in vitro morphogenic edited

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] &

[email protected]


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


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


4/31


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.


6/31


7/31


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


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


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


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


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


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)


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 ).


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


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.


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


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


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 )


20/31

1 2

3

4


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


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..


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


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.


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.


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


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


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.


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.


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


31/31

in vitro morphogenic edited

Documents