In vitro shoot regeneration in Myracrodruon urundeuva Fr. All

Regeneração in vitro de brotações em Myracrodruon urundeuva Fr. All.

Tecla dos Santos Silva Rosembrando Sosthenes Leite Carvalho Filho Priscila Tavares Fonseca José Raniere Ferreira de Santana About the authors

ABSTRACT

Myracrodruon urundeuva Fr. All. is a tree threatened with extinction, which has wood and medicinal potential. This study aimed to analyze the in vitro shoot regeneration in M. urundeuva, in order to increase the species multiplication. Two experiments were conducted: 1) concentrations of 6-benzylaminopurine (BAP) (0.0, 2.0, 4.0, 8.0 and 16.0 µM), in association with naphthaleneacetic acid (NAA) (0.0, 1.5 and 3.0 µM), in explants (cotyledon, hypocotyl and cotyledonary node); 2) concentrations of meta-topolin (mT) (0.0, 2.0, 4.0, 8.0, 16.0 and 32.0 µM) in explants (biaxillary, medial uniaxillary and apical basal nodal segment). The percentage of explants responsive to shoot regeneration, percentage of callus explants, number of shoots and shoot length were evaluated. In the first experiment, the shoot regeneration occurred only in explants of the cotyledonary node and hypocotyl type, with the highest responsiveness percentage (76.67 %) and number of shoots (1.97 and 1.63) obtained for the cotyledonary node in the presence of 3.0 µM of NAA in association with 2.0 (1.97 shoots/explant) and 4.0 µM (1.63 shoots/explant) of mT. In the second experiment, the resolution of the obtained quadratic equation indicates that the use of basal explant with 24.59 µM of mT added to the culture medium leads to the highest number of shoots (1.86). However, despite the mT having increased the mean number of shoots, all treatments containing this cytokinin showed callus formation. As a conclusion, it is possible to regenerate shoots in M. urundeuva from the cotyledonary node using BAP in association with NAA.

KEYWORDS:
Aroeira-do-sertão; in vitro cultivation; medicinal plant

RESUMO

Myracrodruon urundeuva Fr. All. é uma árvore ameaçada de extinção, que possui potencial madeireiro e medicinal. Objetivou-se estudar a regeneração de brotos in vitro de M. urundeuva, visando aumentar a porcentagem de multiplicação da espécie. Foram realizados dois experimentos: 1) concentrações de 6-benzilaminopurina (BAP) (0,0; 2,0; 4,0; 8,0; e 16,0 µM), combinadas com ácido naftalenoacético (ANA) (0,0; 1,5; e 3,0 µM) em explantes (cotilédone, hipocótilo e nó cotiledonar); 2) concentrações de meta-topolina (mT) (0,0; 2,0; 4,0; 8,0; 16,0; e 32,0 µM) sob explantes (segmento nodal basal biaxilar, mediano uniaxilar e apical). Avaliaram-se a porcentagem de explantes responsivos para regeneração de brotações, porcentagem de explantes que formaram calos, número de brotos e comprimento da parte aérea. No primeiro experimento, observou-se regeneração de brotações apenas nos explantes do tipo nó cotiledonar e hipocótilo, sendo a maior porcentagem (76,67 %) de responsividade e número de brotos (1,97 e 1,63) obtidos com o nó cotiledonar na presença de 3,0 µM de ANA, combinado com 2,0 (1,97 brotos/explante) e 4,0 µM (1,63 brotos/explante) de mT. No segundo experimento, a resolução da equação quadrática obtida indica que o maior número de brotos (1,86) é alcançado utilizando-se o explante basal com 24,59 µM de mT adicionado ao meio de cultura. No entanto, apesar de a mT ter elevado o número médio de brotações, observou-se formação de calos em todos os tratamentos contendo essa citocinina. Conclui-se que é possível regenerar brotações em M. urundeuva a partir do nó cotiledonar com o uso de BAP combinado com ANA.

PALAVRAS-CHAVE:
Aroeira-do-sertão; cultivo in vitro; planta medicinal

INTRODUCTION

Myracrodruon urundeuva Fr. All. is a tree belonging to the Anacardiaceae family, popularly known in Brazil as ‘aroeira-preta’ or ‘aroeira-do-sertão’ (Lima 2011LIMA, B. G. Caatinga: espécies lenhosas e herbáceas. Mossoró: Edufersa, 2011.). The species is part of the Caatinga biome, which occupies about 11 % of the Brazilian territory and comprises herbaceous, shrub and arboreal plants (Drumond et al. 2016DRUMOND, M. A.; KIILL, L. H. P.; RIBASKI, J., AIDAR, S. T. Caracterização e usos das espécies da Caatinga: subsídio para programas de restauração florestal nas Unidades de Conservação da Caatinga (UCCAs). Petrolina: Embrapa Semiárido, 2016.). It has a great potential for the recovery of degraded areas and for the composition of agroforestry systems (Maia 2012MAIA, G. N. Caatinga: árvores e arbustos e suas utilidades. 2. ed. Fortaleza: Printcolor, 2012.). Its wood is highly valued in external works and workpieces (Lima 2011LIMA, B. G. Caatinga: espécies lenhosas e herbáceas. Mossoró: Edufersa, 2011.). Moreover, the plant has medicinal use in the treatment of hemorrhages, respiratory and urinary infections, and digestive system disorders (Matos 1999MATOS, F. J. A. Plantas de medicina popular do Nordeste: propriedades atribuídas e confirmadas. Fortaleza: Ed. UFC, 1999.).

The multiple uses of the species and the high indiscriminate exploitation of its resources threaten its genetic variability. Thus, methodologies and strategies for its conservation must be adopted (Cardoso et al. 2012CARDOSO, N. S. N.; OLIVEIRA, L. M.; FERNANDEZ, L. G.; PELACANI, C. R.; SOUZA, C. L. M.; OLIVEIRA, A. R. M. F. Osmocondicionamento na germinação de sementes, crescimento inicial e conteúdo de pigmentos de Myracrodruon urundeuva Fr. Allemão. Revista Brasileira de Biociências, v. 10, n. 4, p. 457-461, 2012.).

Biotechnological processes such as plant tissue culture play an important role in the preservation and multiplication of species threatened with extinction (Suzuki et al. 2009SUZUKI, R. M.; MOREIRA, V. C.; NAKABASHI, M.; FERREIRA, W. M. Estudo da germinação e crescimento in vitro de Hadrolaelia tenebrosa (Rolfe) Chiron & V.P. Castro (Orchidaceae), uma espécie da flora brasileira ameaçada de extinção. Hoehnea, v. 36, n. 4, p. 657-666, 2009., Oseni et al. 2019). One of the techniques for plant tissue culture is micropropagation, which allows obtaining seedlings from a large number of plants in a short space of time and at any time of the year, with adequate phytosanitary quality (Nagao et al. 1994NAGAO, E. O.; PASQUAL, M.; RAMOS, J. D. Efeitos da sacarose e do nitrogênio inorgânico sobre a multiplicação “in vitro” de brotações de porta-enxerto de citros. Bragantia, v. 53, n. 1, p. 25-31, 1994.).

Phytoregulators such as auxins and cytokinins are used to obtain in vitro morphogenic processes (Pasa et al. 2012PASA, M. S.; CARVALHO, G. L.; SCHUCH, M. W.; SCHMITZ, J. D.; TORCHELSEN, M. M.; NICKEL, G. K.; SOMMER, L. R.; LIMA, T. S.; CAMARGO, S. S. Qualidade de luz e fitorreguladores na multiplicação e enraizamento in vitro da amoreira-preta ‘Xavante’. Ciência Rural, v. 42, n. 8, p. 1392-1396, 2012., Soares et al. 2014SOARES, T. C.; SALES, F. M. S.; SANTOS, J. W.; CARVALHO, J. M. F. C. Quitosana e fitorreguladores na indução da organogênese direta em cultivar de algodão colorido. Revista Brasileira de Engenharia Agrícola e Ambiental, v. 18, n. 8, p. 839-843, 2014.). The main objective of these agents is to overcome possible deficiencies in the endogenous levels of hormones in the tissues of explants isolated from the production sites in the matrix plant (Grattapaglia & Machado 1998GRATTAPAGLIA, D.; MACHADO, M. A. Micropropagação. In: TORRES, A. C.; CALDAS, L. S.; BUSO, J. A. Cultura de tecidos e transformação genética de plantas. Brasília, DF: Embrapa, 1998. p. 183-260.). Cytokinins are essential during the multiplication phase, as they control cell division and correlate with cell differentiation, especially during stem bud formation (Kerbauy 2008KERBAUY, G. B. Fisiologia vegetal. 2. ed. Rio de Janeiro: Guanabara Koogan, 2008.). In turn, auxins promote cell elongation and differentiation, in addition to being responsible for apical dominance and promoting in vitro rooting (Taiz & Zeiger 2017TAIZ, L.; ZEIGER, E. Fisiologia vegetal. 6. ed. Porto Alegre: Artmed, 2017.).

The cytokinin type and its concentration are the factors that most influence the success of in vitro multiplication (Grattapaglia & Machado 1998GRATTAPAGLIA, D.; MACHADO, M. A. Micropropagação. In: TORRES, A. C.; CALDAS, L. S.; BUSO, J. A. Cultura de tecidos e transformação genética de plantas. Brasília, DF: Embrapa, 1998. p. 183-260.). Researchers have been using the cytokinin meta-topolin (mT) in in vitro propagation (Ahmad & Anis 2019AHMAD, A.; ANIS, M. Meta-topolin improves in vitro morphogenesis, rhizogenesis and biochemical analysis in Pterocarpus marsupium Roxb.: a potential drug-yielding tree. Journal of Plant Growth Regulation, v. 38, n. 3, p. 1007-1016, 2019., Nowakowska & Pacholczak 2020NOWAKOWSKA, K.; PACHOLCZAK, A. Comparison of the effect of meta-topolin and benzyladenine during Daphne mezereum L. micropropagation. Agronomy, v. 10, n. 12, e10121994, 2020.). Firstly isolated from poplar leaves (Populus x canadensis Moench, cv. Robusta), mT is an aromatic cytokinin that differs from isoprenoids in its biochemistry, receptors and biological activity (Strnad et al. 1997STRNAD, M.; HANUS, J.; VANEK, T.; KAMÍNEK, M.; BALLANTINE, J. A.; FUSSEL, B.; HANKE, D. E. Meta-topolin, a highly active aromatic cytokinin from poplar leaves (Populus x Canadensis moench., cv. Robusta). Phytochemistry, v. 45, n. 2, p. 213-218, 1997.).

Except for 6-benzylaminopurine (BAP), purine cytokinins are chemically unstable (Vinayak et al. 2009VINAYAK, V.; DHAWAN, A. K.; GUPTA, V. K. Efficacy of non-purine and purine cytokinins on shoot regeneration in vitro in sugarcane. Indian Journal of Biotechnology, v. 8, n. 2, p. 227-231, 2009.). In turn, nonpurine types are generally stable and therefore advantageous in procedures that involve culture medium sterilization (Vinayak et al. 2009VINAYAK, V.; DHAWAN, A. K.; GUPTA, V. K. Efficacy of non-purine and purine cytokinins on shoot regeneration in vitro in sugarcane. Indian Journal of Biotechnology, v. 8, n. 2, p. 227-231, 2009.). Some authors have reported the use of mT as an alternative to reduce physiological disorders caused by other cytokinins (Bairu et al. 2008BAIRU, M. W.; STIRK, W. A.; DOLEZAL, K.; STADEN, J. V. The role of topolins in micropropagation and somaclonal variation of banana cultivars ‘Williams’ and ‘Grand Naine’ (Musa spp. AAA). Plant Cell, Tisse and Organ Culture, v. 95, n. 3, p. 373-379, 2008., Aremu et al. 2012AREMU, A. O.; BAIRU, M. W.; DOLEZ’AL, K.; FINNIE, J. F.; STADEN, J. V. Topolins: a panacea to plant tissue culture challenges? Plant Cell, Tissue and Organ Culture, v. 108, n. 1, p. 1-16, 2012., Souza et al. 2019SOUZA, L. M.; SILVA, M. M. A.; HERCULANO, L.; ULISSES, C.; CÂMARA, T. R. Meta-topolin: an alternative for the prevention of oxidative stress in sugarcane micropropagation. Hoehnea, v. 46, n. 3, e1072018, 2019.). For example, Mirabbasi & Hosseinpour (2014)MIRABBASI, S. M.; HOSSEINPOUR, B. Prevention of shoot tip necrosis, hyperhydricity and callus production associated with in vitro shoot culture of Ulmus glabra. Journal of Novel Applied Sciences, v. 3, n. 6, p. 683-689, 2014., in a study with Ulmus glabra Huds, found that explants treated with mT produced more vigorous shoots than those treated with BAP.

In a study with M. urundeuva, Andrade et al. (2000)ANDRADE, M. W.; LUZ, J. M. Q.; LACERDA, A. S.; MELO, P. R. A. Micropropagação da aroeira (Myracrodruon urundeuva Fr. All). Ciência e Agrotecnologia, v. 24, n. 1, p. 174-180, 2000. evaluated the effect of using concentrations of BAP in explants (nodal and apical segments) during in vitro multiplication via direct organogenesis. The authors reported explant regeneration; however, these explants developed in a single shoot. In this sense, the present study analyzes the in vitro shoot regeneration in M. urundeuva, aiming to increase the multiplication percentage of adventitious buds.

MATERIAL AND METHODS

Two experiments were conducted at the Universidade Estadual de Feira de Santana, Bahia state, Brazil, in 2015 and 2016. For in vitro culture establishment, M. urundeuva seeds from the Embrapa Semiárido (Petrolina, Pernambuco state, Brazil) were used.

The in vitro culture establishment relied on the methodology described by Andrade et al. (2000)ANDRADE, M. W.; LUZ, J. M. Q.; LACERDA, A. S.; MELO, P. R. A. Micropropagação da aroeira (Myracrodruon urundeuva Fr. All). Ciência e Agrotecnologia, v. 24, n. 1, p. 174-180, 2000., with modifications. Fruit seeds of the species were superficially disinfested in running water with a few drops of neutral detergent, for 20 min. Then, the endocarp was removed by using a sieve. Finally, the fruit seeds remained for another 10 min in running water. The seeds were disinfested in a laminar flow chamber by immersion in 70 % ethanol for 30 sec, followed by immersion in sodium hypochlorite (1 % active chlorine) for 10 min, being then washed for three consecutive times with sterile distilled water. Subsequently, the seeds were inoculated in a culture medium to be used as an explant source at 30 and 60 days after sowing.

Test tubes (25 x 150 mm) containing 10 mL of woody plant medium (Lloyd & McCown 1980LLOYD, G.; MCCOWN, B. Use of microculture for production and improvement of Rhododendron ssp. HortScience, v. 15, n. 3, p. 416-420, 1980.) supplemented with 87.64 mM of sucrose and solidified with 7 g L-1 of agar were used in the experiments. The pH was adjusted to 5.7 ± 0.1 (using 0.1N NaOH or HCl) before autoclaving at 121º C, for 15 min. After inoculation, the tubes were sealed with a polyvinylchloride (PVC) film.

In the first experiment, explants [cotyledon (whole), hypocotyl (± 10 mm) and cotyledonary node (± 10 mm)] from 30-day-old plants were inoculated in test tubes containing culture medium supplemented with concentrations of BAP (0.0, 2.0, 4.0, 8.0 and 16.0 µM), in association with naphthaleneacetic acid (NAA) (0.0, 1.5 and 3.0 µM). The explants were inoculated vertically, except for the cotyledon (inoculated horizontally with the abaxial region in contact with the culture medium). The statistical design was completely randomized, in a 3 x 5 x 3 factorial arrangement (types of explants x BAP concentrations x NAA concentrations), totaling 45 treatments with 6 replications, each consisting of 5 plots (test tubes).

In the second experiment, explants of the basal nodal segment (biaxillary) (± 10 mm) (first pair of leaves), medial nodal segment (uniaxillary) (± 10 mm) and segment containing the apical bud (± 8 mm) from 60-day-old plants were excised in ascorbic acid solution (1.14 mM). The explants remained immersed in this solution for 10 min and were then inoculated vertically in culture medium containing concentrations of mT (0.0, 2.0, 4.0, 8.0, 16.0 and 32.0 µM). The statistical design was completely randomized, in a 3 x 6 factorial arrangement (explants x mT concentrations), totaling 18 treatments with 6 replications, each consisting of 5 plots (test tubes).

The cultures were kept in a growth room at a temperature of 25 ± 3 ºC, 16-h photoperiod and active photosynthetic radiation of 60 μmol m-2 s-1, except for the material from the second experiment, which remained under dark conditions during the first seven days of culture. After 45 days of inoculation, the percentage of explants responsive to shoot regeneration, number of shoots, shoot length (mm) and percentage of callus explants were evaluated.

The data were statistically analyzed using the Sisvar software (Ferreira 2011FERREIRA, D. F. Sisvar: a computer statistical analysis system. Ciencia e Agrotecnologia, v. 35, n. 6, p. 1039-1042, 2011.). These were submitted to the Shapiro-Wilk normality test, followed by analysis of variance, being then transformed by the function (x + 1)0.5, except for the percentage data, which were submitted to transformation into arcsine √%. When the “F” value was significant, qualitative data and adjustments were subjected to a comparison of means using the Tukey test, and quantitative data were submitted to polynomial regression analysis. The presented results are the original means obtained.

RESULTS AND DISCUSSION

The tests showed high oxidation percentages (data not shown). Tissue oxidation can cause direct death of explants or make the tissue unfeasible for morphogenesis. This problem is particularly serious in the isolation of explants of woody species. This is because their tissues are richer in secondary metabolism substances, more precisely phenolic compounds, which play an important role in defense against predators and microorganisms (Freitas et al. 2009FREITAS, R. M. O.; OLIVEIRA, M. K. T.; DOMBROSKI, J. L. D.; CÂMARA, F. A. A.; SILVA NETO, R. V. Efeito dos tratamentos de oxidação em Aloysia virgata. Revista Caatinga, v. 22, n. 1, p. 151-154, 2009.). Other authors have reported the oxidation of explants of woody species such as blueberry (Vaccinium spp.) (Rosa et al. 2009ROSA, L. P. P.; ETCHEVERRIA, C.; DÁVILA, E. S.; MARTINS, C. R. Efeito de antibiótico e do período de escuro no estabelecimento in vitro de mirtilo Vacciniun spp. Revista da Faculdade de Zootecnia, Veterinária e Agronomia, v. 16, n. 2, p. 265-277, 2009.), pomegranate (Punica granatum L.) (Dias et al. 2013DIAS, M. M.; NIETSCHE, S.; PEREIRA, M. C. T. Carvão ativado e estiolamento no estabelecimento in vitro de romãzeira. Tecnologia & Ciência Agropecuária, v. 7, n. 1, p. 1-5, 2013.), rosewood [Dalbergia nigra (Vell.) Allemão ex Benth.] (Sartor et al. 2013SARTOR, F. R.; ZANOTTI, R. F.; PÔSSA. K. F.; PILON, A. M.; FUKUSHIMA, C. H. Diferentes meios de cultura e antioxidantes no estabelecimento in vitro do jacarandá da Bahia. Bioscience Journal, v. 29, n. 2, p. 408-411, 2013.) and sabiá (Mimosa caesalpiniifolia Benth.) (Bezerra et al. 2014BEZERRA, R. M. F.; ALOUFA, M. A. I.; FREIRE, F. A. M.; SANTOS, D. D. Efeito de 6-benzilaminopurina sobre a propagação in vitro de Mimosa caesalpiniifolia Benth. (Fabaceae). Revista Árvore, v. 38, n. 5, p. 771-778, 2014.).

In the first experiment, the triggering of responses for shoot regeneration occurred only in the cotyledonary node and hypocotyl type explants. The lack of responsiveness of the cotyledon for shoot production may have occurred due to the lack of cellular competence to respond to the stimuli for dedifferentiation and redifferentiation with the aim to provide new abilities to explant cells. In this sense, the analyzed statistical data only regarded shoots originating from the hypocotyl and the cotyledonary node.

The analysis of variance showed a highly significant effect (p < 0.01) of the triple interaction among the factors ‘BAP x NAA x Explant’ for percentage of explants responsive to shoot regeneration, number of shoots and shoot length (Table 1).

For the percentage of explants responsive to shoot regeneration, the use of the cotyledonary node in the presence of 3.0 µM of NAA, combined with 2.0 and 4.0 µM of BAP, led to the highest responsiveness percentage (76.67 %). These results corroborate Cordeiro et al. (2014)CORDEIRO, G. M.; BONDANI, G. E.; OLIVEIRA, L. S.; ALMEIDA, M. Meio de cultura, BAP e ANA na multiplicação in vitro de clones de Eucalyptus globulus Labill. Scientia Forestalis, v. 42, n. 103, p. 337-344, 2014., who studied Eucalyptus globulus Labill clones and found a positive effect of the auxin/cytokinin ratio for shoot regeneration. The hypocotyl explant showed a low percentage of regeneration in most of the treatments under study, reaching the highest percentage (36.67 %) with the use of 2.0 µM of BAP in the absence of NAA (Table 2).

Regarding the mean number of shoots, the use of the cotyledonary node in culture medium added with 3.0 µM of NAA, in association with 2.0 and 4.0 µM of BAP, led to the best results (1.97 and 1.63 shoots per explant, respectively) (Table 2). The values of the present study for number of shoots in M. urundeuva are higher than those observed by Andrade et al. (2000)ANDRADE, M. W.; LUZ, J. M. Q.; LACERDA, A. S.; MELO, P. R. A. Micropropagação da aroeira (Myracrodruon urundeuva Fr. All). Ciência e Agrotecnologia, v. 24, n. 1, p. 174-180, 2000.. These authors conducted a micropropagation study with the same species and observed a single shoot using nodal and apical segment explants in the presence of 4.5 µM of BAP.

Table 1
Summary of the analysis of variance for percentage of explants responsive to shoot regeneration (%RE), number of shoots (NS) and shoot length (SL), at 45 days after inoculation. Values obtained from the hypocotyl and cotyledonary node explants (EXP) of Myracrodruon urundeuva Fr. All. under concentrations of 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA).
Table 2
Percentage of explants responsive to shoot regeneration (%RE), number of shoots (NS) and shoot length (SL), at 45 days after inoculation. Values obtained from the hypocotyl (HYP) and cotyledonary node (CN) explants of Myracrodruon urundeuva Fr. All. under concentrations of 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA).

The highest mean number of shoots occurring from the combination of BAP and NAA factors shows the importance of the synergistic effect of these regulators for regeneration from the cotyledonary node. Notwithstanding, the auxin-free medium also showed shoot formation, probably due to the way BAP works. This cytokinin can break the apical dominance and dormancy of lateral buds, thus forming new shoots (George 2008GEORGE, E. F. Plant tissue culture procedure: background. In: GEORGE, E. F.; HALL, M. A.; KLERK, G.-J. (ed). Plant propagation by tissue culture: the background. 3. ed. Dordrecht: Springer, 2008. p. 2-28.). This corroborates the results of other authors in micropropagation studies of woody species such as Eremanthus erythropappus (DC.) MacLeish (Prudente et al. 2016PRUDENTE, D. O.; NERY, F. C.; PAIVA, R.; GOULART, V. L. A.; ANSELMO, A. C. N. Micropropagação de candeia, uma espécie nativa do Cerrado brasileiro. Scientia Agraria Paranaensis, v. 15, n. 3, p. 305-311, 2016.) and Pterodon emarginatus Vogel (Canatto et al. 2016CANATTO, R. A.; ALBINO, B. E. S.; CORDEIRO, A. T. Propagação in vitro de sucupira branca (Pterodon emarginatus Vogel): uma espécie florestal nativa. Fórum Ambiental, v. 12, n. 3, p. 76-88, 2016.).

For the variable shoot length, in the absence of NAA, the highest means occurred when using the hypocotyl explant in culture medium added with 2.0 µM of BAP (4.33 mm), and when using the cotyledonary node explant in culture medium added with 16.0 µM of BAP (3.37 mm). The addition of 1.5 µM of NAA did not result in statistical differences between the resulting means. In turn, at the concentration of 3.0 µM of NAA, the highest mean values (8.67 and 6.73 mm) occurred with 2.0 and 4.0 µM of BAP, respectively, using the cotyledonary node (Table 2).

Using BAP in the culture medium in association with the highest NAA concentration under study significantly increased the shoot size, showing the importance of the hormonal interaction under this variable. This result differs from that of Silva et al. (2013)SILVA, T. S.; NEPOMUCENO, C. F.; BORGES, B. P. S.; ALVIM, B. F. M.; SANTANA, J. R. F. Multiplicação in vitro de Caesalpinia pyramidalis (Leguminosae). Sitientibus Ciências Biológicas, v. 13, n. 1, p. 1-6, 2013., who verified that the addition of plant growth regulators [BAP, NAA, kinetin (KIN) and thidiazuron (TDZ)] to the culture medium reduces the shoot length in Caesalpinia pyramidalis. Ashraf et al. (2014)ASHRAF, M. F.; AZIZ, M. A.; KEMAT, N.; ISMAIL, I. Effect of cytokinin types, concentrations and their interactions on in vitro shoot regeneration of Chlorophytum borivilianum Sant. & Fernandez. Electronic Journal of Biotechnology, v. 17, n. 6, p. 275-279, 2014. reported that high concentrations of cytokinins increase the shoot proliferation, although they can reduce the shoot size. This is because cytokinins stimulate sprouting by breaking apical dominance, thus reducing the plant size.

The analysis of variance for the second experiment showed a highly significant effect (p < 0.01) of the interaction ‘mT x explants’ for the variables percentage of explants responsive to shoot regeneration, number of shoots and shoot length. Furthermore, the effect was significant (p < 0.05) for the variable percentage of callus explants (Table 3).

When evaluating the percentage of explants responsive to shoot regeneration in the absence of mT, the highest mean (92.5 %) occurred with the use of the apical segment explant. However, the mean did not differ statistically from the other means obtained with the use of the same explant as a function of mT concentrations (Table 4).

The shoot regeneration in M. urundeuva from the increase of mT in the culture medium may be due to cytokinins regulating cell division, i.e., acting on factors that govern the passage of the cell through the cell division cycle (Taiz & Zeiger 2017TAIZ, L.; ZEIGER, E. Fisiologia vegetal. 6. ed. Porto Alegre: Artmed, 2017.). Cytokinins are thus indispensable for breaking apical dominance and inducing axillary buds (Grattapaglia & Machado 1998GRATTAPAGLIA, D.; MACHADO, M. A. Micropropagação. In: TORRES, A. C.; CALDAS, L. S.; BUSO, J. A. Cultura de tecidos e transformação genética de plantas. Brasília, DF: Embrapa, 1998. p. 183-260.), which possibly favored shoot proliferation.

The results of the present study for M. urundeuva corroborate those by Andrade et al. (2000)ANDRADE, M. W.; LUZ, J. M. Q.; LACERDA, A. S.; MELO, P. R. A. Micropropagação da aroeira (Myracrodruon urundeuva Fr. All). Ciência e Agrotecnologia, v. 24, n. 1, p. 174-180, 2000., who also reported a high percentage of regeneration (90 %) for the same species, when using nodal and apical segment explants in the presence of 4.5 µM of BAP. Benmahioul et al. (2012)BENMAHIOUL, B.; DORION, N.; KAID-HARCHE, M.; DAGUIN, F. Micropropagation and ex vitro rooting of pistachio (Pistacia vera L.). Plant Cell, Tissue and Organ Culture, v. 108, n. 2, p. 353-358, 2012. compared the cytokinins benzyladenine (BA), KIN and mT in cultures of Pistacia vera and observed the highest percentages of shoot regeneration in explants treated with BA and mT. For the latter, all concentrations under study led to 100 % of shoot regeneration.

Noteworthy, shoot regeneration occurred even in a cytokinin-free medium. This behavior indicates that there was no need for an exogenous source of cytokinin to stimulate the formation of shoots for the species. However, the regeneration of shoots from the basal and medial segments significantly increased with the addition of mT to the culture medium (Table 4). Dimitrova et al. (2016)DIMITROVA, N.; NACHEVA, L.; BEROVA, M. Effect of meta-topolin on the shoot multiplication of pear rootstock OHF-333 (Pyrus communis L.). Acta Scientiarum Polonorum Hortorum Cultus, v. 15, n. 2, p. 43-53, 2016. did not obtain lateral shoots in cultures of Pyrus communis L. when using an mT-free medium, and the multiplication percentage increased as the concentrations of this cytokinin increased.

Other authors have also reported shoot formation in the absence of plant regulators in woody species such as Hancornia speciosa (Oliveira et al. 2016OLIVEIRA, K. S.; FREIRE, F. A. M.; ALOUFA, M. A. I. Efeito de 6-benzilaminopurina e ácido naftalenoacético sobre a propagação in vitro de Hancornia speciosa Gomes. Floresta, v. 46, n. 3, p. 335-342, 2016.), Caesalpinia pyramidalis (Silva et al. 2013SILVA, T. S.; NEPOMUCENO, C. F.; BORGES, B. P. S.; ALVIM, B. F. M.; SANTANA, J. R. F. Multiplicação in vitro de Caesalpinia pyramidalis (Leguminosae). Sitientibus Ciências Biológicas, v. 13, n. 1, p. 1-6, 2013.), Tapirira guianensis Aubl. (Gutiérrez et al. 2013GUTIÉRREZ, I. E. M.; NEPOMUCENO, C. F.; SILVA, T. S.; FONSECA, P. T.; CAMPOS, V. C. A.; ALVIM, B. F. M.; CARNEIRO, F. S.; ALBUQUERQUE, M. M. S.; SANTANA, J. R. F. Multiplicação in vitro de Tapirira guianensis Aubl. (Anacardiaceae). Revista Ceres, v. 60, n. 2, p. 143-151, 2013.) and Luehea divaricata (Flôres et al. 2011FLÔRES, A. V.; REINIGER, L. R. S.; CURTI, A. R.; CUNHA, A. C. M. C. M.; GOLLE, D. P.; BASSAN, J. S. Estabelecimento e multiplicação in vitro de Luehea divaricata Mart & Zucc. Ciência Florestal, v. 21, n. 1, p. 175-182, 2011.).

For number of shoots, the basal and medial nodal explants showed an increasing quadratic behavior (p < 0.01), as a function of mT concentrations. However, the apical explant had no representative mathematical model. The quadratic equation obtained for the basal segment indicates that the use of 24.59 µM of mT reaches the highest estimate (1.86 shoots per explant) (Figure 1A). Considering the medial explant, the response curve suggests that the calculated value of 20.05 µM results in 1.30 shoots per explant. The mean values for this variable were 1.68 and 1.43 shoots/explant for the basal and medial explants, respectively (Figure 1A).

Table 3
Summary of the analysis of variance for percentage of explants responsive to shoot regeneration (%RE), percentage of callus explants (%CE), number of shoots (NS) and shoot length (SL), at 45 days after inoculation. Values obtained from the basal nodal segment (first pair of leaves; biaxillary), medial nodal segment (uniaxillary) and apical segment explants (EXP) of Myracrodruon urundeuva Fr. All. under concentrations of meta-topolin (mT).
Table 4
Means for percentage of explants responsive to shoot regeneration (%RE) and percentage of callus explants (%CE), at 45 days after inoculation. Values obtained from basal nodal, medial nodal and apical segment explants of Myracrodruon urundeuva Fr. All. under concentrations of meta-topolin (mT).

These results surpass those of a previous study with the same species by Andrade et al. (2000)ANDRADE, M. W.; LUZ, J. M. Q.; LACERDA, A. S.; MELO, P. R. A. Micropropagação da aroeira (Myracrodruon urundeuva Fr. All). Ciência e Agrotecnologia, v. 24, n. 1, p. 174-180, 2000.. The authors reported a single shoot from the nodal and apical segment explants with the use of the cytokinin BAP (4.5 µM).

M. urundeuva probably has a strong apical dominance, and the use of mT increased its mean number of shoots. This represents a positive result for the micropropagation protocol of the species, given the importance of this variable for this purpose (Figure 2). This result corroborates Gentile et al. (2017)GENTILE, A. FRATTARELLI, A.; NOTA, P.; CONDELLO, E.; CABONI, E. The aromatic cytokinin meta-topolin promotes in vitro propagation, shoot quality and micrografting in Corylus colurna L. Plant Cell, Tissue and Organ Culture, v. 128, n. 3, p. 693-703, 2017., who reported an increase in the multiplication percentage of Corylus column with the use of mT.

Figure 1
Number of shoots (A) and shoot length (B), at 45 days after inoculation. Values obtained from the basal nodal segment, medial nodal segment and apical segment of Myracrodruon urundeuva Fr. All. under concentrations of meta-topolin (mT).ns, * and **: not significant and significant at 5 % and 1 % of probability, respectively, by the F test.

In turn, when studying Ulmus glabra, Mirabbasi & Hosseinpour (2014)MIRABBASI, S. M.; HOSSEINPOUR, B. Prevention of shoot tip necrosis, hyperhydricity and callus production associated with in vitro shoot culture of Ulmus glabra. Journal of Novel Applied Sciences, v. 3, n. 6, p. 683-689, 2014. found that explants treated with mT produced more vigorous shoots than those treated with BAP. Notwithstanding, unlike what occurred for M. urundeuva, mT did not increase the multiplication percentage, with the highest values occurring in the absence of this cytokinin in medium added with 1.78 µM of BAP (5.05 and 4.39 shoots per explant, respectively).

Gentile et al. (2014)GENTILE, A.; GUTIÉRREZ, M. J.; MARTINEZ, J.; FRATTARELLI, A.; NOTA, P.; CABONI, E. Effect of meta-topolin on micropropagation and adventitious shoot regeneration in Prunus rootstocks. Plant Cell, Tissue and Organ Culture, v. 118, n. 3, p. 373-381, 2014. compared concentrations of mT with 2.1 µM of benzyladenine in rootstocks of Prunus domestica L. and Prunus insititia x domestica and did not record an increase in the number of shoots with the addition of mT. However, when evaluating P. insititia x domestica alone and using BA and mT at the same concentration (2.1 µM), these authors realized that, despite not having improved shoot proliferation, mT positively influenced the shoot growth and quality.

In the assessment of shoot length, the regression model showed an increasing quadratic behavior (p < 0.01) for the medial nodal explant. This explant is estimated to reach the maximum length value (10.50 mm) with 15.25 µM of mT. Cytokinin increments greater than the estimated maximum value tend to disfavor shoot length (Figure 1B). In turn, the apical explant showed a decreasing linear behavior (p < 0.01). For the basal explant, the authors could not obtain a mathematical model with biological significance.

These results are inferior to those by Andrade et al. (2000)ANDRADE, M. W.; LUZ, J. M. Q.; LACERDA, A. S.; MELO, P. R. A. Micropropagação da aroeira (Myracrodruon urundeuva Fr. All). Ciência e Agrotecnologia, v. 24, n. 1, p. 174-180, 2000., in micropropagation studies with the same species, who reported shoots with a mean height of 13 mm from nodal and apical segments inoculated in culture medium supplemented with 4.5 µM of BAP.

Gentile et al. (2017)GENTILE, A. FRATTARELLI, A.; NOTA, P.; CONDELLO, E.; CABONI, E. The aromatic cytokinin meta-topolin promotes in vitro propagation, shoot quality and micrografting in Corylus colurna L. Plant Cell, Tissue and Organ Culture, v. 128, n. 3, p. 693-703, 2017. also recorded mean values higher than those of the present study in cultures of Corylus colurna. For this species, the authors observed shoots 30 mm long in the presence of the highest concentration of mT under study (8.2 µM). Moreover, Gentile et al. (2014)GENTILE, A.; GUTIÉRREZ, M. J.; MARTINEZ, J.; FRATTARELLI, A.; NOTA, P.; CABONI, E. Effect of meta-topolin on micropropagation and adventitious shoot regeneration in Prunus rootstocks. Plant Cell, Tissue and Organ Culture, v. 118, n. 3, p. 373-381, 2014., using the cytokinins mT and benzyladenine at a concentration of 2.1 µM in Prunus insititia x domestica, found an increase in shoot length, which reached 31.9 mm with the use of mT.

All treatments under study showed callus formation. When the culture medium was added with 32 µM of mT, 80 % of the explants formed calluses with the use of the basal nodal segment; however, without statistical difference from the medial explant (75 %) in the same mT concentration. The value for the first explant did not differ from the percentages (55.56 and 66.33 %) for the same explant at 2.0 and 16 µM of mT, respectively (Table 4).

The increase in the percentage of explants that formed calluses with the addition of the cytokinin to the culture medium, especially at the highest concentrations, is possibly due to hormonal imbalances. Noteworthy, the explants used for shoot regeneration came from plant material germinated in vitro, possibly containing high concentrations of endogenous auxins.

Callus formation in M. urundeuva explants negatively influenced the shoot quality, especially for callus formed in explant regions very close to the buds (data not shown). Callus formation at the base of explants during the multiplication phase may compromise axillary bud proliferation and shoot elongation, affecting the in vitro development (Pereira et al. 2015PEREIRA, M. O.; NAVROSKI, M. C.; REINIGER, L. R. S. Multiplicação in vitro de ipê-amarelo (Handroanthus chrysotrichus). Nativa, v. 3, n. 1, p. 59-63, 2015.).

Figure 2
Myracrodruon urundeuva Fr. All. shoots at 45 days after inoculation. Values obtained from explants under concentrations of meta-topolin (mT). A) 16.0 µM of mT (medial segment); B) 2.0 µM of mT (basal segment); C) 16.0 µM of mT (basal segment); D) 32.0 µM of mT (basal segment).

Photos: Tecla dos Santos Silva

Some authors reported callus formation in explants during in vitro propagation in cultures of other species, including Mimosa caesalpiniifolia Benth. (Bezerra et al. 2014BEZERRA, R. M. F.; ALOUFA, M. A. I.; FREIRE, F. A. M.; SANTOS, D. D. Efeito de 6-benzilaminopurina sobre a propagação in vitro de Mimosa caesalpiniifolia Benth. (Fabaceae). Revista Árvore, v. 38, n. 5, p. 771-778, 2014.) and Handroanthus chrysotrichus (Pereira et al. 2015PEREIRA, M. O.; NAVROSKI, M. C.; REINIGER, L. R. S. Multiplicação in vitro de ipê-amarelo (Handroanthus chrysotrichus). Nativa, v. 3, n. 1, p. 59-63, 2015.).

The results of this research can be applied to the in vitro propagation of the species under study, also serving as a basis for further research seeking to optimize micropropagation protocols for M. urundeuva and other woody plants.

CONCLUSIONS

  1. It is possible to regenerate shoots in Myracrodruon urundeuva from cotyledonary node explants using BAP in association with NAA;

  2. The use of mT favors the shoot proliferation in basal and medial nodal segments; however, the large formation of calluses influences the shoot quality.

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Publication Dates

  • Publication in this collection
    01 Dec 2021
  • Date of issue
    2021

History

  • Received
    10 June 2021
  • Accepted
    23 Aug 2021
  • Accepted
    29 Sept 2021
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