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Article

Multiplication of Axillary Shoots of Adult Quercus robur L. Trees in RITA® Bioreactors

by
Paweł Chmielarz
1,†,
Conchi Sánchez
2,†,
João Paulo Rodrigues Martins
1,
Juan Manuel Ley-López
1,
Purificación Covelo
2,
María José Cernadas
2,
Anxela Aldrey
2,
Saleta Rico
2,
Jesús María Vielba
2,
Bruce Christie
3 and
Nieves Vidal
2,*
1
Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland
2
Misión Biológica de Galicia, Consejo Superior de Investigaciones Científicas, Avda de Vigo s/n, 15705 Santiago de Compostela, Spain
3
The Greenplant Company, Palmerston North 4410, New Zealand
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2025, 16(8), 1285; https://doi.org/10.3390/f16081285
Submission received: 31 May 2025 / Revised: 7 July 2025 / Accepted: 23 July 2025 / Published: 6 August 2025
(This article belongs to the Section Genetics and Molecular Biology)
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Abstract

Adult trees of pedunculate oak (Quercus robur L.) are recalcitrant to vegetative propagation. In this study, we investigated the micropropagation of five oak genotypes corresponding to trees aged 60–800 years in a liquid medium. We used commercial RITA bioreactors to study the influence of the explant type, the culture medium, shoot support and number of immersions. Variables evaluated included the number of normal and hyperhydric shoots, shoot length, multiplication coefficient and number of rootable shoots per explant. All genotypes could be cultured in temporary immersion. Basal stem sections attached to callus grew better than apical sections and developed less hyperhydricity. For long-term cultivation, Gresshoff and Doy medium was the best of the three media evaluated. All genotypes produced vigorous shoots suitable for rooting and acclimation. This is the first protocol to proliferate adult oak trees in bioreactors, representing significant progress towards large-scale propagation of this and other related species.

1. Introduction

In recent years, the public knowledge about the importance of trees has experienced an enormous growth in awareness as a consequence of the current situation of climate change. The ecosystem services provided by forests are of paramount importance to mitigate negative effects on biodiversity, environmental parameters and human well-being [1]. In addition to forests’ ecological role, FAO estimates that timber demand will continue to increase significantly in the next decades [2]. It has been claimed that planted forests with well-managed exotic species can contribute to ecosystem services such as carbon capture and storage, avoidance of erosion and water regulation as much as native forests [3], but at present, there is no scientific consensus about their role in biodiversity conservation. While in highly fragmented landscapes they may increase connectivity between native forest areas and promote the movement of species, their reduced suitability as habitats can make them ecological traps for specialized niche species and vulnerable species, with reports of lower abundance of plants, insects and birds compared with native forests [4,5].
In contrast, the propagation of selected trees native to the zones of production will lead to more cost-effective plantations, more respectful of the environment and aligned with the sustainable development goals. Pedunculate oak (Quercus robur L.) is a deciduous tree belonging to the Fagaceae family. Native to most of Europe, Q. robur can be found as a main component of temperate deciduous mixed forests [6]. In addition to its ecological role in Europe, it is extensively cultivated in this and other temperate regions for both timber production and ornamental landscaping. Its wood is highly valued for its durability, mechanical strength and resistance to decay, making it a preferred material for many industrial applications, including the manufacture of barrels for aging alcoholic beverages [7,8]. These desirable properties underscore the importance of large-scale propagation of the species, which—using conventional methods—relies primarily on seed-based reproduction. However, Q. robur produces acorns in abundance only during mast years, which occur unpredictably and at irregular intervals [9]. Furthermore, its seeds are recalcitrant to cold storage, rendering them unsuitable for long-term preservation [10]. Consequently, there is often a limited supply of high-quality seeds for continuous nursery production and ecological restoration efforts. This constraint emphasizes the urgent need to develop alternative propagation techniques such as in vitro culture to ensure the reliable reproduction of elite genotypes [11].
During the juvenile phase, traits such as tree architecture and wood quality are not fully expressed or stable, making the early selection of individuals in propagation programs difficult. As a result, oaks are generally considered suitable for selection only after reaching maturity, following the juvenile-to-adult phase transition. Mature trees—particularly those over 300 years old—are especially valuable, as their longevity under diverse environmental conditions reflects a high degree of adaptive resilience. These individuals serve as important reservoirs of superior genetic material [12]. Micropropagation methods for oaks began more than 30 years ago [13,14,15,16,17,18,19], but the commercial exploitation of this technology is not yet a reality for this species. The effectiveness of oak micropropagation is significantly influenced by both the genotype and the physiological maturity of the donor plant. Most trees pose specific challenges to vegetative propagation, especially when they have reached maturity [20]. In the case of oak, feasibility for micropropagation decreases sharply during the first months after seed germination, contributing to recalcitrance to culture establishment, shoot multiplication and rooting [11,16,18].
Propagation in temporary immersion system (TIS) bioreactors has improved proliferation and quality of many plants [21,22,23]. The increased absorption of nutrients from a liquid medium together with the renewal of the air inside the bioreactors may improve the physiological response of the explants, making them more competent to undergo rooting and acclimation [21], which can be especially relevant for recalcitrant tree species [24].
TISs have increased micropropagation of several Fagaceae species cultured either by somatic embryos or axillary shoots. Somatic embryo production in bioreactors has been studied in Q. robur [25,26], cork oak (Q. suber L.) [27] and American chestnut (Castanea dentata Borkh. (Marshall)) [28,29]. The authors of these studies reported more embryo production and better plant quality using RITA® with Quercus spp. and airlift bioreactors in the case of American chestnut. Also, for oak and American chestnut, bioreactors facilitated the recovery of genetic transformants as well [26,28,29].
Regarding axillary shoots, the first report in the Fagaceae family occurred for the genus Castanea, with the micropropagation of hybrids of European chestnut (Castanea sativa Mill.) with Chinese chestnut (C. mollissima Blume) or Japanese chestnut (C. crenata Siebold & Zucc.) [30]. Shoots were cultured in RITA® or PlantformTM bioreactors, with the latter yielding more rootable shoots. More recently, a protocol developed for multiplication of C. dentata shoots in rocker bioreactors yielded longer shoots than the agar-based system [31], whereas preliminary studies on Fagus sylvatica L. in PlantformTM are showing promising results [32].
The first attempt to propagate Q. robur shoots in bioreactors was carried out by Gatti et al. [33], who cultured plant material of juvenile origin (seedlings) in PlantformTM vessels. The authors demonstrated that oaks could be cultured in a liquid medium and obtained rooted shoots. Although significant improvements compared to semisolid medium were not reported, the authors identified critical challenges such as the occurrence of hyperhydricity (HH). This disorder represents one of the most common problems in plant micropropagation. The affected shoots exhibit anatomical and morphological abnormalities, like translucent and brittle stems and leaves, as well as physiological and metabolic dysfunctions that make them unsuitable for multiplication and rooting [34,35]. In this pioneering study of propagation of juvenile oak material by TIS, HH was associated with prolonged exposure to a liquid medium and could be reduced by manipulating the frequency and duration of immersion [33].
Regarding the cultivation of adult oak material in bioreactors, to the best of our knowledge, the first experiments were carried out with a 300-year-old Polish tree by one of the research groups involved in the present study. However, the main objectives were not achieved as oak nodal segments cultured in RITA® did not grow as well as in semisolid medium [11].
The present study represents a collaboration in the frame of the COST Action CA21157, “European Network for Innovative Woody Plant Cloning”. We exchanged plant material and put together the previous experience of our research groups in cultivating old oaks in semisolid medium [11] and in the development of efficient protocols for growing Fagaceae trees in bioreactors [26,30]. Our aim was to use RITA® bioreactors to culture old oaks by temporary immersion. We selected five oak genotypes established in vitro from trees collected in Spain and Poland and aged from 60 to 800 years. The factors investigated included the number of immersions, culture medium, explant type and the use of shoot support (rockwool cubes). As a result, the five genotypes could be proliferated by TIS and produced shoots that could be rooted and acclimated, which is a significant step forward towards developing large-scale production of clonally selected Q. robur.

2. Materials and Methods

2.1. Plant Material and Micropropagation in Semisolid Medium

Initial explants for experiments in bioreactors were shoots established in vitro from five oaks aged 60–800 years (Table 1; Figure 1). Four trees—Chaian 1 and 2, Carballo das Mentiras and San Lourenzo—were located in Galicia (Spain), in the municipalities of Trazo, Vedra and Santiago de Compostela, whereas the fifth tree—RUS—was located in Rogalin, Poland. All of them were previously established in vitro from basal or epicormic branches as described by Vieitez et al. [18] for the Spanish oaks and by Chmielarz et al. [11] for the Polish oak. All genotypes were maintained by regular subcultures in semisolid medium for more than three years before the initiation of the experiments in a liquid medium. Spanish oaks were routinely cultured in Gresshoff and Doy (GD) medium [36] with the macronutrients, micronutrients and vitamins detailed in Supplementary Table S1. The medium was supplemented with N6-benzyladenine (BA), 3% sucrose and 0.65% (w/v) Plant Propagation Agar (Condalab, Madrid, Spain). Explants were cultured for 2 weeks with 0.44–0.88 µM BA and transferred for another 2 weeks to a fresh medium with half of the previous BA. The Polish oak had been maintained on Woody Plant Medium (WPM) [37] with 1.76 µM BA, 3% sucrose and 0.7% (w/v) agar [11], but six months before the experiments were started, the RUS material was subcultured either in GD or WPM with 0.88 µM BA and transferred to a fresh medium as with Spanish oaks.

2.2. Experiments in Bioreactors

Experiments with a liquid medium were carried out in temporary immersion (TIS) in commercial RITA® bioreactors (Vitropic, Saint Mathieu de Tréviers, France), which were prepared, sterilized and operated as detailed for chestnut [31].
Explants were cultured with 150 mL of a medium supplemented with 0.44 to 0.88 µM BA and 3% sucrose. The pH was adjusted to 5.6–5.7 before autoclaving at 120 °C for 20 min. Cultures were incubated under a 16 h photoperiod provided by LED lamps providing a photosynthetic photon flux density of 50–60 µmol m−2 s−1 at 25 °C light/20 °C dark. Explants were cultured for 2 weeks and transferred for another 2 weeks to a fresh medium with the same BA concentration. After these four weeks, data was recorded, and shoots were harvested for multiplication or rooting.
Experiments were carried out to test the effects of variables such as explant type, support, frequency of immersion and medium composition, which includes the formulations of macronutrients, micronutrients and vitamins as well as the effect of silver nitrate (SN) and activated charcoal (AC). As previous reports of oak propagation in agar demonstrated that the explant type has significant effects in propagation [16,18], this factor was included in all the experiments. A summary of the experimental layout is presented at the end of the Materials and Methods section.

2.2.1. Explant Type and Support

The initial explants used for TIS were obtained from explants growing on semisolid medium and consisted of 15–20 mm apical or basal sections, the latter still attached to the callus [16,38] (Figure 2a,b). Explants—twelve to sixteen per RITA®—were placed directly on the bioreactor net or maintained in a vertical position by placing them between 1 cm3 rockwool cubes (Grodan, Roermond, The Netherlands). Frequently, RITA® baskets were divided into two sections with aluminum foil to hold explants of different types (apical or basal sections) or with or without support (Figure 2c–e).

2.2.2. Frequency of Immersion

Apical and basal explants were immersed for one minute 3 or 6 times per day. These experiments were performed with genotypes RUS and CM.

2.2.3. Media Composition

Initially, explants for TIS were obtained from shoots growing on GD semisolid medium and were cultured in a TIS with the same formulation. Once the basic parameters for shoot multiplication were established, plant material of clones Ch1BS, Ch2BS, RUS and CM cultured in GD semisolid medium were used to investigate the effect of three types of media in bioreactors: GD, WPM and Murashige and Skoog (MS) medium [39] with half-strength nitrates (MS ½ N) and MS vitamins. The detailed composition of these media is provided in Supplementary Table S1. To identify a possible carryover influence of mineral salts from the previous medium (GD), part of the shoots growing in jars for these experiments were previously subcultured in the new media (WPM or MS ½ N) for at least one subculture before the experiment in the bioreactors. In some experiments with genotypes CM and RUS, the medium was supplemented with 1 g/L of activated charcoal (AC) (Sigma Aldrich®, Merck KGaA, Darmstadt, Germany) that was added before autoclaving, with 30 µM of filtered AgNO3—added after autoclaving—or with the combination of both.

2.3. Rooting

To assess the quality of the shoots produced in a TIS, we conducted preliminary rooting trials with all genotypes in the study. Vigorous shoots of 18–30 mm height were inoculated in 300 mL jars with 50 mL of half-strength macronutrients MS (MS ½ Macro, Supplementary Table S1) supplemented with 24.6 µM indole-3-butyric acid (IBA), 0.65% Plant Propagation Agar and 3% sucrose. After 5 d, shoots were transferred to an IBA-free medium supplemented with 1 g/L AC for rooting expression. After 6 weeks, the rooted shoots were transferred to plug trays filled with a peat/perlite (3:1). Plantlets were acclimatized for 4 weeks in a controlled environmental chamber (Fitotron SGC066, Sanyo Gallencamp PLC, Leicestershire, UK) with a photoperiod of 16 h light/8 h dark, a photosynthetic photon flux of 240–250 µmol m−2 s−1, a temperature of 25 °C (day) and 20 °C (night) and a relative humidity of 85%, being transferred to the greenhouse afterwards.

2.4. Data Recording and Statistical Analysis

For data recording, the parameters analyzed after the multiplication step were as follows: (a) the number of shoots longer than 8 mm per explant, differentiating between normal and abnormal hyperhydric shoots; (b) the length of the longest shoot per explant; (c) multiplication coefficient, defined as the number of apical or nodal sections useful for multiplication obtained per initial explant; (d) the number of rootable shoots obtained per initial explant, defined as non-hyperhydric shoots longer than 18 mm and with an active apex. For rooting experiments, the parameters recorded were (e) the percentage of rooted shoots, (f) number of roots, (g) length of the longest root and (h) shoot viability.
Data correspond to 32–48 explants per treatment in shoot multiplication experiments (at least two RITA® vessels per repetition and two repetitions per experiment) and to 16 shoots per genotype in preliminary and rooting experiments. The data were analyzed by Levene’s test (to verify the homogeneity of variance) and the Shapiro–Wilk test of normality. The data were then subjected to two-way analysis of variance (ANOVA), followed by comparison of group means (Tukey-b test). When an interaction between two factors was indicated by the two-way ANOVA, Bonferroni’s adjustment was applied to detect simple main effects. When the conditions of ANOVA could not be met, data were analyzed by the Kruskal–Wallis non-parametric test. Statistical analyses were performed using SPSS 29.0 (IBM Corp., Armonk, NY, USA).
Table 2 shows a summary of the multiplication experiments performed in this study.

3. Results

The five genotypes responded positively to the culture in RITA® bioreactors (Figure 3a). In the first experiments with a liquid medium, oaks frequently developed HH. Hyperhydric shoots were translucent, with distorted and abnormally wrinkled or curled leaves (Figure 3b), and were unsuitable for further multiplication. In this context, the main challenge of this study was to adjust the experimental conditions to control HH while maintaining suitable multiplication coefficients.

3.1. Effect of the Support

The results of the experiment 1 are shown in Table 3. Rockwool cubes supporting oak explants in bioreactors significantly affected the quality of the new shoots. Basal and apical sections were placed directly on the bioreactor basket or placed between the cubes so they could maintain the vertical orientation during the cultivation period. Without the cubes (Figure 2e), apical sections showed a high occurrence of HH, with percentages ranging from 80 to 87%. With the cubes, this problem was significantly reduced, although with a clear influence of the genotype.
Basal sections benefited from the use of a support as well, even if yield losses caused by HH when they were placed directly on the basket were not so marked. In the next experiments (except for number 6), apical sections were always cultured with cubes, whereas basal sections were either cultured alone or with cubes.

3.2. Effect of Immersion Frequency

The effect of immersion frequency applied to apical and basal sections was investigated in experiment 2 with genotypes RUS and CM; the results are shown in Table 4. For both clones, the explant type contributed to most of the differences in the parameters tested, with a superior multiplication coefficient of basal explants compared to apical sections. The frequency of immersion only significantly affected the multiplication coefficient and the number of rootable shoots in apical shoots of the CM genotype. For these explants, the multiplication coefficient with three immersions per day was less than 1, meaning that real multiplication was not achieved. In contrast, with six immersions, it was possible to obtain a slight but significant improvement, and this frequency was used in the rest of the experiments.

3.3. Effect of the Culture Media

One of the limitations we found when culturing oak explants in bioreactors was the low multiplication coefficients of apical explants (Table 4). Apical explants are necessary to produce explants with callus, which frequently show higher multiplication capacity (Table 4). In an attempt to increase the proliferation of this material, we studied the effect of culture media (experiments 3 to 5). Experiment 3 was carried out with clone SLo, with explants that had been obtained in GD in jars that were cultivated in bioreactors with GD and MS ½ N (Table 5).
We observed that MS ½ N produced significantly longer shoots than GD, and the MC was significantly higher for apical sections (2.2 compared to 1.3). However, in the second experiment—using the shoots produced in the first experiment—the results did not follow the same trend, and we observed a sharp decline in the cultures treated with MS ½ N, with MC values of 0.9 and 1.7 for apical and basal sections, respectively.
Suspecting the occurrence of a carryover effect of the previous medium, we further evaluated the effect of culture medium in bioreactors with clones Ch1BS, Ch2BS and CM (experiment 4) and RUS (experiment 5). In these experiments—as explained in Materials and Methods—the initial explants for TIS came either directly from GD media or had been pre-cultured in the new media for at least one subculture, with the objective of minimizing a possible carryover effect. The explants that came directly from GD medium were designated as “1st Subc” in the destination media, whereas the explants that had been pre-cultured in jars in the new media were designated as “2nd Subc”, meaning first subculture and second subculture, respectively. In the case of GD medium, both subcultures are repetitions of the same condition (from GD to GD) and have been designated as “GD Rep1” and “GD Rep2”.
As observed in Table 6 (exp. 4), apical sections of genotypes Ch1BS, Ch2BS and CM grew longer when cultivated in MS ½ N compared to GD, which increased the number of rootable shoots in these explants. However, basal sections showed a significant decrease in shoot length and in multiplication coefficient when they were cultivated more than once in MS ½ N. This trend was also observed with Carballo das Mentiras cultured in WPM. In the three genotypes, it was noticed that the stems of explants cultured in MS ½ N or WPM did not have as many axillary buds as with GD medium, and also, the size of the basal callus was reduced.
After analyzing the results of these experiments, it seems that for long-term cultivation of these genotypes, GD medium was a better option than MS ½ N or WPM in terms of multiplication coefficient and rootable shoots.
Experiment 5 was carried out with RUS cultured in GD and WPM. To prevent the carryover effect detected before with Ch1BS, Ch2BS and CM clones, the initial explants of RUS had been cultivated at least twice in the treatment media (Figure 4). ANOVA analysis showed an interaction between the explant type and the culture medium for the three variables SL, MC and RSN, with p values of 0.028, 0.0001 and 0.002, respectively, meaning that apical and basal explants will develop better in different media. Apical sections of RUS could grow in both media but produced a better response in WPM for the three variables analyzed. On the contrary, basal sections grew well in GD, whereas, when cultured in WPM, they experienced a significant reduction in growth and quality. In WPM, basal shoots showed an MC of 0.75, which cannot support multiplication (Figure 4b). A sharp decrease was also noted in the competence to develop rootable shoots (Figure 4c), which, with an average of 0.02 rootable shoots per explant, was almost negligible.

3.4. Effect of Silver Nitrate and Activated Charcoal

Experiment 6 aimed to test the effect of two compounds with a putative role in managing hyperhydricity: AgNO3 (silver nitrate, SN) and activated charcoal (AC). We applied these compounds either independently or in combination to three explant types of the RUS clone: apical sections without support, apical sections with rockwool cubes and basal sections without support. The results are shown in Figure 5 and Figure 6. Silver nitrate at 30 µM had a positive effect on the development of the apical sections of RUS and significantly increased their multiplication coefficient (Figure 5b) when cultured with rockwool cubes (Figure 6a), achieving values of 2.20 instead of 1.30 (about 60% higher than without this treatment). However, without cubes, it did not eliminate HH (Figure 6b). Treatments with AC produced large leaves (Figure 6c) and decreased the multiplication coefficient of basal shoots compared to the control and the treatment with SN alone (Figure 5b). Surprisingly, AC induced spontaneous root development in apical and basal sections in the multiplication medium (without auxin). Roots formed mainly in apical explants and started to be visible during the third week of culture (Figure 5d and Figure 6d). These rooted shoots had an actively growing apex and could be transferred to pots for acclimation. Subsequently, AC treatment was applied to Ch1BS, Ch2BS and CM, but in these genotypes, AC did not produce rooted shoots. We observed a decrease in the number of new shoots compared with the controls (Figure 6e–j) that was especially evident in basal explants of clone CM (Figure 6f).

3.5. Rooting and Acclimation

The last experiment in this study was a preliminary assessment of the rooting and acclimation capacity of oak shoots cultured in bioreactors. We transferred to pots plantlets of clone RUS that had rooted spontaneously during the multiplication step in bioreactors treated with AC (Figure 5d and Figure 6d). In addition, we selected vigorous shoots (Figure 7a) of the five genotypes, treated them with MS ½ Macro supplemented with 24.6 µM IBA for 5 days (Figure 7b) and transferred them to jars with an auxin-free medium with 1 g/L AC (Figure 7c). Rooted shoots were obtained in all the clones and are currently being acclimated (Figure 7d–f). These preliminary results reinforce the feasibility of using a temporary immersion system to propagate old oak genotypes.

4. Discussion

This study aimed to develop an efficient protocol that enabled the propagation of old oaks in bioreactors. We selected trees that differed significantly in age, geographical origin and morphogenic capacity, as our goal was to propose a protocol that could be applied to a wide range of genotypes.
The first challenge we had to confront was the prevalence of hyperhydricity. This disorder affects a wide range of plants during micropropagation in semisolid and liquid media [30,34], but at present, there is no simple and universal remedy for avoiding it [40]. In the present study, we recorded the number of hyperhydric shoots separately from the normal shoots, and abnormal propagules were not considered for calculating the shoot length, multiplication coefficient or number of rootable shoots. This approach takes more time and effort than weighing the fresh biomass, but in turn, it provides a clear and more accurate estimate of the real multiplication corresponding to each treatment and the quality of the plant material.
Hyperhydricity ranged between 5 and 90% depending on the genotype, the explant type, the medium and the use of a support. It could be reduced significantly by using rockwool cubes (for apical sections) or by selecting basal shoots as starting material. On the contrary, reducing the frequency of immersion from six to three times per day did not have a significant effect on decreasing hyperhydricity, while it markedly reduced the multiplication coefficient of apical sections in the CM genotype.
The beneficial effect of using rockwool cubes as a support to control hyperhydricity by avoiding the complete immersion of the explants in the liquid medium was previously reported for chestnut and alder (Alnus glutinosa (L.) Gaertn.) [30,41], whereas for other woody plants as willow (Salix viminalis L.), plum (Prunus domestica L.) and hazelnut (Corylus avellana L.), supports were not necessary to ensure a good yield [42,43,44,45].
Basal sections attached to the callus developed fewer symptoms of hyperhydricity and provided a consistent production of good-sized shoots useful for further multiplication or rooting. The superior performance of oak basal sections with callus had been highlighted in pioneer studies in semisolid medium [16,38,46]. The higher multiplication capacity compared with apical shoots can be related to the presence of a higher number of axillary buds than produced in upper sections of the shoot, which can develop quickly as soon as they are released from the apical dominance, but undoubtably, the extra vigor of these explants is also related to the presence of the callus itself, as the callus acts as a reservoir or source of nutrients and plant growth regulators. Plant hormones are not distributed homogeneously through the whole shoot, and the specific balance of auxins, cytokinins and other regulators, as well as their influence in the response to micropropagation treatments, is still not completely understood [47].
Mineral nutrition has a critical role in the success of a culture [48]. For Q. robur, the controversy about the best mineral medium has been ongoing for more than 30 years [13,14,15,16,17,18,19,46]. Recently, Q. dumosa Nutt, an oak native to North America, has been established in tissue culture in a new medium designed specifically to meet its nutritional needs [49,50]. In the present study, we compared three media that supported woody plant growth but differed in their mineral composition, particularly nitrogen, calcium, sulfur and magnesium. These three media have been used for Q. robur micropropagation [11,14,16,18,46]. We found that GD was the best option for the basal sections of the five genotypes tested, whereas for apical sections, WPM can be a good option for some genotypes, and it could be interesting to use MS ½ N for one or two subcultures when increased shoot length is required. Alternating media with different nitrogen concentrations was already suggested by Favre and Juncker in 1987 [13].
In our study, one of the outcomes we observed was a carryover effect from the previous medium. Carryover effects can last for weeks or months and should be studied carefully to avoid the incorrect attribution of effects to treatments. In our case, the first time that an explant was cultured in a new medium showed a response markedly different from the next repetitions in which the carryover effect had disappeared or decreased. These differences were more significant in basal shoots. When shoots were cultivated in GD, basal shoots showed the best performance. During the first subculture in a new medium, some characteristics were improved, but after several subcultures in MS ½ N, the decline was dramatic, rendering the subsequent cultures unsuitable for ongoing studies.
Our hypothesis is that this decline is directly related to the small size of basal callus (if any) that oaks form in this medium [51]. The normal callus would be functioning not as a passive reservoir but as an active metabolic transformer for the shoot, providing the required concentration of minerals and other nutrients or hormones for growth. The reduction in or practical absence of callus minimizes this beneficial effect, and the shoot will no longer be “protected” from an unsuitable medium. On the other hand, the fact that some features benefited from a short exposure to different formulae suggests there may be some real benefits from investigating the chemical requirements of oaks in more detail, as reported for Q. dumosa [49]. The outcome could be the design of a specific medium for this species that optimizes performance during in vitro culture.
We have found that rockwool cubes were easy to use, effective and a relatively cheap material that helped control HH, especially in apical sections. The main disadvantages of rockwool are that it is not easily disposable and that it increases both the time to place the apical sections in the bioreactors and the risk of contamination. For these reasons, it would be a positive step to find alternative and more sustainable approaches to reduce hyperhydricity. Silver nitrate has been proposed as a method for preventing this disorder in several plants cultivated in semisolid medium, like Dianthus chinensis L., Antirrhinum majus L. and Moringa oleifera Lam. [52,53,54,55,56]. In shoots of oak clone RUS, it showed a positive effect on the multiplication coefficient of apical sections, but only if cubes were present. Without cubes, HH was still observed. Other authors have reported better results with lower concentrations of silver nitrate [55] or a combination with other compounds such as cobalt salts [54] or salicylic acid [56]; these strategies—as well as the use of other supports—will be evaluated in future research.
Activated charcoal has been related to improvements in multiplication by the control of HH [57,58]. For RUS, it did not increase the multiplication coefficient of apical explants and even reduced the performance of basal explants. However, it had a positive influence on this genotype as it shortened the time required to obtain rooted shoots, with apical sections producing plantlets ready to be transferred to pots during the first four weeks after being inoculated in the bioreactors. AC had already been used for RUS to induce roots in semisolid medium as an alternative to auxin treatment, but in that study, roots appeared 5 weeks later than in the present study in bioreactors [12]. In this context, it seems the root-promoting effect of charcoal in bioreactors was genotype-specific, as the rest of the clones did not respond in the same way and developed roots only after auxin treatment. Genotypic effects mask factors that influence plant physiology and remain to be discovered for each clone/genotype. The goal of the present study was to develop a protocol for culturing mature oaks in a liquid medium, with the aim of using it for mass propagation and for basic research related to morphogenic processes of recalcitrant woody plants.

5. Conclusions

We developed a protocol suitable for the multiplication of five adult genotypes of Quercus robur. The proposed protocol consists of selecting 15–20 mm apical sections or basal sections with attached callus and inserting them in RITA bioreactors using rockwool cubes as a support (optional for basal sections), immersing them for 1 min 6 times per day in GD with 0.44–0.88 µM BA and transferring them to a fresh medium after 2 weeks. Shoots should be ready for subculturing 4–5 weeks after the initiation of the experiment. Preliminary rooting experiments indicate that the shoots produced in bioreactors have the required quality for being rooted and acclimated. Future research will focus on the characterization of the rooting process and the establishment of a procedure for the photoautotrophic propagation of this species.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/f16081285/s1, Table S1: Macronutrients, micronutrients and vitamins of the media used for the multiplication and rooting steps of Quercus robur.

Author Contributions

Conceptualization, P.C. (Paweł Chmielarz), C.S. and N.V.; Data Curation, J.M.V.; Funding Acquisition, P.C. (Paweł Chmielarz), C.S. and N.V.; Investigation, P.C. (Paweł Chmielarz), C.S., J.P.R.M., J.M.L.-L., P.C. (Purificación Covelo), M.J.C., A.A., S.R., J.M.V. and N.V.; Methodology, P.C. (Paweł Chmielarz), C.S. and N.V.; Project Administration, C.S. and N.V.; Resources, P.C. (Paweł Chmielarz), C.S. and N.V.; Supervision, P.C. (Paweł Chmielarz), C.S. and N.V.; Validation, P.C. (Paweł Chmielarz), C.S. and N.V.; Visualization, C.S., B.C. and N.V.; Writing—Original Draft, P.C. (Paweł Chmielarz) and N.V.; Writing—Review and Editing, B.C. and N.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partially funded by Xunta de Galicia (Spain) (project IN607A 2021/06) and the General Directorate of State Forests (Poland), grant number EO-2717-4/13, and the Institute of Dendrology Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland. Nieves Vidal acknowledges a grant for a Short-Term Scientific Mission from COST action CA21157 “COPYTREE, European Network for Innovative Woody Plant Cloning” supported by COST (European Cooperation in Science and Technology; E-COST-GRANT-CA21157-7f870649).

Data Availability Statement

The datasets presented in this article are not readily available because they are part of an ongoing study. Requests to access the datasets should be directed to nieves@mbg.csic.es.

Acknowledgments

The authors acknowledge David Barba and Julia Serrano for technical assistance and Antonio Vázquez Turnes, Henrique Neira, José García and Asociación Cultural San Campio for their collaboration regarding the collection of plant material of Carballo das Mentiras. This article is based upon work from COST Action CA21157 “European Network for Innovative Woody Plant Cloning”, supported by COST (European Cooperation in Science and Technology) www.cost.eu (accessed on 15 May 2025).

Conflicts of Interest

Author Bruce Christie is an employee of the company The Greenplant Company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Three of the five oaks used in this study. (a) Carballo das Mentiras, (b) San Lourenzo, (c) RUS.
Figure 1. Three of the five oaks used in this study. (a) Carballo das Mentiras, (b) San Lourenzo, (c) RUS.
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Figure 2. Initial explants of oak used in bioreactors. (a) Basal section of clone CM with attached callus. (b) Apical section. (c) Apical sections of Ch2BS placed between 1 cm3 rockwool cubes. (d) Apical sections of RUS placed between cubes (left) and basal sections with attached callus without support (right). (e) Apical sections of RUS without support (left) and placed between cubes (right). Bars = 1 cm.
Figure 2. Initial explants of oak used in bioreactors. (a) Basal section of clone CM with attached callus. (b) Apical section. (c) Apical sections of Ch2BS placed between 1 cm3 rockwool cubes. (d) Apical sections of RUS placed between cubes (left) and basal sections with attached callus without support (right). (e) Apical sections of RUS without support (left) and placed between cubes (right). Bars = 1 cm.
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Figure 3. Normal (a) and hyperhydric shoots (b) of Q. robur genotypes Ch1BS, CM and RUS cultured in RITA® bioreactors. Bars = 1 cm.
Figure 3. Normal (a) and hyperhydric shoots (b) of Q. robur genotypes Ch1BS, CM and RUS cultured in RITA® bioreactors. Bars = 1 cm.
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Figure 4. Effect of culture medium on multiplication characteristics of apical and basal sections of Quercus robur clone RUS. (a) Shoot length; (b) Multiplication coefficient; (c) Number of rootable shoots. Initial explants were cultivated in GD or WPM supplemented with 0.88 µM BA for at least 2 subcultures before the experiment. Apical sections with cubes and basal sections without support were immersed for 1 min 6 times per day. Data represent the mean ± standard error of 32 explants. For each variable, different uppercase letters indicate significant differences (p ≤ 0.05) in relation to the explant type, and different lowercase letters indicate significant differences in relation to the medium.
Figure 4. Effect of culture medium on multiplication characteristics of apical and basal sections of Quercus robur clone RUS. (a) Shoot length; (b) Multiplication coefficient; (c) Number of rootable shoots. Initial explants were cultivated in GD or WPM supplemented with 0.88 µM BA for at least 2 subcultures before the experiment. Apical sections with cubes and basal sections without support were immersed for 1 min 6 times per day. Data represent the mean ± standard error of 32 explants. For each variable, different uppercase letters indicate significant differences (p ≤ 0.05) in relation to the explant type, and different lowercase letters indicate significant differences in relation to the medium.
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Figure 5. Effect of silver nitrate (SN; 30 µM), activated charcoal (AC; 1 g/L), their combination (SN + AC) and their absence (Control) on the growth response of Quercus robur clone RUS. (a) Length of the longest shoot per explant (SL). (b) Multiplication coefficient (MC). (c) Number of rootable shoots per explant (NRS). (d) Number of shoots per explant spontaneously rooted during the multiplication step (SRS). Apical and basal sections were cultivated in GD with 0.88 µM BA and immersed for 1 min 6 times per day. Data represent the mean ± standard error of 16 explants. For SL, MC and SRS, different uppercase letters indicate significant differences (p ≤ 0.05) in relation to the treatment, and different lowercase letters indicate significant differences in relation to the explant type. For SRS, the two-way ANOVA was performed only with the two treatments that produced roots during the multiplica-tion step. NRS data could not be analyzed by ANOVA, and different lowercase letters indicate significant differences between treatments following the non-parametric Kruskal–Wallis test.
Figure 5. Effect of silver nitrate (SN; 30 µM), activated charcoal (AC; 1 g/L), their combination (SN + AC) and their absence (Control) on the growth response of Quercus robur clone RUS. (a) Length of the longest shoot per explant (SL). (b) Multiplication coefficient (MC). (c) Number of rootable shoots per explant (NRS). (d) Number of shoots per explant spontaneously rooted during the multiplication step (SRS). Apical and basal sections were cultivated in GD with 0.88 µM BA and immersed for 1 min 6 times per day. Data represent the mean ± standard error of 16 explants. For SL, MC and SRS, different uppercase letters indicate significant differences (p ≤ 0.05) in relation to the treatment, and different lowercase letters indicate significant differences in relation to the explant type. For SRS, the two-way ANOVA was performed only with the two treatments that produced roots during the multiplica-tion step. NRS data could not be analyzed by ANOVA, and different lowercase letters indicate significant differences between treatments following the non-parametric Kruskal–Wallis test.
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Figure 6. Effect of silver nitrate (SN) and activated charcoal (AC) on the growth of Quercus robur shoots of clones RUS, CM, Ch1BS and Ch2BS cultured in bioreactors with or without support. (a) Apical section of clone RUS cultured with SN and cubes showing normal aspect. (b) Apical section of clone RUS cultured with SN and without cubes showing hyperhydricity. (c) Apical sections of clone RUS cultured with AC and with cubes (left) and without cubes (right). (d) Apical shoot of RUS rooted spontaneously in a bioreactor with AC and cubes. (e) Apical and basal sections of clone CM without SN or AC. (f) Apical and basal sections of clone CM with AC. (g,h) Apical and basal sections of clone Ch1BS with (g) and without AC. (i,j) Apical and basal sections of clone Ch2BS with (i) and without AC (j). Bars = 1 cm.
Figure 6. Effect of silver nitrate (SN) and activated charcoal (AC) on the growth of Quercus robur shoots of clones RUS, CM, Ch1BS and Ch2BS cultured in bioreactors with or without support. (a) Apical section of clone RUS cultured with SN and cubes showing normal aspect. (b) Apical section of clone RUS cultured with SN and without cubes showing hyperhydricity. (c) Apical sections of clone RUS cultured with AC and with cubes (left) and without cubes (right). (d) Apical shoot of RUS rooted spontaneously in a bioreactor with AC and cubes. (e) Apical and basal sections of clone CM without SN or AC. (f) Apical and basal sections of clone CM with AC. (g,h) Apical and basal sections of clone Ch1BS with (g) and without AC. (i,j) Apical and basal sections of clone Ch2BS with (i) and without AC (j). Bars = 1 cm.
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Figure 7. Rooting of oak shoots cultured in bioreactors. (a) Shoots of SLo used for root induction. (b) Shoots of Ch1BS treated with IBA. (c) Roots formed in clone RUS 20 days after transfer to auxin-free medium with activated charcoal. (d–f) Plantlets of RUS (d), SLo (e) and CM (f) during acclimation in the greenhouse. Bars = 1 cm.
Figure 7. Rooting of oak shoots cultured in bioreactors. (a) Shoots of SLo used for root induction. (b) Shoots of Ch1BS treated with IBA. (c) Roots formed in clone RUS 20 days after transfer to auxin-free medium with activated charcoal. (d–f) Plantlets of RUS (d), SLo (e) and CM (f) during acclimation in the greenhouse. Bars = 1 cm.
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Table 1. Description of the plant material used in this study.
Table 1. Description of the plant material used in this study.
Tree CharacteristicsLines Established In Vitro
NameAge (Estimated)OriginAbbreviation
Chaián 160 yBasal shootsCh1BS
Chaián 260 yBasal shootsCh2BS
Carballo das Mentiras200 yEpicormic branchesCM
San Lourenzo300 yBasal shootsSLo
RUS800 yEpicormic branchesRUS
Table 2. Experimental layout of this study, indicating the factors investigated, the levels of each factor, the genotypes involved, as well as other factors studied in the same experiment.
Table 2. Experimental layout of this study, indicating the factors investigated, the levels of each factor, the genotypes involved, as well as other factors studied in the same experiment.
Experiments
FactorLevelsGenotypeExperiment No Other Factors in the Same Exp.
SupportRockwool cubes Ch1BS, Ch2BS, CM, RUSExp. 1Explant type
No supportRUSExp. 6Explant type; SN, AC
Immersions 3/6 per dayCM, RUSExp. 2Explant type
Explant type Apical sections
Basal sections with
callus		Ch1BS, Ch2BS, CM, RUSExp. 1Support
CM, RUSExp. 2Immersions
SLoExp. 3Culture medium
Ch1BS, Ch2BS, CMExp. 4Culture medium
RUSExp. 5Culture medium
RUSExp. 6Support; SN, AC
Culture mediumGD, MS ½ NSLoExp. 3Explant type
GD, MS ½ N, WPMCh1BS, Ch2BS, CMExp. 4Explant type
GD, WPMRUSExp. 5Explant type
Silver Nitrate (SN)Present, Absent, RUSExp. 6Explant type; Support
Act. Charcoal (AC)Combined
Table 3. Effect of the explant type (apical and basal sections) and the use of support (rockwool cubes) on the hyperhydricity of oak shoots of genotypes Ch1BS, Ch2BS, CM and RUS cultured in bioreactors and immersed for 1 min 6 times per day. Explants were cultured in GD medium with 0.44 or 0.88 µM BA (depending on the genotype) for four weeks and transferred to a fresh medium after the second week. Data correspond to the percentage of hyperhydric shoots per explant.
Table 3. Effect of the explant type (apical and basal sections) and the use of support (rockwool cubes) on the hyperhydricity of oak shoots of genotypes Ch1BS, Ch2BS, CM and RUS cultured in bioreactors and immersed for 1 min 6 times per day. Explants were cultured in GD medium with 0.44 or 0.88 µM BA (depending on the genotype) for four weeks and transferred to a fresh medium after the second week. Data correspond to the percentage of hyperhydric shoots per explant.
Genotype
Explant TypeSupportCh1BSCh2BSCMRUS
Apical sectionsNo support80 ± 13.3 a 1,285 ± 7.4 a83 ± 17.3 a87 ± 19.2 a
Cubes5 ± 3.5 c10 ± 7.3 c7 ± 2.8 c17 ± 9.6 c
Basal sectionsNo support26 ± 4.8 b27 ± 14.1 b21 ± 7.7 b42 ± 12.1 b
Cubes17 ± 5.4 b10 ± 1.9 c7 ± 6.0 b13 ± 7.4 c
1 Values are presented as mean ± standard error, n = 16. 2 Within each genotype, different letters indicate significant differences at p ≤ 0.05.
Table 4. Effect of the frequency of immersion on oak performance in bioreactors. Apical and basal sections of clones Carballo das Mentiras and RUS were placed between rockwool cubes in bioreactors and immersed for 1 min 3 or 6 times per day (3 imm; 6 imm) in GD medium with 0.88 µM BA.
Table 4. Effect of the frequency of immersion on oak performance in bioreactors. Apical and basal sections of clones Carballo das Mentiras and RUS were placed between rockwool cubes in bioreactors and immersed for 1 min 3 or 6 times per day (3 imm; 6 imm) in GD medium with 0.88 µM BA.
Apical Sections Basal Sections
GenotypeVariable3 imm6 imm3 imm6 imm
Carballo das Mentiras
Normal Shoot No.0.8 ± 0.25 Ba 1,21.2 ± 0.19 Ba1.8 ± 0.21 Aa1.6 ± 0.27 Aa
Hyperhydric Shoot No.0.38 ± 0.18 Aa0.13 ± 0.07 Ab0.03 ± 0.01 Ba0.20 ± 0.14 Ba
Shoot Length (mm)17.2 ± 3.79 Aa17.9 ± 1.59 Aa24.6 ± 1.74 Aa19.0 ± 2.21 Aa
Multiplication Coefficient0.9 ± 0.30 Bb1.6 ± 0.20 Ba2.5 ± 0.32 Aa2.1 ± 0.32 Aa
Rootable Shoot No.0.4 ± 0.18 Ab1.0 ± 0.12 Aa1.1 ± 0.11 Aa0.7 ± 0.45 Aa
RUS
Normal Shoot No.1.7 ± 0.26 Aa1.7 ± 0.25 Aa2.3 ± 0.36 Aa2.3 ± 0.35 Aa
Hyperhydric Shoot No.0.23 ± 0.11 Aa0.14 ± 0.07 Aa0.33 ± 0.13 Aa0.13 ± 0.09 Aa
Shoot Length (mm)16.7 ± 1.25 Aa19.4 ± 1.17 Aa14.7 ± 0.93 Ba15.5 ± 1.22 Ba
Multiplication Coefficient1.9 ± 0.28 Ba1.7 ± 0.24 Ba2.5 ± 0.35 Aa2.4 ± 0.36 Aa
Rootable Shoot No.0.6 ± 0.17 Aa0.6 ± 0.11 Aa0.7 ± 0.16 Aa0.8 ± 0.16 Aa
1 Columns and bars represent the mean ± standard error of 24 explants. 2 For each genotype, different uppercase letters indicate significant differences (p ≤ 0.05) in relation to the explant type and different lowercase letters indicate significant differences in relation to frequency of immersion.
Table 5. Effect of the culture media and explant type (apical sections with cubes, basal sections without support) on the multiplication of oak shoots of genotype San Lourenzo cultured in bioreactors and immersed for 1 min 6 times per day. Explants previously subcultured in GD were cultured in GD or MS medium with half-strength nitrates (MS ½ N) with 0.44 µM BA for four weeks and transferred to a fresh medium after the second week. Data correspond to the length of the longest shoot per explant (SL), multiplication coefficient (MC) and number of rootable shoots (NRS).
Table 5. Effect of the culture media and explant type (apical sections with cubes, basal sections without support) on the multiplication of oak shoots of genotype San Lourenzo cultured in bioreactors and immersed for 1 min 6 times per day. Explants previously subcultured in GD were cultured in GD or MS medium with half-strength nitrates (MS ½ N) with 0.44 µM BA for four weeks and transferred to a fresh medium after the second week. Data correspond to the length of the longest shoot per explant (SL), multiplication coefficient (MC) and number of rootable shoots (NRS).
Variable
Medium ExplantSL (mm) MCNRS
GDApical + Cubes31.3 ± 5.06 Bb 1,21.3 ± 0.34 Bb0.3 ± 0.12 Bb
Basal − Cubes41.6 ± 2.80 Ba7.3 ± 1.06 Aa2.3 ± 0.45 Aa
MS ½ NApical + Cubes37.7 ± 2.53 Ab2.2 ± 0.32 Ab0.9 ± 0.13 Bab
Basal − Cubes50.6 ± 3.20 Aa6.3 ± 0.53 Aa1.5 ± 0.19 Ba
1 Values are presented as mean ± standard error, n = 16. 2 Within each variable, different uppercase letters indicate significant differences (p ≤ 0.05) in relation to the medium, and different lowercase letters indicate significant differences in relation to the type of explant.
Table 6. Effect of the culture medium on multiplication characteristics of clones Ch1BS, Ch2BS and CM. For Ch1BS and Ch2BS, apical and basal sections were immersed for 1 min 6 times per day in GD or MS ½ N supplemented with 0.44 µM BA. For clone CM, GD, MS ½ N and WPM were supplemented with 0.88 µM BA. Rep1 and Rep2 indicate repetitions of the same condition (cultivation in GD), whereas for MS ½ N and WPM, “1st Subc” or “2nd Subc” indicates the first or second time the explants were exposed to these media. Data correspond to the length of the longest shoot per explant (SL), multiplication coefficient (MC) and number of rootable shoots (NRS).
Table 6. Effect of the culture medium on multiplication characteristics of clones Ch1BS, Ch2BS and CM. For Ch1BS and Ch2BS, apical and basal sections were immersed for 1 min 6 times per day in GD or MS ½ N supplemented with 0.44 µM BA. For clone CM, GD, MS ½ N and WPM were supplemented with 0.88 µM BA. Rep1 and Rep2 indicate repetitions of the same condition (cultivation in GD), whereas for MS ½ N and WPM, “1st Subc” or “2nd Subc” indicates the first or second time the explants were exposed to these media. Data correspond to the length of the longest shoot per explant (SL), multiplication coefficient (MC) and number of rootable shoots (NRS).
FactorsVariables
GenotypeMediumExplantSubcultureSL (mm)MC NRS
Ch1BSGDApicalRep117.3 ± 1.89 Ba 1,21.3 ± 0.19 Ba0.3 ± 0.12 Ba
Rep216.7 ± 0.96 Ba1.8 ± 0.14 Ba0.5 ± 0.10 Ba
BasalRep120.8 ± 2.75 Aa3.4 ± 0.60 Aa0.9 ± 0.30 Aa
Rep222.0 ± 1.25 Aa4.4 ± 0.47 Aa0.9 ± 0.16 Aa
MS ½ N Apical1st Subc25.6 ± 1.56 Aa2.1 ± 0.24 Aa0.9 ± 0.07 Aa
2nd Subc24.9 ± 2.35 Aa1.6 ± 0.20 Ab0.8 ± 0.11 Aa
Basal1st Subc26.3 ± 3.30 Aa2.4 ± 0.43 Aa0.8 ± 0.23 Ba
2nd Subc11.1 ± 1.74 Ab0.8 ± 0.21 Ab0.1 ± 0.09 Bb
Ch2BSGDApicalRep116.7 ± 1.93 Ba1.7 ± 0.25 Ba0.3 ± 0.12 Ba
Rep213.8 ± 1.14 Ba2.2 ± 0.30 Ba0.3 ± 0.10 Ba
BasalRep126.0 ± 2.13 Aa3.4 ± 0.42 Aa1.4 ± 0.46 Aa
Rep222.4 ± 1.22 Ab4.2 ± 0.46 Aa0.9 ± 0.15 Aa
MS ½ NApical1st Subc28.4 ± 1.79 Ba2.0 ± 0.19 Aa0.9 ± 0.13 Aa
2nd Subc25.9 ± 1.64 Ba1.4 ± 0.18 Ab0.7 ± 0.12 Aa
Basal1st Subc33.9 ± 2.34 Aa3.1 ± 0.43 Aa1.1 ± 0.20 Aa
2nd Subc9.8 ± 1.69 Ab1.1 ± 0.24 Ab0.3 ± 0.17 Ab
CMGDApicalRep125.7 ± 1.75 Aa2.0 ± 0.31 Aa0.7 ± 0.10 Aa
Rep217.1 ± 1.09 Ab1.5 ± 0.13 Aa0.8 ± 0.28 Aa
BasalRep125.3 ± 1.88 Aa2.3 ± 0.31 Aa0.6 ± 0.11 Aa
Rep221.2 ± 1.58 Ab1.9 ± 0.22 Aa0.6 ± 0.12 Aa
MS ½ NApical1st Subc30.3 ± 2.96 Aa2.1 ± 0.28 Aa0.7 ± 0.11 Aa
2nd Subc26.6 ± 2.24 Ab1.6 ± 0.22 Ab0.6 ± 0.09 Aa
Basal1st Subc24.8 ± 2.16 Ba1.7 ± 0.35 Ba0.5 ± 0.12 Ba
2nd Subc15.4 ± 3.53 Bb0.6 ± 0.14 Bb0.2 ± 0.09 Ba
WPMApical1st Subc25.0 ± 1.57 Aa1.9 ± 0.19 Aa0.7 ± 0.09 Aa
2nd Subc21.4 ± 1.16 Ab1.5 ± 0.12 Ab0.8 ± 0.09 Aa
Basal1st Subc24.7 ± 2.12 Aa2.7 ± 0.40 Aa0.8 ± 0.16 Aa
2nd Subc15.4 ± 2.64 Ab1.4 ± 0.33 Ab0.3 ± 0.10 Ab
1 Values are presented as mean ± standard error, n = 32. 2 For each medium and genotype, different uppercase letters indicate significant differences (p ≤ 0.05) in relation to the explant type, and different lowercase letters indicate significant differences in relation to the times the explants were exposed to the medium.
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Chmielarz, P.; Sánchez, C.; Martins, J.P.R.; Ley-López, J.M.; Covelo, P.; Cernadas, M.J.; Aldrey, A.; Rico, S.; Vielba, J.M.; Christie, B.; et al. Multiplication of Axillary Shoots of Adult Quercus robur L. Trees in RITA® Bioreactors. Forests 2025, 16, 1285. https://doi.org/10.3390/f16081285

AMA Style

Chmielarz P, Sánchez C, Martins JPR, Ley-López JM, Covelo P, Cernadas MJ, Aldrey A, Rico S, Vielba JM, Christie B, et al. Multiplication of Axillary Shoots of Adult Quercus robur L. Trees in RITA® Bioreactors. Forests. 2025; 16(8):1285. https://doi.org/10.3390/f16081285

Chicago/Turabian Style

Chmielarz, Paweł, Conchi Sánchez, João Paulo Rodrigues Martins, Juan Manuel Ley-López, Purificación Covelo, María José Cernadas, Anxela Aldrey, Saleta Rico, Jesús María Vielba, Bruce Christie, and et al. 2025. "Multiplication of Axillary Shoots of Adult Quercus robur L. Trees in RITA® Bioreactors" Forests 16, no. 8: 1285. https://doi.org/10.3390/f16081285

APA Style

Chmielarz, P., Sánchez, C., Martins, J. P. R., Ley-López, J. M., Covelo, P., Cernadas, M. J., Aldrey, A., Rico, S., Vielba, J. M., Christie, B., & Vidal, N. (2025). Multiplication of Axillary Shoots of Adult Quercus robur L. Trees in RITA® Bioreactors. Forests, 16(8), 1285. https://doi.org/10.3390/f16081285

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