Ethnobotany and Ethnomedicinal Uses , Chromosomal Status and Natural Propagation of Some Plants of Lahaul-Spiti and Adjoining Hills

The present study documented the ethnobotanical and medicinal uses of plants from an ecologically fragile cold desert area of Lahaul-Spiti (Himachal Pradesh, India). Local people use plants for curing the stomach troubles, pain reliever, cough, gastric disorders, and aphrodisiac and other household purposes. In addition, chromosome numbers, male meiosis, and natural propagation were also investigated in these ethnobotanically used plants. Present investigations also form the basis for exploitation of intraspecific chromosomal variation/new cytotypes recorded in some of the presently studied species to detect biochemical diversity in the medicinally important plants. For documentation of ethnobotanical information, personal observations and interviews were conducted withmedicinemen, hakims, farmers, shepherds, local healers, and old aged people.This study identified 40 plant species under 33 genera belonging to 17 families which have been used locally for curing various diseases and other purposes. All the chromosome counts are new to the study area. On worldwide basis, meiotic chromosome counts of n = 14 and n = 8 in Rosularia alpestris and Corydalis govaniana, respectively, are the first ever reports. The present study indicates that the people of the area possess good knowledge about the different uses of plants in the area. It has been noticed that due to the lack of interest among younger generations in the preservation of invaluable ethnic knowledge, there is every possible chance of losing such a rich heritage of knowledge. It is very urgent to conserve such invaluable ethnic knowledge before it gets lost.


Introduction
Present study was conducted in Lahaul-Spiti, a cold and desolate alpine region in northwest Himalayas which is known for its seclusion, Buddhist culture, hostile climate, and unexplored, formidable, and breathtaking scenic beauty of the high snow clad mountains.It is a part of the Indian cold deserts which is situated between 31 ∘ 44  57  and 32 ∘ 59  57  N latitudes and between 76 ∘ 29  46  and 78 ∘ 41  34  E longitudes.Climatic conditions are typical of dry temperate and alpine zones.The vegetation which is mainly composed of perennial herbs and prostrate shrubs survives extreme variation in temperature including months buried under several feet of snow.Using plants for different purposes and in curing and healing is as old as man himself [1].It was very well known to primitive people that plants contain beneficial and medicinal properties which they used in some form or the other.Even in the ancient time, people classified plants on the basis of their medicinal uses.No comprehensive attempt has been made so far to gather ethnobotanical information and to assess the cytological diversity and natural propagation of the plants growing in the higher altitude and cold desert regions of Lahaul-Spiti.Presently an attempt has been made to collect information on the ethnobotanical uses and to examine the cytomorphological diversity and natural propagation in the species of Lahaul-Spiti and adjoining high hills.
1.1.Topography and Vegetation.The topography of the area is entirely different from the adjoining regions of northwest Indian Himalayas.The cold desert plants of Lahaul-Spiti show a wide range of habitat diversity which includes alpine pastures and slopes, moist shady and exposed places, forests and forest undergrowth, water courses, scree slopes, rock crevices, rocks/boulders, roadsides, riverbeds, and sandy   2).For documentation of ethnobotanical information, personal observations and interviews were conducted with medicine men, hakims, farmers, shepherds, local healers, and old aged people.Plant identification was done by consulting the Flora of Lahaul-Spiti [2], the Flora of Kullu district [3], and Flora of Chamba district [4].Besides, the plant accessions were also compared to the samples lying in the Herbaria of the Department of Botany, Punjabi University, Patiala (PUN), Botanical Survey of India (BSI), and Forest Research Institute (FRI) at Dehra Dun.Voucher specimens of the cytologically worked-out specimens were deposited in the Herbarium, Department of Botany, Punjabi University, Patiala (PUN).For male meiotic studies, floral buds of suitable sizes from the wild plants were fixed in Carnoy's fixative (mixture of ethanol, chloroform, and glacial acetic acid in volume ratio of 6 : 3 : 1) for 24 h and preserved in 70% ethanol in a refrigerator until analysed.For chromosome counts and detailed meiotic behavior in pollen mother cells (PMCs) at early prophase-I, metaphase-I (MI), anaphase-I/II (AI/II), telophases-I/II (TI/II), and sporad stage developing anthers were squashed in 1% acetocarmine.Pollen fertility was estimated through stainability tests by smearing mature anthers from different flowers in glycerol-acetocarmine mixture (1 : 1) and aniline blue dye (1%).Best plates of chromosome counts, meiotic abnormalities, sporads, and pollen grains were photographed from the temporary mounts with digital imaging system of Nikon Eclipse 80i microscope and Leica QWin.Field photographs of plants and landscape were taken with Nikon Coolpix L3 camera.

Abnormal Cytological Events during Male Meiosis and
Pollen Fertility.Besides new chromosome reports and additional cytotypes, in some of the species meiosis was abnormal and was characterized by the presence of multivalents and/or univalents, impaired synapsis, cytomixis, and its associated meiotic aberrations such as laggards and bridges, chromatin stickiness, pycnotic chromatin, irregular distribution of chromosomes, and micronuclei at telophases/sporads consequently leading to abnormal microsporogenesis and variable amount of pollen sterility (Figures 5(h)-5(p)).Among the abnormal taxa, 12x cytotype of P. atrosanguinea Lodd.var.argyrophylla (Wall.ex Lehm.)Griers.and Long is an imbalanced polyploid.Owing to the presence of high chromosome number (2 = 84), it depicted a

Discussion
A steady stream of papers over the years have recognized the fact that the reproductive stage in the life cycle of plants is highly sensitive to different abiotic stresses [5][6][7][8] especially the low temperature conditions [9][10][11][12][13][14][15][16].It has been observed that when plants of this area are about to enter the flowering stage, the temperature is very low (sub-zero to 15 ∘ C) which seems to disrupt the meiotic process by causing various meiotic abnormalities and consequently reduced viability of gametes.Ultimately these events hinder natural propagation of plants through sexual means.
Natural propagation observations revealed that majority of the species are perennial and propagate through asexual  means such as rootstocks, offsets, root suckers, and stolons.Thus, it is assumed that in order to survive the harsh environmental conditions and gametic sterility caused by irregular meiotic events, the plants of the area have adopted alternate mechanism of reproduction through vegetative means.Vegetative means of propagation are important for species like Meconopsis aculeata Royle, Potentilla atrosanguinea Lodd.var.argyrophylla (Wall.ex Lehm.)Griers.and Long, Bergenia stracheyi Engl., Rosularia alpestris (Kar and Kir) Boriss., and so forth, where high pollen grain sterility have been observed.Occurrence of sterility on female side in the species may pose serious problem for species existence, especially, which are rare and engendered and are overexploited for medicinal purposes.Problem becomes more serious in species like Bergenia stracheyi, Engl.and Rosularia alpestris (Kar and Kir) Boriss.which propagate through vegetative means, and perennating parts are directly collected from the wild sources.Till now these species have not been included in any of the local cultivation programme.
To date numerous valuable herbal drugs have been discovered by following the knowledge of ancient folk healers [17].The medicinal plants used to cure various diseases by the traditional healers are gradually becoming extinct.The two factors which have been identified as major threats to the floristic diversity are the overexploitation and habitat [18][19][20][21].Majority of the plants (Aconitum heterophyllum Wall.ex Royle, Angelica glauca Edgew., Biebersteinia odora Steph.ex Fish., Bergenia stracheyi Engl., Delphinium denudatum Wall.ex Hook.f. and Thomson, Indigofera gerardiana Wall.ex Baker, Meconopsis aculeata Royle, Podophyllum hexandrum Royle, and Spiraea canescens D. Don) which have been studied presently are restricted to specific pockets with very low population density and belong to rare, critically endangered, endangered, vulnerable, threatened, and newly threatened category because of the fragile ecological conditions of this Himalayan region [20][21][22].Some of the other factors which can also lead to the extinction of the local valuable herbs include agricultural expansion, heavy grazing, road building, increasing entry of tourists, and transport vehicles.So to study and examine different aspects of survival of the plants of the area in their natural habitats will be of colossal significance.Thus it becomes very urgent to collect information about the plants growing in such ecologically fragile region before it gets too late to respond.Immediate steps must be taken up on priority basis to preserve the ethnic knowledge and the plants of the region.The gradual exposure of younger generations to western civilization and limited employment opportunities are important threats that may lead to serious losses of traditional knowledge, and it will not be surprising that after a decade or so nobody will be able to tell about the plants that were once used traditionally for ethnobotanical and medicinal purposes.In view of the above points it is suggested that an institute dealing with the conservation of plants and their traditional uses must be set up in the region.The benefits arising from ethnic knowledge must be shared among the local people and employment must be provided to local youth.Present investigations also form the basis for exploitation of intraspecific chromosomal variation/new cytotypes recorded in some of the presently studied species to detect biochemical diversity in these medicinally important plants.
Cytological Studies, Identification of Plants, and Male Meiotic Chromosome Counts.As the valley remains cut off from other parts of country for more than six months due to heavy snow fall, observations on ethnobotanical information and natural propagation and collection of material for cytological studies were made between June and October in the years 2006-2009.During the intensive cytomorphological and ethnobotanical surveys, 40 plant species used by the local people of Lahaul-Spiti had been collected from different localities (Figure

Figure 3 :Figure 4 :
Figure 3: (a) Biebersteinia odora plants in a group, coming out of withered remains of stems.(b) Cicer microphyllum growing on rock.(c) An individual of Meconopsis aculeata growing among the boulders.(d) Podophyllum hexandrum.(e) Aconitum heterophyllum with greenish blue flowers having purple veins growing on moist slopes.(f) Caltha palustris with bright yellow flowers and large reniform leaves growing near water.(g) A shrub of Rosa webbiana with pink flowers.

Figure 6 :
Figure 6: Natural propagation.(a) A plant grown from old rootstock (arrowed) in Astragalus rhizanthus.(b) New plants of Bergenia stracheyi coming out of the rootstocks (encircled) underlying withered leaves.(c) B. stracheyi plants in full bloom with white pinkish flowers.(d) A new sprout can be seen growing on the old rootstock (arrowed) of B. stracheyi.
Based on the information gathered from different sources, the local people used a good number of plant species for curing the stomach troubles, pain reliever, cough, gastric disorders, and aphrodisiac; as fodder and fuel; to obtain different dyes; and in religious ceremonies and as offerings to deities (Figures 4(a)-4(d)).

Table 1 :
Family, species and its vernacular name, meiotic chromosome number, ploidy level, locality and altitude, habit and habitat, natural regeneration, flowering and fruiting period, plant part used and ethnobotanical uses in the wild plants of Lahaul-Spiti, and adjoining high altitude hills.