Ethylene Inhibition Reduces De Novo Shoot Organogenesis and Subsequent Plant Development from Leaf Explants of Solanum betaceum Cav.

In de novo shoot organogenesis (DNSO) plant cells develop into new shoots, without the need of an existing meristem. Generally, this process is triggered by wounding and specific growth regulators, such as auxins and cytokinins. Despite the potential significance of the plant hormone ethylene in DNSO, its effect in regeneration processes of woody species has not been thoroughly investigated. To address this gap, Solanum betaceum Cav. was used as an experimental model to explore the role of this hormone on DNSO and potentially extend the findings to other woody species. In this work it was shown that ethylene positively regulates DNSO from tamarillo leaf explants. Ethylene precursors ACC and ethephon stimulated shoot regeneration by increasing the number of buds and shoots regenerated. In contrast, the inhibition of ethylene biosynthesis or perception by AVG and AgNO3 decreased shoot regeneration. Organogenic callus induced in the presence of ethylene precursors showed an upregulated expression of the auxin efflux carrier gene PIN1, suggesting that ethylene may enhance shoot regeneration by affecting auxin distribution prior to shoot development. Additionally, it was found that the de novo shoot meristems induced in explants in which ethylene biosynthesis and perception was suppressed were unable to further develop into elongated shoots. Overall, these results imply that altering ethylene levels and perception could enhance shoot regeneration efficiency in tamarillo. Moreover, we offer insights into the possible molecular mechanisms involved in ethylene-induced shoot regeneration.


Introduction
Ethylene is a gaseous plant hormone involved in several physiological processes, including plant growth and development, fruit ripening and seed germination [1]. Its effect on plant development encompasses inhibition on primary root growth [2] and lateral root formation [3] and a positive modulation of root hair formation and growth [4,5]. Ethylene is also involved in the inhibition of leaf growth due to its regulation of cell division and cell expansion (reviewed in [1]). Within the most varied roles of ethylene, its involvement in plant stress responses is notable. Ethylene is a key regulator of stress adaptation, mediating both abiotic [6] and biotic [7] stresses. This hormone acts as a signaling molecule, inducing a variety of physiological and biochemical changes and enabling plants to cope with environmental stress [8].
Plant regeneration systems, such as de novo shoot organogenesis (DNSO) or somatic embryogenesis, rely on plant cell plasticity, which is inherent to mechanisms of pluripotency/totipotency. It can be induced or enhanced by exogenous stress stimulus, such as wounding and hormonal treatments [9,10]. These plant regeneration processes are divided of different ethylene modulators and their effects evaluated on shoot regeneration. The impact of this modulation on subsequent plant development from the regenerated shoots was also assessed. Finally, the expression of genes related to ethylene biosynthesis, in particular ACS1 and ACO1, of the transcription factor ERF061 and of the auxin efflux carrier PIN1, were evaluated to unveil possible molecular mechanisms behind the ethylene modulation effect.

Effect of Ethylene Modulation on De Novo Shoot Organogenesis
To test the effect of ethylene on DNSO in tamarillo, leaf explants were cultured in the presence of 10 µM of each different ethylene modulator (Figure 1a). Silver nitrate (AgNO 3 ) was used to inhibit ethylene perception and aminoethoxyvinylglycine (AVG) to inhibit ethylene biosynthesis. To stimulate ethylene action on plant tissues, the ethylene precursors, 1-aminocyclopropane-1-carboxylic acid (ACC) and 2-chloroethylphosphonic acid (commercially known as ethephon; ETH) were applied. The protocol for shoot regeneration involves 3 weeks in the dark followed by 5 weeks in a 16 h photoperiod (Figure 1b). These different ethylene modulators were present in culture medium during all the induction process. At the end of 8 weeks, the effect of each modulator on shoot regeneration percentage, the number of buds and shoots developed per explant and the morphology of the regenerated shoots ( Figure 1c) were evaluated.
Callus formation was observed at the end of the third week at wounding sites ( Figure 1d). Leaf explants exposed to inhibitors of ethylene biosynthesis or perception presented a reduced capacity for callus induction (Figure 1d(ii,iii)). Further shoot development and elongation was also compromised in ethylene inhibition treatments (Figure 1c(xi)). Furthermore, some abnormal leaf shape in shoots regenerated in the presence of AgNO 3 (Figure 1c(iv)) were noticed. Few shoots regenerated in ACC treatments presented signs of hyperhydricity (Figure 1d(iv-left)), which was completely reversed when shoots were subcultured in hormone-free MS medium.
Regeneration percentage was not significantly affected by any treatment (Table 1). However, this parameter seemed to decrease across all conditions, with a marked reduction in AVG treatment (around 48%). The number of buds and shoots regenerated per responsive explant was the parameter significantly affected by ethylene modulation. We found a statistically significant increase in the number of buds and shoots developed when ethylene perception was enhanced by ACC and ETH treatments. The inhibition of ethylene perception or biosynthesis by AgNO 3 and AVG significantly reduced the number of the buds and shoots regenerated per explant. AgNO 3 and AVG treatments decreased almost two-fold the number of buds and shoots regenerated per explant (around six/explant) relative to control conditions (around 12/explant). When the enhancement of ethylene availability by ACC and ETH was compared with the inhibition of its perception and biosynthesis by AgNO 3 and AVG, it was found a three-fold increase in the number of buds and shoots regenerated per explant when ethylene perception is enhanced (19/explant for ACC and 18/explant for ETH) and vice-versa. Interestingly, opposite effects on ethylene modulation reduced or increased the number of buds or shoots per explant by around six explants compared to the control condition. Moreover, similar modulation treatments contributed to similar effects on regeneration.   For regeneration percentage data are represented as mean ± SEM of three biological replicates (n = 3, N = 24 explants per replicate). For number of buds and shoots data are represented as mean ± SEM of three replicates (n = 3, 15 < N < 20 explants analyzed per replicate for control, 13 < N < 18 for AgNO 3 , 11 < N < 12 for AVG, 13 < N < 18 for ACC, 12 < N < 17 for ETH). Letters indicate statistically significant differences between treatments (one-way ANOVA with Tukey multiple comparison test, p < 0.05).

Effect of Ethylene Modulation on the Expression of Ethylene-Related Genes and Auxin Efflux Carrier
The effect of ethylene modulation on the expression of specific genes was evaluated in two culture timepoints. Samples from the third week of culture, right before the transition to light and at the end of 8 weeks, defined as the final point of regeneration protocol (Figure 2a), were collected and analyzed.
Regarding ethylene biosynthesis, the expression of ACS1 and ACO1 (Figure 2b-c) was assessed. These genes encode for two specific isoforms of the main enzymes involved in ethylene biosynthesis, ACC synthase and ACC oxidase, respectively. In both timepoints, we found a statistically significant increase in ACS1 expression for the ETH condition (2.93 and 1.88 log2 fold). At the end of the eighth week, AVG promoted the opposite effect, with a statistically significant decrease of ACS1 expression (−0.85 log2 fold). AgNO 3 and ACC treatments also showed a tendency to upregulate ACS expression at both timepoints.
For ACO1 expression (Figure 2c), a statistically significant downregulation in AgNO 3 treatments for the first time point (−0.39 log2 fold) was found. At the end of 8 weeks, ACO1 expression tends to be upregulated in all conditions.
The expression of the gene ERF061, encoding for a transcription factor of the AP2/ERF superfamily was also evaluated. ERF061 was significantly upregulated in the presence of the ethylene precursor ETH, but also in the presence of the ethylene biosynthesis inhibitor AVG, post 3 weeks of culture ( Figure 2d). However, the upregulation of this gene was more notorious in ETH treatments (6.36 log2 fold) than in AVG treatments (1.95 log2 fold). ERF061 expression remained significantly upregulated in the presence of ETH post 8 weeks (3.72 log2 fold). Similarly, we observed a statistically significant upregulation of this gene in ACC presence (1.92 log2 fold) for the same culture timepoint. The upregulation observed previously in AVG treatment for the first timepoint did not remain at the end of the eighth week, in contrast to ETH treatment. Moreover, in this second timepoint, although not statistically significant, ERF061 expression seemed to be downregulated when ethylene perception and biosynthesis were inhibited.
in PIN1 expression was found in the presence of ethylene precursors ACC (1.24 log2 fold) and ETH (1.06 log2 fold). Furthermore, PIN1 expression was significantly downregulated in the presence of the ethylene biosynthesis inhibitor AVG (−1.72 log2 fold). PIN1 showed a tendency to be also downregulated when ethylene perception is inhibited by AgNO3. No significant statistical differences were found at the end of the eighth week. Nevertheless, PIN1 expression values showed a tendency to remain upregulated or downregulated in the ACC and AVG conditions, respectively.  . Asterisks indicate statistically significant differences between treatments and control (Student's t test, * p < 0.05, ** p < 0.01 and *** p < 0.001).
Finally, the effect of ethylene modulation in the expression of the auxin efflux carrier PIN1 (Figure 2e) was assessed. Before transition to light, a statistically significant increase in PIN1 expression was found in the presence of ethylene precursors ACC (1.24 log2 fold) and ETH (1.06 log2 fold). Furthermore, PIN1 expression was significantly downregulated in the presence of the ethylene biosynthesis inhibitor AVG (−1.72 log2 fold). PIN1 showed a tendency to be also downregulated when ethylene perception is inhibited by AgNO 3 . No significant statistical differences were found at the end of the eighth week. Nevertheless, PIN1 expression values showed a tendency to remain upregulated or downregulated in the ACC and AVG conditions, respectively.

In Vitro Rotting, Plant Development and Acclimatization
After 8 weeks, the 1 cm shoots regenerated in each condition were cultured in hormonefree MS medium for 1 month to induce rooting and shoot development (Figure 3a). Occasionally, shoots can be cultured in MS medium with low BAP concentrations to induce shoot development and elongation, before the rooting induction step. Nevertheless, this step was skipped to avoid additional hormonal stimuli that could mask ethylene modulation in the subsequent development of the regenerated shoots.  Letters indicate statistically significant differences between treatments (one-way ANOVA with Tukey multiple comparison test, p < 0.05).
After 1 month in hormone-free MS medium, all shoots regenerated in control conditions and in the presence of ethylene precursors were successfully rooted with welldeveloped and elongated roots (Figure 3b,c). Interestingly, shoots regenerated in treatments in which ethylene perception and biosynthesis were inhibited had their capacity to develop adventitious roots disrupted (Figure 3b,c). Likewise, shoot development and elongation was also negatively affected. No significant differences in morphological parameters such as plant height, root length and number of roots per shoots, between control and both ACC and ETH treatments were found (Figure 3c).
Acclimatization was successfully achieved for plants regenerated from control and both ACC and ETH treatments (Figure 3d), reaching 83%, 89%, and 100%, respectively. No differences were found in plant height between treatments after 1-and 3-months of ex vitro growth (Figure 3d-f). Nevertheless, the plants developed from shoots regenerated in the presence of ACC presented a significantly higher dry matter percentage when compared to the control plants (Figure 3d) after 3 months in ex vitro conditions.

Ethylene Positively Modulates Shoot Regeneration from Callus Induction to Shoot Development
Our results bring out a positive effect of ethylene on DNSO from tamarillo leaves, especially notorious in the number of buds and shoots regenerated per explant. While ethylene enhancement contributed to an effective increase in the number of regenerated shoots, the inhibition of its perception and biosynthesis had the opposite effect. A positive effect of ethylene was already described on DNSO from leaf explants of other Solanaceae, such as Solanum pennellii [25] and Petunia hybrida L. [40]. ACC and AgNO 3 treatments affected S. pennellii regeneration in a similar manner as reported in our study. Likewise, exogenous ethylene applications or AgNO 3 treatments increased or reduced the number of shoots per explant in P. hybrida, respectively. Ethylene is also essential to induce shoot regeneration from cotyledons explants in Arabidopsis [26].
Ethylene perception seems to be required to enhance pluripotent callus formation at the cutting sites of tamarillo leaf explants. In fact, less callus formation found in AgNO 3 and AVG treatments negatively impacted further regeneration. Knowing pluripotent callus is formed at wounding sites in leaf explants, which later lead to shoot development [41], a positive correlation between ethylene perception, pluripotent callus formation, and the explant ability to regenerate can be assumed.
Our protocol for DNSO from tamarillo leaves only englobes an exogenous source of cytokinin (BAP, 8.8 µM) and wounding as stress stimulus, although it is sufficient to promote callus formation and subsequent shoot regeneration. Callus development relies on cytokinin accumulation at cutting sites [13] and also in exogenous auxin supplementation [42]. Organ regeneration requires pluripotent acquisition in the middle of the cell layer of the callus promoted by auxin production and enhancement of cytokinin sensitivity [43]. It is known wounding and cytokinin enhance ethylene biosynthesis [14][15][16][44][45][46][47][48] and, in turn, ethylene also increases cytokinin levels [49]. Likewise, ethylene also enhances auxin biosynthesis and vice-versa [2,17,50,51]. In tomato leaves, wounding stimulates ethylene production [44] and the cytokinin BAP upregulates the ACO-like gene [52]. TDZ and BAP treatments also increase ethylene production in cotton leaves [45]. In our study, BAP treatment (in the control condition) is effective to achieve shoot regeneration from tamarillo leaf explants. Furthermore, other studies have already demonstrated TDZ and BAP as good plant growth regulators to promote DNSO from leaf explants in tamarillo [31][32][33][34]. Recently, Shin et al. [53] demonstrated that ethylene facilitates cell dedifferentiation and auxin-induced callus formation by regulating the abundance of transcripts for auxin receptor genes. This background supports our results and suggests that ethylene can positively affect regeneration in a crosstalk between cytokinin and auxins. Further research is needed to confirm this assumption, but we can hypothesize that, in control conditions, cytokinin and wounding stimulates ethylene production, which in turn enhance auxin biosynthesis contributing to pluripotent callus formation, without the requirement of exogenous auxin supplementation. In the presence of AgNO 3 and AVG treatments the effect of cytokinin and wounding on ethylene production is reduced and consequently auxin biosynthesis is downregulated leading to a decrease in callus formation and subsequent shoot regeneration. In addition, this regulation can be addictively regulated by the presence of the ethylene precursors justifying the enhancement of shoot regeneration.

Ethylene Modulation Differentially Regulates Gene Expression Related to Ethylene Biosynthesis and ERF061 Depending on Regeneration Stage
We explored the expression of two genes encoding for ethylene biosynthetic enzymes, ACC synthase and ACC oxidase (ACS1 and ACO1), and the transcription factor ERF061 that potentially affects regeneration and could be regulated by ethylene. We based our decision to analyze ERF061 expression on the knowledge that it has been considered a putative candidate gene related to shoot regeneration in Arabidopsis [54]. ERF061 belongs to the same AP2/ERF subfamily of WIND1, which promotes callus formation and shoot regeneration in Arabidopsis [55]. The acquisition of regeneration competency is heavily dependent on the role of WIND1, and its ectopic expression increases de novo shoot regeneration from Arabidopsis root explants, without the need for either wounding or auxin pre-treatment [56].
An upregulation of ACS1 expression by ETH treatment was found. In addition, ACC and AgNO 3 also showed a tendency to upregulate its expression. On the contrary, a downregulation of ACS1 was found in AVG treatments post 8 weeks. AVG is a strong inhibitor of ACC synthase activity [57,58] while AgNO 3 inhibits ethylene action at the receptor level [59]. Besides the negative effect on ethylene action by both modulators, these compounds seem to affect ACS1 gene expression differently. In agreement with our results, several ACS genes are downregulated by AVG in Cucurbita maxima [60] and Pyrus bretschneideri [61] while AgNO 3 seems to only downregulate some ACS genes [60]. Both ethylene precursors and exogenous ethylene treatments also seem to upregulate some ACS genes [60,[62][63][64], supporting our observations. In turn, the effect of ethylene modulation on ACO1 expression was less notorious compared to those of ACS1. Only AgNO 3 treatment significantly downregulated ACO1 expression post 3 weeks of culture.
ERF061 seems to be involved in biotic and abiotic stress responses [65,66]. Some studies also point that ERF061 is upregulated by exogenous ethylene and ethephon treatments [67][68][69]. Effectively, we found in both ACC and ETH treatments an upregulation of ERF061 expression at the eighth week. Contradictory results were observed at the third week, in which ERF061 expression was not only upregulated by ETH, but also by the ethylene biosynthesis inhibitor AVG, albeit to a lesser extent. Nevertheless, at the end of the eighth week, the AVG effect shows the opposite trend while its upregulation by ETH is preserved. Recently, ERF061 was postulated to be a possible transcription factor involved in regulating plant flower development and flower tissue formation in Actinidia eriantha [70]. Our results do not allow us to affirm ERF061 expression is induced by ethylene. However, ERF061 upregulation by ACC and ETH found at the end of 8 weeks, in which shoot-buds are being developed raises some questions about the possible involvement of ERF061 in shoot regeneration, posterior to the callus formation stage.

Ethylene Precursors Upregulate PIN1 Expression in Cytokinin-Induced Callus
PIN1 is an auxin efflux carrier required for an efficient shoot meristem induction in cytokinin-rich medium [71]. Interestingly, ACC upregulates PIN1 expression in Arabidopsis roots [2]. Thus, we considered that a possible modulation of its expression by ethylene could impact shoot regeneration. In fact, one of the outstanding observations in our results was the significant upregulation of PIN1 at the end of the third week in the presence of ethylene precursors ACC and ETH while its expression was significantly downregulated by AVG treatment. The presence of AgNO 3 tends to also downregulate PIN1 in the same culture timepoint , although not significantly. In shoot regeneration from Arabidopsis root explants, PIN1 is locally upregulated marking future sites of primordium initiation [71] and further development of shoot promeristem also requires its upregulation [9]. Moreover, PIN1 loss of function mutation reduces shoot regeneration [71]. Therefore, we can assume the downregulation of PIN1 in AVG and AgNO 3 treatments explain their negative impact on shoot regeneration and postulate the requirement of ethylene for DNSO in tamarillo leaf explants.

Inhibition of Ethylene Biosynthesis and Perception during Shoot Regeneration Negatively Impacts Subsequent Plant Development
Ethylene also seems to be essential to plant development in tamarillo as the shoots regenerated in treatments where ethylene perception or biosynthesis was inhibited were visibly less developed. Subsequent development, such as shoot elongation and adventitious root (AR) formation was also disrupted. In S. pennellii, shoots regenerated in AgNO 3 treatments were also less developed [25]. In agreement with our observations, in the woody species Populus tremula, AVG treatments also inhibited shoot elongation, induction and development of buds and root formation; in turn, ACC and ETH treatments stimulated these parameters [72]. The positive effect of ethylene in shoot proliferation of P. tremula raises the prospect of micropropagation protocols based on the action of ethylene produced by the plant itself instead of exogenous hormone treatment, such as the use of small-volume vessels with gas exchange restriction [73].
For biotechnological purposes subsequent AR formation is fundamental to a successful acclimatization. Studies have shown that ethylene can affect plant development differently, depending on the plant species, tissue type, and hormone supplementation [39,74]. In tomato and cucumber, ethylene also increases AR formation through an auxin-ethylene crosstalk [75][76][77], supporting our results. The inhibition of AR induction in shoots regenerated from AgNO 3 and AVG impacted further acclimatization and ex vitro adaptation. Unknown molecular patterns previously induced in regenerated shoots seems to impact further AR initiation which is not reverted in the hormone-free MS medium.

Plant Material
Leaves of red tamarillo shoots previously established from in vitro germinated seeds were used for shoot regeneration assays. Tamarillo shoots were in vitro propagated in MS medium [78]

Shoot Regeneration and Culture Conditions
Apical leaves of tamarillo shoots (3 weeks subcultures) cut in approximately 0.25 cm 2 square segments including the midrib were used for shoot regeneration. MS medium supplemented with sucrose (0.07 M, Duchefa), BAP (8.8 µM, Sigma), plant agar (0.7%, w/v, Duchefa) and pH adjusted to 5.7 before autoclaving at 121 • C for 20 min was used to induce regeneration. Leaf explants were cultured with the abaxial side down in dark conditions at 24 • C for 3 weeks. After 3 weeks, the cultures were transferred to a 16 h photoperiod at 25-35 µmol m −2 s −1 and 25 • C for 5 weeks.
To test the effect of ethylene modulation on shoot regeneration, 10 µM of AgNO 3 (Merck, Darmstadt, Germany), AVG (Sigma), ACC (Sigma) or ETH (Sigma) were added to the medium. All these modulators were sterilized by filtration with a 0.2 µm filter and added to the medium after autoclaving to avoid thermal degradation. At the end of 8 weeks, regeneration percentage ((number of responsive explants/total number of initial explants) × 100) and the number of shoots and buds developed in responsive explants were analyzed. Three biological replicates were made for the control and each treatment. Each replicate consisted of 8 glass jars fully closed with 3 explants (N = 24), in a total of 72 explants per condition.

Total RNA Isolation and Quantitative PCR Analysis
Samples from responsive explants with 3 and 8 weeks of culture were selected, frozen in liquid N 2 and stored at −80 • C until RNA extraction. Samples (80 mg) were carefully collected from the visible regeneration sites of the explant. RNA was extracted using the kit NucleoSpin ® RNA Plant (MACHEREY-NAGEL GmbH & Co. KG, Duren, Germany) following the manufacturer's instructions. The final concentration of RNA of each sample was measured using a spectrophotometer (NanoDrop One, Thermo Scientific, MA, USA) and its purity was confirmed with the A 260 /A 280 and A 260 /A 230 ratios. RNA integrity was further validated using the Qubit™ RNA IQ Assay Kit (Invitrogen™, Thermo Fisher Scientific, MA, USA).
First-strand cDNA synthesis was produced from 1 µg of total RNA from 3 biological replicates for each treatment and time-point using the NZY First-Strand cDNA Synthesis Flexible Pack (NZYTech, Lda.-Genes and Enzymes, Lisbon, Portugal) according to the manufacturer's instructions. Quantitative PCR gene expression analysis of two genes coding for ethylene biosynthetic enzymes, 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE1 (ACS1) and 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE-HOMOLOG 1 (ACO1), the transcription factor ETHYLENE-RESPONSIVE TRANSCRIP-TION FACTOR 61 (ERF061) and the auxin efflux carrier PIN-FORMED1 (PIN1), was made using NZYSpeedy qPCR Green Master Mix (2×) (NZYTech, Lda.-Genes and Enzymes, Lisbon, Portugal), following the instructions provided with 50-fold diluted cDNA template. Reactions were performed in a 96-well plate, with two technical replicates measured in CFX96 Real-Time System (Bio-Rad, CA, USA). Gene expression was normalized for both IRON SUPEROXIDE DISMUTASE, FeSOD and ACTIN, ACT reference genes [79]. All the primers (Table 2), except for PIN1 gene primers, were designed from Solanum betaceum transcript sequences obtained from embryogenic cell RNA-seq libraries (unpublished data), using the NCBI primer design tool. PIN1 gene primers were designed from the reference sequence of Solanum lycopersicum (NM_001247234.2) after the selection of conserved coding regions based on the alignment of Solanum sp. sequences (S. lycopersicum; S. pennellii, XM_015212230.2 and S. tuberosum, XM_006341465.2). The relative expression was calculated according to the Pfaffl method [80], using non-treated explants as a control for each timepoint.

Rooting and Acclimatization
Tamarillo shoots regenerated from leaf explants with at least 1 cm were in vitro rooted in hormone-free MS medium supplemented with sucrose (0.07 M, Duchefa), plant agar (0.7%, w/v, Duchefa) and pH adjusted to 5.7 before autoclaving at 121 • C for 20 min. After 1 month, rooting percentage ((number of shoots with roots/number of initial shoots) × 100) for each treatment was analyzed. Plants were acclimatized in a walk-in chamber (FitoClima 10000 HP, Aralab) under 16 h photoperiod at 40 µmol m −2 s −1 , 25 • C and 70% humidity. Briefly, plant roots were carefully washed to remove agar debris and placed on covered containers (70 cm 3 ) with Siro Royal substrate (SIRO, Mira, Portugal). The cover was removed after 2 weeks, and after 1 month, the plants were transferred to individual containers (500 cm 3 ) and the acclimatization percentage ((number of survival plants/number of initial plants) × 100) was analyzed. Plant height was evaluated after 1-and 3-months ex vitro. At the end of 3 months, dry matter percentage ((dry weight/fresh weight) × 100) was also assessed. For this purpose, 3-months acclimatized plants were carefully washed, weighted, and dried for 48 h at 70 • C.

Statistical Analysis
All data are presented as mean ± SEM and statistical analysis was performed using GraphPad Prism 9. The differences between treatments were analyzed using one-way ANOVA, followed by a Tukey's multiple comparison test. For gene expression analysis, differences of each treatment relative to the control were analyzed by the Student's t test.

Conclusions
In conclusion, our findings indicate that ethylene plays a crucial role in DNSO in ta-marillo. When ethylene action is inhibited, both the formation of organogenic callus and the regeneration of shoot-buds are reduced. These results suggest the possibility of a cyto-kinin-ethylene-auxin crosstalk that promotes callus formation and subsequent shoot regeneration. The upregulation of ERF061 suggests that ethylene can affect shoot regeneration through stress-response signaling. Additionally, the upregulation of PIN1 by ethylene supports previous reports and implies that ethylene may enhance shoot regeneration by affecting auxin distribution prior to shoot development. To enhance our comprehension of the molecular mechanisms driving the impact of ethylene on regeneration, it would be valuable to investigate the distribution and quantification of auxins, alongside other regeneration-related genes. Overall, this first approach of the ethylene effect on in vitro regeneration of tamarillo sheds light on the possible molecular mechanisms involved in the induced shoot regeneration of woody species.