Vegetative propagation of Apuleia leiocarpa (Vogel) J. F. Macbr

José Gabriel de Souza Silva ·Diêgo Faustolo Alves Bispo ·

David Pessanha Siqueira1 ·Giovanna Campos Mamede Weiss de Carvalho1 ·Deborah Guerra Barroso1

Abstract Apuleia leiocarpa is an environmentally and economically significant Atlantic Forest species threatened by ongoing exploitation. The vegetative propagation of the species could be a step forward to enable implantation of clonal seed orchards and multiplication of selected trees but remains unknown to date. This study assessed the mini-cutting technique for propagation of A. leiocarpa and determined the time required for rooting and viable minicutting type and sizes. The results show that it is possible to propagate A. leiocarpa with mini-cuttings derived from mini-stumps produced with seeds; the mini-stumps presented regrowth capacity and remained constantly productive for 1 year; 10-cm A. leiocarpa mini-cuttings should remain under misting conditions for at least 40 days, and the vegetative propagation of A. leiocarpa with intermediate 7- to 10-cm mini-cuttings resulted in more vigorous clonal seedlings than smaller mini-cuttings.

Keywords Mini-cutting·Tropical woody species·Adventitious rooting·Fabaceae

Introduction

The reforestation of degraded areas with Atlantic forest species, for economic purposes or environmental conservation, is a relatively recent and growing activity in Brazil. Because of its highly diverse wood products, the Brazilian Atlantic Forest has been one of the country’s most exploited biomes since the country’s founding and its floristic and structural composition is currently fragmented (Baynes et al. 2016).Apuleia leiocarpa(Vogel) J. F. Macbr is an Atlantic Forest tree well-adapted to hillsides and well-drained soils with a wide geographic distribution in South America: Brazil, Argentina, Bolivia, Paraguay, Peru, and Uruguay (Carvalho 2003). However, due to its high value in the timber market,A. leiocarpahas been logged extensively, included on the list of endangered tree species, and it is currently classified as vulnerable (Martinelli and Moraes 2013).

One consequence of the logging and exporting of highquality Brazilian hardwoods has been increased pressure on existing native forest fragments, which made the production ofA. leiocarpaseedlings a profitable enterprise and helped to establish it as one of Brazil’s most traded species (SNIF 2018). In addition, the large portions of degraded areas in the country, as well as a global effort towards mitigating greenhouse gas emissions and promoting forest conservation have helped increase the demand for native species seedlings overall.

The ability to achieve success in the production of forest species seedlings requires high-quality seedlings derived from superior sources, or seedling that meet diversity requirements that satisfy to the plantation objectives. In addition, since the propagation ofA. leiocarpahas traditionally been carried out via seeds, the commercial production of this species’ seedlings requires ready access to seed sources, high seed viability, ideal seedling storage conditions, and extensive phenological knowledge of the species.

Besides these factors, Felippi et al. (2012) reported irregular yearlong seed productivity of the species associated with genetic and environmental factors such as precipitation and temperature, highlighting the need for further regional studies. Vegetative propagation with mini-cuttings from mini-stumps in small multi-clonal gardens could offer a solution to the problems reported with the added benefit of increasing selected genotypes. This requires the species to overcome apical dominance, produce vigorous shoots, and develop root structures adapted to new conditions (Pimentel et al. 2019).

Lencina et al. (2018) observed favorable sprouting capacity inA. leiocarpamicro-strains produced from seeds in vitro in a 6-benzilaminopurina (BAP) enriched medium, even in a subculture without cytokinin. The authors, however, did not assess the rooting capacity of the micro-cuttings.

Several studies of native forest species seedling production through vegetative propagation, carried out in the Atlantic Forest and elsewhere, have reported good results with the mini-cutting technique (Silva et al. 2010; Ciriello and Mori 2015; Mantovani et al. 2017; Freitas et al. 2018). It is noteworthy that results varied according to species, rooting period, substrate, site at which mini-cuttings were removed from the plant, among other factors. ForPlathymenia reticulata, for instance, while mini-stumps had a high sprouting rate over collections, the mini-cuttings exhibited low rooting rates (Pessanha et al. 2018). Silva et al. (2010), Wendling et al. (2015), and Ciriello and Mori (2015) found differences in survival and rooting rates associated with cutting sizes and types as well rooting chamber time permanence period. According to Frassetto et al. (2010), the specific location from which cuttings are removed from plants might influence auxin levels and consequently rooting rates.

Despite the known benefits of mini-cutting technique, the literature still lacks reports onA. leiocarpaand there is a need to define protocols for the species for commercial seedling production. The present study sought to assess the potential of mini-cutting as a vegetative propagation technique forA. leiocarpaand to determine mini-cutting rooting time, type, and size requirements for the species.

Materials and methods

Materials

The experiment was carried out in a greenhouse of the Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF). In total, 150 seedlings were produced fromA. leiocarpaseeds to form a mini-garden. The seeds were obtained from the State Environmental Institute (INEA), in the municipality of Santa Maria Madalena, RJ, and placed in sand to germinate (Fig. 1a) after immersion in water for 12 h (room temperature).

Fig. 1 Establishment of the A. leiocarpa multiclonal mini-garden. a Sowing in sand, b seedlings after transplanting to tubes of 280 cm3, and c, d Mini-stumps with apexes pruned at 10 cm above the collar in tubes of 280 cm3

To form the mini-garden, the seedlings were established in tubes of 280 cm3containing commercial substrate and slow-release fertilizer (Fig. 1b). The commercial forest substrate was Basaplant® used with slow-release fertilizer (8 g kg-1of substrate) formulated at 15-9-12 (N-P-K). Three months after transplanting the seedlings were pruned at a height of 10 cm above the collar to stimulate sprouting and mini-stump formation (Fig. 1c, d).

Mini‑stumps survival rates and productivity

The survival and productivity rates of the multiclonal minigarden mini-stumps were assessed at 150, 270, and 360 days after the apex pruning. The productivity considered the number of sprouts and mini-cuttings of 7 cm produced per mini-stump.

Time for rooting under mist chamber

In order to determine the suitable time periodA. leiocarpamini-cuttings should remain in mist chamber for rooting, 10-cm-long mini-cuttings were produced. The mini-cuttings were distributed in 32 plots (10 mini-cuttings per plot; 320 mini-cuttings in total) with no distinction made in relation to sprouting site position. The mini-cuttings were produced without reduction in leaf area and placed in 55 cm3tubes containing commercial forest substrate and slow-release fertilizer N-P-K (15-9-12) at 8 g kg-1of substrate (Fig. 2b).

The plots remained in the rooting sector under intermittent misting in a complete randomized design, and four plots (40 mini-cuttings each) at 25 days after cutting were randomly selected and rooting progress was evaluated. This procedure was repeated every 5 days until 60 days (Fig. 2a).

Upon assessment, plants were washed in sieves under running tap water to clean the roots (Fig. 2c), and survival (S), rooting (R), number of first-order roots (FRN), total root length (RL), and root system dry mass (RDM) were determined.

Mini‑cutting type and size

The influence of the type and length of the mini-cuttings on the biometric parameters ofA. leiocarpawas studied through a complete randomized design experiment design (2 × 3 factorial scheme). Two types (apical and intermediate) and three lengths (4, 7 and 10 cm) of mini-cuttings were tested with 4 replicates per treatment and 20 mini-cuttings per replicates (480 mini-cuttings in total).

The mini-cuttings were placed in tubes of 180 cm3containing commercial forest substrate and slow-release fertilizer (as mentioned in the previous experiment). After 40 days under intermittent misting, based on previous tests, the plants were evaluated with respect to the following variables: S; root collar diameter cutting (CD); R; FRN; RL; and RDM.

At 190 days after staking, the clonal seedlings were evaluated for the following parameters: S; CD; FRN; RL; RDM; height (H); leaf area (LA); shoot dry mass (SDM); total first-order roots length (FRL); root superficial area (RSA); root diameter (RD); and root volume (RV).We measured LA using bench area meter (LI-3000 LI-COR Inc). The root system was washed under tap water and we evaluated FRN and RL. In sequence the samples were oven-dried at (65 ± 2) °C for 72 h to determine SDM and RDM. The determinations of RSA, RD and RV were made with Winrhizo software (Regent Instruments Inc., Quebec, Canada, 2000). Finally, the system quality production was expressed in terms of the Production Index (PI), calculated through the product of the S obtained at 40 and 190 days after staking and the Dickson Quality Index (DQI), calculated through the “g” equation described in Dickson et al. (1960).

To aid orientation, the steps of theA. leiocarpaminicutting experiments are summarized in Fig. 3.

Fig. 2 Apuleia leiocarpa mini-cuttings produced from mini-stumps. a Collection, b rooting in tubes of 55 cm3, and c substrate removal for rooting assessments

Fig. 3 Steps of the A. leiocarpa minicutting experiment

Fig. 4 Survival and productivity (mini-cuttings per mini-stump) of A. leiocarpa mini-stumps during the mini-garden lifecycle (ministumps produced with seeds). Vertical bars represent the confidence interval (p ＜ 0.05)

Data analysis

Mini-stump survival rates and productivity were compared by confidence interval (p＜ 0.05), considering there is no statistical design and no fill requirement for parametrical analysis.

The data from the experiment focused on the better time under mist chamber and from the final quality of theA. leiocarpaseedlings was tested for normality and homoscedasticity prior to analysis of variance (ANOVA) and differences between the treatments were compared with the Scott-Knott test (5%). S and FRN data were transformed intoarcsenrespectively, meeting ANOVA requirements.

Results

Mini‑stump survival rate and productivity

Around 20% of the mini-stumps died during the assessment period. There was no difference in mini-stump productivity during this period, andA. leiocarpaproduced, on average, 3.4-4.6 mini-cuttings per mini-stump in each collection (Fig. 4).

Fig. 5 Dickson Quality Index (DQI) of A. leiocarpa seedlings produced with apical (API) and intermediate (INT) mini-cuttings of varying sizes. Vertical bars represent standard median error (n = 4). The same upper-case and lower-case letters did not differ significantly (Scott-Knott test at 5%) between the types and sizes of the mini-cuttings, respectively

Time for rooting under mist chamber

Mini-cuttings can be removed from the mist chamber after 30 days, when rooting stabilization has taken place. However, an increase in RDM was observed at 40 days, as well as maintenance in the increment observed in FRN and RL at 35 days (Table 1). It is also important to note the high S values (p＜ 0.05) of the mini-cuttings regardless of time under misting conditions.

Table 1 Survival rate and quality of A. leiocarpa mini-cuttings as a function of time in the mist chamber

Mini‑cutting type and size

The intermediate mini-cuttings showed higher CD and FRN values in comparison with the apical mini-cuttings (Table 2). On the other hand, the apical mini-cuttings significantly outperformed the intermediate mini-cuttings in terms of rooting percentage (7-cm mini-cuttings). The intermediate 10-cm mini-cuttings exhibited higher R and RL values, but lower survival rate when compared to smaller sizes 40 days under misting chamber (Table 2).

Among the apical mini-cutting sizes, the highest R and RL values were associated with those with 7 cm and 10 cm in length, respectively. Also the highest CD and FRN values corresponded to those pruned at 7 and 10 cm. There was no significant difference between the treatments with respect to RDM, which indicated root thickness variation, by the variation observed in length.

At the end of the clonal seedling production cycle, the dendrometric variables (CD, H, FA, SDM, RPO, FRL, RL, and RDM) of seedlings produced with intermediate minicuttings were generally superior to those produced from apical mini-cuttings (Table 3). However, apical mini-cuttings did not differ significantly with respect to all of the variables analyzed and the different mini-cutting sizes.

Among the intermediate mini-cutting sizes, the lowest values of S, H, SDM, and RDM referred to those of 4 cm; in contrast, those of 10 cm were greater than the smaller sizes in terms of RSA. There was no significant difference among treatments for the other variables.

Despite the low rooting rate of the intermediate position with 7 cm (Table 2), the mini-cuttings rooted afterwards and had strong productivity index values (Table 4). The same pattern held for the API treatment with 4 cm.

Table 2 Survival and quality of A. leiocarpa clonal seedlings 40 days after staking, maintained in a mist chamber and produced with apical (API) and intermediate (INT) mini-cuttings of varying size

Table 3 Survival and quality of A. leiocarpa clonal seedlings produced through apical (API) and intermediate mini-cuttings (INT) of varying sizes 190 days after transplanting

Table 4 Productivity Index (PI) of A. leiocarpa clonal seedlings produced with apical (API) and intermediate (INT) mini-cuttings of varying sizes

Figure 5 objectively expresses better quality (higher DQI values) of the seedlings produced with intermediate minicuttings of 7 or 10 cm in length.

Discussion

Survival and productivity of mini‑stumps

The survival rate of mini-stumps submitted to successive collections of shoots depends on the management and set of practices adopted. In addition, inter and intraspecific differences in the capacity to emit shoots and tolerate periodic pruning may exist within the mini-cutting system, especially due to the genetic variability of allogamous species.

Several studies have shown that species adapt well to production management making use of mini-cuttings, following e.g.,Cedrela fissilis(Xavier et al. 2003),Grevillea robusta(Souza Junior et al. 2008),Handroanthus heptaphyllus(Oliveira et al. 2015), andPeltophorum dubium(Mantovani et al. 2017).

Likewise, 100% ofIlex paraguariensismini-stumps survived at 120 (4 collections), 385 (15 collections), 300 (8 collections), 245 (5 collections), and 365 days (4 collections) of production, respectively (Pimentel et al. 2019). Similarly, 99.35% ofErythrina falcata(Cunha et al. 2008) and 95% ofSapium glandulatum(Ferreira et al. 2010) of mini-stump had survived at 120 (8 collections) and 395 days (4 collections) of management, respectively.

The ability of other species to adapt to mini-cutting warrants further investigation. In the case ofP. reticulata, for instance, while Pessanha et al. (2018) obtained results indicating a 65% mini-stump survival rate after 210 days with 3 collections, Neubert et al. (2017) reported a survival rate ranging from 25 to 64.5%, due to the genetic factors, after four collections at 120 days. Conversely, Carvalho (2020) observed survival rate of 91.5% for the same species after 270 days over monthly collections.

Similar results to the productivity rates obtained forA. leiocarpaclonal seedlings (Fig. 4) were also observed for other tropical woody species belonging to the Fabaceae family, such asP. reticulata(Pessanha et al. 2018) andP. dubium(Mantovani et al. 2017). This correlation might be supported by the species’ genetic regrowth potential regardless of the collection period.

However, mini-stump productivity offers no guarantee of a high rate of clonal seedling productivity, as the latter depends on the rooting competence of the propagules. Preliminary tests indicated a low survival rate and rooting forA. leiocarpa(Silva et al. 2019), then we hypothesized that the permanence time of theA. leiocarpamini-cuttings under mist chamber could influence the clonal seedlings productivity index.

Rooting time in the mist chamber

There was a high survival rate (≥ 95%) of theA. leiocarpamini-cuttings (Table 1), demonstrating the technique’s high potential for the vegetative propagation of the studied species. It will inform nursery managers regarding the quantities of propagules require to fulfillA. leiocarpaclonal seedling production according to market demand.

Similar survival rates were observed for stem mini-cuttings ofI. paraguariensis(Wendling et al. 2007),Calophyllum brasiliense(Ciriello and Mori 2015),Anadenanthera macrocarpa(Dias et al. 2015),Araucaria angustifólia(Pires et al. 2015), andHymenaea courbaril(Moura et al. 2019).

Although the highest rooting rates had settled for 30 days after transplanting, at 40 days the mini-cuttings presented promising results regarding FRN, RL, and RDM (Table 1), variables most closely associated with final seedling quality. These results thus indicated that it is possible to achieve a high number of clonal seedlings of good root length and dry root mass after 40 days in the mist chamber. It is noteworthy that approximately the equivalent increase in RL and RDM can be reached at 55 days, but without significant gains with respect to the other parameters assessed.

This information helps determine the optimal timeframe clonal seedlings should remain in the misting sector, preventing their premature transfer to less controlled conditions before roots are fully developed, which could result in reduced productivity.

However, variations in rooting rates can result from genetic differences in the source materials used, as observed by Melo et al. (2011), among hybrid clones ofEucalyptus grandis, and Pessanha et al. (2018) and Carvalho (2020) withP. reticulata. Another cause of variation may occur depending on the type of cutting performed and its position in the sprouting, due to the physiological variations that exist among plant structures (Silva et al. 2010; Wendling et al. 2015). This phenomenon does not hold for all species, as Pimentel et al. (2016) reported in their investigation forH. heptaphyllusmini-cuttings.

Mini‑cutting type and size

The lowest survival rate in the rooting sector was observed in 10-cm long apical mini-cuttings. Among the apical minicutting sizes, those of 7 cm had the highest rooting rate, while the highest was observed in 10-cm mini-cuttings among the intermediate group. Conversely, 2.5 cmI. paraguariensismini-cuttings had lower survival rates compared to longer sizes (up to 10 cm), but the final morphophysiological quality was similar among mini-cutting sizes (Pimentel et al. 2020). The longer the mini-cutting the higher the water requirements for mini-cuttings turgor maintenance, which could explain the lower survival rate for 10 cm mini-cuttings compared to the first experiment which was conducted in mild temperatures.

Because survival and rooting rates play an important role in seedling production capacity in forest nursery (Table 2), it is expected that the most positive results will be obtained with the vegetative propagation either of apical mini-cuttings of 7 cm (S = 98% and R = 88%) or intermediate minicuttings of 10 cm (S = 100% and R = 70%) obtained through processes performed in the rooting sector.

It is important to highlight the importance of the survival rate and rooting for commercial seedling production, as it will reflect on the quantity of seedlings that will be produced through mini-cutting, they facilitate the planning process with respect to the mini-garden dimensions required for the number of seedlings intended.

However, even with the low rooting rate after the rooting sector (Table 2), the seedlings produced with 7-cm intermediate mini-cuttings had a high survival rate (Table 3), indicating an elevated rooting rate thus a strong Productivity Index value (Table 4). Similar behavior was observed with 4-cm apical mini-cuttings. We recommend that apical minicuttings should be shorter than intermediate ones.

It is known that plant capacity for the exploitation and uptake of water and available soil nutrients is closely related to root length. With this in mind, and considering the results of survival and rooting, there is evidence that 10-cm minicuttings exhibited improved development in later stages of the production steps. This assertion is supported by the higher RSA values observed for seedlings derived from these mini-cuttings (Table 3).

There was no RDM difference as a function of the treatments even though intermediate 7- and 10-cm mini-cuttings exhibited a greater number of roots than the 4-cm mini-cuttings (Table 2). It indicates thicker roots in the 4-cm minicuttings, which tends to be less effective in absorbing water and nutrients when compared with thinner and longer root systems, as observed in 10-cm mini-cuttings.

The higher CD of the intermediate mini-cuttings 40 days after transplanting may be related to increased quantity of shoots and availability of reserves (Steffens and Rasmussen 2016). These factors also probably contributed to the higher FRN values, as well as the other variables (CD, H, LA, ADM, FRL, RL, and RDM), obtained with seedlings generated from intermediate mini-cuttings and assessed at a late stage (Table 3).

The lack of apical bud in the intermediate mini-cuttings leads to increased cytokinin concentration in lateral buds due to absence of apical dominance (where auxin is produced). This fact explains higher growth of lateral shoots and hence H, CD, LA (Pimentel et al. 2020).

It is important to emphasize this is the first report of the effects the type and size of mini-cuttings on the quality ofA. leiocarpaclonal seedlings, whose DQI varied from 0.13 to 0.27 with greater values for seedlings produced with intermediate and 7- or 10-cm mini-cuttings (Fig. 5). However, the DQI has been used in several studies, such asP. dubium(Souza et al. 2013),H. heptaphyllus(Oliveira et al. 2015),Toona ciliata(Silva et al. 2016),P. reticulata(Pessanha et al. 2018), andPinus sylvestris(Eskandari et al. 2019), especially for comparing management practice effects on the quality of clonal seedlings produced in nurseries.

The specific range of values for each quality category varies according to the species and local environmental conditions. Thus, the results of the present work can prove useful for forestry and nursery managers, as well as serving as a reference for future studies on the vegetative rescue of forest species of significant economic-ecological value.

Since mist chamber timeframe was determined for 10-cm-long mini-cuttings, assessments of less than 40 days should be performed to verify whether shorter periods under misting chamber could elicit promising responses for minicuttings of 7 cm. Such assessment would not preclude the need of management adjustments (fertility, light, water, etc.) to increase the quality and especially survival rates of clonal seedlings after the misting chamber period, when mortality caused a significant reduction in the Productivity Index in all of the treatments.

Conclusion

It is possible to propagateA. Leiocarpawith mini-cuttings derived from mini-stumps produced from seeds. The ministumps presented sprouting capacity and maintained productivity constant throughout three annual collections.A. leiocarpamini-cuttings with 10-cm in length should be maintained for at least 40 days under misting. The vegetative propagation ofA. leiocarpathrough the use of intermediate mini-cuttings of 7-10 cm resulted in more vigorous clonal seedlings.