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

Maridass, Muthia

Thangavel, K.

English

OpenSIUC

Ethnobotanical Leaflets 12: 419-424. 2008.  Conservation Efforts through In Vitro Seed Germination of a HepatoprotectivePlant - Phyllanthus beddomie (Gamble) Mohanan Maridass, M1 and K. Thangavel2 1Animal Health Research Unit, St. Xavier’s College, Palayamkottai, TN, India2Depart of Biotechnology, Sri Paramakalyani College, Alwarkurichi,Thirunelveli (D.t), Tamil Naud, IndiaAuthor for correspondence: E-mail: orchideyadass@yahoo.com Issued 22 June 2008  IntroductionWhole Plants and plant parts used in ethnomedicine contain curative agents that are used in many modernmedicines. The Phyllanthus genus contains over 600 species of shrubs, trees and annual or biennial herbs distributedthroughout the tropical and subtropical regions of both hemispheres (Figueira et al., 2006), and have long been usedin traditional medicine to treat chronic liver diseases (Dhiman et al., 2005). Phyllanthus appears to be promising inpatients with chronic hepatitis B virus (HBV) infection (Thyagarajan et al.1988; 1990;1999). Bioactive principleslike alkaloids, tannins, flavonoids, lignans, phenols and terpenes have been isolated from various species ofPhyllanthus 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 speciesthat are at risk, that have limited reproductive capacity and exist in threatened habitats (Fay, 1992). In vitropropagation methods are essential components of plant genetic resources management and they are becomingincreasingly 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 themicropropagation 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 theflowering 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 forthe conservation of this species. There are no reports on the in-vitro micropropagation of P. beddomie. The presentstudy represents a preliminary approach for the development of a rapid and reproducible protocol for in vitromultiplication of P. beddomie, which may be helpful in the conservation of this medicinal plant. Moreover, this typeof approach may be a vital tool to have a continuous supply of raw materials for the pharmaceutical industrythroughout the year to formulate antihepatitic medicines. Materials and MethodsIn vitro culture of Phyllanthus beddomiePhyllanthus beddomie seeds were collected from Kalakad Mundanthurai Tiger Reserve forest area (Kodhaiyar) andsterilized by immersion in  70% ethanol for 5sec, followed by immersion in  0.5% aqueous solution of  sodiumhypochlorite with one drop of Tween 20 per 500ml for 20min, and rinsed three times in sterile double distilledwater.  Surface sterilized seeds were sowed for germination in MS medium. Ten duplicate sets were maintained (onein light and one in complete dark condition). Prior to this, an attempt was made to test the viability of the seeds bysowing 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 growthcabinet at 24 o C.  Varying levels cytokinins like BAP and Kinetin (0.10 – 4.00 µM) 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 werecalculated and tabulated (Table 1).Results and ConclusionAmong the tested concentrations and combinations of PGRs, auxins were able to facilitate the rooting phenomenonthan that of shooting (Benson, 2000). Even in case of the duplicates supplemented with cytokinins there was nonotable percentage of shooting.  This may be due to the improper placement of plumule during the initial stage ofgermination. Among the auxins tested, IBA was able to enhance the rooting ability of the implants at the maximumlevel (90%) in the concentration of 4µM.  These observations were indicating the indigenous level of hormonesespecially 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 ofauxins like IBA and NAA (Fig.2).  In case of the roots developed from other sets i.e. media combinationssupplemented 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 µM). 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 andsuch 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 mediumsupplemented with PGRs in different concentrations. PGRs used ConcentrationµMShooting (%) Rooting (%) Callusing(%) BAP 0.100.501.002.003.004.00------------------ Kinetin 0.100.501.002.003.004.00------ 02040506060------ IAA0.100.501.002.003.004.00------------ ------ IBA0.100.501.002.003.004.00------203540608090------NAA0.100.501.002.003.004.00------101020202540------ Acknowledgement:The authors wish to thank the Department of Science and Technology, Govt. of India, New Delhi for providingfinancial assistance. ReferencesBenson, E.E., Danaher J.E., Pimbley I.M., Anderson C.T., Wake J.E., Daley S. and Adams, L.K. 2000. In vitromicropropagation of Primula scotica: a rare Scottish plant. Biodiversity and Conservation, 9: 711–726. Bhatia P., Bhatia N.P. and Ashwath N. 2002. In -vitro propagation of Stackhousia tryonii Bailey (Stackhousiaceae):a rare and serpentine-endemic species of central Queensland, Australia. Biodiversity and Conservation, 11:1469–1477. Cechinel Filho, V., Santos, A.R.S., Campos, R.O.P., Miguel, O.G., Yunes, R.A., Ferrari, F., Messana, J. & Calixto,J.B. 1996. Chemical and pharmacological studies of Phyllanthus caroliniensis in mice. Journal ofPharmaceutical Pharmacology, 48:1231-1236. Fay, M. 1992. Conservation of rare and endangered plants using in -vitro methods. In Vitro Cellular DevelopmentalBiology, 28: 1-4. Gamborg, O.L., Miller, R.A, Ojima, K.1968. Nutrient requirement of suspension cultures of soybean root cells. Exp.Cell. Res., 50:150–158. Iankova E., Cantos M., Lin, J., Robeva P. and Troncoso A. 2001. In vitro propagation of Angelica pancicii Vauds.,an endangered plant species in Bulgaria. Seed Science and Technology, 29: 477–482. Ishimaru, K, Yoshimatsu, K, Yamakawa, T, Kamada, H and Shimomura, K (1992) Phenolic constituents in tissuecultures of Phyllanthus niruri. Phytochemistry, 34: 2015 –2016. Liu, J.,Lin, H, McIntosh, H.2001. Genus Phyllanthus for chronic hepatitis B virus infection: a systematic review. J.Viral Hepat., 8:358–366. Murashige T. and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures.Physiologia Plantarum,15: 473–497. Santos, A.R.S., Cechinel Filho, V., Viana, A.M, Moreno, F.N, Campos, M.M, Yunes, R.A and Calixto, J.B.1994.Analgesic effects of callus culture extracts from selected species of Phyllanthus in mice. J. Pharm.Pharmacol. 46: 755–759. Sudha, C.G., Krishnan, P.N. and Pushpahgadan, P.1998. In-vitro propagation of Holostemma annulare (Roxb.) K.Schum, a rare medicinal plant. In Vitro Cellular and Developmental Biology – Plant, 34: 57–63. Thyagarajan, S.P., Subramanian, S., Thirunaksundari, T, Venkateswaran, P.S., Blumberg, B.S.1988. Effects ofPhyllanthus amarus on chronic carriers of hepatitis B virus. Lancet, 2:764–766. Thyagrajan, S.P., Jayaram, S,Valliammae, T, Madangopalan, N., Pal, V.G., Jayaraman, K.1990. Phyllanthus amarusand hepatitis B.  Lancet, 336:949– 950. Unander, D.W.1991. Callus induction in Phyllanthus species and inhibition of viral DNA polymerase and reversetranscriptase by callus extracts. Plant Cell Rep., 10: 461-466. Viswanathan, M. B., Ramesh, N., Maridass, M. and Manikandan, U. 2002. Rediscovery of a critically endangeredspecies Phyllanthus beddomei (Gamble) Mohanan, Euphorbiaceae, from Kalakkad – Mundanthurai TigerReserve in India. Bombay Natural History Society, 99(3): 560-562.  

Conservation Efforts Through \u3cem\u3eIn Vitro\u3c/em\u3e Seed Germination of a Hepatoprotective Plant - \u3cem\u3ePhyllanthus beddomie\u3c/em\u3e (Gamble) Mohanan

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Conservation Efforts Through \u3cem\u3eIn Vitro\u3c/em\u3e Seed Germination of a Hepatoprotective Plant - \u3cem\u3ePhyllanthus beddomie\u3c/em\u3e (Gamble) Mohanan

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