Micropropagation of Caralluma sarkariae Lavranos _ Frandsen An

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					Micropropagation of Caralluma sarkariae Lavranos & Frandsen : An
Important Medicinal Plant

V Raja Sreelatha, S Karuppusamy and T Pullaiah

Department of Botany, Sri Krishnadevaraya University, Anantapur 515 003, AP, India

Key words: Caralluma sarkariae, micropropagation, nodal explants

A protocol for micropropagation of an endemic medicinal plant Caralluma sarkariae
(Asclepiadaceae) from nodal explants is described. The highest shoot multiplication rate
of 13 shoots/explant was achieved in 90 % of 25-day-old culture on Murashige and
Skoog medium supplemented with 2.0 mg/l BAP+ 1.0 mg/l 2-iP + 1 mg/l NAA.
Excised shoots rooted on half strength MS medium with 0.1 mg/l NAA. Some 75% of the
rooted shoots survived in the field.

Caralluma sarkariae is a wild, succulent, perennial endemic medicinal herb distributed in
dry parts of Nagamalai hills, Madurai district, Tamil Nadu in India. Plants belonging to
this genus are rich in esterified polyhydroxy pregnane glycosides, some of which showed
anti-tumour activity and others were postulated as precursors of cardenolides (Deepak et
al. 1989 a,b). The genus is also characterized by the presence of flavone glycosides
(Ramesh et al. 1999; Rizwani et al. 1990). The hypoglycemic effect of aqueous and
alcoholic extracts of the whole plant of Caralluma species was investigated by Jayakar et
al. (2004) in both normal and alloxan induced diabetic rats.
       The species of Caralluma found in India are edible and its medicinal properties
include anti-inflammatory, anti-nociceptive, anti-ulcer, anti-diabetic, carminative,
febrifugal, antipyretic and antioxidant effects. Caralluma extracts have also been found
to be appetite suppressant, a property which is well known to Indian tribals and hunters.
Indian folklore records its use as a potent appetite suppressant and weight loss promoter.

Some Caralluma species are used in the treatment of obesity. The extract of Caralluma
sp. in the form of capsules has been released under the trade name GENASLIM for the
control of body weight (Lawrence and Choudary 2004). Conventional propagation
through seeds is too slow to provide the answer to meet an ever increasing demand for
this plant species. Seed-derived progenies of C. sarkariae are not true-to-type due to
cross pollination. Propagation through seeds is an inadequate solution due to low
viability, poor seed germination rate, scanty and delayed rooting of the seedlings.
Vegetative propagation by stem is a very tediouds procedure. Until now, no in vitro
studies have been reported on C. sarkariae. The aim of the present investigation was to
develop a system for in vitro propagation and to conserve the wild, endemic this species.

Material and Methods
       C. sarkariae was collected from Nagamalai hills of Madurai district, Tamil Nadu.
The stems were maintained in pots at the Botanical Garden, Botany Department, Sri
Krishnadevaraya University, Anantapur. Actively growing shoots were collected from
the garden to excise the explants. The shoots were defoliated and cut into small pieces of
about 5-8 cm length. The nodal segments were initially disinfected by rinsing in 1% (v/v)
Tween 20 (Merck) for 5 min with constant agitation, rinsed with distilled water thrice and
finally rinsed with sterile double distilled water under laminar air-flow chamber. The
explant material was immersed in 70% ethanol for 1 min followed by surface sterilization
in aqueous solution of 0.1% (w/v) HgCl2 for 5 min and rinsed 3 or 4 times in sterilized
double-distilled water. Stem segments (0.5-1.0 cm) containing the nodal region with
axillary buds were aseptically cut from the sterilized shoot pieces and inoculated on to the
nutrient medium. MS medium was used as the basal medium and was supplemented with
3% (w/v) sucrose and gelled with 0.8% (w/v) agar. Depending on the experiments, the
basal medium was supplemented with cytokinins (BAP, 2-iP), gibberellin (GA3) and
auxins (NAA, IAA, IBA) at different concentrations alone or in combinations as shown
in Tables 1, 2 and 3. The pH of the medium was adjusted to 5.8 before gelling with
bacteriological agar and autoclaved at 1.06 kg cm-2 pressure and 121ºC for 15 min. The
cultures were incubated in a growth room at 25 ± 2°C with 16/8h (light/dark) photoperiod
under fluorescent tubes (25 μmol m-2 s-1). During hardening, the regenerated plantlets

with well developed shoots and roots were transferred to pots. The potted plantlets were
covered with polythene bags to maintain high relative humidity around the plantlets.
       The plantlets were watered every two days with half strength MS salt solution.
After 25 days, hardened plants were transferred to a greenhouse. Rooted shoots were
transferred to small pots containing soil, sand and manure (1:1:1) mixture and
subsequently to the field. For each experiment 15 replicate cultures were raised and all
experiments were repeated thrice. The data were statistically analyzed using one way
analysis of variance and means were compared using the Tukey Test at 0.05% level of
significance, and presented as mean + standard error (SE).

Results and Discussion
       Shoot organogenesis was exhibited best by terminal nodes. More multiple shoots
developed from terminal nodes than basal ones.
       Out of the various concentrations of BAP and 2-iP tested for shoot proliferation
from mature nodal explants, 2.0 mg/l BAP produced 85% shoot sprouting frequency with
2.09 shoots/explant and 2.0 cm of shoot length (Table 1), whereas in all the
concentrations of 2-iP tested, 1.0 mg/l 2-iP was found to be effective by inducing bud
break in 75% of the explants with a maximum of 2.18 cm shoot length (Table 1).
Application of exogenous hormones, was found necessary for bud breaking. MS medium
containing BAP (2.0 mg/l) was more effective than 2-iP for inducing proliferation of
axillary buds, but shoots were longer on the medium supplemented with 2-iP than BAP.
According to Ripley and Preece (1986), among cytokinins, best shoot induction was
obtained when BAP rather than 2-iP was used as a supplement. This finding is in
agreement with an earlier report that BAP is needed for micropropagation of latex
producing plants.
       Assuming that 2-iP was important in promoting shoot elongation it was combined
with BAP (2.0 mg/l ) at different concentrations. Depending on the concentrations of 2-iP
the explants showed 50-68% bud break in 8-15 days. The best response was obtained in
the medium containing 1.0 mg/l 2-iP and 2.0 mg/l BAP. The number of shoots/explant
was 2.14 shoot lengths on the average were 5.0 cm. Higher concentrations of cytokinins
in the medium decreased the per cent of bud break and mean shoot lengths.

       Out of various concentrations of GA3 used in combinations with BAP and 2-iP, a
combination of 2.0 mg/l BAP + 0.5 mg/l GA3 produced maximum shoot lengths of 6.32
cm with 1.83 shoots/explant (Fig. 1A) (Table 2), whereas GA3 combination with 1.0 mg/l
2-iP resulted in short shoot length compared to 2.0 mg/l BAP + 0.5 mg/l GA3
combination (Data not shown). Further increase in the concentration of GA3 reduced the
length and number of shoots (Table 2). The combination of GA3 and cytokinin is less
effective for the induction of shoots than that of auxin and cytokinin combinations.
However, GA3 in combination with BAP gave maximum shoot lengths in C. sarkariae
(Fig. 1A).
       The shoot number increased when auxins NAA, IAA and IBA combined with
optimized BAP and 2-iP (Table 2), whereas auxins (NAA, IAA and IBA), combined with
either 2.0 mg/l BAP or 1.0 mg/l 2-iP resulted in a low propagation rate (Data not shown).
       Of different concentrations of auxins (NAA, IAA and IBA) tested with
combination of 2.0 mg/l BAP + 1.0 mg/l 2-iP, the best result was observed in the medium
supplemented with NAA 1.0 mg/l with 90% of response after 25 days of culture. In
above medium explants produced the maximum of 13.16 shoots/explant with an average
shoot length of 2.52 cm compared to other concentrations of NAA (Table 2; Fig. 1B).
Similar results were observed on Holostemma annulare (Sudha et al. 1998). The
experiments conducted to improve the regeneration performance of terminal nodes with
cytokinins (BAP and 2-iP) and other auxins (IAA and IBA) did not yield a favorable
response (Table 2). Addition of GA3, to the above combination produced a greater
number of shoots among different concentrations. These shoots formed in the medium
with 2.0 mg/l BAP + 1.0 mg/l 2-iP + IAA and IBA and combination with GA3 at
different concentrations were subcultured on to the medium supplemented with 2.0 mg/l
BAP + 1.0 mg/l 2-iP + 1.0 mg/l NAA combinations. But these concentrations had no
significant effect on length (Table 2). Further subculture of these shoots on shoot
elongation medium containing 2.0 mg/l BAP and 0.5 mg/ l GA3 gave an optimal response
in a period of 6 weeks (Fig. 1C).
       The present study exemplifies the improvement in shoot induction efficiency by
low concentrations of an auxin in combination with cytokinins. Excision of the in vitro
derived shoots and sub-culturing them on the same medium facilitated the development

of 25 shoots. All cultures formed some callus at the base from which new shoots
emerged. During sub-cultures basal axillary buds also gave shoot initiation. The shoot
multiplication at an enhanced pace in subsequent cultures is in agreement with reports on
other Asclepiadacean medicinal plants (Sudha et al. 1998).
       Axillary buds derived in vitro shoots (5-6 cm long) were rooted on ½ MS medium
supplemented with IAA, IBA and NAA. A low frequency of rooting was noticed in
hormone-free half MS medium. Half MS medium supplemented with NAA (0.1 mg/l)
was found to be most effective for root induction followed by IAA and IBA. At 30 days
the best response was observed on NAA 0.1 mg/l with 10 roots/shoot (Fig. 1D), whereas
in 0.5 mg/l IBA the maximum root length was observed.
       Reduction of MS salts to ½ enhances root formation in shootlets. Rooting of the
shoot cuttings occurred easily with traces or no callusing at the cut ends when the ½
strength MS medium was used. The use of full strength MS medium induced pronounced
callusing. The favorable effects of low concentrations of macro- and micro-nutrients on
rooting are probably due to smaller requirements of nitrogen for rhizogenesis. Rooting
was observed in different auxins media and rooting was more effective when isolated
shoots were maintained on medium containing NAA (0.1 mg/l). Higher concentrations of
auxin, lower the rooting percentage as well as root number. In general, root formation
was much better with one auxin than in combinations.
       Rooted shoots were transferred directly to small pots filled with sterile sand, soil
and manure (1:1:1); growth resumed after 20 days after transplantation. Of the 200
plantlets transferred to soil with in a period of 150 days, 75% of the plantlets survived
and exhibited uniform morphological characters similar to those of the source plant
material (Fig. 1E).
       The outline procedure for in vitro propagation of C. sarkariae offers a potential
regeneration system for improvement and conservation of this important endemic
medicinal plant from mature nodal explants. Using this method it is possible to produce
30-40 plants from a single explant within 50 days.


Deepak D, Khare A and Khare M P (1989) Plant Pregnanes. Phytochemistry 28: 3255-

Deepak D, Srivastav S and Khare A (1989) Pregnane glycosides. Progr. Chem. Org.
      Natural Prod. 71: 169-325

Jayakar B, Rajkapoor B and Suresh B (2004) Effect of Caralluma attenuata in normal
      and alloxan induced diabetic rats. J. Herb. Pharmacother. 4: 35-40.

Lawrence R M and Choudary S (2004) Caralluma fimbriata in the treatment of obesity.
      12th Annual World Congress on Antiaging Medicine, December 2-5, USA.

Ramesh M, Rama Kumar M, Krishna Mohan G, Ravi kumar B, Appa Rao AVN, Radha
      Kishan M and Madhava Reddy B (1999) Flavone glycosides from three
      Caralluma species. Biochem Syst & Eco, 27: 85-86.

Ripley K and Preece J E (1986) Micropropagation of Euphorbia lathyris. Plant Cell
      Tiss. Org. Cult. 5: 213-218.

Rizwani GH, Usmanghani K, Ahmed M and Ahmad V U (1990) Flavone glycosides of
      Caralluma tuberculata. J Pharm Sci. 3: 27-32.

Sudha CG, Krishnan, PN and Pushpangadan P (1998) In vitro propagation of
      Holostemma annulare (Roxb.) K.Schum., a rare medicinal plant. In vitro Cell Dev
      Biol Plant 33: 57-63.

Table 1. Effect of various concentrations of BAP and 2-iP on multiple shoot induction
from mature nodal explant of C. sarkariae cultured on MS medium

 Plant growth    Shoot regenerating                                    Shoot length (cm) per
                                         No. of shoots/ explant
   regulator          cultures                                                explant
                                             (Mean  SE)
    (mg/l)              (%)                                               (Mean  SE)

    0.1                   0                        NR                           NR
    1.0                  70                   1.61  0.04d                 1.10  0.02f
    2.0                  85                   2.09  0.02b                 2.00  0.03b
    3.0                  60                   1.84  0.01cd                1.48  0.02c
    5.0                  55                   1.56  0.02de                1.28  0.02de
    8.0                  40                   1.32  0.03f                 1.21  0.05 a
    0.1                   0                       NR                            NR
    1.0                  75                   1.92  0.02c                  2.18  0.04b
    2.0                  68
                                              1.60  0.01d                  1.46  0.02c
    3.0                  60
                                              1.43  0.01e                  1.30  0.03d
    5.0                  45
    8.0                  40                   1.22  0.02g                  1.20  0.02e
 BAP + 2-iP                                   1.14  0.01h                  1.18  0.03e

   2.0 + 0.1            65                    1.49 ±0.02e                  1.25 ± 0.02de
   2.0 + 0.5            70                    2.14 ± 0.01b                 1.45 ± 0.01c
   2.0 + 1.0            85                    2.91 ± 0.01a                 5.13 ± 0.15a
   2.0 + 2.0            72                    1.50 ± 0.01e                 1.33 ± 0.02d

Note: Means value with the same letter not significantly different as determined by the
Tukey Test at 0.05%.

NR – No Response

 Table 2. Effect of different combinations of growth regulators in shoot regeneration in mature nodal explants of
                                                  C. sarkariae

           Plant growth regulator (mg/l                                   No. of shoots/
                                                        sprouting                              Shoot length (cm)
                                                        frequency                                (Mean + SE)
                                                                          (Mean + SE)
    BAP          2-iP   NAA     IAA     IBA     GA3        (%)
     2.0          -       -       -        -    0.5         78         1.83 + 0.01f           6.32 + 0.04a

     2.0          -       -       -        -    1.0         65         1.54 + 0.02g           4.18 + 0.02b

     2.0          -       -       -        -    2.0         60         1.34 + 0.03h           3.32 + 0.04bc

     2.0         1.0     0.1      -        -     -          65         7.35 + 0.07bc          1.80 + 0.02d
     2.0         1.0     0.5      -        -     -          78         9.26 + 0.05ab          2.16 + 0.02cd

     2.0         1.0     1.0      -        -     -          90        13.16 + 0.04a           2.52 + 0.01c

     2.0         1.0     1.0      -        -    0.5         70         3.32 + 0.05d           3.25 + 0.04bc

     2.0         1.0      -      0.1       -     -          75         8.34 + 0.05b           2.04 + 0.01cd

     2.0         1.0      -      0.5       -     -          63         4.24 + 0.05cd          1.81 + 0.01d

     2.0         1.0      -       -       0.1    -          60         3.51 + 0.02d           1.70 + 0.02d

     2.0         1.0      -       -       0.5    -          74         5.26 + 0.04c           2.14 + 0.30cd

     2.0         1.0      -      0.1       -    0.5         65         2.63 + 0.04e           2.82 + 0.01c

     2.0         1.0      -       -       0.5   0.5         60         2.28 + 0.04e           2.46 + 0.03c

Note: Mean value with the same letter are not significantly different by the Tukey Test at 0.05%.

Table 3. Effect of various auxins on rooting of in vitro regenerated C. sarkariae shoots
cultured on half strength MS medium (culture age 30 days)

   Concentration of
    auxin (mg/l)        Response       Number of roots/shoot       Root length (cm)
                         (%)              (Mean ± SE)               (Mean ± SE)
 NAA     IAA     IBA

 0.10      -       -         75         10.37 ±       0.05a       3.61 ±     0.02ab

 0.50      -       -         65          6.24 ±       0.02bc      2.12 ±     0.02b

 1.00      -       -         50          4.19 ±       0.03cd      1.42 ±     0.02cd

 2.00      -       -          -                   C                      C

 3.00      -       -          -                   C                      C

   -     0.10      -         60          5.25 ±       0.04c       1.50 ±     0.02c

   -     0.50      -         70          7.12 ±       0.02b       2.48 ±     0.02b

   -     1.00      -         55          3.25 ±       0.03d       1.40 ±     0.02cd

   -     2.00      -         50          2.16 ±       0.02e       1.22 ±     0.02d

   -     3.00      -          -                   C                          C

   -       -     0.10        45          3.19 ±       0.03d       2.24 ±     0.03b

   -       -     0.50        68          5.28 ±       0.03c       4.09 ±     0.02a

   -       -     1.00        60          2.19 ±       0.03e       1.24 ±     0.02d

   -       -     2.00         -                   C                          C

   -       -     3.00         -                   C                          C
Note: Means by the same letter not significantly different by the Tukey Test at 0.05%.
C – Callus

Fig. 1 A-E: Micropropagation of C. sarkariae. (A) Maximum shoot elongation
observed on MS+ 2.0 mg/l BAP + 0.5 mg/l GA3 (after 25 days of culture), (B) Multiple
shoots formed on MS+ 2.0 mg/l BAP + 1.0 mg/l 2-iP + 1.0 mg/l NAA (after 15 days of
culture), (C) Enhanced shoot lengths during sub culture on the above medium (after 25
days of culture), (D) Rooting of regenerated shoots on ½ strength MS medium containing
0.1 mg/l NAA (after 30 days) (bar in A,B,C,D 1 cm) and (E) An acclimated plant in a
pot (After 15 days).


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