HTML

ePub

PDF

Introduction

Acca sellowiana (O. Berg) Burret (Myrtaceae), known as feijoa or pineapple-guava, is a Brazilian native species of the Atlantic Forest, naturally occurring in the States of Santa Catarina and Rio Grande do Sul, with secondary dispersion in Uruguay (Guerra, Cangahuala-Inocente, Dal Vesco, Pescador, & Caprestano, 2013; Cristofolini et al., 2014). This species is typical of the understory of mature formations of the Mixed Ombrophilous Forest, at altitudes above 1,000 m (Finatto et al. 2011).

The high market value of A. sellowiana fruits and flowers makes this species an option for farmers located in high altitude regions. The development of its production would allow the processing of the fruits in products with high commercial value, such as juices, fermented and distilled beverages, ice cream and candied fruits (Vuotto et al., 2000; Ruberto & Tringali, 2004; Beyhan, Elmastaş, & Gedikli, 2010; Weston, 2010). Moreover, this species impacts directly on the recovery of degraded land, riparian forests, permanent preservation areas, legal reserves, and constitution of agro-forest systems (Gomes, Oliveira, Ferreira, & Batista, 2016).

Despite being a native species of Latin America, its cultivation is predominant in countries such as New Zealand, Colombia and Turkey (Guerra et al., 2016), and the increase of cultivation in Brazil is conditioned to the improvement of propagation techniques. Tissue culture techniques, mainly associated to somatic embryogenesis (SE), has the advantage of allowing mass and clonal micropropagation for selected genotypes of this species (Guerra et al., 2016).

SE is an analogous process to zygotic embryogenesis and is based on the totipotency of plant cells to form embryos from somatic cells (Karami & Saidi, 2010). This technique has been extensively studied and improved in the last two decades for A. sellowiana; however, somatic embryos maturation and conversion still present low rates (Guerra et al., 2016).

The γ-glutamylcysteinylglycine or glutathione (GSH-reduced form) is a thiol tripeptide involved in the process of cell division and differentiation, with effect on the induction and control of SE in several plant species (Belmonte, Donald, Reid, Yeung, & Stasolla, 2005; Vieira et al., 2012; Fraga et al., 2016). GSH supplemented to culture medium during the early stages of SE creates a reducing environment, enhancing cell proliferation and the development of immature somatic embryos (Stasolla, 2010). In Araucaria angustifolia, GSH supplementation in the culture medium increased the somatic embryos formation in the initial phase of development, related to increased nitric oxide emission (Vieira et al., 2012). Similarly, GSH supplementation also increased the number of somatic embryos of Podocarpus lambertii obtained during maturation stage, besides improving morphological features (Fraga et al., 2016).

Somatic embryo development is a complex multi-step process, which demands high energy (Konrádová, Lipavská, Albrechtová, & Vreugdenhil, 2002; Dinakar, Djilianov, & Bartels, 2012). During early stages, establishment and growth of embryo structures prevails, while later stages, such as maturation, are characterized by deposition of storage compounds (Konrádová et al., 2002). Thus, the transition phases during SE is accompanied by changes in carbohydrate metabolism (Iraqi & Tremblay, 2001). In A. angustifolia, the use of maltose as a carbohydrate source promoted the embryonic tissues differentiation, which was attributed to its slow hydrolysis (Steiner, Vieira, Maldonado, & Guerra, 2005).

In this context, the aim of the present study was to evaluate the effect of different GSH concentrations on the SE induction in A. sellowiana and to compare different carbon sources (sucrose and maltose) supplementation during the step of somatic embryos conversion to plantlets.

Material and methods

Plant material

Seeds of A. sellowiana (accession 101) were collected from fruits of the germplasm collection of the Research and Extension Agency of Santa Catarina State (Epagri), São Joaquim, south Brazil (latitude 28º 17' 39", longitude 49º 55' 56", altitude 1415 m). The fruits were transported in plastic boxes and the seeds were extracted and disinfested in laminar flow chamber with ethanol (70%) for 1 min and sodium hypochlorite (2%) for 20 min, as described by Guerra, Dal Vesco, Ducroquet, Nodari, and Reis (2001).

Somatic embryogenesis induction

Mature zygotic embryos were excised and incubated in a solution with 2,4-dichlorophenoxyacetic acid (200 μM) for 1 hour, according to Fraga et al. (2012), and then inoculated into a Petri dish containing 25 mL of culture medium consisting of LPm macro and microsalts (von Arnold & Eriksson, 1981) supplemented with Morel vitamins (Morel & Wetmore, 1951), maltose (30 g L-1) and glutamic acid (1.35 g L-1). In this step, different concentrations of GSH (0, 0.1, 0.5 and 1 mM) were tested, consisting of 4 treatments. The pH of culture medium was adjusted to 5.8, gelled with Phytagel® (2 g L-1) and autoclaved at 121°C for 15 min. Cultures were maintained in the absence of light at 25 ± 2°C.

The experimental design was completely randomized, consisting of 4 treatments and 6 replicates, and each sample unit corresponded to 5 zygotic embryos. The percentage of SE induction after 50 and 80 days in culture as well as the number of somatic embryos obtained at each embryonic stage after 80 days in culture were evaluated. The number of responsive and non-responsive explants was submitted to chi-square analysis at 5% probability. These results are presented as induction percentage throughout the manuscript. For the number of somatic embryos at different developmental stages, analysis of variance was performed followed by SNK means separation test at 5% of probability.

Somatic embryos conversion

Torpedo- and pre-cotyledonary-staged somatic embryos were selected, isolated and inoculated in Petri dishes containing 25 mL of conversion culture medium. This culture medium was composed by LPm macro- and micro-salts, supplemented with Morel vitamins, glutamic acid (1.35 g L-1), gibberellic acid (1 μM), fluridone (0.05 μM), 6-benzylaminopurine (0.5 μM) and activated charcoal (1.5 g L-1). In this step, two different carbon sources were tested: sucrose or maltose (30 g L-1). The pH of culture medium was adjusted to 5.8, gelled with Phytagel® (2 g L-1) and autoclaved at 121°C for 15 min. Cultures were maintained in a growth room at 25 ± 2°C and 16 hour photoperiod, with a light intensity of 40-50 µmol m2 s-1 provided by white LED lamps (GreenPower TLED W; PhilipsTM).

The experimental design was completely randomized blocks, with 2 treatments and 8 replicates, with each sample unit consisting of 30 somatic embryos. The number of somatic embryos converted to normal chlorophyllous, normal achlorophyllous and abnormal plantlets after 4 weeks in culture was evaluated. We considered normal plantlets those with two cotyledons, radicle protrusion and presence of chlorophyll. Abnormal somatic plantlets were those which showed only one cotyledon, fused cotyledons, more than two cotyledons, and/or no radicle protrusion. Data were submitted to analysis of variance followed by SNK means separation test at 5% of probability.

Results and discussion

Effects of different GSH concentrations on SE induction

GSH-supplemented treatments resulted in improved SE induction rates (~70%) as compared to the control GSH-free (~35%) after 50 days in culture, with no statistical difference between GSH-supplemented treatments. After 80 days in culture there was no statistical difference regarding the induction rate (Table 1).

Although the total number of somatic embryos did not differ between the treatments evaluated after 80 days in culture, significant differences were observed in relation to the embryonic stages obtained in the different treatments (Table 2 and Figure 1). At day 80 in culture, 0.5 and 1 mM GSH-supplemented treatments indicated the largest portion of somatic embryos obtained in torpedo stage (Figure 2). Differently, treatments supplemented with 0 and 0.1 mM GSH showed equal amounts of somatic embryos at all embryonic stages.

Effects of different carbon sources on somatic embryos conversion

During the somatic embryos conversion step, different plantlets phenotypes were observed: normal chlorophyllous, normal achlorophyllous and abnormal somatic plantlets (Figure 3). The percentage of conversion to normal somatic plantlets of both treatments evaluated, supplemented with sucrose or maltose, did not indicate significant differences, with both treatments showing about 20% conversion rate (Table 3). However, a higher percentage of normal achlorophyllous plantlets were observed in maltose-supplemented treatment. Among the rate of abnormal somatic plantlets obtained, no statistical differences were observed among the evaluated treatments (Table 3).

Effects of different GSH concentrations on SE induction

Our results indicated an improved induction rate in GSH-supplemented treatments at day 50 of induction. Fraga et al. (2016) performed experiments with different GSH concentrations (0, 0.1 and 0.5 mM) during the initial maturation stage of Podocarpus lambertii embryogenic cultures and obtained a higher rate of somatic embryos formation at 0.5 mM GSH treatment after 10 days of maturation. The results of these authors are in agreement with those found in the present study, suggesting that GSH has an effect on somatic embryos formation of A. sellowiana, by accelerating this process.

Vieira et al. (2012) observed an increased initial somatic embryo formation of A. angustifolia in culture medium supplemented with low GSH concentrations (0.01 and 0.1 mM). However, when embryogenic cultures were maintained in the same culture medium for more than seven days of culture, there was a decrease in polarization and reduction of normal somatic embryos.

Results found in the present study also indicated that GSH supplementation improves the synchrony in the somatic embryo formation of A. sellowiana, increasing the number of somatic embryos in more advanced developmental stages (Table 2 and Figure 2). Similarly, GSH supplementation in the somatic embryo maturation stage increased the number and quality of Podocarpus lambertii somatic embryos (Fraga et al., 2016). In Picea glauca, GSH addition to the maturation culture medium increased the subsequent somatic embryo conversion rate, and the authors related this result to the expression of genes associated to embryonic development and morphological changes during the maturation stage (Stasolla et al., 2004).

The manipulation of GSH/GSSG (glutathione disulfide) ratio during the final maturation process may be an interesting strategy to improve the rate of normal embryos at maturation stage (Stasolla, 2010; Vieira et al., 2012; Fraga et al., 2016). According to Stasolla (2010), the somatic embryos transfer during the maturation stage to a more oxidized culture medium supplemented with GSSG favors the tissue differentiation and further somatic embryos maturation.

In experiments with Picea glauca, an increased formation of somatic embryos in culture medium supplemented with 0.1 mM GSH was observed, and when somatic embryos were subsequently transferred to culture medium supplemented with GSSG, the number of cotyledonary-staged somatic embryos was improved compared to GSSG-free treatment (Belmonte & Yeung, 2004; Belmonte et al., 2005). Similarly, Pullman et al. (2015) also obtained increased mature somatic embryos of Pinus taeda in culture medium supplemented with GSSG. Thus, the manipulation of GSH/GSSG ratio during maturation stage may be an appropriate strategy to improve SE in A. sellowiana.

Effects of different carbon sources on somatic embryos conversion

In the present work, the conversion percentage in normal chlorophyllous plantlets was about 20% in both treatments evaluated (Table 3), which is equivalent to previous reports for this species, varying between 20 and 35% (Cangahuala-Inocente, Dal Vesco, Steinmacher, Torres, & Guerra, 2007; Fraga et al., 2012). Furthermore, no differences could be observed between conversion treatments regarding the number of normal chlorophyllous plantlets obtained.

Conflicting results can be found in the literature regarding the use of sucrose or maltose in the somatic embryos conversion. Different carbon sources, including maltose and sucrose, were evaluated in the conversion culture medium of Catharanthus roseus somatic embryos into plantlets, and the best results were obtained with 3-6% sucrose or 3% fructose (Junaid, Mujib, Bhat, & Sharma, 2006). In contrast, the highest number of mature somatic embryos from Ahirs nordmanniana and the highest conversion percentage into plantlets were obtained in maltose-supplemented culture medium, as compared to sucrose (Norgaard, 1997). Similarly, an improved number of mature somatic embryos and conversion rate of Castanea sativa were obtained in culture medium supplemented with maltose (Corredoira, Ballester, & Vieitez, 2003).

In the present study, no differences were observed between treatments for the percentage of abnormal and abnormal achlorophyllous somatic embryos, indicating that the use of maltose instead of sucrose does not interfere in abnormal somatic embryos formation. However, when the number of normal achlorophyllous somatic embryos was evaluated, maltose-supplemented treatment resulted in higher rates (19.47%, as compared to 10.94% in sucrose-supplemented treatment). This result suggests that maltose may have a detrimental effect on the chlorophyll biosynthesis during somatic embryos conversion in A. sellowiana.

There are no reports in the literature about inhibition of chlorophyll formation due to maltose supplementation, and according to Thorpe et al. (2008), sugars do not inhibit the chlorophyll synthesis, except for sucrose in high concentrations. In the present work, the highest percentage of normal chlorophyllous somatic plantlets in response to sucrose treatment can be explained by the fact that sucrose represents the most efficient and abundant translocated sugar in plants, being the most important carbon source for non-photosynthetic tissues and for plant respiration (Taiz & Zeiger, 2009).

In addition to being essential carbon sources, sugars also act as osmotic regulators in the culture medium, which may also have an effect on the percentage of chlorophyllous somatic plantlets observed in the present study. In an experiment to convert wheat somatic embryos, Zhou, Zheng, and Konzak (1991) observed a higher number of chlorophyllous plantlets in the sucrose-supplemented treatment compared to maltose. These authors attributed this result to the increased osmolarity of the culture medium in the sucrose-supplemented treatment due to its rapid hydrolysis and conversion to fructose and glucose (Zhou et al., 1991). Thus, the sucrose supplementation during somatic embryo conversion of A. sellowiana may be more suitable for chlorophyllous somatic plantlets formation.

Conclusion

The results indicated that GSH supplementation to the induction culture medium accelerates the process of somatic embryos formation and increases the embryonic synchrony in A. sellowiana. The use of maltose during somatic embryos conversion into plantlets showed no superior effect on the obtainment of normal chlorophyllous somatic plantlets compared to sucrose-supplemented treatment; however, this supplementation increased the formation of normal achlorophyllous somatic plantlets. Thus, the use of sucrose during somatic embryos conversion step has an apparently physiological advantage over maltose, besides its low cost and easy accessibility. As future perspectives, the manipulation of GSH/GSSG ratio during somatic embryo maturation may be an appropriate strategy to improve SE in this species. In addition, the use of other carbon sources and in different concentrations is seen as a possibility to improve the rate of somatic embryos conversion for this species.

Acknowledgements

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Proc. 478393/2013-0, and 306126/2013-3), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), and Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina (Fapesc, Proc. 2780/2012-4). The authors are also grateful to Dr. Clarissa Alves Caprestano for all contributions during the experiments implementation, and to Raul Solano Ribeiro for reviewing the manuscript.

References

Belmonte, M. F. & Yeung, E. C. (2004). The effects of reduced and oxidized glutathione on white spruce somatic embryogenesis. In Vitro Cellular and Developmental Biology-Plant, 40(1), 61-66. doi: 10.1079/IVP2003483

Belmonte, M. F., Donald, G., Reid, D. M., Yeung, E. C., & Stasolla, C. (2005). Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca). Journal of Experimental Botany, 56(419), 2355-2364. doi: 10.1093/jxb/eri228

Beyhan, O., Elmastaş, M., & Gedikli, F. (2010). Total phenolic compounds and antioxidant capacity of leaf, dry fruit and fresh fruit of feijoa (Acca sellowiana, Myrtaceae). Journal of Medicinal Plants Research, 4(11), 1065-1072. doi: 10.5897/JMPR10.008

Cangahuala-Inocente, G. C., Dal Vesco, L. L., Steinmacher, D., Torres, A. C., & Guerra, M. P. (2007). Improvements in somatic embryogenesis protocol in Feijoa (Acca sellowiana (Berg) Burret): induction, conversion and synthetic seeds. Scientia Horticulturae, 111(3), 228-234. doi: 10.1016/j.scienta.2006.10.030

Corredoira, E., Ballester, A., & Vieitez, A. M. (2003). Proliferation, maturation and germination of Castanea sativa Mill. somatic embryos originated from leaf explants. Annals of Botany, 92(1), 129-136. doi: 10.1093/aob/mcg107

Cristofolini, C., Vieira, L. N., Fraga, H. P. F., Costa, I. R., Guerra, M. P., & Pescador, R. (2014). DNA methylation patterns and karyotype analysis of off-type and normal phenotype somatic embryos of Feijoa. Theoretical and Experimental Plant Physiology, 26(3), 217-224. doi: 10.1007/s40626-014-0020-4

Dinakar, C., Djilianov, D., & Bartels, D. (2012). Photosynthesis in desiccation tolerant plants: Energy metabolism and antioxidative stress defense. Plant Science, 182, 29-41. doi: 10.1016/j.plantsci.2011.01.018

Finatto, T., Santos, K. L., Steiner, N., Bizzocchi, L., Holderbaum, D. F., Ducroquet, J. P. H. J., … Nodari, R. O. (2011). Late-acting self-incompatibility in Acca sellowiana (Myrtaceae). Australian Journal of Botany, 59(1), 53-60. doi: 10.1071/BT10152

Fraga, H. P. F., Vieira, L. N., Caprestano, C. A., Steinmacher, D. A., Micke, G. A., Spudeit, D. A., ... Guerra, M. P. (2012). 5-Azacytidine combined with 2,4-D improves somatic embryogenesis of Acca sellowiana (O. Berg) Burret by means of changes in global DNA methylation levels. Plant Cell Reports, 31(12), 2165-2176. doi: 10.1007/s00299-012-1327-8

Fraga, H. P. F., Vieira, L. N., Puttkammer, C. C., Santos, H. P., Garighan, J., & Guerra, M. P. (2016). Glutathione and abscisic acid supplementation influences somatic embryo maturation and hormone endogenous levels during somatic embryogenesis in Podocarpus lambertii Klotzsch ex Endl. Plant Science, 253, 98-106. doi: 10.1016/j.plantsci.2016.09.012

Gomes, J. P., Oliveira, L. M., Ferreira, P. I., & Batista, F. (2016). Substratos e temperaturas para teste de germinação em sementes de Myrtaceae. Ciência Florestal, 26(1), 285-293. doi: 10.5902/1980509821120

Guerra, M. P., Cangahuala-Inocente, G. C., Dal Vesco, L. L., Pescador, R., & Caprestano, C. A. (2013). Micropropagation systems of feijoa (Acca sellowiana (O. Berg) Burret). In M. Lambardi, E. A. Ozudogru, & S. M. Jain (Eds.), Protocols for micropropagation of selected economically-important horticultural plants (p. 45-62). New York, NY: Springer.

Guerra, M. P., Dal Vesco, L. L., Ducroquet, J. P. H. J., Nodari, R. O., & Reis, M. S. (2001). Somatic embryogenesis in Goiabeira serrana: genotype response, auxinic shock and synthetic seeds. Revista Brasileira de Fisiologia Vegetal, 13(2), 117-128. doi: 10.1590/S0103-31312001000200001

Guerra, M. P., Fraga, H. P. F., Vieira, L. N., Ree, J. F., Heringer, A. S., & Maldonado, S. B. (2016). Fundamentals, advances and applications of somatic embryogenesis in selected Brazilian native species. Acta Horticulturae, 1113, 1-12. doi: 10.17660/ ActaHortic.2016.1113.

Iraqi, D., & Tremblay, F. M. (2001). The role of sucrose during maturation of black spruce (Picea mariana) and white spruce (Picea glauca) somatic embryos. Physiologia Plantarum, 111(3), 381-388. doi: 10.1034/j.1399-3054.2001.1110316.x

Junaid, A., Mujib, A., Bhat, M. A., & Sharma, M. P. (2006). Somatic embryo proliferation, maturation and germination in Catharanthus roseus. Plant cell, Tissue and Organ Culture, 84(3), 325-332. doi: 10.1007/s11240-005-9041-7

Karami, O., & Saidi, A. (2010). The molecular basis for stress-induced acquisition of somatic embryogenesis. Molecular Biology Reporter, 37(5), 2493-2507. doi: 10.1007/s11033-009-9764-3

Konrádová, H., Lipavská, H., Albrechtová, J., & Vreugdenhil, D. (2002). Sucrose metabolism during somatic and zygotic embryogeneses in Norway spruce : content of soluble saccharides and localisation of key enzyme activities. Journal of Plant Physiology, 159(4), 387-396. doi: 10.1078/0176-1617-00624

Morel, G., & Wetmore, R. H. (1951). Tissue culture of monocotyledons. American Journal of Botany, 38(2), 138-140. doi: 10.2307/2437836

Norgaard, J. V. (1997). Somatic embryo maturation and plant regeneration in Abies nordmanniana Lk. Plant Science, 124(2), 211-221. doi: 10.1016/S0168-9452(97)04614-1

Pullman, G. S., Zeng, X., Copeland-Kamp, B., Crockett, J., Lucrezi, J., May, S. W., & Bucalo, K. (2015). Conifer somatic embryogenesis: improvements by supplementation of medium with oxidation–reduction agents. Tree Physiology, 35(2), 209-224. doi: 10.1093/treephys/tpu117

Ruberto, G., & Tringali, C. (2004). Secondary metabolites from the leaves of Feijoa sellowiana Berg. Phytochemistry, 65(21), 2947-2951. doi: 10.1016/j.phytochem.2004.06.038

Stasolla, C. (2010). Glutathione redox regulation of in vitro embryogenesis. Plant Physiology and Biochemistry, 48(5), 319-327. doi: 10.1016/j.plaphy.2009.10.007

Stasolla, C., Belmonte, M. F., Van Zyl, L., Craig, D. L., Liu, W., Yeung, E. C., & Sederoff, R. R. (2004). The effect of reduced glutathione on morphology and gene expression of white spruce (Picea glauca) somatic embryos. Journal of Experimental Botany, 55(397), 695-709. doi: 10.1093/jxb/erh074

Steiner, N., Vieira, F. N., Maldonado, S., & Guerra, M. P. (2005). Effect of carbon source on morphology and histodifferentiation of Araucaria angustifolia embryogenic cultures. Brazilian Archives of Biology and Technology, 48(6), 895-903. doi: 10.1590/S1516-89132005000800005

Taiz, L., & Zeiger, E. (2009). Fisiologia vegetal (4 ed.). Porto Alegre, RS: Artmed.

Thorpe, T., Stasolla, C., Yeung, E. C., Klerk, G.-J., Roberts, A., & George, E. F. (2008). The components of plant tissue culture media II: organic additions, osmotic and pH effects, and support systems. In E. F. George, M. A. Hall, & G. J. Klerk (Eds.), Plant propagation by tissue culture – volume 1. The background (p. 105-176). Dorderecht, NL: Springer.

Vieira, L. N., Santa-Catarina, C., Fraga, H. P. F., Santos, A. L. W., Steinmacher, D. A., Schlogl, P. S., … Guerra, M. P. (2012). Glutathione improves early somatic embryogenesis in Araucaria angustifolia (Bert) O. Kuntze by alteration in nitric oxide emission. Plant Science, 195, 80-87. doi: 10.1016/j.plantsci.2012.06.011

von Arnold, S., & Eriksson, T. (1981). In vitro studies of adventitious shoot formation in Pinus contorta. Canadian Journal of Botany, 59(5), 870-874. doi: 10.1139/b81-121

Vuotto, M. L., Basile, A., Moscatiello, V., Sole, P., Castaldo-Cobianchi, R., Laghi, E., & Ielpo, M. T. L. (2000). Antimicrobial and antioxidant activities of Feijoa sellowiana fruit. International Journal of Antimicrobial Agents, 13(3), 197-201. doi: 10.1016/S0924-8579(99)00122-3

Weston, R. J. (2010). Bioactive products from fruit of the feijoa (Feijoa sellowiana, Myrtaceae): A review. Food Chemistry, 121(4), 923-926. doi: 10.1016/j.foodchem.2010.01.047

Zhou, H., Zheng, Y., & Konzak, C. F. (1991). Osmotic potential of media affecting green plant percentage in wheat anther culture. Plant Cell Reports, 10(2), 63-66. doi: 10.1007/BF00236458

Author notes

miguel.guerra@ufsc.br

IR