In vitro establishment of cambuci [Campomanesia phaea (O. Berg) Landrum], a native fruit tree from the Brazilian Atlantic ForestAbstract:
The cambuci [Campomanesia phaea (O. Berg) Landrum] is an endemic native fruit tree from the Brazilian Atlantic Forest. This study aimed to evaluate the in vitro response of nodal and leaf-derived explants collected from green house grown cambuci plants, including the explant in vitro establishment, and explant responses to culture media composition and to plant growth regulators. Nodal explants exhibited high contamination and oxidation rates, which can be reduced by collecting material during colder seasons. In vitro culture of nodalexplants with media supplemented with 2.0 mg L-1 of AgNO3 reduced the abscissionof new shoots, and the subculture of the material within 20-day intervals enhanced survival rates and decreased oxidation percentages. Cambuci leaf-derived explants treated with 0.75% sodium hypochlorite had low contamination and high oxidation rates, which, in turn, can be also reduced by supplementing of 1.0 g L-1 of polyvinylpyrrolidone(PVP). Leaf-derived explants exhibited no morphogenetic responses, while nodal explants had an increase in the number of new shoots with the supplementation of 11.1 µM BAP. The MS, 1/2 MS, 1/4 MS, WPM,1/2 WPM, JADS, and modified SPculture media were suitable for nodal explant cultivation. Subcultured cambuci shoots exhibited yellowing and necrosis, impacting survival rates.
Index terms
culture medium; growth regulators; micropropagation; Myrtaceae; woody plant
Resumo:
O cambuci [Campomanesia phaea (O. Berg) Landrum] é uma frutífera nativa e endêmica da Mata Atlântica. Este estudo teve como objetivo avaliar a resposta in vitro de explantes derivados de segmentos nodais e foliares coletados de plantas de cambuci, cultivadas em estufa, incluindo a avaliação do estabelecimento e das respostas dos explantes à composição do meio de cultura e aos reguladores de crescimento. Explantes derivados de segmentos nodais apresentaram altas taxas de contaminação e oxidação, que podem ser reduzidas pela coleta do material durante as estações mais frias. O cultivo in vitro de explantes nodais, em meio suplementado com 2,0 mg L-1 de AgNO3, reduziu a abscisão de novos brotos, e osubcultivo do material em intervalos de 20 dias aumentou as taxas de sobrevivência e diminuiu os percentuais de oxidação. Explantes foliares de cambuci tratados com 0,75% de hipoclorito de sódio, apresentaram baixas taxas de contaminação, mas altas taxas de oxidação, que, por sua vez, podem ser reduzidas com a suplementação de 1,0 g L-1 de polivinilpirrolidona (PVP). Explantes foliares não apresentaram respostas morfogenéticas, enquantoexplantes nodais tiveram aumento no número de novos brotos com a suplementação de 11,1 µM de BAP. Os meios de cultura MS; 1/2 MS; 1/4 MS; WPM; 1/2 foram adequados para o cultivo de explantes nodais. Brotos de cambuci sub cultivados apresentaram amarelecimento e necrose, impactando as taxas de sobrevivência.
Termos para indexação
meio de cultura; reguladores de crescimento; micropropagação; Myrtaceae; planta lenhosa
Introduction
The Brazilian Myrtaceae family is considered one of the most important botanical families of the Brazilian flora, having many different species belonging to the Brazilian Atlantic Forest, a biome considered a global terrestrial hotspot for biodiversity (MITTERMIER et al., 2011; WAGNER et al., 2020). In general, 140 genera with, approximately, 5,800 species, compose the Myrtaceae family (FRAUCHES et al., 2016).
In Brazil, around 1,205 plant species and 29 genera are cataloged, and 798 species are endemic to the country (FLORA; FUNGA DO BRASIL, 2024).
Some Myrtaceae Brazilian native fruit species are already commercially explored and well known by consumers, such as guava (Psidium guajava L.), pitanga (Eugenia uniflora L.), and jabuticaba (Myrciaria caulifora (Mart.) O. Berg) (NOGUEIRA et al., 2016). However, this family has many other fruit trees species with high potential for commercial use and exploration, such as cambuci [Campomanesia phaea (O. Berg) Landrum] (TEIXEIRA et al., 2019).
The genus Campomanesia has 35 cataloged species, which 25 are endemic to Brazil (OLIVEIRA et al., 2024).
Campomanesia phaea (O. Berg) Landrum, known as cambuci, is a Brazilian endemic semi-deciduous tree with a dense canopy of up to 9 m in height. Flowering occurs from August to November (in the Southern Hemisphere).
Cambuci fruits are shaped like flying saucers, smooth and greenish, even when ready to be eaten, ripening from January to February, and the pulp presents an acidic but pleasant flavor (LORENZI et al., 2015).
This fruit species occurs naturally in the Atlantic Forest, along the states of São Paulo, Rio de Janeiro, and Minas Gerais, and shows excellent potential for industrialization.
Local farmers commercialize frozen whole fruits or frozen pulp to restaurants in large cities. Products and preserves derived from the fruit, such as jellies, liqueurs, mousses, and ice creams can also be produced (TOKAIRIN et al., 2018).
The cambuci fruits have a great potential to be included in the human diet due to their nutraceutical and medicinal properties, once they contain high amounts of antioxidants, fiber, and pectin (TOKAIRIN et al., 2018; STAFUSSA et al., 2021).
Furthermore, studies have proven that cambuci-fruit-derived products can help to reduce oxidative stress and can be a potential inhibitor of enzymes related to the incidence of type 2 diabetes mellitus (T2DM) (DONADO-PESTANA et al., 2018; DONADO-PESTANA et al., 2021).
Despite its relevance, in the near past, cambuci was registered at risk of extinction and was considered a vulnerable species by the Red List of the International Union for Conservation of Nature (IUCN) of threatened species (IUCN, 2024).
Nonetheless, through historical and cultural rescue actions, this scenario has been reversed, and this species is now included in the category of “least concern” (LC) by the Official Red List of Brazil (CNCFLORA, 2024).
The main propagation method for cambuci is through seeds since vegetative multiplication methods are inefficient. Grafting, for instance, has reached only 6% success (SANTORO et al., 2021), which, in turn, may lead to serious difficulties in cloning an elite genotype.
In vitro techniques could be a successful tool to the large-scale propagation of this species. Demétrio et al. (2021) demonstrated that it was possible to obtain acclimated rooted plants of cambuci through micropropagation process.
However, in that study, in vitro germinated seedlings were used as explants source, leading to the regeneration of plants with great genetic variability, and, consequently, high variability of the agronomic characteristics.
The use of explants collected from greenhouse- grown cambuci plants has not been previously explored, making this study the first to evaluate their in vitro establishment and response to culture media components.
Therefore, the purpose of this research was to evaluate herbaceous nodal and leaf-derived explants of cambuci plants [Campomanesia phaea (O. Berg) Landrum] during their in vitro establishment, and their responses to disinfestation treatments and different culture media compositions, to develop an in vitro multiplication and regeneration protocol for this species.
Material and Methods
Explant collection and preparation
The explants were collected from twoyear- old cambuci seedling-derived plants obtained from a commercial nursery in Rio Claro, SP, Brazil.
Plants were transferred into 30-L plastic containers containing pine bark-based potting mix (Topstrato® HT hortaliças) and were kept in a greenhouse in the Department of Crop Science of the Luiz de Queiroz College of Agriculture, University of São Paulo, Piracicaba, São Paulo, Brazil.
For maintenance, cambuci plants were daily irrigated, weekly fertigated [calcium nitrate 0.8 g L-1 and Kristalon™ (6-12-36+1,8%Mg+8%S), 0.8 g L-1], and periodically pruned, according to explant collection schedules.
Plants used to explant collection were weekly sprayed with a Kasumin® (kasugamycin) solution (3.0 mL L-1), and biweekly, with a Nativo® (tebuconazole+ trifloxystrobin) solution (0.2 mL L-1) to reduce the initial contamination.
Herbaceous branches, with about six nodes each, were collected in the early morning (8:00 through 10:00 am), kept in humid plastic bags, and immediately taken to the Horticulture Biotechnology Lab of the Department of Crop Science. The branches were washed under tap water with neutral detergent, for 2 minutes, then rinsed with distilled water five times for 50 seconds each time.
For nodal explant experiments, the apical parts of the branches were cut off, and the leaves were detached.
The branches were reduced into about 15-cm pieces, and then immersed in 70% alcohol (v/v) for 1 minute, followed by three rinses with distilled water, for 50 seconds each time.
The segments were then immersed, for 15 minutes, in agitation, in the disinfestation solution (according to selected treatment), with the addition of 3 drops of Tween 20®. In a laminar flow chamber, the segments were rinsed five times with autoclaved distilled water, for 50 seconds each time. The extreme parts of the segments that were in contact with the solution were excised. Finally, the branch segments were cut into 1.5 cm-long explants, each one containing one node.
Experiments involving leaf-derived explants were set up with leaves from the middle portion of the herbaceous branches.
Leaves were immersed in 70% alcohol (v/v) for 1 minute and rinsed three times with distilled water, for 50 seconds each time, followed by the immersion in disinfestation solution (according to selected treatment), with the addition of 3 drops of Tween 20®, for 15 minutes, in agitation. The leaf discs were prepared using a 0.8 cm diameter cylindrical cutter.
Nodal and leaf-derived explant disinfestation
Nodal explants were used to evaluate two times of the year, Summer (December) and Fall (March), and different disinfestation solutions during the explant asepsis.
The following disinfestation solutions were used: 1.25% (v/v) calcium hypochlorite (Ca(ClO)2); 1.25% (v/v) sodium hypochlorite (NaClO); 2.5% (v/v) calcium hypochlorite (Ca(ClO)2) and 2.5% (v/v) sodium hypochlorite (NaClO). After disinfestation, the explants were cultivated in MS medium (MURASHIGE; SKOOG, 1962) supplemented with 30 g L-1 of sucrose, solidified with 2.0 g L-1 Phytagel™ (Sigma-Aldrich), and the pH adjusted to 5.8.
The material was kept in a growth room at 25°C, in a photoperiod of 16 h light (70 μmol m-2 s-1), provided by artificial light (20w white fluorescent lamps). The experiment followed a 4 x 2 factorial design, composed by four disinfestation solutions and two collection seasons, with five replications, and six test tubes (25 x 150 mm) per replication, containing one explant per test tube.
General contamination (GC %), percentage of fungal, bacterial, or combined (fungal + bacterial) presence; fungal contamination (FC %), percentage of fungal presence; bacterial contamination (BC %), percentage of bacterial presence; explant survival percentage (ES %), percentage of contamination- free explants exhibiting non-oxidized axillary buds; and oxidation rate (OR) were accessed after 40 days of culture.
The effect of disinfestation solutions in the explant asepsis was also evaluated for leaf-derived explants. The leaves were treated with the following disinfestation solutions: 0.75% (v/v) calcium hypochlorite (Ca(ClO)2); 0.75% (v/v) sodium hypochlorite (NaClO); 1.25% (v/v) calcium hypochlorite (Ca(ClO)2) and 1.25% (v/v) sodium hypochlorite (NaClO), and explant preparation was previously described.
The explants were then cultivated in MS medium, supplemented with 30 g L-1 of sucrose, solidified with 2.0 g L-1 Phytagel™ (Sigma-Aldrich), and the pH adjusted to 5.8.
The material was kept in a BOD incubator at 26°C±1 in the dark. The experiment comprised four treatments (disinfestation solutions), with five replications (100 x 15 mm Petri dishes) containing eight explants each.
As described in the previous experiment, general contamination (GC %), fungal contamination (FC %), bacterial contamination (BC %), explant survival percentage (ES %), and oxidation rate (OR) were accessed after 20 days of culture. The oxidation rate was evaluated based on an oxidation rate scale developed for this research (Figure 1).
Oxidation rate scale of cambuci [Campomanesia phaea (O. Berg) Landrum]. A Nodal explants oxidation rate scale. B Leaf-derived explants oxidation rate scale. 0 Explant without visible oxidation; 1 Explant presenting 25% oxidized tissue; 2 Explant presenting 50% oxidized tissue; 3 Explant presenting 75% oxidized tissue and 4 Explant presenting 100% oxidized tissue. Bars = 1 cm (A – B).
Explant survival rate and explant oxidation
The nodal and leaf-derived explants were used to evaluate different antioxidants and adsorbing agent added to the culture medium aiming to yield higher rates of explant survival and to reduce explant oxidation.
The explant collection and preparation were carried out as previously described, with the use of 1.25% and 0.75% NaClO disinfestation solutions for nodal and leaf-derived explants, respectively.
The MS culture medium, supplemented with 30 g L-1 of sucrose, solidified with 6 g L-1 agar, and pH adjusted to 5.8 was used in this set of experiments, with the supplementation of the adsorbing agent: 2.0 g L-1 of activated charcoal; and different antioxidants, as follows: 1.0 g L-1 of polyvinylpyrrolidone (PVP40) (Sigma-Aldrich); or 0.2 g L-1 of ascorbic acid, or no addition of antioxidant.
Nodal explants were maintained in a growth room at 25°C in a photoperiod of 16 h light (70 μmol m-2 s-1) provided by artificial light (20w white fluorescent lamps). Leaf-derived explants were maintained in a BOD incubator, at 26°C±1 in the dark.
Data on nodal explant survival rate was assessed by the calculation of the percentage of explants that survived in the culture medium after 40 days. Explants with new shoot abscission were included in data set as non-surviving explants. The oxidation rate was evaluated based on an oxidation rate scale developed for this research (Figure 1).
The experiment involving nodal explants was composed by four treatments (one adsorbing agent, two different antioxidants, and control), with five replications, and six test tubes (25 x 150 mm) per replication containing one explant per tube.
The experiment using leaf-derived explants was composed by four treatments (one adsorbing agent, two different antioxidants, and control), with five replications (100 x 15 mm Petri dishes) containing eight explants each. Explant survival percentage (ES %) and oxidation rate (OR) were accessed after 40 and 20 days of culture, for nodal and leaf-derived explants, respectively.
Due to the persistent oxidation and lower survival rates (including new shoot abscission of nodal explants) (Figure 2), two other experiments were carried out to mitigate such negative impacts, with the use of two alternative treatments.
New shoot abscission of cambuci [Campomanesia phaea (O. Berg) Landrum]. Arrows indicating new shoots abscission. Bars = 1 cm (A); 0.2 cm (B); 0.1 cm (C).
In the first experiment, nodal explants (collected and prepared as previously de scribed), were cultivated in MS medium with the supplementation of silver nitrate (AgNO3) at the concentrations of 0.0, 2.0, 4.0, and 6.0 mg L-1.
The experiment comprised four treatments (three different AgNO3 concentrations or no addition of AgNO3), with five replications, and six test tubes (25 x 150 mm) per replication containing one explant per test tube.
The second experiment consisted of nodal explant cultivation (collected and prepared as previously described) in MS medium, without supplementation of any antioxidant for 40 days, with two treatments: transferring the explants to a fresh medium after 20 days of cultivation, and no transferring.
This experiment comprised two treatments (transferring the explants to a fresh medium after 20 days of cultivation, and no transferring), 15 replications, with six test tubes (25 x 150 mm) per replication containing one explant per test tube. The material was kept in a growth room at 25°C in a photoperiod of 16 h light (70 μmol m-2 s-1) provided by artificial light (20w white fluorescent lamps). Explant survival percentage (ES %) and oxidation rate (OR) were accessed after 40 days of culture.
Effect of culture media composition on the response of nodal explants
Nodal explant response was also evaluated according to different media compositions.
Explant collection and preparation followed the same protocol as previously described.
Explants were then introduced in test tubes (25 x 150 mm) containing 25 mL of one of the following culture media: MS;1/2 of the salt of MS; 1/4 of the salt of MS; WPM (LLOYD; MCCOWN, 1980), 1/2 of the salt of WPM,1/4 of the salt of WPM; JADS (CORREIA et al., 1995); SP (CID et al., 1999) and modified SP.
The description of all the culture media compositions used in the experiment has been previously detailed in our previous research (OLIVEIRA JUNIOR et al., 2024).
The material was kept in a growth room at 25°C in a photoperiod of 16 h light (70 μmol m-2 s-1) provided by artificial light (20w white fluorescent lamps) and was subcultured once, to a fresh new media, 20 days after the beginning of the experiment.
The experiment comprised nine treatments, with six replications, and six test tubes (25 x 150 mm) per replication containing one explant per test tube.
Explant survival percentage (ES %), shoot length (SL), percentage of sprouted buds (SB %), number of shoots per explant (NSPE), number of leaves per shoot (NLPS), number of senescent leaves per shoot (NSS), and oxidation rate (OR) were accessed after 40 days of culture.
After data collection, shoots were excised and subcultivated to new fresh media containing the same previous treatments. Explant survival percentage (ES %) was accessed after 30 days of subculture.
Effect of plant growth regulators on axillary bud induction in nodal explants and morphogenesis in leaf-derived explants
Three experiments were carried out to evaluate the effect of BAP (6-benzylaminopurine) and NAA (naphthaleneacetic acid) supplementation on axillary bud induction in nodal explants or on morphogenesis of leaf-derived explants.
Explant collection and preparation followed the same protocol as previously described, including the use of 1.25% and 0.75% NaClO disinfestation solution for nodal and leaf-derived explants, respectively.
Nodal explants were cultivated in MS culture medium supplemented with 30g L-1 of sucrose, solidified with 6g L-1 agar and pH adjusted to 5.8 with the supplementation of different concentrations of 6-benzylaminopurine (BAP) (0.0; 4.4; 11.1 and 22.2 μM) in combination with naphthaleneacetic acid (NAA) (0.0 and 0.5 μM).
The material was kept in a growth room at 25°C in a photoperiod of 16 h light (70 μmol m-2 s-1) provided by artificial light (20w white fluorescent lamps).
The experiment followed a 4 x 2 (BAP concentrations x NAA concentrations) factorial design, with five replications, and six test tubes (25 x 150 mm) per replication containing one explant per test tube.
Explant survival percentage (ES %), shoot length (SL), percentage of sprouted buds (SB %), number of shoots per explant (NSPE), and oxidation rate (OR) were accessed after 40 days of culture.
Morphogenesis was assessed in leaf-derived explants of cambuci after two experiments were carried out.
In the first experiment, explants were cultivated in MS culture medium supplemented with 30g L-1 of sucrose and 1g L-1 of PVP, solidified with 6g L-1 agar, and pH adjusted to 5.8, with the supplementation of BAP (0.0, 2.2, 4.4, and 8.8 μM).
In this experiment, the explant response was also evaluated according to explant position. Therefore, the leaf discs were introduced in two different positions concerning the culture medium (adaxial and abaxial).
The material was kept in a BOD incubator at 26°C±1 in the dark, for 40 days (with one subculture to a fresh medium after 20 days of cultivation).
The experiment followed a 4 x 2 (BAP concentrations x explant positions) factorial design, with five replications (100 x 15 mm Petri dishes) containing eight explants each.
At the end of the experiment, responsive explant percentage (RE %), expressed as explant percentage with callus formation, was assessed.
In the second experiment, leaf discs were introduced with the abaxial part in contact with the MS culture medium supplemented with 30g L-1 of sucrose and 1g L-1 of PVP, solidified with 6g L-1 agar, and pH adjusted to 5.8, supplemented with BAP (0.0; 4.4; 8.8 μM) in combination with NAA (0.0; 0.05 and 0.5 μM).
The material was kept in a BOD incubator at 26°C±1 in the dark, for 40 days (with one subculture to a fresh medium after 20 days of cultivation). The experiment followed a 3 x 3 (BAP concentrations x NAA concentrations) factorial design, with five replications (100 x 15 mm Petri dishes) containing eight explants each.
At the end of the experiment, responsive explant percentage (RE %), expressed as explant percentage with callus formation, was assessed.
Statistical analysis Normality and homogeneity of variances were tested, and when these assumptions were satisfied, two-way or one-way ANOVA was performed, followed by Tukey’s test (p<0.05), using SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA).
Results and Discussion
Nodal and leaf-derived explant disinfestation
Nodal explants of cambuci showed low survival rates after the disinfestation procedures (Table 1) due to several causes, such as high general contamination percentage and high oxidation rate.
On the other hand, the time of the year interfered significantly on explant disinfestation, mitigating some of the initial in vitro establishment problems for this species.
In this way, nodal explants collected in the Fall season conferred lower values of general contamination percentage, fungal contamination percentage, bacterial contamination percentage, and oxidation rate (Table 2).
Leaf-derived explants did not exhibit bacterial contamination; however, fungal contamination was observed in the material treated with all disinfestation solutions, with lower values registered in explants exposed to sodium hypochlorite disinfestation solutions (Table 3). Finally, leaf-derived explants showed low survival rates, mainly due to high tissue oxidation (Table 3).
Explant and culture media contaminations represent one of the greatest difficulties in establishing an efficient protocol for in vitro plant multiplication, especially in woody species (DAGNE et al., 2023).
In vitro contaminants can originate from explant surface (MAHANANDA et al., 2023), material manipulation (SILVEIRA et al., 2022), or endophytic sources (MOSQUEIRA et al., 2023).
Moreover, contamination rates may vary according to the physiological and phytosanitary condition of the explant source and the environment in which it is cultivated, to explant type, and the time of the year of explant collection (ESPOSITO-POLESI, 2020).
In the present study, general contamination rates in nodal explants were very high, which may be expected in the case of explants derived from ex-vitro-grown plants.
However,the time of the year of explant collection led to significant differences in contamination rates in this type of explant, probably because temperature affects the fluctuation of endophytic and exophytic microorganisms, i.e., high temperatures may lead to increasing in microorganism titles, and vice versa (JU et al., 2006; CHENG et al., 2019).
Our results corroborate with other previous findings for yerba mate (Ilex paraguariensis St.Hil.) (ROSA et al., 2006) and guava (Psidium guajava L.) (SINGH; SINGH, 2018).
As previously mentioned, in vitro contamination rates may vary according to the explant type. Cambuci leaf-derived explants had low values of general contamination rate, and the use of sodium hypochlorite solution was considered adequate for disinfestation of this explant type.
Furthermore, higher values of phenolic oxidation represent another important challenge to the in vitro culture of woody species (XU et al., 2023).
As reported in the present study, nodal and leaf-derived explants exhibited high oxidation rates. Phenolic exudates, resulting from the explant excisions, oxidize through the action of polyphenol oxidase enzymes, leading to browning and tissue death (OLIVEIRA et al., 2013; ASSIS et al., 2018).
In addition, most species of the Myrtaceae family are known to have very high rates of phenolic compounds in their composition (HAMINIUK et al., 2014), including the species studied herein (SANTORO et al., 2022).
Nevertheless, in our study, cambuci nodal explants collected in the Fall revealed lower oxidation rates when compared to those collected during the Summer.
Similar studies involving other species indicated that guava explants collected in early Spring were the most suitable for tissue culture due to their lower oxidation rates (SINGH; SINGH, 2018).
In the same way, yerba mate (Ilex paraguariensis St. Hil.) explant oxidation rates were reduced when collection occurred during the colder seasons compared to the Summer, due to lower plant physiological activity and, consequently, lower production of phenolic compounds (ROSA et al. 2006).
Explant survival rate and explant oxidation
The response of nodal explants was not favored by the addition of different antioxidants and adsorbing agent (Table 4).
However, in the second experiment, involving only the use of different concentrations of silver nitrate (AgNO3), a significant increase in explant survival rate was recorded in explants treated with any concentration AgNO3 as compared with non-treated explants (Table 5).
Finally, the third experiment involving the frequency of medium renewal on explant response revealed that nodal explants had a higher survival rate and lower oxidation rate when they were transferred to a new fresh medium after 20 days (Table 6).
Despite no response of nodal explants to different antioxidants and adsorbing agent has been registered (Table 4), cambuci leaf-derived explants showed significantly higher values of explant survival rate when cultivated in a medium supplemented with PVP or activated charcoal (Table 7).
The oxidation rate was also significantly lower in medium supplemented with PVP (Table 7).
In our study, the abscission of new shoots in nodal explants was considered an additional challenge faced during the in vitro establishment, hindering the development of the following in vitro culture stages.
This new-shoot abscission response may be linked to the high ethylene concentrations produced by the explant in the test tube. Similar responses have been reported in kalanchoe (Bryophyllum calycinum Salisb) (MARASEK-CIOLAKOWSKA et al., 2020) and moringa (Moringa spp.) in vitro culture (RAVI et al., 2019).
Ethylene is a phytohormone involved in different plant responses, such as growth and senescence, according to its interaction with other hormones, to the hormone concentration in the tissue, and primarily, to the species under study (IQBAL et al., 2017).
On in vitro culture systems, high values of ethylene concentration may result in leaf abscission, leaf yellowing, and growth inhibition (CARDOSO, 2019).
The ethylene production under tissue culture conditions is significantly increased by the stress imposed on the explant during its introduction, such as explant cutting and surface disinfestation.
Furthermore, the use of small, sealed containers under in vitro conditions intensifies the adverse effects caused by the high concentration of ethylene (YASMIN et al., 2014).
Considering the limiting issues encountered during the explant establishment stage, experiments to reduce explant oxidation and new-shoot abscission were carried out to favor the explant survival percentage.
In tissue culture, products with oxidative inhibition effects have been used, such as antioxidants and PPO (polyphenoloxidase) inhibitors.
The most common used products with this effect include ascorbic acid, polyvinylpyrrolidone (PVP), and activated charcoal, acting on the inhibition of the synthesis and action of enzymes that cause oxidation or serving as adsorbents of these substances in the culture medium (GOULART et al., 2010; CHAI et al., 2018).
Our findings did not indicate any positive effect of antioxidants to reduce oxidation of cambuci nodal explants.
On the other hand,in vitro cultured leaf-derived explants with the supplementation of PVP (1 g L-1) led to significantly lower oxidation rates when compared to those cultivated with the supplementation of activated charcoal, ascorbic acid, and no addition of antioxidant, reaching 63% of survival rate.
Studies performed with casaqueira [Campomanesia rufa (O. Berg) Nied.] indicated that the supplementation of PVP was essential to reduce tissue oxidation, by approximately 30%, leading to an increase in the callogenesis of leaf-derived explants (SANT’ANA et al., 2018). Similar results were reported for Eremanthus incanus (Less.) Less,nodal explants (MIRANDA et al., 2018).
The supplementation of the culture media with silver ions, such as silver nitrate (AgNO3), has been described for a range of species, suggesting its potential positive effect in the in vitro culture process (BELLO-BELLO; SPINOSO-CASTILLO, 2023).
In this way, the supplementation of AgNO3 reduced the hyperhydricity, improving the in vitro culture in snapdragon (Antirrhinum majus L.) (LEE et al., 2023).
Furthermore, silver nitrate effectively reduced oxidation and controlled necrosis in Seabuckthorn (Hippophae salicifolia D. Don) (TRIVEDI et al., 2023).
Silver nitrate has also a potential inhibition of ethylene action by competing with its receptor binding sites on the membrane (KUMAR et al., 2009).
In our study, silver nitrate favored the final survival percentage in nodal explants. This positive effect was related to the reduction of new-shoot abscission, which may suggest that cambuci nodal explants are sensitive to ethylene action.
The simple process of material transferring to a fresh new culture medium results in benefits to the in vitro establishment, especially in those species with high phenolic oxidation rates and ethylene accumulation, such as the one studied herein. Frequent culture medium renewal allows the removal of toxic metabolites that affect the cultivation process (GEORGE et al., 2008).
In the present study, renewing the culture medium on the 20th day favored the survival percentage and resulted in lower oxidation of cambuci nodal explants.
Effect of culture media composition on the response of nodal explants
The nodal explants of cambuci exhibited different responses according to the cultured media (Table 8,Figure 3).
In vitro development of cambuci [Campomanesia phaea (O. Berg) Landrum] nodal explants on different culture media at 40 days after cultivation. A MS complete medium. B ½ MS medium. C ¼ MS medium. D WPM complete medium. E ½ WPM medium. F ¼ WPM medium. G JADS medium. H SP medium. I Modified SP medium. Bars = 1cm
Explant survival percentage (ES %); shoot length (SL); percentage of sprouted buds (SB %); number of shoots per explant (NSPE); number of leaves per shoot (NLPS); number of senescent leaves per shoot (NSS) and oxidation rate (OR) after 40 days of in vitro culture of cambuci nodal explants in relation to the culture media.
Survival rates were significantly higher in all culture media formulations, except on SP medium (Table 8).
Shoot length ranged from 0.33 to 0.61cm, with higher values recorded in material cultivated on the modified SP medium, and the lowest, on the 1/4 WPM. Significantly high values of sprouted buds were registered in most culture media evaluated, except on the 1/4 WPM and SP (Table 8).
Similarly, high numbers of shoots per explants were obtained in material cultivated in most culture media, except in that cultured in SP (Table 8).
No significant differences in number of leaves per shoot, number of senescent leaves per shoot and oxidation rate 40 days after in vitro culture of these explants on different culture media compositions (Table 8).
Explant survival percentage (ES%), accessed after 30 days of subculture indicated no significant differences among treatments (culture media compositions), with the general average of ES% equal to 17%.
The explant response to the culture medium composition is very important to optimize the in vitro culture protocols (PHILLIPS; GARDA, 2019).
On the other hand, despite the different nutrient requirements for each species, some established culture media are used (RAMAGE; WILLIAMS, 2002).
MS medium (MURASHIGE; SKOOG, 1962) and WPM (LLOYD; MCCOWN, 1980), this last one specifically designed for woody species (PHILLIPS; GARDA, 2019), are among the most used in vitro culture media.
Specific culture media compositions developed for the in vitro culture of Myrtaceae species have also been reported, such as the SP (CID et al., 1999) and the JADS culture media (CORREIA et al., 1995).
In our study, all the evaluated culture media compositions lead to adequate in vitro responses for cambuci nodal explants, except the SP and 1/4 WPM. SP medium is composed by 1/2 MS salt concentrations and 1/4 MS FeEDTA concentration.
The SP medium also has reduced carbohydrate concentrations compared to those of the other media compositions, containing 0.05 mg L-1 of myo-inositol and 10 g L-1 of sucrose (CID et al., 1999), which, in turn, are considered essential substances for the in vitro plant development (GAGO et al., 2023; O’BANION et al., 2023).
The carbohydrates are supplemented to the culture medium as a carbon source, because the in vitro culture conditions are limited for the photosynthetic process; therefore, the absence or deficiency of carbohydrates under these conditions does affect the photosynthetic potential (SUMARYONO et al., 2012).
In other reports, the sucrose supplementation is crucial for in vitro development of candeia [Eremanthus incanus (Less.)], and the concentration of 30 g L-1 is the most indicated in this case (MIRANDA et al., 2018).
Regarding the effect of culture media composition on explant response, it is worth to emphasize that the lower nitrogen concentrations in 1/4 WPM may also have contributed to the low performance of this culture medium composition in our study.
High nitrogen concentrations promote greater shoot development (POOTHONG et al., 2018). For the in vitro culture of camu-camu (Myrciaria dubia), a Brazilian native species from the Myrtaceae family, higher salt concentrations of the WPM led to increase in shoot length (ARAUJO et al., 2016).
Effect of plant growth regulators on axillary bud induction in nodal explants and morphogenesis in leaf-derived explants
No significant statistical interaction was revealed between BAP and NAA concentration regarding nodal explant response (Table 9).
Explant survival percentage (ES %); shoot length (SL); percentage of sprouted buds (SB %), number of shoots per explant (NSPE) and oxidation rate (OR) 40 days of in vitro culture of cambuci nodal explants in relation to different 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) concentrations added in the MS medium.
Moreover, the isolated effect of BAP was not significantly different for most of the accessed variables, except for the number of shoots per explant, where the supplementation of 11.1 μM BAP led to higher values than those cultured with no addition of this plant growth regulator (Table 10).
No significant morphogenesis responses of leaf-derived explants were registered with the supplementation of BAP and NAA.
The morphogenic responses in tissue culture depend on external and internal factors in which the donor plant and the explant are conditioned.
The environment, type of explant, culture medium, degree of juvenility of the donor plant, and the ratio of auxin/ cytokinin added to the culture medium are some examples of elements that, along with the tissue’s responsive capacity, influence the in vitro morphogenesis (HARTMANN et al., 2014).
Additionally, woody species commonly exhibit recalcitrance in responding to given in vitro conditions, a phenomenon observed in Brazilian native species as well (OLIVEIRA et al., 2013).
In our study, cambuci leaf-derived explants did not show any morphogenic response to BAP and ANA supplementations, as opposed with the results reported for casaqueira [Campomanesia rufa (O. Berg) Nied], where the leaf-derived explants were responsive to callus formation in culture medium supplemented with 2.2 mg L-1 of 2,4-D (SANT’ANA et al., 2018).
However, in that study, leaf discs from young seedlings germinated under in vitro conditions were used, differing from the experiments in our study, which used plants kept in greenhouses as explant donors.
On the other hand, nodal explants of cambuci were responsive to BAP supplementation, inducing higher numbers of shoots per explant with supplementation of 11.1 μM BAP.
The cytokinin 6-benzylaminopurine (BAP) acts on breaking apical dominance, inducing development of axillary buds, increasing the in vitro multiplication rates (BISERICARU et al., 2025).
Similar results were observed for Eugenia pyriformis Cambess, where the use of 4.4 μM BAP enabled to obtain 2.0 shoots per explant (NASCIMENTO et al., 2008).
Conclusion
This is the first report of in vitro culture of explants collected from greenhouse-grown cambuci plants. Morphogenic responses were more evident in nodal explants as compared with leaf-derived explants.
High contamination rates observed in nodal explants were reduced with the explant collection during colder season and transferring the explants to a fresh new culture medium diminished oxidation.
Cambuci explants exhibited recalcitrance to in vitro conditions despite differential responses in different culture media compositions and the supplementation of plant growth regulators.
Acknowledgements
The authors thank CNPq and CAPES for financial support. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Grant N°. 429044/2018-6, 134406/2018-4, 133734/2020-0 and 306677/2023-7), and Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
References
-
ARAUJO, M.C.R.; CHAGAS, E.A.; GARCIA, M.I.R.; PINTO, S.T.S.; CHAGAS, P.C.; VENDRAME, W.; MOTA FILHO, A.B.; SOUZA, O.M. Micropropagation of caçari under different nutritive culture media, antioxidants, and levels of agar and pH. African Journal of Biotechnology, Nairobo, v.15, p.1771-80, 2016. https://doi.org/10.5897/AJB2016.15417
» https://doi.org/10.5897/AJB2016.15417 -
ASSIS, F.A.; RODRIGUES, F.A.; PASQUAL, M.; ASSIS, G.A.; LUZ, J.M.Q.; JANONI, F.; COSTA, I.J.S.; COSTA, B.N.S.; SOARES, J.D.R. Antioxidants in the control of microorganism contamination and phenol oxidation in Eugenia pyriformis Bioscience Journal, Uberlândia, v.34, p.49-58, 2018. https://doi.org/10.14393/BJ-v34n1a2018-36311
» https://doi.org/10.14393/BJ-v34n1a2018-3631 -
BELLO-BELLO, J.J.; SPINOSO-CASTILLO, J.L. Utilización de nanopartículas de plata en la micropropagación de plantas. Mundo Nano, v.16, n.1, e-14e, 2023. https://doi.org/10.22201/ceiich.24485691e.2023.30.69692
» https://doi.org/10.22201/ceiich.24485691e.2023.30.69692 -
BISERICARU, D.M.; PÉREZ-JIMÉNEZ, M.; ROMERO-MUÑOZ, M.; CALDERA, F.; BORDIGNON, S.; TROTTA, F.; MATENCIO, A. Optimizing 6-benzylaminopurine for micropropagation: a cyclodextrin monomers and polymers approach. Carbohydrate Polymers, London, v.362, p.123658, 2025. https://doi.org/10.1016/j.carbpol.2025.123658
» https://doi.org/10.1016/j.carbpol.2025.123658 -
CARDOSO, J.C. Silver nitrate enhances in vitro development and quality of shoots of Anthurium andraeanum Scientia Horticulturae, New York, v.253, p.358-63, 2019. https://doi.org/10.1016/j.scienta.2019.04.054
» https://doi.org/10.1016/j.scienta.2019.04.054 -
CHAI, J.; GAO, Y.; DONG, Y.; KONG, L.; ZHANG, Y. Browning treatment in tissue culture of ‘Hongyang’ kiwifruit. IOP Conference Series: Materials Science and Engineering, Bristol, v.452, p.022075, 2018. https://doi.org/10.1088/1757-899X/452/2/022075
» https://doi.org/10.1088/1757-899X/452/2/022075 -
CHENG, Y.T.; ZHANG, L.; HE, S.Y. Plant-microbe interactions facing environmental challenge. Cell Host and Microbe, Cambridge, v.26, p.183-92, 2019. https://doi.org/10.1016/j.chom.2019.07.009
» https://doi.org/10.1016/j.chom.2019.07.009 -
CID, L.P.B.; MACHADO, A.C.M.G.; CARVALHEIRA, S.B.R.C.; BRASILEIRO, A.C.M. Plant regeneration from seedling explants of Eucalyptus grandis x E.urophyll Plant Cell, Tissue and Organ Culture, Dordrecht, v.56, p.17-23, 1999. https://doi.org/10.1023/A:1006283816625
» https://doi.org/10.1023/A:1006283816625 -
CNCFLORA - Centro Nacional de Conservação da Flora. Campomanesia phaea in lista vermelha da flora brasileira 2024. Disponível em: http://www.cncflora.jbrj.gov.br/portal/pt-br/profile/Campomanesia%20phaea Accesso em: 10 set. 2024.
» http://www.cncflora.jbrj.gov.br/portal/pt-br/profile/Campomanesia%20phaea - CORREIA, D.; GONÇALVES, A.N.; COUTO, H.T.Z.; RIBEIRO, M.C. Efeito do meio de cultura líquido e sólido no crescimento e desenvolvimento de gemas de Eucalyptus grandis x Eucalyptus urophylla na multiplicação in vitro IPEF, Piracicaba, v.48/49, p.107-16, 1995.
-
DAGNE, H.; PRABHU, V.; PALANIVEL, H.; YESHITILA, A.; BENOR, S.; ABERA, S.; ABDI, A. Advanced modeling and optimizing for surface sterilization process of grape vine (Vitis vinifera) root stock 3309C through response surface, artificial neural network, and genetic algorithm techniques. Heliyon, London, v.9, e18628, 2023.https://doi.org/10.1016/j.heliyon.2023.e18628
» https://doi.org/10.1016/j.heliyon.2023.e18628 -
DEMÉTRIO, C.A.; JACOB, J.F.O.; AMBROSANO, G.B.; OLIVEIRA, Ê.T.; ROSRIGUES, P.H.V. In vitro propagation of cambuci (Campomanesia phaea): An endangered exotic fruit and ornamental plant from Brazilian Atlantic Forest. Plant Cell, Tissue and Organ Culture, The Hague, v.145, p.203-8, 2021. https://doi.org/10.1007/s11240-020-02002-1
» https://doi.org/10.1007/s11240-020-02002-1 -
DONADO-PESTANA, C.M.; MOURA, M.H.C.; ARAUJO, R.L.; SANTIAGO, G.L.; BARROS, H.R.M.; GENOVESE, M.I. Polyphenols from Brazilian native Myrtaceae fruits and their potential health benefits against obesity and its associated complications. Current Opinion in Food Science, Amsterdam, v.19, p.42-49, 2018. https://doi.org/10.1016/j.cofs.2018.01.001
» https://doi.org/10.1016/j.cofs.2018.01.001 -
DONADO-PESTANA, C.M.; PESSOA, E.V.M.; RODRIGUES, L.; ROSSI, R.; MOURA, M.H.C.; SANTOS-DONADO, P.R.; CASTRO, E.; FESTUCCIA, W.T.; GENOVESE, M.I. Polyphenols of cambuci (Campomanesia phaea (O.Berg.)) fruit ameliorate insulin resistance and hepatic steatosis in obese mice. Food Chemistry, Amsterdam, v.340, p.128169, 2021. https://doi.org/10.1016/j.foodchem.2020.128169
» https://doi.org/10.1016/j.foodchem.2020.128169 -
ESPOSITO-POLESI, N.P. Contaminação versus manifestação endofítica: implicações no cultivo in vitro de plantas. Rodriguésia, Rio de Janeiro, v.71, e00562018, 2020. https:// doi.org/10.1590/2175-7860202071072
» https:// doi.org/10.1590/2175-7860202071072 -
FLORA e funga do Brasil. Myrtaceae in flora e funga do Brasil. Rio de Janeiro: Jardim Botânico, 2024. Disponível em: https://floradobrasil.jbrj.gov.br/FB171 Acesso em: 10 set. 2024.
» https://floradobrasil.jbrj.gov.br/FB171 -
FRAUCHES, N.S.; AMARAL, T.O.; LARGUEZA, C.B.D.; TEODORO, A.J. Brazilian Myrtaceae fruits: a review of anticancer properties. Journal of Pharmaceutical Research International, West Bengal, v.12, 2016. https://doi.org/10.9734/BJPR/2016/26782
» https://doi.org/10.9734/BJPR/2016/26782 -
GAGO, J.; MARTÍNEZ-NÚÑEZ, L.; LANDÍN, M.; FLEXAS, J.; GALLEGO, P.P.Modeling the effects of light and sucrose on in vitro propagated plants: A multiscale system analysis using artificial intelligence technology. PloS One, San Francisco, v.9, p.e85989, 2023. https://doi.org/10.1371/journal.pone.0085989
» https://doi.org/10.1371/journal.pone.0085989 - GEORGE, E.F.; HALL, M.A.; KLERK, G.J. Plant propagation by tissue culture3 ed. New York: Springer, 2008. 479p.
-
GOULART, P.B.; XAVIER, A.; DIAS, J.M.M. Efeito de antioxidantes no enraizamento de miniestacas de clones de Eucalyptus grandis X E.urophylla Revista Árvore, Viçosa, MG, v.36, p.961-72, 2010.https://doi.org/10.1590/S0100-67622010000600001
» https://doi.org/10.1590/S0100-67622010000600001 -
HAMINIUK, C.W.; PLATA-OVIEDO, M.S.V.; MATTOS, G.; CARPES, S.T.; BRANCO, I.G. Extraction and quantification of phenolic acids and flavonols from Eugenia pyriformis using different solvents. Journal of Food Science and Technology, Mysore, v.51, p.2862-6, 2014. https://doi.org/10.1007/s13197-012-0759-z
» https://doi.org/10.1007/s13197-012-0759-z - HARTMANN, H.T.; KESTER, D.E.; DAVIES JUNIOR, F.T.; GENEVE, R.L. Plant propagation: principles and practices 8.ed. Upper Saddle River: Prentice Hall, 2014. 928p.
-
IQBAL, N.; KHAN, N.A.; FERRANTE, A.; TRIVELLINI, A.; FRANCINI, A.; KHAN, M.I.R. Ethylene role in plant growth, development and senescence: interaction with other phytohormones. Frontiers in Plant Science, Lausanne, v.4, p.475, 2017. https://doi.org/10.3389/fpls.2017.00475
» https://doi.org/10.3389/fpls.2017.00475 -
IUCN – International Union for Conservation of Nature.The IUCN Red List of Threatened Species 2024. Disponível em: https://www.iucnredlist.org/species/35332/9927420 Acesso em: Sept.2024.
» https://www.iucnredlist.org/species/35332/9927420 -
JU, H.J.; HILL, N.S.; ABBOTT, T.; INGRAM, K.T. Temperature influences on endophyte growth in tall fescue. Crop Science, Madison, v.46, p.404-12, 2006. https://doi.org/10.2135/cropsci2005.0282
» https://doi.org/10.2135/cropsci2005.0282 -
KUMAR, V.; PARVATAM, G.; RAVISHANKAR, G.A. AgNO3 - a potential regulator of ethylene activity and plant growth modulator. Electronic Journal of Biotechnology, Valparaiso, v.12, p.1-15, 2009. https://doi.org/10.4067/S0717-34582009000200008
» https://doi.org/10.4067/S0717-34582009000200008 -
LEE, J.; NAING, A.H.; PARK, K.I.; KIM, C.K. Silver nitrate reduces hyperhydricity in shoots regenerated from the hypocotyl of snapdragon cv. Maryland apple blossom. Scientia Horticulturae, New York, v.308, p.111593, 2023. https://doi.org/10.1016/j.scienta.2022.111593
» https://doi.org/10.1016/j.scienta.2022.111593 - LLOYD, G.; MCCOWN, B.H. Commercially-feasible micropropagation of Mountain Laurel, Kalmia latifolia, by shoot tip culture. Combined Proceedings, International Plant Propagator’s Society, Seattle, v.30, p.421-7, 1980.
- LORENZI, H.; LACERDA, M.T.C.; BACHER, L.B. Frutas no Brasil nativas e exóticas (de consumo in natura) São Paulo: Instituto Plantarum de Estudo da Flora, 2015. 768p.
-
MAHANANDA, B.T.U.; BHARATHI, T.U.; USHARANI, T.R.; KUMAR, R.; KULKARNI, B.S.Standardization of sterilization protocol for explants and its suitability for direct organogenesis in tuberose cv.Arka Vaibhav. Journal of Horticultural Science, Alexandria, v.18, p.173-80, 2023. https://doi.org/10.24154/jhs.v18i1.2160
» https://doi.org/10.24154/jhs.v18i1.2160 -
MARASEK-CIOLAKOWSKA, A.M.; SANIEWSKI, M.; DZIURKA, M.; KOWALSKA, U.; GÓRAJ-KONIARSKA, J.; UEDA, J.; MIYAMOTO, K. Formation of the secondary abscission zone induced by the interaction of methyl jasmonate and auxin in Bryophyllum calycinum: relevance to auxin status and histology. International Journal of Molecular Science, v.21, n.2784, 2020. https://doi.org/10.3390/ijms21082784
» https://doi.org/10.3390/ijms21082784 -
MIRANDA, N.A.; TITON, M.; PEREIRA, I.M.; FERNANDES, J.S.C.; SANTOS, M.M.; OLIVEIRA, R.N. Antioxidants, sucrose, and agar in the in vitro multiplication of Eremanthus incanus Revista Floresta, Curitiba, v.48, p.311-20, 2018. https://doi.org/10.5380/rf.v48i3.51365
» https://doi.org/10.5380/rf.v48i3.51365 - MITTERMIER, R.A.; TURNER, W.R.; LARSEN, F.W.; BROOKS, T.M.; GASCON, C. Global biodiversity conservation: the critical role of hotspots. In: ZACHOS, F.E.; HABEL, J.C. Biodiversity hotspots: distribution and protection of conservation priority areas. Berlin: Springer-Verlag, 2011, p.3-22. https://doi.org/10.1007/978-3-642-20992-5_1
-
MOSQUEIRA, J.G.A.; PAIVA, R.; RIVAS, E.B.; HARAKAVA, R.; SOUZA-DIAS, M.A.G.; REIS, M.V. Isolation, molecular identification, and in vitro control of Campomanesia rufa (O.Berg) Nied.endogenous bacteria. Ciência e Agrotecnologia, Lavras, v.47, e004023, 2023. https://doi.org/10.1590/1413-7054202347004023
» https://doi.org/10.1590/1413-7054202347004023 -
MURASHIGE, T.; SKOOG, F. A review medium for rapid growth and bioassay with tobacco tissue culture. Physiologia Plantarum, Oxford, v.15, p.473-93, 1962. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
» https://doi.org/10.1111/j.1399-3054.1962.tb08052.x - NASCIMENTO, A.C.; PAIVA, R.; PAIVA, P.D.O.; VARGAS, D.P.; SOARES, F.P. Micropropagação de uvaieira (Eugenia pyriformis Cambess): efeitos do BAP e AIB. Revista Verde, Mossoró, v.3, n.2, p.20-6, 2008.
-
NOGUEIRA, A.M.; FERREIRA, A.; FERREIRA, M.F.S. Transferability of Microsatellites from Psidium guajava to Eugenia, Myrciaria, Campomanesia, and Syzygium Species (Myrtaceae). Plant Molecular Biology Reporter, Dordrecht, v.34, p.249-56, 2016. https://doi.org/10.1007/s11105-015-0921-7
» https://doi.org/10.1007/s11105-015-0921-7 -
O’BANION, B.S.; JONES, P.; DEMETROS, A.A.; KELLEY, B.R.; KNOOR, L.H.; WAGNER, A.S.; CHEN, J-G.; MUCHERO, W.; REYNOLDS, T.B.; JACOBSON, D.; LEBEIS, S.L. Plant myo-inositol transport influences bacterial colonization phenotypes. Current Biology, Cambridge, v.33, p.3111-24, 2023. https://doi.org/10.1016/j.cub.2023.06.057
» https://doi.org/10.1016/j.cub.2023.06.057 -
OLIVEIRA JUNIOR, M.A.; COLLI, B.A.B.; STIPP, L.C.L.; LATADO, R.R.; PIEDADE, S.M.D.S.; MOURÃO FILHO, F.A.A. In vitro culture of Rio Grande cherry (Eugenia involucrata DC.). Plant Cell, Tissue and Organ Culture, The Hague, v.157, n.21, 2024. https://doi.org/10.1007/s11240-024-02735-3
» https://doi.org/10.1007/s11240-024-02735-3 -
OLIVEIRA, L.S.; DIAS, P.C.; BRONDANI, G.E. Micropropagação de espécies florestais brasileiras. Pesquisa Florestal Brasileira, Colombo, v.33, p.445-60, 2013. https://doi.org/10.4336/2013.pfb.33.76.481
» https://doi.org/10.4336/2013.pfb.33.76.481 - OLIVEIRA, M.I.U.; COSTA, I.R.; PROENÇA, C.E.B. Campomanesia in Flora e Funga do Brasil. Rio de Janeiro: Jardim Botânico; 2024. Disponível em: https://floradobrasil.jbrj.gov.br/FB10307. Acesso em: 10 set. 2024.
-
PHILLIPS, G.; GARDA, M. Plant tissue culture media and practices: an overview. In Vitro Cellular and Developmental Biology - Plant, Columbia, v.55, p.242-57, 2019. https:// doi.org/10.1007/s11627-019-09983-5
» https:// doi.org/10.1007/s11627-019-09983-5 -
POOTHONG, S.; KHEN, T.; CHUMPHUKAM, O. In vitro mineral nutrition for improving growth and multiplication of stevia. Agriculture and Natural Resources, Amsterdam, v.52, p.477-83, 2018. https://doi.org/10.1016/j.anres.2018.11.007
» https://doi.org/10.1016/j.anres.2018.11.007 -
RAMAGE, C.M.; WILLIAMS, R.R.Mineral nutrition and plant morphogenesis. In Vitro Cellular and Developmental Biology – Plant, Columbia, v.38, p.116-24, 2002. https://doi.org/10.1079/IVP2001269
» https://doi.org/10.1079/IVP2001269 -
RAVI, R.S.D.; SIRIL, E.A.; NAIR, B.R. The effect of silver nitrate on micropropagation of Moringa oleifera Lam.an important vegetable crop of tropics with substantial nutritional value. Physiology and Molecular Biology of Plants, New Delhi, v.25, p.1311-22, 2019. https://doi.org/10.1007/s12298-019-00689-x
» https://doi.org/10.1007/s12298-019-00689-x - ROSA, F.C.; HANSEL, F.A.; DUTRA, L.F.QUADROS, K.M. Micropropagação de erva mate: efeito de diferentes épocas do ano no estabelecimento in vitro de segmentos nodais. Colombo: Embrapa Florestas, 2006. 4p.
-
SANT’ANA, C.R.O.; PAIVA, R.; REIS, M.V.; SILVA, D.P.C.; SILVA, L.C. In vitro propagation of Campomanesia rufa: An endangered fruit species.Ciência e Agrotecnologia, Lavras, v.42, p.372-80, 2018. https://doi.org/10.1590/1413-70542018424011018
» https://doi.org/10.1590/1413-70542018424011018 -
SANTORO, M.B.; BROGIO, B.A.; BUENO, S.C.S.; TANAKA, F.A.O.; JACOMINO, A.P.; SILVA, S.R. Vegetative propagation of Campomanesia phaea by the air-layering and grafting techniques. Pesquisa Agropecuária Brasileira, Brasília, DF, v.56, p.1-6, 2021. https://doi.org/10.1590/S1678-3921.pab2021.v56.02402
» https://doi.org/10.1590/S1678-3921.pab2021.v56.02402 -
SANTORO, M.B.; BROGIO, B.A.; TANAKA, F.A.O.; JACOMINO, A.P.; PEDROSO, R.M.; SILVA, S.R. Adventitious rooting and anatomical aspects of Campomanesia phaea stems. Acta Scientiarum Agronomy, Maringá, v.44, e53602, 2022. https://doi.org/10.4025/actasciagron.v44i1.53602
» https://doi.org/10.4025/actasciagron.v44i1.53602 -
SILVEIRA, E.O.; LOPES, R.; RODRIGUES, F.A.; RAIZER, M.D.M.; DIAS, F.J.; HARADA, P.K.; ARAÚJO, D.S.; PASQUAL, M.; LUZ, J.M.Q.; LOPES, M.T.G. In vitro establishment and multiplication of Echynochloa polystachya Bioscience Journal, Uberlândia, v.38, e38075, 2022. https://doi.org/10.14393/BJ-v38n0a2022-60508
» https://doi.org/10.14393/BJ-v38n0a2022-60508 -
SINGH, K.K.; SINGH, S.P. A review: micropropagation of guava (Psidium Spp.). Horticulture International Journal, Edmond, v.2, p.462-7, 2018. https://doi.org/10.15406/hij.2018.02.00097
» https://doi.org/10.15406/hij.2018.02.00097 -
STAFUSSA, A.P.; MACIEL, G.M.; BORTOLINI, D.G.; MAROLDI, W.V.; RIBEIRO, V.R.; FACHI, M.M.; PONTAROLO, R.; BACH, F.; PEDRO, A.C.; HAMINIUK, C.W.I. Bioactivity and bioaccessibility of phenolic compounds from Brazilian fruit purees. Future Foods, Amsterdam, v.4, 2021. https://doi.org/10.1016/j.fufo.2021.100066
» https://doi.org/10.1016/j.fufo.2021.100066 -
SUMARYONO, S.; MUSLIHATIN, W.; RATNADEWI, D. Effect of carbohydrate source on growth and performance of in vitro sago palm (Metroxylon sagu Rottb.) plantlets. HAYATI Journal of Biosciences, Bogor, v.19, p.88-92, 2012. https://doi.org/10.4308/hjb.19.2.88
» https://doi.org/10.4308/hjb.19.2.88 -
TEIXEIRA, N.; MELO, J.C.S.; BATISTA, L.F.; PAULA-SOUZA, J.; FRONZA, P.; BRANDÃO, M.G.L. Edible fruits from Brazilian biodiversity: A review on their sensorial characteristics versus bioactivity as tool to select research. Food Research International, Ottawa, v.119, p.325-48, 2019. https://doi.org/10.1016/j.foodres.2019.01.058
» https://doi.org/10.1016/j.foodres.2019.01.058 -
TOKAIRIN, T.O.; BREMER NETO, H.; JACOMINO, A.P.Cambuci – Campomanesia phaea (O.Berg.) Landrum .In: RODRIGUES, S.; SILVA, E.O.; BRITO, E.S. Exotic fruits reference guide. Cambridge: Academic Press, 2018, p.91-5. https://doi.org/10.1016/B978-0-12-803138-4.00013-7
» https://doi.org/10.1016/B978-0-12-803138-4.00013-7 -
TRIVEDI, V.L.; SEMWAL, P.; CHANDRA, S.; NAUTIYAL, M.C.; ATTRI, D.C.; SINGH, Y. Impact of silver nitrate on the survival and regeneration potential of explants of Hippophae salicifolia (Seabuckthorn) D. Don. Materialstoday: Proceedings, v.73, n.1, p.1-8, 2023. https://doi.org/10.1016/j.matpr.2022.08.111
» https://doi.org/10.1016/j.matpr.2022.08.111 -
WAGNER, F.H.; SANCHEZ, A.; AIDAR, M.P.M.; ROCHELLE, A.L.C.; TARABALKA, Y.; FONSECA, M.G.; PHILLIPS, O.L.; GLOOR, E.; ARAGÃO, L.E.O.C. Mapping Atlantic rainforest degradation and regeneration history with indicator species using convolutional network.PLoS One, San Francisco, v.15, p.1-24, 2020. https://doi.org/10.1371/journal.pone.0229448
» https://doi.org/10.1371/journal.pone.0229448 -
XU, X.; ZHU, D.; HUAN, Z.; GENG, X.; RAN, J.Mechanisms of tissue culture browning in five Magnoliaceae family species. Plant Cell, Tissue and Organ Culture, The Hague, v.155, p.183- 95, 2023. https://doi.org/10.1007/s11240-023-02568-6
» https://doi.org/10.1007/s11240-023-02568-6 -
YASMIN, S.; MENSUALI-SODI, A.; PERATA, P.; PUCCIARIELLO, C. Ethylene influences in vitro regeneration frequency in the FR13A rice harbouring the SUB1A gene.Plant Growth Regulation, Netherlands, v.72, p.97-103, 2014. https://doi.org/10.1007/s10725-013-9840-5
» https://doi.org/10.1007/s10725-013-9840-5
Edited by
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Scientific Editor
Alexandre Pio Viana
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Associate Editor
Eduardo Augusto Girardi
Data citations
IUCN – International Union for Conservation of Nature.The IUCN Red List of Threatened Species 2024. Disponível em: https://www.iucnredlist.org/species/35332/9927420 Acesso em: Sept.2024.
