected to mist irrigation with spraying bottles for 3-4 weeks (Fig.9).
The field establishment rate was very low. 60% of the plantlets could
survive in the field after hardening process. The tissue cultured
plantlets showed no morphological difference than the parent plant.
There was uniformity in growth pattern among the TC raised plants
than the seed grown plants (Fig. 10). However, micropropagation of
Gymnema syivestre could be established successfully in MS medium
(Fig 11). The callus harvested was studied for the Gymnemagenin
content and compared with the parent plant.
96
Chapter-V
Fig. 11 Flow chart for clonal multiplication of Gymnema sylvestre
97