Introduction

Pitaya or dragon fruit (Selenicereus spp., sin. Hylocereus spp.) is an important species in Peru and internationally due to its high content of ascorbic acid (4-25 mg∙100 g^-1 of fresh pulp, depending on the species), minerals, fiber, betalains, betanin, and betacyanins, as well as its remarkable antioxidant capacity (Cañar-Sena et al., 2014; Oney-Montalvo et al., 2023; Verona-Ruiz et al., 2020). The hybridization of this species has led to the development of new varieties with wide adaptability to diverse environmental conditions and low water requirements (Mercado-Silva, 2018; Montesinos-Cruz et al., 2015; Yadav et al., 2023). In arid and semi-arid regions, its adaptation is evident in features such as an extensive horizontal root system, thick-cuticled cladodes, a reduced number of stomata, water-storing parenchyma, and fleshy aerial organs (Zimmerman et al., 2017).

Pitaya is primarily propagated through cuttings to produce a greater number of uniform plants suitable for the establishment of commercial orchards and to shorten the time to initial fruit production (Balaguera-López et al., 2010; Anushi et al., 2024). This process relies on the formation of adventitious roots and is influenced by several factors, including the degree of lignification, cutting age and size, substrate type, and hormonal balance (Chhetri et al., 2021; Dhruve et al., 2018; Sun et al., 2023). Among these, substrate and the application of growth regulators are key external factors affecting both rooting success and root quality in cuttings.

The substrate must meet specific criteria for porosity, moisture retention, and nutrient availability (Andrade et al., 2006; Bastos et al., 2006) to support vigorous and uniform cutting development (de Sousa-Antunes et al., 2021). Substrates are composed of varying proportions of organic inputs (such as vermicompost, moss, compost, manure, rice husks, and sawdust) and inorganic materials (such as sand, pumice, perlite, and vermiculite) (de Sousa-Antunes et al., 2021; ElObeidy, 2006; López-Gómez et al., 2000).

Auxins stimulate nearly all aspects of plant growth and development, particularly the induction of lateral roots (Benková & Hejátko, 2009; Haim et al., 2021; Overvoorde et al., 2010). These molecules are transported over long distances within the plant via the phloem, and because they share the same transport pathway as carbohydrates, they show efficient and rapid mobility (Overvoorde et al., 2010). This behavior supports the formation of adventitious roots, as auxin reserves can originate from both shoots and pre-existing roots.

Among the most commonly used auxins, indole-3-butyric acid (IBA) stands out for its effectiveness in inducing root formation in pitaya cuttings (Ahmad et al., 2016; Balaguera-López et al., 2010; Chhetri et al., 2021). Similarly, naphthaleneacetic acid (NAA) has proven effective in stimulating cell division and expansion, contributing to increased cutting survival rates (Fàbregas & Fernie, 2021; Haim et al., 2021). However, while growth regulators offer significant advantages for vegetative propagation, it is essential to determine optimal conditions for pitaya, because their effectiveness depends on several factors, including the type of hormone (Bozkurt et al., 2022), concentration (Irfan-Ali et al., 2022), tissue age (cutting size) (Potente & Manigo, 2023; Rodrigues et al., 2021), and type of rooting substrate (Galvão et al., 2016), among others.

Despite being a common practice in the propagation of other fruit crops, the effectiveness of combining IBA and NAA for rooting pitaya cuttings remains uncertain. Therefore, this study aimed to assess the effects of auxins (NAA + IBA) and different substrate types on the rooting performance of cuttings of various lengths in red-fleshed pitaya ‘Physical Graffiti’ (Selenicereus guatemalensis × S. undatus).

Materials and methods

Study area

The study was carried out from March to June 2023 in plot 425 of San Juan El Alto Pedregal, located in the Majes Irrigation District, Arequipa, Peru (16° 30’ 12’’ S and 73° 20’ 62’’ W, 1 280 m a. s. l.). The average temperature recorded during the study period was 18.5 °C, with a minimum of 8 °C and a maximum of 29.8 °C. The average relative humidity was 57.4 %, ranging from 38.2 % to 73.5 %, with the lowest values recorded in June.

Experimental design

A completely randomized experimental design with three replications was used. The treatment structure followed a 3×2×3 factorial arrangement, resulting in 18 treatments (Table 1). The evaluated factors were three levels of auxin concentration (NAA + IBA at 0 ppm, 20 + 5 ppm, and 40 + 10 ppm), two cutting lengths (20 and 40 cm), and three substrate types (sand; sand:vermicompost [1:1, w/w]; and vermicompost).

Table 1. Description of the treatments evaluated in ‘Physical Graffiti’ pitaya.

Study development

Vegetative material

The pitaya variety evaluated was the red-fleshed ‘Physical Graffiti’ (S. guatemalensis × S. undatus) (Korotkova et al., 2017; Pagliaccia et al., 2015). Cuttings were collected from a commercial plantation with 1 year of production, located in the Pedregal district, Majes Irrigation District, Caylloma province, Arequipa region, Peru. Only cuttings from visually healthy plants, showing no signs of nutritional deficiencies or stress, were selected. These cuttings were classified into two sizes: 20 and 40 cm in length. After collecting, the cuttings were allowed to heal for 15 days in shaded conditions (75 %), under ambient temperature and relative humidity.

Substrates

The sand used as a substrate was collected from the Siguas River (Arequipa, Peru), and it was washed to remove any potential impurities. The vermicompost was purchased from a local company. The physicochemical properties determined for both substrates included pH, electrical conductivity (EC), organic matter (OM), total N, P, K, bulk density, and total porosity. These determinations were carried out following the methodology described by Bazán-Tapia (2017), using the following techniques: pH measurement with a potentiometer (HI9126, Hanna® Instruments, EU) in a soil:water ratio of 1:1, EC measured with a conductimeter (HI993310, Hanna® Instruments, USA) in a saturated soil extract, the Walkley-Black method for OM, the micro-Kjeldahl method for total N, the modified Olsen method for P, spectrophotometry with ammonium acetate extraction for K, the metal cylinder method for bulk density, and indirect estimation of total porosity based on bulk density.

The substrates were placed in black nursery bags measuring 20 cm in width × 30 cm in height, with a thickness of 120 gauge and a capacity of 1 kg. For the mixed substrates, 500 g of sand and 500 g of vermicompost were weighed, homogenized, and bagged.

Auxin application

Auxins IBA (≥98 % purity) and NAA (≥95 % purity) were purchased from Sigma-Aldrich (USA). Solutions were first diluted in analytical-grade ethanol (≥95 % purity) and then in distilled water adjusted to a pH of 5.5. Three auxin solutions containing IBA + NAA were prepared: 0 ppm (control without auxins), 20 + 5 ppm, and 40 + 10 ppm. The basal end (3 cm) of each cutting was immersed in the corresponding solution for 30 seconds. Afterwards, each cutting was placed into a nursery bag at an approximate depth of 5 cm and kept under a green shade net providing 75 % shade.

Irrigation

Irrigation frequency varied according to the type of substrate: every 5 days for sand, and every 10 days for vermicompost. At each irrigation event, 200 mL of water was applied to sand substrates, 250 mL to the sand:vermicompost mixture, and 300 mL to vermicompost. The experiment lasted for 90 days, after which the rooting period was considered complete and the corresponding evaluations were carried out.

Evaluations

Evaluations were performed on each rooted cladode or cutting. The variables analyzed were: 1) Root length (cm), measured from the base of the cutting to the tip of the longest root; 2) Number of roots, including only the most developed roots with visible rootlets; 3) Fresh and dry root biomass (g); and 4) Fresh and dry biomass of the cuttings (g). To determine dry biomass, samples were placed in a drying oven at 105 °C until constant weight (Cañar-Sena et al., 2014).

Statistical analysis

The data obtained were subjected to an analysis of variance and Tukey’s multiple mean comparison tests (significance level of 99%; P < 0.001). In addition, Pearson’s linear correlation analyses were performed between quantitative variables (significance level of 95 %; P < 0.05). The assumptions of parametric statistics were verified using the Shapiro-Wilk test to assess the normality of residuals (P < 0.05) and Bartlett’s test to confirm homoscedasticity (P < 0.05). All statistical analyses were carried out using R software (R Core Team, 2013).

Results and discussion

The evaluated data met the assumptions of homogeneity of variances and normality of residuals required for the application of parametric statistical analyses. The p-values obtained from the analysis of variance revealed highly significant differences in the three-way interaction among the evaluated factors, except for fresh and dry root biomass, for which the interaction effects were not significant (Table 2).

Table 2. Significance levels of the variables evaluated in ‘Physical Graffiti’ pitaya.

The interaction effects indicated that the most effective rooting occurred with 20 cm cuttings treated with 20 + 5 ppm of NAA and IBA and grown in the sand:vermicompost mixture (T8). In contrast, the poorest performance was observed in treatment T4, which involved 40 cm cuttings without auxin application, established in sand (Table 3).

Table 3. Effect of auxin application in three substrate types on the rooting of two cutting lengths in ‘Physical Graffiti’ pitaya.

The results corresponding to the main effects revealed statistically significant differences in all evaluated variables in response to the applied treatments (Table 4). In general terms, the sand and vermicompost mixture promoted significantly greater root development (P < 0.05), as reflected in increased root length, number of roots, and biomass accumulation. Regarding auxin treatments, the best response was observed when cuttings were immersed in the 20 + 5 ppm NAA and IBA solution, respectively.

Table 4. Main effect response of auxin application in three types of substrates for rooting ‘Physical Graffiti’ pitaya cuttings.

Although previous studies have reported that pitaya cuttings between 35 and 45 cm in length show better rooting performance (Anushi et al., 2024), in this study, the 20 cm cuttings showed greater root development and biomass accumulation compared to the 40 cm cuttings (Table 4). This difference may be attributed to factors such as the crop used, weight, maturity and age of cuttings (Chhetri et al., 2021; Irfan-Ali et al., 2022; Anushi et al., 2024). Moreover, interactions with external factors, such as substrate and the use of growth regulators, also influence the efficiency of the rooting process (Table 3).

The results of the Pearson correlation analysis indicate a significant, though low to moderate, association between auxin concentration and the root growth and biomass variables of cuttings. Positive correlations were observed for root length (r = 0.36; P = 0.01), number of roots (r = 0.29; P = 0.03), fresh biomass of cuttings (r = 0.65; P < 0.0001), and dry biomass of cuttings (r = 0.60; P < 0.0001) (Figure 1). Although the best results were obtained with the sand:vermicompost substrate, the Pearson correlation showed a stronger positive association between auxin concentration and root growth when using the simple substrates (sand or vermicompost alone).

The interplay between auxin synthesis, transport, and degradation generates concentration gradients that determine local cell differentiation events (Fàbregas & Fernie, 2021). However, the response of a plant cell to a specific auxin concentration also depends on its biochemical, physiological, and molecular capacity to perceive and interpret that hormonal signal (Alcántara et al., 2019; Casanova-Sáez & Voß, 2019). These responses may be continuous or follow distinct phases of increase, stabilization (or threshold), and subsequent decrease. Due to cell-specific responses, it is possible for the same auxin concentration to promote cell expansion in shoots while simultaneously inhibiting cell elongation to favor cell division in the root meristem (Alcántara et al., 2019; Haim et al., 2021; Overvoorde et al., 2010). This behavior is reflected in root growth and cutting biomass results, because the solution containing 40 + 10 ppm of auxins caused a significant decrease in root growth (length, number, and biomass), without negatively affecting the dry biomass of the cuttings. This represents a partially favorable effect on the rooting process (Table 3 and Table 4).

Regarding the fresh and dry biomass of the cuttings, auxin concentration was the only factor with a significant effect, as lower biomass accumulation was observed in the absence of auxins (Figure 1). This pattern can be explained by the close relationship between root system development and the cuttings’ ability to absorb water and nutrients, which is essential for the growth of aboveground structures (Anushi et al., 2024; Balaguera-López et al., 2010; Overvoorde et al., 2010). The distribution of dry matter between cuttings and roots is a dynamic process regulated by phytohormones, and any external factor can alter the biomass concentration in both organs (Anushi et al., 2024). In this study, root growth in pitahaya was not enhanced by the use of auxin concentrations of 40 + 10 ppm of NAA and IBA.

This contrasts with the findings reported by Balaguera et al. (2010) in Colombia, who observed that 60 cm pitahaya cuttings treated with 4 500 ppm of IBA had the highest values for root length (13.6 cm), number of roots (11), fresh biomass (5.56 g), and dry biomass (0.79 g) after 90 days. Similarly, Seran and Thiresh (2015) in Sri Lanka reported that immersing pitahaya cuttings in 6 000 ppm IBA solutions and growing them in a sand:topsoil:cow manure substrate (2:1:1, w/w/w) resulted in longer (9.5 cm) and heavier shoots (10.25 g fresh and 0.59 g dry weight) after 60 days.

Although other studies have reported positive results using high concentrations of IBA, in the present study, the concentration that produced the best response was 20 + 5 ppm of NAA and IBA, while the highest dose (40 + 10 ppm of NAA and IBA) resulted in a significant decrease. This response may be related to a synergistic interaction between auxins with complementary effects.

Regarding the role of the substrate in rooting, the sand:vermicompost mixture (1:1, w/w) yielded the best results, showing higher values of OM, P, K, and porosity compared to the single-component substrates (Table 5). According to Andrade et al. (2007), substrate properties such as porosity, moisture retention, organic matter content, and nutrient availability favor the vegetative propagation of pitahaya. The sand:vermicompost combination proved suitable for root system development, providing an optimal balance of aeration, water retention, pH, salinity, and nutrient availability, which supported the establishment and growth of the cuttings (Table 5).

Table 5. Physicochemical characteristics of substrates for rooting of ‘Physical Graffiti’ Pitahaya Cuttings.

Conclusions

The factors studied significantly influenced root and shoot growth in red-fleshed ‘Physical Graffiti’ pitahaya cuttings, highlighting the importance of selecting the appropriate rooting substrate, auxin concentration, and cutting size. Specifically, the combined use of NAA (20 ppm) and IBA (5 ppm) promoted rooting in pitahaya cuttings, particularly when using sand:vermicompost (1:1, w/w) substrate.