Screening of Important Medicinal Plants in Madhupur Tract and Determination of Their Potentials for In Vitro Conservation and Cell Culture

Abstract

Madhupur Tract is a large upland area in the central part of Bangladesh with an area about 4,144 sq km. The tribal people living in this area hold the century old indigenous knowledge of using medicinal plants growing in this Tract for their health remedies. In the present investigation an Ethnobotanical survey was carried out in order to explore the indigenous knowledge of the tribal people and to select the most important medicinal plants growing in this ecosystem. Experiments were also conducted to establish micropropagation and in vitro conservation for some selected medicinal plants. The Ethnobotanical survey reveals that the existence of 192 medicinal plants species belonging to 68 families in the Madhupur ecosystem. Among these Fabaceae, Asteraceae, Euphorbiaceae, Apocynaceae, Lamiaceae, Liliaceae, Zingiberaceae, Malvaceae, Solanaceae and Amaranthaceae were identified as major families contributing the medicinal plants in the Madhupur Tract. The highest 21 species of medicinal plants were belonging to the family Fabaceae. Ethnobotanical survey also reveals that 107 types diseases were treated with herbal medicine by the village doctors. Herbal remedies for the diseases like cough, fever, weakness, worm, dysentery, stomach complaints, sexual debility, jaundice, skin disease, piles, urinary trouble, diarrhoea, rheumatism and diabetes were found more frequent than other diseases. Field survey also indicated that the highest 36 plant species were used for cough and cold, 30 species for fever, 27 species for weakness, 26 species for warm, 25 species for dysentery and stomach complaints, 20 species for sexual debility, 18 species for jaundice and skin disease, 17 species for piles and urinary trouble, 16 species for diarrhoea and rheumatism, 15 species for diabetes, 13 species for dyspepsia, 12 species for constipation, gleet bruises, menstrual troubles and sore, 11 species for each of Itch and ulcer,10 species for asthma, 9 species for cuts, heart disease and wounds. Among the 192 medicinal plant species 5 plants viz., Centella asiatica, Commelina benghalensis, Curcuma zedoaria, Mucuna pruriens and Vitex negundo selected for the experiments on plant tissue culture for micropropagation and in vitro conservation. Different types of explants viz. nodal, shoot tip, axillary bud, internodal and leaf segments cultured in MS medium fortified with different formulation of auxins (NAA, IAA, IBA and 2,4–D) and cytokinins (BAP and Kn). In case of C. asiatica the growth regulator formulation 2.0 mg/l Kn+0.5 mg/l IAA was the most inducive culture medium for multiple shoot proliferation. Whereas, 1.0 mg/l BAP+0.5 mg/l NAA was found the best formulation for multiple shoot proliferation from nodal explant for C. benghalensis. In case of C. zedoaria good response to microshoot proliferation was noticed in liquid MS medium supplemented 2.0 mg/l BAP+1.0 mg/l IBA+20% cocoanut water. The highest multiple shoot proliferation was achieved for M. pruriens in 0.2 mg/l BAP + 0.2 mg/l Kn + 0.2 mg/l IAA. Multiple shoot proliferation for V. negundo was also the maximum in 2.0 mg/l Kn + 0.5 mg/l GA3 supplemented MS medium. In general nodal explant showed better response to multiple shoot proliferation than shoot tip explant. Root development of microcuttings of all five species of medicinal plants were achieved when individual shoot were subculture in auxin supplemented MS medium. However, concentration and type of auxin requirement was found to vary with plant species. For C. asiatica MS + 2.0 mg/l IAA was found most effective rooting medium. Whereas, half strength MS with 1.5 mg/l IBA, ½MS+2.0 mg/l IBA + 0.5 mg/l BAP, ½ MS +2.0 mg/l IBA + 0.5 mg/l NAA and ½MS +1.0 mg/l IAA were respectively found the most effective rooting media formulation for C. benghalensis, C. zedoaria, M. pruriens and V. negundo. Attempt was also made to produce artificial seed by encapsulating the in vitro grown shoot tip and nodal segments of two medicinal plants viz. Centella asiatica and Rauvolfia serpentina. In C. asiatica the highest 90% shoot formation of artificial seeds was observed in MS medium containing 2.0 mg/l Kn + 0.5 mg/l IAA from nodal segments. In R. serpentina the highest 76% shoot proliferation was observed from shoot tip in MS medium containing 1.0 mg/l BAP + 0.2 mg/l NAA. Survivality test reveals encapsulated shoot tip and nodal segments of C. asiatica, the highest % of artificial seed survived up to 60 days of storage at 4±1ºC. However, highest survival percentage was noted up to 60 days of storage at 4±1ºC for R. serpentina. Under the present investigation four selected medicinal plant viz. C. asiatica, Coccinia cordifolia, M. pruriens and R. serpentina were used to develop cell suspension culture. In case of C. asiatica, 0.5 mg/l BAP + 2.0 mg/l NAA found to be most effective culture medium formulation for cell suspension culture.Whereas, 1.0 mg/l BAP+0.5 mg/l NAA was found to be most effective formulation for cell suspension culture of C. cordifolia. In M. pruriens, the best response was found in 3.0 mg/l BAP + 0.5 mg/l IAA supplemented culture medium for cell suspension culture. In case of R. serpentina, 2.0 mg/l 2,4–D+0.5 mg/l BAP found to be most effective formulation for cell suspension culture.The present study demonstrate that for all targeted species, the cultured cells begin to divide within 8-12 days of culture proving their potential for developing cell culture industry for the production of important alkaloid/secondary metabolites.