Introduction

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., 2011Bindumadhava H, Tamak J, Mahavishnan K, Upadhyay AP, Varghese M, Sharma N, 2011. Clonal propagation in Eucalyptus camaldulensis using mini-cutting technique. CurrSci 101 (12): 1578-1585.; Shanthi et al., 2015Shanthi K, Bachpai VKW, Anisha S, Ganesan M, Anithaa RG, Subashini V, Chakravarthi M, Sivakumar V, Yasodha R, 2015. Micropropagation of Eucalyptus camaldulensis for the production of rejuvenated stock plants for microcuttings propagation and genetic fidelity assessment. New For 46: 357–371. 10.1007/s11056-014-9465-1). 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., 2021Amrutha S, Parveen ABM, Muthupandi M, Vishnu K, Bisht SS, Sivakumar V, Ghosh Dasgupta M, 2021. Characterization of Eucalyptus camaldulensis clones with contrasting response to short-term water stress response. Acta Physiol Plant 43 (14): 1-13. 10.1007/s11738-020-03175-0).

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, 2012Shanmugam PS, Seenivasan R, 2012. Standardization of Mini Cutting Clonal Propagation Technique in Eucalyptus Camaldulensis and E. Terticornis Spp.Indian For 138 (4): 333-338.). Mass multiplication of E. camaldulensis clones has been previously reported using various propagation techniques, including micropropagation by direct organogenesis (Girijashankar, 2012Girijashankar V, 2012. In vitro regeneration of Eucalyptus camaldulensis. Physiol Mol BiolPlants 18 (1): 79-87. 10.1007/s12298-011-0092-4), as well as cutting and mini-cutting methods (Bindumadhava et al., 2011Bindumadhava H, Tamak J, Mahavishnan K, Upadhyay AP, Varghese M, Sharma N, 2011. Clonal propagation in Eucalyptus camaldulensis using mini-cutting technique. CurrSci 101 (12): 1578-1585.; Shanmugam & Seenivasan, 2012Shanmugam PS, Seenivasan R, 2012. Standardization of Mini Cutting Clonal Propagation Technique in Eucalyptus Camaldulensis and E. Terticornis Spp.Indian For 138 (4): 333-338.). 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., 2013Xavier A, Wendling I, Silva RL, 2013. Silvicultura clonal: Princípios e Técnicas. pp: 279. Viçosa, Minas Gerais.; Kuppusamy et al., 2019Kuppusamy O, Ramanathan S, Sengodagounder S,Senniappan C, Brindhadevi K, Kaliannan T, 2019. Mini-cutting - A powerful tool for the clonal propagation of the selected species of the Eucalyptus hybrid clones based on their pulpwood studies. Biocat Agric Biotechnol 22: 101357. 10.1016/j.bcab.2019.101357).

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., 2014Wendling I, Trueman SJ, Xavier A, 2014. Maturation and related aspects in clonal forestry-part II: reinvigoration, revitalization and juvenility maintenance. New For 45 (1): 473-486. 10.1007/s11056-014-9415-y; Vilasboa et al., 2022Vilasboa J, Da Costa CT, Fett-Neto AG, 2022. Environmental Modulation of Mini-Clonal Gardens for Cutting Production and Propagation of Hard- and Easy-to-Root Eucalyptus spp.Plants 11 (23): 3281-3305. 10.3390/plants11233281). Juvenile branches in good nutritional condition tend to exhibit better rooting ability (Wendling et al., 2015Wendling I, Brooks PR, Trueman SJ, 2015. Topophysis in Corymbia torelliana × C. citriodora seedlings: adventitious rooting capacity, stem anatomy, and auxin and abscisic acid concentrations. New for 46 (1): 107-120. 10.1007/s11056-014-9451-7), which is further enhanced by the application of plant growth regulators (Wendling et al., 2015Wendling I, Brooks PR, Trueman SJ, 2015. Topophysis in Corymbia torelliana × C. citriodora seedlings: adventitious rooting capacity, stem anatomy, and auxin and abscisic acid concentrations. New for 46 (1): 107-120. 10.1007/s11056-014-9451-7; Stuepp et al., 2017Stuepp CA, Wendling I, Trueman SJ, Koehler HS, Zuffellato-Ribas KC, 2017. The use of auxin quantification for understanding clonal tree propagation. Forests 8 (1): 27. 10.3390/f8010027; Lima et al., 2022Lima MS, Araujo MM, Berghetti ALP, Aimi SC, Costella C, Griebeler AM, Somavilla AM, Santos OP, Valente BMRT, 2022. Mini-cutting technique application in Corymbia and Eucalyptus: effects of mini-tunnel use across seasons of the year. New For 53 (1): 161-179. 10.1007/s11056-021-09851-4). Environmental conditions, such as the collection season, also play a critical role in propagation success (Batista et al., 2015Batista AF, dos Santos GA, Silva LD, Quevedo FF, de Assis TF, 2015. The use of mini-tunnels and the effects of seasonality in the clonal propagation of Eucalyptus in a subtropical environment. AustFor 78 (2): 65-72. 10.1080/00049158.2015.1039162).

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, 2021Díaz-Sala C, 2021. Adventitious Root Formation in Tree Species. Plants 10 (3): 486-489. 10.3390/plants10030486). They are essential regulators of adventitious root induction (Díaz-Sala, 2020Díaz-Sala C, 2020. A Perspective on Adventitious Root Formation in Tree Species. Plants 9 (12): 1789-1726. 10.3390/plants9121789). 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., 2017Stuepp CA, Wendling I, Trueman SJ, Koehler HS, Zuffellato-Ribas KC, 2017. The use of auxin quantification for understanding clonal tree propagation. Forests 8 (1): 27. 10.3390/f8010027). 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., 2017Stuepp CA, Wendling I, Trueman SJ, Koehler HS, Zuffellato-Ribas KC, 2017. The use of auxin quantification for understanding clonal tree propagation. Forests 8 (1): 27. 10.3390/f8010027; Díaz-Sala, 2020Díaz-Sala C, 2020. A Perspective on Adventitious Root Formation in Tree Species. Plants 9 (12): 1789-1726. 10.3390/plants9121789), 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., 2011Hartmann HT, Kester DE, DaviesJunior FT, Geneve RL, 2011. Plant propagation: principles and practices. pp: 915. Prentice-Hall, New Jersey.).

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., 2010Brondani GE, Grossi F, Wendling I, Dutra LF, Araujo MA, 2010. IBA application for rooting of Eucalyptus benthamii Maiden & Cambage x Eucalyptus dunnii Maiden mini-cuttings. Acta Sci. Agron 32 (4): 667-674. 10.4025/actasciagron.v32i4.4879). Additionally, the origin of the propagule along the branch can influence rooting success due to ontogenetic and physiological factors (Wendling et al., 2014Wendling I, Trueman SJ, Xavier A, 2014. Maturation and related aspects in clonal forestry-part II: reinvigoration, revitalization and juvenility maintenance. New For 45 (1): 473-486. 10.1007/s11056-014-9415-y). 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, 2015Bryant PH, Trueman SJ, 2015. Stem anatomy and adventitious root formation in cuttings of Angophora, Corymbia and Eucalyptus. Forests 6 (4): 1227-1238. 10.3390/f6041227).

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, 2015Bryant PH, Trueman SJ, 2015. Stem anatomy and adventitious root formation in cuttings of Angophora, Corymbia and Eucalyptus. Forests 6 (4): 1227-1238. 10.3390/f6041227; Vilasboa et al., 2021). However, the anatomy of adventitious rooting remains variable and not fully understood (Bryant and Trueman, 2015Bryant PH, Trueman SJ, 2015. Stem anatomy and adventitious root formation in cuttings of Angophora, Corymbia and Eucalyptus. Forests 6 (4): 1227-1238. 10.3390/f6041227), despite its critical role in maximizing rooting rates (Pimentel et al., 2020Pimentel N, Pedroso MF, Lencina KH, Oliveira JMS, Bisognin DA, 2020. Anatomical Characterization of the Adventitious Roots of Mate (Ilex paraguariensis A. St.-Hil.) Mini-cuttings. Braz Arch Bio Technol 63:1-13. 10.1590/1678-4324-2020190359). 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.

Material and methods

Plant Material

 ⌅

A five-year-old genotype of E. camaldulensis was carefully selected based on its survival rate (90%) and growth performance (height: 15.3 m; diameter at 1.30 m: 10.4 cm) for cultivation in the semiarid region of Mossoró, located at coordinates 05°03’13.1’’S, 37°23’34.9’’W, and an altitude of 78 m, in the state of Rio Grande do Norte, Brazil. This region experiences a dry semiarid climate, with an average temperature of 27.8°C and a relative humidity of 68.9%. Precipitation is highly irregular, averaging 740 mm/year and often accompanied by prolonged drought periods (Fig. 1). The experiments were conducted in March, May, June, July, August, and September.

  

Figure 1 Monthly averages of temperature (°C), relative humidity (%) and pluviosity (mm) for 2018, 2019, 2021 and 2022 in Mossoró, Rio Grande do Norte, Brazil. 

Statistical analysis

 ⌅

To identify pruning effects in a mini-clonal garden for mini-cutting production and propagation of E. camaldulensis clone selected in a semiarid condition, we examined the effects of propagullos collections on survival, number of sprouts per mini-stump and yield of sprouts at 5 successive collections. For statistical analysis, the assumptions of normality (Shapiro-Wilk test) and homoscedasticity (Bartlett test) were calculated and tested, at a level of 5% significance and then submitted to ANOVA.

The quantitative data were checked for normality and then submitted to factorial ANOVA followed by estimated marginal means, Tukey’s,

In sequence, to investigate whether mini-cuttings from mini-stumps of E. camaldulensis clone are responsive to adventitious rooting and the auxin can improve mini-cutting rooting, we setup a experiment analizing the effect of different doses of IAA and IBA auxins on mini-cutting rooting. Next, we investigated the optimum time for mini-cutting rooting inside the greenhouse, as well we identified different morphological events that result in adventitious rooting. To explain the rooting response of auxin-treated mini-cuttings to concentrations and days, and differentiate the morphological responses of mini-cuttings, we applied a generalized linear model (GLM) having a Poisson distribution and a log link function. In experiment II, we set auxin types and concentrations as fixed effects. For experiment III, we included both auxins and days as fixed effects. We ran all our analyses in R software version 4.3.3 (R Development Core Team, 2017R Development Core Team, 2017. R Foundation for Statistical Computing R: A Language and Environment for Statistical Computing. https://www.R-project.org/. [19 March 2024]) using packages dplyr, tibble and ggplot2.

A comprehensive overview of each step, from the clonal mini-garden to the experimental design sequence, is illustrated by Fig. 2. An overall picture of the mini-stumps is shown in Fig. 2B before the shoot collections, while Figure 2C shows the mini-stumps after shoot collection events, and Fig. 2D with mature propagules on mini-stumps that were excluded from propagation.

  

Figure 2 Clonal propagation of Eucalyptus camaldulensis through mini-cutting. (A) General view of the mini-stumps after breaking apical dominance. (B) Mini-stumps before the different shoot collection events with juvenile propagules. (C) the mini-stumps after shoot collection events. D Mini-stumps with mature propagules that were not utilized for propagation. Sequency of the experimental design: experiment I, II, III and IV. 

Results

Mini-stump survival and production

 ⌅

The mini-stump prunings showed no significant differences across the five propagule collections in terms of survival, the number of sprouts per mini-stump, and sprout yield (sprouts/m²/month) (Fig. 3A, B, C). Survival was consistently high at 100% (Fig. 3A). The mini-stumps of the E. camaldulensis clone demonstrated efficient propagule production, yielding more than 13 propagules per mini-stump per month (Fig. 3B) and 900 sprouts per m² per month (Fig. 3C).

  

Figure 3 Impact of mini-stump pruning on propagule production in the Eucalyptus camaldulensis clone across successive sprout collections. (A) Mini-stump survival, (B) number of sprouts per mini-stump, and (C) the yield of sprouts (sprouts/m²/month). Bars indicate the standard error of the mean of four biological replicates (n=40). ^ns: not significant. 

Influence of IAA and IBA

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There was a significant interaction between auxins and their concentrations for most morphological variables, except for longest root length (cm per mini-cutting) and number of roots (units per mini-cutting), p < 0.05 (Table 1). Auxins significantly affected only base oxidation (%) and callus formation (%). Conversely, concentrations significantly influenced rooting (%), survival (%), base oxidation (%), and callus formation (%).

  

Table 1 Effects of auxin types (IAA and IBA), concentrations (0 - control; 1,000; 2,000; and 4,000 mg·L^-1), and their interactions (Auxins types:Concentrations) on rooting (%), survival (%), base oxidation (%), callus formation (%), longest root length (cm mini-cutting ^-1) and number of roots (unit mini-cutting ^-1) in Eucalyptus camaldulensis mini-cuttings. ANOVA of generalized linear model (Poisson function) to rooting (%), survival (%), base oxidation (%), presence of callus (%), of Eucalyptus camaldulensis mini-cuttings subjected to auxins (IAA and IBA) and concentrations (0 - control; 1,000; 2,000; and 4,000 mg.L^-1). 

Effect	Pr (>Chi)
	Rooting	Survival	Base Oxidation	Presence of Callus	Longest Root Length	Number of Roots
Auxins types	0.0567	0.0566	0.0292	1.5-09	0.1789	0.9364
Concentrations	4.5-14	1.6-14	< 2.2-16	< 2.2-16	0.5919	0.9462
Auxins types: Concentrations	0.0062	0.0179	1.8-12	1.6-12	0.7854	0.4426

[i] 

Statistical analysis was performed using ANOVA based on a generalized linear model (Poisson function). The numbers in table indicate the p-value.

The mini-cuttings of the E. camaldulensis clone showed responsiveness to adventitious rooting, with more than 55% rooting and survival in the control treatment (Figs. 4A, B). Both IAA and IBA improved rooting and survival equally, up to a concentration of 2,000 mg·L⁻¹. At a concentration of 4,000 mg·L^-1, the auxin IAA resulted in higher rooting rates (75%) compared to IBA (50%) (Fig. 4A, B). The highest percentages of rooting and survival were observed at 2,000 mg·L^-1, reaching 80% (Fig. 4A) and 85% (Fig. 4B), respectively.

  

Figure 4 Effects of Indole-3-Acetic Acid (IAA) and Indole-3-Butyric Acid (IBA) on adventitious rooting of Eucalyptus camaldulensis clone propagated by mini-cuttings. (A) Mini-cutting rooting (%), (B) mini-cutting survival (%), (C) base oxidation (%), (D) presence of callus (%), (E) longest root length (cm mini-cutting ^-1), and (F) number of roots (unit mini-cutting ^-1). 95% confidence interval predicted by generalized linear model (Poisson function). 

Base oxidation was higher (14%) in mini-cuttings treated with 1,000 mg·L^-1 of IAA (Fig. 4C), while callus formation was more prominent in the control and at 4,000 mg·L^-1 of IBA (Fig. 4D). However, different concentrations of IAA and IBA did not influence the number of roots (units per mini-cutting) or the longest root length (cm per mini-cutting) (Table 1 and Figs. 4E, F).

Collectively, these results suggest that the mini-cuttings of the E. camaldulensis clone are highly responsive to adventitious rooting and that the auxins IAA and IBA can significantly enhance rooting and survival.

Rotting speed

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There was a significant interaction between auxin application and days for rooting (%), callus formation (%), and oxidation (%), p < 0.05. Auxin application significantly affected rooting (%) and callus formation (%). However, the number of days in the greenhouse produced significant differences (p < 0.05) across all morphological variables (Table 2).

  

Table 2 Effects of auxin application (control, IAA, and IBA), evaluation days, and their interaction (Auxin application:Evaluation days) on swelling (%), rooting (%), callus (%), oxidation (%), number of roots (unit mini-cutting ^-1) and longest root length (cm mini-cutting ^-1) in Eucalyptus camaldulensis mini-cuttings. ANOVA of generalized linear model (Poisson function) to swelling (%), rooting (%), callus (%), oxidation (%), number of roots (unit mini-cutting ^-1) and longest root length (cm mini-cutting ^-1) of mini-cuttings of Eucalyptus camaldulensis clone subjected to auxin application (control, IAA and IBA) and days. The numbers in table indicate the p-value. 

Effect	Pr (>Chi)
	Swelling	Rooting	Callus	Oxidation	Number of Roots	Longest Root Length
Auxins application	0.9307	5.8-12	2.1-05	1.0000	0.0647	0.2089
Evaluation days	<2-16	< 2.2-16	< 2.2-16	<2-16	< 2-16	<2-16
Auxins application: Evaluation days	0.9712	2.4-13	6.9-05	0.0122	0.7560	0.9990

[i] 

Statistical analysis was performed using ANOVA based on a generalized linear model (Poisson function). The numbers in table indicate the p-value.

As expected, initial morphological changes in the basal region of the mini-cuttings were observed seven days after their introduction to the greenhouse (Fig. 5A, C). Adventitious root formation began with swelling at the bases of the mini-cuttings, followed by callus formation. Over time, the swelling response was similar among treatments with IAA, IBA, and the control, with higher averages (70%) recorded between 7 and 14 days (Fig. 5A). In contrast, greater callus formation (60%) was observed in the control treatment at 14 days (Fig. 5C). The mini-cuttings of the E. camaldulensis clone also exhibited low base oxidation, with the highest percentage (6%) occurring in the IAA treatment at 28 days (Fig. 5D).

Rhizogenesis in the mini-cuttings began on Day 14 for IAA and IBA treatments, while it was delayed for the control treatment (Fig. 5B). Thus, IAA and IBA treatments increased the rooting speed of the mini-cuttings, resulting in the highest rooting percentages throughout the evaluation period. For both IAA and IBA treatments, up to 45% of the mini-cuttings had rooted by Day 21, with rooting stabilization occurring only after 28 days (Fig. 5B). Therefore, the time required for mini-cuttings to remain in the greenhouse to achieve adequate rooting percentages depends on auxin application, with a longer duration required in the absence of IAA or IBA (Fig. 5B).

  

Figure 5 Indole-3-Acetic Acid (IAA) and Indole-3-Butyric Acid (IBA) increased the rooting speed of mini-cuttings from propagules of the Eucalyptus camaldulensis clone. (A) Mini-cutting swelling (%), (B) mini-cutting rooting (%), (C) presence of callus (%), (D) base oxidation (%), (E) number of roots (unit mini-cutting ^-1), and (F) longest root length (cm mini-cutting ^-1). 95% confidence interval predicted by generalized linear model (Poisson function). 

Root length and the number of roots did not differ significantly among the control, IAA, and IBA treatments (Table 2) and remained relatively stable until Day 21. By Day 28, the longest root reached 4.8 cm, with an average of 2.6 roots per mini-cutting (Figs. 5E, F).

Histological analyses

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The shoots of the E. camaldulensis clone contained a central pith surrounded by xylem, vascular cambium, phloem, cortex, and epidermis (Fig. 6A). The stems were rectangular in transverse section, with a prominent ring of sclerenchyma cells at the periphery of the vascular tissue. Additionally, the cortex, near the epidermis, contained cavities with oil (idioblasts) (Fig. 6A). Secondary growth was evident in all studied mini-cuttings, characterized by cambium activity and well-developed secondary phloem and xylem.

Adventitious roots originated from the xylem, phloem, or vascular cambium in mini-cuttings treated with IAA and IBA (Figs. 6B, C). However, in untreated mini-cuttings (without auxins), roots arose from the phloem or vascular cambium (Fig. 6B). Root formation was direct, with the same behavior observed in auxin-treated and untreated mini-cuttings; no simultaneous formation of callus and adventitious roots occurred. The sclerenchyma ring was ruptured by root primordia formation (Fig. 6C). After seven days, groups of dividing cells (meristemoids) became oval in shape and differentiated into root primordia (Fig. 6C). These primordia extended perpendicularly to the main axis toward the epidermis.

At approximately 14 days, young white roots began to emerge from the epidermis and became visible outside the propagule (Fig. 6D). Adventitious roots developed from both corner and side positions, up to 2 cm above the base of the mini-cuttings (Fig. 6D). In the following weeks, the roots continued to grow and initiated the production of secondary lateral roots (Fig. 6E).

  

Figure 6 Transverse sections of the basal regions of Eucalyptus camaldulensis mini-cuttings and their role in adventitious root formation. (A) Mini-cutting base overview, (B) detail showing the formation of adventitious root primordium from vascular and phloem tissue, (C) developing adventitious root from pith tissue, (D) external adventitious root primordia, and (E) root elongation. c: cortex, e: epidermis, ph: phloem, p: pith, rc: sclerenchyma ring, ca: vascular cambium, cv: cavity containing oil (idioblasts), x = xylem. 

These results suggest that root primordia formation occurred within one to two weeks, and the sclerenchyma ring did not prevent adventitious rooting. In mini-cuttings treated with IAA and IBA, adventitious root primordia originated from vascular, phloem, and pith tissues. In contrast, in untreated mini-cuttings, roots developed from the vascular cambium and phloem.

Discussion

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., 2014Wendling I, Trueman SJ, Xavier A, 2014. Maturation and related aspects in clonal forestry-part II: reinvigoration, revitalization and juvenility maintenance. New For 45 (1): 473-486. 10.1007/s11056-014-9415-y; Batista et al., 2015Batista AF, dos Santos GA, Silva LD, Quevedo FF, de Assis TF, 2015. The use of mini-tunnels and the effects of seasonality in the clonal propagation of Eucalyptus in a subtropical environment. AustFor 78 (2): 65-72. 10.1080/00049158.2015.1039162; Vilasboa et al., 2022Vilasboa J, Da Costa CT, Fett-Neto AG, 2022. Environmental Modulation of Mini-Clonal Gardens for Cutting Production and Propagation of Hard- and Easy-to-Root Eucalyptus spp.Plants 11 (23): 3281-3305. 10.3390/plants11233281; Lima et al., 2022Lima MS, Araujo MM, Berghetti ALP, Aimi SC, Costella C, Griebeler AM, Somavilla AM, Santos OP, Valente BMRT, 2022. Mini-cutting technique application in Corymbia and Eucalyptus: effects of mini-tunnel use across seasons of the year. New For 53 (1): 161-179. 10.1007/s11056-021-09851-4).

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., 2013Trueman SJ, McMahon TV, Bristow M, 2013. Production of Eucalyptus cloeziana Cuttings in Response to Stock Plant Temperature. J Trop For Sci 25 (1): 60–69.). 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., 2022Lima MS, Araujo MM, Berghetti ALP, Aimi SC, Costella C, Griebeler AM, Somavilla AM, Santos OP, Valente BMRT, 2022. Mini-cutting technique application in Corymbia and Eucalyptus: effects of mini-tunnel use across seasons of the year. New For 53 (1): 161-179. 10.1007/s11056-021-09851-4). 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., 2015Batista AF, dos Santos GA, Silva LD, Quevedo FF, de Assis TF, 2015. The use of mini-tunnels and the effects of seasonality in the clonal propagation of Eucalyptus in a subtropical environment. AustFor 78 (2): 65-72. 10.1080/00049158.2015.1039162; Lima et al., 2022Lima MS, Araujo MM, Berghetti ALP, Aimi SC, Costella C, Griebeler AM, Somavilla AM, Santos OP, Valente BMRT, 2022. Mini-cutting technique application in Corymbia and Eucalyptus: effects of mini-tunnel use across seasons of the year. New For 53 (1): 161-179. 10.1007/s11056-021-09851-4; Vilasboa et al., 2022Vilasboa J, Da Costa CT, Fett-Neto AG, 2022. Environmental Modulation of Mini-Clonal Gardens for Cutting Production and Propagation of Hard- and Easy-to-Root Eucalyptus spp.Plants 11 (23): 3281-3305. 10.3390/plants11233281), is unnecessary in semiarid conditions due to naturally high temperatures.

In E. camaldulensis, regrowth ability from stumps (Heth et al., 1986Heth D, Fanger-Vexler L, Reuveni O, 1986. Mass production of cuttings of Eucalyptus camaldulensis Dehn. The Commonw For Rev 65 (3): 215-225.; Reuveni et al., 1990Reuveni O, Fanger-Vexler L, Heth D, 1990. The effect of rooting environment, kind and source of cuttings on rooting of Eucalyptus camaldulensis Dehn. cuttings. CommonwFor Rev 69 (2): 181-189.), mini-stumps (Bindumadhava et al., 2011Bindumadhava H, Tamak J, Mahavishnan K, Upadhyay AP, Varghese M, Sharma N, 2011. Clonal propagation in Eucalyptus camaldulensis using mini-cutting technique. CurrSci 101 (12): 1578-1585.), and micro-stumps (Shanthi et al., 2015Shanthi K, Bachpai VKW, Anisha S, Ganesan M, Anithaa RG, Subashini V, Chakravarthi M, Sivakumar V, Yasodha R, 2015. Micropropagation of Eucalyptus camaldulensis for the production of rejuvenated stock plants for microcuttings propagation and genetic fidelity assessment. New For 46: 357–371. 10.1007/s11056-014-9465-1) 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., 2011Bindumadhava H, Tamak J, Mahavishnan K, Upadhyay AP, Varghese M, Sharma N, 2011. Clonal propagation in Eucalyptus camaldulensis using mini-cutting technique. CurrSci 101 (12): 1578-1585.; Brondani et al., 2012Brondani GE, Wendling I, Brondani AE, Araujo MA, da Silva ALL, Gonçalves AN, 2012. Dynamics of adventitious rooting in mini-cuttings of Eucalyptus benthamii x Eucalyptus dunnii. Acta Sci. Agron 34 (2): 169-178. 10.4025/actasciagron.v34i2.13059) and is comparable to productivity achieved using mini-tunnels in different seasons (Batista et al., 2015Batista AF, dos Santos GA, Silva LD, Quevedo FF, de Assis TF, 2015. The use of mini-tunnels and the effects of seasonality in the clonal propagation of Eucalyptus in a subtropical environment. AustFor 78 (2): 65-72. 10.1080/00049158.2015.1039162; Lima et al., 2022Lima MS, Araujo MM, Berghetti ALP, Aimi SC, Costella C, Griebeler AM, Somavilla AM, Santos OP, Valente BMRT, 2022. Mini-cutting technique application in Corymbia and Eucalyptus: effects of mini-tunnel use across seasons of the year. New For 53 (1): 161-179. 10.1007/s11056-021-09851-4; Vilasboa et al., 2022Vilasboa J, Da Costa CT, Fett-Neto AG, 2022. Environmental Modulation of Mini-Clonal Gardens for Cutting Production and Propagation of Hard- and Easy-to-Root Eucalyptus spp.Plants 11 (23): 3281-3305. 10.3390/plants11233281). Moreover, our data closely align with productivity levels reported for mini-stumps of other species grown under semiarid conditions (Souza et al., 2023Souza LS, Diógenes FEG, Silveira GVS, da Silva CJ, Araujo PCD, 2023. Mini-cutting as a technique to propagate Tabebuia aurea, an important tree found in tropical dry forests. Aust Jl Crop Sci, 17 (8): 631-638. 10.21475/ajcs.23.17.08.p3892; Silva et al., 2022Silva AKV, Aguiar TS, Santos MEC, Araujo JKP, Freire AC, Salami G, Araujo PCD, 2022. Vegetative propagation of Mimosa Caesalpiniifolia BY mini-cuttings technique. RevÁrvore 46: 4631. 10.1590/1806-908820220000031). 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, 2022Gudynaitė-Franckevičienė V, Pliūra A. 2022. Performance and Genetic Parameters of Poplar Hybrids and Clones in a Field Trial Are Modified by Contrasting Environmental Conditions during the Vegetative Propagation Phase. Plants 11(18):2401. 10.3390/plants11182401). 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., 2014Wendling I, Trueman SJ, Xavier A, 2014. Maturation and related aspects in clonal forestry-part II: reinvigoration, revitalization and juvenility maintenance. New For 45 (1): 473-486. 10.1007/s11056-014-9415-y; Gianguzzi et al., 2020Gianguzzi V, Barone E, Sottile F, 2020. In Vitro rooting of Capparis spinosa L. as affected by genotype and by the proliferation method adopted during the multiplication phase. Plants 9 (3): 398. 10.3390/plants9030398). Together, these factors influence rooting capacity and determine the success of clonal propagation by mini-cuttings (Wendling et al., 2014Wendling I, Trueman SJ, Xavier A, 2014. Maturation and related aspects in clonal forestry-part II: reinvigoration, revitalization and juvenility maintenance. New For 45 (1): 473-486. 10.1007/s11056-014-9415-y; De Almeida et al., 2017De Almeida MR, Aumond M, Da Costa CT, Schwambach J, Ruedell CM, Correa LR, Fett-Neto AG, 2017. Environmental Control of Adventitious Rooting in Eucalyptus and Populus Cuttings. Trees 31: 1377-1390. 10.1007/s00468-017-1550-6).

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., 2013Trueman SJ, McMahon TV, Bristow M, 2013. Production of Eucalyptus cloeziana Cuttings in Response to Stock Plant Temperature. J Trop For Sci 25 (1): 60–69.). 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, 2019Pizarro A, Díaz-Sala C, 2019. Cellular dynamics during maturation-related decline of adventitious root formation in forest tree species. Physiol plant 165 (1): 73-80. 10.1111/ppl.12768).

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., 2017De Almeida MR, Aumond M, Da Costa CT, Schwambach J, Ruedell CM, Correa LR, Fett-Neto AG, 2017. Environmental Control of Adventitious Rooting in Eucalyptus and Populus Cuttings. Trees 31: 1377-1390. 10.1007/s00468-017-1550-6). For instance, Vilasboa et al. (2022Vilasboa J, Da Costa CT, Fett-Neto AG, 2022. Environmental Modulation of Mini-Clonal Gardens for Cutting Production and Propagation of Hard- and Easy-to-Root Eucalyptus spp.Plants 11 (23): 3281-3305. 10.3390/plants11233281) 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, 2020Díaz-Sala C, 2020. A Perspective on Adventitious Root Formation in Tree Species. Plants 9 (12): 1789-1726. 10.3390/plants9121789).

In mini-cutting, IAA and IBA are commonly used to enhance rooting rates (Hartmann et al., 2011Hartmann HT, Kester DE, DaviesJunior FT, Geneve RL, 2011. Plant propagation: principles and practices. pp: 915. Prentice-Hall, New Jersey.). However, the choice of auxin depends largely on the species or genotype (Steffens and Rasmussen, 2016Steffens B, Rasmussen, A, 2016. The Physiology of Adventitious Roots. Plant Physiol, 170: 603–617. 10.1104/pp.15.01360; Ayala et al., 2022Ayala PG, Acevedo RM, Luna CV, Rivarola M, Acuña C, Marcucci Poltri S, González AM, Sansberro PA, 2022. Transcriptome Dynamics of Rooting Zone and Leaves during In Vitro Adventitious Root Formation in Eucalyptus nitens. Plants, 11(23):3301. 10.3390/plants11233301). 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., 1986Heth D, Fanger-Vexler L, Reuveni O, 1986. Mass production of cuttings of Eucalyptus camaldulensis Dehn. The Commonw For Rev 65 (3): 215-225.; Reuveni et al., 1990Reuveni O, Fanger-Vexler L, Heth D, 1990. The effect of rooting environment, kind and source of cuttings on rooting of Eucalyptus camaldulensis Dehn. cuttings. CommonwFor Rev 69 (2): 181-189.). Using the mini-cutting technique, some genotypes achieved up to 97% adventitious rooting (Bindumadhava et al., 2011Bindumadhava H, Tamak J, Mahavishnan K, Upadhyay AP, Varghese M, Sharma N, 2011. Clonal propagation in Eucalyptus camaldulensis using mini-cutting technique. CurrSci 101 (12): 1578-1585.). However, for recalcitrant genotypes, micropropagation techniques inducing somatic embryos improved propagation capacity, enabling large-scale multiplication of elite trees (Girijashankar, 2012Girijashankar V, 2012. In vitro regeneration of Eucalyptus camaldulensis. Physiol Mol BiolPlants 18 (1): 79-87. 10.1007/s12298-011-0092-4). 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., 2015Shanthi K, Bachpai VKW, Anisha S, Ganesan M, Anithaa RG, Subashini V, Chakravarthi M, Sivakumar V, Yasodha R, 2015. Micropropagation of Eucalyptus camaldulensis for the production of rejuvenated stock plants for microcuttings propagation and genetic fidelity assessment. New For 46: 357–371. 10.1007/s11056-014-9465-1). 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., 2013Xavier A, Wendling I, Silva RL, 2013. Silvicultura clonal: Princípios e Técnicas. pp: 279. Viçosa, Minas Gerais.), 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., 2012Brondani GE, Wendling I, Brondani AE, Araujo MA, da Silva ALL, Gonçalves AN, 2012. Dynamics of adventitious rooting in mini-cuttings of Eucalyptus benthamii x Eucalyptus dunnii. Acta Sci. Agron 34 (2): 169-178. 10.4025/actasciagron.v34i2.13059), 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, 2015Bryant PH, Trueman SJ, 2015. Stem anatomy and adventitious root formation in cuttings of Angophora, Corymbia and Eucalyptus. Forests 6 (4): 1227-1238. 10.3390/f6041227). 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., 2013Xavier A, Wendling I, Silva RL, 2013. Silvicultura clonal: Princípios e Técnicas. pp: 279. Viçosa, Minas Gerais.). 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., 2014Goulart PB, Xavier A, Iarema L, Otoni WC, 2014. Morpho-anatomy of adventitious rhizogenesis in mini-cuttings of Eucalyptus grandis x Eucalyptus urophylla. CiêncFlorest 24 (3): 521-532. 10.5902/1980509815721; Bryant and Trueman, 2015Bryant PH, Trueman SJ, 2015. Stem anatomy and adventitious root formation in cuttings of Angophora, Corymbia and Eucalyptus. Forests 6 (4): 1227-1238. 10.3390/f6041227), 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 (2015Bryant PH, Trueman SJ, 2015. Stem anatomy and adventitious root formation in cuttings of Angophora, Corymbia and Eucalyptus. Forests 6 (4): 1227-1238. 10.3390/f6041227), 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., 2014Goulart PB, Xavier A, Iarema L, Otoni WC, 2014. Morpho-anatomy of adventitious rhizogenesis in mini-cuttings of Eucalyptus grandis x Eucalyptus urophylla. CiêncFlorest 24 (3): 521-532. 10.5902/1980509815721), Corymbia torelliana F.Muell., and E. camaldulensis (Bryant and Trueman, 2015Bryant PH, Trueman SJ, 2015. Stem anatomy and adventitious root formation in cuttings of Angophora, Corymbia and Eucalyptus. Forests 6 (4): 1227-1238. 10.3390/f6041227).

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., 2007Sánchez C, Vielba JM, Ferro E, Covelo G, Solé A, Abarca D, de Mier BS, Díaz-Sala C, 2007. Two SCARECROW-LIKE genes are induced in response to exogenous auxin in rooting-competent cuttings of distantly related forest species. Tree Physiol 27 (10): 1459–1470. 10.1093/treephys/27.10.1459; Wang et al., 2022Wang Y, Khan MA, Zhu ZL, Hai TM, Sang ZY, Jia ZK, Ma LY, 2022. Histological, morpho-physiological, and biochemical changes during adventitious rooting induced by exogenous auxin in Magnolia wufengensis cuttings. Forests 13 (6): 925. 10.3390/f13060925). Auxin has been shown to induce xylem differentiation in Arabidopsis (Ohashi-Ito et al., 2013Ohashi-Ito K, Matsukawa M, Fukuda H, 2013. An atypical bHLH transcription factor regulates early xylem development downstream of auxin. Plant Cell Physiol. 54 (3): 398–405. 10.1093/pcp/pct013). 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., 2015Wendling I, Brooks PR, Trueman SJ, 2015. Topophysis in Corymbia torelliana × C. citriodora seedlings: adventitious rooting capacity, stem anatomy, and auxin and abscisic acid concentrations. New for 46 (1): 107-120. 10.1007/s11056-014-9451-7). 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.

Conclusions

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.