Vegetative propagation of Phytolacca acinosa Roxb. by rhizome cuttings: a step towards conservation and cultivation approach

Abstract

Introduction:

Phytolacca acinosa Roxb. is a highly valuable multipurpose herb native to the Himalayan region. Unsustainable harvesting of this species due to its diverse uses has resulted in a rapid decline in its population across natural habitats, thereby necessitating its propagation and conservation. To overcome this challenge, the potential of P. acinosa rhizomes for ex situ regeneration was evaluated.

Methods:

The current study aims to develop a standard propagation protocol for P. acinosa. Rhizome cuttings derived by splitting whole rhizomes were used to study the effect of various hormones and soil compositions on their sprouting and growth performance.

Results:

Soil compositions SC10 and SC5 consisting of soil, sand, pebbles, and vermicompost (1:1:1:1) and soil, sand, and vermicompost (1:1:1), respectively, were the most suitable compositions for the optimum growth of this species. The rhizome segments treated with GA₃ (150ppm) induced the highest sprouting percentage (91.67%), with a minimum sprouting time of 23.25 days. The maximum root length (9.25 cm), shoot length (16.5 cm), and leaf number (11.25) were recorded for GA₃ (150ppm) treated rhizome cuttings.

Conclutions:

Overall, the results of the present study helped in establishing a cost-effective, rapid, efficient, and simple mass propagation method for the target species. The results of this study will serve as a guide for the large-scale cultivation, effective conservation, and sustainable utilization of this economically valuable medicinal herb.

Introduction

The conservation of plant genetic resources involves two main approaches: in situ, which involves protecting populations in their natural habitat, and ex situ, which involves protecting these resources outside their natural environment. The rising demand from pharmaceutical industries for medicinal plants has led to their increased harvesting from the wild. Anthropogenic threats including overexploitation, habitat degradation, overgrazing, and illegal trade practices have pushed many medicinal plant species to the verge of extinction (Ganie et al., 2019; Chisale et al., 2021; Wani et al., 2022; Wani et al., 2024a). Given the urgency of conserving such species, ex situ conservation methods are recognized as a viable and effective option for the conservation and sustainable utilization of threatened plant species (Dávila-Aranda et al., 2016). This serves as both a backup conservation strategy and, in certain instances, a temporary substitute for in situ conservation.

Vegetative propagation is an important and promising method for mass cultivation of economically important species, especially those posing difficulty when raised through seeds. Propagation through rhizomes is a convenient and effective method for the rapid multiplication of elite plant populations and for conserving their essential genetic characteristics (Lata et al., 2017; Reddy et al., 2022). It is an easy, cost-effective, and successful technique for the large-scale cultivation, conservation, and regeneration of threatened plant species. While seed germination offers the advantage of producing numerous individuals from a single mother plant, its success is constrained by the need for complex stratification treatments and the slow growth of seedlings in most forest understory plant species (Baskin and Baskin, 1998; Luna, 2001). Thus, vegetative propagation in plants, especially alpine species, is considered more important than sexual reproduction (Brožová et al., 2019). Furthermore,vegetative propagation, as compared to sexual reproduction, enables the development of mature individuals within a year of cultivation. It stands out as a highly effective method for conserving economically important overexploited species, contributing to advancements in propagation techniques, and fostering large-scale commercial cultivation.

Phytolacca acinosa Roxb., commonly known as pokeberry,belongs to the family Phytolaccaceae. It is an economically valuable perennial medicinal plant native to the East Asian and Himalayan regions (POWO, 2023). In the traditional medicine system, it is widely employed to treat eye disorders, body aches, swelling, edema, sores, and as a diuretic drug (Basnet and Kalauni, 2020). Rhizomes of P. acinosa contain numerous bioactive compounds, including chochliophilin (A and B), hypaphorine, esculentosides, β-sitosterol, monoglyceride, daucosterol, phytolacacinoside (A, B, and E), phytolaccoside (A, B, and E), esculentoside G, and palmitic acid (Gao et al., 2009; Krishan et al., 2022; Li et al., 2023). Consequently, it serves as an important medicinal herb with diverse pharmacological properties like antibacterial, anti-fungal, anti-inflammatory, antiviral, anti-oxidative, anticancer, immunity-enhancing, anti-parasitic, and insecticidal properties (Gao et al., 2009; Cheng et al., 2017; Bailly, 2021). It has been reported to exhibit cytotoxicity in human cancer cell lines and antimicrobial activity in bacterial culture (Ma et al., 2017, 2019; Krishan et al., 2022). Besides being used as medicine, P. acinosa is also used as a source of food, red dye for coloring wool fabric, and for phytoremediation of heavy metals like cadmium (Cd), lead (Pb), manganese (Mn), and zinc (Zn) (Zhao et al., 2014; Wu et al., 2016; Liu et al., 2022). During the last few decades, climate change, habitat destruction, and unsustainable harvesting of P. acinosa due to its diverse uses have resulted in a rapid decline in its population across natural habitats (Cheng et al., 2017; Magray et al., 2023; Wani et al., 2024b).

The present study aimed to develop an agro-technique protocol using rhizome cuttings for the vegetative propagation of P. acinosa, a multipurpose medicinal plant native to the Himalaya (Magray et al., 2022). Although the species reproduces through both sexual and asexual means, the time-consuming nature of seed germination and vegetative propagation under natural conditions poses a challenge in generating well-developed seedlings (Magray et al., 2023).Vegetative propagation through rhizomes under in vitro conditions offers an alternative method for rapid and mass multiplication of the target plant and enables the cultivation of plants with the desired clone. Furthermore, different plant growth regulators are widely used in vegetative propagation to stimulate rooting and improve the growth of cuttings (Yoon et al., 2021).

To the best of our knowledge, the vegetative propagation of P. acinosa has not been carried out to date. In this context, the current study was conducted with the following specific objectives: (i) to investigate the effect of different growth hormones on the vegetative propagation potential of P. acinosa; (ii) to determine the effect of different soil compositions on sprouting percentage and related growth parameters; and, based on the scientific insights gained, (iii) to outline a reproducible protocol for vegetative propagation of the target species for its conservation, restoration, and mass production for the pharmaceutical industries. This study may serve as a fundamental guide for the restoration of this valuable plant species in Kashmir Himalaya and also provide scientific insights for the restoration of other valuable medicinal plants growing elsewhere in the world.

Materials and methods

Sample collection and experimental location

The fresh rhizomes of P. acinosa were harvested during February 2022 from the wild populations of Drung and Gogaldara, Jammu and Kashmir, India (Figure 1). The collected rhizomes were washed thoroughly with running tap water and kept in a dark and cool room (4°C) to avoid the emergence of buds until the experiments started. The experiment was conducted at Kashmir University Botanical Garden (KUBG), University of Kashmir, Srinagar (1590m asl; 34.13254722°N, 74.83755278°E), with a humid temperate climate and a high mean annual rainfall of 1005.5 mm (Dad et al., 2021).

Figure 1

Effect of different soil compositions

Freshly collected rhizomes were cut longitudinally into different sections, each 4 ± 0.5 g and with 2 buds (Figures 2A, B). Prepared rhizome cuttings were disinfected by a surface wash with a 2% solution of the systemic fungicide, Bavistin. The rhizome cuttings were sown in different soil compositions prepared by putting weighed amounts of soil, sand, pebbles, vermicompost (VC), farmyard manure (FYM), and peat in pots (Table 1). In each composition, five replicates of three cuttings were used. The pots were transferred to the pot house under 60% shade, and irrigation was usually performed by daily watering for the first week of the experiment and subsequently when the soil on the surface started drying. The total sprouting percentage was calculated at the culmination of the experiment for all the treatments. Plants were harvested destructively and were analyzed for various morphological parameters like rhizome length, rhizome breadth, plant height, leaf number, leaf length, leaf breadth, and biomass allocation towards above and belowground parts (Figure 2F).

Figure 2

Growth hormone experiment

To study the effect of different growth hormones on the clonal propagation of P. acinosa, the rhizomes were divided longitudinally into 2-6 sections (each 4 ± 0.5 g and 2 buds) with a sterilized sharp knife, depending on the size and number of apical buds on the parental rhizome. The rhizome cuttings were disinfected by a surface wash with a 2% Bavistin solution followed by treatment with different growth hormones including Kinetin, Indole Butyric Acid (IBA), Indole Acetic Acid (IAA), and Gibberellic Acid (GA₃) (50ppm, 100ppm, 150ppm, and 200ppm), and distilled water (control) for 48 hours (Figure 2C; Supplementary Material S1). The treated rhizome cuttings were planted in pots containing a mixture of soil and sand in a 1:1 ratio. The pots were placed under partial shade (60%) in pot-house at Kashmir University Botanical Garden and irrigated daily for the first few days (10 days) and after that every 3^rd day without fertigation. The experiment consisted of 19 treatments (Supplementary Material S1), each repeated four times in a randomized block design with three cuttings per treatment. The experiment was monitored regularly, and growth parameters including root length, shoot length, leaf number,time taken for sprouting (days), and sprouting percentage were recorded (Figure 2F). The average number of leaves per plant was counted manually, while root length (cm) and shoot length (cm) were measured using a scale.

Statistical analysis

Data from the experiment were subjected to analysis of variance (ANOVA). The analyses were carried out using IBM SPSS Statistics software (version 23). ANOVA was performed to evaluate the effect of different soil compositions and growth hormones on different growth parameters and morphological traits like sprouting percentage, time taken for sprouting, rhizome length, rhizome breadth, plant height, leaf number, leaf length, leaf breadth, and biomass allocation towards above and belowground parts. Tukey’s test (at p ≤ 0.05) was conducted for statistical mean comparisons.

Results

Effect of different soil compositions on growth and survival of rhizome cuttings

The sprouting percentage varied significantly among different soil compositions (Table 2). The highest sprouting percentage of rhizome cuttings was recorded in SC10 treatment (100%) consisting of soil, sand, pebbles, vermicompost (1:1:1:1) followed by SC5 (91.67%) having soil, sand, vermicompost in the ratio of 1:1:1, and SC9 (83.33%) treatment having soil, pebbles, vermicompost in 1:1:1 ratio (Table 2; Figure 2E,). The sprouting time of rhizome cuttings also varied significantly with different soil compositions. The shortest sprouting time was recorded in the SC10 treatment (26 days), followed by the SC5 treatment (28.75 days) (Table 2). All morphological parameters also showed significant variation among the various treatments (Table 2; Figures 3A, B). Maximum rhizome length and rhizome breadth of 15.38±0.59cm and 3.13±0.31cm, respectively, were observed for soil composition SC10, followed by composition SC5 (Table 2; Figure 3A). Similarly, maximum plant height (34.2±0.86 cm) and leaf number (49.65±1.03) were recorded in SC10 composition(Table 2; Figure 3A). The leaf length was highest in the SC10 treatment (8.83±0.44 cm), followed by SC5 (7.53±0.51 cm). Likewise, the leaf breadth was highest in the SC5 treatment (2.73±0.4 cm), followed by SC10 (2.63±0.13 cm) (Table 2; Figure 3A). The analysis of variance also exhibited a significant variation in the above and belowground biomass among all the treatments (Table 2). Both the highest aboveground biomass of 11.65±0.66 g and belowground biomass of 31.79±0.88 g were estimated in SC10 composition followed by SC9 with above and belowground biomass of 9.95±0.8 g and 30.05±0.86 g respectively (Table 2; Figure 3B).

Figure 3

The rhizome cuttings treated with different phytohormones showed significant variation in all the growth parameters (Table 3; Figures 2D, F). The highest sprouting percentage (91.67%) was observed in the rhizome divisions treated with GA₃ (150ppm), followed by (83.33%) treated with GA₃ (100ppm) as compared to the control with 33.3% of sprouting (Table 3, Figure 2D). The auxins used (IAA and IBA) failed to stimulate the sprouting of rhizome cuttings. The sprouting time varied among different treatments, ranging from 5.25 to 9.25 (Table 3). GA₃ at a concentration of 150ppm was most effective in reducing the sprouting time of rhizome cuttings. GA₃ (150ppm) was most effective in reducing the sprouting time of rhizome cuttings. The highest sprouting percentage was recorded in 150ppm GA₃ (91.67%) and 100ppm GA₃ (83.33%); sprouting time was also attained rapidly with 23.25 and 25 days, respectively; however, the sprouting percentage of 33.33% in control was completed with a sprouting time of 35.75 days (Table 3). Further, treatment of rhizome cuttings with gibberellic acid also significantly increased the shoot length, root length, and leaf number on plants. The maximum number of leaves (11.25±0.63) was observed in GA₃ (150ppm) followed by 50ppm GA₃ (9.75±0.63). The highest root length (9.25±0.48 cm) and shoot length (16.5±0.65 cm) were also recorded in rhizome cuttings treated with GA₃ (150ppm) (Table 3).

Discussion

Vegetative reproduction offers an efficient means for mass multiplication of germplasm and cultivation for species exhibiting poor regeneration through sexual means (Deepak et al., 2016; Sreekissoon et al., 2021). The frequent weather changes at high elevations result in reproductive failures at various developmental stages of plants (Carbognani et al., 2018). Phytolacca acinosa, a typical sub-alpine plant, exhibits poor seed germination within its wild habitats (Magray et al., 2023). The present study demonstrated multi-approaches for the propagation of P. acinosa through rhizome cuttings to compensate for this reproductive bottleneck. In vegetative propagation, the application of plant growth regulators and various chemicals has been extensively employed to enhance the rooting and subsequent growth of rhizome cuttings (Nadeem et al., 2000; Butola and Badola, 2007; Bisht and Bhatt, 2014). During the present study, the vegetative growth and sprouting percentage of the plant was found to be highest at intermediate hormone concentrations (Table 3). The best results were recorded for GA₃ 150ppm and kinetin 150ppm.Gibberellic acid is an important phytohormone, as it is at the core of many plant growth and developmental processes. Gibberellic acid (GA₃)-treated rhizome cuttings showed a significant increase in root and shoot development (Ma and Huang, 2016). It is reported to enhance the sprouting percentage and improve the growth and survival of vegetatively propagated plants under in vitro conditions (Shabir et al., 2010; Hassan et al., 2020). In blueberries, gibberellic acid induces plant growth and enhances leaf development as compared to the control (Zang et al., 2016). Consistent with earlier studies (Tuna et al., 2008; Wen et al., 2010; Bose et al., 2013), GA₃ application enhanced various plant growth characteristics like leaf number, root length, and shoot length in P. acinosa. Our results conform to those of Shabir et al. (2010), who also reported a significant increase in growth-related parameters in Inula racemosa when treating rhizomes with GA₃.Gibberellins can influence plant growth by accelerating the movement of cytokinins to developing buds and also by altering carbohydrate metabolism (Salimi et al., 2010). Further, similar to our findings, cytokinins have been reported to increase the shoot length and leaf number in Phytolacca dodecandra (Chishimba et al., 1999). Kinetin also improved the shooting and leaf numbers in Phytolacca americana (Trunjaruen et al., 2022). The efficacy of applying cytokinins (kinetin) and GA₃ in promoting sprouting responses, root and shoot development among rhizome explants has been reported in several plant species, such as Pongamia pinnata (Sugla et al., 2007); Jurinea dolomiaea (Banday et al., 2014); Talinum triangulare (Swarna and Ravindhran, 2013); Picrorhiza kurroa (Patial et al., 2012) and Euphorbia wallichi (Hassan et al., 2020). In Nardostachys jatamansi, vegetative propagation by rhizome splitting was found to be successful and proved to be a better and more rapid means of multiplication, as well as higher production than cultivation through seedlings (Nautiyal and Nautiyal, 2004).

The current study revealed that different soil combinations significantly affected the sprouting and growth performance of P. acinosa. Rhizome cuttings exhibited maximum sprouting percentage with minimum sprouting time in soil:sand:pebbles:vermicompost (1:1:1:1) composition (Table 2). In natural habitats, the species is mainly found growing along roadsides and forest margins with a soil texture consisting mainly of soil, sand, and pebbles. Porous sandy soil is best for successful mass cultivation as it allows the plant to develop a thick, deep root system (Chauhan, 1999). According to Banday et al. (2014), porous sandy soil allows enough aeration and easy penetration of roots and, therefore, is suitable for propagating and developing robust rootstock in Jurinea dolomiaea. Many researchers (Devkota and Jha, 2009; Deshmukh, 2010; Hassan et al., 2020) also reported that sandy loam soils result in maximum growth and higher yields in various economically important medicinal plant species. Similarly, in Paris polyphylla, maximum sprouting and rooting were found in sandy soils (Kavita et al., 2015). Chauhan and Bhatt (2000) found that sand particles, being larger than soil particles, hold more oxygen and less water due to rapid drainage, resulting in improved aeration compared to compactly packed soil particles.

Our study also reflected a positive growth response of rhizomes to adding vermicompost. Rhizome cuttings planted in the soil, sand, and pebbles mixed with vermicompost lead to a significant increase in morphological parameters such as rhizome length, rhizome breadth, plant height, leaf number, and leaf dimensions. The aboveground and belowground biomass also increased significantly in the soil:sand:pebbles:vermicompost (1:1:1:1) combination (SC10), indicating the importance of vermicompost in generating a higher biomass yield. Our results are in line with those obtained for Angelica glauca and Heracleum candicans (Butola and Badola, 2007), which reported a significant influence of sandy loam soil mixed with vermicompost on the cultivation and growth parameters of these species. The addition of vermicompost to soil enhanced germination, root biomass, and pod number in pea crops (Bhadauria et al., 2014). In Polygonatum cirrhifolium, vermicompost addition to the soil leads to increased biomass production (Lohani et al., 2011). Plant growth is significantly impacted by the availability of soil resources, particularly nutrients (Bazzaz and Bazzaz, 1996). Several researchers (Lohani et al., 2011; Rather et al., 2022) also reported an increase in plant biomass with the application of vermicompost. The composted organic matter supplies key nutrients like nitrogen and phosphorus essential for the proper growth and development of plants (Sánchez et al., 2017). Incorporating organic materials such as compost, manure, and animal dung into the soil not only boosts crop yield but also contributes to maintaining soil fertility for longer periods of time. Additionally, organic fertilizers enhance both soil nutrient retention and water-holding capacity (Ali et al., 2018). These fertilizers also promote microbial growth and activity, which is crucial for breaking down soil nutrients and making them readily available to plants (Chen, 2006).

The development of efficient integrated procedures for the vegetative propagation of plants is of great importance for their cultivation, regeneration, and conservation. Considering the urgent need to restore the diminished natural populations of P. acinosa, our research could serve as a foundational reference. This could enable conservationists to implement a multifaceted approach in devising prompt restoration and regeneration strategies for this multipurpose medicinal herb.

Conclusion

Vegetative propagation is one of the most effective methods to regenerate and sustain plant diversity. The knowledge of vegetative reproduction requirements, growth hormone effects, and soil compositions is important for mass cultivation, restoration, and ex situ conservation of medicinally valuable species. Our study is the first report on the successful propagation of P. acinosa through rhizomes. The current study revealed that the application of hormones, especially Gibberellic acid and Kinetin, proved effective in reducing sprouting time and promoting sprouting percentage, rhizome, and shoot length in rhizome cuttings. The plants also showed maximum growth performance in highly porous, loosely packed soils consisting of soil, sand, pebbles, and vermicompost (1:1:1:1). The addition of vermicompost to the soil enhanced the vegetative growth of the plant. Thus, propagation through rhizome cuttings proves a convenient and cost-effective method for large-scale cultivation and conservation of the target species under ex situ conditions. The present study provides a basic guide for the regeneration and conservation of P. acinosa, along with scientific insights for the restoration of threatened endemic biodiversity elsewhere in the world. Its implications are significant for achieving successful germination, and large-scale cultivation and serve as a sustainable approach for utilizing and conserving the target species, thereby reducing its exploitation in wild habitats.

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