270 IN VITRO PROPAGATION OF ZINGIBER SPECTABILE SCIENTIFIC ARTICLE In vitro propagation of Zingiber spectabile (1) MICHELE VALQUÍRIA DOS REIS(2), FERNANDA CARLOTA NERY(3), DÉBORA DE OLIVEIRA PRUDENTE(2), PATRÍCIA DUARTE DE OLIVEIRA PAIVA(4), RENATO PAIVA(2), RODRIGO THEREZAN FREITAS(5), DIOGO PEDROSA CORRÊA DA SILVA(4 ABSTRACT Zingiber spectabile is a tropical ornamental species with difficulties to obtain efficient propagation system. Thus, this study aimed to assess the in vitro propagation of Zingiber spectabile. Seed characterization was determined by measuring length, width and thickness, the weight of 1000 seeds and imbibition curve. In vitro germination of seeds was at constant (25 °C) or alternating temperatures (20-30 ºC). For optimization of in vitro multiplication, different concentrations of activated charcoal (0.0, 0.1 and 0.3%) and sucrose (0.0, 0.1, 0.3, 0.5 and 0.7 M) were evaluated. Plantlets were inoculated in flasks with different sealing systems (PVC covers with or without filters at the center) and culture media (MS or WPM). The plants were acclimatized in Plantmax® substrate. Seeds were of 6.06 mm length, 3.22 mm wide and 2.83 mm thick. The weight of 1,000 seeds corresponded to 46.4 g. The seed imbibition curve approaches to a tree phase pattern. Alternating temperatures induced high germination rates (68%). The addition of 0.3% activated charcoal provided higher root growth and plants with smaller number of senescent leaves. The best plant growth was obtained by the use of 0.1 M sucrose. All acclimatized plants survived (100%). The results demonstrate that Z. spectabile respond well to in vitro propagation. Keywords: beehive ginger, micropropagation, tropical plants. RESUMO Propagação in vitro de Zingiber spectabile Zingiber spectabile é uma espécie ornamental tropical com dificuldades para se obter um sistema de propagação eficiente. Assim, este estudo teve como objetivo avaliar a propagação in vitro de Z. spectabile. A caracterização das sementes foi determinada pela medição do comprimento, largura e espessura, pelo peso de 1.000 sementes e pela curva de embebição. A germinação in vitro das sementes foi avaliada em temperatura constante (25 °C) ou alternada (20-30 ºC). Para a otimização da multiplicação in vitro, diferentes concentrações de carvão ativado (0,0, 0,1 e 0,3%) e sacarose (0,0, 0,1, 0,3, 0,5 e 0,7 M) foram avaliadas. As plântulas foram inoculadas em frascos com diferentes sistemas de vedação (tampas de PVC com ou sem filtros no centro) e meio de cultura (MS ou WPM). As plantas foram aclimatizadas em substrato Plantmax®. As sementes apresentaram comprimento de 6,06 mm, largura de 3,22 cm e espessura de 2,83 mm. O peso de 1000 sementes correspondeu a 46,4 g. A curva de embebição de sementes se aproxima de um padrão trifásico. Temperaturas alternadas induziram altas taxas de germinação (68%). A adição de 0,3% de carvão ativado proporcionou maior crescimento radicular e plantas com menor número de folhas senescentes. O melhor crescimento de plantas foi obtido pelo uso de sacarose 0,1 M. Todas as plantas aclimatizadas sobreviveram (100%). Os resultados demonstraram que Z. spectabile responde bem à propagação in vitro. Palavras-chave: sorvetão, micropropagação, plantas tropicais. 1. INTRODUCTION medicines (SADHU et al., 2007). Rhizomes are rich in phenolic compounds with antioxidant and antibacterial As an attractive tropical ornamental plant, Zingiber properties, which can be used as a natural preservative in the spectabile (Zingiberaceae) is widely cultivated in Brazil food industry (SIVASOTHY et al., 2012a; 2013). Zerumbone due its high commercial value. This species is known is the most abundant component in the rhizome, which has as beehive ginger or ornamental ginger, and is used for been found to possess multiple biomedical properties, such landscaping and as a cut flower (VIÉGAS et al., 2012; as antiproliferative, antioxidant, anti-inflammatory, and LESSA et al., 2015). anticancer activities (SIVASOTHY et al. 2012a; RAHMAN Besides ornamental uses Z. spectabilie has others et al. 2014). A dimeric flavonol glycoside (Spectaflavoside important applications: leaves and rhizomes are used for A), a potent iron chelating agent, is also found in Z. spectabile culinary purposes and for the preparation of traditional rhizomes (SIVASOTHY et al., 2012b). DOI: http://dx.doi.org/10.14295/oh.v23i3.1035 (1)Received in 06/06/2017 and accepted in 16/09/2017 (2)Universidade Federal de Lavras (UFLA), Departamento de Biologia, Lavras-MG, Brazil. *Corresponding author: [email protected] (3)Universidade Federal de São João del Rei (UFSJ), Departamento de Engenharia de Biossitemas, São João del Rei-MG, Brazil. (4)Universidade Federal de Lavras (UFLA), Departamento de Agricultura, Lavras-MG, Brazil. (5)Universidade de São Paulo, Escola Superior de Agricultura “Luiz de Queiroz” (ESALQ), Piracicaba-SP, Brazil. Licensed by CC BY 4.0 V. 23, No. 3, 2017, p. 270-278 MICHELE VALQUÍRIA DOS REIS et al. 271 Despite its wide use and economic importance, large- documentation carried out using a Powershot A 640 scale propagation methods are not well established for this digital camera (Canon®, Tokyo, Japan). species. Z. spectabile is commonly propagated by plant The seed imbibition curve was determined with use of division or by rhizomes, which are not efficient methods four replicates of 25 seeds each (BRASIL, 2009), inside for commercial propagation of quality planting materials in crystal polystyrene boxes (Gerbox®) with Germtest® (because of possible transmission of diseases and very small paper substrate moistened with distilled water in the number of plants obtained). This species is susceptible to amount of 2.5 times the dry paper mass (BRASIL, 2009). several pests and diseases, nematodes and bacteria, which The seeds were stored in a B.O.D. (Biochemical Oxygen may be easily transmitted by conventional methods of Demand) incubator under constant light 36 μmol m-2 s-1 propagation (BEZERRA and LOGES, 2005). Therefore, irradiancy, provided by fluorescent lamps (20 W, Osram, the use of tissue culture techniques as micropropagated Brasil) at a temperature of 30 °C. Initially, the seeds plants of Z. spectabile is highly recommended to produce a were weighed every 30 minutes during the first 8 hours healthy planting stock in large scale. of imbibition by means of an analytical precision scale Tissue culture techniques have been reported as an (0.0001 g). After this time, the seeds were weighed at efficient method to several species of Zingiber, as Z. intervals of three hours, marked from the beginning of the officinale, Z. rubens, Z. moran and Z. zerumbet (HAMIRAH experiment, ending the weighing after the primary root et al., 2010; MOHANTY et al., 2011; ABBAS et al., 2011; protrusion of 50% of seeds. At every weighing, the seeds DAS et al., 2013; SERAN, 2013; YEPTHOMI and MAITI, were removed from the Gerbox® and placed over paper to 2014; NIRMAL BABU et al., 2016). However, there are absorb external moisture, weighed and then returned to not completed established protocol to in vitro Z. spectabile, the Gerbox® and B.O.D. The percentage increase over the just few studies in some steps of micropropagation of this initial fresh weight was determined. species are available (FARIA and ILLG, 1995; OLIVEIRA To evaluate the germination rate, seeds were placed et al., 2010; MOREIRA et al., 2012; NERY et al., 2015). In on MS medium (MURASHIGE and SKOOG, 1962) none of these studies were used seed to in vitro established with 0.7% agar (w/v), 0.09M sucrose (w/v), pH 5.8, and and propagation. maintained at 100% relative humidity. Germination tests Therefore, this study aimed to determine seed were conducted in B.O.D. either at constant temperature characteristics and protocol for in vitro propagation (seed (25 °C) or alternating temperatures (20-30 ºC) during night germination, growth conditions and acclimatization) of Z. and day, respectively, constant light with photosynthetic spectabile to produce large quantities of quality plants for photon flux density of 36 μmol m-2 s-1, provided by the ornamental market. fluorescent lamps (20 W). The germination parameter was evaluated after 30 days. 2. MATERIALS AND METHODS Germination was assessed using the root protrusion at ± 2.0 mm as criteria (MAGUIRE, 1962). The germination Seeds characterization and germination speed index (GSI) was also determined and calculated Seeds of Z. spectabile with black color (mature seed) according to the equation proposed by Maguire (1962). were randomly selected from a population of 30 plants. The The treatments were arranged in a completely randomized moisture content of seeds was determined by drying the design (CRD) with three replications of 10 seeds per seed in oven at 105 ± 2 ºC for 24 hours (BRASIL, 2009). treatment. Three replicates were used with 10 seeds each. A digital caliper (accurate to 0.5 mm) was used to In vitro multiplication measure length (longitudinal distance between the apex Nodal segments of Z. spectabile in vitro established was and the base - cm), width (measured perpendicularly to the used as explants for axillary shoot induction in MS medium middle region of the seeds - cm) and thickness of the seeds. supplemented with 4 mg L-1 6-benzyladenine (BA), 0.09 M The weight of 1000 seeds was determined using eight sucrose (w/v) and 0.7% agar (w/v), pH 5.8 (NERY et al., replicates of 100 seeds, weighed on analytical balance 2015). The material was stored in B.O.D. under 16-hours accurate to 0.001 g, according to Brasil (2009). Seed photoperiod light, photosynthetic photon flux density coats were subjected to manual cross-section cuts using of 36 μmol m-2 s-1 provided by fluorescent lamps (20W) a scalpel. Sections were stained by a mixture (9:1) of at a temperature of 25 ± 2 °C.