International Journal for Environmental Rehabilitation and Conservation

ISSN: 0975 6272

XI (SP2): 64 — 74 www.essence-journal.com

Original Research Article

Sustainable conservation of endangered alpine medicinal and aromatic grandiflora DC. through top edge cuttings using plant growth regulators (PGR’s)

Dobhal, Pradeep; Purohit, Vijay Kant and Chauhan, Jaidev High Altitude Plant Physiology Research Centre (HAPPRC), H.N.B. Garhwal University (A Central University), Srinagar, Garhwal, Uttarakhand Corresponding Author: [email protected] A R T I C L E I N F O Received: 4 July 2020 | Accepted: 20 August 2020 | Published Online: 30 September 2020 EOI: 10.11208/essence.20.11.SP2.128 Article is an Open Access Publication This work is licensed under Attribution-Non Commercial 4.0 International (https://creativecommons.org/licenses/by/4.0/) ©The Authors (2020): Publishing Rights @ MANU—ICMANU and ESSENCE—IJERC.

A B S T R A C T

Nardostachys grandiflora DC. (Jatamansi) is a small herbaceous species of family (previously ) commonly known as Jatamansi, Indian nard or . It is a perennial, dwarf, hairy, rhizomatous medicinal and aromatic herb, grows in steep, moist, rocky, undisturbed grassy slopes in higher Himalayan region. The causative aim of our designed study was preparative and conservative point of view, prior producing the large number of using the top edge cuttings obtained from plants at uprising senescence and investigating the effect on N. grandiflora, of different concentrations were tried to stimulate sprouting and rooting of IAA, IBA NAA and BAP respectively. The cuttings were dipped in different concentration of PGRs for 24 hours and planted in Styrofoam trays at Greenhouse condition (21.5 ± 3.30°C while night temperature was 8.60 ± 2.45 °C and the relative humidity was between 65-80% during the experiment) containing mixture of Soil, Sand, Farm Yard Manure (FYM), and Forest Litter (FL) at the ratio of 1:1:1:1. The study reveals that IBA (50 µM) showed better result compared to other PGRs. Experiments indicated that the large number of N. grandiflora plants can be obtained through using top edge cuttings (upper part of shoot portion) without damaging the plants before senescence period.

K E Y W O R D S Nardostachys grandiflora DC | Top edge cuttings | Vegetative propagation | Plant Growth Regulators | Conservation

C I T A T I O N

Dobhal, Pradeep; Purohit, Vijay Kant and Chauhan, Jaidev (2020): Sustainable Conservation of Endangered Alpine Medicinal and Aromatic Plant Nardostachys grandiflora DC. Through Top Edge Cuttings using plant growth regulators (PGR’s). ESSENCE Int. J. Env. Rehab. Conserv. XI (SP2): 64 — 74. https://eoi.citefactor.org/10.11208/essence.20.11.SP2.128

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Introduction of rhizomes for medicinal and aromatic use, habitat degradation and other biotic Nardostachys grandiflora DC. One of the interferences, the species has become highly economical species commonly known critically endangered depending on habitats as jatamansi. It is a perennial rhizomatous (Airi et al., 2000; Nautiyal et al., 2003). medicinal and aromatic herb growing in Hence emphasis was given on its subalpine/alpine areas of the north western conservation and multiplication by several Himalayan region of Pakistan, India, , workers (Chauhan and Nautiyal, 2005; China and Tibet, mostly grows in immerse, Chauhan et al., 2008). Effective conservation, moist, stony, slopes between 3000-5000 m asl management and recovery of rare and in higher Himalayan region. It is traditionally endangered species can be deliberated used for its incense, perfume and in ethno through variability analysis (Chauhan et al., medicine as well as modern medicine 2011). A single plant species generally (Chauhan and Nautiyal, 2005). In different consists of genetically differentiated parts of India. The species has been declared populations that are outcomes of the as ‘Endangered’ in Arunachal Pradesh, interactions among natural selection, gene and Himachal Pradesh, ‘Critically flow and genetic drift (Suichi, 2003). There Endangered’ in Uttarakhand and Kashmir are several reports available on distribution district, (Kaul, 2001) and ‘Vulnerable’ in pattern, medicinal properties (Purohit et al., Nepal during Conservation Assessment and 2017), etc. The Governments is making Management Plan (CAMP) workshop held in serious effort to promote conservation and the year 2001 (Bhattarai et al., 2002). It is cultivation of this species through different listed in CITES (Convention on International promotional schemes. In this context, effect Trade in Endangered Species of Wild Flora of various growth regulators was assessed for and Fauna) appendix II and the Red Data effective propagation of this species. Book of Indian Plants (Nayar and Sastry, Vegetative propagation assists in sustaining 1988; Mulliken, 2000). It is widely used for genetic identity of medicinal plants and safe the hair related problems like hair loss, guarding the populations from destruction and greyness, growth and nervous system deterioration (Kharkwal et al., 2008). Plant disorders i.e., insomnia, epilepsy, learning growth regulators are known their role in and memory disorders (Yadav et al., 2011; growth and other lively activities in plants. Rahman et al., 2011; Singh et al., 2013). In Auxins are the group of hormones (IBA, IAA, Ayurveda it is used for cure various elements NAA etc), which stimulates the sprouting and related disorder as Medhya (brain tonic), rooting in cuttings, promotes cell elongation Rasayana (Rejuvenative to the mind), and growth by synthesized in young shoot tips Nidrajnana (promotes sleep) and and transported to different tissues and are Manasrogaghna (alleviates mental diseases) widely used in vegetative propagation of (Sharma et al., 2001). It is also useful in plants. Whereas, cytokinins play an important antibacterial, antifungal, nematicidal, anti- role in cell division. In this experiment auxins malarial and mosquito repellent activity group of hormones (IBA, IAA and NAA) and (Singh et al., 2013). Due to over-exploitation 65

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BAP were used and stock solutions (mg/mL) the cuttings were treated with different of Indole-3-acetic acid (M.W. 175.2), Indole- concentrations of PGRs i.e. IAA, IBA NAA 3-butyric acid (M.W. 203.2), and and BAP (50, 100, 150 and 200 µM) for 24 Naphthalene acetic acid (M.W. 186.2) and hours treated cuttings were planted in Benzylaminopurine (M.W. 225.3) were Styrofoam trays containing mixture of soil, prepared. Hormone solutions such as 50 µM sand, FYM, and FL at the ratio of 1:1:1:1. All 100 µM, 150 µM and 200 µM were prepared the Styrofoam trays containing cuttings were from the stock solutions. In this context, effect kept inside the greenhouse condition at of various growth regulators was assessed on Srinagar (550 m asl) for further observations. vegetative propagation for effective Recording of cuttings parameters conservation of this species. Statistical analysis Material and Methods Data analysis: One-way ANOVA was Plant materials performed to find out the variance among the Top edge cuttings of N. grandiflora were treatments, further Duncan Multiple Range collected from nursery of Tungnath (TN): an Test (DMRT) was applied to find out the alpine zone (3400 m asl) located between variance within groups. Regression plots were 30◦14' N latitude and 79◦13' E longitude in plotted for all PGRs, different concentrations Rudraparyag district of Garhwal, Uttarakhand of PGRs used as predictor and growth Himalaya, India. After collection top edge percentage of morphological parameters as cuttings was tested to propagate the species response variable. Growth performance was under polyhouse conditions at Srinagar calculated for number of leaves, plant height, Garhwal (550 m asl), Uttarakhand (29◦26’-31 stem diameter, leaf area and fresh biomass 28’ N and 77 49’-80 6’ E), India. using the following formula:

Preparation of top edge cuttings (Last observation- First The top edge cuttings (7.11 ±1.69 cm Length; % observation) = 4.34 ±1.59 mm Diameter; 2.93±1.29gm Growth weight), prepared from five-year-old plants Last observation×100 grown in nursery at Tungnath. The top edge or above ground biomass which totally dries during winter months was used for formation First observation was taken after 30 DAP and of new plants. The top edge (twigs) collected last observations after 150 DAP. Readings after complete seed formation and collection were taken on the monthly basis (30 days’ at the level of soil were brought to Srinagar in interval). Dry weight, number of roots, length the first week of December. After that the of roots and diameter of roots were recorded application was composed of 3 replications only once so growth rate for these variables (45 number in each treatment) were washed was not calculated. carefully with tap water to remove soil particles followed by distilled water after that

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Sprouting Percentage Number of cutting survive Survival The total number of top edge cuttings that Percenta = sprouted under each treatment were counted Total number of cutting ge (%) and expressed as sprouting percentage: planted ×100

Number of cutting sprouted Sprouting percentage was taken after 30 DAP Sprouting cuttings and survivability and rooting Percentage = Total number of cutting percentage was calculated after 150 DAP. Dry (%) planted ×100 weight of cuttings was recorded after drying them in electric oven for 48 hours at 80°C and Rooting Percentage weight was taken by electronic weighing The total number of top edge cutting within balance SARTORIUS (CPA224), in the each treatment which showed the growth of laboratory. Thus eight parameters were root (≥ 1mm) were counted and expressed in determined at the end of the experiment, percentage: namely: Plant height (PH), number of leaves (NL), Leaf area (LA), stem diameter (SD), Number of cutting rooted Number of Roots (NR), Length of Roots Rooting (LR), Diameter of Roots (DR), and Dry Percentage = total number of cutting Biomass of Cuttings (DB). Width of cutting (%) planted×100 was recorded by Vernier Digital caliper (YAAYO) and cutting length were recorded Survival Percent (%) by measuring scale in the laboratory of The total number of top edge cutting survives HAPPRC. All chemicals and PGRs (IBA, under each treatment were counted and IAA, NAA, BAP were purchased from expressed as survivability percentage: Himedia and Sigma Aldrich.

120 Sprot% 100 Sur% 80 60 40 20 0

Fig. 1: Effect of hormonal treatment on Sprouting, rooting and survivability percentage of top edge cuttings of N. grandiflora

Cuttings Transplantation after 90DAP. The root parameters and biomass of cutting were uprooted and The well-established top edge cuttings were recorded after 150DAP. The survivability % further transferred to the field under natural after 150 DAP was recorded (Fig. 1). The habitat at Tungnath field nursery (3400masl), 67

ESSENCE—IJERC | Dobhal et al. (2020) | XI (SP2): 64 — 74 mean air temperature during the (88.88%) and NAA 50 µM (84.44%), while transplantation was 20°C, and Relative minimum sprouting percentage was recorded humidity 60% - 70% was recorded. in IAA 100µM (64.44%). After 150 DAP it was recorded that cuttings treated with IBA Results and Discussion 50 µM showed highest percentage of rooting Effect of PGRs on survivability of cuttings (53.33%) with highest survivability (68.88%), The result on sprouting percentage (Graph 1) while IAA 100 µM showed lowest rooting showed that at 30 DAP, the maximum percentage (24.44%) with minimum sprouting percentage (97.77%) was observed survivability percentage (42.22%). in control condition followed by IBA 150 µM

A B

C D

E F

Fig. 2: Nursery growing mature plants of N. grandiflora used for vegetative propagation using top edge cuttings: Mature plants (A), Senescised plants before winter season (B), top edge collected (twigs) for treatments (C),top edge cuttings dipped in solutions of different PGRs (D), sprouting of cuttings of N. grandiflora (E), well-formed roots after five months of transplantation of cuttings in field (F).

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Effect of PGRs on number of leaves and significant for all morphological leaf area of cuttings characteristics; the F value at 17, 36 degree of freedom was between 2.82 and 5.73 for Maximum number of leaves (19±4.58) and different morphological parameters (Table 1). leaf area (24.3±5.16), found in NAA100 and IBA 50 µM respectively, which was 10.33 A Plant growth regulator plays an and 1.25 higher than control respectively, indispensable role in growth and other vital while both parameters number of leaves activities in plants. Auxins are the group of (5±2.65) and leaf area (4.6±1.48) was lowest hormones (IBA, IAA, NAA etc.), which respectively in IAA 100 µM treated cuttings. stimulates sprouting and rooting in cuttings and are widely used in vegetative propagation Effect of PGRs on plant height and stem of plants. (Sevik and Turhan, 2015). Rooting thickness of cuttings and survival of cuttings are highly influenced The result reveal that maximum height of due to initial carbohydrates reserve (Kraus plant (20.8±2.17), and diameter of cuttings and Kraybill, 1918). Similar results were (8.51±0.86) were found maximum for lower agreed by (Wright, 1975) for vegetative concentration of (50 µM) of IBA, which was propagation. Sprouting of cuttings, 2.63 and 1.77 higher than control, while survivability and formation of new roots were minimum in IAA (7.47±0.9) and BAP found significant with age of selected plant, (5.21±0.24) respectively. season and application/solicitation of Effect of PGRs on root numbers, root different PGRs. The experiment showed that length, and root thickness of cuttings top edge cuttings treated with IBA found the highest survival and rooting percentages as The effect of hormone application on number compared to other. The immediate formation of roots (18±2.65), were found maximum for and the growth of roots are the major factors lower concentration (50 µM) of IBA, which which effects the survival of cuttings and it was 2.33 higher than control, while IAA 100 could be influenced by many factors i.e., µM showed least number of root formation rooting media, origin of cuttings, the (4.67±2.52) compared to other PGRs. When environmental conditions and physiological there is no effect of PGRs on root length and status of mother plant, (Mehrabani et al., root thickness and found both maximum in 2002; Dick et al., 2004; Leakey 2004; without treated cuttings. Hartmann et al., 2002). The auxins activated Effect of PGRs on dry biomass of cuttings shoot growth which results the elongation of stems and leaves through cell division Total dry biomass was recorded after 150 accounting in longest sprout. It may be due to DAP was found maximum (1.64±0.36) for species, favourable climatic conditions in lower concentration (50 µM) of IBA, which mist to increase the length of sprout. Similar was 0.54 higher than control, while minimum results were also reported by (Bhatt and recorded in BAP 100 µM (0.35±0.06) treated Tomar, 2011) in Citrus aurantifolia and cuttings. Results from one-way ANOVA (Dutra and Kersten, 1996) in plum (Prunus showed that the main effect of treatments was salicina L.) cutting. The present study 69

ESSENCE—IJERC | Dobhal et al. (2020) | XI (SP2): 64 — 74 revealed that IBA treatments were more result (54%) than the higher concentration. efficient than other PGR’s in inducing rooting Also agreed by (Pandey et al., 2011) and of top edge cuttings. There are several (Butola and Badola, 2007) have suggested researchers also reported that application of that IAA and IBA as promising treatments to auxin treatments mostly IBA and IAA improve rooting, growth and biomass in enhanced root initiation as well as root Angelica glauca and Heracleum candicans. number in many species (Zhang et al., 2010; (Vashistha et al., 2009) found that lower Kumar et al., 2009). The increased number of concentrations of IBA (100ppm) showed roots per cutting resulted in reduced average better results compared to IAA and GA3 in length of roots (Kumaresan et al., 2019). both the species i.e., Angelica glauca and Angelica archangelica. Similar result was During the transplantation of rooted cuttings, also found by (Tarit et al., 2010). it is important to have more number of roots instead of too many longer roots for better Overall growth and development of cuttings results (Singh et al., 2014). In the present were better acquired with IBA 50 µM than study IBA 50µM showed maximum numbers that of NAA, IAA and BAP. Thus IBA 50 µM of roots, survivability percentage and dry is superior amongst all the hormonal weight compared to others PGRs while treatment because due to following reasons thickness and length of roots was found low higher constancy, transportability, capability compared to untreated cuttings, which to produce roots and accordingly results in supports the previous study. The lower lower mortality in plants. concentration of auxin acquired better rooting

Fig. 3: Root formation after six months’ plantation of top edge cuttings of N. grandiflora dipped in different PGR’s concentration

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Avg. No. of Avg. Treatment Avg. Plant Avg. No. of Avg. Leaf Avg. Stem Avg. Length Avg. Dry Roots Diameter of s height leaves area Diameter of Roots biomass Formed Roots 6.74±0.68abc 15.67±4.16cd 1.1±0.45bcdef Control 18.17±3.41cde 8.67±1.15abc 23.05±6.29e 7.77±2.82d 1.3±0.12e de ef g 16.33±2.08de 15.21±5.74b NAA50 14.37±4.01bcd 5.77±0.97abc 9.33±2.52ab 4.17±0.76c 0.94±0.37cde 1.22±0.17cdef f cd 18.25±1.72c 3.33±1.04ab 1.47±0.37abcd NAA100 16.1±1.44bcde 19±4.58f 7.3±0.74cde 15±1bcdef 0.41±0.07abc de c ef 19.28±5.6cd 7.01±0.43abc 1.04±0.54bcde NAA 500 17.67±4.19cde 10±4abcd 14±2.65bcdef 3.83±0.7bc 1.07±0.69de e de fg 12.77±1.94b 6.51±0.44abc 13.67±1.53bc 2.53±0.12 0.71±0.54abc NAA 1000 14.67±2.08bcd 6.33±1.15ab 0.47±0.15ab c d def abc d 14.33±2.31cd 0.87±0.09bcd IBA50 20.8±2.17e 24.3±5.16e 8.51±0.86e 18.33±2.65ef 4.07±1.01c 1.64±0.36g ef e 12.83±4.37b 15.33±2.52bc 3.43±0.93 IBA100 12.73±4.15bc 17.33±6.51ef 6.13±0.78abc 0.7±0.44abcd 0.8±0.18bcde c def abc 12.95±2.91b 14.67±2.52bc 3.27±0.21 0.62±0.16abc IBA500 14.87±0.32bcd 15±3.61cdef 7±0.57abcde 1±0.16bcdefg c def abc d 10.33±2.08ab IBA1000 11.07±2.69ab 7.67±1.53ab 8.3±2.94ab 5.4±0.79ab 2.3±0.92abc 0.31±0.1ab 0.51±0.03def cd 19.17±4.85c 6.87±0.55abc 13.33±3.06bc 0.55±0.15abc 0.84±0.13abcd IAA50 16.3±1.97bcde 6.67±1.15 1.93±0.49ab de de def d ef 7.25±1.53bc 0.86±0.52abcd IAA100 7.47±0.9a 5±2.65a 4.6±1.48a 4.67±2.52a 1.57±0.51a 0.25±0.08a de ef 21.69±6.52d 16.33±4.04de 3.43±0.35 0.8±0.02abcd IAA500 19.5±5.27de 12±5.29bcde 8.17±1.52de 1.3±0.49efg e f abc e 14.67±3.79cd 14.77±1.99b 11.67±1.53bc 0.53±0.25abc IAA1000 14.3±0.3bcd 5.82±1.28abc 2.53±0.5 abc 0.64±0.21abc ef cd de d 10.67±1.53ab 13.22±0.63b 14.33±1.53bc 3.13±0.55 0.64±0.33abc BAP50 13.4±0.85bc 5.36±0.49a 0.77±0.24bcd cd c def abc d BAP100 11.1±2.29ab 6.33±1.15ab 9.77±3.14ab 5.21±0.24a 9.67±3.51abc 1.9±0.1ab 0.33±0.1ab 0.35±0.06a 6.55±1.93abc 16.67±4.16de 3.07±1.29 BAP500 10.83±2.25ab 13±3bcdef 8.34±3.09ab 0.3±0.04a 0.63±0.16 d f abc 12.67±5.69bc 3.33±0.76 BAP1000 18.07±3.96cde 18.5±2.7cde 6.31±0.84abc 18±7.64f 0.47±0.29abc 1.43±0.69fg def abc BAVASTI 16.23±1.62bcd 15.36±1.61b 16.33±2.08de 1.08±0.04bcde 8.67±2.89abc 6.02±0.4abc 3.5±1.32 abc 0.74±0.1abcd NE e cd f fg F-value (Df 4.41** 4.52** 5.73** 2.82* 3.48* 5.40** 3.00* 3.61* =17, 36)

Note: Superscript in the table indicated by the Table 1: Assessment of morphological characteristic of plants same letter (a, b, c and d) showing non- raised through top edge cuttings of Nardostachys significant differences according to DMRT grandiflora

Conclusion conservation is the major priority of this species. This species is becoming endangered As this is economically important and day by day due to over exploitation from critically endangered plant hence higher Himalayan ranges for medicinal use, 71

ESSENCE—IJERC | Dobhal et al. (2020) | XI (SP2): 64 — 74 habitat degradation and other biotic raised plantlets of Ginkgo biloba L. interference. At some places the species gets International Journal of Biodiversity pre harvested from its natural pockets before and Conservation. 3(4): 142–146. its reproductive phase or at juvenile stage Airi, S.; Rawal, R.S.; U. Dhar and A. N. Purohit which acts as a barrier for its natural (2000): Assessment of availability and regeneration. The species require rapid and habitat preference of Jatamansi-A successful methods of vegetative propagation critically endangered medicinal plant and multiplication. The observations of the of west Himalaya. Current Science. 79 present study indicate that the large number of (10): 1467-1470 N. grandiflora plants can be obtained through B. B. Bhatt and Y. K. Tomar (2011): Effect of top edge cuttings, taken right before IBA and growing conditions on senescence, without damaging the vegetative performance of Citrus economically important part i.e., underground aurantifolia (Swingle) cuttings. part of the plants. Journal of Hill Agriculture. 2(1): 98- Acknowledgements 101.

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