Eucalyptus camaldulensis Dehnh. is one of the most widely planted Eucalyptus species for paper pulp, fuelwood, and timber due to its fast growth, high drought tolerance, and adaptability to diverse climatic conditions and soil types (Bindumadhava et al., 2011; Shanthi et al., 2015). Its widespread distribution, economic potential, and ability to survivve in rainfall zones ranging from 250 to 1,600 mm make it a crucial species for improvement programs. Furthermore, E. camaldulensis has been used in hybrid breeding programs to enhance drought and salinity tolerance (Amrutha et al., 2021).

Clonal propagation is a widely employed strategy to explore the economic potential of Eucalyptus species and hybrids by multiplying desirable genotypes. Consequently, the efficient development of vegetative propagation protocols is essential for the large-scale production of E. camaldulensis clones (Shanmugam & Seenivasan, 2012). Mass multiplication of E. camaldulensis clones has been previously reported using various propagation techniques, including micropropagation by direct organogenesis (Girijashankar, 2012), as well as cutting and mini-cutting methods (Bindumadhava et al., 2011; Shanmugam & Seenivasan, 2012). Among these techniques, mini-cutting has proven to be the most efficient and widely used method for cloning superior genotypes of Eucalyptus within the framework of clonal forestry (Xavier et al., 2013; Kuppusamy et al., 2019).

Clones propagated using the mini-cutting technique generally produce highly uniform plants. However, the success of this method depends on the development of adventitious roots, a process influenced by various factors such as plant genotype, physiological status, nutritional condition, and the juvenile state of vegetative shoots (Wendling et al., 2014; Vilasboa et al., 2022). Juvenile branches in good nutritional condition tend to exhibit better rooting ability (Wendling et al., 2015), which is further enhanced by the application of plant growth regulators (Wendling et al., 2015; Stuepp et al., 2017; Lima et al., 2022). Environmental conditions, such as the collection season, also play a critical role in propagation success (Batista et al., 2015).

Auxins are widely used as growth regulators to enhance plant propagation from cuttings by promoting the formation of adventitious roots from non-root tissues (Díaz-Sala, 2021). They are essential regulators of adventitious root induction (Díaz-Sala, 2020). An imbalance in auxin levels can adversely affect rooting, leading to low rooting rates, reduced numbers of roots, root system asymmetry, and a reduced root-to-shoot ratio (Stuepp et al., 2017). These features significantly influence tree stability, survival, and trunk volume in nurseries and plantations. When natural auxin production in apical meristems is insufficient to induce adventitious root formation in stem tissues (Stuepp et al., 2017; Díaz-Sala, 2020), exogenous auxins, particularly indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA), are commonly used to induce adventitious rooting during clonal propagation (Hartmann et al., 2011).

The application of IAA and IBA has been shown to accelerate adventitious rooting in Eucalyptus mini-cuttings, with optimal concentrations varying by genotype (Brondani et al., 2010). Additionally, the origin of the propagule along the branch can influence rooting success due to ontogenetic and physiological factors (Wendling et al., 2014). The morphoanatomy of adventitious rooting is important for identifying anatomical features, such as lignification and the presence of a sclerenchyma ring, that may negatively affect root induction, initiation, and emergence (Bryant and Trueman, 2015).

Anatomical studies of transverse sections have shown that adventitious roots can originate from various tissues, including the cambium zone (phloem, vascular cambium, or pericycle) or from callus (Bryant and Trueman, 2015; Vilasboa et al., 2021). However, the anatomy of adventitious rooting remains variable and not fully understood (Bryant and Trueman, 2015), despite its critical role in maximizing rooting rates (Pimentel et al., 2020). While IBA has been reported to accelerate anatomical changes at the bases of cuttings (Vilasboa et al., 2021), its effects on E. camaldulensis genotypes remain unexplored.

The emergence of adventitious roots is essential for clonal propagation and the production of high-quality E. camaldulensis plants. This process is influenced by genetic and ontogenetic factors, as well as anatomical and physiological characteristics, which contribute to the root development of mini-cuttings. However, further research is needed to establish a solid mini-cutting propagation methodology for E. camaldulensis clones, facilitating the clonal propagation of selected genotypes in breeding programs.

Therefore, this study aimed to evaluate the effectiveness of the mini-cutting technique for propagating a E. camaldulensis genotype selected for semiarid conditions. Additionally, it aiemd to determine the impact of IAA and IBA on the rooting of apical mini-cuttings and included an anatomical evaluation of propagules at different stages of adventitious rooting. The hypotheses tested were as follows: (I) the E. camaldulensis genotype has potential for clonal propagation using the mini-cutting technique; (II) IAA and IBA stimulate rooting and enhance the vigor of the mini-cutting root system; (III) low relative humidity and high temperatures in the growing environment affect the productivity and survival of the mini-garden; and (IV) mini-cutting anatomical features are related to rooting speed and regeneration capacity.

The productivity of the mini-stumps stands out as a key deciding factor for the success of mini-cuttings, considering the productivity of mini-stumps as an objective parameter of the mini-garden’s performance. The productivity of the mini-stumps is influenced by variables such as the type of clonal mini-garden, the nutritional management employed, seasonality, light quality and irradiance, air humidity and temperature, mineral nutrition, and water availability (Wendling et al., 2014; Batista et al., 2015; Vilasboa et al., 2022; Lima et al., 2022).

Here, we present evidence that the mini-stumps of the E. camaldulensis clone, cultivated under semiarid conditions, exhibited consistent performance due to 100% survival and the production of 13 propagules per mini-stump per month in establishing a clonal mini-garden. This performance was maintained even in collections III and IV, conducted in August and September—months characterized by low relative humidity (60%) and high temperatures (28°C). Our results suggest that the mini-stumps of this specific genotype have excellent regrowth potential after pruning. In this sense, both the genotype and environmental control optimized the survival and productivity of the mini-stumps.

Regarding environmental conditions in semiarid regions, temperature and water availability stand out as variables that can influence mini-garden performance. However, water limitations can be mitigated with an efficient irrigation system, such as the drip irrigation used in the present study. Temperature is another factor influencing the productivity of mini-stumps and the quality of mini-cuttings (Trueman et al., 2013). The significant productivity of the mini-garden in this study at a temperature close to 30°C can be attributed to the complete adaptation of the mini-stumps to the environment, which favors the process of cell division. The processes of cell proliferation, sprout emission, and root formation are favored by higher temperatures, up to a certain extent (Lima et al., 2022). In this context, the application of mini-tunnels, a recent forestry practice used to increase the temperature of the mini-stump propagation environment during colder seasons (Batista et al., 2015; Lima et al., 2022; Vilasboa et al., 2022), is unnecessary in semiarid conditions due to naturally high temperatures.

In E. camaldulensis, regrowth ability from stumps (Heth et al., 1986; Reuveni et al., 1990), mini-stumps (Bindumadhava et al., 2011), and micro-stumps (Shanthi et al., 2015) has been documented, supporting the results of the present study. These studies demonstrate the resistance of this species to successive pruning, with a favorable response in the emission of epicormic shoots. Furthermore, in our study, both mini-stumps and shoots were free of pathogens, ensuring their suitability for subsequent collection of mini-cuttings.

Notably, the productivity of mini-stumps observed in the present study exceeds values reported for Eucalyptus mini-stumps in clonal mini-gardens (Bindumadhava et al., 2011; Brondani et al., 2012) and is comparable to productivity achieved using mini-tunnels in different seasons (Batista et al., 2015; Lima et al., 2022; Vilasboa et al., 2022). Moreover, our data closely align with productivity levels reported for mini-stumps of other species grown under semiarid conditions (Souza et al., 2023; Silva et al., 2022). This reaffirms that high temperatures and low relative humidity, typical of semiarid regions, are not barriers to vegetative propagation by mini-cutting when proper management practices are employed. However, it is critical that plants undergo acclimatization and hardening after the rooting phase before being exposed to field conditions.

In greenhouses, where planting material for forests and plantations is rooted, air humidity is typically higher than in field conditions. Consequently, transplanting to the field can stress the trees, potentially affecting growth and development (e.g., height and diameter), as well as physiological and biochemical processes, including survival. Therefore, acclimatization after the rooting phase is essential, and the plant’s reaction in the field depends on the clone’s phenotypic plasticity (Gudynaitė-Franckevičienė & Pliūra, 2022). The maintenance of survival and sprout production potential observed during August and September (periods of high temperature and low humidity) for the mini-garden in full sun suggests high survival and vigor rates are expected under field conditions for these plants.

Additionally, shoots from mini-stumps exhibited adventitious rooting potential. The high rooting rate observed in this study reflects a complex interaction between various factors, including the genetic control of the propagule donor plant (genotype), the nutritional and water balance of the mini-stumps, and the genotype’s high regrowth capacity after pruning. The production of juvenile propagules with high vigor, combined with appropriate control of rooting conditions, contributes to these results (Wendling et al., 2014; Gianguzzi et al., 2020). Together, these factors influence rooting capacity and determine the success of clonal propagation by mini-cuttings (Wendling et al., 2014; De Almeida et al., 2017).

Efforts to increase the rooting rate of vegetative cuttings often involve optimizing environmental conditions, hormone applications, and physiological aspects of the hedge stock (Trueman et al., 2013). In this study, we identified auxin as a key factor for improving rooting potential in the selected E. camaldulensis clone. Adventitious rooting development is regulated by a crosslinked network of hormone signaling, with auxin playing a central role. Auxin accumulation at rooting sites, mediated by polar transport, is a specific initial response of rooting-competent tissues (Pizarro and Díaz-Sala, 2019).

Procedures to improve adventitious root induction often involve auxin treatments and other factors, such as light or temperature, which interact with auxin activity (De Almeida et al., 2017). For instance, Vilasboa et al. (2022) reported differentially expressed transcripts during adventitious root formation in response to auxin and other factors, highlighting the molecular regulation of this process, which involves auxin-, gibberellin-, jasmonic acid-, and ethylene-mediated responses, wounding, sugar signaling, and cell cycle regulation. Therefore, the application of exogenous auxin is necessary for stimulating adventitious root development in most tree species (Díaz-Sala, 2020).

In mini-cutting, IAA and IBA are commonly used to enhance rooting rates (Hartmann et al., 2011). However, the choice of auxin depends largely on the species or genotype (Steffens and Rasmussen, 2016; Ayala et al., 2022). Our work describes the application of both IAA and IBA at an optimal concentration of 2,000 mg·L⁻¹ to improve rooting.

At the onset of clonal propagation of E. camaldulensis, rooting rates ranged between 4% and 50%, depending on the cloned genotype (Heth et al., 1986; Reuveni et al., 1990). Using the mini-cutting technique, some genotypes achieved up to 97% adventitious rooting (Bindumadhava et al., 2011). However, for recalcitrant genotypes, micropropagation techniques inducing somatic embryos improved propagation capacity, enabling large-scale multiplication of elite trees (Girijashankar, 2012). The adventitious rooting recorded from 33 superior performing E. camaldulensis clones propagated by microcutting was between 24.8% to 100%, depending on the genotype (Shanthi et al., 2015). The adventitious rooting rates observed in this study (>80%) underscore the high propagation potential of this genotype under semiarid conditions.

As noted, the genetic background of the plant material is of great importance for the rooting of E. camaldulensis. The results of the present study show that the E. camaldulensis genotype selected in a semiarid conditions has a high potential for adventitious rooting, with rates exceeding 80% when applying 2,000 mg·L⁻¹ of IAA or IBA. In this sense, rooting is not a limiting factor for the large-scale multiplication of this genotype. A common practice in mass propagation systems is to select clones with rooting levels of 70% or higher (Xavier et al., 2013), while clones with poorer rooting performance are discarded. Furthermore, the application costs of IAA and IBA are offset by the benefits of these auxins, as their application can reduce the time required for rooting. Optimizing the rooting duration in the greenhouse can maximize the utilization of facilities, mitigate the adverse impacts of high humidity on the adventitious root system, prevent post-rooting mortality (Brondani et al., 2012), and consequently reduce the costs of genotype multiplication.

Regarding the origin of adventitious roots in stem cuttings, it can vary significantly according to the plant species and types of propagules (Bryant and Trueman, 2015). Anatomical analysis of mini-cutting stems showed similarities in structural development patterns across treatments with IBA, IAA, and the control. There are two patterns of root development in vegetative propagules: direct and indirect rhizogenesis. In the direct pattern, competent cells initiate cell division, and root formation occurs directly from the original stem tissues (Xavier et al., 2013). On the other hand, the indirect pattern involves the formation of irregular masses of parenchyma cells (callus formation), which is then followed by rooting (Hartmann et al., 2011). As described for other Eucalyptus species (Goulart et al., 2014; Bryant and Trueman, 2015), the rooted mini-cuttings of the E. camaldulensis genotype presented a direct pattern of adventitious root formation.

However, as previously described, auxin accelerated anatomical changes at the bases of mini-cuttings, most notably at the root primordia formation stage. Unlike what was reported by Bryant and Trueman (2015), the precise location of adventitious root initiation was easy to identify in E. camaldulensis mini-cuttings. Anatomical analyses showed that roots were emitted close to the vascular cambium in both control and IAA- or IBA-treated mini-cuttings, a pattern also observed in other forest tree species, such as Eucalyptus grandis × Eucalyptus urophylla (Goulart et al., 2014), Corymbia torelliana F.Muell., and E. camaldulensis (Bryant and Trueman, 2015).

Only in IAA- and IBA-treated mini-cuttings was disorganization of the pith and xylem tissue observed, which could be related to the capacity of these phytoregulators to promote cell proliferation (Sánchez et al., 2007; Wang et al., 2022). Auxin has been shown to induce xylem differentiation in Arabidopsis (Ohashi-Ito et al., 2013). The application of exogenous auxin is expected to stimulate the activation of cells in the vascular cambium, thus facilitating cell multiplication and rhizogenesis (Sá et al., 2022).

Some literature reports suggest that, in certain cases, the presence of a sclerenchyma ring may delay or completely obstruct root emission in vegetative propagules (Wendling et al., 2015). However, the continuous and discontinuous sclerenchyma layers observed in the mini-cuttings of the E. camaldulensis genotype exhibit different behavior and do not appear to inhibit the emission and development of adventitious roots. Taken together, our results demonstrate that the genotype selected in a semiarid conditions has a natural competence for adventitious rooting.

The mini-cutting technique applied has notable applications in propagating the selected genotype. For example, the productivity and absence of mortality in mini-stumps during successive pruning, combined with the rooting rate, ensure the genotype’s potential to be multiplied via mini-cutting. Rooting rates can be further increased by applying IAA and IBA, as these auxins exhibit equivalent rooting performance. This provides substantial benefits for commercial production, including reduced time to root formation, potentially due to the activation of multiple tissues (pith and xylem) for the direct origin of the root system.

The findings from this study offer valuable insights for mini-garden management and adventitious rooting, facilitating the effective application of the mass mini-cutting technique in commercial nurseries to propagate the E. camaldulensis genotype selected in a semiarid conditions.

Altogether, the present data support the following conclusions: (I) The mini-cutting technique is applicable and viable for the propagation of the E. camaldulensis genotype under semiarid conditions. (II) The survival and productivity of mini-stumps were not influenced by low relative humidity (60%) and high temperature (28ºC), as well as the response of propagules to adventitious rooting. (III) The pattern of root tissue initiation is directly from the vascular cambium and consequently the sclerenchyma ring surrounding the vascular system was not a mechanical barrier to root formation. However, IAA and IBA treatment can contribute to the disorganization of pith and xylem tissues, leading to the formation of adventitious roots. (IV) The application of rooting hormones, IAA and IBA, on mini-cuttings enhanced the rooting ability of the E. camaldulensis genotype. Additionally, auxin application at the base of the mini-cuttings, also induces faster rooting compared to the control. The accelerated rooting process shortened the duration required for mini-cuttings to root in the greenhouse, making it a viable option for adoption in commercial nurseries to enhance operational efficiency and optimize their facilities.