Indian Journal of Traditional Knowledge Vol 18 (1), January 2019, pp 68-75

Nepenthes khasiana Hook f., an endangered tropical pitcher of

,1,+ 2 1 1 3 Soibam Purnima Devi* , Suman Kumaria , Ph. Ranjit Sharma , Pramesh Khoyumthem & Pramod Tandon 1Department of Genetics & Plant Breeding, Central Agricultural University, Imphal 795 004, India. 2Department of Botany, North-Eastern Hill University, Shillong 793 022, India. 3Biotech Park, Lucknow 226 021, India. E-mail: [email protected]

Received 30 July 2018; revised 17 September 2018

Nepenthes khasiana Hook f., is the only representative member of the Nepenthes found in India. The species has great ethnomedicinal importance in addition to its ornamental value. It is used for treatment of several diseases by the local people of . The plant is also characterized by synthesis of bioactive compound called naphthoquinones which is a potent antifungal agent. However, it has become threatened in its natural habitat due to several anthropogenic activities. N. khasiana has been included in the Appendix-I of CITES (Convention on International Trade in of Wild Fauna and Flora). The potential for long term survival of this species in the wild is uncertain, but will surely depend upon the continuing efforts of the local communities to preserve its habitats. The present review highlights the significance of the species and also elucidates the recent findings on the species on various aspects.

Keywords Conservation, Endangered, Insectivorous, Nepenthes khasiana IPC Code: Int. Cl.18 A01D 91/00, A61K 38/00, A01G 13/00, A01C 11/04

In course of evolution, some of the have Droseraceae, Nepenthaceae and . acquired special characteristics in order to adapt to the They have unique structural specialized organs such harsh environmental conditions. Plants inhabiting as pitcher-like leaves or analogous leaf arrangements the nutrient limited environments are specialized to trap insects 3,7. for trapping a wide group of insects in order to The genus Nepenthes popularly known as tropical supplement a part of their nutrition and hence are pitcher plants, belonging to the monotypic family popularly known as insectivorous plants. This insects- Nepenthaceae is one of the largest genus among the trapping mechanism might have evolved as an insectivorous plants. It comprises about 134 species adaptation to grow in nutrient deficient acidic soils, so including numerous natural and many cultivated as to provide a complemental source of nutrients, hybrids8. Nepenthes species certainly attract and kill especially nitrogen1,2. Insectivorous plants are, their prey through active production of attractive therefore, among the curiosities of nature being colours, sugary nectar, and even sweet scents. The different from the normal plants in their mode of plants primarily gain nitrogen and phosphorus nutrition. These plants are generally poor competitors through the trapped insects to supplement their for light and nutrients as compared with normal nutrient requirements for growth, given that these soil plants3,4,5. These plants have fascinated evolutionary nutrients are typically lacking. The most frequent prey ecologists, botanists and horticulturists for centuries. belongs to abundant and diverse group of arthropods, Darwin provided the evidence for carnivory in with ants and other insects. Most of its species are several genera for the first time6. Carnivorous plants characterized by an ontogenetic pitcher dimorphism represent members of five orders including both with young rosette, self-supporting plants exhibiting monocotyledons and eudicotyledons: , terrestrial pitchers of the “lower” type and older climbing plants exhibiting aerial pitchers of the Oxalidales, Ericales, and Poales. Over 600 9,10 species of carnivorous plants belonging to 9 families “upper” type . are found growing all over the world. Insectivorous Indo-Malaysia is considered as the center of plants of India belong to mainly three families: evolution of the genus Nepenthes. The genus is —————— mostly distributed in the Malay Archipelago with the *Corresponding author greatest diversity in Borneo and Sumatra and the DEVI et al.: NEPENTHES KHASIANA HOOK F., AN ENDANGERED TROPICAL PITCHER PLANT OF INDIA 69

Philippines with many endemic species8. It is also Habit found in Madagascar (N. madagascariensis and N. khasiana is a climbing under shrub which N. masoalensis), the Seychelles (N. pervillei), Sri ranges from a few centimeters to several meters in Lanka (N. distillatoria), India (N. khasiana), Australia height (Fig. 1a). Two heights of plant are noticeable, (N. mirabilis, N. rowanae, and N. tenax) and New dwarf plant which grows on rocky or sandy pockets Caledonia (N. vieillardii) in the South east. attaining a height of 10-15 cm only and tall plant N. mirabilis is the most widely distributed species in which grows along hill streams or on moist soil strata the genus, ranging from Indo-China to Australia7,11. of substantial depth, straggling up on to small trees or Many of the species occur in hot and humid lowland large shrubs with the help of tendrils and attaining a areas, but most of the species are found growing in height of 15-20 m. The plant has superficial root the tropical regions receiving warm and humid system penetrating only a few centimeters into the climate. Nepenthes species usually grow in acidic soil. The stem is cylindrical, green in colour when soils composed of peat, white sand, sandstone, or young and ultimately turns brown in the older parts. volcanic soils. A few species thrive in soils with high The leaf is very interesting and of great morphological heavy metals (N. rajah) and in sandy beaches importance as parts of it undergoes different (N. albomarginata)7. Nevertheless, the present review modifications to carry out different functions. The focuses on the significance of the species Nepenthes midrib of the leaf extends from the tip and modify khasiana Hook f., the only representative member of into attractive pitchers to catch insects so as to the pitcher plant in India which is one of the best balance the limited nutrients acquired from the soil studied Nepenthes. Also, this review highlights the (Fig. 1b)16. Flowering season is from June to October. recent advancement in research on this rare species The plant is dioecious bearing male and female from various aspects so as to provide a remarkable flowers on separate plants. The inflorescence is a opportunity for further investigations in future.

Nepenthes khasiana, an endemic insectivorous plant of Meghalaya, India Nepenthes khasiana Hook f., being an insectivorous plant is one of the botanical curiosities of nature belonging to the family Nepenthaceae with polyploid chromosome number of 2n = 8012. The species has been named after the Khasi Hills of the state of Meghalaya, India. It is a scandent insectivorous shrub of the tropical and subtropical climatic regions. The local communities of Meghalaya call the plant by different names viz., tiew-rakot in Khasi, kset phare in Jaintias, memang- koksi in Garo which means demon-flower or devouring- plant.

Distribution The plant species has a very localized distribution. It is endemic to Meghalaya and is found growing from West Khasi Hills to East Khasi Hills, Jaintia Hills, East to West and South Garo Hills from 1000 to 1500 m altitude13. It occurs in the Jarain area of Jaintia Hills and the Baghmara, Bandari, Chokpot area of Garo Hills, and few more localities, such as Nongstoin, Mukthapur, Bhagmara, Lawbah and Sonapahar in Meghalaya14. It is believed that the species represents ancient endemic remnants of older Fig. 1 — (a) Nepenthes khasiana in its natural habitat, (b) Well flora which usually occur in land masses of geological developed pitchers, (c) Male inflorescence, (d) Female 15 inflorescence, (e) Mature capsules, (f) Chromosome complement antiquity (Paleoendemics) . in N. khasiana, 2n = 80 (Bar = 10 μm) 70 INDIAN J TRADIT KNOWLE, JANUARY 2019

raceme consisting of 2-flowered cymes approximately strategy for identification of more useful bioactive 25-60 cm long14. The male inflorescence is twice as compounds32,33. In addition to its ethno-medicinal long and denser compared to the female inflorescence values, N. khasiana is also in great demand for its (Fig. 1c,d). Multicellular trichomes are present on the ornamental value on account of its fascinating abaxial side of perianth lobes of both male and female pitchers. The species is, therefore, being collected flowers17. The anthers are bisporangiate in which the from the wild and sold at the rate of ₹ 40-50 per plant archesporial initials are arranged in uniseriate rows of in the markets of Meghalaya13. cells18. Pollen grains are non-aperturate with thick spinuliferous sexine which are dispersed in tetrads N. khasiana as a source of bioactive compound, with a sparsely echinate sculpture. The size of the naphthoquinone tetrad (30.7 μm) is closer to two Bornean species such N. khasiana is characterized by the synthesis of as N. rafflesiana and N. mirabilis (30.5 and 31.0 μm bioactive compounds called naphthoquinones which 19 possess anti-malarial, antimicrobial, antifungal and respectively) . The fruits are elongated capsules 34-36 ranging from 20 to 25 mm long (Fig. 1e). The species anticancer activities . Droserone and its methylated is also characterized by high polyploid chromosome derivative 2-methyl-3-hydroxy- 5-methoxy-1, 4- number (2n=80) (Fig. 1f). naphthoquinone (5-O-methyldroserone) were identified as the natural naphthoquinone synthesized in closed Ethnomedicinal values of N. khasiana pitchers of N. khasiana following chitin injection N. khasiana is one of the most collected medicinal which mimic the prey capturing conditions in the plant species in Meghalaya. It is traditionally used by insect trap37. In addition to these naphthoquinones, different indigenous communities of Meghalaya for presence of plumbagin in the root of N. khasiana was treatment of various ailments20. The fluid of the detected and plumbagin content in potted plants of unopened pitcher is used by local Khasis and Garos N. khasiana with limited growth exhibited higher as an eye drop for redness, itching, cataract, night levels of plumbagin accumulation (1.9260.02 %, dry blindness14. The fluid is also taken for stomach wt.) as compared with field plants (1.3360.02 %, dry ailments and female diseases21,22. The unopened wt.) which was the highest found in any natural pitcher with its contents is made into a paste and source38. However, plumbagin was never detected in applied for various skin diseases, including leprosy23. the pitcher fluid whereas both its derivatives viz., The local herbalists of Khasi and Jaintia Hills droserone and 5-O-methyldroserone were found only prescribe the fluid of the pitcher for the treatment of in the pitcher fluid on chitin induction or prey capture. diabetes and painful urination24,25. The liquid present Chitin induction enhanced plumbagin levels in roots in the pitcher is also consumed in the morning as a of potted plants (2.1760.02 %, dry wt.) as well as field digestive tonic by local people of Meghalaya and the plants (3.3060.21 %, dry wt.) of N. khasiana. Chitin fine powder of N. khasiana pitchers mixed with water induction might act as insect exoskeleton which is taken orally in the treatment of cholera26,27. The induces the biosynthesis of naphthoquinone39. The liquid inside the closed pitcher is also used as ear three naphthoquinones, plumbagin, droserone, and 5- drop27. Pitcher extract of N. khasiana has been O-methyl droserone, act as molecular triggers in prey reported to reduce the level of glucose and lipid capture and digestion in N. khasiana. significantly in rats after oral administration of The pitcher liquid containing naphthoquinones was methanolic abstract of the pitchers confirming the reported to possess antifungal effect on a wide range traditional use of this species in the treatment of of both plant and human fungal pathogens. Plumbagin diabetes28. N. khasiana can also be used as natural is the most efficient secondary metabolite isolated so protective against liver damage as the methanolic far from carnivorous plants. However, its application extract of its leaves showed potent hepatoprotective29. is limited due to its high cytotoxicity and relatively Staining properties of plumbagin, a kind of chemical low therapeutic selectivity40. On the other hand, the naphthoquinone present in the leaves of the genus minimum concentrations of droserone and Me Nepenthes has also been studied30. Naphthoquinones droserone required for effective inhibition of human are allelopathic substances and exhibit high biological fungal pathogens viz., Candida albicans CBS 562, activities such as insecticidal, molluscidal, antifeedant C. albican mas, C. krusei, C. glabrata, Aspergillus and antifungal activities31. The potentialities of flavus, A. niger, A. fumigatus were significantly lower ethnomedicinal studies can provide us an effective than those causing cytotoxicity in cells of the human DEVI et al.: NEPENTHES KHASIANA HOOK F., AN ENDANGERED TROPICAL PITCHER PLANT OF INDIA 71

embryonic kidney cell line, 293T36. These bioactive cultured on ½ MS medium fortified with 1.5 mg/L compounds were also found to inhibit the growth NAA. It was also observed that 60% of the plants as well as spore germination of the common plant introduced in their natural habitat were survived. pathogens such as Rhizoctonia. solani, Fusarium Subsequently, a rapid regeneration protocol oxysporum, and Mycosphaerella graminicola suggesting through multiple shoot induction in axenic seedling a general antifungal effect. Therefore, these derived nodal segment culture of N. khasiana was naphthoquinone derivatives may offer a new lead to developed45. Multiple shoots were proliferated on the develop alternative antifungal drugs with reduced WPM containing 2.2 μM BA (Benzyladenine) and the selectable pressure for potentially evolved resistance. shoots induced were rooted on medium containing 2.7 The species is also rich in antioxidant phytochemicals μM NAA. An efficient in vitro protocol using nodal such as flavanoids and polyphenols which play a segments for large-scale multiplication of vital role in synthesis and stabilization of gold N. khasiana was also developed46. The highest shoot nanoparticles41,42. The reductive activity of plant proliferation of 19.16 ± 0.23 shoots/explant was biomolecules is responsible for reduction of gold achieved in ½ MS medium supplemented with resulting in the formation of nanoparticles. This 2.5 mg/L KN and 2.0 mg/L BAP. The best rooting efficient and ecofriendly method for synthesis of gold was recorded in ½ MS medium supplemented with nanaoparticles using plant extracts is advantageous 2.0 mg/L NAA with an average of 9.04 ± 0.46 due to biocompatibility and non toxicity of the roots/shoot. The plantlets were successfully transferred nanoparticles even at higher concentration providing to the greenhouse with survival rate of 92%, exhibiting new prospects for safe applications in biomedical42. normal development. However, micropropagation leads to genetically uniform plants which would not preserve In vitro propagation of N. khasiana using tissue the genetic diversity required for the conservation. On culture techniques the other hand, multiplication through seed would In vitro propagation has emerged as a powerful maintain genetic diversity which will in turn increase technique for large-scale propagation and conservation the adaptability to the stochastic environment. In vitro of germplasm of several plant species. Multiplication multiplication for large-scale propagation of of this endangered insectivorous plant is difficult N. khasiana has also been achieved using seeds to under natural conditions as the seeds take 223 days to conserve this rare and unique pitcher plant of India47. germinate and the percentage of germination is quite 20 Different nutrient media were used for seed low . Factors affecting in vitro regeneration of germination to ascertain the nutritional requirements N. khasiana from nodal segments of in vitro raised of germinating seeds. The study showed that the seedlings and the development of pitchers were 43 highest seed germination was achieved in the seeds studied . The study revealed that multiple shoots cultured in ¼ MS medium. Pitchers were found to were induced on MS (Murashige and Skoog) medium have developed after 150 days of in vitro culture supplemented with 0.1 mg/L IAA (Indole-3- acetic in seedlings cultured on ¼ MS devoid of growth acid) and 2.0 mg/L BAP (6-Benzyl aminopurine). regulators. Growth regulators in the medium were The number of shoot was increased by incorporation also found to have a pronounced effect on the of ascorbic acid, citric acid, arginine and adenine development of pitchers on in vitro raised seedlings. sulphate to the culture medium. The in vitro induced Well-developed and healthy pitchers were observed shoots were rooted on ½ MS medium supplemented after 120 days of culture on ¼ MS medium with 2.0 mg/L NAA (α-Naphthaleneacetic acid) and supplemented with 2.68 µM NAA. 0.1 mg/L KN (Kinetin). Addition of activated charcoal was found to reduce leaching from the Assessment of genetic fidelity in micropropagated explants which may be due to their ability to adsorb plants of N. khasiana phenolic compounds produced by the explants. Clonal Plant tissue culture is recognized as one of the key multiplication of N. khasiana has been achieved areas of biotechnology because of its potential to through shoot bud culture of the mature field grown regenerate elite and conserve valuable plant genetic plants44. In this study, multiple shoots were resources. Plant tissue culture techniques have been differentiated from the axillary buds cultured on ½ successfully applied for rapid clonal multiplication of MS medium supplemented with 2.5 mg/L KN and many rare and endangered plant species48. However, healthy roots were produced when the shoots were genetic stability cannot be guaranteed in the tissue 72 INDIAN J TRADIT KNOWLE, JANUARY 2019

culture-raised plants as there are reports of genetic decreased genetic variability leading the population to variations in micropropagated plants 49,50. Many of the extinction55. regenerated plantlets may not be the clonal copies of Prey capture and digestion their donor genotype when passaged through in vitro Different parts of pitcher play different roles in cultures due to a phenomenon known as somaclonal attracting and capturing insects, digesting the prey variation. Genetic stability of the micropropagated and absorbing the nutrients. The ultrastructure studies plants of N. khasiana maintained for three consecutive have confirmed that the pitchers possess extraordinary regenerations was assessed using single primer 51 architecture with exceptional anti-adhesive properties amplification reaction (SPAR) . The cumulative for trapping insects56-59. High level of CO dissolved analysis of SPAR methods reflected a genetic 2 in acidic pitcher fluids of N. khasiana act as a cue for variation of 5.65% in the first regeneration which attracting insects60. It has also been demonstrated that increased subsequently to 7.77% in the second the most unique features of Nepenthes pitchers such regeneration and 10.87% in the third regeneration. as enhanced growth rate, high level of carbohydrate, Genetic variations in micropropagated plants has also low level of protein, low photosynthetic capacity, been determined using chromosome counts and elevated respiration rate and evolved stomata, heterochromatin distribution pattern wherein changes are influenced by the dissolved CO in pitcher fluid. in chromosomes number and the distribution of AT 2 52 Pitcher peristome in N. khasiana emits blue and GC base pairs were observed . 23.33% of the fluorescence attracting the insects as they can cells studied in the plantlets of the first regeneration perceive the blue light ring in the pitcher peristome as showed deviant chromosome number which was a landing pad indicating that the blue emission plays a increased to 33.33% and 40% in the plantlets of the critical role in guiding the visitors to the pitcher second and the third regenerations, respectively. traps61. Also, changes in the number of 4′, 6-diamidino-2- + + Two different types of endochitinases which phenylindole (DAPI) and chromomycin A (CMA) 3 involved in prey capture were identified and their binding sites were observed in the micropropagated + differential expression during digestion of prey in plants. On an average, 5.74±0.47 DAPI and 36 + N. khasiana was investigated . Four chitinase 5.00±0.30 CMA binding sites were observed in the + genes (Nkchit1b-1 and 2, and Nkchit2b-1 and 2) plantlets of the first regeneration. DAPI binding sites representing two subgroups of basic chitinases, were subsequently increased to 6.61±0.39 and 6.74± denoted as Nkchit1b and Nkchit2b, were isolated 0.57 in the plantlets of the second and the third from the secretory region of pitchers of N. khasiana. regenerations, respectively, with a corresponding Both NkCHIT1b and NkCHIT2b share 66% amino decrease in the CMA+ binding sites (4.63±0.45 and acid homology while NkCHIT1b is characterized by 4.16±0.47 CMA+ sites in the plantlets of the second the presence of a proline-rich hinge region with eight and the third regenerations, respectively). putative glycosylation sites whereas a C-terminal

putative vacuole targeting extension is present only in Genetic diversity existing among the different NkCHIT2b which indicates that these two enzymes populations of N. khasiana have different compartments. Reverse transcription– Genetic diversities of N. khasiana Hook. f. polymerase chain reaction (RT-PCR) evaluation at collected from different parts of Meghalaya was mRNA levels showed that transcription of Nkchit1b assessed using RAPD and ISSR markers53. RAPD genes take place under the condition induced by chitin (88.50%) showed a higher level of polymorphism injection while Nkchit2b genes are constitutively than ISSR (69.16%) and this high genetic diversity expressed in the secretory cells of the pitchers. was attributed to isolation of the population at different locations and allogamous nature of the Status in its natural habitat species. RAPD revealed high genetic variation The majority of N. khasiana habitats have been (78.04%) within the population of N. khasiana due to destroyed, and remaining populations have declined its dioceous in nature54. However, there is a tendency severely as a result of unsustainable poaching and to lose intrapopulation genetic variability due to low indiscriminate collection even by the students of frequency of gene exchange among the population in Botany62. Unsustainable harvests due to phenomenal fragmented habitats and also inbreeding results in increase of prescription by the local medical DEVI et al.: NEPENTHES KHASIANA HOOK F., AN ENDANGERED TROPICAL PITCHER PLANT OF INDIA 73

practitioners have also led to rapid depletion of the accelerated pace within a short period of time43,44,46. species in its natural habitat. The species is also Propagation using in vitro seed germination has exported by local plant collectors to other states of also been achieved to obtain a large number of India and has, thus, led to its further exploitation53. plants for the conservation of this rare and unique The rampant coal mining in Jaintia Hills of endemic pitcher plant of India47. The populations of Meghalaya has drastically affected the regeneration of N. khasiana need to be conserved at small protected N. khasiana in nature63. Extraction of coal in Jaintia areas for proper management and allowing transfer of hills is carried out using a primitive method genes from one population to another so that commonly known as ‘rat-hole’ mining in which variability may be maintained among the populations. vertical pits ranging from 5-100 m2 are dug in the ground after clearing the vegetation causing large- Conclusion scale destruction to the surrounding vegetation, often Besides being a curious and attractive ornamental beyond replenishment. Geographical distribution plant, N. khasiana is also an important source of pattern of threatened and endemic species of naphthoquinone which possess several pharmaceutical Meghalaya were studied and the priority sites for activities. The species has the potential for anti-fungal conservation were also identified64. Nokrek area drugs with broad spectrum of activities against human which is one of the dwelling places of N. khasiana fungal pathogens. Further research on the available was found to be one of the eleven priority sites for information is required in developing novel clinical conservation. The population of the species in Nokrek therapeutics. Moreover, effective strategies should be Biosphere Reserve has been affected by various implemented for its conservation and sustainable factors such as coal mining, limestone mining, utilization as several anthropogenic activities stone quarrying, forest fire, overexploitation of the are constantly disturbing the wild populations of species leading to severe destruction of the species65. N. khasiana which would lead to extinction of this , deforestation, urban development, endangered insectivorous plant of India. developmental projects and modern agriculture, fragmentation of large contiguous populations into Acknowledgment isolated small and scattered ones have rendered the Corresponding author gratefully acknowledges species increasingly vulnerable to environmental the financial support provided by Department of stochasticity, which, if unchecked, would ultimately Biotechnology, Government of India, New Delhi, lead to its extinction. India in the form of Research Associateship. And acknowledges Dr JM Laishram, The Dean, College of Conservation strategies Agriculture, Central Agriculture University, Imphal In an attempt to protect the existing stands of for his support. N. khasiana in the wild, the Government of India banned its export during the 1970s. N. khasiana has References also been included in the Appendix-I of CITES 1 Givnish TJ, Burkhardt EL, Happel RE & Weintraub JD, (Convention on International Trade in Endangered Carnivory in the bromeliad Brocchinia reducta, with a Species of Wild Fauna and Flora) and Negative cost-benefit model for the general restriction of carnivorous 66 plants to sunny, moist, nutrient-poor habitats, Am Nat, 124 List of Exports of the Government of India . The (4) (1984) 479-49. potential for long term survival of this species in 2 Gallie DR & Chang SC, Signal transduction in the the wild is uncertain, but will surely depend upon the carnivorous plant Sarracenia purpurea -regulation of continuing efforts of the local communities to secretory hydrolase expression during development and preserve its habitats. It is of paramount importance in response to resources, Plant Physiol, 115(4) (1997) 1461-1471. that all lineages of N. khasiana are retained in cultivation 3 Juniper BE, Robbins RJ & Joel DM, The carnivorous plants, and propagated to preserve the reproductive potential (Academic Press, London), 1989. of this species. Under natural conditions, N. khasiana 4 Brewer JS, Why don’t carnivorous pitcher plant’s compete propagates mostly through seedlings produced from with noncarnivorous plants for nutrients?, Ecology, 84 the rhizomatous basal portion of the stem as the (2003) 451-462. 20 5 Kesler HC, Trusty JL, Hermann SM & Guyer C, percentage of seed germination is very low . Demographic responses of ionantha to prescribed However, in vitro multiplication of N. khasiana has fire: a regression-design LTRE approach, Oecologi, 156 been successfully attempted for its propagation at an (2008) 545–557. 74 INDIAN J TRADIT KNOWLE, JANUARY 2019

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