# In Vitro Conservation of Twenty-Three Overexploited Medicinal Plants Belonging to the Indian Sub Continent

Authors: Priyanka Verma; Ajay Kumar Mathur; Sheetal Prasad Jain; Archana Mathur
Journal: The Scientific World Journal
 (2012)

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## Abstract

Twenty-three pharmaceutically important plants, namely,Elaeocarpus spharicus, Rheum emodi, Indigofera tinctoria, Picrorrhiza kurroa, Bergenia ciliata, Lavandula officinalis, Valeriana wallichii, Coleus forskohlii, Gentiana kurroo, Saussurea lappa, Stevia rebaudiana, Acorus calamus, Pyrethrum cinerariaefolium, Aloe vera, Bacopa monnieri, Salvia sclarea, Glycyrrhiza glabra, Swertia cordata, Psoralea corylifolia, Jurinea mollis, Ocimum sanctum, Paris polyphylla, and Papaver somniferum, which are at the verge of being endangered due to their overexploitation and collection from the wild, were successfully established in vitro. Collections were made from the different biodiversity zones of India including Western Himalaya, Northeast Himalaya, Gangetic plain, Western Ghats, Semiarid Zone, and Central Highlands. Aseptic cultures were raised at the morphogenic level of callus, suspension, axillary shoot, multiple shoot, and rooted plants. Synseeds were also produced from highly proliferating shoot cultures of Bacopa monnieri, Glycyrrhiza glabra, Stevia rebaudiana, Valeriana wallichii, Gentiana kurroo, Lavandula officinalis, and Papaver somniferum. In vitro flowering was observed in Papaver somniferum, Psoralea corylifolia, and Ocimum sanctum shoots cultures. Out of 23 plants, 18 plants were successfully hardened under glasshouse conditions.


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## Body

## 1. Introduction

Numerous drugs or their precursors used in the current pharmacopoeias originate from plant sources. Natural products or natural product-derived drugs comprise nearly 28% of all the new chemical entities launched into the market in the last 20 years [1].  Medicinal plant-based drugs have the added advantage of being simple, effective, and offering a broad spectrum of activity with well-documented prophylactic or curative actions. Medicinal plant products have also proved useful in minimizing the adverse side effects of various chemotherapeutic agents [2, 3].  The World Health Organization (WHO) has estimated that the present demand for medicinal plants is approximately US $14 billion per year. The demand for medicinal plant-based raw materials is growing at the rate of 15 to 25% annually, and according to an estimate of WHO, the demand for medicinal plants is likely to increase more than US $5 trillion in 2050.  In India, the medicinal plant-related trade is estimated to be approximately US $1 billion per year [4].  Medicinal plants are living resource, exhaustible if overused and sustainable if used with care and wisdom. At present 95% collection of medicinal plant is from the wild. Current practices of harvesting are unsustainable, and many studies have highlighted depletion of resource base.  Medicinal plants-based industries although old and vast are still being managed on traditional ethos and practices and lack a proactive and socially responsible image.  Many studies have confirmed that pharmaceutical companies are also responsible for inefficient, imperfect, informal, and opportunistic marketing of medicinal plants.  As a result, the raw-material supply situation is shaky, unsustainable, and exploitative. There is a vast, secretive, and largely unregulated trade in medicinal plants, mainly from the wild which continue to grow dramatically in the absence of serious policy attention with environmental planning. Confusion also exists in the identification of plant materials where the origin of a particular drug is assigned to more than one plant, sometimes having vastly different morphological and taxonomical characters; therefore, adulteration is common in such cases [5–7].India is rich in medicinal plant diversity.  All known types of agroclimatic, ecologic, and edaphic conditions are met within India [8]. The biogeographic position of India is so unique that all known types of ecosystems range from coldest place like the Nubra Valley with −57°C, dry cold deserts of Ladakh, temperate and Alpine and subtropical regions of the North-West and trans-Himalayas, rain forests with the world’s highest rainfall in Cherrapunji in Meghalaya, wet evergreen humid tropics of Western Ghats, arid and semiarid conditions of Peninsular India, dry desert conditions of Rajasthan and Gujarat to the tidal mangroves of the Sunderban. India is rich in all the three levels of biodiversity—such as species diversity, genetic diversity, and habitat diversity [8]. There are about 426 biomes representing different habitat diversities that gave rise to one of the richest centres in the world for plant genetic resources [9].  Concerning the total number of flowering plant species, although only 18,665, the intraspecific variability found in them make it one of the highest in the world.  Out of 18,665 plants, the classic systems of medicines like Ayurveda, Siddha, and Unani make use of only about 3000 plants in various formulations [7].The continued growth of human populations and ofper capita consumption has resulted in unsustainable exploitation of Earth’s biological diversity, exacerbated by climate change, ocean acidification, and other anthropogenic environmental impacts [10].  Conservation of medicinal plants can be accomplished by the ex situ, that is, outside natural habitat by cultivating and maintaining plants through long-term preservation of plant propagules in plant tissue culture repositories [10]. Although species conservation is achieved most effectively through the management of wild populations and natural habitats (in situ conservation), ex situ techniques can be used to complement in situ methods and, in some instances, may be the only option for some species [11–13].In vitroapproaches for the conservation and the use of plant germplasm can offer some distinct advantage over alternative strategies.  Some of these are as follows: (1) collection may occur at anytime independent of flowering period for each species (this assumes that seed material is not required), (2) there is the potential of virus elimination from contaminated tissue through meristem culture, (3) clonal material can be produced where this is useful for the maintenance of elite genotypes, (4) rapid multiplication may occur at any time where stocks are required using micropropagation procedures, (5) germination of difficult or immature seed or embryo may be facilitated for breeding programmes, and (6) distribution across the border may be safer, in terms of germplasm health status using in vitrocultures. Some more general positive advantages of in vitro techniques include the fact that storage space requirements are vastly reduced compared with field storage. Storage facilities may be established at any geographical location and cultures are not subject to environmental disturbances such as temperature fluctuation, cyclones, insect, pests, and pathogen [14, 15].The present investigation deals with collection andin vitro conservation of twenty-three overexploited medicinal plants belonging to different biodiversity zones of Indian subcontinent, at various morphogenic levels and their glasshouse hardening. These plants are at the verge of being endangered, so the work presented here will be beneficial for the biological conservation as well as for the worldwide pharmaceutical industry.


## 2. Methods

### 2.1. Collection of Plant Material

The plant material of twenty-three medicinal plants [16], namely, (1) Elaeocarpus spharicus, (2)Rheum emodi, (3)Indigofera tinctoria, (4)Picrorhiza kurroa, (5)Bergenia ciliata, (6)Lavandula officinalis, (7)Valeriana wallichii, (8)Coleus forskohlii, (9) Gentiana kurroo, (10) Saussurea lappa, (11)Stevia rebaudiana, (12) Acorus calamus, (13) Pyrethrum cinerariaefolium, (14)Aloe vera, (15)Bacopa monnieri, (16)Salvia sclarea, (17)Glycyrrhiza glabra, (18)Swertia cordata, (19)Psoralea corylifolia, (20)Jurinea mollis, (21)Ocimum sanctum, (22)Paris polyphylla, and (23)Papaver somniferum,were collected from different biodiversity zones of India (Figure 1; Table 1). Collections were made in terms of different plant part like rhizome,  tuber,  leafy shoot,  proliferating bud,  rooted plant,  seeds,  and so forth.Table 1
Details of the plants collected, medicinal value and their site of collection forin vitro conservation.
S. no.Plant speciesFamilyPart usedMedicinal valueZone of collectionCoordinates/altitude of site of collection(1)Acorus calamusAraceaeRhizomeAntiasthmatic, antibronchitis, antidiarrhoea, used in dysentery, stomach ache, and constipationWestern Himalayas29°54′36′′N 79°37′46′′E;1,500 mt(2)Aloe veraAloaceaeLeavesPurgative, liver tonic, and used against skin allergiesCentral Highlands21°54′N 83°24′E;  215 mt(3)Bacopa moneeiriScrophulariaceaeWhole plantDiuretic, cardiac, nervine, and tonic, improve intellect, used against asthma, hoarseness, insanity, epilepsy, potent nervous tonic, and antianxiety agentWestern Ghats10°31′N 76°13′E;  2.83 mt(4)Bergenia ciliataSaxifragaceaeWhole plantUsed against pimples, sun burn, stomach ache, muscular pain, and running eyesWestern Himalaya25°34′N 91°53′E;  1,525 mt(5)Coleus forskohliiLabiateaeRootsAntiglaucoma, antiplatelet, bronchospasmolytic, cardiotonic, hypotensive, antiaging, antiallergic, smooth muscle, and arterial relaxant, antiasthmaticWestern Himalaya29°54′36′′N 79°37′46′′E;  1,500 mt(6)Elaeocarpus spehericusElaeocarpaceaeFruits/seed kernelExpectorant, used against nervous weakness, insanity, epilepsy, melancholia, mania, other mental disorders, insomnia, headache, jaundice, hypertension, and feverCentral Highlands21°08′N 81°23′E; 298 mt(7)Gentiana kurrooGentianaceaeRootsAnthelmintic, anti-inflammatory, antiseptic, bitter tonic, cholagogue, emmenagogue, febrifuge, refrigerant, stomachicNortheast Himalaya27°0′N 88°16′E; 5,307 mt(8)Glycyrrhiza glabraFabaceaeRoots/rhizomesExpectorant, diuretic, mild laxative, antiarthritic, antiinflammatory, antibiotic, antiviral, antiulcer, memory stimulant, antitussive, aphrodisiac, antimitotic, estrogenic, antioxidant, anticaries agent, antineoplastic, anticholinergic, antidiureticGangetic plain26°51′N 80°55′E; 128 mt(9)Indigofera tinctoriaFabaceaeLeavesBlood purifier (detoxify) reduces inflammation and alleviate pain antiseptic, stimulantGangetic plain26°51′N 80°55′E; 128 mt(10)Jurinea mollisCompositaeRootEruptions, decoction is given in colic, puerperal feverWestern Himalaya31°58′N 77°06′E; 1220 mt(11)Lavandula officinalisLabiateaeWhole plantKnown for essence, stimulant, sternutatory and tonicSemiarid Zone30.83°N 76.93°E; 656 mt(12)Ocimum sanctumLabiateaeLeaves/seedsDiaphoteric, antiperiodic, stimulating, expectorant, and anticatarrhalGangetic plain26°51′N 80°55′E; 128 mt(13)Papaver somniferumPapaveraceaeCapsuleNarcotic, analgesic, muscle relaxant, and antimicrobial agentGangetic plain26°51′N 80°55′E;128 mt(14)Paris polyphyllaTrilliaceaeRootsAnalgesic, anthelmintic, antiphlogistic, antispasmodic, antitussive, narcoticWestern Himalaya30°25′N 79°20′E; 2,500 mt(15)Picrorrhiza kurroaScrophulariaceaeRoot/stemUsed in fever, gastralgia, as laxative, liver complaints, anaemia. and jaundiceWestern Himalaya29°49′23′′N 79°26′54′′E;2,286 mt(16)Pyrethrum cinerariefoliumCompositaeWhole plantFebrifuge, stimulatory, and stimulant of nervous system, insecticideWestern Himalaya29°54′36′′N 79°37′46′′E;1,500 mt(17)Psoralea corylifoliaPapilionaceaeSeedsAgainst bacteria causing skin diseases, leucoderma, leprosy, and psoriasisWestern Ghats12°58′N 77°34′E; 920 mt(18)Rheum emodiiPolygonaceaeRootsStomach pains, constipation, dysentery, swelling of throat, and tonsillitisWestern Himalaya32°22′N 77°14′E;3,979 mt(19)Salvia sclareaLabiateaeLeaves/flower topsAntidepressant, anticonvulsive, antispasmodic, antiseptic, aphrodisiac, astringent, bactericidal, carminative, deodorant, digestive, emmenagogue, euphoric, hypotensive, nervine, sedative, stomachic, uterine and nerve tonicWestern Himalaya31°58′N 77°06′E; 1,220 mt(20)Saussurea lappaCompositaeRootAntiseptic, disinfectant, bronchitis, asthma, flatulence, and cardiac complaintsNortheast Himalaya27°0′N 88°16′E; 5,307 mt(21)Swertia cordataGentianaceaeWhole plantBlood purifier, febrifuge, anthelmintic, antimalarial, antidiarrhoeaWestern Himalaya29°49′23′′N 79°26′54′′E;2,286 mt(22)Stevia rebaudianaAsteraceaeLeavesSweetener (regulate blood sugar), increase energy level, mental alertness, appetite, vasodilator, skin careWestern Ghats10°31′N 76°13′E; 2.83 mt(23)Valeriana wallichiiValerianaceaeRootsUseful in disease of eye, blood, liver, and spleen enlargement. Also useful in cleaning voice, headacheWestern Himalaya30°27′N 78°05′E; 1,876 mtFigure 1
Different biodiversity zones of Indian subcontinent from where 23 plant species were collected.


### 2.2. Raising Aseptic Cultures and Multiplication

Various explants, namely, leaf, node, bulb, basal disc, and seeds, were first washed thoroughly for 2-3 hours under tap water (seeds left overnight for soaking).  These were then washed with a detergent (cetrimide) which removes dust particles and clears the surface of the explant by removing hairs.  Washed material was then treated with (4%) savlon for 2-3 minutes, followed by exposure to absolute alcohol for 30 seconds, and finally subjected to surface sterilization by 0.1% mercuric chloride. Treatment time varied from plant to plant and from explant to explant. While underground parts and basal discs were treated for 8–10 min, seeds, leaves, and nodes were treated for 1-2, 2-3,  and 4-5 min, respectively. The explants were then thoroughly washed with sterile water (3-4 times). The explants were cut and inoculated on different media combinations.More than 100 media combinations were tested for raising aseptic cultures of all the 23 plant species. According to the morphogenetic response required, medium recipes were made by using basal media [17] fortified with different ranges of  2,4-diphenoxyacetic acid (2,4-D), 2,isopentenyl adenine (2IP), ascorbic acid (AA), 6-benzylamino purine (BAP), casein hydrolysate (CH), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), kinetin (KN), and α naphthalene acetic acid (NAA).  Cultures were incubated at 25 ± 2°C under 8 hours/16 hours photoperiod light 2500–3000 lux intensity and 75–85%  relative humidity.


### 2.3. Synseed Production

The nodal segments and shoot tips of highly proliferating shoot cultures of different plant species were dissected fromin vitro raised mother plant and used as explants for the synseed production. For encapsulation explants dispersed in 4% sodium alginate solution were suspended dropwise in 75 mM calcium chloride solution so that each drop contained single explant. The complexation time was 1 hr, and then the synseeds were thoroughly washed with sterile water and plated on MS basal medium for germination.


### 2.4. Glasshouse Hardening

Before transplantation, shoots were shifted to rooting medium. To establish root proliferation, actively growing shoots were transferred to respective rooting media (Table2). When adequate rooted shoots were obtained, the rooted plantlets (mostly 20–30 days old) were transferred to glasshouse conditions.  For that plantlets were carefully taken out without injuring the roots, thoroughly washed with running tap water to remove the adhered agar by gentle shaking in water filled plastic tray, and planted in a mixture of sterilized soil and vermicompost (1 : 1) in earthen pots. Temperature and humidity was gradually lowered according to the native conditions of the plant.Table 2
Morphogenic level along with the optimised medium and glasshouse hardening status of 23 plant species processed forin vitro conservation.
S. no.Plant speciesExplant usedCallusCultures established at the level ofSuspensionAxillary/multiple shootsFloweringSynseedRooted plantsTime taken/success rate during hardening(1)Acorus calamusNode−−+ 5.0KN*−−+ 3.0IBA3-4 wks/60%(2)Aloe veraBasal disc−−+ 5.0KN**−−+ 1.0IAA3-4 wks/90%(3)Bacopa moneeiriLeaf+ 3.0BAP; 1.0IAA−+ MS0***−++ MS02 wks/100%(4)Berginia ciliataNode−−+ 3.0BAP;  0.1IAA;  40AA−−+ 3.0IAA5-6 wks/30%(5)Coleus forskohliiNode+ 2.5NAA−+ 0.2BAP−−+ 2.5IAA3-4 wks/70%(6)Elaeocarpus spehericusLeaf+ 2.0 NAA; 0.5KN−−−−−−(7)Gentiana kurrooNode−−+ 5.0KN−++ 2.0IAA5-6 wks/50%(8)Glycyrrhiza glabraNode+ 0.5NAA; 0.5-2,4-D; 1000CH+ 0.5NAA; 0.5-2,4-D; 1000CH+ 2.5KN−++ 1.0IAA2-3 wks/80%(9)Indigofera tinctoriaSeeds−−+ 1.0BAP−−+ 0.1IAA5-6 wks/40%(10)Jurinea mollisLeaf+ 3.0BAP; 1.0IAA)−−−−−−(11)Lavandula officinalisSeeds+ 0.5 NAA; 0.5-2,4-D; 0.5 KN+ 0.5NAA; 0.5-2,4-D; 1000CH+ 2.5KN; 0.1NAA−++ 0.5IAA5-6 wks/50%(12)Ocimum sanctumNode−−+ 3.0BAP; 1.0IAA+−+ 1.0IAA3-4 wks/100%(13)Papaver somniferumSeeds+ 1.0-2,4-D; 0.1KN; 40AA+ 2.0–2,4-D; 0.25KN+ 0.5-2IP; 0.1 IAA+++ 1.0IAA5-6 wks/70%(14)Paris polyphyllaBulb−−+ 2.0BAP−−−−(15)Picrorrhiza kurroaLeaf/node+ 1.0-2,4-D; 0.5BAP−+ 5.0KN−−+ 3.0IBA5-6 wks/40%(16)Pyrethrum cinerariefoliumLeaf/node+ 2.0-2,4-D; 1.0KN−+ 5.0KN; 2.0BAP; 1.0IBA−−+ 1.0IBA5-6 wks/60%(17)Psoralea corylifoliaNode−−+ 5.0KN; 2.0BAP; 1.0IBA+−+ 1.0IBA3-4 wks/90%(18)Rheum emodiiBasal disc−+ 3.0BAP; 1.0IAA−−−−(19)Salvia sclareaNode−−+ 5.0KN; 2.0BAP; 1.0IBA−−+ 1.0IBA5-6 wks/100%(20)Saussurea lappaLeaf+ 1.0-2,4-D; 0.5BAP−−−−−−(21)Swertia cordataLeaf/node+ 2.0-2,4-D; 1.0KN−+ 5.0KN; 2.0BAP; 1.0IBA−−+ 3.0IBA5-6 wks/50%(22)Stevia rebaudianaLeaf/node+ 2.0-2,4-D; 0.25KN−+ 0.2BAP−++ 0.1IAA4-5 wks/100%(23)Valeriana wallichiiNode−−+ 2.5KN−++ MS03-4wks/100%*All the medium recipes were formulated using Murashige and Skoog [17] basal medium; **concentration of all the used hormone is expressed in mg/I; ***MS0: MS basal without hormone supplementation.2,4-D: 2,4-diphenoxyacetic acid; 2IP: 2,isopentenyl adenine; AA: ascorbic acid; BAP: 6-benzylamino purine; CH: casein hydrolysate; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; KN: kinetin; NAA:α naphthalene acetic acid; wks: weeks.


## 2.1. Collection of Plant Material

The plant material of twenty-three medicinal plants [16], namely, (1) Elaeocarpus spharicus, (2)Rheum emodi, (3)Indigofera tinctoria, (4)Picrorhiza kurroa, (5)Bergenia ciliata, (6)Lavandula officinalis, (7)Valeriana wallichii, (8)Coleus forskohlii, (9) Gentiana kurroo, (10) Saussurea lappa, (11)Stevia rebaudiana, (12) Acorus calamus, (13) Pyrethrum cinerariaefolium, (14)Aloe vera, (15)Bacopa monnieri, (16)Salvia sclarea, (17)Glycyrrhiza glabra, (18)Swertia cordata, (19)Psoralea corylifolia, (20)Jurinea mollis, (21)Ocimum sanctum, (22)Paris polyphylla, and (23)Papaver somniferum,were collected from different biodiversity zones of India (Figure 1; Table 1). Collections were made in terms of different plant part like rhizome,  tuber,  leafy shoot,  proliferating bud,  rooted plant,  seeds,  and so forth.Table 1
Details of the plants collected, medicinal value and their site of collection forin vitro conservation.
S. no.Plant speciesFamilyPart usedMedicinal valueZone of collectionCoordinates/altitude of site of collection(1)Acorus calamusAraceaeRhizomeAntiasthmatic, antibronchitis, antidiarrhoea, used in dysentery, stomach ache, and constipationWestern Himalayas29°54′36′′N 79°37′46′′E;1,500 mt(2)Aloe veraAloaceaeLeavesPurgative, liver tonic, and used against skin allergiesCentral Highlands21°54′N 83°24′E;  215 mt(3)Bacopa moneeiriScrophulariaceaeWhole plantDiuretic, cardiac, nervine, and tonic, improve intellect, used against asthma, hoarseness, insanity, epilepsy, potent nervous tonic, and antianxiety agentWestern Ghats10°31′N 76°13′E;  2.83 mt(4)Bergenia ciliataSaxifragaceaeWhole plantUsed against pimples, sun burn, stomach ache, muscular pain, and running eyesWestern Himalaya25°34′N 91°53′E;  1,525 mt(5)Coleus forskohliiLabiateaeRootsAntiglaucoma, antiplatelet, bronchospasmolytic, cardiotonic, hypotensive, antiaging, antiallergic, smooth muscle, and arterial relaxant, antiasthmaticWestern Himalaya29°54′36′′N 79°37′46′′E;  1,500 mt(6)Elaeocarpus spehericusElaeocarpaceaeFruits/seed kernelExpectorant, used against nervous weakness, insanity, epilepsy, melancholia, mania, other mental disorders, insomnia, headache, jaundice, hypertension, and feverCentral Highlands21°08′N 81°23′E; 298 mt(7)Gentiana kurrooGentianaceaeRootsAnthelmintic, anti-inflammatory, antiseptic, bitter tonic, cholagogue, emmenagogue, febrifuge, refrigerant, stomachicNortheast Himalaya27°0′N 88°16′E; 5,307 mt(8)Glycyrrhiza glabraFabaceaeRoots/rhizomesExpectorant, diuretic, mild laxative, antiarthritic, antiinflammatory, antibiotic, antiviral, antiulcer, memory stimulant, antitussive, aphrodisiac, antimitotic, estrogenic, antioxidant, anticaries agent, antineoplastic, anticholinergic, antidiureticGangetic plain26°51′N 80°55′E; 128 mt(9)Indigofera tinctoriaFabaceaeLeavesBlood purifier (detoxify) reduces inflammation and alleviate pain antiseptic, stimulantGangetic plain26°51′N 80°55′E; 128 mt(10)Jurinea mollisCompositaeRootEruptions, decoction is given in colic, puerperal feverWestern Himalaya31°58′N 77°06′E; 1220 mt(11)Lavandula officinalisLabiateaeWhole plantKnown for essence, stimulant, sternutatory and tonicSemiarid Zone30.83°N 76.93°E; 656 mt(12)Ocimum sanctumLabiateaeLeaves/seedsDiaphoteric, antiperiodic, stimulating, expectorant, and anticatarrhalGangetic plain26°51′N 80°55′E; 128 mt(13)Papaver somniferumPapaveraceaeCapsuleNarcotic, analgesic, muscle relaxant, and antimicrobial agentGangetic plain26°51′N 80°55′E;128 mt(14)Paris polyphyllaTrilliaceaeRootsAnalgesic, anthelmintic, antiphlogistic, antispasmodic, antitussive, narcoticWestern Himalaya30°25′N 79°20′E; 2,500 mt(15)Picrorrhiza kurroaScrophulariaceaeRoot/stemUsed in fever, gastralgia, as laxative, liver complaints, anaemia. and jaundiceWestern Himalaya29°49′23′′N 79°26′54′′E;2,286 mt(16)Pyrethrum cinerariefoliumCompositaeWhole plantFebrifuge, stimulatory, and stimulant of nervous system, insecticideWestern Himalaya29°54′36′′N 79°37′46′′E;1,500 mt(17)Psoralea corylifoliaPapilionaceaeSeedsAgainst bacteria causing skin diseases, leucoderma, leprosy, and psoriasisWestern Ghats12°58′N 77°34′E; 920 mt(18)Rheum emodiiPolygonaceaeRootsStomach pains, constipation, dysentery, swelling of throat, and tonsillitisWestern Himalaya32°22′N 77°14′E;3,979 mt(19)Salvia sclareaLabiateaeLeaves/flower topsAntidepressant, anticonvulsive, antispasmodic, antiseptic, aphrodisiac, astringent, bactericidal, carminative, deodorant, digestive, emmenagogue, euphoric, hypotensive, nervine, sedative, stomachic, uterine and nerve tonicWestern Himalaya31°58′N 77°06′E; 1,220 mt(20)Saussurea lappaCompositaeRootAntiseptic, disinfectant, bronchitis, asthma, flatulence, and cardiac complaintsNortheast Himalaya27°0′N 88°16′E; 5,307 mt(21)Swertia cordataGentianaceaeWhole plantBlood purifier, febrifuge, anthelmintic, antimalarial, antidiarrhoeaWestern Himalaya29°49′23′′N 79°26′54′′E;2,286 mt(22)Stevia rebaudianaAsteraceaeLeavesSweetener (regulate blood sugar), increase energy level, mental alertness, appetite, vasodilator, skin careWestern Ghats10°31′N 76°13′E; 2.83 mt(23)Valeriana wallichiiValerianaceaeRootsUseful in disease of eye, blood, liver, and spleen enlargement. Also useful in cleaning voice, headacheWestern Himalaya30°27′N 78°05′E; 1,876 mtFigure 1
Different biodiversity zones of Indian subcontinent from where 23 plant species were collected.


## 2.2. Raising Aseptic Cultures and Multiplication

Various explants, namely, leaf, node, bulb, basal disc, and seeds, were first washed thoroughly for 2-3 hours under tap water (seeds left overnight for soaking).  These were then washed with a detergent (cetrimide) which removes dust particles and clears the surface of the explant by removing hairs.  Washed material was then treated with (4%) savlon for 2-3 minutes, followed by exposure to absolute alcohol for 30 seconds, and finally subjected to surface sterilization by 0.1% mercuric chloride. Treatment time varied from plant to plant and from explant to explant. While underground parts and basal discs were treated for 8–10 min, seeds, leaves, and nodes were treated for 1-2, 2-3,  and 4-5 min, respectively. The explants were then thoroughly washed with sterile water (3-4 times). The explants were cut and inoculated on different media combinations.More than 100 media combinations were tested for raising aseptic cultures of all the 23 plant species. According to the morphogenetic response required, medium recipes were made by using basal media [17] fortified with different ranges of  2,4-diphenoxyacetic acid (2,4-D), 2,isopentenyl adenine (2IP), ascorbic acid (AA), 6-benzylamino purine (BAP), casein hydrolysate (CH), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), kinetin (KN), and α naphthalene acetic acid (NAA).  Cultures were incubated at 25 ± 2°C under 8 hours/16 hours photoperiod light 2500–3000 lux intensity and 75–85%  relative humidity.


## 2.3. Synseed Production

The nodal segments and shoot tips of highly proliferating shoot cultures of different plant species were dissected fromin vitro raised mother plant and used as explants for the synseed production. For encapsulation explants dispersed in 4% sodium alginate solution were suspended dropwise in 75 mM calcium chloride solution so that each drop contained single explant. The complexation time was 1 hr, and then the synseeds were thoroughly washed with sterile water and plated on MS basal medium for germination.


## 2.4. Glasshouse Hardening

Before transplantation, shoots were shifted to rooting medium. To establish root proliferation, actively growing shoots were transferred to respective rooting media (Table2). When adequate rooted shoots were obtained, the rooted plantlets (mostly 20–30 days old) were transferred to glasshouse conditions.  For that plantlets were carefully taken out without injuring the roots, thoroughly washed with running tap water to remove the adhered agar by gentle shaking in water filled plastic tray, and planted in a mixture of sterilized soil and vermicompost (1 : 1) in earthen pots. Temperature and humidity was gradually lowered according to the native conditions of the plant.Table 2
Morphogenic level along with the optimised medium and glasshouse hardening status of 23 plant species processed forin vitro conservation.
S. no.Plant speciesExplant usedCallusCultures established at the level ofSuspensionAxillary/multiple shootsFloweringSynseedRooted plantsTime taken/success rate during hardening(1)Acorus calamusNode−−+ 5.0KN*−−+ 3.0IBA3-4 wks/60%(2)Aloe veraBasal disc−−+ 5.0KN**−−+ 1.0IAA3-4 wks/90%(3)Bacopa moneeiriLeaf+ 3.0BAP; 1.0IAA−+ MS0***−++ MS02 wks/100%(4)Berginia ciliataNode−−+ 3.0BAP;  0.1IAA;  40AA−−+ 3.0IAA5-6 wks/30%(5)Coleus forskohliiNode+ 2.5NAA−+ 0.2BAP−−+ 2.5IAA3-4 wks/70%(6)Elaeocarpus spehericusLeaf+ 2.0 NAA; 0.5KN−−−−−−(7)Gentiana kurrooNode−−+ 5.0KN−++ 2.0IAA5-6 wks/50%(8)Glycyrrhiza glabraNode+ 0.5NAA; 0.5-2,4-D; 1000CH+ 0.5NAA; 0.5-2,4-D; 1000CH+ 2.5KN−++ 1.0IAA2-3 wks/80%(9)Indigofera tinctoriaSeeds−−+ 1.0BAP−−+ 0.1IAA5-6 wks/40%(10)Jurinea mollisLeaf+ 3.0BAP; 1.0IAA)−−−−−−(11)Lavandula officinalisSeeds+ 0.5 NAA; 0.5-2,4-D; 0.5 KN+ 0.5NAA; 0.5-2,4-D; 1000CH+ 2.5KN; 0.1NAA−++ 0.5IAA5-6 wks/50%(12)Ocimum sanctumNode−−+ 3.0BAP; 1.0IAA+−+ 1.0IAA3-4 wks/100%(13)Papaver somniferumSeeds+ 1.0-2,4-D; 0.1KN; 40AA+ 2.0–2,4-D; 0.25KN+ 0.5-2IP; 0.1 IAA+++ 1.0IAA5-6 wks/70%(14)Paris polyphyllaBulb−−+ 2.0BAP−−−−(15)Picrorrhiza kurroaLeaf/node+ 1.0-2,4-D; 0.5BAP−+ 5.0KN−−+ 3.0IBA5-6 wks/40%(16)Pyrethrum cinerariefoliumLeaf/node+ 2.0-2,4-D; 1.0KN−+ 5.0KN; 2.0BAP; 1.0IBA−−+ 1.0IBA5-6 wks/60%(17)Psoralea corylifoliaNode−−+ 5.0KN; 2.0BAP; 1.0IBA+−+ 1.0IBA3-4 wks/90%(18)Rheum emodiiBasal disc−+ 3.0BAP; 1.0IAA−−−−(19)Salvia sclareaNode−−+ 5.0KN; 2.0BAP; 1.0IBA−−+ 1.0IBA5-6 wks/100%(20)Saussurea lappaLeaf+ 1.0-2,4-D; 0.5BAP−−−−−−(21)Swertia cordataLeaf/node+ 2.0-2,4-D; 1.0KN−+ 5.0KN; 2.0BAP; 1.0IBA−−+ 3.0IBA5-6 wks/50%(22)Stevia rebaudianaLeaf/node+ 2.0-2,4-D; 0.25KN−+ 0.2BAP−++ 0.1IAA4-5 wks/100%(23)Valeriana wallichiiNode−−+ 2.5KN−++ MS03-4wks/100%*All the medium recipes were formulated using Murashige and Skoog [17] basal medium; **concentration of all the used hormone is expressed in mg/I; ***MS0: MS basal without hormone supplementation.2,4-D: 2,4-diphenoxyacetic acid; 2IP: 2,isopentenyl adenine; AA: ascorbic acid; BAP: 6-benzylamino purine; CH: casein hydrolysate; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; KN: kinetin; NAA:α naphthalene acetic acid; wks: weeks.


## 3. Results

Twenty-three medicinally important plants were successfully establishedin vitro at various morphogenic levels. Details pertaining to optimised media combination and morphogenic levels are given in the Table 2.P. kurroa, L. officinalis, C. forskohlii, S. rebaudiana, P. cinerariefolium, B. moneeiri, G. glabra, S. cordata, and P. somniferumshowed both induction and proliferation of callus as well as shoots. E. spehericus, J. mollis, and S. lappa were established only at the callus level, while I. tinctoria, B. ciliata, V. wallichii, G. kurroo, A. calamus, A. vera, S. sclarea, P. corylifolia, O. sanctum, P. polyphylla, and R. emodiiwere maintained only at shoot level (Figure 2). All the shoot cultures showed first bud break within 20–30 days, but P. polyphylla bulb took longest time of six months to show first bud emergence. Out of these cultures, friable callus of only G. glabra, L. officinalis, and  P. somniferumwere able to give rise to fine suspensions.  Cultures of B. ciliata, P. somniferum,andJ. mollis showed excessive leaching of phenolics in the medium; therefore, they were subcultured frequently. 40–45-day old shoot cultures of  P. somniferum, P. corylifolia,andO. sanctumwere shown in in vitro flowering in 100%, 40%, and 70% cultures, respectively (Figure 2).  In P. Somniferum, terminal white flowers were obtained which were either pentamerous or tetramerous. The five-lobed persistent stigma was prominent. Yellow coloured anthers were elliptical, ovoid, and numerous. Characteristic unilocular capsule formation was observed within 15 days of flower initiation. Similarly in P. corylifoliacapitulate cylindrical inflorescence with white purplish flowers having unequal corolla, inserted stamens, and feathered stigma was observed. In case of O. sanctum, pentamerous flowers with white petals, four stamens surrounded single style, arranged in verticillaster manner were obtained. There were six flowers in each whorl. The shoots of B. moneeiri, G. glabra, S. rebaudiana, V. wallichii, G. kurroo, L. officinalis, and P. somniferum were highly proliferating; therefore, nodal segments of above plants were easily encapsulated to produce synthetic seeds. B. moneeiri, S. rebaudiana, V. wallichii, L. officinalis, and P. somniferum showed 100% germination rate of synseeds, while only 50% and 70% germination was observed in G. glabraandG. kurroo, respectively. Rooting was observed in all the plant species except R. emodii and P. polyphylla. Rooted plants were successfully transferred to the glasshouse conditions with a success rate of 30–60% (B. ciliata, P. kurroa, I. tinctoria, G. kurroo, L. officinalis, S. cordata, A. calamus, andP. cinerariefolium,), 70–90%  (C. forskohlii, P. somniferum, G. glabra, A. vera, andP. corylifolia), and 100% (B. moneeiri, O. sanctum, S. sclarea, S. rebaudiana, andV. wallichii).In vitro established cultures of 23 plant species. (a)Elaeocarpus spehericus, (b)Rheum emodii, (c)Indigofera tinctoria, (d)Picrorrhiza kurroa, (e)Bergenia ciliata, (f)Lavandula officinalis, (g)Valeriana wallichii, (h)Coleus forskohlii, (i) Gentiana kurroo, (j) Saussurea lappa, (k)Stevia rebaudiana, (l) Acorus calamus, (m) Pyrethrum cinerariefolium, (n)Aloe vera, (o)Bacopa moneeiri, (p)Salvia sclarea, (q)Glycyrrhiza glabra, (r)Swertia cordata, (s)Psoralea corylifolia, (t)Jurinea mollis, (u)Ocimum sanctum, (v)Paris polyphylla, (w)Papaver somniferum, and (x)-(y) synseeds of Bacopa moneeiri.  Arrows showing in vitro flowering.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)(o)(p)(q)(r)(s)(t)(u)(v)(w)(x)(y)


## 4. Discussion

India has been in focus for its high biodiversity, and this region has been a priority for leading conservation agencies of the world. World Wildlife Fund (WWF) has identified the entire Eastern Himalayas as a priority Global 200 Ecoregion while Conservation International has up scaled the Eastern Himalaya Hotspot [4, 7]. The region is rich in medicinal plants and many other rare and endangered taxa. Its high endemism in higher plants has qualified it to be a biodiversity “hotspot” [18]. Presently biological diversity of India is facing various threats like deforestation, degradation, agriculture, commercial plantations, and replacement of indigenous species with exotics [10]. Encroachment of forestland is a serious threat to forests and its conservation. It is estimated that 60% of the domestic herbivore population graze in the forest. The grazing causes soil compaction and heavy damage to the forest plantations and natural regeneration. Forest fires are also common and frequent affecting about 20% of the total forest area. Ex situ conservation of plants through in vitro technique is an ideal approach to combat above threats and restore biodiversity. In vitro techniques have been found to be useful in the propagation of large number of threatened species [11].  The first constrain for in vitro culture is the contamination which can be exogenous or endogenous. Exogenous contamination can be removed by thorough detergent wash while contamination from the internal sources can be potentially serious as many plant species harbour endophytic bacteria and fungi. In our study such condition was encountered in B. ciliata, P. kurroa, G. kurroo, S. cordata, and A. calamus and was successfully overcome by the incorporation of antibiotics (Ampicillin/Chloramphenicol 100 mg/I) in the respective media composition. Another major obstacle found during the establishment of the cultures was medium browning and excessive leaching in case of B. ciliata, P. somniferum, andJ. mollis. Supplementation of 40 mg/I ascorbic acid in the respective media ofB. ciliata, P. somniferum was able to control the leeching and browning up to certain extent, but frequent subculturing was the only feasible option observed in all the three plant species. The flowering process is one of the critical events in the life of a plant. This process involves the switch from vegetative stage to reproductive stage of growth and is believed to be regulated by both internal and external factors [19].   In vitro flowering observed in P. somniferum, P. corylifolia,  andO. sanctum was highly significant as it is considered to be a convenient tool to study specific aspects of flowering like floral initiation, floral organ development, and floral senescence. Encapsulation technique used in the present study is very promising for conservation purposes as the protection provided to the plant material by encapsulation could increase its resistance to dehydration and low temperature, thus opening new possibilities for medium-term storage [20].  Lastly, 18 plant species were successfully hardened under the glasshouse conditions. During the acclimatization of different plant species in the glasshouse, two different stages were observed. In the first stage, plants adapted to the new environment with slow shoot and root growth. This stage is comparatively longer, but time period varies with the species to species. In the later stage, plants showed fast growth phase which was almost equal in most of the species.The present microcloning/micropropagation protocols of 23 important medicinal plants will provide backup of collected germplasmin vitro at various levels of morphogenesis to supplement field gene bank. This tissue culture interface provides genetic enhancement of collected germplasm in terms of disease clearance and clonal propagation.


## 5. Conclusion

Medicinal plants are under the threat of overexploitation and biodiversity depletion. There is urgent need of theirex situ conservation. Collection and in vitro conservation of 23 pharmaceutically important plants in the present study opens fresh avenues towards the conservation and resource management of the overexploited medicinal plants.



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*Source: 929650-2012-04-19.xml*