Micropropagation of Crataeva religiosa Hook. f & Thoms.

M. Jothi Basu¹*^†, R. Ramanathan,N. Yogananth¹ and S. Baburaj

¹J.J. College of Arts and Science, Pudukkottai-622 422,Tamilnadu,India.

Thiagarajar College (Autonomous), Madurai-625 009,Tamilnadu,India.

^† Present Address: Alagappa Uiversity, Karaikudi-630 003,Tamilnadu,India

*For Correspondence -  jothibasu77@gmail.com

Current Trends in Biotechnology and Pharmacy , Volume 3 (3) July - 2009

Abstract

 A protocol was developed for in vitro propagation by multiple shoot induction of  Crataeva religiosa Hook. f. & Thoms, a medicinal tree having high medicinal values belonging to the family Capparidaceae. High frequencies of multiple shoot regeneration were achieved from apical bud on MS medium fortified with 8 mg/L BAP alone. Five to seven shoots per explant were obtained. The elongated shoots were subcultured for rooting on half strength MS supplemented with various concentrations of IBA and IAA. The in vitro raised plantlets were acclimatized in green house and successfully transplanted to natural condition with 72% survival.   

Key words: Crataeva religiosa, Medicinal plant, Micropropagation, Acclimatization.

Abbreviations: MS-Murashige Skoog medium, NAA-Napthalene Acetic Acid, BAP-Benzyl Amino Purine, IAA-Indole Acetic Acid, IBA-Indole Butric Acid. 

Introduction

Tissue culture techniques are being increasingly exploited for clonal multiplication and in vitro conservation of valuable indigenous germ -plasm threatened with extinction. Greater demand for these plants especially for the purpose of food and medicines which is one of the causes of their rapid depletion from primary habitats. Micropropagation offers a great potential for large scale multiplication of such useful species and subsequent exploitation (1). Crataeva  religiosa Hook. f. & Thoms., non Frost. F. f belongs to the family Capparidaceae is a moderate-sized deciduous tree (Tamil: Mavilankai, Telugu: Magalingam, Malayalam: Nirmatalam, Hindi: Barun). C. religiosa is characterized with astringent, bitter, acrid, diuretic, anthelmintic, carminative, laxative and stomachic. Root and park bark promote appetite, and increase biliary secretion. Leaves are stomachic and tonic. Juice of leaves is given internally in to cure rheumatism. Bark is demulcent, alterative, tonic, stomachic, laxative, diuretic, antipyretic, and useful in calculus affections and for disorders of urinary organs. Powdered bark is useful in urinary and renal troubles, gastro-intestinal and uterine affections infections (2). There has been progress in tissue culture studies in many Capparidaceae members such as C. nurvala (3), C. magna (4) and Capparis decidua (5)to propage them. But no such in vitro culture studies have been carried out in this valuable medicinal tree, C. religiosa. The present investigation elucidates in vitro multiple shoot regeneration through apical bud segments of C. religiosa for better exploitation and also preservation of this valuable germ-plasm which has already undergone a severe biotic pressure.

Materials and Methods

Apical buds of C. religiosa were collected from the campus ofThiagarajar College,Madurai, washed thoroughly in running tap water and treated with detergent solution. The apical buds were initially disinfected by rinsing it in 90% ethanol for 15 seconds followed by surface sterilization in an 0.1% (w/v) HgCl₂ aqueous solution of 0.1% (w/v) HgCl₂ for 2-3 minutes. The explants were once again rinsed thrice with sterile distilled water. The sterilized explants were inoculated in MS medium containing 3% sucrose and 0.8% agar with supplemented with BAP alone (0.5 – 10.0 mg/L) and different concentrations (1.0 – 5.0 mg/L) of BAP along with NAA (0.25 mg/L). The pH of the media was adjusted to 5.8 and autoclaved at 1.06 kg/cm² pressure and 121º C temperature for 15 min. For root induction, the well developed shoots were transferred in to half strength MS medium with different concentrations of IAA (3.0 mg/L) and IBA (3.0 mg/L). All subsequent subculturing e were performed at four week of interval to fresh medium. The cultures were incubated in a culture room maintained at 25 ± 2ºC under 16 hours photoperiod with light intensity of 3000 lux. For each treatment 14 replicate cultures were maintained and all the experiments were replicated thrice. The data were statistically analyzed using one way analysis of variance and means were compared using theDuncan’s Multiple Rank Test at the 0.05% level of significance.

Results And Discussion

The use of pre-existing buds for propagation reduces the possibility of variation among the progeny and therefore can be safely applied for rapid propagation of Crataeva religiosa. We optimized shoot multiplication conditions and novel rooting techniques for mass clonal propagation without interference of callus. This method is quite common for the propagation of Fragaria indica (6), and Acacia mearnsii (7) and Sandalam album(8). The apical bud explants showed slight swelling prior to the emergence of shoot buds developing from the pre-existing material 20 days after inoculation. Initiallytwo to four shoot buds per explant emerged 30 days after inoculation and gradually the number of shoot buds per explant increased up to 5 – 7 (Table 1; Plate 1a) on MS media fortified with 8 mg/L BAP. But low number of buds developed in the concentration of 0.5 mg/L BAP. Superiority of BAP over other cytokinin has been reported and discussed in relation to shoot proliferation in cultures of trees (9, 10) and the regeneration of shoots from nodal explants has also been encountered in Capparidaceae plants like C. nurvala (3), C. magna (4) C. adansonii (11) and Capparis deciduas (5). Sometimes callus formation from the basal cut ends of the apical bud explant was also observed (Plate 1b).

The in vitro developed shoots from the apical bud cultures were harvested and transferred to half strength MS medium added with various concentrations of IBA and IAA. A maximum number of 4 - 5 roots were observed when the medium was supplemented with 3.0 mg/ L IBA or IAA after 5 weeks , irrespective of the type of auxins used (Fig. 1). Similarly, pulse treatments of IBA were given for root induction in shoots produced in cultures from nodal explants of adult plants of Camellia sinensis (12), Maytenus emayginata (13) and Prosopis cineraria (14) and Sandalum album (8). IBA is the most commonly used auxin for root formation from shoots of woody trees (15).  The in vitro regenerated rooted plantlets were washed with sterile water and transferred to small plastic pots containing sand, garden soil and digested coir pith (1:1:1) mixture (Plate 1c). Then the plants were maintained under shade and partial shade for one more week. The regenerated plants were transferred to the soil with 72% survival.

The present investigation has demonstrated a potentially efficient technique for the large scale micropropagation of C. religiosa from apical bud explant. In the present investigation it was observed The data indicated that BAP at 8.0 mg/L on in MS medium is more effective for shoot multiplication from the shoot apical bud. Half strength MS medium supplemented with 3.0 mg/L IBA or IAA is best for root induction.

The protocol described is an efficient and could be used as a means of propagation and multiplication of Crataeva religiosa – a potential medicinal plant for commercial exploitation while previously published protocols are having some complications like incubation in dark for 6 days (5).

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Table1: Effect of NAA and BAP on in vitro shoot formation from apical shoot bud .

explants of Crataeva religiosa after 30 days of culture

± - Standard Error. Means followed by the same letter not significantly different by theDuncan’s Multiple Rank Test at P< 0.05 level of significance.

Where CS – Callus + Shoot,   S - Shoot

Fig.1: Effect of various auxins on rooting response from in vitro regenerated shoots of Crataeva religiosa cultured on half strength MS medium after 5 weeks of culture.

Vertical line on the bar indicates Standard Error. Means followed by the same letter not significantly different by theDuncan’s Multiple Rank Test at P< 0.05 level of significance.

a).Shoot proliferation from apical bud on MS media fortified with 8 mg/L BAP

b).Direct shoot formation from callus on MS media supplemented with 0.25 mg/L NAA and 1.0 mg/L BAP

c).Successful establishment of rooted plants in plastic cup.