In vitro Propagation for Rapid and Efficient Regeneration...

Current Trends in Biotechnology and PharmacyVol. 10 (4) 368-373 October 2016, ISSN 0973-8916 (Print), 2230-7303 (Online)

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AbstractCardiospermum halicacabum, a

medicinial plant r ich in β-sitosterol, D-glucoside, saponin and quebrachitol. Directregeneration was achieved only from the nodalexplants which showed 100 per cent sproutingon MS medium supplemented with BAP (0.5ppm) + NAA (1.0 ppm) in 12 days, while MSmedium supplemented with BAP (0.5 ppm) +IBA (1, 2 or 3 ppm) showed early but decreasedsprouting. The half strength MS basal mediumwithout any growth regulator proved to be bestmedium for root induction for excised shoots.The in vitro raised plantlets were transplantedto mixture of sterile dune sand and FYM (3:1)with moderate (80 %) success.

Key words: Cardiospermum halicacabum,nodal explants, axi l lary buds, growthregulators.

Abbreviations : MS, Murashige and Skoog;2, 4-D, 2, 4-dichlorophenoxy acetic acid; IAA,indole acetic acid; IBA, α-indole 3-butyric acid;FYM, Farm Yard Manure; NAA, α-naphthaleneacetic acid, BAP, 6-benzyl amino purine; Kn,kinetin.

IntroductionThe World Health Organization (WHO)

has enlisted over 21,000 plants having

medicinal and therapeutic values. Out of this,more than 2000 plant species are used intraditional medicines of India, as described intexts Charak Samhita and Sushruta Samhita.Globally, about 3.5 billion people rely on thesetraditional medicines for their health care needs(2). Cardiospermum halicacabum L. (BallonVine) is an annual or perennial cl imberbelonging to the family Sapindaceae. It iscommonly found throughout India up to anelevation of 1200 m. It is recognized as amedicinal plant of repute in Ayurvedic andHomoeopathic system of medicine (12). It hasdiuretic, stomachic, antispasmodic andrubefacient properties and is used to treatrheumatism, nervous disorders and dropsy (5).Its leaves contain β-sitosterol and D-glucoside,saponin and quebrachitol as pharmaceuticallyimportant compounds. Besides, it also containsmany important alkaloids like stigmasterol,proanthocynidia and apigenin (1). The plantextract shows vasodepressant activity and hasblood purifying properties. The juice of leavesis given to reduce obesity. The decoction ofroots is useful in curing gonorrhea, nervousdiseases, haemorrhoids and erysipelas. Thepowder of seeds is useful for the treatment ofcancer (1).

Micropropagation plays an important rolein the sustainable utilization of plants and is

In vitro Propagation for Rapid and EfficientRegeneration of Cardiospermum halicacabum

L. – An important Medicinal Plant

Ashwani Kumar1, 2*, S.C. Goyal1, Pooja1, Jagdish Parshad3, Anshuman Singh2,Parvender Sheoran2, Charu Lata2, Rajkumar2 and Meenakshi Jain4

Department of Botany & Plant Physiology1, Department of Microbiology3, Department of MolecularBiology and Biotechnology4, CCS HAU, Hisar – 125004, Haryana, India

2ICAR-Central Soil Salinity Research Institute, Karnal-132001, Haryana, India*For Correspondence: [email protected]

Micropropagation studies of Ballon vine


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especial ly valuable for the commercialcultivation of such plants. Often these plantsare accessed from the wild areas and foreststhat generally results in excessive harvesting.In many cases, the entire plant is cut off foruse and this practice may threaten theexistence of overexploited species. In order toprevent overexploitation as well as to promoteconservation of such medicinal speciesthrough cultivation, availability of elite plantingmaterial is a prerequisite. Conventionally,Cardiospermum halicacabum is multipliedthrough seeds, which have low viability, poorgermination and show insufficient and delayedrooting in seedlings. This has necessitated thedevelopment and standardization of alternativeprotocols for the rapid multiplication of thisvaluable medicinal plant (5). Plant propagationthrough sexual (seed) means is not only a slowprocess but the seedling population also oftenexhibits considerable variability for importanttraits. In medicinal plants, this variability mayresult in poor levels and inferior quality ofdifferent active ingredients in the harvestedtissues. Plant tissue culture techniques providean attractive option for the rapid and efficientmultiplication of different plants (8, 9). InCardiospermum halicacabum, only a fewreports are available on plant regenerationthough callus culture but practical ly noinformation is available regarding directorganogenesis (5). In this study we reportprotocol for an efficient in vitro multiplicationof Cardiospermum halicacabum.

Materials and Methods

Plant Material: The nodal explants obtainedfrom the field grown plants of Cardiospermumhalicacabum were thoroughly washed in therunning tap water followed by washing with 1.0% teepol solution for 10 min by vigorousstirring. Subsequent operation was performedaseptically under a laminar air flow cabinet. Theexplants were surface sterilized with 0.1%mercuric chloride (2 min) followed by 70%ethanol (1 min) and thereafter washed 4-5times with sterile distilled water to remove the

traces of mercuric chloride. The surfacesterilized explants were inoculated on MS (10)basal medium alone and with variousconcentrations of different auxins (2, 4-D, IBA,NAA) and cytokinins (BAP, Kn) - either aloneor in different combinations.

Experimental Conditions: The 30-40 ml ofMS medium containing 3% sucrose and 0.8%agar along with growth regulator(s) was pouredinto 150 ml flasks. The pH of the medium wasadjusted to 5.8 with 1N sodium hydroxide(NaOH) and/or 1N hydrochloric acid (HCl), priorto the addition of agar and the medium wassubsequently autoclaved at 121ºC (1.2 Kg cm-

2 pressure) for 15 min. All the chemicals usedwere of analytical grade. All the cultures wereincubated at 26 ± 2 ºC under white fluorescentlight with a photoperiod intensity of 2000 lux(16 h of light and 8 h of darkness).

All experiments were repeated twice,using 10 replicates (flasks) each containingthree explants. The data were analyzedstatistically using completely randomizeddesign and the significance was tested at 5%level of critical difference using OPSTATsoftware (CCS HAU, Hisar).

Results and Discussion

Shoot Regeneration: Direct regeneration inCardiospermum halicacabum cultures wasachieved only from the nodal explants. On MSmedium devoid of growth regulators, 66%axillary bud sprouting took place in 21 daysafter inoculation (Table 1). On MS mediumsupplemented with 2, 4-D (0.5, 1, 1.5 and 2ppm), no sprouting was observed even after 4weeks of inoculation. In contrast,supplementation with Kn (1.0, 1.5 and 2.0 ppm)or use of BAP in higher concentration (4 ppm),resulted in 100% sprouting between 13-17 daysafter inoculation (Table 1; Figs 1a, 1b). Shootproliferation directly from the nodal explantcould be attributed to the presence of pre-existing axillary primordial, which proliferatedinto shoots under adequate concentration ofcytokinin supplied in the nutrient medium.

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Synergistic effects of plant growth regulatorshave influenced the cultural response in shootproliferation and elongation. However, thenumber and percentage of sprouting of axillarybuds varies with the species as well as withhormone concentration. Similar results havebeen reported by Singh (16) in Chlorophytumborivilianum, Sharma et al. (15) in Vitexnegundo and Kumar et al. (9) in Dioscoreaalata.

When BAP (0.5 and 1 ppm) along withIBA (1, 2 and 3 ppm) was used in the medium,75-82% sprouting was obtained between 7-11days after inoculation. It was also noted thatincrease in concentration of IBA in the mediumresulted in delayed but significantly highersprouting, i.e. from 75% in IBA (1 ppm) to 82%in IBA (3 ppm). In MS medium supplementedwith BAP (1 ppm) and NAA (1 ppm), shootswere formed between 7-12 days of inoculationwith 100% sprouting. Use of higherconcentrations of NAA (above 1 ppm) alongwith BAP (1 ppm), resulted in no sprouting. Itwas also observed that only one plantlet wasnormally formed from each axillary bud (Table

1; Fig 1c). It appeared that supplementation ofnutrient medium with adequate concentrationsof cytokinin and trace amounts of auxinsovercame the effects of apical dominance andthus enhanced the proliferation of axillary buds(7, 9). These results are in accordance withearl ier reports on Mose rose (14) andDioscorea alata (9).

Rooting of Shoots: The regenerated shoots(3-5 cm in length) derived from the axillary budsof nodal explants (direct regeneration) weretransferred on the MS basal mediumsupplemented with different strengths ofmineral salts (¼MS, ½MS and full strength MSbasal medium) for rooting. The rooting was inrange of 67-100% under media with differentstrength of mineral salts and the maximumnumber of roots (8-9; 3.0-4.0 cm in length)were formed at ½MS strength basal medium(Table 2; Fig 2a). Similar results have earlierbeen reported in Acacia catechu (17),Centarium erythraea (11) and Hedychiumspicatum (6), where ½ strength MS mediumwas also used for rooting.

Acclimatization of in Vitro Raised Plantlets:The plantlets developed were taken out of themedium after sufficient development of rootsand were subsequently transferred to smallpots containing sterilized sand and FYM (3:1).The pots, covered with polythene bags tomaintain the high humidity and kept in cultureroom at 26 ± 2ºC, were irrigated with ½ strengthMS salt solution from time to time. After about2 weeks, the potted plants were taken out fromthe culture room and polythene bags wereremoved for 3-4 h daily to expose the plant tothe natural humidity condit ions foracclimatization (Fig 2b). These acclimatizedplants were then transferred to the bigger potsunder field conditions. During this process ofhardening some plants could not withstand thetransplanting shock and died after few weeks.After about 4 weeks, these plants weresubsequently shifted to the pots containing soiland sand mixture in the Botanical Gardenunder natural light, temperature and humidity

Fig. 1. Direct shoot regeneration from nodalexplants on MS medium supplemented with 4.0ppm BAP (Figure 1a); 1.0 ppm Kn (Figure 1b); and1.0 ppm BAP + 1.0 ppm NAA (Figure 1c).



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Table 1. In vitro response of nodal explants of C. halicacabum on MS medium supplementedwith auxins and cytokinins (in different combinations).

Medium + growth Explant Days required Visual Colour andregulator (ppm) callusing for callus induction/ growth of texture of callus

(%) sprouting of callus after (sprouting ofaxillary buds* 4 weeks axillary bud)

Control 66 36/21 + Green, fragile (sprouting)2,4-D (0.5) 87 11/— + Light green, compact

(1.0) 100 11/— + Light green, compact(1.5) 100 11/— + Light green, compact(2.0) 100 11/— Light green, compact

Kn (0.5) 75 11/— + Light green, compact(1.0) 100 11/15 + Light green, compact(1.5) 100 11/15 + (sprouting)(2.0) 100 11/13 + Light green, compact

(sprouting)Light green, compact

(sprouting)

BAP (1.0) 75 30/— + Green, compact(2.0) 80 26/— + Green, compact(3.0) 82 25/— + Green, compact(4.0) 100 22/17 + Green, compact (sprouting)

IBA (1.0) +BAP (0.5) 75 21/7 + Green, compact (sprouting) IBA (2.0) +BAP (0.5) 80 21/9 + Green, compact (sprouting) IBA (3.0) +BAP (0.5) 82 21/11 + Green, compact (sprouting) NAA (1.0) +BAP (1.0) 100 36/12 + Green, compact (sprouting) NAA (1.5) + BAP (1.0) 100 36/— + Green, compact NAA (2.0) + BAP (1.0) 97 36/— + Green, compact

Data presented are means of 5 replicates* — No sprouting, + Poor callus, ++ Moderate callus.

Table 2. Formation of roots from excised shoots regenerated in vitro from axillary bud breakfrom nodal segments on various strengths of MS medium (observation recorded upto4-weeks after inoculation).

Medium strengths No. of days required % root No. of root per shoot/for root induction induction (length in cm)

Full MS basal medium 6 67 2-3/(1.5-2.0) ½ MS basal medium 4 100 8-9/(3.0-4.0) ¼ MS basal medium 4 100 4-5/(2.5-3.5)

Data presented is mean of three replicates

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Fig. 2 Root induction from in vitro regenerated shoots on half strength MS medium in C. halicacabum(Figure 2a); 2 months old plant in pot before transferring to the field conditions (Figure 2b); and fullymatured plants of Cardiospermum halicacabum (Figure 2c).

conditions (Fig 2c). Similar acclimatizationprocedure for successful establishment of invitro regenerated plants has beendemonstrated by Purohit et al. (13),Chukwujekwu et al. (4) and Singh (16).

ConclusionAn efficient and repeatable method for

was developed in vitro micropropagation of C.halicacabum. Regeneration was found to bedependent on synergistic effect of cytokininsand auxin in the MS medium and the best resultof multiple plantlets regeneration was obtainedfrom nodal explants when BAP (1.0 ppm) alongwith NAA (1.0 ppm) supplemented to MSmedium. The rooted shoots were hardened forsurvival after transplantation to mixture ofsterile dune sand and FYM (3:1) in pots byexposing them to subsequent humidityconditions. These were then transfer to the soilwith moderate (80 %) success. The plantregeneration protocols described here can beused for mass propagation and to provide plantmaterial for improvement programs of thecultivated species. Those based on thedevelopment of preexisting meristems anddirect organogenesis is particularly useful forin vitro conservation.

AcknowledgementsThe first author is thankful to the Head,

Department of Botany and Plant Physiology,CCS HAU, Hisar for providing the requiredresearch facilities.

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