Micropropagation ’ s Complete Protocol of Red Araçá ( Psidium cattleianum , Myrtaceae ) from Germinated Seeds in vitro

Red Araçá’s (Psidium cattleianum) micropropagation processes have shown enormous potential both in terms of research and as a sustainable native resource to be used in the areas of food production, ecology, and pharmacology. Currently, however, despite that potential, research efforts involving this myrtaceae, native to the Brazilian Atlantic Forest, have been scarce. With that in mind, this study set out to establish micropropagation techniques that would allow the development of a feasible protocol to be used with Red Araçá, achieving its mircropropagation from in vitro germinated seeds. Different types of explants were tested for in vitro establishment. For the multiplication of nodal segments, different concentrations of BAP and IAA combinations were tested in an MS medium. Using the same medium, different concentrations of ampicillin were applied in order to determine its influence on the decontamination of the apical segments. The BAP and IAA combinations were also used to test their effects on the in vitro explants’ development and rooting. During pre-acclimatization, survival of in vitro rooted plants was tested in a nebulizer chamber, using a commercial substrate and that same substrate mixed with washed sand (1:1). In essence, it was indeed possible to develop a complete protocol for the micropropagation of the Red Araçá from seedlings obtained by in vitro germination. The in vitro introduction of the Red Araçá was rather efficient, independently of the type of explants used. As the BAP and IAA concentrations increased, so did the in vitro seedlings’ development (7 leaves explant) and rooting (67%). Additionally, the in vitro rooted plants exhibited a high rate of survival (80%) in the pre-acclimatization phase, independently of the substrate used.


Introduction
Red Araçá (Psidium cattleianum Sabine) is a species native to the Mata Atlântica (Atlantic Forest), a Brazilian ecosystem hotspot (RBMA, 2017;Myers et al., 2000), and can be found throughout the Brazilian territory (Vibrans, 2013), as well as in other ecosystems in the world (Tng et al., 2015).bioactive compounds (Silva, Rodrigues, Mercadante, & De Rosso, 2014), essential oils and volatile substances that play an important role in pharmacology (Marin et al., 2008).Additionally, its capacity to attract pollinators (Gressler, Pizo, & Morelatto, 2006) and produce food for the native fauna (H.G. Baker, I. Baker, & Hodges, 1998;Kuhlmann, 1975) makes it an attractive species to be used as a secondary tree in reforestation efforts of degraded areas (Embrapa, 2006).
The array of potential exhibited by the Red Araçá has drawn the attention of the Brazilian Ministry of the Environment, which included it in their list of food producing native species with immediate or future potential uses in tropical and subtropical ecosystems (Kinupp, 2011).It has also been identified as an important species in the apiculture industry (Falkenberg & Simões, 2011).In spite of its importance and potential in the above mentioned fields, research efforts involving the biological diversity, conservation, and in vitro propagation of this species are rather scarce (Pasqual, Chagas, Soares, & Rodrigues, 2012).
Research efforts involving the different micropropagation phases of the P. cattleianum are still rare in many aspects.Some studies involving in vitro introduction and multiplication have already been developed, and yielded evidence of problems with phenolic oxidation and high rates of microbial contamination (Freire, Oliveira, & Vieira, 2014;Rodríguez, 2013).Furthermore, complete protocols for the in vitro propagation of other species of the Psidium genus, such as the Red Guava (P.guajava) (Liu & Yang, 2011), are already available and have been widely used in several fields in Brazil and around the world (IEA, 2017).All of this has made it quite clear that there is a need for further studies involving P. cattleianum micropropagation techniques, in order to better understand this genetic resource native to the Atlantic Forest.The purpose of this study, therefore, was to devise micropropagation techniques for the in vitro introduction, establishment, multiplication, rooting and pre-acclimatization of P. cattleianum, and develop a feasible protocol related to the specie.

Materials and Methods
This study used different experiments to test the establishment, multiplication, rooting and pre-acclimatization of Red Araçá from seedlings obtained by in vitro germination (see Figure 1).

General Conditions for the Experiments
The germination, establishment, multiplication and rooting experiments with Red Araçá were all performed at the Plant Tissue Cultures Laboratory belonging to the Experimental Station of the Agricultural Research and Rural Extension Company from Santa Catarina (Epagri), in the City of Caçador.There, the experiments were assembled in a plant growth chamber, set at a temperature of 25±2 ºC, where glass flasks (230 mL capacity) or test tubes (25 × 150 mm) were exposed to a 16-hour photoperiod, with a luminous intensity of 75 μmol m -2 s -1 , obtained using cool white fluorescent bulbs.
An MS medium was used (Murashige & Skoog, 1962), with all its mineral salts and vitamins, and supplemented by 30 g L -1 of sucrose and phytohormones, both dependent on each experiment.The pH factor was always adjusted to 5.8±0.05before adding the solidifying agent [6 g L -1 of agar (Merck®)].
Different assessments of the explants' characteristics were performed after each experiment.Bacterial and filamentous fungal contaminations were quantified according to Scherwinski-Pereira (2010).Partially or totally darkened explants were considered oxidized, whereas the explants that were greenish in color or totally green, and had developed at least one bud or leaf were considered to be established.Rooting was assessed by visually analyzing the growth of adventitious roots, according to Grattapaglia and Machado (1998).The number of completely formed and expanded leaves were counted.The growth of the in vitro explants was assessed using the relative growth rate (RGR) established by Briggs, Kid, and West (1920), according to Equation 1: where, Ln is the natural or Naperian logarithm for the length values obtained from the different assessments; and C2 and C1 represent the growth (in mm) recorded at the specific times t2 (final) and t1 (initial).prior to au The cultur were indiv The follow percentage day -1 ).

This exper Bearing Pl
In

In Vitro Establishment of Red Araçá Seedlings
Table 1 shows results for bacterial and fungal percentages, oxidation percentages and establishment percentages.
As can be seen, results didn't vary for the different types of explant (p > 0.05), but were efficient in establishing the species in vitro.Table 1 shows that only treatment D had bacterial contamination (8%) and not one of the types of explant tested had fungal contamination.All types of explant also exhibited low percentages of oxidation, and an average of about 95% establishment.
Differences between the types of explant were expected, especially considering the variations in size, and the presence or absence of paracotyledons and radicle roots.Such factors can influence, for example, the quantity of nutritional reserves present in the explants, which could affect in vitro establishment (Hartmann, Kester, & Davies Junior, 1990).Grattapaglia and Machado (1998) also determined that bigger explants with leaves have a better possibility of in vitro development.By the same token, Souza et al. (2007) used different sized explants (0.5 cm; 1.0 cm; 1.5 cm) during the in vitro introduction of the "pitangueira" plant (Eugenia uniflora, Myrtaceae), and determined that the bigger explants showed significantly higher results than their smaller counterparts, with approximately 83% having been established by the 45th day of cultivation.
Previous studies had already tested different methods of in vitro introduction for the P. cattleianum, but the results were less than satisfactory.Freire, Oliveira, and Vieira (2014) tested different asepsis methods (with ethanol 70% v/v and sodium hypochlorite 1.5%) on P. cattleianum explants obtained from stock plants that had gone through previous fungal treatment.These authors had problems with bacterial contamination (average of 50%, with 95% for the control group) and fungal contamination (average of 13.34%, with 55% for the control group).Similar data was observed by Souza, Schuch, and Silva (2006) who, when testing the introduction of P. cattleianum cv."Irapuã" using semi-lignified and herbaceous shoots, found that the average percentage of bacterial and fungal contaminations were 89.68% and 46.85%, respectively.
Furthermore, the in vitro establishment of native Myrtaceae trees, such as the Red Araçá, also poses problems due to oxidation processes that take place in explants obtained from stock plants (Pasqual, Chagas, Soares, & Rodrigues, 2012).The high concentration of phenolic compounds in these species and the use of different disinfectants during the decontamination process increase oxidation and decrease in vitro survival rates (Grattapaglia & Machado, 1998).In general, these negative factors impair or make the subsequent phases of micropropagation unfeasible for different myrtaceae, as has been determined for the "cerejeira-do-mato" (Eugenia involucrata or cherry of the Rio Grande) (Golle, Reiniger, Belle, & Curti, 2013) and for the "pitangueira" (Eugenia uniflora) (Lattuada, 2010), for example.
Contrary to those efforts, in this study, the use of explants derived from in vitro germinated seedlings has proven a rather feasible technique to study the micropropagation capabilities of the Red Araçá.As a matter of fact, the percentages related to bacterial contamination and oxidation were virtually null, and no explant fungal contamination was detected after the 30th day of in vitro introduction (Table 1).Such data is of great importance, especially considering the fact that the Red Araçá is a myrtaceae tree with a high incidence of contamination and oxidation during in vitro introduction (George, Hall, & De Klerk, 2008).
When studying the micropropagation of Myrtaceae species, other authors have also used explants obtained from in vitro germinated seedlings.Souza (2010), for example, yielded high in vitro establishment and multiplication rates with "guabijuzeiro" (Myrcianthes pungens) explants obtained from in vitro germinated seeds.Similarly, Nascimento (2006) germinated "uvaieira" (Eugenia pyriformis) seeds and reported rather high establishment, multiplication, and rooting percentages for that species' explants.

Growth Phytohormones during the Morphogenesis of Nodal Shoot Segments
Table 2 shows results for Psidium cattleianum explants after 15 days in vitro.Note.Averages appended by the same lower case letter within a column do not differ statistically from each other according to the Scott-Knott test, within a 5% significance level.ns Not significant.
The nodal segments used in this experiment were excised from in vitro introduced explants that remained over 90 days without visible contamination.Despite that, after the 12th day of the in vitro experiment, many of the segments (Table 2) showed bacterial contamination with the same appearance and coloration as was observed at the base of the explants, eventually developing towards the outside of the culture medium within the test tubes.Thus, a high bacterial contamination percentage (p < 0.0001) was observed (see Table 2), with the exception of the trial involving treatment T5 (1.5 mg L -1 of BAP + 0.5 mg L -1 of IAA), which yielded a value (28.57%) significantly lower than the rest.
Comparable to what was seen in this P. cattleianum study, other research efforts, such as the ones involving the in vitro establishment of "cerejeira-do-mato" (Eugenia involucrata) (Paim, 2011) and "camu-camuzeiro" (Myrciaria dubia) (Araújo, 2012), have detected the proliferation of bacterial contamination after a few weeks in vitro.According to these authors, such contaminations were very possibly due to the presence of endophytic microorganisms.
As stated by Camara, Willadino, and Albuquerque (2010), many in vitro plant cultures can synchronously develop bacterial growths during several of their micropropagation phases.This can occur even if the cultures were apparently pure after several sub-cultivations, in general due to the presence of endophytic or fastidious microorganisms.Therefore, the bacterial contaminations that occurred during this study are believed to have evolved from latent endophytic microorganisms or low concentrations of these microorganisms that are present in the P. cattleianum tissues that later proliferate in the culture medium.
On the 15th day after the in vitro introduction of the explants, the number of buds (p = 0.0724) and oxidation percentage (p = 0.3804) showed no significant effects for the different treatments, with values ranging from 0.14 and 0.57 for buds per explant, and from 17.14% and 34.29% for oxidation (Table 2).An assessment on the 45th day of contact with the culture medium, however, showed a significant effect on the bacterial contamination percentages (p < 0.0001), oxidation percentages (p = 0.0020) and in vitro establishment of the explants (p = 0.0012), as can be seen on Figure 4.For myrta Jaiswal, an L -1 , also in higher con Ribeiro, S higher num they also n Note.For each variable, recorded values appended by the same letter, lower case within a row and upper case within a column, do not differ statistically from each other according to the Tukey test, within a 5% significance level.ns Not significant.

Differe
The concentrations of ampicillin added to the medium for the purposes of this study efficiently suppressed contaminations (p = 0.0240) and were not phytotoxic to the explants.Throughout the experiment, no bacterial contamination was detected when the medium was treated with 500 mg L -1 of ampicillin, with its contamination percentage being statistically inferior (p = 0.0240) to that obtained when treated with 250 mg L -1 of ampicillin (Table 3).
After 45 days of being kept in a medium with 500 mg L -1 of ampicillin, the explants went through a development phase that made them grow in length (RGR = 0.011 mm mm day -1 ) and generated leaves (5.50 per explant), with 20% of them experiencing rooting, even though the medium was not specific for that process (Table 3).This was evidence that the cultivation of P. cattleianum explants, supposedly contaminated with bacteria, in a medium containing 500 mg L -1 of ampicillin resulted in a culture that was free of microorganism, also making its development feasible.
Pasqual, Dutra, Araújo, and Pereira (2010) reported that, when using culture tissue techniques, contaminated seedlings and explants are generally discarded; that, however, shouldn't always be the case, since there are microorganism controlling techniques that can be used.Pereira, Mattos, and Fortes (2003), for example, determined that ampicillin, chloramphenicol, streptomycin, and tetracycline (between 32 mg L -1 and 256 mg L -1 ) efficiently inhibited the growth of endophytic bacteria or contaminants in potato tissue cultures (Solanum tuberosum).Similarly, Leifert et al. (1991) cultivated the Delphinium sp. and also obtained cultures that were free of pathogens and showed in vitro growth increases when adding different antibiotics (gentamicin, streptomycin, carbenicillin, cephalothin, rifampicin and polymyxin) to their MS culture medium.
Besides having been quite effective in decontaminating the Red Araçá in this study, other studies and their results have also attested to the effectiveness of adding ampicillin in the culture medium.Araújo (2012), for example, tested different concentrations (100 mg L -1 , 200 mg L -1 , 300 mg L -1 , 400 mg L -1 , 500 mg L -1 and 600 mg L -1 ) of ampicillin in the culture medium for the in vitro establishment of the "camu-camuzeiro" (Myrciaria dubia, Myrtaceae), which, after 30 days, yielded an establishment rate of 50% and 0% bacterial and fungal contaminations.Additionally, Pereira and Fortes (2003) reported that culture mediums with concentrations of up to 1,024 mg L -1 of ampicillin are not phytotoxic to the in vitro cultivation of potatoes (Solanum tuberosum) because it decontaminates the explants and does not prevent their development.

Growth Phytohormones during the Morphogenesis of Apical Segments
Treatments of the full strength MS medium with different concentrations of BAP and IAA phytohormones did not significantly affect oxidation percentages (p = 0.4242), rooting percentages (p = 0.3910), number of leaves per explant (p = 0.2537) and relative growth rate per explant (RGR) (p = 0.4679) (Table 4).Significant differences were only detected between dates of assessment for the variables rooting percentages (p = 0.0004) and number of leaves per explant (p = 0.0002), both being higher on the 40th day of in vitro cultivation (Table 4). Pre-acclimatization of in vitro multiplied and rooted Red Araçá plants is efficient when using a commercial substrate, or a combination of commercial substrate and sand;  In essence, it was indeed possible to develop a complete protocol for the micropropagation of the Red Araçá (establishment, multiplication, rooting, and acclimatization) from seedlings obtained by in vitro germination, just as it could be done for other Myrtaceae.

Figure
Figure 4.Red Ara


Fig eedlings obtain de with bud gr IAA; E, rooted ed plants, afte ltures Laborato

Table 1 .
Bacterial and fungal contamination percentages, oxidation percentages, and establishment percentages for Red Araçá explants, 30 days after in vitro introduction of seedlings obtained by germination 1Radicle root between 4-6 mm; 2 Bacterial contamination; 3 Fungal contamination; 4 Explant oxidation; 5 In vitro explant establishment.ns Not significant, based on the analysis of variance's F test.

Table 2 .
Bacterial contamination percentages, oxidation percentages, and number of buds per explant for Psidium cattleianum nodal segments cultivated in different concentrations of BAP and IAA, after 15 days in vitro

Table 3 .
Bacterial and fungal contamination percentages, oxidation percentages, rooting percentages, number of leaves, and relative growth rate (RGR) for contaminated Psidium cattleianum explants after the 15th and 45th day of in vitro cultivation in an MS medium, with different concentrations of ampicillin

Table 4 .
Oxidation percentages, rooting percentages, number of leaves and relative growth rates (RGR) for Red Araçá (Psidium cattleianum) apical segments after the 18th and 40th day of in vitro cultivation in an MS medium containing different concentrations of phytohormones nsNote.For each variable, recorded values appended by the same letter, lower case within a row and upper case within a column, do not differ statistically from each other according to the Tukey test, within a 5% significance level.ns Not significant.jas.ccsenet.