Callogenesis and in vitro Regeneration of Baru (Dipteryx alata Vog.) Esprouts

The aim of this study was to evaluate the effect of different concentrations of 6-benzylaminopurine (BAP) and naphthalene acetic acid (ANA) on calogenesis and regeneration from baru leaf and apex segments. The explants were obtained from baru plants previously established in vitro from almonds and cauline apices. The leaf segments were placed in Petri dishes containing MS medium (Murashige & Skoog, 1962) with concentrations (0.0, 2.0, 3.0, 4.0 and 5.0 mg L) BAP combined with ANA (0.0 and 2.0 mg L). The shoot apices were inoculated in test tubes with the same medium using the concentrations (0.0, 0.5, 1.0 and 1.5 mg L) BAP combined (0.0 and 0.1 mg L) ANA. After 25 days of inoculation, the percentages of callus and texture in leaf explants and apices were evaluated. The number of shoots was also evaluated by the Scott-Knott test at 5% of probability. The most efficient concentration in the formation of callus in leaves was 3.0 mg L BAP + 2.0 mg L ANA (68.88%), at apexes the most efficient concentration was 1.0 mg L BAP without ANA with 100% calogenesis. The most effective concentration was 1.0 mg L BAP without ANA with an average of 1.90 of shoots in relation to the concentrations evaluated.


Introduction
Cerrado is one of the main Biomas of Brazil, both in area and in biodiversity (Ribeiro & Walter, 2008). Native to this biome, the barueiro (Dipteryx alata Vog.) is an economically promising species with multiple uses, such as the exploitation of wood, fruits and seeds (Ratter et al., 2000;Durigan et al., 2011). It is a fruitful arboreal, highly valued for its economic potential, with various possibilities for use in food, forage, forestry, silvopastoral system, landscape and reforestation of degraded areas (Larson, 2014).
Baru has been studied due to lipid and protein properties, as well as the production of bioactive molecules used in the food and pharmaceutical industry. The baru fruit also serves for human consumption, both pulp and baru almond can be used in human nutrition, and the pulp consists mainly of carbohydrates (63%), predominantly starch, insoluble fibers and sugars (Alves et al., 2010). Almond is consumed in nature or toast, has high lipid levels (42%), protein (30%), calcium, phosphorus, manganese and potassium, in addition to iron, zinc, selenium and considerable carbohydrate and fibre level (Souza et al., 2011).
Few reports regarding the micropropagation of Dipteryx alata as seed germination in vitro, but with inconclusive results (Mamedes & Araújo, 2010). The germination rate of seeds is high, however, the fruits can present seeds with pathogens that will give rise to unhealthy seedlings, since their fruits can remain for a long period in the field. For the purpose of obtaining pathogen-free seedlings, plant tissue culture has been used (Perez, 2004).
Plant tissue cultivation techniques have been effectively used for the clonal propagation and genetic improvement of different crops (Rocha et al., 2012;Sousa et al., 2019), providing higher productive and pathogen-resistant plants, higher vigour and adaptability at environments heterogeneous. Cultivation in vitro is an important strategy to solve problems crop using classical genetic improvement and wood biotechnology (Erigs & Schuch, 2005).
Zygotic embryos culture is a very common procedure to regenerate seed embryos that do not germinate under conventional sowing conditions. However, its greatest applicability is through the rescue of immature embryos from developing seeds (Raghavan, 2003).
In vitro embryo cultivation is a promising technique for advancing knowledge about certain species, because, based on such activity, is possible reproduction and embryonic development, dormancy breakdown and plant production (Raghavan, 2003).
Calogenesis is the methodologie more applied to cultivation in vitro, also used for production of secondary metabolites and genetic transformation (Santos, 2004). Studies with callus can also serve as initial point for determining necessary conditions of development (Landa et al., 2000). For induction of callus, virtually any part of the plant can be used as an explant. However, it is sought to use those who contain the highest proportion of meristematic tissues or who have the greatest ability to express totipotence where each plant cell had the genetic potential to regenerate a plant (Flores, 2006).
In regeneration of woody plants, usually leaves and internodes developed in vitro are explants more used (Pérez-Tornero et al., 2000;Cassana et al., 2007). Significant differences in organogenic capacity in vitro are found when varying the type of explant and the nutritional composition of the culture medium. However, most components optimised in the growth medium are the phytoregulators, particularly auxin/cytokinin balance (Erig & Schuch, 2005).
Auxins act in cell division and tissue expansion, while cytokinins are used regularly to stimulate multiple sprouts (auxins and cytokinins act as growth regulators, presenting an important work in vitro, cell division and tissue expansion), while cytokinins are regularly used to stimulate multiple sprouts (Morais, 2012).
The physiological effect of each regulator depends on its concentration in the medium, and each part of the plant has a different response to changes in the concentrations of auxins and cytokinins (Pozo et al., 2005). BAP is a cytokinin added to right concentrations to the culture medium and promotes increase in the number buds, leaves and sprouts and increase the production of fresh mass and quality of cultivated plants. ANA is auxin that inhibit the proliferation of sprouts, however, it is widely used in association with BAP, because the interaction between both it can further favor the quality of micropropagated plants (Torres et al., 1998). Therefore, studies aiming at the regeneration of baru plants in vitro, formation of corns, production of multiple shoots aiming at the production of seedlings micropropagated and studies of the production of secondary metabolites may be used by pharmaceutical industry for extracting the active ingredient.
In view of the lack of specific information regarding the in vitro propagation of baru, the study aims to evaluate the effect of different concentrations of 6-benzylaminopurine (BAP) and naphthalene acetic acid (ANA), and their combinations, in callogenesis and plant regeneration from leaf segments and stem apex of baru in vitro.

Materials and Methods
Baru fruits were collected between August and September 2018 in the Campo Grande city, MS.
These fruits were stored at room temperature at the school farm of Catolic University Dom Bosco, Campo Grande, MS.
The seeds were removed from fruits with a hydraulic press, selecting the major size, and the plants in vitro were used to remove the leaf segments.

Callogenesis Procedure From Leaf Segments
Leaves segments (1 cm 2 ) were placed in Petri dishes containing 30 ml of MS culture medium (Murashige & Skoog, 1962), supplemented with different concentrations of BAP and ANA (Table 1), totaling four treatments with 10 repetitions. Each plot consisted of a Petri dish containing three explants. Table 1. Treatments used to induce callogenesis from leaf explants baru (Dipteryx alata Vog.) Treatments BAP and ANA concentrations T 1 0.0 mg L -1 BAP and 0.0 mg L -1 ANA T 2 2.0 mg L -1 BAP and 2.0 mg L -1 ANA T 3 3.0 mg L -1 BAP and 2.0 mg L -1 ANA T 4 4.0 mg L -1 BAP and 2.0 mg L -1 ANA T 5 5.0 mg L -1 BAP and 2.0 mg L -1 ANA The MS medium was enriched with 30.0 g L -1 sucrose and 7.0 g L -1 agar and pH adjusted to 5.8. After inoculation, the material was subjected to seven days of darkness and subsequently transferred to photoperiod of 16 hours and temperature of 27±2 °C under irradiation of 36 μmol of m -2 s -1 .
After 25 days of inoculation with leaf explants, the average percentage of callus formation and texture was evaluated.
Regarding texture the callus were classified into four types: A) compact (tightly bound cells); B) semi-compact (moderately bound cells); C) friable (loosely bound cells); and D) without reaction.
The callus from leaf explants were transferred to test tubes to obtain regeneration.
Tubes contained 15 ml of MS medium with 2.0 mg L -1 BAP combined with 0.5 mg L -1 ANA, totaling a treatment with 10 repetitions (each plot consisted of a test tube).
After inoculation, the material was subjected to seven days of darkness and subsequently transferred to photoperiod of 16 hours and temperature of 27±2 °C under irradiation of 36 μmol m -2 s -1 .

Stem Callogenesis From Apexes and Regeneration
After disinfestation, the seeds remained embedded for 24 hours in Petri dishes containing sterile distilled water to facilitate the removal of the embryos. After the imbibition period, the embryos were removed with the aid of forceps and scalpel.
The embryos were distributed in tubes containing 110 ml of MS culture medium (Murashige & Skoog, 1962), 30 g L -1 sucrose, 100 mg L -1 inositol and 7 g L -1 of agar with pH adjusted to 5.8±0.1 For one month, the embryos were kept in growth room located in the tissue culture laboratory, under temperature conditions of 25±2 °C under 16 hour photoperiod and irradiation of 36 µmol m -2 s -1 .

Stem Regeneration From Apexes
Seedlings germinated in vitro were used to evaluate the effect of phytoregulators on callus from stem apexes.
Stem tips containing approximately two leaves, after one month of inoculation were placed in test tubes containing 15 ml of MS medium (Murashige & Skoog, 1962), supplemented with different concentrations of 6-benzylaminopurine (BAP) and naphthalene acetic acid (ANA), (Table 2), totaling seven treatments and 8 repetitions, each experimental unit represented by one test tube, each tube containing one explant. Table 2. Treatments used to induce callogenesis and stem regeneration from apexes of baru (Dipteryx alata Vog.) Treatments Concentrations BAP and ANA T 1 0.0 mg L -1 BAP and 0.0 mg L -1 ANA T 2 0.5 mg L -1 BAP and 0,0 mg L -1 ANA T 3 0.5 mg L -1 BAP and 0.1 mg L -1 ANA T 4 1.0 mg L -1 BAP and 0.0 mg L -1 ANA T 5 1.0 mg L -1 BAP and 0.1 mg L -1 ANA T 6 1.5 mg L -1 BAP and 0.1 mg L -1 ANA T 7 1.5 mg L -1 BAP and 0.0 mg L -1 ANA After inoculation of stem tips, they were placed in the dark after 20 days of inoculation of stem tips in medium and evaluated in relation the percentage of callus formation and its texture and the average number of shoots.
Regarding texture, the callus were classified into four types: A) compact (tightly bound cells); B) semi-compact (moderately bound cells); C) friable (loosely bound cells); and D) without reaction.
Average sprouts formed in Dipteryx alata Vog. after 20 days of cultivation in medium MS supplemented with different concentrations of phytoregulators BAP and ANA. Shooting averages were compared Scott-Knott test (1974) at 5% probability using the Sisvar® statistical program.

Evaluation of Foliar Calogenesis in Segments and Callus Regeneration
Callus can be obtained from fragment of tissues that have the capacity to differentiate into tissues, organs and even embryos (R. Paiva & P. D. O. Paiva, 2001). This process occurs because plant tissues have a high degree of plasticity for cell differentiation (Ikeuchi et al., 2013).
Callus production started on seventh day of cultivation. Concentration 3.0 mg L -1 BAP and 2.0 mg L -1 ANA, was the one that provided highest number of explants callogenic (68.88%). Concentration 5.0 mg L -1 BAP and addition 2.0 mg L -1 ANA promoted 24.44% of callus (Table 3). Other treatments evaluated did not provide callus formation (Table 3).  Positive influence of auxin and cytokinin combination was also verified in trials involving calogenesis in several coffee cultivars (Santos et al., 2000). Similarly, in studies of calogenesis in coffee testing different doses of 2.4-D and 2.0 mgL -1 kinetin. Maciel (2001) also found greater formation of primary nodular callus with high concentrations of 2.4-D.
The auxins are indispensable for the formation of callus, since they are responsible to start cellular division and control cellular growth and stretching processes (Taiz & Zeiger, 2004). Cytokines are also necessary for plant cell division with positive results in embryogenic callus induction (Pasqual, 2001), confirming callogenesis observed in this experiment was probably favored by the joint action of these phytoregulators. After fifteenth day of cultivation, it was also observed regarding the texture of the callus, and there was only formation of friable and compact callus. The texture of callus was dependent on the concentrations of BAP and ANA used. Higher percentage of friable callus occurred in concentrations of 3.0 mg L -1 BAP combined with 2.0 mg L -1 ANA, totaling 35.55 % callus ( Figure 1A, Table 4).
However, the highest percentage of compact callus was fifteenth day of cultivation, it was also observed regarding the texture of callus that there was only formation of friable and compact callus. The texture of callus was dependent on the concentrations of BAP and ANA used. Higher percentage of friable callus occurred in concentrations of 3.0 mg L -1 BAP combined with 2.0 mg L -1 ANA, totaling 35.55 % callus ( Figure 1A, Table 4). However, highest percentage of compact callus was observed in the same concentration of 3.0 mg L -1 BAP and 2.0 mg L -1 ANA totaling 66.66% callus ( Figure 1B, Table 4). In Tables 3 and 4 (Flores, 2006) was observed a ts formed from the fifteenth d ally at the 5% p lture medium rmation of ear k, where baru uts.
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