In vitro multiplication of Eugenia calycina Cambess. (Myrtaceae) under the influence of BAP and NAA

Eugenia calycina Cambess. (Myrtaceae) is a fruit species native to Brazil indicated for the recovery of degraded areas. Generally its propagation occurs by seeds, but leads to an increase in the juvenile period of the plants, problems in maintaining seed viability and pathogen incidence. However, with the application of tissue culture techniques it is possible to produce seedlings in large scale, uniformity of the plants obtained and reduction in the period of seed germination. Thus, the objective of this research was to define the best medium for in vitro multiplication of E. calycina . Nodal segments of 1 cm, extracted from E. calycina plants already established in vitro , were used as explants. The treatments consisted of different concentrations of BAP (0; 0.5; 1 and 2 mg L -1 ) and NAA (0; 0.5; 1 and 2 mg L -1 ). The experimental design was completely randomized, in a 4x4 factorial scheme, with four concentrations of BAP and four concentrations of NAA, with four replications and three tubes per replication. After 90 days, shoot number, length and fresh mass of the aerial part, production and fresh mass of callus were evaluated. The MS medium plus 2.0 mg L -1 of BAP is ideal for in vitro multiplication of E. calycina .


Introduction
The Cerrado is one of the largest Brazilian biomes and with the presence of a large amount of native fruit trees.
However, some species have difficulty propagating via seeds, as they have heterogeneity in the maturation process of fruits and seeds with some type of dormancy.Factors that can harm the production of seedlings on a commercial scale (Pinhal et al., 2011).
Eugenia calycina Cambess., known as pitanga vermelha or pitanga do Cerrado, belongs to the Myrtaceae family and comprises approximately 100 genera and 3,500 species, which are found mainly in tropical and subtropical regions of the world.This species can occur in the shrub form, commonly found in the Cerrado, or in the arboreal or subarboreal form, which is typical of riparian forests.Its propagation can be done by seeds, grafting and cutting methods.The production of seedlings from seeds is the most used because the species has good germination condition.However, there are problems regarding seed storage of the species due to low longevity and high incidence of pathogens (Von Bulow et al., 1994).
Tissue culture is characterized by the in vitro cultivation of plants from cells, tissues, organs, embryos or fragments of living tissues in a nutrient medium under aseptic conditions and a controlled environment.In addition, this biotechnological tool is of great importance in plant genetic improvement, germplasm conservation, industrial propagation and plant conservation, as well as research in plant physiology and in vitro production of secondary compounds (Carvalho et al., 2003).
Thus, with the application of tissue culture techniques to fruit trees in the Cerrado, it becomes possible to solve or minimize some of the problems regarding the propagation of these species; through the systematic multiplication of plants, exchange of genetic material, reduction in the germination period, germplasm maintenance, standardization of the plants obtained, among other techniques (Melo et al., 2002).
Therefore, the objective of this research to define the best medium for in vitro multiplication of E. calycina through the use of BAP and NAA.

Methodology
The experiment was carried out at the Plant Tissue Culture Laboratory of the Department of Agriculture (DAG) of the Federal University of Lavras (UFLA), Lavras-MG, Brazil.
Nodal segments of 1 cm in length, extracted from Eugenia calycina plants already established in vitro, were used as explants.
The culture medium used in the experiment was MS (Murashige & Skoog, 1962) containing 30 g L -1 sucrose, 5.5 g L - 1 agar and pH 6.0.The treatments consisted of concentrations of BAP (0; 0.5; 1 and 2 mg L -1 ) and NAA (0; 0.5; 1 and 2 mg L - 1 ) in possible combinations.The culture medium was distributed in test tubes (25 mm x 150 mm) containing 15 mL of medium per tube, and autoclaved at 121ºC and 1.0 atm pressure for 20 min.After autoclaving, the tubes were taken to a laminar flow chamber, where the nodal segments were inoculated.The tubes were kept in a growth room at a temperature of 25 ± 1 °C, irradiance of 47.6 µmol m -2 s -1 and photoperiod of 16 h.Research, Society and Development, v. 12, n. 10, e40121043380, 2023 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v12i10.43380 3 The experimental design was completely randomized, in a 4x4 factorial scheme, with four concentrations of BAP and four concentrations of NAA, with four replications and three tubes per repetition.After 90 days, the number of shoots, shoot length (cm), shoot fresh mass (g), production and calli fresh mass (g) were evaluated.Data were subjected to analysis of variance and, when significant, regression analysis was performed at a 5% probability level, using the statistical program SISVAR (Ferreira, 2011).

Results and Discussion
Many researchers have already worked on the use of BAP aiming at the in vitro multiplication of various species (Arruda et. al., 2011;Asmar et al., 2019;Salem et al., 2022;Villa et al., 2005;Villa et al., 2006).From the present study, it was possible to establish the most suitable medium for in vitro multiplication of Eugenia calycina, and thus, in Figure 1 it is possible to observe the behavior of the species in different treatments containing the growth regulators BAP and NAA.A) 0.0 mg L -1 BAP + 0.0; 0.5; 1.0 and 2.0 mg L -1 NAA; B) 0.5 mg L -1 BAP + 0.0; 0.5; 1.0 and 2.0 mg L -1 NAA; C) 1.0 mg L -1 BAP + 0.0; 0.5; 1.0 and 2.0 mg L -1 NAA; and D) 2.0 mg L -1 BAP + 0.0; 0.5; 1.0 and 2.0 mg L -1 NAA.Fonte: Filipe Almendagna Rodrigues.
Significant interaction was observed in the association between BAP and NAA factors for shoot length, callus number and fresh mass of callus of E. calycina.However, a significant effect was observed for the number of shoots and fresh mass of shoots between the factors alone (BAP and NAA).
BAP provided a greater increase in the number of shoots as its concentration increased, whereas NAA contributed to a reduction in the number of shoots with the increase in its concentration in the culture medium (Figure 2).While the highest number of shoots (5.85) was observed at the concentration of 2.0 mg L -1 BAP in the absence of NAA (Figure 3).Nascimento et al. (2008) studied the effect of the addition of BAP in cauline segments of uvaieira (E.pyriformis Cambess) and did not observe significant differences for the number of shoots when the explants were submitted to different concentrations of BAP.Greater shoot lengths were obtained, respectively, in the combination of 2.0 mg L -1 BAP and 2.0 mg L -1 NAA (1.88 cm) or 2.0 mg L -1 BAP in the absence of NAA (2.17 cm) (Figure 4).Furthermore, higher concentrations of BAP provided greater lengths, regardless of the concentration of NAA used (0 or 2.0 mg L -1 NAA).A significant effect was observed between BAP concentrations for shoot fresh mass, with the highest increase (0.0543 g) occurring at the BAP concentration of 2.0 mg L -1 (Figure 5).However, when evaluating the effect of NAA, it was verified that the 2.0 mg L -1 concentration promoted a smaller increase in mass (0.0152 g) (Figure 6).In the control treatment, in which BAP was not used, the shoot fresh mass was 0.003 g, while in the 2.0 mg L -1 concentration it reached 0.0543 g (Figure 6).Greater callus production by explants occurred in the significant interaction between BAP and NAA factors (Figure 7).In the combinations of 1.0 mg L -1 BAP and 0.5 mg L -1 NAA and 1.0 mg L -1 BAP and 1.0 mg L -1 NAA, respectively, 2.5 and 3.0 calli per explant.Fonte: Filipe Almendagna Rodrigues.Bordignon et al. (2022) evaluated the presence and absence of callus in the explants, and were able to observe significant differences between treatments supplemented with BAP and NAA related to the production of callus by the explants.Thus, the researchers observed that the greatest callus production occurred in media containing 0.22; 0.33 and 0.44 mg L -1 BAP and 0.1 mg L -1 NAA.Significant interaction was observed between BAP and NAA factors for calli fresh mass (Figure 8).Greater increases were observed in the combinations of 0.5 mg L -1 BAP and 0.5 mg L -1 NAA (0.5632 g); 1.0 mg L -1 BAP and 1.0 mg L -1 NAA (1.0245 g) and 1.0 mg L -1 BAP and 2.0 mg L -1 NAA (1.3698 g).pyriformis Cambess.) in the different concentrations of BAP tested, however the highest average referring to the number of leaves was also found in the treatment supplemented with BAP, at a concentration of 1.0 mg L -1 .Furthermore, it is worth mentioning that the researchers in the present study have been carrying out research with the species E. calycina aiming to obtain plants using in vitro indirect organogenesis and somatic embryogenesis techniques (Figure 9).Through this technique, it will be possible to maintain the genetic characteristics of the species, as well as prevent its extinction process from occurring.

Conclusion
The MS medium added with 2.0 mg L -1 BAP is ideal for the in vitro multiplication of Eugenia calycina plants.

Figure 1 -
Figure 1 -In vitro multiplication of E. calycina under the influence of BAP and NAA.

Figure 2 -
Figure 2 -Number of shoots from explants of E. calycina under the influence of BAP.

Figure 3 -
Figure 3 -Number of shoots from explants of E. calycina under the influence of NAA.

Figure 4 -
Figure 4 -Shoot length of E. calycina explants under the influence of BAP and NAA.

Figure 5 -
Figure 5 -Shoot fresh mass of E. calycina explants under the influence of BAP.

Figure 6 -
Figure 6 -Shoot fresh mass of E. calycina explants under the influence of NAA.

Figure 7 -
Figure 7 -Calli per explant from E. calycina under the influence of BAP and NAA.

Figure 8 -
Figure 8 -Calli fresh mass from explants of E. calycina under the influence of BAP and NAA.