A Regenerative Route for Eugenia Uniflora L. (Myrtaceae) Through in Vitro Germination and Micropropagation
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Ann. For. Res. 57(1): 39-45, 2014 ANNALS OF FOREST RESEARCH DOI: 10.15287/afr.2014.179 www.afrjournal.org A regenerative route for Eugenia uniflora L. (Myrtaceae) through in vitro germination and micropropagation P.R. Diniz da Silva, R.G. Rispoli, M.M. Minozzo, L.H. Jobim, M. Junges, V.M. Stefenon Diniz da Silva P.R., Rispoli R.G., Minozzo M.M., Jobim L.H., Junges M., V.M. Ste- fenon 2014. A regenerative route for Eugenia unifl ora L. (Myrtaceae) through in vitro germination and micropropagation. Ann. For. Res. 57(1): 39-45, 2014. Abstract. Eugenia uniflora is a tree species native from Central and South America, largely employed in the popular medicine, in the cosmetics and pharmaceutical industries and also consumed in natura. Aiming to pro- vide plant material with high sanity and genetic uniformity for the estab- lishment of commercial plantations, we developed a protocol for seeds disinfestation, in vitro germination and in vitro propagation of this spe- cies through organogenesis. Fruits of E. uniflora were obtained from wild trees growing in the Pampa biome, Southern Brazil. Seeds were disinfest- ed using ethanol 70% (10 min) and NaOCl 1.25% (10 or 25 min). Shoot apexes and nodal segments of non-contaminated plantlets were cultivated in verification medium AS30 during 20 days, posteriorly in ½MS medium supplemented with sucrose, IBA and BAP during 45 days and acclimatized in greenhouse. Disinfesting seeds with ethanol 70% (10 min) and NaOCl 1.25% (25 min) allowed germination with significantly lower contami- nation (2.0%) and production of healthy explants for the micropropaga- tion. No difference concerning size and contamination was observed for the propagation using shoot apexes or nodal segments as explant. Accli- matized plants revealed normal phenotype and healthy appearance. This regenerative route can be applied for mass clonal propagation from seeds of cross-pollinated or self-pollinated selected trees aiming the establish- ment of commercial plantations of E. uniflora and other Myrtaceae species. Keywords Brazilian cherry, organogenesis, tree breeding, conservation of genetic resources. Authors. Paulo Roberto Diniz da Silva, Rosa Gomes Rispoli, Mônica Munareto Minozzo, Laura Honório Jobim, Marina Junges, Valdir Marcos Stefenon (val- [email protected]) - Federal University of the Pampa, Av. Antonio Trilha, 1847, 97300-000, São Gabriel, RS, Brazil. Manuscript received February 12, 2014; revised May 06, 2014; accepted May 12, 2014; online fi rst May 15, 2014. 39 Ann. For. Res. 57(1): 39-45, 2014 Research article Introduction ed and deforested areas, as well as in urban forestry and in non-wood products forestry. Eugenia unifl ora L. (Myrtaceae, Myrtoideae) Although in vitro techniques have been is a tree species native to Central and South recommended for the reproduction and germ- America. In Brazil, it is found in different bi- plasm conservation of E. unifl ora (Pilatti et omes along rivers in riparian forests, in coastal al. 2011), only a few studies (Uematsu et al. forest formations and in semidecidual forests 1999, Souza et al. 2008, Lattuada 2010) have (Margis et al. 2002, Salgueiro et al. 2004). This focused on the micropropagation of shoots species is an important source of food for a va- from plants growing in greenhouse. In addi- riety of birds and mammals in the forests and tion, obtaining explants free of bacterial and has major signifi cance in the natural regenera- fungal contamination is a major concern for tion (Almeida et al. 2012). the micropropagation of selected plants of E. Due to the small to medium size and the unifl ora (Lattuada 2008). multi-branched stem, E. unifl ora lacks value Since, in developing countries, rural popu- as a timber species, but is largely employed lation derive a signifi cant part of their food in the popular medicine, in the cosmetics and requirements and economic profi ts from for- pharmaceutical industries and also consumed est species (FAO 1986), the micropropagation in natura, as juice and ice cream (Almeida may advance the forestry use of E. unifl ora in et al. 2012, Costella et al. 2012). In addition, South America, through the establishment of this light-demanding tree species can be em- clonal plantations of selected trees. In such a ployed in the restoration of degraded areas and case, the development of genetic improvement deforested zones. The potential of E. unifl ora programs is important and the use of controlled for forestry includes a growing concern in col- cross-pollination or self-pollination of selected lecting and conserving the species germplasm trees followed by in vitro germination of the across the world. Over 300 selected genotypes seeds and micropropagation of the plantlets is are conserved in germplasm banks in Brazil, an attractive alternative. USA, Australia, Africa, Central America and Following this rationality, towards the ad- Asia (Lattuada 2008). vance of genetic improvement programs for E. Despite the broad utility and commercial unifl ora and the establishment of commercial perspectives for this species, the establish- plantations of this species, this study aimed to ment of E. unifl ora orchards in South Brazil develop a protocol for disinfestation of select- has been practiced mainly for household con- ed seeds, in vitro germination and in vitro sumption, without commercial aspirations. multiplication of E. unifl ora through organo- Moreover, the nursery stocks of E. unifl ora are genesis. typically propagated through randomly col- lected seedlings, so that such plantations lack morphological, physiological and genetic uni- Material and methods formity, making the commercialization of the fruits diffi cult. The in vitro micropropagation Studied species and sample collection is a viable method for mass propagation of tree species, providing plants with high sanity and Eugenia unifl ora is a rather versatile species clonal homogeneity. Thus, the development of and grows in several different habitats includ- in vitro regenerative protocols in E. unifl ora ing riparian forests, arid and semiarid environ- has direct applications for clonal mass propa- ments. It can be found as a medium tree 4-5 m gation of selected plants and may be integrated tall, reaching up to 12 m. The plants are her- into breeding programs, restoration of degrad- maphrodites, with small white fl owers having 40 Diniz da Silva et al. A regenerative route for Eugenia uniflora L. ... four petals and several stamens. Insects polli- In vitro multiplication nate the species and fl owering happens twice a year, in January and September. Fruit ripening After 45 days, the non-contaminated plantlets occurs in February and October, approximate- from each treatment (10 min and 25 min im- ly fi ve to six weeks after fl owering. Seeds are mersion in NaOCl) that presented more than recalcitrant (Pilatti et al. 2011) and their dis- 3.0 cm length were incised in 1.5 cm segments persion occurs through animals, mainly birds as proposed by Souza et al. (2007). The ex- (Almeida et al. 2012). Natural self-pollination plants (shoot apex or nodal segment) were in- seems to occur, but the fruit development is oculated in 180 x 20 mm test tubes containing not effective, reaching less than 7.0% of the 10 mL of verifi cation medium AS30, composed self-pollinated fl owers (Franzon 2008). by agar 7.5 g L-1 and sucrose 30 g L-1 (Figure The seeds employed in this study were ob- 1B). Before explant inoculation, test tubes tained from four wild genotypes growing in were sealed with plastic fi lm and autoclaved the Brazilian Pampa, Rio Grande do Sul State, during 17 min at 121ºC and 1.2 atm. This me- Southern Brazil. These genotypes were select- dium was employed to verify the existence of ed based on the large size and appealing color endogenous bacteria and fungi, which could of the fruits, which reach commercial require- emerge after germination. The explants were ments. For this experiment, 100 healthy ripe evaluated after one, nine and 20 days of inocu- fruits were randomly collected during the sum- lation in this medium. The size of the explants mer season of the year 2012, stored in paper and the number of buds were registered in the bags and transported to the laboratory within 20th day of culture. 24 hours. After 20 days in the verifi cation medium, non-contaminated shoot apices and nodal seg- Seeds disinfestation and in vitro germina- ments were transferred to test tubes containing tion 10 mL of ½MS medium (Murashig & Skoog 1962) pH 5.8 (Figure 1C). Based on our prior After removed from the fruits, seeds were di- tests of the culture medium composition for E. vided in two samples of 50 seeds each. Seeds unifl ora, the ½MS medium was supplemented were surface disinfected in a fl ow chamber with 30 g L-1 of sucrose, 0.1 mg L-1 indolebutyr- with ethanol 70% (1 min), 1.25% sodium hy- ic acid (IBA), 0.2 mg L-1 6-benzylaminopurine pochlorite solution (NaOCl) during 10 min (50 (BAP) and solidifi ed with 6.0 g L-1 of agar. All seeds) or 25 min (50 seeds) and rinsed three reagents were purchased from Sigma-Aldrich. times in sterile distillated water. Each seed was The ½MS medium was distributed in 180x20 individually inoculated in test tubes containing mm test tubes (10 mL/tube), tubes were sealed 10 mL of germination medium (Figure 1A), with plastic fi lm and autoclaved as described composed by 6.0 g L-1 agar (Sigma-Aldrich, above. St Louis, MO) in ultrapure water. Before ex- Explants were cultured during 45 days in a plant inoculation, the germination medium completely randomized blocks design, with was distributed in 180 x 20 mm test tubes (10 two treatments (shoot apices and nodal seg- mL/tube), which were sealed with plastic fi lm ments) and 40 replicates.