Conservation and domestication of young tongkat ali (Eurycoma longifolia Jack) via evaluating root and shoot traits by combining different light intensities and water levels

The effects of three light intensities (represented by different shade levels) and three water levels on plant growth parameters, including root mass ratio, root/shoot ratio, height ratio, plant height, stem diameter, and total biomass of young tongkat ali (Eurycoma longifolia Jack) plants, were investigated. The two-factor experiment was laid out in a split-plot design based on a randomized complete block design (RCBD) with three replications. The factors were organized as follows: main plot, irradiance intensity; subplot, water treatments – used to determine the interactions among water availability, various proportions of irradiance, different durations of shading on the root system, and tongkat ali root growth performance. Results showed that plant growth parameters, including plant height, stem diameter, and total biomass, were reduced by only the light scarcity condition (25% FC) by 22%, 25%, and 20%, respectively, compared to the non-shaded condition after 4 1 2 months of treatment. Meanwhile, lower values were recorded for root mass ratio (10%–12.5%) and root/shoot ratio (24%–29%), while height ratio (8%–11%) was higher in 70% shading compared to 50% shading and the control, thus exhibiting lower ability for tongkat ali seedlings to compete for water and light.

Eurycoma longifolia Jack (tongkat ali, Genus: Eurycoma; Family: Simaroubaceae) is one of the most prominent tropical herbal plants, indigenous to Southeast Asian countries such as Malaysia, Indonesia, and Vietnam. Some plant species are also found in specific patches in Cambodia, Myanmar, and Thailand. This plant is locally known as tongkat ali, wherein “ali” means “walking stick”, assigned due to the presence of long twisted roots. The plant extract, especially roots, is exclusively used (traditionally) for enhancing testosterone levels in men and, similar to other herbs, is also widely used as a basic ingredient, whether in traditional treatment or in modern medication (Khanam et al., 2015; Rehman et al., 2016; Antonio et al., 2024; Kousalya et al., 2025).

There has been increased attention to understanding the mechanisms of plant acclimation to environmental stresses (Dixon, 1983; Heath and Boller, 2002; Nawaz et al., 2023), as a fundamental of climate-smart agriculture to reduce the impact of climate stress. Plants respond to adverse environmental conditions by modifying their morphology, physiology, and biochemistry (Heath and Boller, 2002), and light is the primary energy source and a crucial environmental factor in plant growth. As such, for good growth, morphogenesis, and other physiological plant responses, light intensity and quality are essential variables (Fukuda et al., 2008; Li and Kubota, 2009; Paradiso and Proietti, 2021).

As for conservation, domestication, and enhanced crop growth rate under a protected environment, sustainable agriculture depends on optimal environmental factors (Petitpierre et al., 2023). However, reports indicate that plants also require sufficient shade for optimal growth rate and yield, especially under the high solar radiation and temperatures of the tropics (Rylski and Spigelman, 1986).

Recently, significant focus has been placed on the impact of the interplay between irradiance and water on plant performance, encompassing individual leaves (Quero et al., 2008; Mikić et al., 2016), entire plants (Sánchez-Gómez et al., 2006; Desoky et al., 2020), and plant communities. Indeed, in a variety of natural ecosystems, both irradiance and water availability can be altered independently or may exhibit either a positive or negative relationship (Abrams and Mostoller, 1995; Niinemets, 2002a; Valladares and Pearcy, 2002). Responses of tongkat ali seedlings to increasing or decreasing shade levels have not been reported in other scientific research, and, to the best of this researcher’s knowledge, there is a lack of information on this superior herb. In addition, not much information is available on how varying light intensities affect the growth and development of this seedling. Moreover, tongkat ali’s responses to drought may vary widely depending on stress severity and environmental conditions (Rowshanaie et al., 2014). These responses may include changes in the plant’s physiological and morphological traits, including aboveground and underground organs.

Nearly all previously reported experiments showing benefits of increased irradiance have involved plants growing in glasshouses in temperate regions, even though low levels of radiation are more critical during the seedling stage (Quero et al., 2008; Desoky et al., 2020). However, reports indicate that plants also require sufficient shade for optimal growth rate and yield, especially under the high solar radiation and temperatures of the tropics (Jumrani et al., 2024; Wang et al., 2024).

Some recent research has placed considerable focus on the impact of irradiance and water on the overall performance of plants, from leaves to plants and to communities. In fact, across diverse natural systems, irradiance and water availability may be independently varied or positively or negatively correlated (Niinemets, 2002b; Costa et al., 2015; Dhakar et al., 2023). Zhao et al (Zhao et al., 2024). found that an appropriate light intensity [40 μmol/(m 2 ·s)] can enhance seedling growth rate, increase stomatal density, and improve photosynthetic capacity. Liao et al (Liao et al., 2005). studied the impacts of light concentration on the growth of four Mosla species. They found decreases in root mass (M root), stem mass, leaf mass, total mass, root mass ratio (RMR), and root/shoot ratio (R/S) from saturation irradiance to 70% of ambient irradiance. Moreover, specific leaf area (SLA), leaf area ratio (LAR), and height ratio (HR) increased with decreasing growth light. Miao et al (Miao et al., 2023). found that light intensity is an essential factor and should be optimized for specific crop species and cultivars to achieve healthy growth in plant factories, such as lettuce and spinach. The light intensities of 240 and 180 μmol m −2 s −1 were observed to be optimal for Crunchy and Deangelia (a semi-heading lettuce variety), respectively. They would provide a relative balance between growth and morphogenesis. Yasar et al (Yasar et al., 2022). reported that the highest root, stem, and leaf weights were observed in plants with high light exposure. There was higher uptake under high-light application than under optimum-light application in tomato plants. Tang et al (Tang et al., 2022). demonstrated that increased light intensity significantly improved the orientation of Alfalfa leaves toward the sun. Furthermore, high light (400 and 500 µmol m −2 s −1) significantly enhanced carbohydrate accumulation; these results advance the understanding of morphological and physiological regulation in shade avoidance in Alfalfa, which would guide the identification of suitable spatial planting patterns in the agricultural system.

Tongkat ali is highly demanded in Malaysia, and since its source is mainly in the forest, it may be endangered by non-selective harvesting at an alarming rate of extinction. The responses of tongkat ali to abiotic stress may vary greatly depending on stress severity and environmental conditions. These responses may encompass alterations in the physiological and structural characteristics of plants, affecting both aboveground and belowground parts (Khanam et al., 2015). Although water deficit is known to retard all aspects of growth, including morphological and physiological traits, irradiance also plays a significant role in plant growth and development, especially in young seedlings. The effect of irradiance levels on tongkat ali seedling growth needs to be examined to determine their performance, which is crucial at the nursery stage. In this study, the experiment was laid out in a split-plot design based on a randomized complete block design (RCBD) with three replications. The objective of this study was to investigate the effects of different shading durations and varying water availability on the root system and the morphological and physiological traits of E. longifolia. We evaluated the optimum level of shading – representing three different levels of light intensity (uncovered, 50% shading, and 70% shading) combined with three water levels (100% FC, 75% FC, and 25% FC) – with respect to root mass ratio, root shoot ratio, plant height, stem diameter, and plant total biomass. Thus, the study establishes the best levels of shading and light intensity given ideal water levels for the propagation and domestication of young tongkat ali.

Experimental site preparation

Growth conditions

Experiments were conducted in the Greenhouse unit at farm 2, Universiti Putra Malaysia (UPM) (3°0′30′′ N latitude; 10°42′18′′ E longitude; altitude of 63 m above sea level). The ambient average temperature, RH, VPD, and atmospheric pressure were 30°C, 70%, 1.05 kPa, and 1.013 kPa, respectively. The average daily air temperature in the greenhouse was 28–30°C, with a relative humidity of 80%–90% and a photosynthetic photon flux density (PPFD) of 700–800 µmol m −2 s −1 as light intensity (control). Six-month-old uniform tongkat ali seedlings were planted in white polyethylene bags measuring 20 cm × 25 cm. The topsoil (selected from the local Serdang area of Malaysia), river-washed and air-dried sand, and coco peat (organic substrate) were thoroughly hand-mixed before potting. Each poly bag contained 6 kg of soil mix (topsoil:sand:coco peat, in a ratio of 3:2:1). The combined treatments including three water levels and three different light intensities replicated three times were arranged using RCBD with a split-plot layout consisting of two factors of three different light intensities using black tilde net shading (no shading, 50% shading, and 70% shading) provided by the UPM supplier and three other water treatments using the field capacity (FC) technique (100% FC, 75% FC, and 25% FC) based on actual volumetric water content, which means nine combination treatments using the drip irrigation system for 4.5 months (Figure 1 and Tables 1, 2, also illustrated in Figures 2, 3). Because of the limitations in selecting uniform seedlings, three replications were conducted in the study. Still, to implement this project, we faced a scarcity of uniform seedlings of these herbal plants, since collecting uniform-age seedlings for this endangered species is crucial to us. Light intensity was arranged as the main plot, and water treatment was arranged as the subplot. For shading preparation, metal frames were constructed to provide shade for the shading treatments. Two types of the Sarlon ® shade, made of black polypropylene fabric (50% and 70%), were mounted to cover the frames, providing 30%, 50%, and 100% sunlight transmission, respectively. Each shade frame, measuring 2 m by 2 m, was mounted 1.5 m above the ground (2 × 2 × 1.5 m). Before experimenting, soil analysis was conducted, and field capacity was measured by the UPM Agricultural Faculty’s analytical laboratory. Proportional distribution and adaptive developmental reactions of specific morphological characteristics, such as RMR, R/S, height ratio, and overall biomass, also demonstrate the plasticity of plants in response to varying treatments.

Morphological plasticity

The measurement of growth parameters in this study is calculated as follows:

• Root mass ratio (g g −1): RMR = Root Dry Weight Total Dry Biomass

• Root–shoot ratio (g g −1): RS = Root Mass Shoot Mass

• Height ratio (cm g −1): HR = Height Total Dry Biomass

Measurements

Plants were harvested every 6 weeks, with three plants per treatment and replication sampled. There are three harvests; at each harvest, 81 pots from the various blocks and replications were randomly selected for sampling. Plants were carefully removed from the soil per treatment; roots were washed, placed in polybags, and transferred to the lab for measurement. The leaves, stems, and roots of the plants were manually separated. All these were then dried in a forced-dry oven at 40°C until a constant dry weight was achieved. The dried samples were then weighed.

Statistical analysis

Data were analyzed using analysis of variance (ANOVA) in the Statistical Analysis System (SAS Institute Inc., 2007). A mean separation test was conducted between treatments using Duncan’s new multiple range test (DNMRT), and p -values ≤ 0.05 were considered significant.

The RMR (fraction) was affected by the combination of three light regimes and three water levels in tongkat ali seedlings (p < 0.05; Figure 4). Tongkat ali had a significantly lower RMR under 70% shading compared to other shading treatments, particularly when combined with 75% and 100% FC, recorded at weeks 6 and 12 after treatment imposition. Plants under 70% shade recorded significantly (p < 0.05) lower RMR than those in the control (no shading) and 50% shade treatments, when combined with water levels at 100% and 75% FC (Figure 4). Further decreasing the water level to 25% FC, however, did not lower the RMR; instead, the RMR increased and was similar to that recorded in the other shade treatments at 25% FC. Under prolonged shade and water-level treatments at week 18 (18WAT), significant increases in RMR values occurred with decreasing water level at 25% FC, especially under controlled and 70% shade, followed by 50% shade. Other harvesting times [after 6 and 12 weeks after treatments (6WAT and 12WAT)] recorded similar results.

It is essential to note that, with a change in water regime from 100% to 25% field capacity, mild-stressed plants (75% FC) had a higher RMR than those grown under non-stress conditions (Figure 4). Furthermore, under severe drought stress (25% FC), this parameter showed the highest value among the two other water regimes at each applied light intensity. Root growth was significantly affected by irradiance, as indicated by the shade treatments (Figure 5). Root growth was favored under strong light conditions (A1 and A2) compared with A3, and there were no significant differences between unshaded and 50% shaded plants. There is an interaction effect between applied treatments in this research in all three harvesting stages (Tables A1-A3).

Tongkat ali seedlings grown in unshaded and 50% conditions, under limited water level at 25% FC (severe water stress condition), exhibited a higher R/S followed by 70% shaded treatments (p ≤ 0.05) as compared to seedlings that were exposed to other treatments of combined different shading and water level after 18-week treatment (Figure 6). The result indicates that the highest R/S is associated with access to water for plant use when light is not limiting (A1 and A2). It is important to note that root growth favored relatively strong light (A1 and A2 light conditions compared with A3 light conditions) (Figure 7). Water treatments and light treatments depicted interaction at 100% FC (Figure 6 and Table A3).

The plant height ratio (H/R) of the tongkat ali seedling was significantly affected by age and light conditions. With increasing severity of access to light from unshaded to shaded conditions, regardless of the shading percentage (50% and 70%), the plant height-to-biomass ratio in tongkat ali seedlings increased significantly (p ≤ 0.05) at both 6WAT and 12WAT (Figure 8). Increasing plant age to 18WAT demonstrated the effect of a longer duration of low irradiance under 70% shading, with a significantly higher height-to-biomass ratio than in the unshaded and 50% shaded treatments. This was consistent with the shade and moisture in the natural habitat of tongkat ali at the end of this research (18-week treatment). Also, in Table A3, the interaction effect between treatments was revealed.

Figure 8 shows significant differences among the treatments. At the end of the experiment (18WAT), the greatest H/R was observed in 70% shading seedlings, with approximately 3.95, followed by moderate shading and fully ambient light (uncovered).

The height of the tongkat ali seedling was significantly affected by different shading levels combined with varying water levels. With increasing severity of water stress from 75% FC to 25% FC, stem height decreased steadily and significantly across all light regimes, especially under the 70% shading treatment. Upon completion of this experiment, moderate water stress (75% FC) resulted in greater stem height across all light treatments (Figure 9). By 18 weeks of treatment, the most significant difference in length was observed in seedlings in 50% shade, with approximately 25.75 cm, followed by unshaded and 70% shaded plants (Figure 10). Also, the interaction effect was revealed in Table A4.

The significant effect of different shading treatments combined with varying water levels on stem diameter in tongkat ali seedlings is shown in Figure 11. Light and water deficits significantly reduced stem diameter in all seedlings. Seedlings exposed to 70% shading (A3) under severe water availability at 25% FC showed a considerably reduced stem diameter from 12 weeks of growth onward. However, a noticeable increase in stem diameter was observed under slight shading (50%) and slight water stress (50% FC) throughout the experiment. The stem diameter of the 6-month-old tongkat ali seedling was significantly affected by the different shading levels, as shown in Figure 12. Throughout the experiment, the seedlings consistently showed the thickest stem diameter in plants exposed to the 50% shade treatment, followed by unshaded plants, and the thinnest in 70% shaded plants. After 18-week treatments, seedlings exposed to 50% shade exhibited the largest stem diameter, which was 7.3% and 23.39% greater than those recorded in the unshaded and 70% shaded plants, respectively.

Biomass accumulation of tongkat ali seedlings during the experimental period significantly affected interactions between shading treatments and water levels (Figure 13). Plant total dry biomass was also considerably affected by the shading treatments (Figure 14).

At 6WAT and 12WAT, there was no significant difference between 100% FC well-watered treatment and 75% FC water level of moderate stressed seedling grown under both unshaded whole ambient light and 50% shaded conditions. Increasing the water level during this period to severe stress at 25% FC water availability decreased plant total biomass in all unshaded and shaded treatments, but after 4.5 months of treatment, it was observed that the best result of biomass belonged to seedlings that were grown under mild water stress and mild percentage of shading, with 9.35 g, also followed by well-watered plants under 50% shading, well-watered plants under whole light ambient, and mild water stressed seedlings under uncovered conditions with 9.1, 8.95, and 8.82 g, respectively (Figure 13). The biomass of seedlings grown under 70% shading was 5.9 g, compared with 50% shading and unshaded conditions, with 7.57 and 7.31 g after the third harvest, and showed an interaction with light and water treatments in Tables 3, 4.

The results indicate that the ability to acquire aboveground and belowground resources is constrained, caused by biomass allocation. It can thus be concluded that the main factor constraining root growth parameters under severe environmental stress is diffusional limitation through aboveground organs, such as leaves, and underground organs, such as roots, as previously reported (Aranda et al., 2003; Gallé and Feller, 2007; Robson et al., 2008). Furthermore, the impact of drought should be more substantial in deeper shade, as other researchers have indicated (Aranda et al., 2003). Results in this study confirm that the plasticity index is reduced by light scarcity condition as shown in Figures 5, 7, and 8, which present tongkat ali having lower RMR, R/S, and plant height ratio in 70% shading and thus exhibiting lower ability to compete for water and light, which could be the reason for the reduced ability in extending their population, therefore endangering their survival as a plant species in the same light condition. These concur with the current findings of this research.

Plants’ roots will receive more biomass if the growth-limiting factor is belowground (e.g., nutrients and water), but biomass allocation will be directed to shoots if the growth-limiting factor is aboveground (e.g., light and CO 2) (Poorter et al., 2011). Plants grown at low light intensities have reduced biomass and R/S (Mašková and Herben, 2018). Therefore, alongside shoot response to aboveground conditions, root biomass is influenced by belowground conditions, where low availability of either water or nutrients commonly leads to greater root (A1B3 and A2B3).

It is important to note that drought effects were ameliorated at intermediate irradiance (A1B2, A2B2, and A3B2) (Figure 6), becoming more severe at higher or lower light levels in parallel, in many ecosystems with exposure of plants to water and heat stress. At the same time, shade generally improves plant growth and survival. This indicates that, under conditions of this experiment, moderately stressed plants in combination with two critical environmental factors, such as light intensity and water, save water and capture maximum light and ensure long-term survival. Under such conditions, plants may develop a stress tolerance mechanism. These results are in line with the recommendations of Cahill and Gómez‐Aparicio (Cahill, 2008; Gómez-Aparicio, 2009). Furthermore, reports indicate that plants require sufficient shade to optimize growth and yield, especially under high solar radiation and temperatures (Rylski and Spigelman, 1986). This fact aligns with A1 and A2 in Figure 7.

In the case of HR, many researchers believe that this parameter increases as growth light decreases (Rowshanaie et al., 2014). Results presented in this research confirm previous observations on other plants, showing that different light regimes have a marked effect on morphological characteristics and growth parameters. Also, the proper intensity of light is crucial for plant growth, morphogenesis, and other physiological processes.

Higher or lower light intensity negatively affected plant growth and development, consistent with the results of the second experiment of this study. As shown in Figures 9 and 10, the highest range of shoot height belongs to A2 compared with A1 and A3. Furthermore, in line with this result, other researchers have reported similar findings regarding light acclimatization strategies and mechanisms for effective light harvesting under low-light conditions and protection against excessive light (Grimshaw et al., 2002).

From the results, severe shading reduced tongkat ali seedling growth, with shoot height and stem diameter significantly reduced at shading levels of 50%–70%. Low-light conditions impede plant growth and productivity and affect gas exchange (Zavala and Ravetta, 2001). In conclusion, data from all treatments and harvests were collected to show a strict allometric relationship between plant height and stem diameter in tongkat ali.

The dry matter distribution between different plant components reveals that they were exposed to the nursery stage (6 months to less than 1 year) of growth for tongkat ali plants during all three light regimes in combination with three water levels as was done in the first experiment and applied in the second experiment. Similarly, the tap roots and shoots acted as sources of dry matter accumulation, and also leaves worked as a source of assimilates and the dry matter accumulated was transferred to the root system to enhance growth; this is in the same line with the finding of two researchers who found that shade causes increased leaf area ratio, i.e., leaf area per total plant dry mass (Thompson et al., 1992; Reich et al., 1998; Rowshanaie et al., 2014). This study found that the high PPFD of 450–550 mol m −2 s −1 (50% shading compared with 70% shading) promoted higher fresh weight, dry weight, and health index in young plants, enabling them to reach the highest total biomass in a moderate light intensity condition. On the other hand, the data show that shading significantly reduced the uptake capacity of plants exposed to a severe shading regime (70%) and affected biomass (Konvalinková et al., 2015). All in all, stress lowers the photosynthetic rate by imposing stomatal or nonstomatal constraints. Moreover, the regulation of leaf stomatal conductance (g s) is a crucial phenomenon in plants, as it is vital both for preventing desiccation and for survival, and this supports the observations of Medici et al (Medici et al., 2007).

In the conservation and domestication of E. longifolia, recognizing and predicting its water requirements and optimal light intensity is paramount. Therefore, three water treatments based on field capacity, combined with three different light regimes, were conducted, and the plasticity index and morphological traits of tongkat ali were measured as shown below.

• The plasticity index is reduced by light scarcity conditions, which presents tongkat ali having lower RMR, R/S, and HR in 70% shading and thus exhibiting lower ability to compete for water and light, which could be the reason for the reduced ability to extend their population, therefore endangering their survival as a plant species in the same light condition. In contrast, high light intensity can be favorable for root growth. From the results of this experiment, significant differences were observed between 30% and 50% at 100% light intensity (A1 and A2 light conditions compared with A3).

• In terms of root-shoot ratio and the combination of shade and drought effects, plant roots will receive more biomass if the growth-limiting factor is belowground. Plant roots will receive more biomass if the growth-limiting factor is belowground (e.g., nutrients and water), but biomass allocation will be directed to shoots if the growth-limiting factor is aboveground (e.g., light and CO 2). Plants grown at low light intensities have reduced biomass and R/S. Alongside shoot responses to aboveground conditions, root biomass is influenced by belowground conditions, where low availability of either water or nutrients commonly leads to greater root biomass (A1B3 and A2B3).

• In the case of HR, this parameter increases with decreasing growth light. The lowest height ratio is observed in seedlings exposed to 70% shading. It is worth noting that, in terms of HR, there were no significant differences between 50% shading and control (un-shading condition) at the end of the experiment (18 weeks after treatment); moreover, for plant height and stem diameter, the optimum result was achieved under 50% shading. Regarding the positive relationship between aboveground and belowground organs, the optimal root parameters also presented under 50% shading. Biomass allocation results appear optimal in seedlings induced with 50% shading; thus, as mentioned above, because there is no significant difference between 50% shading and non-shading, for the sake of water conservation, 50% shading is recommended as optimum light intensity for the farming and domestication of tongkat ali.

• The highest ranges of shoot height and stem diameter are observed in A2 compared with A1 and A3 under light-acclimatization strategies, for effective light harvesting under low-light conditions and protection against excessive light. In addition, irradiance is a case in which shoot growth takes priority in low light. In contrast, the results show that severe shading reduces tongkat ali seedling growth; at shading levels of 50%–70%, shoot height and stem diameter were significantly reduced.

• The results indicate that the ability to acquire aboveground and belowground resources is constrained, caused by biomass allocation, and also reveal that low-light conditions hinder plant growth and productivity by affecting gas exchange, while excessive light intensity damages the photosynthetic apparatus. On the other hand, the data show that shading significantly reduced the uptake capacity of plants exposed to a severe shading regime (70%) and reduced biomass.

Information on other requirements of growth and development of this superior herb is still scarce. Therefore, investigation and identification of growth parameters of tongkat ali under other biotic and abiotic environmental stresses (salinity, heat stress, different levels of CO 2 enrichment, and different levels of fertilizer) are also recommended. Furthermore, another medicinal plant, Kacip Fatimah, was recommended for identifying its growth requirements.