Histological study of adventitious rooting in Acacia mearnsii and Ilex paraguariensis mini-cuttings: insights into the so-called anatomical barrier

Abstract

Acacia mearnsii and Ilex paraguariensis are tree species of great social and economic importance in Brazil, demanding clonal cultivars. Their shoots possess a reservoir of totipotent cells with suitable potential for adventitious rooting, essential for mass production of high-quality seedlings. This study aimed to gain new insights into how anatomical barriers, i.e. sclerified tissues in the cortical region, may affect the adventitious rooting of cuttings from these species through histological examinations. For both species, histological analysis revealed significant diagnostic features. Tissue decay appears to be equivalent to an anatomical barrier in A. mearnsii. Starch abundance was notable in clones with higher rooting competence of I. paraguariensis, but they were not observed in the fundamental tissues of A. mearnsii, regardless of the rooting competence of the clone. The main differences in adventitious rooting were associated with the speed of response, initiated from cortical meristems, followed by differentiation of conductive tissue from newly formed tissue, connecting the periphery with the secondary vascular tissue. Thus, this newly formed tissue with parenchymatic structure provides the necessary structural basis for radial vascular connections. For both studied species, rhizogenesis presents distinct barriers to rooting, nevertheless these are not necessarily of anatomical nature.

Key words:
Cylindrocladium sp.; forest species; mini-cutting; rooting competence; starch.

Resumo

Acacia mearnsii e Ilex paraguariensis são espécies arbóreas de grande importância social e econômica no Brasil, que demandam cultivares clonais. Seus brotos possuem estoque de células totipotentes com potencial adequado para enraizamento adventício, essencial para a produção em massa de mudas de alta qualidade. Este estudo teve como objetivo obter novas perspectivas sobre como as barreiras anatômicas, ou seja, tecidos esclerificados na região cortical, podem afetar o enraizamento adventício de miniestacas dessas espécies por meio de estudos histológicos. Para ambas as espécies, a análise histológica revelou características diagnósticas significativas. O apodrecimento de tecidos parece ser equivalente a uma barreira anatômica em A. mearnsii. A abundância de amido foi notável em clones com maior competência de enraizamento de I. paraguariensis, porém não foram observados grãos de amido nos tecidos fundamentais de A. mearnsii, independentemente da competência de enraizamento do clone. As principais diferenças ao enraizamento adventício estavam associadas à velocidade de resposta, que se inicia a partir de meristemoides corticais, seguida pela diferenciação do tecido condutor a partir de um tecido neoformado com estrutura parenquimática, que conecta a periferia com o tecido vascular secundário. Portanto, esse tecido neoformado promove a base estrutural necessária para conexões vasculares radiais. Para ambas as espécies estudadas, a rizogênese apresenta barreiras distintas ao enraizamento, contudo estas não são necessariamente de natureza anatômica.

Palavras-chave:
Cylindrocladium sp.; espécies florestais; miniestacas; competência de enraizamento; amido.

Introduction

Most forest species are propagated by using seedlings. Sexual propagation results in highly variable populations for important traits, such as plant height, diameter, and yield potential, which hinders the use of adequate management practices. Asexual propagation is the best choice for species in which seedling production is dependent on either seed availability or quality. Asexual propagation results in uniform populations and ensures favorable combinations of traits, high levels of heterozygosity, and superior interactions of genetic variance (Pimentel et al. 2019). Among the asexual propagation techniques, cuttings are largely used for propagating horticultural and forest species because they are an efficient technique for maximizing genetic gain (Naidu & Jones 2016). Cuttings are prepared from stems, leaves, or roots, with stem cuttings being the most extensively used.

Mini-cutting is a variation of the cuttings technique. Mini-cuttings are the most important technique for cloning forest species, as demonstrated in the genus Eucalyptus L'Hér. (Naidu & Jones 2016), with a well-established protocol, and are widely used commercially by forestry companies (de Lima et al. 2022). It has been successfully used to improve the rooting competence of forest species, such as Pinus pinaster Endl. (Majada et al. 2011). Experimental protocols have been developed for other species, including Ilex paraguariensis A. St.-Hil., a species native to Brazil (Pimentel et al. 2019). The success of mini-cutting technology is directly associated with competence in the development of an adventitious root system.

Adventitious root formation is a highly controlled process that includes dedifferentiation and founder cell formation, cell division and primordia formation, and adventitious root growth (Lakehal & Bellini 2019). Adventitious root formation has a genetic component depending on the species and genotypes within the species, and an environmental component. Genetic differences among clones have been reported in the literature, such as rooting competence in Eucalyptus (Naidu & Jones 2016), Cabralea canjerana (Vell.) Mart. (Burin et al. 2020), and at the optimum time for mini-cutting rooting in hybrids of E. benthamii × E. dunnii (Brondani et al. 2012) and Sequoia sempervirens Endl. (Pereira et al. 2018).

Reserve accumulation in plant tissues, which is necessary for adventitious rooting, is affected by plant metabolism. Soluble sugar and protein consumption is associated with callus formation, whereas their accumulation is associated with adventitious rooting in Ziziphus jujube Mill. (Shao et al. 2018). Annual changes in soluble sugar and protein concentrations have been observed in the leaf and bark tissues of Olea europaea L. (Eris et al. 2007). The presence of leaves improves cutting rooting (Hartmann et al. 2011). Leaf reduction to half of the original area has been indicated for I. paraguariensis propagation by single-bud mini-cuttings (Pimentel et al. 2020). In this species, a significant inverse relationship was found between mini-cutting diameter and the percentages of survival and rooting (Gazzana et al. 2020a).

Adventitious rooting is a biological phenomenon that is commonly discussed in histological terms. Histology, anatomy, and structural studies in a broad way continue to be extremely important as, in terms of plant production, adventitious rooting resulting from experimentation remains a structural manifestation (Lovell & White 1986). Histological and anatomical studies have established a basis that has improved the understanding of adventitious rooting. In this context, the analysis of rooting potential as a function of the structure of the stems is remarkable, especially with respect to anatomical barriers. The study by Sachs et al. (1964), among others (Amissah et al. 2008; Monder et al. 2019), reinforced the need for histological studies, as the importance given to anatomical barriers, due to rooting failure was greatly reduced, in addition to the establishment of morphological markers of reference.

We explored the genetic component of the adventitious root formation by selecting competent genotypes for propagation by mini-cuttings to maximize the gain from selection. We also performed cloning and histological studies of few forest species during this process, as A. mearnsii De Wild. and I. paraguariensis. Acacia mearnsii is a fabaceous tree that is native to Australia. It has high economic and social importance in Rio Grande do Sul state, Brazil, where it is cultivated for tannin extraction, and cellulose and energy production. Ilex paraguariensis is an aquifoliaceous tree that naturally occurs in southern Brazil. Its thin branches and leaves are the main raw material for the tea and energy drinks industry (Cardozo Junior & Morand 2016), and cosmetic and pharmaceutical industries (Dartora et al. 2013), which has specific quality requirements and demands a continuous supply (Gazzana et al. 2020b).

Therefore, it is evident that knowledge of the species-specific barriers is the key to commercial success of mini-cuttings propagules. The aim of this study was to carry out histological studies to provide new insights into how barriers may affect adventitious rooting of A. mearnsii and I. paraguariensis mini-cuttings.

Material and Methods

The experiments were performed with propagules collected in a clonal mini-garden established in an acclimatized greenhouse at the Plant Breeding and Vegetative Propagation Center (MPVP), Department of Plant Science, Federal University of Santa Maria (UFSM), Santa Maria (RS), Brazil. The clonal mini-gardens of A. mearnsii and I. paraguariensis were established on benches in a closed soilless cultivation system, with coarse sand as the substrate and nutrient solution supplied to the culture bed by flood fertigation. Nutrient solution composition, distribution, and management practices were maintained as described by Gazzana et al. (2020b). For analyzes on A. mearnsii clones AB6, B5 and IA45 were used. For the analyzes on Ilex paraguariensis, clones 10SM07 (difficult-to-root) and 06SM15 (easy-to-root) were used.

Mini-cutting preparation and cultivation

Mini-cuttings were prepared by sectioning the shoots into single buds, and the leaves were reduced to 50% of the original area. Mini-cuttings were treated with a hydroalcoholic solution (1:1 v/v) of indolebutyric acid (IBA) at a concentration of 2,000 mg L^-1 for 10 s, as defined in preliminary studies. Few I. paraguariensis mini-cuttings were not treated with IBA. The mini-cuttings were grown in 100-well trays containing equal proportions of a commercial substrate with pine bark base, medium vermiculite, and coarse-grained sand. They were placed on a bench inside a humidity chamber, with the relative humidity maintained above 95% by automated nebulization. Mini-cuttings of I. paraguariensis were collected at 0, 30, and 60 d, and those of A. mearnsii were collected at 0, 1, 2, 5, 10, 15 and 30 d of cultivation in a humidity chamber for anatomical investigations.

Sample preparation

The basal region of the mini-cuttings was first sectioned into ~ 1 cm pieces. Freehand cuts were made using a razor blade. The samples were fixed in two chemical solutions. A solution containing 3% glutaraldehyde and 0.1 M of sodium phosphate buffer with pH 7.2 (McDowell & Trump 1976), or a 6% solution of glyoxal and the same buffer (Dapson 2007). The samples were washed for 15 min, first in the buffer (pH 7.2) and then in distilled water, respectively.

Samples were subjected to continuous rotation in a microcentrifuge for 5-7 d in tubes containing 2 mL L^-1 of Tween 20 or 5% Extran solution to remove epicuticular waxes and any lipophilic substances to facilitate subsequent infiltration in 2-hydroxyethyl methacrylate (HEMA). The samples were dehydrated in an ethyl alcohol series (O’Brien & McCully 1981), pre-infiltrated in HEMA and absolute ethanol solution (1:1 v/v) for 12 h, followed by infiltration in HEMA for ~ 4 h, and embedded in a Teflon holder until polymerization was completed (Gerrits & Smid 1983).

The samples were sectioned to 3, 4, and 5 μm thickness using a Leica RM2245 rotary microtome. Histological slides were stained with toluidine blue in 0.05% sodium benzoate buffer of pH 4.4 (Sidman et al. 1961). Total phenolic compounds were detected with toluidine blue (O’Brien et al. 1964), starch with Lugol’s solution (Johansen 1940), starch and total polysaccharides with Schiff´s reagent and periodic acid (O’Brien & McCully 1981), and lipophilic substances with Sudan red 7 B (Brundrett et al. 1991). Schiff’s reagent and periodic acid and Sudan red 7B histochemical tests were performed individually and in combination with toluidine blue.

To prevent rot disease, symptomatic mini-cuttings of A. mearnsii were disinfected in a 70% ethanol solution, followed by 1% solution of sodium hypochlorite. The mini-cuttings were transferred to Petri dishes containing potato dextrose agar medium, sealed, and cultivated at 25 °C under a 12 h photoperiod for 48 h.

Photographic documentation and analyses were performed using a Leica^® DM 2000 and M80 microscope with a DFC295 image capture system, Zeiss AxioImager A2 microscope with a Zeiss MCr digital capture system, and ZEN (Zeiss^TM) software. The mini-cuttings were observed using a stereoscopic microscope (Olympus SZ51). Slides were prepared with fungal structures and visualized using an optical microscope (Zeiss Primo Star). Photomicrographic registration was performed with a Canon Rebel T5i camera with a 105 mm macro lens.

Results

General aspects of the mini-cuttings at time zero

In cross sections, the base of A. mearnsii mini-cuttings showed a combined structure of primary and secondary tissues. The primary tissues were the epidermis, fundamental tissue in the cortical region and pericycle, phloem, xylem, and the internal pith (Fig. 1a-b). The secondary tissues were represented by the cambium, phloem, and xylem (Fig. 1b). Based on the results presented below, the cortical region, sclerified and lignified pericycle, and primary and secondary phloem are highlighted. Callus formation occurred in these regions and tissues, through which the vascular connection between the adventitious roots and the stem vascular tissue was established, as well as the recruitment of cells for the formation of adventitious roots. Thus, the outermost cortical layer was typically composed of alternating collenchyma and chlorenchyma (Fig. 1a-b). Internally, the parenchymatic tissue differentiated into one or a few layers (Fig. 1b). The endoderm was composed of a layer of cells with a parenchymatic structure, with several cells being crystalliferous idioblasts (Fig. 1b). The pericycle was deep inside and composed of septate and sclereid fibers, both of which were usually alive (Fig. 1c). Sclerification and lignification of fibers was centripetal and gradual, cells were alive in most mini-cuttings, and higher number of cells was observed in young internodes. Even with relative lignification, the fibers divided transversally, becoming septate, in which, it was possible to distinguish basic aspects such as cytoplasm, vacuoles, and nucleus, in addition to pecto-cellulosic transverse walls (Fig. 1c). Although the pericycle was continuous, sclerification and lignification did not occur in all cells, as observed by the parenchymatic structure in some pericycle cells (Fig. 1b).

The stem structure of I. paraguariensis among the mini-cuttings investigated always presented with secondary xylem and phloem, although with a limited formation of tissues. Therefore, it still retained all the primary tissues (Fig. 2a), and in some cases, even presented general aspects of the primary organ. In the context of the present study, it was important to highlight the structure of the epidermis, pericyclic sclerenchyma, cortical region, and primary and secondary phloem (Fig. 2a-e).

Figure 1
a-c. Cross-sectional (a, b) and longitudinal (c) sections of the stem of Acacia mearnsii clone B5 at time zero - a. general appearance with indications of different primary and secondary tissues (arrow, epidermis; dotted line, vascular cambium); b. detail of the discontinuous pericyclic sclerenchyma region containing fibers (f), sclereids (ec) and cells with parenchyma structure (red arrow) [black arrow indicates crystalliferous idioblasts in endoderm (en)]; c. detail of a portion of the cortical and vascular region separated by septate fibers with emphasis on the transverse walls (red arrows) and nuclei (black arrows). Short forms: col = collenchyma; clo = chlorenchyma; Phf = phloem fiber bundle; Ph1 = primary phloem; Ph2 = secondary phloem; X2 = secondary xylem; X1 = primary xylem; p = pith. Scale bars = 50 μm.

The epidermis contained a single layer of cells when mini-cuttings were produced. In the cortical region, a layer of chlorenchyma was observed, and more internally, parenchymatic tissue (Fig. 2a-b), with an internal limit in the endoderm (Fig. 2a,b). The pericyclic sclerenchyma was generally not continuous (Fig. 2c,d) and was composed of fibers and sclereids (Fig. 2b-d). The thickness of this sclerenchyma varied from one to six layers, alternating with cells with a parenchymatic structure or crystalliferous idioblasts (Fig. 2c-d). The phloem region, comprising primary and secondary tissues, contained many parenchyma and companion cells (Fig. 2c-d). Small amounts of starch grains were identified in the chlorenchyma, endodermis, xylem rays, and medullary regions.

Figure 2
a-d. Transverse (a-b) and longitudinal (c-d) sections of Ilex paraguariensis clones 10SM07 and 06SM15 at time zero (a,c,d. clone 10SM07; b. clone 06SM15) - a. general anatomy of the stem showing primary and secondary tissues (arrow = epidermis; red arrow = sclerenchyma discontinuity; dotted line = vascular cambium); b. detail of a portion of the pericyclic sclerenchyma with emphasis on the external parenchyma (pair) and internal primary phloem (Ph1) and secondary phloem (Ph2) and the discontinuity of sclerification (red arrow) and endoderm (black arrow); c. detail of fibers (f) of the pericyclic region and endoderm (black arrow); d. detail of sclereids (ec) of the pericyclic region, endoderme (black arrow) and crystals (red arrow). Short forms: clo = chlorenchyma; par = parenchyma; Pf = primary phloem fibers; f = pericyclic fibers; ec = sclereids; Ph1 = primary phloem; Ph2 = secondary phloem; X2 = secondary xylem; X1 = primary xylem; p = pith. Scale bars = 50 μm.

Adventitious rooting process of the mini-cuttings

Among the evaluated clones of A. mearnsii, a clear reaction to cultivation was observed in the first 20-26 h. The endoderm is the tissue that responds initially with dedifferentiation and cell proliferation, although primary phloem cells may also respond (Fig. 3a) or proliferate (Fig. 3b) in both the primary and secondary phloem regions. Such proliferation occurred in several samples without the due accompaniment of the external portion of the cortex and epidermis, and these tissues were therefore compressed, with extensive accumulation of phenolic compounds in the remaining tissue in these regions (Fig. 3b). In locations where reactions were observed in the primary phloem, discontinuity of the sclerenchyma was common (Fig. 3a).

In general, after the initial process of endodermal cell proliferation in A. mearnsii, the phloem region actively contributes to new cells through primary and secondary tissues. Thus, at 30 d of cultivation, the phloem proliferation process, together with cortical tissue, generated important structural consequences for the process with projection of new tissue between sclerenchyma groups and the formation of conductive tissues, which were already connected to the stem vascular tissue (Fig. 3c). Endoderm and phloem cells around the sclerenchymatic clusters remained divisible (Fig. 3d). The tissues derived from the phloem and endoderm differentiated into groups of vascular elements (Fig. 3c) and roots were formed (Fig. 3d). This process took place within 30 d, although samples were not collected and the exact period is still unknown. In samples of clone AB6 of A. mearnsii, only the reaction of the cortical region to the culture was common (Fig. 4a), without impeding root formation (Fig. 4b). Although the volumes of fundamental tissue between sclerenchyma clusters were smaller, there was differentiation of conductive elements between adventitious roots and stem vascular tissue (Fig. 4b). Furthermore, in the region of the vascular connection between adventitious roots and stems, cambial activity was lost (Fig. 3d). The understanding of how the vascular groups in the callus are spatially differentiated is limited due to the loss of orientation within the axial and radial planes of symmetry of the cutting.

Figure 3
a-d. Cross sections in the base region of the of mini-cuttings of Acacia mearnsii clones - a. clone IA45 after 24 h of cultivation. Black triangle = endoderm reacting to cultivation; black square = primary phloem cells reacting to cultivation. Fiber groups (*); b. clone AB6 after 24 h of cultivation. Fiber groups (*); c. clone B5 after 30 d of cultivation. Neoformed vascular tissue (nvt) has a cambium line through the vascular and cortical regions (dashed line). Dotted line = vascular cambium; fiber groups (*); d. clone B5 after 30 d of culture showing a region with adventitious root (AR) and established vascular connection (nvt). Fiber groups (*) far apart with space filled with newly formed tissue. Arrows indicate the direction of proliferation from fiber clusters. Dotted line = vascular cambium. Short forms: Short forms: clo = chlorenchyma; Phf = primary phloem fibers; Ph1 = primary phloem; Ph2 = secondary phloem; X2 = secondary xylem; X1 = primary xylem. Scale bars: a-c = 50 μm; d = 200 μm.

The rooting process of Acacia mearnsii mini-cuttings (Fig. 5a) was accompanied by hyphal proliferation of Cylindrocladium sp. (Fig. 5b-c) with extensive tissue dissolution, mainly of the vascular cambium (Fig. 5d,f) that was observed at 48 h of cultivation (Fig. 5d). Hyphae were observed within the dissolved tissues and inside cells (Fig. 5e). Consequently, a discontinuity occurred between the live tissues that surrounded the cambium and through which vascular connections to the adventitious roots would be built (Fig. 5f). This tissue dissolution is acropetal (Fig. 5a), and ended up being colonized in all or almost the entire circumference. In affected mini-cuttings, it is common for the roots to emerge far above the basal region where the infection occurred (Fig. 5a), in addition to the emergence of a few roots facing the same direction, or even the emergence of a single root (Fig. 5a).

Figure 4
a-b. Cross sections in the region of the base of the mini-cuttings of clone AB6 of Acacia mearnsii - a. 30 d of cultivation. Neo-formed vascular tissue (nvt) in the cortical region; b. general aspects showing region with emergence of two adventitious roots (AR). Short forms: Black triangle = endoderm reacting to culture; Phf = primary phloem fibers; Ph1 = primary phloem; Ph2 = secondary phloem; X2 = secondary xylem; Cc = central cylinder. Scale bars: a = 100 μm; b = 200 μm.

In Ilex paraguariensis, both the evaluated clones (06SM15 and 10SM07) exhibited three common characteristics: (a) both produced roots, (b) exhibited identical processes of callogenesis and differentiation of adventitious roots, and (c) the roots always formed at the base of the mini-cuttings, that is, through the regions exposed by the cut at the time of the mini-cutting preparation (Fig. 6a-b). In rare situations, there was a combined emergence of adventitious roots above the region of the cut, and therefore, through the cortical region and epidermis (Fig. 6b), presenting aspects of direct rhizogenesis.

The initial process of mini-cutting differentiation in I. paraguariensis was marked by cell proliferation on both sides of the pericyclic sclerenchyma, mainly cells of the primary and secondary phloem, and cells of the inner portion of the cortical region, including the endoderm (Fig. 6c). Rapid proliferation produced two effects. One of these is macromorphological, which makes the base of the mini-cuttings bulky (Figs 6b). The second effect, histologically, is the discontinuity of the pericyclic sclerenchyma, that is interrupted synchronously by the cortical and phloem tissues (Fig. 6c). Thus, the previously isolated cortical and vascular regions (Fig. 2a) became continuous. In general, cortical tissues react within the first millimeter of the wound together with phloem tissues. Above the base, the main contribution to callus is the phloem. Cells from these new tissues were recruited and differentiated into conductive elements that connected the adventitious roots to the stem conductive tissues (Fig. 6c-d), and the process was completed before 30 d of culture.

Detection of starch grains

In Acacia mearnsii, only a few starch grains were observed, and other polysaccharides were not observed in the fundamental tissues of either pre-(shoot) or neo-formed (callus) tissues, regardless of the rooting competence of the studied clone (Fig. 7a-b). In the 06SM15 clone of I. paraguariensis with greater rooting competence, the presence of large amounts of starch grains in cells was generalized to pre- and neo-formed tissues (Fig. 7c), unlike clones with lower rooting competence (Fig. 7d). In both the species studied, there was no accumulation of polysaccharides in vacuoles.

Figure 5
a-f. Side view (a-b), cross-sections of mini-cuttings (d-f) of Acacia mearnsii clones, and (c) side view of conidiophores and conidia of Cylindrocladium spp - a. clone B5 at 30 d of cultivation; b. general appearance of the mini-cutting showing presence of Cylindrocladium spp. sporulation; c. conidiophores and conidia of Cylindrocladium spp. under differential and interferential contrast; d. clone B5, 48 h of culture showing dissolution of the cambial region (d); e. clone AB6, detail of infected region with the presence of extracellular hyphae (black arrows) and intracellular hyphae (white arrow); f. clone B5 at 30 d of culture with dissolution of the cambial region (d). Short forms: AR = adventitious root; Phf = primary phloem fibers; Ph1 = primary phloem; X2 = secondary xylem. Scale bars: a = 200 μm; b = 500 μm; c = 50 μm; d = 50 μm, e = 25 μm; f = 50 μm.

Figure 6
a-d. Side views of stems from clone 13SM05 (a)(b) Longitudinal sections of stems from clone 10SM07 (c)(d) of Ilex paraguariensis at 30 days of cultivation - a. adventitious roots emerged from the base in the mini-cutting; b. adventitious roots emerged at the mini-cutting base and adventitious root emerged above the base (arrow); c. black dotted lines indicate continuity of newly formed vascular tissues through sclerenchyma clusters connecting the callus to the stem vascular tissues, red dotted lines indicate pericyclic fibers; d. black dotted lines indicate continuity of newly formed vascular tissues through the callus with a central cylinder (Cc). Short forms: nvt = neo-formed vascular tissue; X2 = secondary xylem; cls = callus. Scale bars: a-b. 10 mm; c-d. 400 μm.

Discussion

General aspects of the mini-cuttings at time zero

Regarding anatomical aspects, it should be noted that the stems of A. mearnsii and I. paraguariensis were highly similar. Some tissue and cytological aspects varied, mainly in terms of tissue distribution in the cortical region, sclerification, and lignification in the pericycle and primary phloem fibers.

Adventitious rooting process of the mini-cuttings

In the present study, there was a discontinuous sclerenchymatic layer in the pericyclic region of the internodes, and this discontinuity was amplified as a function of callus formation. Sclerenchyma discontinuity is one of the variations, considering continuous (Bryant & Trueman 2015; Sá et al. 2022) or absence of sclerenchyma (Harbage et al. 1993; Ahkami et al. 2009) is also reported. Beakbane (1961) and Goodin (1965) considered the sclerified layer as a factor that reduces the rooting potential, including in A. mearnsii (Beakbane 1961), thus acting as a barrier to rooting. In the present study, there was no evidence of its effects on the rhizogenic potential of clones already tested in either species, especially in those considered to have low rooting potential. Altamura (1996), in general, considered that in species with greater rhizogenic potential, the sclerenchymatic barrier may be present, but discontinuously, when comparing such potentials in species whose sclerenchymatic layer is continuous. Loss of continuity of peripheral sclerified tissue can be achieved by culture conditions that induce cell proliferation and growth, such as exposure to mist and hormone treatment (Sachs et al. 1964), or hormone application alone (Altamura 1996), increasing potential sites of vascularization and/or root emergence. Sachs et al. (1964) present, as part of their argument against the sclerenchymatic layer being the cause of non-rooting or potential reduction, that the mini-cutting structure does not have any barrier in the cut region, which is the point of root emergence, having proven such a possibility in Anacardium occidentale L., according to Jásik & De Klerk (1997) in Olea europaea L. (Altamura 1996), Coffea robusta L.Linden (Reaño 1940) and Dianthus caryophyllus L. (Stangler 1956), where “root primordia are initiated, grow down inside the fiber band and emerge through the cut base” (Lovell & White 1986).

Figure 7
a-d. Cross-sections of mini-cuttings. (a)(b) Acacia mearnsii. (c)(d) Ilex paraguariensis - a. clone B5. General aspect of a stem region with the presence of callus. Few cells with starch grains are seen. Rectangle indicates site of occurrence of starch cells derived from phloem callogenesis; b. detail of starch cells (arrow); c. detail of the shoot region after callogenesis in clone 10SM07 with starch-free tissues; d. detail of a portion of callus in clone 06SM15 with tissues containing a large numbers of amiliferous cells (arrows). Scale bars: a,c = 200 μm; d = 400 μm.

The proliferation of cortical and phloem tissues is relatively discrete in the species studied, mainly in A. mearnsii, once callus formation imposes a lower morphological alteration. Rhizogenesis is therefore indirect and necessarily passes through the stages of rapid stimulation, tissue reaction with proliferation and dedifferentiation, formation of meristemoids, initial differentiation of adventitious roots, and differentiation of conductive tissue, which is concomitant with the growth of adventitious roots. In addition, the process is acropetously active and vertical series of new tissue are formed, which are relatively longer in A. mearnsii. In this context, in genotypes with lower rooting competence, if rooting is indirect, the anatomical barrier becomes discontinuous with tissue filling, it is possible that the anatomical barrier or structural barrier does not exist.

For both species, the sclerified tissue in the cortical region seems to be important to the process, as intense cell proliferation occurred in the parenchymatic tissues surrounding them, a process that is considered essential for the formation of vascular tissue, which is similar to that described for Malus domestica (Suckow) Borkh. “Jork 9” (Jásik & De Klerk 1997), among others (Altamura 1996). Therefore, sclerified clusters appear to contribute to callus formation, vascularization and rooting.

A comparison of the rooted mini-cuttings showed a marked difference in A. mearnsii due to the easy separation of the bark, the absence of rooting at the base of the mini-cuttings, and the formation of few roots or only one root. The adventitious root structure of A. mearnsii is predominantly different from that observed in other studies, including I. paraguariensis, both due to the smaller number of roots formed and due to its asymmetric distribution. This deficiency in root system formation is maintained during the growth and development of plants in the field and is associated with the high percentage of dislodging observed in some clones in the first years of cultivation. The inclusion of histological studies was also motivated by these characteristics, although the study hypothesis was that only intrinsic structural differences could be associated with both the type and rooting potential. Therefore, the context is very similar to the usual assessments of rooting potential associated with structural barriers.

In A. mearnsii the same conditions necessary for maximizing the rooting process favor disease development, in this case caused by the fungus Cylindrocladium sp. To our knowledge, this is not the first report of rot disease in A. mearnsii mini-cuttings. Twenty-nine endophytic fungi have been isolated from asymptomatic mini-cuttings (Duin et al. 2018), and the genus Calonectria De Not. is the most common cause of rot disease (Duin et al. 2017).

It should be noted that among the investigated clones of A. mearnsii differing in rooting competence, there was a single and robust radial vascular connection with an adventitious root. Notably, fungal infection is not present in these places, which may indicate that hyphal proliferation may be inhibited.

Detection of starch grains

In the studied species, among the different clones, starch in a reasonable amount was identified only in the mini-cuttings of the 06SM15 clone of I. paraguariensis, which has already been highlighted for presenting the best rooting rates (Pimentel et al. 2019). In general, results related to starch are usually considered controversial because of the lack of a pattern regarding rooting. Thus, in some studies, similar to the present one, there is a positive relationship between starch and adventitious rooting (Reuveni & Raviv 1980; Aslmoshtaghi & Shahsavar 2010; Pimentel et al. 2020), whereas other studies observed the opposite (Denaxa et al. 2012), or no correlation between the presence of starch and adventitious rooting (Ferriani et al. 2008; Lima et al. 2011), as also observed in the present study for A. mearnsii. Thus, starch would be an interesting marker only for I. paraguariensis. In the cases of A. mearnsii and I. paraguariensis, complementary studies integrating soluble sugars, among other factors, and adventitious rooting are necessary.

Although there is a positive relationship between starch and/or other sugars and rhizogenesis, this is not a cause-and-effect relationship (Del Rio et al. 1991; Rahman et al. 2002). However, excess starch may indicate an excess of carbohydrates and consequent availability in at least one clone of I. paraguariensis. There does exist a demand for structural sugars and energy sources, mainly for species with indirect rooting, as the calluses themselves are important drains, and their differentiation ends up overlapping the rooting itself. In addition to the fact that the species in the present study, even considering their different rooting potentials, present a relatively fast process, as they already had functional roots 30 d after the start of cultivation. Thus, there is a demand for several different morphogenetic processes over a short period of time.

The use of the periodic acid-Schiff (PAS) method in the present study was associated with the possibility of detecting sugars, mainly hexoses in the cytoplasm or vacuoles (Kiernan 1981), which has been successful in plant reproductive cells (Oliveira et al. 2015). However, for both species in the present study, the analyses did not allow for conclusions regarding the presence of other sugars. In general, there is a correlation between starch, its mobilization, and other sugars, which has been demonstrated in adventitious rooting using biochemical methods (Reuveni & Raviv 1980; Del Rio et al. 1991; Rahman et al. 2002; Ferriani et al. 2008; Aslmoshtaghi & Shahsavar 2010; Lima et al. 2011).

For both the species, histological analysis contributed substantially to diagnosis. In Acacia mearnsii, the absence of tissue appears to be an anatomical barrier. For Ilex paraguariensis, the abundance of starch was remarkable in clones with the greatest rooting competence. In A. mearnsii, starch grains were not observed in fundamental tissues regardless of the rooting competence of the clone.

Although there are some differences, the formation of adventitious roots in both species and the studied clones followed a similar pattern, from the recruitment of cells during callus formation, adventitious vascularization, and the indirect formation of functional roots. The main differences were associated with the response speed for adventitious rooting that starts from cortical meristemoids, followed by the differentiation of conductive tissue from a neo formed one, which connects the periphery with the secondary vascular tissue. Therefore, this neo-formed tissue promotes the structural basis necessary for radial vascular connections. Both the studied species have barriers to rhizogenesis, but these are not necessarily anatomical in nature.

Acknowledgements

The authors are grateful to the Coordination for the Improvement of Higher Education Personnel (CAPES - Finance Code 001); the National Council for Scientific and Technological Development (CNPq - Grant 302388/2019-2) of the Ministry of Science and Technology of Brazil; and Tanagro SA (Agreement AGTT 011/2019), for scholarships and partially supporting this research.

Data availability statement

In accordance with Open Science communication practices, the authors inform that all data are available within the manuscript.

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Edited by

Area Editor:

Dr. João Paulo Basso-Alves

Publication Dates

• Publication in this collection
  17 June 2024
• Date of issue
  2024

History

• Received
  28 Aug 2023
• Accepted
  04 Mar 2024