Supervisors:
Prof. L.M.S. Palni, Senior Scientific Advisor - Biotechnology, Government of Uttaranchal
Summary: The Indian Himalayan Region (IHR) is home to a variety of important medicinal plant species. These medicinal plants have been in use since Vedic times, for the cure of various ailments. A number of biologically active compounds have been identified and new formulations based on these compounds are being produced at a fast rate. Due to recent increase in the demand of herbal drugs in India and abroad, these are being harvested in large quantities, mostly by illegal means, well over the regeneration capacity, to meet the demand of raw materials by the pharmaceutical firms.
The medicinal plants of the alpine Himalaya are subjected to extreme climatic conditions and the actual period of growth is confined to a few summer months. The seed production as well as seed viability is poor and most of these plants, being herbaceous in nature, are prone to grazing and trampling by the cattle. As a result of overexploitation, unscientific harvesting and lack of organized cultivation, many species have already been listed under the threatened or endangered category.
Some highly valued species of the alpine region, including Picrorhiza kurrooa, Aconitum heterophyllum, Aconitum balfourii, Aconitum violaceum and Podophyllum hexandrum are among the over harvested species from the wilds. These 5 species have been listed under the threatened category, as per IUCN criteria. Due to their importance in traditional system of medicine, threatened status, and high commercial demand in the herbal market, these were selected as target species for conducting seed germination studies, clonal propagation, agronomic trials and active content estimation in the present study. The methodology, results and observations have been described separately in individual (4) chapters, along with conclusions and recommendations.
Chapter 1. This section consists of general introduction and description of the selected (target) species. A general account of work carried out by earlier workers on these species in particular, and on medicinal plants in general, has been reviewed in the context to the objectives of the present study. Habitat conditions, traditional uses, present market price and the active principles for the target species have been described. A brief taxonomic description of each species is also presented. The difficult habitat conditions, threatened status and high market price of these species indicates the need for conducting studies related to mass multiplication, agronomy and subsequent estimation of active contents.
Chapter 2. Germination trials, under the laboratory conditions, were conducted in order to assess the effects of certain plant growth regulators, nitrogenous compounds and temperature conditions on seed germination in P. kurrooa and A. violaceum. Seeds of P. kurrooa, from four different populations, and that of A. violaceum, from a single location, were collected during the months of OctoberNovember. Germination trials using freshly harvested seeds, and after storage of seeds, were carried out for both the species at 20 șC in the dark. Germination of freshly harvested seeds of P. kurrooa from Phurkia population (Pindari area, Kumaun Himalaya) was found to be low as compared to more than 50% germination of seeds collected from three other populations. Above 50% germination of freshly collected seeds of A. violaceum was also recorded. Storage of seeds up to 3 months at low temperature (4 șC) was found to improve the percent germination in both the species; it was however, found to decrease during further storage. Seed treatment with GA3 was found to stimulate and synchronize uniform germination in P. kurrooa. Enhancement in germination was also found in seeds treated with two nitrogenous compounds namely, CH4N2S and KNO3. Among various PGSs applied, only GA3 treatment was found to improve germination of low temperature stored seeds in A. violaceum. Incubation of seeds at 20 șC (germination temperature) was found to improve germination in both the species.
On the basis of observations of the present study, it can be concluded that seed germination can be improved by simple seed treatments with PGSs and low cost nitrogenous compounds. These methods can be applied for propagation of materials and in the establishment of nurseries of these high value medicinal plants.
Chapter 3. Clonal propagation through rooting of runner cuttings was carried out in P. kurrooa. Small runner segments (with 2-3 nodes in each) were easily rooted in high humidity chamber inside a greenhouse. Pretreatment of the basal portions of runner cuttings with IBA (50 mM) resulted in promotion of rooting. Treatment of cuttings with NAA alone or in combination with BAP also enhanced rooting by more than 80%. Above 50% untreated cuttings were also found to root.
Tissue culture studies were carried in P. kurrooa and A. violaceum and complete micropropagation protocols were developed, including hardening and field transfer of plantlets. Shoot multiplication in P. kurrooa was obtained through nodal explants, taken from field grown plants, and juvenile explants (cotyledonary nodes and shoot tips) derived from in vitro germinated seeds. Higher shoot production per explant (12 shoots) was obtained through shoot tip culture on MS medium supplemented with BAP. These shoots during further subcultures were grown on MS medium variously supplemented with BAP, Kn or 2-iP and NAA. Higher multiplication rate was observed on medium containing 1.0 mm Kn. Different light sources (PAR and CFL) and two gelling agents (agar and phytagel) were evaluated in terms of shoot production, shoot quality, chlorophyll content and rooting of microshoots. Rooting of microshoots was obtained on MS medium supplemented with IBA, NAA or IAA. Cent percent rooting, without callus formation at the basal end of shoots, was obtained on IBA and NAA supplemented medium. Micropropagated plants were hardened under greenhouse (25 ± 3 °C, 85% RH) conditions. The high mortality of plants encountered during initial weeks of transfer, due to fungal attack was successfully controlled by inoculation with two known antagonistic bacterial strains (biological hardening). More than 90% survival of plants was observed in inoculated soils.
After a total period of 13 months, following hardening and growth under field conditions, the micropropagated plants were evaluated in terms of growth, dry mass production and leaf characteristic. The morphological, physiological and anatomical details of micropropagated plants were found to be similar to those in seed raised plants of similar age.
In vitro shoot regeneration and multiplication was achieved in A. violaceum through callus formation using shoot tip explants. Shoot tips were harvested from greenhouse maintained plants from the natural population. Callus was obtained on MS medium containing 1.0 mm BAP and 0.5 mm NAA. Shoot bud formation and subsequent shoot regeneration was obtained on MS basal medium. Multiplication of shoots was found to be better on MS medium without any plant growth regulator, and spontaneous rooting of microshoots was also observed on this medium. The rooted shoots were hardened under greenhouse conditions. Performance evaluation of 12-months-old micropropagated and seed raised plants of similar age was carried out. The morphological and anatomical features of micropropagated plants were found to be similar to that of seed raised plants. A normal tuber formation and higher tuber dry mass was observed in micropropagated plants compared to seeds raised plants. Cytological examination also revealed similar chromosome number (2n = 16) in both types of plants.
Chapter 4. Agronomic trials were conducted in four species, namely P. kurrooa, A. heterophyllum, A. balfourii and P. hexandrum at an experimental field in a high altitude village (Khaljhuni, 2450 m amsl). Growth behaviour and biomass were estimated for two successive seasons following soil amendments with three levels of combined NPK treatments and a single dose of FYM. The plants used were cutting raised in case of P. kurrooa, seedling raised in case of A. heterophyllum, from daughter tubers in case of A. balfourii, and from rhizome segments in case of P. hexandrum. The higher levels of combined NPK treatment (120:60:40 kg/ha) was found to improve overall vegetative growth in all the species; aboveground and belowground biomass was also affected in this treatment. Except for P. hexandrum, the biomass of all the three species was found to be higher in response to highest level of NPK. The next level of NPK fertilizer treatment was also found to increase growth and biomass in some plots. In general application of FYM (2 kg/ha) was not found to exert any effect; except in P. hexandrum the treatment was found to improve the belowground biomass.
Three well-known GA inhibitors, namely ancymidol, CCC and uniconazole were found to reduce the vegetative growth (shoot height) of A. violaceum seedlings grown inside a greenhouse at Kosi-Katarmal. Maximum inhibition of aerial parts was observed in response to uniconazole treatment (100 mm) without any negative effect on overall yield. The tuber size and biomass was higher in CCC treated seedlings.
Aconitine content in tubers of A. heterophyllum and aconitine and pseudoaconitine content in tubers of A. balfourii was quantified by high performance liquid chromatography (HPLC) analyses. Aconitine content in A. heterophyllum tubers was found to be sufficiently higher. It was higher than the content earlier reported from natural populations. No significant effect of applied NPK and FYM treatments was observed in the second year of harvest in respect of both aconitine and pseudoaconitine content. Podophyllotoxin content in the rhizome and roots of P. hexandrum was also quantified in samples collected from agronomic trials. Higher podophyllotoxin content was recorded in the roots of plants from fertilizer treated as well as in control plants. The podophyllotoxin content was slightly higher in response to some NPK treatments, but the same was reduced following the second season of growth.
Chapter 5. It is evident from the observations of the present study that the high altitude villages of subalpine ranges in this part of Himalaya provided ample, suitable locations for the cultivation of medicinal herbs. The growth and biomass can be improved by soil amendments with fertilizers. For more information on this aspect, extensive studies conducted over a large area are needed. Cultivation of these highly valued medicinal herbs is the only practical and effective means of improving the conservation status of these plants. This can also be a major source of increasing the economic returns, from a limited land, for the betterment of local inhabitants and sustainable development of region.
Bhuwan Chandra
Subject : Botany
Ph. D - Botany Kumaun University, Nainital
Correspondence Address:
Bhuwan Chandra, EPB/GBPIHED, Kosi-Katarmal, Almora,Uttaranchal-263 643
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