IJCRR

IJCRR - 4(21), November, 2012

Pages: 43-47

Date of Publication: 15-Nov-2012

EFFICIENT MICROPROPAGTION PROTOCOL FOR A VALUED ENDANGERED MEDICINAL PLANT - CHITRAK (PLUMBAGO ZEYLANICA LINN.)

Author: Bharti Dohare, Kirti Jain, Bharti Jain, Swati Khare

Category: General Sciences

Abstract:An efficient protocol for clonal propogation through embryogenesis were established for Chitrak Plumbago zeylanica Linn. (Plumbaginaceae), which is endangered medicinal plant for high commercial value. Seeds from healthy grown plants were cultured on standard Murashige and Skoog (MS) medium supplemented with different concentrations of Benzyl amino purine (BAP) or Naphthalene acetic acid (NAA). The best shoot proliferation 22.12 shoots per explants with 93% induction, shoot length (7.43\?0.53) was observed in MS medium containing 1.5 mgl-1 BAP + 0.5 mgl-1 NAA. This study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of the elite medicinal plant.

Keywords: Plumbago zeylanica, medicinal plants, embryogenesis, Micropropagation.

Full Text:

INTRODUCTION
Plumbago zeylanica Linn. (Chitrak) belongs to Plumbaginaceace is an important medicinal plant grown as a perennial herb in most parts of India, but on larger scale in the plains of West Bengal and Southern India. The whole plant contain plumbagin, a natural napthaquinone, possessing various pharmacological activities such as antimalarial, antimicrobial (Didry et al. 1994), anticancer, cardiotonic, antifertility action, antibiotic and antineoplastic (Kirtikar and Basu 1975; Modi 1961, Krishnaswamy and Purushottamam 1980, Pillai et al. 1981). This active chemical has properties of vitamin K, and is antibiotic on several human pathogens (Burkill, 1985). Its roots have been used as folk medicine specially in the treatment of rheumatism, dysmenorrhea, carbuncles, contusion of the extremities, ulcers and elimination of intestinal parasites (Chiu NY et al.,) skin disease, piles, anasarca, syphilis and carcinoma. P. zeylanica has been reported that when given orally or applied to ostium uteri, causing abortion (Azad Chowdhury et al., 1982; Premakumari et al., 1977). It is also used as an appetizer. In traditional Indian medicine, P. zeylanica L. has been assigned medicinal properties and is used in formulations of a number of ayurvedic compounds (Kirtikar KR et. Al). It is widely used in the treatment of diarrhoea, leprosy and anasarca (Anonymous). Collection of its roots from wild stock to furnish the demand of different pharmaceutical companies is posing threat to this important plant species and has been categorized as threatened plant species as per IUCN criteria (Jain AK and Vairale MG.). Propagation through seed is unreliable due to poor seed quality, erratic germination and seedling mortality as under natural field conditions. Despite of all these there is a need to generate a much more reliable regeneration protocol by exploiting newer combinations of different growth hormones and adjuvants. In the present study the effect of different hormonal combinations and different growth adjuvents on in vitro regeneration of Plumbago zeylanica has been worked out.

Application of tissue culture to plant conservation in india has been largery restricted to economically important species. However, the approach could usefully be extended to conserve all threatened plants so that vital biodiversity and the ecological network is sustains can be preserved. (Jiten Chandra et al., 2011). Plumbago zeylanica is categorized as a endangered species and is on the IUCN Red list of Endangered species (2001).

METHOD AND MATERIAL
The Materials and Methods of Plant tissue culture were the standard methods as described in Plant Cell, Tissue and Organ Culture Fundamental methods (Gamborg and Phillips, 2004). General techniques involve the preparation of nutrient media, sterilization, inoculation, maintenance of culture. The explants viz. seeds was excised from plant and were washed thoroughly in running tap water for 30 minutes were further treated with an antifungal agent (Bavistin) for 1 hour and were further with liquid detergents for 20 minutes and rinsed properly with sterile distilled water or in running tap water for 30 min and then surface sterilized with (0.1%) mercuric chloride for 2 minutes followed by washing with distilled water 5 times for surface sterilization under aseptic conditions in laminar chamber. The seed coats is carefully dissected with sterile forceps in sterile Petri dishes. The embryo were aseptically implanted vertically on the sterile culture medium in glass bottles using sterile forceps. After inoculation in the different nutrient media, bottles were sealed with and labelled. The sterilized explants were cultured on the surface of MS basal medium (Murashige and Skoog, 1962) supplemented with 3% (w/v) sucrose, 6% of agar. The pH of the media was adjusted to 5.6-5.8 with 0.1 N NaOH or 0.1 N HCl before autoclaving at 15 lb pressure at 121°C for 15-20 minutes. The cultures were incubated under 16 hours of photo period (2000 Lux) cool, white fluorescent light at 25±2°C. Media was used with various concentrations of plant growth regulators or without growth hormones for culture initiation and multiplication. After 20 days of ovservation embryos swell in initial condition and then regenerated shoots (>2 cm long), excised after 10 of culture initiation, were individually transferred to MS medium supplemented with BAP (0.1-1.5 mg/L), NAA (0.1-0.5 mg/L) KN (0.1-1.5 mg/l) for shoot multiplication, The cultures were maintained by regular subculture at 2-week intervals on fresh medium with the same composition.

MS = Murashige and Skoog salt base (Murashige and Skoog, 1962); NAA = 1-napthalene acetic acid; BAP = Benzyl aminopurine; KN =Kinetin, mg/L = milligram per litter. The data is based on 15 replicate cultures, results were recorded after 15 days and are presented as mean ±SD= Standard deviation.

RESULTS AND DISCUSSION
Growth of Plumbago zeylanica embryos was achieved on MS medium are shown in Table respectively. Direct regeneration of shoots from the explants were tested using MS medium without any growth hormons. When MS supplemented with different concentrations of BAP and NAA was used, the growth and shoot production of P. zeylanica embryos significantly enhanced leaf formation and elongation within two weeks of incubation. Among different concentrations of growth hormones tested, the combination of cytokinnins and auxins stimulate the in-vitro multiplication and growth of shoots of several plant species. A common feature of auxins is the property of inducing cell division. In nature the hormones of this group are involved with such activities as elon- gation of stem, internodes, tropism, apical dominance, abscission and rooting. Cytokinins are adenine deriva- tives which are mainly concerned with cell division, modification of apical dominance and shoot differentiation in the tissue culture (Razdan, 2003). To enhance shoot multiplication different concentrations of NAA was combined with the optimized BAP concentration of 1.5 mg l-1. When an auxin was used in combination with BAP, numerous shoot buds were produced after 15 days of inoculation. There was a increase in both percentage of shoot induction and average number of shoots per explants. when explants were cultured on MS media supplemented with 1.5 mg/l BAP and different concentration of NAA. Addition of 0.5 mg/l NAA to MS media containing 1.5 mg/l BAP was capable of increasing shoot induction up to 93% with an average of 22.12 shoots per explant. The maximum length of shoots (7.43±0.53) was observer on MS medium containing 1.5 mg/L BAP and 0.5 mg/L NAA. On an average within three subcultures. The highest percentage of shoot growth was also observed in the same combination. The MS medium devoid of cytokinins was used as control. Of the two cytokinins tested, BAP was superior for multiple shoot induction than KN for seedling growth and experiment, reported kinetin did not improve significantly the shoot length and the number of proliferating shoots, superiority of BA and KN in combination has been found for micropropagation of other woody perennials reported by Das et al. (1996). The standardized protocol contributes improvement for the propagation of multipurpose medicinal plant as the rates of conventional seeds germination method. Results of seed germination on different medium show in table and fig- A,B,C and D.

CONCLUSION
The present investigation describes a procedure for invitro micropropagation through embryogenesis and successful regeneration of Plumbago zeylanica in invitro condition. We have developed a simple and efficient protocol for direct regeneration from seeds of P.zeylanica. This protocol can be exploited for conservation and commercial propagation of this medicinal plant in the Indian subcontinent. Mass multiplication of Plumbago zeylanica is feasible for field plantings to produce roots as the chief source of Plumbagin (2-methyl-5-hydroxy- 1,4- naphthoquinone) for the pharmaceutical industry. P. zeylanica has been collected by the indigenous people without replanting; this study provides a rapid method of propagation to supplement natural propagation and prevent the plant from extinction due to frequent collection. These in vitro protocol would provide an effective strategy for the conservation and building up of nuclear base populations of this widely exploited plant species. Tissue culture offers means not only for rapid and mass multiplication of existing stock of germplasm but also for conservation of important, elite and endangered plants (Razdan, 2003).

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