In vitro callus co-cultivation strategy for prospecting growth regulators to induce in vivo grapevine graft union

Cocultivo de calos in vitro como estratégia na prospecção de reguladores de crescimento para indução da enxertia in vivo da videira

Abstract

The use of growth regulators (GRs) in a mechanized bench grafting system is essential for the final quality of grafted grapevine plants. In Brazil, the GRs used (commercial waxes) are imported, increasing the production cost for nurseries. This work aimed to test the in vitro callus co-cultivation technique as a strategy for prospecting effective doses and mixtures of GRs for in vivo graft union on grapevines. Two genotypes were used as scions (‘Bordo’ and ‘Riesling Italico’) and two as rootstocks (‘SO4’ and ‘P1103’). Callus from different scion/rootstock combinations were co-cultivated, varying the doses (0.25 to 20 mg l^-1) of auxins (DBA, IAA and IBA) and cytokinin (BAP), following the Doehlert design. The best in vitro treatments were validated in vivo, in plants grafted with an ‘omega’ cutter machine. Multivariate and non-parametric methods were applied to integrate the results. In vitro prospecting combined with the in vivo validation strategy allowed us to select effectively the IAA(2.50 mg l^-1) plus BAP(1.00 mg l^-1) formulation with the potential to induce graft callus union similar to commercial waxes. The induction intensity varied among the genetic combinations but was similar in the in vitro and in vivo conditions.

Index terms
Vitis spp.; micropropagation; grafting; phytohormones; nurseries

Resumo

A utilização de reguladores de crescimento (RC) no sistema de enxertia de mesa da videira são fundamentais para a qualidade final das mudas. No Brasil, os RC utilizados (ceras comerciais) são importados, aumentando o custo de produção para os viveiristas. Este trabalho teve como objetivo testar a técnica do cocultivo de calos in vitro na prospecção de doses e misturas de RC para indução da enxertia in vivo da videira. Foram utilizados dois genótipos como copas (‘Bordo’ e ‘Riesling Italico’) e dois como porta-enxertos (‘SO4’ e ‘P1103’). Calos das combinações copa/porta-enxerto foram cocultivados, variando-se as doses (0,25 a 20mg l^-1) de auxinas (DBA, IAA e IBA) e citocinina (BAP), seguindo o delineamento Doehlert. Os melhores tratamentosin vitro foram validados in vivo, em plantas enxertadas com máquina deenxertia tipo ‘omega’. Métodos multivariados e não paramétricos foram aplicados naintegração dos resultados. A prospecção in vitro combinada com a validação in vivo foi eficaz na seleção da formulação IAA (2,50mg l^-1) mais BAP (1,00mg l^-1) com potencial parainduzir o calo da enxertia de forma semelhante à cera comercial. A intensidade dessa indução variou entre as combinações genéticas de forma similar tanto na condição in vitro como na in vivo.

Termos para indexação
Vitis spp; micropropagação; enxertia; fito-hormônios; viveiros

Introduction

In Brazil, vitiviniculture is distributed over an area of 74.826 ha (MELLO; MACHADO, 2021), ranking 19^th in cultivated areas worldwide and 14^th in grape production (MELLO, 2016). Despite being small compared to traditional grape-producing countries, Brazilian products have gradually been recognized by domestic and foreign markets (MELLO; MACHADO, 2021). Aiming at the sustainable growth of this sector, several practices are being improved, such as the production of grafted grapevine plants. Traditionally, in the preparation of grafted plants, grape growers used cuttings of scion cultivars and rootstocks collected directly from commercial vineyards (NACHTIGAL; SCHNEIDER, 2007).

However, recently, nurseries have intensified the offer of commercially grafted grapevines, using cuttings collected from certified mother plants, with identity and sanitary control (GROHS et al., 2017). The number of grafted plants produced in commercial nurseries in Rio Grande do Sul, the largest grape-producing state in Brazil, grew by 22% between 2008 and 2015 (MELLO; MACHADO, 2017).

This scenario is associated with both social aspects, due to the reduction of workers in the field, and technical aspects, such as the early death of plants due to fungal diseases when grafting is conducted in the field.

Despite this advance, the grafting systems are still far from ideal in Brazilian nurseries, as they primarily adopt hand bench grafting.

In contrast, in most traditional wine-producing countries, the mechanized ‘omega’ bench grafting system has been the most widespread and used since the 1960s (GROHS et al., 2017). Compared to hand bench grafting, the mechanical system results in fewer labor requirements, and higher yield and percentage of grafts with a uniform morphological pattern, thus improving the final quality of the grafted plants (WAITE et al., 2015).

To be considered functional, the graft union requires a vascular connection between the scion and rootstock, initialized from the formation of the callus bridge (JONARD et al., 1990). The callus is defined as an irregular mass of parenchyma cells at different stages of lignification (PINA et al., 2009). Callus formation (callogenesis) is determined especially by the balance of endogenous levels of phytohormones between the scion and rootstock.

Among phytohormones, the relationship between auxin and cytokinin is the most important, since any imbalance between these two phytohormones induces an incomplete callus bridge, restricting the vascular connections of the xylem and phloem (NANDA; MELNYK, 2018; BARON et al., 2019). For this reason, in the mechanized bench grafting system, the use of paraffin with waxes rich in growth regulators has been recommended to modulate the balance between auxins and cytokinin (WAITE et al., 2015; ZHOU et al., 2020).

In general, these commercial waxes are produced in Europe and are mainly enriched with an auxinic growth regulator (DBA, 2,5-dichlorobenzoic acid). In addition to being expensive, the grafting quality of these imported commercial waxes exhibits variable results in Brazil due to the different genotype combinations used as a scion (S) and rootstock (R). As an example, using the omega cutter machine and commercial waxes enriched with growth regulators, Regina et al. (2012) obtained grafted plants productivity ranging from 8 to 85% for the European cultivar Chardonnay, grafted on thirteen different rootstock varieties. These indexes tend to vary even more when considering the diversity of scion cultivars that are grafted in Brazil, involving genotypes of Vitis vinifera, Vitis labrusca, and hybrids. This diversity is also observed among rootstocks, as there are genotypes with high rooting capacity and grafting affinity to recalcitrant genotypes in both processes (MAYER et al., 2006). Similar results were also reported in Bulgaria, where Iliev et al. (2014) used waxes enriched with auxins from France and Spain and obtained a viable grafted plant yield between 90% and 66%, respectively, with local grape cultivars.

Therefore, these results show that the benefits of formulations with growth regulators depend on the scion/rootstock combinations of each location and must be adjusted to achieve maximum efficiency in the production of grafted plants with mechanized grafting.

Despite the practical importance of these formulations for the grafting quality and yield of quality grafted plants, it is noteworthy that tests for prospecting types, doses and mixtures of growth regulators are very complex and costly regarding time, space and resources.

This fact is evident in the design of in vivo tests based on complete factorials, which demand costly experiments, with many interactions (GABRIELSSON et al., 2002). To circumvent this problem, in vitro tests (tissue culture) have been performed in other cultures and presented as an effective strategy for prospecting chemical compounds with high analytical performance while demanding less space and time (MATOS, 2009). Among the in vitro techniques, the callus co-culture between species or cultivars has been used successfully in anatomical (PINA et al., 2009) and molecular studies (LIU et al., 2018). Cocultivated callus consists of non-differentiated agglomerated tissues, obtained from vegetative explants, with physiological uniformity and closer to the pure genetic effect (JONARD et al., 1990). Traditionally, co-culture trials are important tools in studies for predicting and characterizing graft incompatibility in fruit species (ERREA et al., 2001; PEDERSEN, 2006).

In these studies, full callus fusion requires adjusting the culture medium, especially types and doses of growth regulators. This in vitro approach is innovative and there are no records in the world literature on the application of this technique for adjusting the management of grapevine grafting. Therefore, this work aims to adjust and test the viability of the in vitro callus co-culture technique in the preliminary prospection of effective formulations with growth regulators (GRs) for mechanized grapevine grafting systems.

Materials and Methods

This study adopted the coupled strategy: stage 1: in vitro prospection of GR candidate formulations in two experimental steps, and stage 2: in vivo validation of GR candidate formulations in one step. The study was conducted in Bento Gonçalves, Brazil, in 2018 and 2019. Each experimental step defined the treatments that should be carried on to the next step, up to the in vivo validation.

The experimental steps conducted in each stage are described in the following sections.

Stage 1. In vitro trials

The callus used in the trial were obtained by the in vitro co-culture technique. First, the in vitro culture of isolated callus was obtained for each genotype, following a protocol adapted from Khan et al. (2015). Cuttings were collected from the mother plants of each cultivar in the dormancy phase (July) and rooted in pots until budburst (Figure 1A).

Figure 1
Details ofin vitro callus co-cultivation for grafting compatibility tests with different scion/rootstock combinations and treatments with growth regulators: cuttings from mother plants for explant production (A); developmental stage for explant removal (B); in vitro establishment of explants in medium for basal callus induction (C); basal callus formation (D); removal of the basal callus from the scion (S) and rootstock (R), placing together in same plate with the specific medium for each treatment (growth regulator) (E); detail of scion/rootstock callus co-cultivation (F); plate with scion/rootstock callus fusion, recorded after 30 days of co-cultivation (G). Three levels of callus fusion compatibility: incompatible (H), partially compatible (I) and compatible (J). Details of in vivo grafting: omega cutter for scion/rootstock union (K); first application of candidate formulations in treatments not submitted to paraffinization (L). After 30 days of forcing (acclimatization period) (M), three callus formation rate around the graft union (frontal and lateral views) in treatmens not submitted to paraffinization: low (N), moderate (O), and high (P).

Individual nodal segments were obtained from the shoot apical tips (≈10 mm) (Figure 1B) and placed in tubes (five segments per plant on average, Figure 1C) with 12 mL MS medium (Murashige and Skoog 1962), supplemented with 30 g L^-1 sucrose and 2.0 mg l^-1 6-benzylaminopurine (BAP), and solidified with 0.6% agar (pH 5.8). The segments were kept in a growth chamber at 23 ± 2°C and 16-h photoperiod until the basal callus was formed (Figure 1D). After this period, the callus at the basal end was removed and stored in the same medium described above until the co-culture trial. For the trials, 3 to 5 mm tissues were removed from the internal part of the callus (Figure 1E) following the methodology described by Jonard et al. (1990).

To start the co-culture, the callus pieces from the scion and rootstock were placed together in the same medium described above (but without BAP), with or without a GR according to treatment (Figure 1F-1G). The trials were carried out for 30 days in a growth chamber at 28 ± 1°C, without additional light. The growth conditions were based on the settings used as standard during forcing grafted cuttings (in vivo), seeking to emulate those in which the prospective formulations were validated. It is noteworthy that, in the following two steps, the experimental unit consisted of each callus scion/rootstock combination, with eight replications per treatment.

Step 1.1. Obtaining GR formulations

The trial was conducted on fusion callus obtained from the ‘Riesling Italico’ (Vitis vinifera) scion on ‘SO4’ (Vitis berlandieri x Vitis riparia hybrid) rootstock, abbreviated as R/S.

This R/S combination is economically important in wine-producing countries, including Brazil, while the grafting capacity and grafted plant yield have been historically classified between low and moderate. (VRŠIČA et al. 2015). As there are no reports of specific formulations of culture medium for fusion callus in the R/S, the GRs and reference doses were obtained from isolated callus induction works in different cultivars (CLOG, 1990; MII et al., 1991; SRNGH; BRAR, 1993; JIMÉNEZ; BANGERTH, 2000; AAZAMI, 2010; KHAN et al., 2015; KUMSA, 2017). From these protocols, the following list of GRs to be tested was established: IBA (indolebutyric acid), IAA (indoleacetic acid), DBA (2.5-dichlorobenzoic acid), BAP (6-benzylaminopurine).

For the test of each GR, the Doehlert design (GABRIELSSON et al., 2002) was applied, starting from the reference dose and progressively increasing, considering the minimum and maximum limits of the most effective dose of the previous trial (details in Figure 2). Thus, the tests were progressively repeated in time, until reaching the adjusted dose of each GR (GR formulation), defined as the greatest callus fusion concerning the CONTROL (without the addition of GR in the culture medium).

Figure 2
Example of sequential steps for analyzing the response of a given callus variable in vitro for the obtaing GR (growth regulator) formulation (dose adjustment), following Doehlert's design: first test - three initial doses (x) were used, which were selected from literature data: minimum dose (x_min), average dose (x₀) and maximum dose (x_max) plus CONTROL (C_t1) (without added GR in the culture medium) (A); Tr_{quality variable} obtation (T_r0), considering the responses of the different doses of the first test compared to the CONTROL, with x₀ being selected as the optimal dose (B); second test - analysis of two new doses close to x₀ (x₁ and x₂) (C) to confirm if x₀ remains the optimal dose (D) or if there is an indication of a new optimal dose (x₂) (E); third test - applying two more doses close to x₂ (x₃ and x₄), ending the test by confirming x2 as the optimal dose (F). This is just a sequence of tests, as an example, and if the optimal dose is not clearly confirmed, new rounds are necessary, following the same test design.

Step 1.2. Obtaining GR candidate formulations

The GR formulation (obtained from the results of step 1.1) were mixed (auxin plus cytokinin), aiming to identify possible synergistic or additive effects in the R/S callus co-culture.

In parallel, these formulations were also applied to other genetic scion/rootstock combinations to verify the breadth use. The most promising composite formulations (dose and mixture) in all genetic combinations were named GR candidate formulation to validate in the stage 2.

The treatments were applied following a completely randomized design arranged as a factorial scheme, with factor 1 (formulation): a) isolated GR formulations and b) mixed GRs formulations; factor 2 (genetic combinations): a) R/S; b) ‘Riesling Italico’ (Vitis vinifera)/ ‘P1103’ (Paulsen 1103, Vitis berlandieri X Vitis rupestris), abbreviated as R/P; c) ‘Bordo’ (Vitis labrusca)/‘SO4’ (Vitis berlandieri X Vitis riparia), abbreviated as B/S and d) ‘Bordo’/‘P1103’, abbreviated as B/P.

Stage 2. In vivo trials

The GR candidate formulations (obtained from the results of step 1.2) were validated in the cuttings grafting step (in vivo) to select the ideal formulation to improve the quality of the graft union. The mechanized grafting process, summarized below, was based on the protocol defined by Regina (2002) and Waite et al. (2015). Cuttings of dormant hardwood were collected in July, wrapped in plastic film, and stored in a cold chamber (3 ± 1ºC) for 60 days until grafting, using a semi-automated omega cutter machine (Figure 1K). Then, the grafted cuttings were placed in boxes with a 3cm layer of moistened vermiculite on the bottom (Figure 1L) and wrapped with plastic film to keep humidity above 85%. The boxes were placed in a forcing chamber for 30 days, without lighting, between 27 and 28°C. The grafted cuttings from the scion/rootstock combinations submitted to the STANDARD formulation were treated with commercial wax containing 0.01% oxyquinoline and 0.00175% DBA, which was previously melted (67°C) and applied right after grafting by immersing grafts in the wax (1 to 2s), followed by the same forcing conditions. The grafted cuttings treated with the GR formulations were not submitted to paraffinization. The doses of each GR formulations were previously dissolved in 1 M NaOH (for auxins) and 1 M HCl (for cytokinins), diluted in 1 L distilled water, and kept under refrigeration (± 3ºC). Starting on the grafting date (winter bud stage), each GR formulation was sprayed onto the grafted cuttings, in the boxes (Figure 1L), at threeday intervals throughout the forcing period.

The treatments were applied following a completely randomized design arranged as a factorial scheme, with factor 1 (formulations): a) GR candidate formulations, b) STANDARD and c) CONTROL (without candidates or STANDARD formulations); factor 2 (genetic combinations) (the same ones used in step 1.2). It is noteworthy that isolated and mixed formulations were validated only for the R/S genetic combination, while only those defined as candidates were validated for the all genetic combinations (R/S, R/P, B/S and B/P). Each genetic combination was represented by 30 replicates per treatment (grafted cutting was considered as the experimental unit.).

Assessment and statistical analysis

After 30 days of in vitro co-culture, callus quality was evaluated by the following five variables: (1) degree of callus fusion (JONARD et al., 1990), scored from 1 = nonfused callus (Figure 1H), 2 = incomplete fusion (Figure 1I) to 3 = complete fusion (fused) (Figure 1J); development of the (2) scion and (3) rootstock callus, scored from 1 = no development (Figure 1H), 2 = moderate (Figure 1I) to 3 = high (Figure 1J); (4) color (adapted from SRNGH; BRAR, 1993), determined by the percent without oxidation, where the high percentage values represent a more solid, white, and shiny mass of fused callus (Figure 1J); and, (5) mass of the co-fused callus (mg). After 30 days of in vivo forcing during the acclimatization period (five days) before planting (Figure 1M), the quality of the grafted plants was evaluated by five variables: percentage of plants with (1) visible graft callus union; (2) with developed shoots (budburst stage); (3) callus formation rate around the graft union (%) (CELIK, 2000), scored from low (below 25%) (Figure 1N), moderate (between 25% and 75) (Figure 1O) to high (between 75 and 100%) (Figure 1P); (4) callus diameter (mm) at the graft union; and (5) phenological stage of shoots (EICHORN; LORENZ, 1977).

The statistical analysis was based on meta- analysis concepts. Thus, one indicator was generated to aggregate quality variables.

The ‘Tr indicator’ (treatment response) was adapted from Lajeunesse (2011) and characterizes the performance of each formulation on the fusion quality of callus or grafted plants, compared to the CONTROL.

First, ‘Tr_{quality variable}’ was obtained (exemple in Figure 2A and 2B). The absolute value of a quality variable for a given formulation was divided by the absolute value in the CONTROL of the same genetic combination.

Subsequently, the average of all ‘Trquality variable’ was calculated to obtain the ‘Tr indicator’ for each formulation. ‘Tr indicator’ value above zero and close to 1 characterized the high capacity of the formulation to induce and fuse callus in vitro (called Tr_vitro) or to induce grafting callogenesis and union in plants (called Tr_vivo) of a scion/rootstock combination.

After calculating the Tr_vitro, a multivariate exploratory analysis was performed by applying the Principal Components Analysis (PCA) method. To adjust the T_rvitro values to the PCA, these were mean-centered, integrating the scores of GR with the quality variable loadings.

For the contrast of independent groups in the PCA spatialization, multiple standard deviation bars (± 2σ) were adjusted. The groups that did not overlap vertically or horizontally were considered distinct. The multivariate analysis was performed using the ChemoStat software (HELFER et al., 2015).

Besides, tests were applied to explore, contrast and relate the quality variables with Tr_vitro and Tr_vivo. Therefore, after checking data normality using the Kolmogorov-Smirnov (p<0.05), the non-parametric Chi-square test (p<0.05) was applied, followed by the Kruskal-Wallis of independent samples (for comparison of means), Q-Cochran of related samples (for comparison of frequencies) and Spearman (for correlation analysis).

Statistical tests were performed using R software, Agricolae and Kendall packages (R CORE TEAM, 2023).

Results and Discussion

In general, the in vitro callus co-cultivation strategy was deemed an effective tool for a broad prospection of GR formulation that may be useful for the in vivo grafting of different grapevine genotypes. Until the writing of this work, the literature references on callus co-culture are found only for other species, such as Citrus spp. (JONARD et al., 1990) and Prunus spp. (ERREA et al., 2001; PEDERSEN, 2006; PINA et al., 2009), but not for grapevines. In addition, these references do not address the use of callus co-culture for prospecting GR types, doses, or mixtures to increase callus fusion and, consequently, the quality of callogenesis in plant grafting.

The co-cultivation assays (step 1.1) showed that the Doehlert design (Figure 2) provided a maximum response for the callus fusion values compared to the CONTROL with a small number of tests for each compound (from 10 to 12) (Figure 3). The Doehlert design has already been successfully applied for selecting new chemical drugs (MATOS, 2009) and this work showed that it can be an effective strategy in the prospection of GR formulations under in vitro conditions.

Figure 3
Principal component analysis (PCA) of Tr_{quality variables}: scion (Tr_scion) and rootstock (Trrootstock) development, color (Tr_color), fusion degree (Tr_fusion) and mass (Tr_mass) of ‘Riesling Italico’ and ‘SO4’ callus co-cultivated. Callus co-fusioned was treated with doses ranging from 0.25 to 20 mg l^-1 of DBA, IAA, IBA and BAP. Non-overlapping multiple standard deviation bars (±2s), indicate discrimination between doses with different effect levels in the fusion callus quality.

The optimal doses of each GR were easily characterized with Tr _{quality variables} (Trmass, Tr_fused, Tr_color, Tr_rootstock, Tr_scion) employing a multivariate principal component analysis (PCA) (Figure 3). This combined approach (design, meta-analysis indicator and multivariate method) avoids applying more costly full factorial experiments, whose data evaluation and interpretation involve a large number of interactions and complexity (GABRIELSSON et al., 2002).

The total variance of the in vitro results was explained by PC1 (58.87%) and PC3 (16.12%).

According to their responses compared to CONTROL, three independent clusters were spatially constituted: low (negative scores on PC1), moderate (intermediate and positive scores on PC1), and high (positive and higher scores on PC1) effect (Figure 3).

Can et al. (2014) reported that high concentrations of growth regulators in a culture medium can inhibit cell division and biochemical reactions, inducing explant death. This fact was observed in clusters with low and moderate effects, where callus fusion was limited (doses lesser than 1.00 mg l^-1) or inhibited by the phytotoxic effect (doses from 5.00 to 20.00 mg l^-1) of DBA, IAA, IBA and BAP.

Therefore, the doses in the low and moderate effect groups were disregarded for the following steps, but the doses of IBA and BAP (1.00 mg l^-1), DBA (1.75 mg l^-1) and IAA (2.50 mg l^-1), grouped with better effect in step 1.1, were selected (GR formulations) for to step 1.2.

Detailing PC3, while BAP and IBA were associated with mass, degree of fusion and callus color, IAA and DBA were more related to the development of scion and rootstock callus (Figure 3). Indeed, these compounds have been previously reported to effectively induce isolated callus formation in Vitis spp.(MII et al., 1991; KHAN et al., 2015; KUMSA, 2017). Both auxins and cytokinin at adjusted doses trigger callus cell proliferation (JASKANI et al., 2008). Auxins containing culture medium change the gene expression of callus cell mass and the dose defines the continuity of globular growth or cell differentiation (JIMÉNEZ, 2001). Whereas cytokinin promotes the synthesis of RNA and proteins in the callus, increasing the enzymatic activity related to cell division and the loosening of the cell wall (KHAN et al., 2015). Highlighted, among the compounds used for in vitro tests, some GR traditionally used in tissue culture protocols were not tested in this study for being inadequate for in vivo use. The 2,4D was disregarded for being toxic to humans and the environment (JUNIOR et al., 2002). Likewise, despite not being an auxin traditionally used for in vitro callus cultivation, DBA was tested since it is commonly used as a component of the main commercial grafting waxes (WAITE et al., 2015; LIU et al., 2018).

In the selection of candidate GR formulations (step 1.2), approaching contrasts between isolated or mixtures of GR formulations, the mixed of IAA(2.50 mg l^-1) plus BAP(1.00 mg l^-1) (abbreviated IAA+BAP) and DBA(1.75 mg l^-1) plus BAP(1.00 mg l^-1) (abbreviated DBA+BAP) were observed to provide additive and superior effects over isolated applications of each compound (Figure 4AD).

The effects of these mixtures were more expressive on callus color (Tr_color) and callus development of the rootstock (Tr_rootstock), especially in grafting combinations on ‘P1103’ (Figure 4B and 4D). Moreover, to a lesser extent, the same trend can be observed with the ‘SO4’ combinations (Figures 4A and 4C).

Figure 4
Tr_vitro accumulated for the five Tr_{quality variables} (sum of Tr_{quality variables}) of callus fusion (A, B, C and D) and Tr_vivo accumulated for the five Tr_{quality variables} of grafted plants (E, F, G and H), for growth regulators formulations (mg l^-1): BAP(1.00), IBA(1.00), IBA(1.00)+BAP(1.00), DBA(1.75), DBA(1.75)+BAP(1.00), IAA(2.50) and IAA(2.50)+BAP(1.00) in the following genetic combinations: (A, E) ‘Riesling Italico’/‘SO4’; (B, F) ‘Riesling Italico’/‘P1103’; (C, G) ‘Bordo’/‘SO4’; (D, H) ‘Bordo’/‘P1103’. 1Same letters (± two standard error bars) do not differ statistically by the Kruskal-Wallis test (p<0.05). ^nsnot significant. GR candidate formulations (F, G, H) selected in 1.2 step and validaded in stage 2.

In the metabolism of plant tissue damage repair, such as cuttings for grafting, auxin always acts in combination with cytokinin for the callogenesis process (NANDA; MELNIK, 2018), which corroborates this synergistic effect of the mixtures compared to isolated applications. Srngh and Brar (1993) also reported that in grapevine tissue culture, the isolated use of auxins favors high cell proliferation, but the callus produced are friable and unstable over time. However, when associated with BAP, the resulting callus are more compact since this cytokinin favors lignification through the phenylpropanoid pathway (KAPARAKIS; ALDERSON, 2003).

This combined and synergistic effect requires the right balance between the doses of both GR, with BAP generally being used in a lower proportion than auxin (KHAN et al., 2015; MASTUTI et al., 2017; LIU et al., 2018).

At higher concentrations compared to auxin, BAP usually induces callus differentiation and leaf regeneration (KHAN et al., 2015; MASUTI et al., 2017).

The physiological responses of growth regulators are tissue-specific (ALONI, 1980; ENDER; STREIDER, 2015) so the candidate GR formulations obtained in stage 1, were specific to callus tissues. In the grafting, the development of callus (resulting from the multiplication of secondary xylem cells and phloem parenchyma cells close to the graft cut), is an initial and priority step for obtaining functional vascular connections (MIYASHIMA et al., 2013; COOKSON et al., 2013). Therefore, the in vivo validation becomes essential (stage 2). For this stage, the validated GR formulations are required to meet the following three requirements.

Firstly, they must be effective not only to induce callus at the grafting point but also to favor scion budburst. Nanda and Melnyk (2018) reported that grapevine graft union is only considered effective and functional when the shooting and rooting processes are initially integrated by callogenesis, followed by active vascular connection. Second, the responses of each scion/rootstock combination to the validated GR formulation should be significant and advantageous over the CONTROL. The CONTROL treatment indicate the natural capacity of a scion/rootstock combination to form viable and quality grafted plants, due to the endogenous hormonal balance that these genotypes present.

Third, the formulations selected and rated as promising must perform at least comparable to commercial formulation of grafting waxes (STANDARD treatment).

Analyzing the first requirement, the candidate GR formulations (IAA+BAP and DBA+BAP) improved the quality of grafting in combinations of ‘Riesling Italic’ and ‘Bordo’ with ‘SO4’ or ‘P1103’ (Figura 4EH).

However, the isolated application of BAP or mixed with IBA provided the lowest Tr_vivoacumulated in the ‘Riesling’/‘SO4’ grafting (Figures 4E) as observed in the in vitro assays (Figure 4A). Highlighted it is, in all genetic combinations, IAA+BAP stood out compared to DBA+BAP because it induces a higher Tr_vivoacumulated (Figure 4EH). Thus, the IAA+BAP was chosen as the validated GR formulation for the second requirement.

The effectiveness of this auxin/cytokinin mixture for inducing callogenesis and, consequently, obtaining grafted grapevine plants is in agreement with recent studies on the use of GR. The most significant effect of cytokinin mixed with auxins lies in its interaction with the auxin metabolic pathway (BARON et al., 2019). Cytokinin enhances the regulation and distribution of PIN proteins, favoring the transport of auxins between callus cells during the formation of vascular bundles.

Bidabadi et al. (2018) obtained satisfactory quality in grafting callogenesis for a formulation based on ANA and BAP, with a simultaneous increase in the concentration of antioxidant enzymes (catalase, peroxidases, and ascorbate peroxidase) and less accumulation of H₂O₂ in the tissues of the graft union.

Analyzing the second requirement, the responses of each treated graft compared to the CONTROL shows that IAA+BAP induced a positive effect on different in vivo quality variables, especially for R/S, B/P and B/S combinations (Table 1). The benefit of IAA+BAP becomes more evident to the phenological stage of shoots (Table 1B), callus formation rate (Table 1D) and callus diameter (Table 1E). The contrast with the CONTROL was more expressive when using the ‘SO4’ rootstock. The CONTROL showed a high percentage of plants with shoots (Table A) and plants with visible callus at the grafting point (Table 1C), especially for R/P, B/P and R/S combinations. The benefit of IAA+BAP becomes less evident in these variables, provided a superior effect in B/S combination.

Thus, the IAA+BAP effects were more expressive in the genetic scion/rootstock combinations that present the lowest natural capacity for callogenesis in the grafting union. The Tr_{in vivo} (Table 1F), indicated that IAA+BAP presented gain compared to the CONTROL in B/S (1.79), R/S (1.49) and B/P (1.18) combinations.

In R/P, the gain about 1.0 (0.95), not justifying the use of formulation.

Table 1
Five quality grafted plants variables and Trvivo from the six combinations of ‘Riesling Italico’ and ‘Bordo’ scions on ‘P1103’ and ‘SO4’ rootstocks, treated with growth regulators formulations (IAA(2.50)+BAP(1.00); STANDARD) and CONTROL.

Treatment	P1103				SO4
	Riesling		Bordo		Riesling		Bordo
	A. Plants with shoots (%)
IAA+BAP	75	Bb	94	Ans	96	Ans	92	Ans
STANDARD	80	Bb	100	A	93	A	97	A
CONTROL	100	Aa	100	A	84	B	79	B
Mean	86		96		88		89
	B. Phenological stage of shoots (Scale)
IAA+BAP	5.5	Bb	8.8	Aa	7.3	Aa	4.5	Ba
STANDARD	5.1	Ab	6.0	Ab	6.4	Aab	5.1	Aa
CONTROL	8.0	Aa	5.4	Bb	5.5	Bb	3.3	Cb
Mean	6.0		7.3		6.4		3.9
	C. Plants with visible callus at graft union (%)
IAA+BAP	100	NSa	100	ns	100	ª	100	ª
STANDARD	100	Aa	100	A	100	Aa	100	Aa
CONTROL	100	Aa	100	A	96	Aa	64	Bb
Mean	92		100		93		76
	D. Callus formation rate around graft union (%)
IAA+BAP	95	Aa	89	Aab	97	Aa	68	Bb
STANDARD	94	Aa	96	Aa	100	Aa	93	Aa
CONTROL	85	Aa	66	Bb	51	Bb	46	Bc
Mean	75		76		72		59
	E. Callus diameter at the graft union (mm)
IAA+BAP	4.9	Ans	3.5	Bb	4.8	Aa	3.0	Bab
STANDARD	6.0	A	5.8	Aa	5.2	Aa	5.4	Aa
CONTROL	4.2	A	3.6	ABb	2.4	BCb	0.9	Cb
Mean	3.7		3.5		3.2		1.9
	F. Trvivo
IAA+BAP	0.95	Bns	1.18	ABns	1.49	Ans	1.79	Ab
STANDARD	1.00	Bns	1.23	ABns	1.48	Ans	2.47	Aa
Mean	0.98		1.21		1.49		2.13

• ^*
  Values followed by the same uppercase (row) or lowercase (column) letter do not differ statistically (p<0.05) by the Q-Cochran (A, C, D) and Kruskal-Wallis (B, E, F) tests. ^NS/nsnot significant.

Several factors linked to the genetic origin of these scion/rootstock combinations, such as anatomical and biochemical contrasts, may be modulating the level of effect of each GR formulation on callogenesis and grafting quality. Among the anatomical factors, it is noteworthy that the variation in the vascularization pattern can occur even between cultivars of the same species, mainly in the xylem area and in the number of vessel elements (SANTAROSA et al., 2016). For example, the vascular alignment between the scion and rootstock interferes with chemical and molecular transport, affecting the functioning of the graft union (VAHDATI et al., 2010).

Regarding the biochemical aspects of the scion/rootstock combinations, the contrasts that can occur in the accumulation of phenolic compounds (ASSUNÇÃO et al., 2019), in the isoenzyme activity (GÖKBAYRAK et al., 2007), or in the phytohormone (HE et al., 2018) are emphasized. Changes in the balance of these compounds or the accumulation of their phytotoxic forms can determine the success of grafting or the incompatibility response. Possibly, these biochemical variations may also be present in the different combinations of American cultivars (e.g. ‘Bordo’) on hybrid rootstocks (e.g. ‘P1103’ or ‘SO4’), typical in Brazil, which may increase callogenesis restrictions and grafting incompatibility without the use of GRs.

Analyzing the third requirement, the validate GR formulation must similiar the result obtained with the commercial waxes availables.

According to Regina et al. (2012), the GR treatment is satisfactory only when in post-forcing the grafted plants present more than 90% of budburst/shoots and callogenesis.

Furthermore, Hunter et al. (2013) classify post-forcing grafted plants as high quality if they present callus diameter at grafting union between 2.0 and 3.0 mm. Following these references, the IAA+BAP was similar to the STANDARD in the same parameters.

Compared to STANDARD, IAA+BAP promoted higher or similar gains in the percentage of shoots (Table 1A), phenological stage (Table 1B), and plants with visible callus (Table 1C) to all genetic combinations. Callus diameter (Table 1D) and callus formation rate (Table 1E) were especially higher in the STANDARD to B/P and B/S combinations. However, the Tr_vivo (Table 1F) indicated that STANDARD and IAA+BAP formulations presented similar results compared to the CONTROL in R/P, B/P and R/S. Only in the B/S combination, the STANDARD presented higher Tr_vivo compared to IAA+BAP.

The commercial wax possibly offers the GR to the grafting region at a different rate compared to the direct application of the liquid formulation of IAA+BAP. The Tr_vivo result in B/S combination does not invalidate applying the liquid IAA+BAP formulation. The direct spraying of the IAA+BAP, during the forcing period, ensured adequate graft plant quality limits, thus becoming an alternative to commercial waxes. But, highlights the need for new adjustments of in vivo application in this genetic combination to reach maximize the IAA+BAP effects. This selection strategy and GR application method to produce grafted plants are highly important for the grape production sector, especially in countries that do not produce waxes. Although not evaluated in this work, these advances can provide direct benefits for nurseries, reducing the cost of grafted plant production.

This study demonstrated that callus induction and GR responses are determined by the genetic potential of the grafted cultivars.

A joint analysis of Figure 4 and Table 1 allows us to correlate the indicators/variables (Table 2 and Figure 5) of the two experimental conditions (in vitro and in vivo). Both Tr_vitro and Tr_vivo increased similarly (ƿ=0.89**, Table 2, Figure 5A) in R/S combination and are therefore suitable for characterizing and correlating the overall quality of cofused callus in vitro and grafting in vivo. Furthermore, both Tr_vitro and Tr_vivo were highly influenced and, consequently, correlated with the callus diameter evaluated in vitro (ƿ=0.89**) and in vivo (ƿ=0.99**) (Table 2, Figures 5B and 5C, respectively). This high correlation shows that the increase in callus in the grafting region in vivo, occurred proportionally to the increase in callus that were cofused in vitro and treated with the same GR formulation.

Table 2
Matrix correlation analysis of callus fusion quality variables (fusion – F; development of the (– S) scion and (– R) rootstock; mass – M; color – C) and Trvitro with grafted plants quality variables (plants with shoots – PWS; phenological stage of shoots – PS; Plants with visible callus at graft union – GP; Callus formation rate around graft union - CF; Callus diameter at the graft union – CD) and Trvivo in the ‘Riesling Italico’/‘SO4’ combination, treated with seven growth regulators formulations (mg l-1): BAP(1.00), IBA(1.00), IBA(1.00)+BAP(1.00), DBA(1.75), DBA(1.75)+BAP(1.00), IAA(2.50) and IAA(2.50)+BAP(1.00).

	PWS (%)	PS (Scale)		GP (%)		CR (%)		CD (mm)		Trvivo		Trvitro
F (Scale)	-0.28	-0.94	**	-0.28		-0.71		-0.55		-0.55		-0.38
S (Scale)	-0.41	-0.77	**	-0.41		-0.26		-0.29		-0.29		0.46
R (Scale)	-0.51	-0.30		-0.15		-0.31		-0.12		-0,12		-0,09
M (mg)	-0.42	<0.0		-0.34		-0.18		-0.32		-0.32		-0.32
C (%)	-0.18	0.23		0.52		0.32		0.57		0.57		-0.43
Trvivo	0.53	0.69		0.79	*	0.82	*	0.99	**	-		0.89	**
Trvitro	0.29	0.71		0.74		0.79	*	0.89	**	0.89	**	-

• ^*
  */**Significant correlation by the Spermann test (p<0.05 and p<0.01, respectively).

Figure 5
Correlation analysis of ‘Tr indicator’ of grafted plants (Tr_vivo) and fusion callus (Tr_vitro) (A), callus diameter at the graft union and Tr_vivo (B); callus diameter at the graft union and Tr_vitro (C) for seven growth regulators formulations (acronyms are described in the caption of Figure 4), just in the ‘Riesling Italico’/‘SO4’ combination. Correlation analysis of callus diameter at the graft union and fusion callus indicator (Tr_vitro) for validate formulation IAA(2.50)+BAP(1.00) to following genetic combinations: ‘Riesling Italico’ or ‘Bordo’ as scions on ‘SO4’ or ‘P1103’ as rootstocks (D). **Significant correlation by the Spermann test (p<0.01).

Although tissue cutting for in vivo grafting activates a complex cascade of molecular reactions (ENDERS; STRADER, 2015; NANDA; MELNYK, 2018), these positive correlations show that the responses of the in vitro cofused callus interaction were similar, despite involving different physiological conditions.

In addition, the association strength and tendency of such variables in callogenesis were modulated by the genetic combination.

The Tr_vitro correlation with callus diameter (ƿ=0.73*, Figure 5D) compared to the different genetic combinations, exposes the combination B/S with the lowest values of Tr_vitro and callus diameter, while R/P and B/P reached the highest values for the same variables.

These results confirm the contrasts observed in Table 1F, indicating that the B/S combination tends to be responsive to GR.

Therefore, despite the contrasts in response intensity between the different genetic combinations, it is noteworthy that there was a direct response pattern between callus diameter and Tr_vitro. In general, the greater the fusion and growth of the callus by the in vitro GR treatment, the greater the in vivo effect of this same treatment on the callus diameter and, consequently, the quality of the grafted plants. This association, between two direct visual analysis variables, proves to be an important tool for the in vitro prediction of future responses of genetic compatibility or grafting quality in grapevine plants. Until the writing of this work, only Aazami (2010) carried out a study with Vitis spp. correlating the developmental characteristics of grafted genotypes in vitro with the vigor responses of plants in vivo. However, the strategy of in vitro analysis to estimate graft compatibility has not been conducted in grapevines so far.

In other perennial cultures, this approach has already been applied with predictive success. For example, with Prunus genotypes, Pina et al. (2009) were able to easily discriminate homografts from heterografts in vivo, through confocal microscopy analysis of the intercellular communication of callus that were co-cultured in vitro.

Conclusion

The in vitro prospecting coupled with the in vivo validation strategy was effective to select and validate GR formulation with the potential to induce graft callus union and assure the quality of grapevine graft plants.

For the ‘Bordo’ or ‘Riesling Italico’ scions on ‘P1103’ or ‘SO4’ rootstocks, the formulation of IAA (2.50 mg l^-1 indoleacetic acid) and BAP (1.00 mg l^-1 6-benzylaminopurine) was the most promising for the ‘omega’ mechanized bench grafting system. Furthermore, the intensity of grafting quality response to the validate GR formulation was more intense in grafts with ‘SO4’, especially with ‘Bordo’.

Finaly, the in vitro cofusion of callus in a medium containing the optimal formulation determined a direct and positive association with the callus diameter that was induced by the same formulation in vivo in the grafted plants.

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Edited by

Viriginia Silva Carvalho/Alexandre Pio Viana

Publication Dates

• Publication in this collection
  15 Apr 2024
• Date of issue
  2024

History

• Received
  27 Feb 2023
• Accepted
  18 Dec 2023