in vitro morphogenic edited
TRANSCRIPT
-
8/8/2019 In Vitro Morphogenic Edited
1/31
IN VITRO MORPHOGENIC RESPONSE OF
DIFFERENT PARTS OF CARDIOSPERMUM
HALICACABUM LINN. (BALLOON VINE)
PLANT WITH TREMENDOUS
THERAPEUTIC POTENTIALS
BY
Dr. ICHHA PURAK & Dr. ANITA MEHTA
DEPARTMENT OF BOTANY, RANCHI WOMENS COLLEGE,RANCHI
Email : [email protected] , [email protected] &
-
8/8/2019 In Vitro Morphogenic Edited
2/31
Cardiospermum halicacabum Linn. belonging to family Sapindaceae is commonly knownas Balloon Vine due to inflated membranous balloon like tri-celled ridged fruits having
black seeds with prominent white heart shaped scar. It is recognized as a medicinal plant of
repute in Ayurvedic and Homoeopathic mode of treatment under different names such as
Jyotishmati, Kanphuti, Lataphatkari, Indravalli, Heart pea, Love- in-a -puff, Heart seed,
Winter cherry etc. The plant possesses active ingredients viz. alkaloids, flavonoids,
proanthocyanide, cyanolipids , glycosides, saponins, tri-terpenes, steroids ec . It is used tocure rheumatism, diarrhoea, chronic bronchitis, nervous diseases, stiffness of limbs, snake
bite, pulmonary troubles, itchy skin, problems in menstrual cycle, gastric ulcer, earache ,
eyesore and piles.
Standardization of protocol was undertaken for achieving callus mass from different parts
ofCardiospermum halicacabum. Different explants responded differently to chemicals andmedia they had been subjected.MS Medium supplemented with low concentration of 2,4 -
D had a tendency to regenerate into shoot and complete plantlet but high concentration
induced callus formation as well as proliferation . Green embryogenic callus from leaf
explants was observed in media containing 2,4-D. Callus tissue is good source of genetic
and karyotypic variability so variants can be regenerated from these genetically variable
cells. Callus culture is useful for obtaining commercially important secondary metabolities.Several biochemical assays can be performed from callus culture.
ABSTRACT
-
8/8/2019 In Vitro Morphogenic Edited
3/31
INTRODUCTION
Cardiospermum halicacabum Linn. belonging to family Sapindaceae is commonly
known as Balloon Vine due to inflated balloon like tri-celled ridged fruits having black
seeds with prominent white heart shaped scar. Cardiospermum is widely distributed in
tropical and sub-tropical Africa and Asia.
In India it is widely distributed in tropical and subtropical plains.The plant climbs with
tendrils and needs some form of support. Leaves are deeply cut trifoliate, up to 4
inches long, with highly lobed leaflets. petioles 3-4 cm; leaflets subsessile; blades thinly
papery, margin sparsely serrate, apex acuminate .
BRIEF MORPHOLOGICALDESCRIPTION
-
8/8/2019 In Vitro Morphogenic Edited
4/31
-
8/8/2019 In Vitro Morphogenic Edited
5/31
Flower stalk has a pair of axial spiral tendrils at the base . The
bisexual flowers are white in colour and possess 4 unequal sepals, 4
narrowly elliptical white petals ,8 stamens ( 4 larger and 4 shorter),
slightly longer than petals; single carpel with 3 lobed ovary and tri-fidstigma.
Each locule of ovary has one black seed with white heart shaped scar.
Capsules green in the beginning turns brown on ripening Disc of two
glands present below ovary. The fruit appears Balloon like due to
inflated membranous seed cover.
-
8/8/2019 In Vitro Morphogenic Edited
6/31
-
8/8/2019 In Vitro Morphogenic Edited
7/31
-
8/8/2019 In Vitro Morphogenic Edited
8/31
It is recognized as a medicinal plant of repute in Ayurvedic and Homoeopathic mode
of treatment under different names such as Jyotishmati, Kanphuti, Lataphatkari,
Indravalli, Heart pea, Love- in-a -puff, Heart seed, Winter cherry etc.
It is used to cure rheumatism, diarrhoea, chronic bronchitis, nervous diseases,
stiffness of limbs, snake bite, pulmonary troubles, itchy skin, problems in menstrual
cycle, gastric ulcer, earache , eyesore and piles etc. Datta et al (2010 ).
Indian system of medicine also recommends Cardiospermum halicacabum leaves for
rheumatism, stiffness of limbs,chronic bronchitis and snakebite ( Chopra,1980,1981 )Cardiospermum is an active ingredient in creams and lotions for dermatitis, eczema,
and psoriasis.
MEDICINAL IMPORTANCE
OF CARDIOSPERMUM
-
8/8/2019 In Vitro Morphogenic Edited
9/31
The plant possesses active ingredients viz. alkaloids, proanthocyanide,
cyanolipids , glycosides, saponins, steroids etc . Leaves contain b-
sitosterol, quebrachitol, saponin, oxalic acid and Apigenjn,
proanthocyanidin,stigmatosterol Dass(1966), Satyavati (1955). Rao et al
2006 mentioned antidiarrhoeal activity of the extracts ofCardiospermum
halicacabum due to presence of phytochemical constituents such as
sterols, tannins, flavonoids and triterpenes.
ACTIVE INGREDIENTS
-
8/8/2019 In Vitro Morphogenic Edited
10/31
Leaves are crushed and made into a tea, which cures itchy skin. Salted leaves are used as
a poultice on swellings.
Young leaves can be cooked as vegetables.
The leaf juice has been used as a treatment for earache . Nadkarni (1976),Asha et al
(1999 ),Gopalkrishnan et al (1976),Sadiqwe et al (1987),Joshi et al (1981).
It is used in nervous diseases and in dropsy.
The fresh decoction of leaves is given to asthmatic patients while its powdered form isused to heal wounds.
Varier (1993) reported the sedative actions of plant on central nervous system probably
due to presence of hydrocynic acid.
THERAPEUTIC APPLICATIONS
-
8/8/2019 In Vitro Morphogenic Edited
11/31
It is used for medicinal purposes as tea,Decoction,Juice, paste,
Poultice,oil, chutney, tooth brush etc.
As Decoction of leaves/root, a teaspoon at a time should be taken
twice a day
Leaves and young shoots are edible
MODE OF APPLICATION AND
DOSE
-
8/8/2019 In Vitro Morphogenic Edited
12/31
Pharmaceutical companies depend largely upon materials procured from
naturally occurring stands that are being rapidly depleted. Plant tissue culture is
an alternative method of propagation based on totipotent nature of cells. It plays
a key role in large scale multiplication of medicinally important plant species
(Pattnaik and Chand 1996, Rout er al., 1999, 2000,Ahmad and Anis,2007 ) For
enhancement of secondary metabolite content through genetic transformation or
by manipulating the constituents of growth media ,an efficient in-vitro
regeneration protocol is necessary. In-vitro mass propagation obviousky has
great potential to fulfill the immediate requirement of plant based
pharmaceutical industries.
CONSERVATION OF IMPORTANT
MEDICINAL PLANT
-
8/8/2019 In Vitro Morphogenic Edited
13/31
.The regeneration of plants from callus culture has proved very useful
commercially. Keeping these things in mind the present investigation was
undertaken. Micropropagation plays a distinct role in conservation of species,
especially those having pharmacological value. Most of the pharmaceutical
plants are collected from the wild and very few from the cultivated stands. In
many cases, the entire plant is severed during harvesting. To overpass this
insufficiency and for safeguarding the species from extinction, it is urgent to
develop protocols which are cost effective and be utilized for scaling-up the
same (Bajaj et al. 1988)
-
8/8/2019 In Vitro Morphogenic Edited
14/31
Conventionally the plant is propagated through seeds but is not feasible due to low
germination rate ( 35-40 % ) , low viability and delayed rooting by seedlings Kirtikar
and Basu (1935 ) There are few earlier reports on regeneration of Cardiospermum
halicacabum Babber et al (2001),Thomas and Maseena (2006), Girish et al,( 2008 )
and Jahan and Anis (2009 ) In the present investigation an attempt has been made to
study micropropagtion of this species. The aim of this Study was to initiate and
maintain the calli produced from tissue of various plant parts ( leaf,stem
internode,node,flower,seed ).
-
8/8/2019 In Vitro Morphogenic Edited
15/31
Culture mediumMS ( Murashig and Skoog,1962 ) basal medium was used in the present investigation .
Concentrated stock solution of major and minor salts, iron source, vitamins and amino acids
were prepared separately and stored under refrigeration. Required volume of stock solution
was pipetted out during media preparation and 0.3% sucrose was added. The chemicals
used for preparing media were of analytical grade from Loba, Merck and Sigma. Medium
was homogenized by boiling and continuous stirring with pH of medium adjusted upto 5.8
before adding 0.8% agar by using 0.1 N NaOH or 0.1N HCL.Desired concentration of
growth regulators was added and mixed properly. 15-20 ml medium was poured into
culture tubes ( 15x2.5cm ) which were washed thoroughly rinsed in distilled water and
oven dried.Sterilizarion of medium was done for 15- 20 minutes at pressure 1.1 kg/cm2 at
temperature 121C .Tubes containing sterilized medium were left in tilted position to
prepare slants in air conditioned room.
MATERIAL AND METHODS
-
8/8/2019 In Vitro Morphogenic Edited
16/31
Preparation of Explant(Culture material )
Some seeds ofCardiospermum halicacabum were soaked in water after removing the black
covering by scraping to facilitate in-vitro germination.Shoots bearing leaves were collected
from plants growing in garden ofP P Compound area of Ranchi. Different vegetative parts
such as stem node,internode,leaf, flower etc which were taken as expant after thoroughly
washing with tap water and were treated with Cetavelon (1:100) solution for about 5
minutes and again washed with running tap water.The explants were then surface sterilized
in 0.2% of HgCl2by immersing for 2-3 minutes and then were rinsed in double distilled
water. These sterilized stem and leaves were then cut into pieces of about 1.0-1.5cm and
made ready for inoculation.
-
8/8/2019 In Vitro Morphogenic Edited
17/31
The explants were in-oculated into test tubes containing sterilized media under Laminar
AirFlow cabinet.Transfer area was sterilized with UV light and by swabbing the foor
surface with 95% ethyl alcohol.Inoculation tools were flamed before transferring the
explants. Each experiment with 10 culture tubes was repeated 4 times and were
observed every day for their morphological response.
INOCULATION
-
8/8/2019 In Vitro Morphogenic Edited
18/31
In the present investigation different explants responded differently to chemicals and
media they had been subjected. All types of explants showed initial hypertrophy. Low
concentration of 2,4-D ( 0.05 mg /l to 0.5 mg/l ) had a tendency to regenerate into
shoot or complete plantlet.But high concentrations of 2,4-D (2.5-5.0mg/l) induced
callus formation as well as its proliferation.
RESULT AND DISCUSSION
-
8/8/2019 In Vitro Morphogenic Edited
19/31
Green embryogenic callus was also observed from leaf explants in medium
containing 2.5mg/l 2,4-D. (Fig . 1 ) after 4 weeks of inoculation. White fragile
callus was observed in 1.5 months old culture obtained from leaf explants.( Fig. 2 )
in medium supplemented with 2.5mg/l 2,4-D.These calli were about 1.5 cm in
diameter. Nodulated and compact calli were induced in 1.5 months old stem
culture in medium containing 2.0-2.5 mg/l 2,4-D.(F
ig 3 & 4 )
-
8/8/2019 In Vitro Morphogenic Edited
20/31
1 2
3
4
-
8/8/2019 In Vitro Morphogenic Edited
21/31
Fig. 1 1.5 months old leaf culture on MS Medium
containing 2,4-D ( 2.5 mg/l ) showing green
embryogenic callus.
Fig. 2 1.5 months old leaf culture on MS Medium
containing 2,4-D ( 2.5 mg/l ) showing white
Fragile callus.
Fig.3 45 days old stem culture on MS medium containing
2,4-D ( 2.0 mg/l ) showing nodulated and compact
callus
Fig. 4 1.5 months old stem culture on MS Medium
supplemented with 2.5 mg/l 2,4-D
showing compact and nodulated callus
-
8/8/2019 In Vitro Morphogenic Edited
22/31
Callus tissue is good source of genetic and karyotypic variability so variants can be
regenerated from these genetically variable cells. Callus culture is useful for obtaining
commercially important secondary metabolities. Several biochemical assays can be
performed from callus culture.
Such callus formation had been reported previously by Girish et al (2008) who
employed MS medium supplemented with auxins viz. 2,4-D,NAA IAA and cytokinins
viz. BAP and Kn alone in different concentrations..
-
8/8/2019 In Vitro Morphogenic Edited
23/31
A protocol for rapid micropropagation of C. halicacabum through plant
regeneration from leaf and nodal explant derived calli has been developed by
Thomas and Maseena (2006). Nodal and leaf explants were cultured on MS
medium supplemented with 0.5-9 M 2,4-D. Highesr frequency of callus formation
was recorded with 5 M 2,4-D.These calli on subculturing with kinetin and IAA
resulted shoot regeneration.Rooting of shoots was recorded when subjected to MS
medium supplemeted with 2.5 M IBA
-
8/8/2019 In Vitro Morphogenic Edited
24/31
Babber et al (2001) were able to produce calli from different explants of
Cardiospermum halicacabum as cotyledon,hypocotyls,leaf,internode and node in MS
medium supplemented with BAP and NAA.Maximum number of shoots were produced
on subculturing calli on MS and BAP ( 17.8 M ).
Recently Jahan and Anis (2009) developed a simple,rapid and efficient protocol for
microprogation of this plant via axillary bud multiplication by using nodal segments and
employing BA(Benzyladenine ), Kn (Kinetin), TDZ(Thidiazuron ) and 2-iP ( 2
isopentenyladenine. Multiple shoots differentiated directly without callus mediation
within 4 weeks.
-
8/8/2019 In Vitro Morphogenic Edited
25/31
As Cardiospermum halicacabum, a noxious weed owes tremendous therapeutic
potentials so is being exploited very frequently from its wild and native habitats. As it is
not a cultivated plant may face danger of extinction. Keeping this scenario in view it is
advisable to develop protocols for rapid in- vitro regeneration of this species through
callus as well as multiple shoot formation.
-
8/8/2019 In Vitro Morphogenic Edited
26/31
Ahmed I,Ahmed M and Ahmed A (1993) Chemical investigation of the genus
Cardiospermum of family Sapindaceae.Lahore: Science International
Ahmad N and Anis M (2007) Rapid clonal propagation of a woody tree,Vitex negundo L.
through axillary shoots proliferation. Agrofor Syst 71:195200.
Asha, V.V. and P. Pushpangadan,(1999). Antipyretic activity ofCardiospermum halicacabum.
Indian J. Exp. Biol., 37: 411-414.
Babber S, Mittal K, Ahlawat R, and Varghese TM (2001) Micropropagation of
Cardiospermum halicacabum. Biol Plant 44:603606.
Bajaj YPS, Furmanowa M and Olszowska O (1988) Biotechnology of micropropagation of
medicinal and aromatic plants. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry
medicinal plants. I, vol 4. Springer, Berlin, pp 60103
Chopra R N (1980) Glossary of Indian Medicinal Plants. New Delhi: Council for Scientific
and Industrial Research,51-55.
Chopra. R. N., Nayar. S. L. and Chopra. I. C. (1986 ) Glossary of Indian Medicinal Plants
(Including the Supplement). Council of Scientific and Industrial Research, New Delhi.
REFERENCES
-
8/8/2019 In Vitro Morphogenic Edited
27/31
Datta S., Ghosh A,Pal P ,Das M and KarP K( 2010) Pharmacognostical,
Phytochemical and Biological evaluation ofCardiospermum halicacabum Int J Pharm
Sci Bio 1(1):37-42.
Dass AK. (1966 ) Chemical examination ofCardiospermum hali-cacabum Linn. Bull
Bot Sur India;8:357-358
Gopalakrishnan, C., Dhananjayan R. and Kameswaran L. (1976) . Studies on the
pharmacological actions ofCardiospermum halicacabum. Indian J Physiol. Pharmacol.,
20: 203-206.
Joshi, S.K., B.D. Sharma, C.R. Bhatia, R.V. Singh and R.S. Thakur ( 1992). The Wealth
of India Raw Materials,Vol. III. Council of Scientific and Industrial Research
publication, New Delhi, pp: 270-271.
Kirtikar KR, and Basu BD (1935) Indian medicinal plants. M/s Bishen SinghMahendrapal, New Delhi, pp 267268
-
8/8/2019 In Vitro Morphogenic Edited
28/31
Murashige T and Skoog F (1962) A revised medium for rapid growth and
bioassays for tobacco tissue culture. Physiol Plant 15:473497.
Nadkarni, K.M. ( 1976). Indian Materia Medica Book Depot, Bombay, pp: 271.
Pattnaik SK and Chand PK (1996) In vitro propagation of medicinal herbs Ocimum
americanum L. syn. Ocimum canum Sims (hoary basil) and Ocimum sanctum (holy
basil). Plant Cell Rep 15:846850.
-
8/8/2019 In Vitro Morphogenic Edited
29/31
Rao,N V.,Prakash,K C and Shanta Kumar SM (2006). Pharmacological investigation of
Cardiospermum halicacabum ( Linn ) in different animal models of diarrhea.Ind. J.of
Pharmacology38( 5 ) : 346-349
Rout GR, Saxena C, Samantray S and Das P ( 1999 ) Rapid clonal propagation ofPlumbago zeylanica L. Plant Growth Regul 2:14.
Rout,GR ,Samantry,S and Das,P (2000.) In vitro manipulation and propagation of
medicinal plants. Biotechnol. Advances 18:91-120.
-
8/8/2019 In Vitro Morphogenic Edited
30/31
Sadique, J., Chandra,T. Thenmozhi V. and Elango V. ( 1987).
Biochemical modes of action ofCassia occidentalis and
Cardiospermum halicacabum in the inflammation. J. Ethnopharmacol.,
19: 201-205
Satyavathi VV. (1995) Medicinal Plants of India. New Delhi: Indian
Council of Medical Research, 183.
Thomas TD and Maseena EA (2006) Callus induction and plant
regeneration in Cardiospermum halicacabum L.a medicinally
important plant. Scientia Horticulture 108:332336
VarierPS (1993) Indian medicinal plants, vol I. Orient
Longman,Madras
-
8/8/2019 In Vitro Morphogenic Edited
31/31