Author: Patil, D A
Date published: January 1, 2012
Cardiospermum halicacabum L., commonly known as Balloon vine, is an important medicinal herb belonging to family Sapindaceae (Fig. 1). The roots of the plant are considered as diaphoretic, diuretic and aperient. Roots are also administered in fever. The whole plant is applied to reduce swellings and hardened tumors . There is a claim that roots are used by some local tribes to treat rheumatoid arthritis inAsian and African communities [2,3].
Scientific studies have provided an explanation for some of the medicinal properties of balloon vine. In vitro antifilarial activity of extracts of C. halicacabum L. against Brugia pahangi has mild but definite direct macrofilaricidal action . C. halicacabum L. also showed antipyretic activity against yeast-induced pyrexia in rats  and cure for gastric ulcers in rats . The whole plant has been used as anti-inflammatory [7,8], antipyretic [7,9], analgesic  antiparasitic , as well as an effective non toxic antifertility herb .
Various forms of products like gel, cream, shampoo, spray etc. of C. halicacabum L. are available in the market. These products are useful for dry itchy skin and scalp. In the globalmarket, balloon vine has been utilized in several products, 'Love in a Puff ', 'Balloon Vine' and 'Heartseed'. It is also one of the ingredients in "Allergy Relief Liquid TM" and "Bioforce Pollinosan® Tabs" marketed by Bioforce USA as a natural relief for hay fever, allergies, sneezing, watery eyes, and allergic reactions. Another US based company, Boericke and Tafel produces "Florasone Cardiospermum Cream" for skin ailments such as swelling, scaling, blisters/vesicles, burning and pain. These products are supported by the various claims concerning with medicinal properties of balloon vine .
Herbal drugs used for treating various diseases represent a substantial proportion of global drug market. Most of the drugs required for pharmaceutical industries are collected from wild resources. Natural reserves of C. halicacabum L. plant are declining due to overexploitation by local people and tribal who harvest the plants for medicinal uses. Therefore, it is a prerequisite to develop a rapid method for large scale propagation of C. halicacabum L. to meet the pharmaceutical industrial demand, consistency in quality and also for its sustainable conservation.
Biotechnology is one of the important tool to select, multiply and conserve the important genotypes of medicinal plants . The whole plant can be regenerated in large number from callus tissue through manipulation of the nutrient and hormonal constituents in the culturemedium. This phenomenon is known as plant regeneration or organogenesis or morphogenesis . There are many reports on the regeneration of various medicinal plants via callus culture and also organogenesis is one of the efficient methods for mass multiplication of medicinal plants [15-19].
Therefore, the appearance of calli is an important aspect in research on callogenic cultures and the associated morphogenic response. Thus callogenic induction can be one of the best alternatives for cloning andmass producing valuable genotypes. The objective of this work was to morphologically characterise the type of calli and histologically analyse the calli produced from in vitro grown seedlings of C. halicacabum L.
MATERIALS AND METHODS
Seeds (Fig. 2) of Cardiospermum halicacabum L. were separated fromthe mature fruits and treated with 1% bavistin with few drops of Tween 20 for 1 hour and then thoroughly washed under running tap water for 30 minutes to remove all traces of detergent. These seeds were sterilized with 0.1% mercuric chloride (HgCl2) for 5 minutes and rinsed thoroughly with autoclaved water. Seeds were then treated with 70% alcohol for 1-2 minutes and finally rinsed thrice with autoclaved water. The seeds were then inoculated on basal Murashige and Skoog  and Gamborg's  media. Different parts of the in vitro grown seedlings (Fig. 3) were used as source of explants viz. leaves, cotyledon, hypocotyl, epicotyl and radicle. The explants were inoculated on MS (Murashige and Skoog) and B5 media supplemented with 2, 4-Dichlorophenoxy acetic acid (2,4-D) and 6-Benzyl amine purine (BAP).
The calli were evaluated qualitatively in terms of colour (cream, brown, greenish white or green) and consistency (friable or compact). For histological analysis thin sections were taken, stained with safranin and observed microscopically.
Callus culture: Various explants inoculated on MS medium fortified with 2, 4-D (0.5- 2.5 mg/l) showed different coloured callus. Leaf explants showed cream coloured compact callus, cotyledon showed brown coloured compact callus in less amount, hypocotyl and epicotyl illustrated moderate to good amount of pale yellow coloured compact callus (Fig. 4) while radicle revealed pale yellow coloured friable callus in good to less amount (Table 1).
Leaf and epicotyl showed less to moderate amount of cream coloured compact and friable calli respectively when inoculated on B5 medium supplemented with 2, 4-D (0.5- 2.5 mg/l), whereas cotyledon and hypcotyl demonstrated less amount of callus. Radicle inoculated onB5mediumfortifiedwith 2, 4- D (0.5- 2.5 mg/l) showed good to less amount of brown friable callus (Table 2, Fig. 5).
Leaf, hypocotyl and epicotyl inoculated on MS medium fortified with BAP (0.5- 2.5 mg/l) showed green coloured compact callus in less amount whereas cotyledon showed green friable callus (Fig. 7). However, brown coloured friable callus was observed from radicle when inoculated on MS medium fortified with BAP (Table 3).
Leaf inoculated on B5 medium fortified with BAP (0.5- 2.5 mg/l) showed direct rhizogenesis (Fig. 8), whereas cotyledon showed green coloured friable callus in less amount. Hypocotyl inoculated on B5 medium fortified with BAP (0.5- 2.0 mg/l) showed green coloured compact callus in moderate to good amount, whereas greenishwhite coloured callus was obtained on B5 medium supplemented with 2.0 and 2.5 mg/l BAP (Fig. 6). However, epicotyl showed green coloured friable callus in less to moderate amount. Radicle showed creamish coloured friable callus in moderate amount (Table 4).
Histology of callus culture: Calli obtained from the above explants were further subjected to histological studies (Figs. 9-14). The histological studies of young non embryogenic callus showed hyaline cells of various sizes and shapes devoid of any chlorophyll (Fig. 9).
Light microscopic observations of hypocotyl derived callus grown on inductionmediumMS supplemented with BAP (0.5-1.5 mg/l) revealed the presence of scattered xylem elements (Fig. 10) which later developed into prominent nest like structures (Fig. 11). However, callus cells obtained from hypocotyl inoculated on B5 medium fortified with BAP (1.5- 2.0 mg/l), dedifferentiated into nodules of meristemoids, whichwere quite distinct fromadjoining callus cells (Fig. 12). This may further develop into shoot tips (Fig. 13). Iodine staining of callus cells obtained from hypocotyl inoculated on B5 medium fortified with BAP (1.5-2.0 mg/l), showed a strong accumulation of starch (Fig. 14).
The most influential factors in the morphogenic response associated with calli culture is the type and concentration of growth regulators and culturemedia used . In the present study less to good amount of calli from various explants inoculated on MS and B5 mediumfortified with 2,4 - D (0.5- 2.5 mg/l) and BAP (0.5- 2.5 mg/l l) were obtained. Direct rhizogenesis from leaf explants was also observed (B5+BAP). Kedra and Bach  found best rhizogenesis on basalMS andMS with BAP. Chandra and Bhanja  also observed rhizogenesis from internode and leaf explants inoculated onMS medium and supplement with 1.0 mg/l NAA.
Variations in calli in terms of colour and consistency depending on the nutritive medium have been recorded in past . Koli et al.  also reported variation in the texture and type of callus with different concentrations of growth regulators, type of media and explants. Kedra and Bach  found formation of three different types of calli depending on the kind and concentration of growth regulators. According to Varshney et al. [26,27] and Fennell et al.  white, compact and organized calli ensured regeneration of viable plants in wheat. On the other hand, callus with slowgrowth rate, friable and watery in appearance has been considered to be of poor quality with low potential for regeneration of wheat plants . In the present study compact and green coloured callus was obtained from hypocotyl inoculated on MS and B5 media supplemented with 2,4-D and BAP, whereas cream to brown coloured friable callus was observed from radicle. However, cotyledon and epicotyl showed green and friable callus when inoculated on B5 medium fortified with BAP.
According to Chen and Galstan  callus culture contains vascular elements and parenchymatous cells, together called vascular nodules, which may be associated with an early stage of the development of shoot meristems. Madhavan and Joseph  depicted that histologicalmarkers like xylem elements and meristemoids can be used to differentiate organogenic calli from non-organogenic calli. They have also suggested that the reason for high xylogenesis in non - organogenic calli is attributed to the PGRs in the medium. Chen and Galstan  and Cassels  also claimed that the nodules containing xylem elements in callus of Pelargonium developed into shoots when moved to auxin free medium.
Fromthe histological analysis of callus obtained from hypocotyl inoculated on MS and B5 media fortified with BAP of C. halicacabum L. vascular nodules as well as meristemoids were observed in callus section. Thus histological study of callus can help to predict fate of callus.
In earlier investigation Fortes and Pais  reported a large deposition of starch in internodal explants of Humulus lupulus var. Nugget. According to them starch play a potential causative role in organogenesis. Thorpe and Murashige  and Mangat et al.  observed that in tobacco callus and in Begonia rex stem explants there is strong accumulation of starch in areas that eventually give rise tomeristemoids and shoot primodia in tissues grown on shoot forming media. Similar observations are made in callus cells obtained from hypocotyl in present study.
The present studies proved that the culture medium (MS and B5) and Plant Growth Regulators (2,4 -D and BAP) influences the morphogenetic and histological characteristics of the callus culture obtained fromleaf, cotyledon, hypocotyl, epicotyl and radicle explants of in vitro grown seedlings of Cardiospermum halicacabum L. A good amount of green coloured compact callus was obtained from hypocotyl, whereas pale yellowcoloured friable callus was observed from radicle inoculated on B5 and MS media supplemented with BAP and 2, 4-D. Direct rhizogenesis was also observed from leaf inoculated on B5 medium fortified with BAP. The histological analysis of hypocotyl showed vascular nodules as well as meristemoids formation, which will also develop into shoot meristems. Thus the results obtained frompresent investigationwill help to induce indirect organogenesis and lead to micropropagation of Cardiospermum halicacabum L.
The authors are thankful to UGC for sanctioning the Minor Research Project.
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PATIL, D. A.1, PATIL,A. G., KOLI, S. P.AND NARESH CHANDRA
Department of Botany (Herbal Sciences), Birla College, Kalyan 421 304,
1Department ofBotany, Smt. C.H.M. College, Ulashnagar 421 303.
E. mail: email@example.com
Received: February 15, 2012;March: 12, 2012