Ethnobotanical Leaflets 12: 419-424. 2008.

 

 

Conservation Efforts through In Vitro Seed Germination of a Hepatoprotective Plant - Phyllanthus beddomie (Gamble) Mohanan

 

Maridass, M1 and K. Thangavel2

 

1Animal Health Research Unit, St. Xavier’s College, Palayamkottai, TN, India

2Depart of Biotechnology, Sri Paramakalyani College, Alwarkurichi,

Thirunelveli (D.t), Tamil Naud, India

Author for correspondence: E-mail: orchideyadass@yahoo.com

 

Issued 22 June 2008

 

 

Introduction

Whole Plants and plant parts used in ethnomedicine contain curative agents that are used in many modern medicines. The Phyllanthus genus contains over 600 species of shrubs, trees and annual or biennial herbs distributed throughout the tropical and subtropical regions of both hemispheres (Figueira et al., 2006), and have long been used in traditional medicine to treat chronic liver diseases (Dhiman et al., 2005). Phyllanthus appears to be promising in patients with chronic hepatitis B virus (HBV) infection (Thyagarajan et al.1988; 1990;1999). Bioactive principles like alkaloids, tannins, flavonoids, lignans, phenols and terpenes have been isolated from various species of Phyllanthus and their compounds showed antinociceptive activity (Cechinel Filho et al.1996). Liu et al. (2001) published a meta-analysis of the effect on and safety of genus Phyllanthus for chronic HBV infection.

In - vitro propagation of endangered plants can offer considerable benefits for the rapid cultivation of species that are at risk, that have limited reproductive capacity and exist in threatened habitats (Fay, 1992). In vitro propagation methods are essential components of plant genetic resources management and they are becoming increasingly important for conservation of rare and endangered plant species (Sudha et al. 1998; Benson et al. 2000; Iankova et al. 2001; Bhatia et al. 2002). The successful use of plant tissue culture techniques for the micropropagation of members of the genus Phyllanthus has been reported for species such as P. emblica, P. urinaria, P. amarus, P. abnormis, P. caroliniensis, P. tenellus, and P. niruri (Unander, 1991; Ishimaru et al., 1992; Santos et al., 1994). Phyllanthus beddomie (Gamble) Mohanan is a sub-shrub found in evergreen forest and the flowering and fruiting season is April - May. Viswanathan et al., (2002) reported the existence of this species (rediscovered) after a lapse of about 73 years in 1998 (collected from Kalakad Mundanthurai Tiger Reserve, Tirunelveli District), and they have treated this species as critically endemic one. Thus, there is an urgent need for the conservation of this species. There are no reports on the in-vitro micropropagation of P. beddomie. The present study represents a preliminary approach for the development of a rapid and reproducible protocol for in vitro multiplication of P. beddomie, which may be helpful in the conservation of this medicinal plant. Moreover, this type of approach may be a vital tool to have a continuous supply of raw materials for the pharmaceutical industry throughout the year to formulate antihepatitic medicines.

 

Materials and Methods

In vitro culture of Phyllanthus beddomie

Phyllanthus beddomie seeds were collected from Kalakad Mundanthurai Tiger Reserve forest area (Kodhaiyar) and sterilized by immersion in  70% ethanol for 5sec, followed by immersion in  0.5% aqueous solution of  sodium hypochlorite with one drop of Tween 20 per 500ml for 20min, and rinsed three times in sterile double distilled water.  Surface sterilized seeds were sowed for germination in MS medium. Ten duplicate sets were maintained (one in light and one in complete dark condition). Prior to this, an attempt was made to test the viability of the seeds by sowing them in fertile soil.  But there was no appreciable rate of germination.

 

 Sowed seeds in MS medium (Murashige and Skoog, 1962) were incubated for 21 days in darkness in a growth cabinet at 24 o C.  Varying levels cytokinins like BAP and Kinetin (0.10 – 4.00 ΅M) and auxins like NAA, IAA, IBA (Sigma Chemicals, USA) supplemented with MS medium.  For all the combinations, 10 duplicates were maintained. During the course of incubation, parameters like shooting, rooting and callusing were observed, average values were calculated and tabulated (Table 1).

Results and Conclusion

Among the tested concentrations and combinations of PGRs, auxins were able to facilitate the rooting phenomenon than that of shooting (Benson, 2000). Even in case of the duplicates supplemented with cytokinins there was no notable percentage of shooting.  This may be due to the improper placement of plumule during the initial stage of germination. Among the auxins tested, IBA was able to enhance the rooting ability of the implants at the maximum level (90%) in the concentration of 4΅M.  These observations were indicating the indigenous level of hormones especially the auxins in the seeds specifically during the initial phases of germination (Table.1)

 

Regarding the nature of emerged roots, sturdy nature of the roots was proportional to the increasing concentration of auxins like IBA and NAA (Fig.2).  In case of the roots developed from other sets i.e. media combinations supplemented with cytokinins and in low levels of auxins, there appeared fragile and less sturdy ones (Fig.1).

No responses in root and shoot formation of MS medium containing BA, and Kinetin in low concentrations (0.10 – 2.00 ΅M). The findings of the present study may be helpful for the development of micropropagation of P. beddomie. Further studies are ongoing for the further establishment and ex situ conservation of P. beddomie and such type of medicinal plants are in need to fulfill the present need of the pharmaceutical industry (Fay, 2002).

 

Table 1. The frequency (%) of seed germination responses of P. beddomie after 21 days on MS medium supplemented with PGRs in different concentrations.

 

PGRs used

 

Concentration

΅M

Shooting (%)

 

Rooting (%)

 

Callusing

(%)

 

BAP

 

0.10

0.50

1.00

2.00

3.00

4.00

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

 

Kinetin

 

0.10

0.50

1.00

2.00

3.00

4.00

-

-

-

-

-

-

 

0

20

40

50

60

60

-

-

-

-

-

-

 

IAA

0.10

0.50

1.00

2.00

3.00

4.00

-

-

-

-

-

-

-

-

-

-

-

-

 

-

-

-

-

-

-

 

IBA

0.10

0.50

1.00

2.00

3.00

4.00

-

-

-

-

-

-

20

35

40

60

80

90

-

-

-

-

-

-

NAA

0.10

0.50

1.00

2.00

3.00

4.00

-

-

-

-

-

-

10

10

20

20

25

40

-

-

-

-

-

-

 



Acknowledgement:

The authors wish to thank the Department of Science and Technology, Govt. of India, New Delhi for providing financial assistance.

 

References

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Liu, J.,Lin, H, McIntosh, H.2001. Genus Phyllanthus for chronic hepatitis B virus infection: a systematic review. J. Viral Hepat., 8:358–366.

 

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Viswanathan, M. B., Ramesh, N., Maridass, M. and Manikandan, U. 2002. Rediscovery of a critically endangered species Phyllanthus beddomei (Gamble) Mohanan, Euphorbiaceae, from Kalakkad – Mundanthurai Tiger Reserve in India. Bombay Natural History Society, 99(3): 560-562.