Influence of root inducers and substrates on the vegetative propagation of pitahaya species (Hylocereus spp.) under nursery conditions

Influence of root inducers and substrates on the vegetative propagation of pitahaya species (Hylocereus spp.) under nursery conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Influence of root inducers and substrates on the vegetative propagation of pitahaya species (Hylocereus spp.) under nursery conditions Beimer Chuquibala-Checan, Jonathan M. Cruz-Malca, Jefferson A. Cubas Sanchez, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9180319/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Pitahaya ( Hylocereus spp.) is an emerging crop for tropical regions; however, its vegetative propagation is strongly influenced by environmental and management factors. Optimizing rooting and establishment under nursery conditions is essential to produce uniform and vigorous seedlings. This study evaluated the effects of root inducers and substrates on the vegetative propagation of three Hylocereus species under nursery conditions. A completely randomized design (CRD) with a 2×2×3 factorial arrangement was implemented, including two root inducers (NAA + IBA solution and seaweed extract), two substrates (substrate 1 and substrate 2), and three species ( H. undatus, H. megalanthus , and H. costaricensis ), resulting in 12 treatments with four replicates each. Seven variables were assessed: shoot emission time (SET), cutting viability (CV), number of shoots (NS), shoot length (SL), number of roots (NR), root length (RL), and vegetative vigor index (VVI). ANOVA revealed significant effects (p ≤ 0.05) in the three-factor interaction for SET, CV, NS, SL, RL, and VVI. Treatment T9 achieved the shortest SET (21.53 days) and highest CV (100%), T7 recorded the highest NS (1.62 shoots plant⁻¹), T3 showed greater SL (68.67 cm) and NR (5.30 roots plant⁻¹), and T12 obtained the highest RL (42.69 cm) and VVI (110.18). The species factor exerted the greatest influence, with H. costaricensis showing the best overall performance. These findings demonstrate that combining H. costaricensis, substrate 1, and NAA + IBA enhances vegetative propagation efficiency, supporting large-scale seedling production and sustainable expansion of pitahaya cultivation in tropical Peru. Biological sciences/Ecology Earth and environmental sciences/Ecology Biological sciences/Plant sciences Phytohormones cuttings rooting organic substrate Hylocereus spp Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Pitahaya ( Hylocereus spp.), commonly known as dragon fruit, is a cactus belonging to the genus Hylocereus within the family Cactaceae. The genus comprises approximately 14 species, of which H. undatus , H. monacanthus (Lem.) Britton & Rose, H. costaricensis , and H. megalanthus are among the most widely cultivated worldwide 1 , 2 . Species of this genus are native to tropical and subtropical regions of the Americas 3 , and have high export potential because of their nutritional value and adaptability to a wide range of soil and climatic conditions 4 Global pitahaya production has increased steadily, with Vietnam, China, and Indonesia together accounting for nearly 93% of total global output 5 . In Latin America, pitahaya has gained importance as an alternative crop that improves rural livelihoods and contributes to agricultural sustainability and diversification 4 . In Peru, the cultivated area expanded markedly from 34.2 ha in 2019 to 419.2 ha in 2023, accompanied by a rapid increase in production from 156.9 t to 4,178.9 t 6 . At the regional level, Lima and Piura lead national production, accounting for 48.4% and 36.1% of total output, respectively, followed by Ica, Amazonas, Moquegua, and La Libertad 6 . These trends indicate growing interest in pitahaya cultivation and its potential as a sustainable and commercially viable crop. However, the rapid expansion of cultivated areas also poses new challenges, particularly regarding the availability of high-quality genetic material to support newly established plantations 7 . Although pitahaya can be propagated by seed, this approach does not ensure genetic uniformity. Therefore, vegetative propagation through stem cuttings is preferred as a more efficient approach 8 . This method is widely used because vegetatively propagated plants exhibit faster growth, earlier fruiting, and greater genetic stability 9 , 10 . Achieving effective vegetative propagation depends on several key factors that play a crucial role during the rooting stage 11 . Among these, the application of auxins and the selection of an appropriate rooting substrate are considered critical 12 , 13 . Understanding and optimizing these factors are essential to ensure successful propagation and enhance productivity at the commercial scale. Previous studies have shown that the choice of rooting agent, whether synthetic or organic, has a significant effect on the formation of adventitious roots in cuttings 14 , as well as root length and shoot development in vegetatively propagated species 10 , 11 , 15 , 16 . Among synthetic rooting agents, the most widely used auxins across a wide range of plant species are indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), which promote cell division and differentiation, although their effectiveness depends on concentration and plant species 11 , 17 . Moreover, seaweed extracts derived from Ecklonia maxima contain phytohormones such as auxins, cytokinins, ethylene, and gibberellins, which can stimulate root formation and overall plant growth 18 – 20 . These extracts have also been reported to improve soil health, plant growth, and stress tolerance through the action of natural phytohormones, amino acids, polysaccharides, and micronutrients 21 , 22 . Accordingly, comparing synthetic and organic rooting agents is essential for developing efficient and sustainable propagation strategies for pitahaya. In addition, substrate composition is a key factor influencing the success of cutting establishment, as it is essential for moisture retention and nutrient availability during the rooting process 15 . One of the most suitable substrates can be formulated by combining organic components such as sawdust, peat, rice husks, and cattle manure, which together provide optimal physical and chemical conditions for root development and cutting establishment 23 – 25 . Given the increasing commercial demand for pitahaya and the limited technical information available on its efficient propagation in tropical regions, there is a need for studies aimed at optimizing seedling production. In this context, the present study evaluated the effect of two types of rooting agents and two types of substrates on the vegetative propagation of three Hylocereus spp. species in a controlled nursery environment. We hypothesized that an appropriate combination of rooting agents and substrates would significantly improve rooting success and promote the development of vigorous and high-quality pitahaya seedlings. 2. Methodology 2.1. Study area The study was conducted at the Center for Plant Production for Research and Technology Transfer, Centro Experimental Yanayacu, Instituto Nacional de Innovación Agraria (INIA), located in the province of Jaén, Cajamarca Department, Peru (5.6771° S, 78.7749° W), at an elevation of 624 m a.s.l. (Fig. 1 ). The site is characterized by a mean annual temperature of 28°C and an average annual precipitation of approximately 1200 mm 26 . 2.2. Biological material Three Hylocereus species were evaluated (Fig. 2 ): two red-skinned species, H. undatus and H. costaricensis , collected from mature pitahaya plantations at Grupo Campo Agro S.A., in the province of Zarumilla, Tumbes department; and one yellow-skinned species, H. megalanthus , sourced from plantations in the province of San Ignacio. Species selection was driven by their adaptability to tropical environments, favorable agronomic and organoleptic traits, and strong commercial demand in both domestic and international markets. 2.3. Experimental design The experiment was conducted using a completely randomized design (CRD) with a 2A × 2B × 3C factorial arrangement, consisting of two rooting inducers (seaweed extract and a solution of NAA and IBA), two substrate types (substrate 1 and substrate 2), and three pitahaya species ( H. undatus , H. costaricensis , and H. megalanthus ). In total, twelve treatment combinations were evaluated with four replicates each, resulting in 48 experimental units. Ten cuttings were assessed per treatment, for a total of 480 cuttings across the entire experiment. Table 1 Description of treatments and interaction of factors. Treatment Combination of factors Rooting agent Substrate Species 1 Seaweed Substrate 1 H. undatus 2 Seaweed Substrate 1 H. megalanthus 3 Seaweed Substrate 1 H. costaricensis 4 Seaweed Substrate 2 H. undatus 5 Seaweed Substrate 2 H. megalanthus 6 Seaweed Substrate 2 H. costaricensis 7 NAA + IBA Substrate 2 H. undatus 8 NAA + IBA Substrate 2 H. megalanthus 9 NAA + IBA Substrate 2 H. costaricensis 10 NAA + IBA Substrate 1 H. undatus 11 NAA + IBA Substrate 1 H. megalanthus 12 NAA + IBA Substrate 1 H. costaricensis 2.4. Setting up the experiment 2.4.1. Preparation of vegetative seed Cuttings were obtained from healthy, pest-free mother plants at the productive stage (Fig. 2 ). Lignified cladodes were selected, and 40 cm segments were taken from the central portion, avoiding both the young apical region and the senescent basal end. Thus, vegetative material was collected from H. undatus , H. megalanthus , and H. costaricensis (Fig. 2 ). At the basal end of each cutting, a V-shaped incision was made to expose 5–7 cm of the vascular tissue. The cuttings were disinfected by immersion in a copper (II) sulfate pentahydrate (CuSO₄5H₂O) solution for 5 min and subsequently allowed to cure for 5 days in a dry, shaded environment under diffuse light. 2.4.2. Preparation of substrates The substrates used in the experiment differed in composition. Substrate 1 consisted of forest soil, compost, and cattle manure mixed in a 3:4:3 ratio, whereas Substrate 2 was composed of river sand and forest soil in a 4:6 ratio. Both substrates were sieved and sterilized using the solarization method with low-density polyethylene (LDPE) plastic sheets, which allow solar radiation to penetrate while trapping heat within the substrate, thereby disrupting the life cycle of soil-borne pathogenic organisms 27 . Following sterilization, the substrates were placed in 10 × 25 cm polyethylene bags with gussets and arranged according to the corresponding treatments and replicates. 2.4.3. Application of rooting inducers Two rooting agents were used: an Ecklonia maxima –based seaweed extract as an organic rooting agent (15 mL L⁻¹) and a synthetic rooting agent composed of 1-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) (2 mL L⁻¹). Both products function as biostimulants by modulating plant physiological responses 28 , 29 . Application was performed by immersing the basal end of each cutting in the respective solution for 10 min in the case of the seaweed extract and for 2 min in the case of the NAA + IBA solution prior to planting. 2.4.4. Planting and maintenance The basal end of each cutting was inserted into polyethylene bags containing sterilized substrate until the V-shaped incision was completely covered. The substrate was lightly compacted to ensure stability the cutting. All bags were labeled and arranged according to the experimental design and replicates. Plants were maintained in a nursery under a Raschel shade net providing 60% shade throughout the evaluation period. Routine maintenance practices, including irrigation, weed removal, and insect pest control, were performed manually, while fungal diseases were prevented through periodic applications of copper (II) sulfate pentahydrate (CuSO₄5H₂O). 2.5. Parameters evaluated Six variables were evaluated during the four months following planting: shoot emission time, cutting viability, number of shoots, shoot length, number of roots, and root length. In addition, a composite variable, termed the Vegetative Vigor Index, was calculated by integrating all evaluated variables except time to shoot emergence, due to its inverse nature. The evaluation period was defined based on the growth dynamics of the studied Hylocereus species and extended until the plants reached an optimal size for transplantation to the final field. Shoot emergence time (SET) This variable was assessed as the number of days from planting to the emergence of the first visible vegetative shoot (≥ 3 mm) from an areole of the cladode. Cutting viability (CV) The percentage of viable cuttings was determined by classifying cuttings that developed at least one shoot by the end of the nursery stage as viable. The calculation was performed using the formula adapted from Jose & Sivaprasad 30 . $$\:VE=\frac{Cuttingswith\ge\:1vegetativeshoot}{Totalnumberofcuttingsevaluated}x100\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(1\right)$$ Number of shoots (NS) This variable was assessed by counting the total number of visible vegetative shoots (≥ 3 mm) per cutting. Evaluations were conducted at 15-day intervals, and the value recorded at the final assessment was used for analysis. Dormant buds and senescent shoots were excluded from this variable. Shoot length (SL) This variable was measured by counting the total number of visible vegetative shoots (≥ 3 mm) per cutting. Assessments were conducted at 15-day intervals, and the value from the final assessment was used for analysis. Dormant buds and senescent shoots were excluded. Number of roots (NR) The number of roots per cutting was recorded at the end of the nursery stage. For this assessment, the substrate was carefully removed, and the root system was gently washed several times to avoid mechanical damage. Only roots emerging directly from the vascular bundle were counted. Root length (RL) The substrate was carefully removed from the root zone, and root length was measured using a tape measure graduated in centimeters, from the point of root emergence to the tip of the longest root. Vegetative vigor index (VVI) The Vegetative Vigor Index (VVI) was calculated using the formula adapted fromBharathKumar et al. 31 and Fetouh & Hassan 32 . $$\:VVI=\frac{\left(CV\right)*\left(NS+SL+NR+RL\right)}{100}\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(2\right)$$ 2.6. Statistical analysis Statistical analyses were conducted using R software version 4.4.3 33 with the packages dplyr 34 , agricolae 35 , ggplot2 36 , and corrplot 37 . All variables were tested for the normality of residuals using the Shapiro Wilk test (p ≤ 0.05) and for homogeneity of variances using Levene’s test. A three-way analysis of variance (ANOVA; p ≤ 0.05) was performed to evaluate the effects of rooting agents, substrates, and pitahaya species. Mean comparisons for individual factors and for treatment interactions were conducted using Tukey’s test. In addition, a Principal Component Analysis (PCA) and a Spearman correlation test were carried out to assess the relationships among the evaluated variables. 3. Results 3.1. Establishment Shoot emission time (SET) and cutting viability (CV) were evaluated during the establishment stage. The three-way ANOVA (A × B × C) indicated significant differences among treatments for both SET and CV (p < 0.05), with coefficients of variation of 5.40% and 16.43%, respectively (Supplementary Table 1). Regarding SET, Tukey’s multiple comparison test distinguished five statistically homogeneous groups (Fig. 3 A). The shortest shoot emission times were observed in treatments T9, T3, T12, and T6 (group d), with mean values of 21.53, 23.31, 23.43, and 24.71 days, respectively. In contrast, treatments T2 and T8 (group a) exhibited the longest emission times, averaging 68.19 and 67.87 days. The remaining treatments showed intermediate SET values, forming groups ab, bc, and c. For cutting viability, Tukey’s test identified four statistically distinct groups (Fig. 3 B). The highest viability was recorded in treatments T9, T3, and T12 (group a), all reaching 100%. Conversely, treatments T8 and T2 (group c) showed lower viability, with mean values of 40% and 42.5%, respectively. Intermediate viability levels were observed in the remaining treatments, which were classified into groups ab and bc. Figure 4 illustrates the response of pitahaya species to the interaction of rooting agents and substrates on shoot emission time (SET) and cutting viability (CV). For SET, the interaction between rooting agents and species (A × C) was not statistically significant, although a marginal trend was detected (p = 0.0518). Tukey’s multiple comparison test identified four statistically homogeneous groups (Fig. 4 A). The shortest SET was observed in H. costaricensis treated with NAA + IBA, with a mean value of 22.48 days, whereas the longest SET corresponded to H. megalanthus treated with the seaweed extract, averaging 68.03 days. H. undatus under both rooting agents showed intermediate SET values (Fig. 4 A). In contrast, the substrate × species interaction (B × C) significantly affected SET (p = 0.0184) (Fig. 4 B). Tukey’s test separated three distinct groups, with the shortest SET recorded in H. costaricensis grown in substrate 2 (23.12 days). Conversely, the longest SET was observed in H. megalanthus grown in substrate 1 (65.63 days), while H. undatus exhibited intermediate SET values under both substrates. Figure 4 C shows cutting viability (CV) as affected by the interaction between rooting agents and species (A × C), which was not statistically significant. Although the interaction was non-significant, Tukey’s multiple comparison test distinguished four statistically homogeneous groups. The highest CV values were observed in H. costaricensis treated with NAA + IBA and the seaweed extract, reaching 100% and 97.5%, respectively (group a). In contrast, H. megalanthus exhibited the lowest viability under NAA + IBA and seaweed extract treatments, with mean values of 52.5% and 61.25%, respectively (groups c and bc). H. undatus showed intermediate CV values under both rooting agents. Likewise, Fig. 4 D presents cutting viability (CV) in relation to the substrate × species interaction (B × C), which was also not statistically significant. Nevertheless, Tukey’s test identified four distinct groups. H. costaricensis achieved the highest viability when grown in substrates 1 and 2, with values of 100% and 97.5%, respectively. Conversely, H. megalanthus showed the lowest viability in substrates 1 and 2, reaching 53.75% and 60%, respectively, whereas H. undatus displayed intermediate CV values under both substrates. The species factor (C) had a significant effect on shoot emission time (SET) and cutting viability (CV) (p < 0.05) (Supplementary Table 1). Tukey’s multiple comparison test identified three statistically distinct groups for both variables. For SET, H. costaricensis and H. undatus exhibited the shortest emission times, whereas H. megalanthus showed the longest SET values (Fig. 5 A). With respect to CV, H. costaricensis displayed the highest viability, H. megalanthus the lowest, and H. undatus intermediate values (Fig. 5 B). 3.2. Vegetative development The variables number of shoots (NS), shoot length (SL), number of roots (NR), and root length (RL) were used to assess the vegetative development of pitahaya seedlings. Figure 6 shows representative plants from the 12 treatments evaluated, where marked morphological differences were observed in both the aerial and root systems. Treatment T10 displayed well-developed aerial and root structures compared to the other treatments. The ANOVA revealed significant differences among treatments (A × B × C) for number of shoots (NS), shoot length (SL), and root length (RL) (p < 0.05), whereas no significant effect was observed for number of roots (NR). The coefficients of variation for these variables ranged from 8.46% to 13.12% (Supplementary Table 2). Figure 7 A shows the number of shoots (NS), where Tukey’s multiple comparison test identified six statistically distinct groups. Treatment T7 presented the highest mean (1.62 shoots per plant; group a). In contrast, T2 and T8 exhibited the lowest values, with means of 1.06 and 1.08 shoots per plant, respectively (groups d and cd). The remaining treatments exhibited intermediate values, forming groups ab, abc, and bcd. For shoot length (SL) (Fig. 7 B), Tukey’s test also separated treatments into six statistically distinct groups. Treatments T3, T9, T12, and T6 showed the greatest mean shoot lengths (68.67, 67.99, 60.94, and 57.83 cm per plant, respectively; group a). Conversely, T5 and T2 had the lowest mean SL values (23.30 and 24.07 cm per plant, respectively; group d), whereas treatments classified into groups b, bc, bcd, and cd exhibited intermediate shoot lengths. Figure 7 C shows the number of roots (NR). Although the overall ANOVA did not detect significant differences among treatments, Tukey’s multiple comparison test distinguished five statistically homogeneous groups. The highest mean NR values were observed in treatments T3 and T12 (5.30 and 5.23 roots per plant, respectively; group a). In contrast, T8, T2, T11, and T5 exhibited the lowest means (3.28, 3.34, 3.43, and 3.49 roots per plant, respectively; group c). The remaining treatments displayed intermediate NR values (groups ab, abc, and bc). For root length (RL) (Fig. 7 D), Tukey’s test identified ten statistically distinct groups. Treatments T12 and T10 presented the greatest mean RL values (42.69 and 41.38 cm per plant, respectively; groups a and ab), whereas T8 and T2 showed the shortest root lengths (14.35 and 14.74 cm per plant, respectively; group g). The remaining treatments exhibited intermediate RL values, classified into groups abc, abcd, bcd, cd, de, ef, and fg. Figure 8 shows the response of the three Hylocereus species in terms of vegetative growth variables. ANOVA indicated significant differences among species for number of shoots (NS) and shoot length (SL) (p < 0.05), with coefficients of variation of 8.46% and 10.48%, respectively (Supplementary Material 2). For NS (Fig. 8 A), Tukey’s multiple comparison test identified three statistically distinct groups. H. undatus and H. costaricensis produced the highest numbers of shoots (1.47 and 1.33 shoots per plant; groups a and b, respectively), whereas H. megalanthus exhibited the lowest mean value (1.21 shoots per plant; group c). For SL (Fig. 8 B), Tukey’s test also distinguished three statistically distinct groups. H. costaricensis presented the greatest mean shoot length (63.83 cm; group a), while H. megalanthus showed the lowest value (25.79 cm; group c). H. undatus displayed an intermediate mean value and was classified in group b. Figures 8 C and 8 D show the response of the pitahaya species for number of roots (NR) and root length (RL), respectively. ANOVA indicated highly significant differences among species for both variables (p < 0.001), with coefficients of variation of 13.12% for NR and 9.61% for RL (Supplementary Material 2). For NR (Fig. 8 C), Tukey’s multiple comparison test identified three statistically distinct groups. H. costaricensis presented the highest mean number of roots (5.17 roots per plant; group a), whereas H. megalanthus exhibited the lowest value (3.38 roots per plant; group c). H. undatus showed an intermediate mean and was classified in group b. For RL (Fig. 8 D), Tukey’s test distinguished two statistically distinct groups. H. costaricensis and H. undatus exhibited the greatest mean root lengths (36.19 and 36.76 cm, respectively; group a), while H. megalanthus recorded the lowest mean value (18.57 cm; group b). 3.3. Vegetative vigor index (VVI) Figure 9 A shows the mean values of the 12 treatments (A × B × C) for the Vegetative Vigor Index (VVI). The ANOVA revealed significant differences (p < 0.05) with a coefficient of variation of 15.61% (Supplementary Table 3). Tukey’s multiple comparison test identified five statistically distinct groups. Treatments T12, T3, T9, and T6 presented the highest VVI values (110.18, 107.52, 105.64, and 98.41, respectively; group a), whereas T2 and T8 exhibited the lowest means (18.19 and 18.20, respectively; group d). The remaining treatments, classified into groups b, bc, and cd, displayed intermediate VVI values (Fig. 9 A). The VVI also differed significantly among Hylocereus species (p < 0.0001). According to Tukey’s test, H. costaricensis showed the highest mean index value (105.44; group a), while H. megalanthus exhibited the lowest mean (28.56; group c). H. undatus displayed an intermediate VVI and was classified in group b (Fig. 9 B). 3.4. Spearman correlation between evaluated variables Spearman’s correlation analysis (Fig. 10 ) showed significant associations among most of the variables evaluated. Due to its inverse nature, shoot emission time (SET) exhibited negative correlations with all other variables: shoot length (SL) (ρ = −0.90*), number of roots (NR) (ρ = −0.77*), root length (RL) (ρ = −0.51*), cutting viability (CV) (ρ = −0.73*), and vegetative vigor index (VVI) (ρ = −0.85*). In contrast, number of shoots (NS) showed weak correlations with most variables (ρ ≈ 0.16–0.25), although a moderate positive correlation was observed with RL (ρ = 0.49*). Among the vegetative growth variables, strong positive correlations were detected between SL and NR (ρ = 0.73*), RL (ρ = 0.57*), CV (ρ = 0.76*), and VVI (ρ = 0.89*). Likewise, NR was positively correlated with CV (ρ = 0.81*), VVI (ρ = 0.80*), and RL (ρ = 0.46*). The strongest positive association was observed between CV and VVI (ρ = 0.92*). 3.5. Principal component analysis (PCA) of the variables evaluated The principal component analysis (PCA) explained 87.3% of the total variance based on the first two dimensions (Dim1 = 69.3% and Dim2 = 18.0%) for the three Hylocereus species (Fig. 11 ). Dim1 represented a vegetative performance gradient, with strong positive loadings for shoot length (SL), number of roots (NR), root length (RL), cutting viability (CV), and vegetative vigor index (VVI), and a negative loading for shoot emission time (SET), reflecting its inverse relationship with the other traits. Thus, higher Dim1 values indicated more vigorous seedlings, characterized by greater rooting capacity and earlier shoot emergence. Dim2 captured secondary variation primarily associated with the number of shoots (NS). The species exhibited distinct distributions along the PCA axes. H. costaricensis was positioned toward higher values of SL, NR, RL, CV, and VVI, whereas H. megalanthus was associated with higher SET values. H. undatus occupied an intermediate position and was associated with higher NS values. 4. Discussion The results indicate that vegetative propagation of pitahaya through cuttings is strongly influenced by rooting agents, substrate type, species-specific responses, and the interaction among these factors. Regarding shoot emission time (SET), H. costaricensis consistently exhibited the shortest emission times, indicating faster shoot emergence compared with the other species evaluated. These findings agree with those reported by Chhetri et al. 8 , who observed earlier sprouting in H. costaricensis cuttings relative to other pitahaya varieties. In contrast, Anagha et al. 38 reported a mean sprouting time of 13.2 days for H. undatus grown in vermicompost, which is considerably shorter than the minimum value recorded in the present study (21.53 days). This discrepancy may be associated with substrate composition, as vermicompost is rich in readily available nutrients and supports high microbial activity, particularly enhancing nitrogen (N) and phosphorus (P) availability 39 – 41 . These conditions can accelerate metabolic processes related to bud activation and shoot emergence, thereby reducing the time required for sprouting. Similarly, Cardoso et al. 42 , who evaluated five Hylocereus species, reported that H. undatus exhibited greater rooting capacity and higher cutting viability than H. costaricensis . This contrasts with the findings of the present study, in which H. costaricensis showed the highest cutting viability (CV), surpassing H. undatus . In addition, H. megalanthus consistently exhibited the lowest CV values regardless of the rooting agent or substrate used. The reduced viability observed in this species may be associated with its ecological requirements, as H. megalanthus is better adapted to altitudinal ranges between 800 and 1800 m a.s.l. 43 . Differences in environmental adaptation may therefore influence rooting responses and propagation success among species. Regarding the variables associated with vegetative development, the responses of number of shoots (NS) and shoot length (SL) varied among treatments. At the interaction level (A × B × C), the highest NS value was observed in H. undatus established in substrate 2 and treated with NAA + IBA (T7), reaching a mean of 1.6 shoots per plant (Fig. 5 A). In comparison, Filho et al. 44 reported an average of 3.25 shoots per plant in H. undatus cuttings treated with IBA, a value considerably higher than that obtained in the present study. This difference, despite involving the same species and a similar rooting inducer, may be attributed to variation in substrate composition and environmental conditions, which play a key role in vegetative propagation processes 13 . For shoot length (SL), the highest mean value was recorded in H. costaricensis under the combination of substrate 1 and seaweed extract (T3), reaching 68.67 cm after four months of evaluation. By comparison, Cardoso et al. 42 reported shoot lengths of 27.25 cm and 22.18 cm for H. undatus and H. costaricensis , respectively, at 82 days, while Chhetri et al. 8 observed 52.1 cm at 90 days. These differences are likely associated with variation in evaluation period, as longer experimental durations generally allow greater shoot elongation to be expressed. Supporting this interpretation, Navarrete Torres et al. 45 reported a total plant height of 263.40 cm in H. undatus after nine months, highlighting the strong influence of developmental time on shoot growth in pitahaya. Successful rooting during vegetative propagation is strongly influenced by substrate moisture conditions and the use of rooting stimulants 46 , 47 . In the present study, the number of roots (NR) variable showed that treatments T3 and T12 produced the highest averages, with 5.30 and 5.23 roots per plant, respectively. This indicates that the species H. costaricensis and substrate 1 were the most decisive factors, while the type of rooting agent had no significant influence. By comparison, Cardoso et al. 42 evaluated the effect of biostimulants on pitahaya cuttings and reported averages of 15.13 and 9.13 roots per plant for H. undatus and H. costaricensis , respectively, using a commercial substrate enriched with river sand. These differences could be attributed to the physical and chemical properties of the substrate, as such formulations are specifically designed to optimize moisture balance and aeration for vegetative propagation. With respect to root length (RL), the highest values were observed in treatments combining NAA + IBA and substrate 1, particularly in H. costaricensis and H. undatus (treatments T12 and T10), which reached mean root lengths of 42.69 cm and 41.38 cm, respectively, after four months of evaluation. Previous studies have reported lower RL values, including 24.07 and 19.00 cm in H. undatus and H. costaricensis at 82 days 42 , 25.2 cm in H. costaricensis at 90 days 8 , and 13.6 cm in H. megalanthus at 90 days 48 . These differences are likely associated with variation in evaluation period, as the cited studies assessed rooting for less than four months. However, Navarrete Torres et al. 45 reported a maximum root length of 22.89 cm in H. undatus after nine months, suggesting that although evaluation time can influence root development, root elongation dynamics may vary depending on species and propagation conditions, particularly during early stages of vegetative establishment. Taken together, these results indicate that root development is influenced by both species-specific responses and the physicochemical properties of the substrate. The vegetative vigor index (VVI) integrates all evaluated variables except shoot emission time (SET), as this variable is inversely related to the other growth parameters. The results showed that treatments involving H. costaricensis consistently exhibited the highest VVI values. In particular, treatment T12 recorded the highest index, indicating that the interaction between H. costaricensis , NAA + IBA, and substrate 1 was associated with the greatest vegetative vigor. Notably, both the highest and lowest VVI values were obtained with substrate 1, suggesting that substrate alone may not fully determine variation in this index. Nevertheless, previous studies emphasize that substrate characteristics play a critical role in the vegetative propagation of pitahaya 49 . According to Campbell et al. 50 , selecting an appropriate substrate is essential, as low porosity can promote waterlogging and reduce oxygen availability in the rhizosphere 13 . In this study, substrate 1 may have contributed to improved vegetative development due to its composition. The combination of forest soil, compost, and cattle manure likely enhances nutrient availability, organic matter content, and microbial activity, which can promote root formation and overall plant growth 13 , 51 . Not all plants respond uniformly to a specific type of stimulant; this response may vary even among individuals of the same species 52 . In the present study, rooting agents significantly influenced the vegetative vigor index (VVI), with the combination of NAA + IBA being associated with higher VVI values compared to the seaweed extract. However, several studies have reported beneficial effects of seaweed-based products on vegetative propagation, attributing their activity to phenolic compounds and hormone-like substances that enhance endogenous auxin activity and nutrient assimilation 53 – 56 . Indeed, 57 observed superior growth responses with seaweed extracts compared to synthetic NAA in lettuce and tomato transplants, reflected in increased biomass accumulation and dry matter content. These contrasting findings suggest that the effectiveness of biostimulants may depend on species-specific responses, physiological status of the cuttings, and environmental conditions during propagation. Auxin based rooting agents containing NAA and IBA, which were associated with higher effectiveness than seaweed extract under the conditions of this study, are among the most widely used compounds in vegetative propagation due to their consistent efficacy across a wide range of plant species 17 . Their activity is primarily related to the stimulation of cell division, elongation, and differentiation processes that are essential for adventitious root initiation and development 58 , 59 . Numerous studies have reported that IBA, in particular, tends to promote superior rooting responses in cuttings compared with other plant growth regulators 60 – 62 . Although species of the genus Hylocereus can be cultivated under marginal agronomic conditions and warm climates 63 , 64 , their physiological responses can vary and may be constrained by thermal conditions, which are a determining factor for plant growth and development 65 . Based on the morphological differences observed across treatments (Fig. 6 ) and the results of the statistical analyses, species identity emerged as the most influential factor affecting vegetative propagation in pitahaya. Among the 12 treatments evaluated, H. costaricensis consistently exhibited shorter shoot emission time (SET), higher cutting viability (CV), greater shoot length (SL), number of roots (NR), and root length (RL), whereas number of shoots (NS) did not follow this same pattern. In contrast, H. megalanthus generally showed the lowest values for most vegetative growth variables, while H. undatus displayed intermediate responses. These differences suggest the presence of species-specific morphological and physiological traits that influence vegetative propagation responses. In particular, H. megalanthus appears to be less adapted to hot and arid environments compared with H. costaricensis and H. undatus 66 , 67 . which may partly explain its reduced rooting and growth responses under the conditions of this study. Furthermore, it has been demonstrated that plant responses to hormonal stimuli depend on the biochemical, physiological, and molecular capacities of each tissue to perceive and transduce these signals 68 , 69 , highlighting the importance of species specific sensitivity to growth regulators during vegetative propagation. These findings indicate that vegetative propagation of pitahaya is governed by a complex interaction among species-specific genetic, physiological, and environmental factors. Although rooting agents and substrate type influenced vegetative development, their effects were strongly modulated by species identity and prevailing propagation conditions. The results provide robust evidence for optimizing propagation protocols in Hylocereus spp. and underscore the need for further research addressing field adaptation, as well as the physiological and molecular mechanisms underlying the interspecific differences observed. Collectively, these insights contribute to improving propagation efficiency, strengthening production strategies, and promoting the long-term sustainability of pitahaya cultivation in tropical regions. 5. Conclusions Vegetative propagation of pitahaya was influenced by the interaction among multiple factors, with species identity emerging as the primary determining factor. Among the species evaluated, Hylocereus costaricensis exhibited the most consistent and favorable responses in establishment and vegetative growth variables, as well as the highest vegetative vigor index (VVI). Regarding rooting agents, the application of NAA + IBA was associated with higher responses than the seaweed extract ( Ecklonia maxima ), particularly in root development and overall seedling vigor, supporting the effectiveness of synthetic auxins in stimulating cell division and elongation processes. With respect to the propagation medium, substrate 1 composed of forest soil, compost, and cattle manure resulted in higher values for root and vegetative growth variables compared with the other substrates evaluated. The interaction between H. costaricensis , substrate 1, and NAA + IBA (treatment T12) produced the highest VVI, identifying this combination as the most favorable for vegetative propagation under nursery conditions. Collectively, these findings provide a solid scientific basis for the development of large-scale propagation programs aimed at producing high-quality pitahaya seedlings, thereby contributing to the sustainability, productivity, and expansion of pitahaya cultivation in tropical regions of Peru. Declarations CRediT authorship contribution statement B.C.C : Writing – original draft, Formal Analysis, Data Curation, Conceptualization; J.M.C.M : Research, Methodology; J.A.C.S : Data Curation, Formal Analysis; M.A.I : Research, Methodology; V.H.T.M : Project Management, Financial Procurement; D.G.F : Visualization, Formal Analysis; J.T.C : Investigation, Methodology; M.G : Visualization, Validation; D.T : Visualization, Validation, Formal Analysis. Declaration of competing interest The authors declare that they have no conflicts of interest. The authors declare that they have no competing financial interests or personal relationships that could influence the work presented in this article. Supplementary material Supplementary material associated with this article can be found, in the online version, at Funding This research was funded by the Instituto Nacional de Innovación Agraria (INIA), through the investment project with CUI N° 2472675 “Mejoramiento de los Servicios de Investigación y Transferencia de Tecnología Agraria en la Estación Experimental Agraria Baños del Inca en la localidad Los Baños del Inca del distrito Los Baños del Inca - provincia de Cajamarca - departamento de Cajamarca” Author Contribution B.C.C: Writing – original draft, Formal Analysis, Data Curation, Conceptualization; J.M.C.M: Research, Methodology; J.A.C.S: Data Curation, Formal Analysis; M.A.I: Research, Methodology; V.H.T.M: Project Management, Financial Procurement; D.G.F: Visualization, Formal Analysis; J.T.C: Investigation, Methodology; M.G: Visualization, Validation; D.T: Visualization, Validation, Formal Analysis. Data Availability Data will be made available on request. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9180319","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":617157365,"identity":"ff4f3517-9a04-4c4f-a7cb-9a444e3bc329","order_by":0,"name":"Beimer Chuquibala-Checan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYBACAwYeECWRwMDOwPgAyOLhI14LMwOzAUgLG5FaGEBa2CRALIJazNnPHpN422aRx9/M/Kzya46dDBsD88NHN/BosezJS5Oc2yZRLHGYzey27LZkoMPYjI1z8DnsBo+ZNG+bRGLDYQaz25LbmIFaeNikidIy/zD7t2LJbfUkaNlwmMeM8eO2w4S1WPbkGFvOOSdRbHiYp1iacdtxHjZmAn4xZz9jeONNWV2e3PH2jR9/bqu252dvfvgYnxYw4IHSzGAGMyHlyFoYfxCjehSMglEwCkYcAAChED9G7T1PfAAAAABJRU5ErkJggg==","orcid":"","institution":"Instituto Nacional de Innovación Agraria","correspondingAuthor":true,"prefix":"","firstName":"Beimer","middleName":"","lastName":"Chuquibala-Checan","suffix":""},{"id":617157369,"identity":"c6477a39-4ffe-491c-b5a7-a5577967747e","order_by":1,"name":"Jonathan M. Cruz-Malca","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"M.","lastName":"Cruz-Malca","suffix":""},{"id":617157372,"identity":"265cffbb-bfe4-489e-9684-b17d15a6a30f","order_by":2,"name":"Jefferson A. Cubas Sanchez","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria","correspondingAuthor":false,"prefix":"","firstName":"Jefferson","middleName":"A. Cubas","lastName":"Sanchez","suffix":""},{"id":617157374,"identity":"65f45166-6a69-41b0-91d7-b740e4cf8808","order_by":3,"name":"Marielita Arce-Inga","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria","correspondingAuthor":false,"prefix":"","firstName":"Marielita","middleName":"","lastName":"Arce-Inga","suffix":""},{"id":617157376,"identity":"db721878-252a-4515-8b33-6122af6bdea3","order_by":4,"name":"Víctor H. Taboada-Mitma","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria","correspondingAuthor":false,"prefix":"","firstName":"Víctor","middleName":"H.","lastName":"Taboada-Mitma","suffix":""},{"id":617157377,"identity":"0645bc87-5d7d-4043-af73-a9e9c2dc0835","order_by":5,"name":"Darwin Gómez-Fernández","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria","correspondingAuthor":false,"prefix":"","firstName":"Darwin","middleName":"","lastName":"Gómez-Fernández","suffix":""},{"id":617157379,"identity":"698855bd-0119-4d92-ba79-b139eebbdb8a","order_by":6,"name":"Josué Tafur-Culqui","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria","correspondingAuthor":false,"prefix":"","firstName":"Josué","middleName":"","lastName":"Tafur-Culqui","suffix":""},{"id":617157380,"identity":"3262e300-1f7a-4383-b047-2ce8d630097e","order_by":7,"name":"Malluri Goñas","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria","correspondingAuthor":false,"prefix":"","firstName":"Malluri","middleName":"","lastName":"Goñas","suffix":""},{"id":617157381,"identity":"adc41218-8b23-4e17-b7d9-d6aba99cadc2","order_by":8,"name":"Daniel Tineo","email":"","orcid":"","institution":"Instituto Nacional de Innovación Agraria","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Tineo","suffix":""}],"badges":[],"createdAt":"2026-03-20 15:24:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9180319/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9180319/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107704762,"identity":"edac5eb5-7b7d-435c-b742-1e383277bc78","added_by":"auto","created_at":"2026-04-24 08:57:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":73799594,"visible":true,"origin":"","legend":"\u003cp\u003eGeographical location of the Center for Plant Production for Research and Technology Transfer at Centro Experimental Yanayacu, province of Jaén, Cajamarca.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/c5f49e3f0749ba1d57d46253.jpg"},{"id":108180710,"identity":"ba033f4e-55e2-40bd-b85f-d3704f48d471","added_by":"auto","created_at":"2026-04-30 08:52:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20735576,"visible":true,"origin":"","legend":"\u003cp\u003eObtaining cuttings from a mother plant and vegetative material from three species of pitahaya. A) \u003cem\u003eH. undatus\u003c/em\u003e, B) \u003cem\u003eH. megalanthus\u003c/em\u003e and C) \u003cem\u003eH. costaricensis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/87b0c63ea473ef139c840ebd.png"},{"id":107480410,"identity":"9f2ba793-f7a6-4838-aa55-fd02c695ffeb","added_by":"auto","created_at":"2026-04-22 02:10:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1012551,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 12 treatments (A × B × C) on cutting establishment variables. (A) Shoot emission time (SET) and (B) percentage of cutting viability (CV, %). Different letters above the bars indicate statistically significant differences according to Tukey’s test (p ≤ 0.05).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/60706261325295d4d4afe0e0.jpg"},{"id":107480331,"identity":"3573890e-5fa7-4f58-9144-050c65e78067","added_by":"auto","created_at":"2026-04-22 02:08:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7117855,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of rooting agents and substrates on three \u003cem\u003eHylocereus\u003c/em\u003especies. (A) Effect of rooting agents on shoot emission time (SET); (B) effect of substrates on shoot emission time (SET); (C) effect of rooting agents on cutting viability (CV); and (D) effect of substrates on cutting viability (CV). Different letters above the bars indicate statistically significant differences according to Tukey’s test (p ≤ 0.05).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/22a6d16c96249bc04ffa038f.jpg"},{"id":107868704,"identity":"5483b791-0a8e-4231-b4c8-79f618c32410","added_by":"auto","created_at":"2026-04-27 07:32:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":825662,"visible":true,"origin":"","legend":"\u003cp\u003ePerformance of three \u003cem\u003eHylocereus\u003c/em\u003e species in establishment variables. (A) Shoot emission time (SET) and (B) cutting viability (CV). Different letters above the bars indicate statistically significant differences according to Tukey’s test (p ≤ 0.05).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/a0610237d116c40c7b032aad.jpg"},{"id":107480412,"identity":"5384c5fa-56af-405d-9939-5083753dddfd","added_by":"auto","created_at":"2026-04-22 02:10:08","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5322932,"visible":true,"origin":"","legend":"\u003cp\u003ePitahaya seedlings corresponding to the 12 treatments evaluated in the study. Scale bar = 5 cm.\u003c/p\u003e","description":"","filename":"Fgure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/9e6bb6868023c8b96ea967a6.jpg"},{"id":107480398,"identity":"b4f20f1b-f64c-45fe-9bab-08f42fc52fa7","added_by":"auto","created_at":"2026-04-22 02:09:51","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1672702,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 12 treatments (A × B × C) on growth variables of pitahaya seedlings. (A) Number of shoots (NS); (B) shoot length (SL); (C) number of roots (NR); and (D) root length (RL). Different letters above the bars indicate statistically significant differences according to Tukey’s test (p ≤ 0.05).\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/deaa1f011898896d932d9e93.jpg"},{"id":108005926,"identity":"d9642fef-5f76-477e-8bcf-47f9985d9489","added_by":"auto","created_at":"2026-04-28 12:50:55","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1356144,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth response of three pitahaya species. A) Number of shoots, B) Shoot length, C) Number of roots, and D) Root length. Different letters indicate statistically different groups according to the Tukey test (p ≤ 0.05).\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/cb5f1fc4eb1b2104ab015558.jpg"},{"id":107480526,"identity":"2d3afbd5-e3f3-48e0-88f0-b04435f4b23e","added_by":"auto","created_at":"2026-04-22 02:11:49","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1034030,"visible":true,"origin":"","legend":"\u003cp\u003eVegetative vigor index (VVI) of (A) 12 treatments and (B) three \u003cem\u003eHylocereus\u003c/em\u003e species. Different letters above the boxes indicate statistically significant differences according to Tukey’s test (p ≤ 0.05).\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/cbfac5683a14f24e5e92159b.jpg"},{"id":107480332,"identity":"f5d85491-eaa5-42f5-806e-cdc69641acbf","added_by":"auto","created_at":"2026-04-22 02:08:44","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":3871910,"visible":true,"origin":"","legend":"\u003cp\u003eSpearman correlation matrix with density and scatter plots by species for the seven variables evaluated in \u003cem\u003eHylocereus\u003c/em\u003e spp.\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/1924c02d7e305463330c1a01.jpg"},{"id":107868586,"identity":"868a4d2e-9e87-4055-bc6c-525827b50236","added_by":"auto","created_at":"2026-04-27 07:28:15","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":798429,"visible":true,"origin":"","legend":"\u003cp\u003ePCA biplot of vegetative growth variables in \u003cem\u003eHylocereus\u003c/em\u003e species, showing the first two principal components (Dim1 and Dim2), variable loading vectors, and 95% confidence ellipses for species grouping.\u003c/p\u003e","description":"","filename":"Figure11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/3d5985cbd1e3b9c9939ca80c.jpg"},{"id":108184933,"identity":"214a7d72-73e8-485c-9ebe-0c3612f7da05","added_by":"auto","created_at":"2026-04-30 09:05:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":121048135,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/2df0f5ab-d4f6-445f-8f52-d63fd88daee2.pdf"},{"id":107480408,"identity":"7724c49b-730f-4b0f-95dc-1dd157032be4","added_by":"auto","created_at":"2026-04-22 02:10:07","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23248,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/4a352f57d49285be3cc5cb72.docx"},{"id":108180823,"identity":"50429523-a727-44c9-8a43-050e081e3f17","added_by":"auto","created_at":"2026-04-30 08:54:07","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":27920269,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-9180319/v1/303c5f1c78aef2da7e9cb10c.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of root inducers and substrates on the vegetative propagation of pitahaya species (Hylocereus spp.) under nursery conditions","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePitahaya (\u003cem\u003eHylocereus\u003c/em\u003e spp.), commonly known as dragon fruit, is a cactus belonging to the genus \u003cem\u003eHylocereus\u003c/em\u003e within the family Cactaceae. The genus comprises approximately 14 species, of which \u003cem\u003eH. undatus\u003c/em\u003e, \u003cem\u003eH. monacanthus\u003c/em\u003e (Lem.) Britton \u0026amp; Rose, \u003cem\u003eH. costaricensis\u003c/em\u003e, and \u003cem\u003eH. megalanthus\u003c/em\u003e are among the most widely cultivated worldwide \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Species of this genus are native to tropical and subtropical regions of the Americas \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, and have high export potential because of their nutritional value and adaptability to a wide range of soil and climatic conditions \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eGlobal pitahaya production has increased steadily, with Vietnam, China, and Indonesia together accounting for nearly 93% of total global output \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In Latin America, pitahaya has gained importance as an alternative crop that improves rural livelihoods and contributes to agricultural sustainability and diversification \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In Peru, the cultivated area expanded markedly from 34.2 ha in 2019 to 419.2 ha in 2023, accompanied by a rapid increase in production from 156.9 t to 4,178.9 t \u003csup\u003e6\u003c/sup\u003e. At the regional level, Lima and Piura lead national production, accounting for 48.4% and 36.1% of total output, respectively, followed by Ica, Amazonas, Moquegua, and La Libertad \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. These trends indicate growing interest in pitahaya cultivation and its potential as a sustainable and commercially viable crop. However, the rapid expansion of cultivated areas also poses new challenges, particularly regarding the availability of high-quality genetic material to support newly established plantations \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough pitahaya can be propagated by seed, this approach does not ensure genetic uniformity. Therefore, vegetative propagation through stem cuttings is preferred as a more efficient approach \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This method is widely used because vegetatively propagated plants exhibit faster growth, earlier fruiting, and greater genetic stability \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Achieving effective vegetative propagation depends on several key factors that play a crucial role during the rooting stage \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Among these, the application of auxins and the selection of an appropriate rooting substrate are considered critical \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Understanding and optimizing these factors are essential to ensure successful propagation and enhance productivity at the commercial scale.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that the choice of rooting agent, whether synthetic or organic, has a significant effect on the formation of adventitious roots in cuttings \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, as well as root length and shoot development in vegetatively propagated species \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Among synthetic rooting agents, the most widely used auxins across a wide range of plant species are indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), which promote cell division and differentiation, although their effectiveness depends on concentration and plant species \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Moreover, seaweed extracts derived from Ecklonia maxima contain phytohormones such as auxins, cytokinins, ethylene, and gibberellins, which can stimulate root formation and overall plant growth \u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These extracts have also been reported to improve soil health, plant growth, and stress tolerance through the action of natural phytohormones, amino acids, polysaccharides, and micronutrients \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Accordingly, comparing synthetic and organic rooting agents is essential for developing efficient and sustainable propagation strategies for pitahaya.\u003c/p\u003e \u003cp\u003eIn addition, substrate composition is a key factor influencing the success of cutting establishment, as it is essential for moisture retention and nutrient availability during the rooting process \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. One of the most suitable substrates can be formulated by combining organic components such as sawdust, peat, rice husks, and cattle manure, which together provide optimal physical and chemical conditions for root development and cutting establishment \u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven the increasing commercial demand for pitahaya and the limited technical information available on its efficient propagation in tropical regions, there is a need for studies aimed at optimizing seedling production. In this context, the present study evaluated the effect of two types of rooting agents and two types of substrates on the vegetative propagation of three \u003cem\u003eHylocereus\u003c/em\u003e spp. species in a controlled nursery environment. We hypothesized that an appropriate combination of rooting agents and substrates would significantly improve rooting success and promote the development of vigorous and high-quality pitahaya seedlings.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Study area\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe study was conducted at the Center for Plant Production for Research and Technology Transfer, Centro Experimental Yanayacu, Instituto Nacional de Innovaci\u0026oacute;n Agraria (INIA), located in the province of Ja\u0026eacute;n, Cajamarca Department, Peru (5.6771\u0026deg; S, 78.7749\u0026deg; W), at an elevation of 624 m a.s.l. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The site is characterized by a mean annual temperature of 28\u0026deg;C and an average annual precipitation of approximately 1200 mm \u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Biological material\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThree \u003cem\u003eHylocereus\u003c/em\u003e species were evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e): two red-skinned species, \u003cem\u003eH. undatus\u003c/em\u003e and \u003cem\u003eH. costaricensis\u003c/em\u003e, collected from mature pitahaya plantations at Grupo Campo Agro S.A., in the province of Zarumilla, Tumbes department; and one yellow-skinned species, \u003cem\u003eH. megalanthus\u003c/em\u003e, sourced from plantations in the province of San Ignacio. Species selection was driven by their adaptability to tropical environments, favorable agronomic and organoleptic traits, and strong commercial demand in both domestic and international markets.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Experimental design\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe experiment was conducted using a completely randomized design (CRD) with a 2A \u0026times; 2B \u0026times; 3C factorial arrangement, consisting of two rooting inducers (seaweed extract and a solution of NAA and IBA), two substrate types (substrate 1 and substrate 2), and three pitahaya species (\u003cem\u003eH. undatus\u003c/em\u003e, \u003cem\u003eH. costaricensis\u003c/em\u003e, and \u003cem\u003eH. megalanthus\u003c/em\u003e). In total, twelve treatment combinations were evaluated with four replicates each, resulting in 48 experimental units. Ten cuttings were assessed per treatment, for a total of 480 cuttings across the entire experiment.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDescription of treatments and interaction of factors.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eCombination of factors\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRooting agent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeaweed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eH. undatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeaweed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eH. megalanthus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeaweed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eH. costaricensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeaweed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eH. undatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeaweed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eH. megalanthus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeaweed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eH. costaricensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNAA\u0026thinsp;+\u0026thinsp;IBA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eH. undatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNAA\u0026thinsp;+\u0026thinsp;IBA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eH. megalanthus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNAA\u0026thinsp;+\u0026thinsp;IBA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eH. costaricensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNAA\u0026thinsp;+\u0026thinsp;IBA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eH. undatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNAA\u0026thinsp;+\u0026thinsp;IBA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eH. megalanthus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNAA\u0026thinsp;+\u0026thinsp;IBA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubstrate 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eH. costaricensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Setting up the experiment\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Preparation of vegetative seed\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCuttings were obtained from healthy, pest-free mother plants at the productive stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Lignified cladodes were selected, and 40 cm segments were taken from the central portion, avoiding both the young apical region and the senescent basal end. Thus, vegetative material was collected from \u003cem\u003eH. undatus\u003c/em\u003e, \u003cem\u003eH. megalanthus\u003c/em\u003e, and \u003cem\u003eH. costaricensis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At the basal end of each cutting, a V-shaped incision was made to expose 5\u0026ndash;7 cm of the vascular tissue. The cuttings were disinfected by immersion in a copper (II) sulfate pentahydrate (CuSO₄5H₂O) solution for 5 min and subsequently allowed to cure for 5 days in a dry, shaded environment under diffuse light.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Preparation of substrates\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe substrates used in the experiment differed in composition. Substrate 1 consisted of forest soil, compost, and cattle manure mixed in a 3:4:3 ratio, whereas Substrate 2 was composed of river sand and forest soil in a 4:6 ratio. Both substrates were sieved and sterilized using the solarization method with low-density polyethylene (LDPE) plastic sheets, which allow solar radiation to penetrate while trapping heat within the substrate, thereby disrupting the life cycle of soil-borne pathogenic organisms \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Following sterilization, the substrates were placed in 10 \u0026times; 25 cm polyethylene bags with gussets and arranged according to the corresponding treatments and replicates.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. Application of rooting inducers\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTwo rooting agents were used: an \u003cem\u003eEcklonia maxima\u003c/em\u003e\u0026ndash;based seaweed extract as an organic rooting agent (15 mL L⁻\u0026sup1;) and a synthetic rooting agent composed of 1-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) (2 mL L⁻\u0026sup1;). Both products function as biostimulants by modulating plant physiological responses \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Application was performed by immersing the basal end of each cutting in the respective solution for 10 min in the case of the seaweed extract and for 2 min in the case of the NAA\u0026thinsp;+\u0026thinsp;IBA solution prior to planting.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4. Planting and maintenance\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe basal end of each cutting was inserted into polyethylene bags containing sterilized substrate until the V-shaped incision was completely covered. The substrate was lightly compacted to ensure stability the cutting. All bags were labeled and arranged according to the experimental design and replicates. Plants were maintained in a nursery under a Raschel shade net providing 60% shade throughout the evaluation period. Routine maintenance practices, including irrigation, weed removal, and insect pest control, were performed manually, while fungal diseases were prevented through periodic applications of copper (II) sulfate pentahydrate (CuSO₄5H₂O).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Parameters evaluated\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSix variables were evaluated during the four months following planting: shoot emission time, cutting viability, number of shoots, shoot length, number of roots, and root length. In addition, a composite variable, termed the Vegetative Vigor Index, was calculated by integrating all evaluated variables except time to shoot emergence, due to its inverse nature. The evaluation period was defined based on the growth dynamics of the studied \u003cem\u003eHylocereus\u003c/em\u003e species and extended until the plants reached an optimal size for transplantation to the final field.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eShoot emergence time (SET)\u003c/em\u003e \u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThis variable was assessed as the number of days from planting to the emergence of the first visible vegetative shoot (\u0026ge;\u0026thinsp;3 mm) from an areole of the cladode.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eCutting viability (CV)\u003c/em\u003e \u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe percentage of viable cuttings was determined by classifying cuttings that developed at least one shoot by the end of the nursery stage as viable. The calculation was performed using the formula adapted from Jose \u0026amp; Sivaprasad \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:VE=\\frac{Cuttingswith\\ge\\:1vegetativeshoot}{Totalnumberofcuttingsevaluated}x100\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(1\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eNumber of shoots (NS)\u003c/em\u003e \u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThis variable was assessed by counting the total number of visible vegetative shoots (\u0026ge;\u0026thinsp;3 mm) per cutting. Evaluations were conducted at 15-day intervals, and the value recorded at the final assessment was used for analysis. Dormant buds and senescent shoots were excluded from this variable.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eShoot length (SL)\u003c/em\u003e \u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThis variable was measured by counting the total number of visible vegetative shoots (\u0026ge;\u0026thinsp;3 mm) per cutting. Assessments were conducted at 15-day intervals, and the value from the final assessment was used for analysis. Dormant buds and senescent shoots were excluded.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eNumber of roots (NR)\u003c/em\u003e \u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe number of roots per cutting was recorded at the end of the nursery stage. For this assessment, the substrate was carefully removed, and the root system was gently washed several times to avoid mechanical damage. Only roots emerging directly from the vascular bundle were counted.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eRoot length (RL)\u003c/em\u003e \u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe substrate was carefully removed from the root zone, and root length was measured using a tape measure graduated in centimeters, from the point of root emergence to the tip of the longest root.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eVegetative vigor index (VVI)\u003c/em\u003e \u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe Vegetative Vigor Index (VVI) was calculated using the formula adapted fromBharathKumar et al.\u003csup\u003e31\u003c/sup\u003e and Fetouh \u0026amp; Hassan \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:VVI=\\frac{\\left(CV\\right)*\\left(NS+SL+NR+RL\\right)}{100}\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(2\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using R software version 4.4.3 \u003csup\u003e33\u003c/sup\u003e with the packages dplyr \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, agricolae \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, ggplot2 \u003csup\u003e36\u003c/sup\u003e, and corrplot \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. All variables were tested for the normality of residuals using the Shapiro Wilk test (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) and for homogeneity of variances using Levene\u0026rsquo;s test. A three-way analysis of variance (ANOVA; p\u0026thinsp;\u0026le;\u0026thinsp;0.05) was performed to evaluate the effects of rooting agents, substrates, and pitahaya species. Mean comparisons for individual factors and for treatment interactions were conducted using Tukey\u0026rsquo;s test. In addition, a Principal Component Analysis (PCA) and a Spearman correlation test were carried out to assess the relationships among the evaluated variables.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Establishment\u003c/h2\u003e \u003cp\u003eShoot emission time (SET) and cutting viability (CV) were evaluated during the establishment stage.\u003c/p\u003e \u003cp\u003eThe three-way ANOVA (A \u0026times; B \u0026times; C) indicated significant differences among treatments for both SET and CV (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with coefficients of variation of 5.40% and 16.43%, respectively (Supplementary Table\u0026nbsp;1). Regarding SET, Tukey\u0026rsquo;s multiple comparison test distinguished five statistically homogeneous groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The shortest shoot emission times were observed in treatments T9, T3, T12, and T6 (group d), with mean values of 21.53, 23.31, 23.43, and 24.71 days, respectively. In contrast, treatments T2 and T8 (group a) exhibited the longest emission times, averaging 68.19 and 67.87 days. The remaining treatments showed intermediate SET values, forming groups ab, bc, and c.\u003c/p\u003e \u003cp\u003eFor cutting viability, Tukey\u0026rsquo;s test identified four statistically distinct groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The highest viability was recorded in treatments T9, T3, and T12 (group a), all reaching 100%. Conversely, treatments T8 and T2 (group c) showed lower viability, with mean values of 40% and 42.5%, respectively. Intermediate viability levels were observed in the remaining treatments, which were classified into groups ab and bc.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates the response of pitahaya species to the interaction of rooting agents and substrates on shoot emission time (SET) and cutting viability (CV). For SET, the interaction between rooting agents and species (A \u0026times; C) was not statistically significant, although a marginal trend was detected (p\u0026thinsp;=\u0026thinsp;0.0518). Tukey\u0026rsquo;s multiple comparison test identified four statistically homogeneous groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The shortest SET was observed in \u003cem\u003eH. costaricensis\u003c/em\u003e treated with NAA\u0026thinsp;+\u0026thinsp;IBA, with a mean value of 22.48 days, whereas the longest SET corresponded to \u003cem\u003eH. megalanthus\u003c/em\u003e treated with the seaweed extract, averaging 68.03 days. \u003cem\u003eH. undatus\u003c/em\u003e under both rooting agents showed intermediate SET values (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, the substrate \u0026times; species interaction (B \u0026times; C) significantly affected SET (p\u0026thinsp;=\u0026thinsp;0.0184) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Tukey\u0026rsquo;s test separated three distinct groups, with the shortest SET recorded in \u003cem\u003eH. costaricensis\u003c/em\u003e grown in substrate 2 (23.12 days). Conversely, the longest SET was observed in \u003cem\u003eH. megalanthus\u003c/em\u003e grown in substrate 1 (65.63 days), while \u003cem\u003eH. undatus\u003c/em\u003e exhibited intermediate SET values under both substrates.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC shows cutting viability (CV) as affected by the interaction between rooting agents and species (A \u0026times; C), which was not statistically significant. Although the interaction was non-significant, Tukey\u0026rsquo;s multiple comparison test distinguished four statistically homogeneous groups. The highest CV values were observed in \u003cem\u003eH. costaricensis\u003c/em\u003e treated with NAA\u0026thinsp;+\u0026thinsp;IBA and the seaweed extract, reaching 100% and 97.5%, respectively (group a). In contrast, \u003cem\u003eH. megalanthus\u003c/em\u003e exhibited the lowest viability under NAA\u0026thinsp;+\u0026thinsp;IBA and seaweed extract treatments, with mean values of 52.5% and 61.25%, respectively (groups c and bc). \u003cem\u003eH. undatus\u003c/em\u003e showed intermediate CV values under both rooting agents. Likewise, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD presents cutting viability (CV) in relation to the substrate \u0026times; species interaction (B \u0026times; C), which was also not statistically significant. Nevertheless, Tukey\u0026rsquo;s test identified four distinct groups. \u003cem\u003eH. costaricensis\u003c/em\u003e achieved the highest viability when grown in substrates 1 and 2, with values of 100% and 97.5%, respectively. Conversely, \u003cem\u003eH. megalanthus\u003c/em\u003e showed the lowest viability in substrates 1 and 2, reaching 53.75% and 60%, respectively, whereas \u003cem\u003eH. undatus\u003c/em\u003e displayed intermediate CV values under both substrates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe species factor (C) had a significant effect on shoot emission time (SET) and cutting viability (CV) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Supplementary Table\u0026nbsp;1). Tukey\u0026rsquo;s multiple comparison test identified three statistically distinct groups for both variables. For SET, \u003cem\u003eH. costaricensis\u003c/em\u003e and \u003cem\u003eH. undatus\u003c/em\u003e exhibited the shortest emission times, whereas \u003cem\u003eH. megalanthus\u003c/em\u003e showed the longest SET values (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). With respect to CV, \u003cem\u003eH. costaricensis\u003c/em\u003e displayed the highest viability, \u003cem\u003eH. megalanthus\u003c/em\u003e the lowest, and \u003cem\u003eH. undatus\u003c/em\u003e intermediate values (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Vegetative development\u003c/h2\u003e \u003cp\u003eThe variables number of shoots (NS), shoot length (SL), number of roots (NR), and root length (RL) were used to assess the vegetative development of pitahaya seedlings. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows representative plants from the 12 treatments evaluated, where marked morphological differences were observed in both the aerial and root systems. Treatment T10 displayed well-developed aerial and root structures compared to the other treatments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe ANOVA revealed significant differences among treatments (A \u0026times; B \u0026times; C) for number of shoots (NS), shoot length (SL), and root length (RL) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas no significant effect was observed for number of roots (NR). The coefficients of variation for these variables ranged from 8.46% to 13.12% (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA shows the number of shoots (NS), where Tukey\u0026rsquo;s multiple comparison test identified six statistically distinct groups. Treatment T7 presented the highest mean (1.62 shoots per plant; group a). In contrast, T2 and T8 exhibited the lowest values, with means of 1.06 and 1.08 shoots per plant, respectively (groups d and cd). The remaining treatments exhibited intermediate values, forming groups ab, abc, and bcd.\u003c/p\u003e \u003cp\u003eFor shoot length (SL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), Tukey\u0026rsquo;s test also separated treatments into six statistically distinct groups. Treatments T3, T9, T12, and T6 showed the greatest mean shoot lengths (68.67, 67.99, 60.94, and 57.83 cm per plant, respectively; group a). Conversely, T5 and T2 had the lowest mean SL values (23.30 and 24.07 cm per plant, respectively; group d), whereas treatments classified into groups b, bc, bcd, and cd exhibited intermediate shoot lengths.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC shows the number of roots (NR). Although the overall ANOVA did not detect significant differences among treatments, Tukey\u0026rsquo;s multiple comparison test distinguished five statistically homogeneous groups. The highest mean NR values were observed in treatments T3 and T12 (5.30 and 5.23 roots per plant, respectively; group a). In contrast, T8, T2, T11, and T5 exhibited the lowest means (3.28, 3.34, 3.43, and 3.49 roots per plant, respectively; group c). The remaining treatments displayed intermediate NR values (groups ab, abc, and bc).\u003c/p\u003e \u003cp\u003eFor root length (RL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), Tukey\u0026rsquo;s test identified ten statistically distinct groups. Treatments T12 and T10 presented the greatest mean RL values (42.69 and 41.38 cm per plant, respectively; groups a and ab), whereas T8 and T2 showed the shortest root lengths (14.35 and 14.74 cm per plant, respectively; group g). The remaining treatments exhibited intermediate RL values, classified into groups abc, abcd, bcd, cd, de, ef, and fg.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows the response of the three \u003cem\u003eHylocereus\u003c/em\u003e species in terms of vegetative growth variables. ANOVA indicated significant differences among species for number of shoots (NS) and shoot length (SL) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with coefficients of variation of 8.46% and 10.48%, respectively (Supplementary Material 2). For NS (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA), Tukey\u0026rsquo;s multiple comparison test identified three statistically distinct groups. \u003cem\u003eH. undatus\u003c/em\u003e and \u003cem\u003eH. costaricensis\u003c/em\u003e produced the highest numbers of shoots (1.47 and 1.33 shoots per plant; groups a and b, respectively), whereas \u003cem\u003eH. megalanthus\u003c/em\u003e exhibited the lowest mean value (1.21 shoots per plant; group c). For SL (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), Tukey\u0026rsquo;s test also distinguished three statistically distinct groups. \u003cem\u003eH. costaricensis\u003c/em\u003e presented the greatest mean shoot length (63.83 cm; group a), while \u003cem\u003eH. megalanthus\u003c/em\u003e showed the lowest value (25.79 cm; group c). \u003cem\u003eH. undatus\u003c/em\u003e displayed an intermediate mean value and was classified in group b.\u003c/p\u003e \u003cp\u003eFigures \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD show the response of the pitahaya species for number of roots (NR) and root length (RL), respectively. ANOVA indicated highly significant differences among species for both variables (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with coefficients of variation of 13.12% for NR and 9.61% for RL (Supplementary Material 2). For NR (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC), Tukey\u0026rsquo;s multiple comparison test identified three statistically distinct groups. \u003cem\u003eH. costaricensis\u003c/em\u003e presented the highest mean number of roots (5.17 roots per plant; group a), whereas \u003cem\u003eH. megalanthus\u003c/em\u003e exhibited the lowest value (3.38 roots per plant; group c). \u003cem\u003eH. undatus\u003c/em\u003e showed an intermediate mean and was classified in group b. For RL (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), Tukey\u0026rsquo;s test distinguished two statistically distinct groups. \u003cem\u003eH. costaricensis\u003c/em\u003e and \u003cem\u003eH. undatus\u003c/em\u003e exhibited the greatest mean root lengths (36.19 and 36.76 cm, respectively; group a), while \u003cem\u003eH. megalanthus\u003c/em\u003e recorded the lowest mean value (18.57 cm; group b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Vegetative vigor index (VVI)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA shows the mean values of the 12 treatments (A \u0026times; B \u0026times; C) for the Vegetative Vigor Index (VVI). The ANOVA revealed significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with a coefficient of variation of 15.61% (Supplementary Table\u0026nbsp;3). Tukey\u0026rsquo;s multiple comparison test identified five statistically distinct groups. Treatments T12, T3, T9, and T6 presented the highest VVI values (110.18, 107.52, 105.64, and 98.41, respectively; group a), whereas T2 and T8 exhibited the lowest means (18.19 and 18.20, respectively; group d). The remaining treatments, classified into groups b, bc, and cd, displayed intermediate VVI values (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThe VVI also differed significantly among \u003cem\u003eHylocereus\u003c/em\u003e species (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). According to Tukey\u0026rsquo;s test, \u003cem\u003eH. costaricensis\u003c/em\u003e showed the highest mean index value (105.44; group a), while \u003cem\u003eH. megalanthus\u003c/em\u003e exhibited the lowest mean (28.56; group c). \u003cem\u003eH. undatus\u003c/em\u003e displayed an intermediate VVI and was classified in group b (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Spearman correlation between evaluated variables\u003c/h2\u003e \u003cp\u003eSpearman\u0026rsquo;s correlation analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e) showed significant associations among most of the variables evaluated. Due to its inverse nature, shoot emission time (SET) exhibited negative correlations with all other variables: shoot length (SL) (ρ = \u0026minus;0.90*), number of roots (NR) (ρ = \u0026minus;0.77*), root length (RL) (ρ = \u0026minus;0.51*), cutting viability (CV) (ρ = \u0026minus;0.73*), and vegetative vigor index (VVI) (ρ = \u0026minus;0.85*). In contrast, number of shoots (NS) showed weak correlations with most variables (ρ\u0026thinsp;\u0026asymp;\u0026thinsp;0.16\u0026ndash;0.25), although a moderate positive correlation was observed with RL (ρ\u0026thinsp;=\u0026thinsp;0.49*).\u003c/p\u003e \u003cp\u003eAmong the vegetative growth variables, strong positive correlations were detected between SL and NR (ρ\u0026thinsp;=\u0026thinsp;0.73*), RL (ρ\u0026thinsp;=\u0026thinsp;0.57*), CV (ρ\u0026thinsp;=\u0026thinsp;0.76*), and VVI (ρ\u0026thinsp;=\u0026thinsp;0.89*). Likewise, NR was positively correlated with CV (ρ\u0026thinsp;=\u0026thinsp;0.81*), VVI (ρ\u0026thinsp;=\u0026thinsp;0.80*), and RL (ρ\u0026thinsp;=\u0026thinsp;0.46*). The strongest positive association was observed between CV and VVI (ρ\u0026thinsp;=\u0026thinsp;0.92*).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Principal component analysis (PCA) of the variables evaluated\u003c/h2\u003e \u003cp\u003eThe principal component analysis (PCA) explained 87.3% of the total variance based on the first two dimensions (Dim1\u0026thinsp;=\u0026thinsp;69.3% and Dim2\u0026thinsp;=\u0026thinsp;18.0%) for the three \u003cem\u003eHylocereus\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). Dim1 represented a vegetative performance gradient, with strong positive loadings for shoot length (SL), number of roots (NR), root length (RL), cutting viability (CV), and vegetative vigor index (VVI), and a negative loading for shoot emission time (SET), reflecting its inverse relationship with the other traits. Thus, higher Dim1 values indicated more vigorous seedlings, characterized by greater rooting capacity and earlier shoot emergence. Dim2 captured secondary variation primarily associated with the number of shoots (NS).\u003c/p\u003e \u003cp\u003eThe species exhibited distinct distributions along the PCA axes. \u003cem\u003eH. costaricensis\u003c/em\u003e was positioned toward higher values of SL, NR, RL, CV, and VVI, whereas \u003cem\u003eH. megalanthus\u003c/em\u003e was associated with higher SET values. \u003cem\u003eH. undatus\u003c/em\u003e occupied an intermediate position and was associated with higher NS values.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe results indicate that vegetative propagation of pitahaya through cuttings is strongly influenced by rooting agents, substrate type, species-specific responses, and the interaction among these factors. Regarding shoot emission time (SET), \u003cem\u003eH. costaricensis\u003c/em\u003e consistently exhibited the shortest emission times, indicating faster shoot emergence compared with the other species evaluated. These findings agree with those reported by Chhetri et al.\u003csup\u003e8\u003c/sup\u003e, who observed earlier sprouting in \u003cem\u003eH. costaricensis\u003c/em\u003e cuttings relative to other pitahaya varieties.\u003c/p\u003e \u003cp\u003eIn contrast, Anagha et al.\u003csup\u003e38\u003c/sup\u003e reported a mean sprouting time of 13.2 days for \u003cem\u003eH. undatus\u003c/em\u003e grown in vermicompost, which is considerably shorter than the minimum value recorded in the present study (21.53 days). This discrepancy may be associated with substrate composition, as vermicompost is rich in readily available nutrients and supports high microbial activity, particularly enhancing nitrogen (N) and phosphorus (P) availability \u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. These conditions can accelerate metabolic processes related to bud activation and shoot emergence, thereby reducing the time required for sprouting.\u003c/p\u003e \u003cp\u003eSimilarly, Cardoso et al.\u003csup\u003e42\u003c/sup\u003e, who evaluated five \u003cem\u003eHylocereus\u003c/em\u003e species, reported that \u003cem\u003eH. undatus\u003c/em\u003e exhibited greater rooting capacity and higher cutting viability than \u003cem\u003eH. costaricensis\u003c/em\u003e. This contrasts with the findings of the present study, in which \u003cem\u003eH. costaricensis\u003c/em\u003e showed the highest cutting viability (CV), surpassing \u003cem\u003eH. undatus\u003c/em\u003e. In addition, \u003cem\u003eH. megalanthus\u003c/em\u003e consistently exhibited the lowest CV values regardless of the rooting agent or substrate used. The reduced viability observed in this species may be associated with its ecological requirements, as \u003cem\u003eH. megalanthus\u003c/em\u003e is better adapted to altitudinal ranges between 800 and 1800 m a.s.l. \u003csup\u003e43\u003c/sup\u003e. Differences in environmental adaptation may therefore influence rooting responses and propagation success among species.\u003c/p\u003e \u003cp\u003eRegarding the variables associated with vegetative development, the responses of number of shoots (NS) and shoot length (SL) varied among treatments. At the interaction level (A \u0026times; B \u0026times; C), the highest NS value was observed in \u003cem\u003eH. undatus\u003c/em\u003e established in substrate 2 and treated with NAA\u0026thinsp;+\u0026thinsp;IBA (T7), reaching a mean of 1.6 shoots per plant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In comparison, Filho et al.\u003csup\u003e44\u003c/sup\u003e reported an average of 3.25 shoots per plant in \u003cem\u003eH. undatus\u003c/em\u003e cuttings treated with IBA, a value considerably higher than that obtained in the present study. This difference, despite involving the same species and a similar rooting inducer, may be attributed to variation in substrate composition and environmental conditions, which play a key role in vegetative propagation processes \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor shoot length (SL), the highest mean value was recorded in \u003cem\u003eH. costaricensis\u003c/em\u003e under the combination of substrate 1 and seaweed extract (T3), reaching 68.67 cm after four months of evaluation. By comparison, Cardoso et al.\u003csup\u003e42\u003c/sup\u003e reported shoot lengths of 27.25 cm and 22.18 cm for \u003cem\u003eH. undatus\u003c/em\u003e and \u003cem\u003eH. costaricensis\u003c/em\u003e, respectively, at 82 days, while Chhetri et al.\u003csup\u003e8\u003c/sup\u003e observed 52.1 cm at 90 days. These differences are likely associated with variation in evaluation period, as longer experimental durations generally allow greater shoot elongation to be expressed. Supporting this interpretation, Navarrete Torres et al.\u003csup\u003e45\u003c/sup\u003e reported a total plant height of 263.40 cm in \u003cem\u003eH. undatus\u003c/em\u003e after nine months, highlighting the strong influence of developmental time on shoot growth in pitahaya.\u003c/p\u003e \u003cp\u003eSuccessful rooting during vegetative propagation is strongly influenced by substrate moisture conditions and the use of rooting stimulants \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In the present study, the number of roots (NR) variable showed that treatments T3 and T12 produced the highest averages, with 5.30 and 5.23 roots per plant, respectively. This indicates that the species \u003cem\u003eH. costaricensis\u003c/em\u003e and substrate 1 were the most decisive factors, while the type of rooting agent had no significant influence.\u003c/p\u003e \u003cp\u003eBy comparison, Cardoso et al.\u003csup\u003e42\u003c/sup\u003e evaluated the effect of biostimulants on pitahaya cuttings and reported averages of 15.13 and 9.13 roots per plant for \u003cem\u003eH. undatus\u003c/em\u003e and \u003cem\u003eH. costaricensis\u003c/em\u003e, respectively, using a commercial substrate enriched with river sand. These differences could be attributed to the physical and chemical properties of the substrate, as such formulations are specifically designed to optimize moisture balance and aeration for vegetative propagation.\u003c/p\u003e \u003cp\u003eWith respect to root length (RL), the highest values were observed in treatments combining NAA\u0026thinsp;+\u0026thinsp;IBA and substrate 1, particularly in \u003cem\u003eH. costaricensis\u003c/em\u003e and \u003cem\u003eH. undatus\u003c/em\u003e (treatments T12 and T10), which reached mean root lengths of 42.69 cm and 41.38 cm, respectively, after four months of evaluation. Previous studies have reported lower RL values, including 24.07 and 19.00 cm in \u003cem\u003eH. undatus\u003c/em\u003e and \u003cem\u003eH. costaricensis\u003c/em\u003e at 82 days \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, 25.2 cm in \u003cem\u003eH. costaricensis\u003c/em\u003e at 90 days \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and 13.6 cm in \u003cem\u003eH. megalanthus\u003c/em\u003e at 90 days \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. These differences are likely associated with variation in evaluation period, as the cited studies assessed rooting for less than four months.\u003c/p\u003e \u003cp\u003eHowever, Navarrete Torres et al.\u003csup\u003e45\u003c/sup\u003e reported a maximum root length of 22.89 cm in \u003cem\u003eH. undatus\u003c/em\u003e after nine months, suggesting that although evaluation time can influence root development, root elongation dynamics may vary depending on species and propagation conditions, particularly during early stages of vegetative establishment. Taken together, these results indicate that root development is influenced by both species-specific responses and the physicochemical properties of the substrate.\u003c/p\u003e \u003cp\u003eThe vegetative vigor index (VVI) integrates all evaluated variables except shoot emission time (SET), as this variable is inversely related to the other growth parameters. The results showed that treatments involving \u003cem\u003eH. costaricensis\u003c/em\u003e consistently exhibited the highest VVI values. In particular, treatment T12 recorded the highest index, indicating that the interaction between \u003cem\u003eH. costaricensis\u003c/em\u003e, NAA\u0026thinsp;+\u0026thinsp;IBA, and substrate 1 was associated with the greatest vegetative vigor.\u003c/p\u003e \u003cp\u003eNotably, both the highest and lowest VVI values were obtained with substrate 1, suggesting that substrate alone may not fully determine variation in this index. Nevertheless, previous studies emphasize that substrate characteristics play a critical role in the vegetative propagation of pitahaya \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. According to Campbell et al.\u003csup\u003e50\u003c/sup\u003e, selecting an appropriate substrate is essential, as low porosity can promote waterlogging and reduce oxygen availability in the rhizosphere \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In this study, substrate 1 may have contributed to improved vegetative development due to its composition. The combination of forest soil, compost, and cattle manure likely enhances nutrient availability, organic matter content, and microbial activity, which can promote root formation and overall plant growth \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNot all plants respond uniformly to a specific type of stimulant; this response may vary even among individuals of the same species \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. In the present study, rooting agents significantly influenced the vegetative vigor index (VVI), with the combination of NAA\u0026thinsp;+\u0026thinsp;IBA being associated with higher VVI values compared to the seaweed extract.\u003c/p\u003e \u003cp\u003eHowever, several studies have reported beneficial effects of seaweed-based products on vegetative propagation, attributing their activity to phenolic compounds and hormone-like substances that enhance endogenous auxin activity and nutrient assimilation \u003csup\u003e\u003cspan additionalcitationids=\"CR54 CR55\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Indeed, \u003csup\u003e57\u003c/sup\u003e observed superior growth responses with seaweed extracts compared to synthetic NAA in lettuce and tomato transplants, reflected in increased biomass accumulation and dry matter content. These contrasting findings suggest that the effectiveness of biostimulants may depend on species-specific responses, physiological status of the cuttings, and environmental conditions during propagation.\u003c/p\u003e \u003cp\u003eAuxin based rooting agents containing NAA and IBA, which were associated with higher effectiveness than seaweed extract under the conditions of this study, are among the most widely used compounds in vegetative propagation due to their consistent efficacy across a wide range of plant species \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Their activity is primarily related to the stimulation of cell division, elongation, and differentiation processes that are essential for adventitious root initiation and development \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Numerous studies have reported that IBA, in particular, tends to promote superior rooting responses in cuttings compared with other plant growth regulators \u003csup\u003e\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough species of the genus \u003cem\u003eHylocereus\u003c/em\u003e can be cultivated under marginal agronomic conditions and warm climates \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, their physiological responses can vary and may be constrained by thermal conditions, which are a determining factor for plant growth and development \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Based on the morphological differences observed across treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) and the results of the statistical analyses, species identity emerged as the most influential factor affecting vegetative propagation in pitahaya.\u003c/p\u003e \u003cp\u003eAmong the 12 treatments evaluated, \u003cem\u003eH. costaricensis\u003c/em\u003e consistently exhibited shorter shoot emission time (SET), higher cutting viability (CV), greater shoot length (SL), number of roots (NR), and root length (RL), whereas number of shoots (NS) did not follow this same pattern. In contrast, \u003cem\u003eH. megalanthus\u003c/em\u003e generally showed the lowest values for most vegetative growth variables, while \u003cem\u003eH. undatus\u003c/em\u003e displayed intermediate responses. These differences suggest the presence of species-specific morphological and physiological traits that influence vegetative propagation responses.\u003c/p\u003e \u003cp\u003eIn particular, \u003cem\u003eH. megalanthus\u003c/em\u003e appears to be less adapted to hot and arid environments compared with \u003cem\u003eH. costaricensis\u003c/em\u003e and \u003cem\u003eH. undatus\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. which may partly explain its reduced rooting and growth responses under the conditions of this study. Furthermore, it has been demonstrated that plant responses to hormonal stimuli depend on the biochemical, physiological, and molecular capacities of each tissue to perceive and transduce these signals \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e, highlighting the importance of species specific sensitivity to growth regulators during vegetative propagation.\u003c/p\u003e \u003cp\u003eThese findings indicate that vegetative propagation of pitahaya is governed by a complex interaction among species-specific genetic, physiological, and environmental factors. Although rooting agents and substrate type influenced vegetative development, their effects were strongly modulated by species identity and prevailing propagation conditions. The results provide robust evidence for optimizing propagation protocols in \u003cem\u003eHylocereus\u003c/em\u003e spp. and underscore the need for further research addressing field adaptation, as well as the physiological and molecular mechanisms underlying the interspecific differences observed. Collectively, these insights contribute to improving propagation efficiency, strengthening production strategies, and promoting the long-term sustainability of pitahaya cultivation in tropical regions.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eVegetative propagation of pitahaya was influenced by the interaction among multiple factors, with species identity emerging as the primary determining factor. Among the species evaluated, \u003cem\u003eHylocereus costaricensis\u003c/em\u003e exhibited the most consistent and favorable responses in establishment and vegetative growth variables, as well as the highest vegetative vigor index (VVI).\u003c/p\u003e \u003cp\u003eRegarding rooting agents, the application of NAA\u0026thinsp;+\u0026thinsp;IBA was associated with higher responses than the seaweed extract (\u003cem\u003eEcklonia maxima\u003c/em\u003e), particularly in root development and overall seedling vigor, supporting the effectiveness of synthetic auxins in stimulating cell division and elongation processes. With respect to the propagation medium, substrate 1 composed of forest soil, compost, and cattle manure resulted in higher values for root and vegetative growth variables compared with the other substrates evaluated.\u003c/p\u003e \u003cp\u003eThe interaction between \u003cem\u003eH. costaricensis\u003c/em\u003e, substrate 1, and NAA\u0026thinsp;+\u0026thinsp;IBA (treatment T12) produced the highest VVI, identifying this combination as the most favorable for vegetative propagation under nursery conditions. Collectively, these findings provide a solid scientific basis for the development of large-scale propagation programs aimed at producing high-quality pitahaya seedlings, thereby contributing to the sustainability, productivity, and expansion of pitahaya cultivation in tropical regions of Peru.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003cb\u003eCRediT authorship contribution statement\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eB.C.C\u003c/b\u003e: Writing \u0026ndash; original draft, Formal Analysis, Data Curation, Conceptualization; \u003cb\u003eJ.M.C.M\u003c/b\u003e: Research, Methodology; \u003cb\u003eJ.A.C.S\u003c/b\u003e: Data Curation, Formal Analysis; \u003cb\u003eM.A.I\u003c/b\u003e: Research, Methodology; \u003cb\u003eV.H.T.M\u003c/b\u003e: Project Management, Financial Procurement; \u003cb\u003eD.G.F\u003c/b\u003e: Visualization, Formal Analysis; \u003cb\u003eJ.T.C\u003c/b\u003e: Investigation, Methodology; \u003cb\u003eM.G\u003c/b\u003e: Visualization, Validation; \u003cb\u003eD.T\u003c/b\u003e: Visualization, Validation, Formal Analysis.\u003c/p\u003e\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflicts of interest. The authors declare that they have no competing financial interests or personal relationships that could influence the work presented in this article.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eSupplementary material\u003c/h2\u003e \u003cp\u003eSupplementary material associated with this article can be found, in the online version, at\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by the Instituto Nacional de Innovaci\u0026oacute;n Agraria (INIA), through the investment project with CUI N\u0026deg; 2472675 \u0026ldquo;Mejoramiento de los Servicios de Investigaci\u0026oacute;n y Transferencia de Tecnolog\u0026iacute;a Agraria en la Estaci\u0026oacute;n Experimental Agraria Ba\u0026ntilde;os del Inca en la localidad Los Ba\u0026ntilde;os del Inca del distrito Los Ba\u0026ntilde;os del Inca - provincia de Cajamarca - departamento de Cajamarca\u0026rdquo;\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eB.C.C: Writing \u0026ndash; original draft, Formal Analysis, Data Curation, Conceptualization; J.M.C.M: Research, Methodology; J.A.C.S: Data Curation, Formal Analysis; M.A.I: Research, Methodology; V.H.T.M: Project Management, Financial Procurement; D.G.F: Visualization, Formal Analysis; J.T.C: Investigation, Methodology; M.G: Visualization, Validation; D.T: Visualization, Validation, Formal Analysis.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKishore, K. 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Effects of NAA and \u003cem\u003eEcklonia maxima\u003c/em\u003e Extracts on Lettuce and Tomato Transplant Production. \u003cem\u003eAgron. 2022\u003c/em\u003e. \u003cb\u003e12, Page 329\u003c/b\u003e (12), 329 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePompelli, M. F. et al. Effect of Indole-3-butyric acid (IBA) and 3-Indoleacetic acid (IAA) on promoting roots on micropropagated \u003cem\u003eDyckia distachya\u003c/em\u003e Hassl. \u003cem\u003eSouth. Afr. J. Bot.\u003c/em\u003e \u003cb\u003e184\u003c/b\u003e, 387\u0026ndash;394 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLudwig-M\u0026uuml;ller, J. \u0026Aacute;cido indol-3-but\u0026iacute;rico en el crecimiento y desarrollo de las plantas. \u003cem\u003ePlant. Growth Regul.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, 219\u0026ndash;230 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePe\u0026ntilde;a-Baracaldo, J. F., Chaparro-Zambrano, H. 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Sci.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e, 741\u0026ndash;754 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlcantara Cortes, J. S., Godoy, A., Alc\u0026aacute;ntara Cort\u0026eacute;s, J., S\u0026aacute;nchez Mora, R. M. \u0026amp; J. D. \u0026amp; Principales reguladores hormonales y sus interacciones en el crecimiento vegetal. \u003cem\u003eRevista Nova publicaci\u0026oacute;n cient\u0026iacute;fica en ciencias biom\u0026eacute;dicas\u003c/em\u003e. \u003cb\u003e17\u003c/b\u003e, 109\u0026ndash;129 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Phytohormones, cuttings, rooting, organic substrate, Hylocereus spp","lastPublishedDoi":"10.21203/rs.3.rs-9180319/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9180319/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePitahaya (\u003cem\u003eHylocereus\u003c/em\u003e spp.) is an emerging crop for tropical regions; however, its vegetative propagation is strongly influenced by environmental and management factors. Optimizing rooting and establishment under nursery conditions is essential to produce uniform and vigorous seedlings. This study evaluated the effects of root inducers and substrates on the vegetative propagation of three \u003cem\u003eHylocereus\u003c/em\u003e species under nursery conditions. A completely randomized design (CRD) with a 2\u0026times;2\u0026times;3 factorial arrangement was implemented, including two root inducers (NAA\u0026thinsp;+\u0026thinsp;IBA solution and seaweed extract), two substrates (substrate 1 and substrate 2), and three species (\u003cem\u003eH. undatus, H. megalanthus\u003c/em\u003e, and \u003cem\u003eH. costaricensis\u003c/em\u003e), resulting in 12 treatments with four replicates each. Seven variables were assessed: shoot emission time (SET), cutting viability (CV), number of shoots (NS), shoot length (SL), number of roots (NR), root length (RL), and vegetative vigor index (VVI). ANOVA revealed significant effects (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) in the three-factor interaction for SET, CV, NS, SL, RL, and VVI. Treatment T9 achieved the shortest SET (21.53 days) and highest CV (100%), T7 recorded the highest NS (1.62 shoots plant⁻\u0026sup1;), T3 showed greater SL (68.67 cm) and NR (5.30 roots plant⁻\u0026sup1;), and T12 obtained the highest RL (42.69 cm) and VVI (110.18). The species factor exerted the greatest influence, with H. costaricensis showing the best overall performance. These findings demonstrate that combining H. costaricensis, substrate 1, and NAA\u0026thinsp;+\u0026thinsp;IBA enhances vegetative propagation efficiency, supporting large-scale seedling production and sustainable expansion of pitahaya cultivation in tropical Peru.\u003c/p\u003e","manuscriptTitle":"Influence of root inducers and substrates on the vegetative propagation of pitahaya species (Hylocereus spp.) under nursery conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 09:35:08","doi":"10.21203/rs.3.rs-9180319/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-04T09:25:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T22:42:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-20T10:19:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27579181393554313551768285052298026180","date":"2026-04-17T13:12:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104338964828501710989433817242214139862","date":"2026-04-15T19:44:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136785428028288540022364048564335520901","date":"2026-04-15T12:54:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305244888667527725200061103896073063670","date":"2026-04-03T12:37:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-03T12:32:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-23T16:06:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-21T06:50:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-21T06:50:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-20T15:15:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"90facb74-928a-4d93-b55a-a0beab73536a","owner":[],"postedDate":"April 9th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-04T09:25:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T22:42:32+00:00","index":30,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":65680433,"name":"Biological sciences/Ecology"},{"id":65680434,"name":"Earth and environmental sciences/Ecology"},{"id":65680435,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-05-04T09:41:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 09:35:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9180319","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9180319","identity":"rs-9180319","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}