Efficient Propagation Methods and Management Implication of Aconitum chasmanthum Stapf ex Holmes: A Critically Endangered and Endemic Medicinal Herb of Kashmir Himalaya

Aconitum chasmanthum (Ranunculaceae) locally called Mohand, Mohand Posh or Mohand gurd is a perennial herb having tuberous root, erect stem, numerous leaves and hermaphrodite flowers aggregated in racemes. The species is restricted to alpine meadowlands of Kashmir and North West Pakistan (Dhar and Kachroo, 1983) and is used for the treatment of fever, rheumatism, cough, asthma and snake bites (Mohi-Ud-Din et al.,  2009). Pulvarized roots are mixed with butter oil and used as an ointment to cure abscess and boils (Rasool, 1998). The underground tubers are known to have antifungal, insecticidal and antibacterial properties (Anwar et al., 2003). Several bioactive compounds including alkaloids, terpenoids, glycosides, flavonoids, saponins, proteins, carbohydrate and fixed oil were detected in the extract of acetone, methanol, chloroform and water solvents from the seeds of the species under study (Perveen et al., 2018). The major chemical constituents of A. chasmanthum root are aconitine, mesaconitine and hypaconitine and their respective hydrolyzed analogues, called monoester alkaloids, which include benzoylaconine, benzoylmesaconine and benzoylhypaconine (Dubey et al., 2009). In view of large-scale exploitation, biotic interferences and increasing demand for A. chasmanthum as herbal drug, its assessment as critically endangered species in nature and consequently the need for its cultivation to salvage the species from loss and also raise the germplasm and provide economic avenues for poor, it becomes obligatory to initiate steps for its large scale cultivation and development of elementary agrotechniques at lower altitudes under ex situ conditions. Owing to its endemic nature, restricted ecological niches, limited reproductive potential through vegetative and sexual means and intensive and unabated extraction for pharmaceutical purposes the herb has been declined alarmingly in its natural populations to the extent that it has been declared “critically endangered” (Anonymous 1997). It is imperative to understand the biology of such species and different aspects of reproduction (Primack, 1980; Ayensu 1981; Manjikola et al., 2005). Although Aconitum chasmanthum has been recently investigated for its pollination mechanisms and breeding behavior (Mohi-Ud-Din et al., 2009), however; least is known in respect of its seed germinability, propagation and ex situ conservation. In this backdrop, the present studies were under taken which may prove useful in up scaling the cultivation of the herb under ex situ conservation.

Five alpine sites of Kashmir Himalaya ranging in altitude from 3,150 to 3,846 m a.s.l were monitored and used as a source material for seed, soil and propagule collection (Table 1).


 
Seed germination
 
For seed germination, mature seeds of A. chasmanthum of the same age (seed cohort) were collected from a selected natural population, Apharwat II, 3250 m a.s.l (Kashmir Himalaya). The seeds were treated with 0.1% mercuric chloride for 5 minutes and washed with double distilled water. Seeds were germinated in petriplates on Whatman filter paper (in vitro) using different media (Mohi-Ud-Din et al., 2007) (Table 2). For each treatment three replicates were used each with a set of control. The mean germination time (MGT) was calculated following Joshi and Dhar (2003) as given below:  Where
‘n’  = Number of seeds germinated on each day.
‘d’  = Number of days from the beginning of the test.
‘N’ = Total number of seeds germinated at the termination of the experiment.


 
Development of plant production technique (ex situ conservation)
 
To make the cultivation of this herb possible in an effective way at lower altitudes, its response was observed in different soil textural classes recommended by Nautiyal et al., (2001) and fertilizer application were given to each treatment (Table 3). The following soil textural classes were used: (i) Natural soil (collected from natural habitat). (ii) Loam: organic manure (2:1). (iii). Loam soil (garden soil) (iv) Sandy loam (2:1) (v) sandy loam (1:2) (vi) Sand: silt: clay (1:2:2).


              
Young seedlings (of the same age) of A. chasmanthum were collected from natural habitats in April-May and sown in the above mentioned soil textural classes in pots. Each set of pots (containing equal amount of soil) were irrigated at different intervals for the first year. In the next growing season the stabilized and adapted plants were treated with various inorganic fertilizers (Table 3) in different concentrations each with a set of control. The plants were kept in diffused light and harvested (at full bloom) to determine dry biomass (after oven dried at 80°C for 72 hours; Singh and Purohit 2003).
 
In vivo propagation
 
The fresh underground tubers of the species were collected from natural population Apharwat II, 3250 m a.s.l (Kashmir Himalaya) in the first week of May and split longitudinally into 2, 3 and 4 pieces depending upon the size of the parental tuber containing a portion of shoot apex. The split cuttings were treated with 100 ppm IAA, IBA and GA₃ for 48 hours and were sown in sandy loam soils under pots at Herbal Garden (1495 m), University of Kashmir, Srinagar (India). The pots were kept in diffused light to monitor the leafy shoot generation, survival and comparison with the control (untreated cuttings) (Table 4).

The present studies on seed germination reveal that even though the seeds germinate nicely without any treatment (control), the speed of germination is slow. The mean germination time (MGT) in control replicas works to 22.11±0.358 days with only 10.66±1.154% germination.
       
Among different treatments only chilling (of varying periods) and GA₃ resulted in germination (Table 2, Fig 1). Chilling at 4^oC for 20, 40 and 100 days, however, respectively led to 90.0±8.66, 63.33±10.40 and 11.66±7.63% seed germination against the control of 10.66±1.154%. The mean germination time (MGT) also decreased in chilled seeds and varied between 13.67±1.87 to 19.55±1.53 days possibily because of the fact that the seeds of A. chasmanthum remain under snow for about five months and requires the long chilling period in order to break the seed dormancy. The seeds chilled for 20 days germinated in 0.25 mM thiourea showing 30.0±22.19 per cent germination with 14.11±1.694 day MGT indicating that the chilling triggers the breaking of seed dormancy.


       
The studies on agro-techniques (Table 3 and Fig 2) reveal that the application of fertilizers increased the herbage yield/biomass and longevity of growth period as compared to control. The present studies reveal that the total biomass per individual works to 12.37±11.25 gm in natural soil. Among different soil types with different fertilizers used, the plants in loamy soils fertilized with nitrogen (300 mg/kg soil) and NPK (nitrogen: phosphorus: potassium; 3:1:1; 500 mg/kg of soil) showed maximum dry biomass of 13.36±2.13 gm and 18.23±1.16 gm per individual respectively as against the control of 4.50±4.24 gm. The loamy texture offers little or no resistance for rooting and less leaching of nutrients than clayey and sandy soils.  


       
The in vivo propagation studies reveal (Table 4, Fig 3) that the cuttings without any treatment showed 62.5% survival and require 18-24 days to regenerate a leafy shoot. However, the cuttings treated with IAA and IBA (100 ppm) exhibited 75% and 70% survival respectively and required 10-15 days for shoot generation in sandy loam soils. GA₃ (100 ppm) treated cuttings showed shoot regeneration in lesser time, but survival was a little lesser than other treatments. The difference even though marginal clearly demonstrates that the tuber cuttings having a piece of apical shoot have the capability to regenerate and form new plants under ex situ conditions. Utilizing this cost effective method of propagation, the species can be multiplied in lesser time with good survival rates.


       
The seeds of the species remain dormant for a pretty long period of time and do not germinate in darker conditions and seem to be photoblastic in nature. The present studies reveal that only chilling and GA₃ concentrations show desirable response. The species under discussion showed 90.0±8.66% germination when chilled for 20 days with mean germination time (MGT) of 13.678±1.874 days. The seeds chilled for 40 and 100 days, however; showed low germination rates than of the 20 day chilled seeds depicting superiority of 20 day chilling as a dormancy breaking technique to improve seed germination. Similar kind of results was obtained by Yousef and Mahmood (2014) on Bunium persicum an important medicinal aromatic plant species of Himalaya. Seeds of alpine plant species mostly hydrated ones commonly germinate when exposed to low temperature (Prakash et al., 2011). The overexploitation of important plant species is the basic reason for their extinction. This has necessitated the development and standardization of agrotechniques for their cultivation and consequent conservation (Joshi et al., 1990). The major conservation strategies recommended are development of germplasm centres, in situ and ex situ conservation of critically endangered species, establishment of high altitude nurseries, systematic collection and domestication etc. (Joshi and Rawat, 1997; Dwivedi, 1999). Considering the increased demand of herbal drugs and consequent depletion of several species, it is important to urgently initiate proper steps for conservation (Nautiyal et al., 2001). The species under discussion showed increased herbage yield/biomass and longevity of growth period as compared to control after application of different fertilizers. The species responded to each nutrient treatment, however, in all the soil textural classes the nitrogen nutrient trials and NPK treated individuals have shown overall best performance and biomass. The plants performed well and gave excellent adaptability when grown on various soils. However, maximum plant survival and vigorous growth was obtained in loamy textured soils, possibly because this texture offers little or no resistance for rooting and less leaching of nutrients than clayey (difficult to root) and sandy (maximum nutrient leaching) soils. Similar kind of results was observed by Ramesh et al., (1989), Khan and Zaidi (1991), Singh and Neoparly (1993), Yugalkishore et al., (2019), Adebayo et al., (2021) and Senthilkumar and Gokul (2021) on several plant species.
       
The application of auxins is known to stimulate the activity of the cambium resulting in the mobilization of reserve food materials to the site of root initiation (Gurumurthi et al., 1984). Several species of the genus Aconitum inhabiting the alpine zones of Indian Himalayan region have been propagated in vitro (Giri et al., 1993; Singh et al., 1998; Nadeem et al., 2001). The experiments on the species under discussion reveal that the species has a potential to propagate through their tuber cuttings in the sandy loam soils. The cuttings treated with IAA and IBA showed best survival and required lesser time duration to develop plantlets as also argued by Giri et al., (1993) who observed that regenerated embryos were successfully converted to plantlets on addition of IBA to MS medium. GA₃ treated cuttings required least time period to regenerate the leafy shoot but showed lesser % survival as compared to IAA and IBA treated cuttings. Such kind of findings was observed earlier by Nautiyal et al., (2001), Naidu et al., (2006) and Rathod et al., (2021). Utilizing these cost effective techniques of propagation, multiplication and conservation at low altitudes can relieve the pressure of extinction (especially for threatened taxa) to a large extent and also can provide an alternate income-generating resource.

Present study demonstrates the efficient methods of seed germination, agro-techniques and vegetative propagation through tubers, therefore, may prove useful for sustainable management of this important and critically endangered medicinal plant species.