In vitro Plantlets Regeneration from Nodal Segments of Murici ( Byrsonima gardneriana )

This study aimed to develop efficient protocols for the in vitro micropropagation of Byrsonima gardneriana. Nodal segments were obtained from seedlings germinated in vitro with 60 days of life. These were inoculated in MS/2 supplemented with 87.64 μM of sucrose and solidified with 0.7% of agar, supplemented with different concentrations of cytokinin 6-benzylaminopurine (0.0; 2.0; 4.0 and 8.0 μM) associated with different concentrations of auxin, indole acetic acid (0.0; 0.5 and 1.0 μM) and naphthaleneacetic acid (0.0; 0.5 and 1.0 μM). The sprouting were individualized and transferred to MS/2 cultures with different concentrations of indole butyric acid (0.0; 1.0; 2.0 and 3.0 μM), and presence and absence of activated charcoal (1.0 g L). The use of concentrations from 2.0 to 4.0 μM 6-benzylaminopurine was efficient in the multiplication of B. gardneriana, given that, using concentrations above these, a decrease in this efficiency occurs. The use of auxin interfered negatively with the results. In vitro rooting occurs even in medium free of auxin. The activated charcoal was insufficient for rooting. The use of growth regulators 6-benzylaminopurine and indole butyric acid are efficient in micropropagation of B. gardneriana, however, further studies should be performed to optimize this protocol.


Introduction
Byrsonima gardneriana A. Juss.(Malpighiaceae) is native and endemic of Brazil (Seixas, Silva, Morais, & Santos, 2011).It is known in some regions of Northeastern Brazil, mainly in the Caatinga biome, as murici and murici pitanga.The fresh fruits are highly appreciated, being consumed in natura, as well as juices, popsicles, liquors, jellies, sweets, preserves and in the form of flour.Local populations live off extractive activities and, this species is one of their sources of income when commercialized in free markets (Donadio, Môro, & Servidone, 2002).In addition to the fruit, the remaining parts of the plant are widely used for therapeutic means, having some of the properties proven in laboratory (Sannomiya et al., 2005).
Despite the significant potential of the species, there are no reports in literature regarding propagation studies.In this context, it is necessary to design strategies to ensure the sustainable multiplication, conservation and use of this species.Tissue culture techniques, especially micropropagation, represents an important alternative for the production of seedlings and the conservation of this genetic resource, allowing us to obtain plants with identical genetic characteristics in large scale and short period of time.In the in vitro propagation, an explant is isolated and cultivated under aseptic conditions, in an artificial medium generally supplemented with plant regulators.The basic principal of tissue culture is cell totipotentiality, i.e., any cell in the plant organism contains all genetic information necessary to regenerate a complete plant (Grattapaglia & Machado, 1998).
For obtaining efficient protocols for in vitro morphogenesis, it is necessary to adjust the existing protocols for each species, due to the variations in the responses obtained.In this dedifferentiation and re-differentiation induction process, responsible for the formation of tissues and organs, the use of plant regulators capable of stimulating the formation of the aerial part and of the roots are necessary.In this aspect, it is almost a rule that the auxin/cytokinin balance, favorable to the first, stimulate rooting; the counter balance favors the formation of the aerial part (Torres, Caldas, & Buso, 1998).
Thus, considering the importance of the species and the absence of propagation studies, the objective of the present study was to establish a micropropagation protocol for this species, by means of direct organogenesis.

Materials and Methods
Murici ripe fruits and inflorescences were sampled from natural population located at the environmental protection area Serra Branca/Raso da Catarina,Jeremoabo,Bahia,Brazil (10º00′49.1″S and 38º22′52.4″W).The botanic identification was performed at the herbarium of the University of State of Bahia, where the exsiccates are deposited under registry number 28450.
After the sampling, the fruits underwent processing, with the removal of the pulp and washing in current water during 10 minutes.After drying, the fruits were stored in paper bags, at 10 o C.
The murici fruits had their endocarps manually opened with the aid of a vise bench for the removal of the seeds.Subsequently, the seeds were disinfected in laminar flow chamber and immersed in alcohol 70% for 30 seconds, followed by immersion in sodium hypochloride at 2.5% added of a drop of neutral detergent for 10 minutes, and washed four times in autoclaved distilled water.Posteriorly, the culture medium used for the seed germination and all trials was MS/2 (Murashige & Skoog, 1962) supplemented with 87.64 mM of sucrose, and solidified with 0.7% of agar.The pH of the culture medium was adjusted to 5.8±0.1 (using NaOH or HCl 0.1 N) before autoclaving.The culture medium was distributed into test tubes (25 × 150 mm) sealed with plastic cover and sterilized by autoclaving for 15 minutes at a temperature of 120 o C and pressure of 1 atm.The inoculations were performed in laminar flow chamber for maintaining the aseptic conditions, and the recipients were closed with polyvinyl chloride (PVC) film.The cultures were maintained in growth room with temperature of 25±3 o C, photoperiod of 16 hours, relative humidity of 60% and active photosynthetic radiation of 40 µmol m -2 s -1 .
For in vitro multiplication, we used plantlets derived from in vitro germination of approximately 60 days.Explants (nodal segments) with length of 0.5 cm, derived from plantlets germinated in vitro, were inoculated in test tubes containing 10 mL of culture medium added with different concentrations of 6-benzylaminopurine (0.0, 2.0, 4.0 and 8.0 µM), associated to different concentrations of indole acetic acid (0.0, 0.5 and 1.0 µM) or different concentrations of naphthaleneacetic acid (0.0, 0.5 and 1.0 µM).
The experimental design was completely randomized in a 4 × 3 factorial scheme, in experiments, with five replicates, and each replicate comprised of four test tubes containing one explant.For all multiplication experiments, after 45 days, we evaluated: percentage of shoots, length of the shoot, number of shoots and leaves per shoot.
For the induction of the roots, shoots presenting the best results from the previous experiment were individualized and transferred to test tubes containing 10 mL of MS/2 culture medium, supplemented with different concentrations of indolbutiric acid (0.0, 1.0, 2.0 and 3.0 µM), in the presence of 1.0 g L -1 and in the absence of activated charcoal.The experimental design was completely randomized in a 4 × 2 factorial scheme (four concentrations of auxin indolbutiric acid × two concentrations of activated charcoal), totalizing eight treatments.Each treatment constituted of five replicates, each comprised of four tubes containing one shoot.After 45 days, we evaluated: survival percentage of the shoots; number of roots; length of the longest root; rooting percentage.
The data were statistically evaluated by means of analysis of variance, comparing the means using the Scott-Knott test (at 5% of significance), when qualitative, and compared by polynomial regression, when quantitative.The data were analyzed with the aid of the SISVAR 4.3 program (Ferreira, 2011).

Results and Discussion
To shooting induction in nodal segments using BAP and IAA, we verified, with the analysis of variance, significant effect (p ≤ 0.05) just for concentration of plant regulator IAA (Figure 1), and the average percentage of shoot induction was 60%.Erig, Schuch, and Braga (2004) with sieve in vitro rooting (Pyrus communis L.), and by Schwalbert, Maldaner, Amaral, Aita, and Tarouco (2015) with Desmodium incanum DC., in which the presence of this component did not alter the rooting rate of this species.This fact can possibly be explained in function of the capacity of the activated charcoal to alter the pH of the culture medium, adsorb a portion of all elements that comprise the medium, causing nutritional deficiency, as well as making a lower amount of free auxin available for the shoots (Grattapaglia & Machado, 1998), damaging the rooting of this species.For the test concentration of IBA, there was no statistical difference between treatments, presenting a general average of 36% of number of roots.However, we verified higher root vigor when the culture medium was supplemented with the highest concentration of IBA.However, for many plant species, the use of IBA was shown to be beneficial, such as with A. cearenses specie, presenting rooting rate of 92% in the presence of 10.0 µM of IBA (Campos, Lima-Brito, Gutierrez, Santana, & Souza, 2013).V. agnus-castus presented 90% (Balaraju, Lima-Brito, Gutierrez, Santana, & Souza, 2008) and Pistaceae vera L. presenting 84% (Tilkat, Onay, Yıldırım, & Ayaz, 2009) of rooting, both species in culture medium supplemented with 0.49 µM and 9.84 µM de IBA, respectively.The IBA is the synthetic auxin most commonly used for inducing rooting for presenting the property of promoting the formation of early roots.This regulator has been used to induce rooting of numerous plant species.According to Souza and Pereira (2007), the responses for induction and initiation of adventitious roots are regulated by the quantitative relation between the levels of auxin and cytokinin in the plant, where these endogenous contents vary according to the studied species.Presence of activated charcoal 0.0 b 0.0 b Absence of activated charcoal 1.0 a 0.9 a Note.Means followed by the same letters do not differ statistically between each other at the level of 5% of probability by the Scott-Knott test.

Conclusion
Combinations of BAP with IAA, as well as with NAA, presented a negative effect on the in vitro multiplication of shoots of B. gardneriana.The use of BAP presents 100% efficiency of shoot induction from nodal segments.The concentration of 2.0 and 4.0 µM of BAP was efficient for multiplication, given that, above these concentrations, the efficiency decreases.In vitro rooting occurred in all treatments evaluated, even in the absence of the IBA regulator, except in the presence of activated charcoal.New studies on in vitro multiplication are recommended in order to optimize shoot formation, and the use of IBA in more elevated concentrations, in order to optimize in vitro rooting protocol for the studied species.

Table 1 .
Number of roots in B. gardneriana sprouts in the presence and absence of activated charcoal