Pak. J. Bot ., 46(6): 2179-2187, 2014. DIRECT ORGANOGENESIS AND LEAF-ANATOMY MODIFICATIONS IN VITRO OF NEOREGELIA CONCENTRICA (VELLOZO) L.B. SMITH (BROMELIACEAE) JOÃO PAULO RODRIGUES MARTINS 1* , EDILSON ROMAIS SCHMILDT 2, RODRIGO SOBREIRA ALEXANDRE 2, EVARISTO MAURO DE CASTRO 1, THAÍS FURTADO NANI 1, MARINÊS FERREIRA PIRES 1AND MOACIR PASQUAL 1 1Federal University of Lavras, Biology Department, Lavras, MG, Brazil 2Federal University of Espírito Santo, Agricultural and Biological Sciences Department, São Mateus, ES, Brazil *Corresponding g author’s e-mail: [email protected]; + 55353829-1783 Abstract Tissue culture can contribute in the multiplication of several species with commercial interest, like the bromeliads. It was aimed to evaluate cytokinins and its concentrations in the multiplication and leaf structure of Neoregelia concentrica (Vellozo) L.B. Smith. Previously In vitro -established N. concentrica plants were inoculated in MS medium supplemented with 6-benzylaminopurine (BAP) or Kinetin (KIN) with concentrations 0.0, 5.0, 10.0 and 15.0 µM. For the anatomic analyses tree plants of each treatment were randomly sampled at 60-day growth. Significant differences were verified in the evaluated characteristics due to the treatments. The raise in cytokinin concentrations induced a higher percentage and average number of explants with shoots. BAP provided higher averages when compared to KIN. The cytokinin use modified the epidermal structure and induced a larger thickening of the water-storage and chlorophyll parenchymas. The use of 15.0 µM BAP was efficient in the In vitro multiplication and in the leaf tissue development of N. concentrica. Key words: Bromeliad, Cytokinin, In vitro multiplication, Tissue culture. Introduction and/or physiological alterations can significantly affect the acclimation chance of success (Majada et al ., 2000; Bromeliads are originated almost exclusively in the Zobayed et al., 2001). American continent and around 50% of the known species The objective was to evaluate the effects of the are found in Brazil. Most of the Brazilian species are cytokinin types and concentrations in the multiplication endemic in the Atlantic Forest (Ceita et al ., 2008; Ribeiro and leaf structure of Neoregelia concentrica , during In et al ., 2009), a threatened biome with approximately 8% vitro growth. of its original area preserved (Myers et al ., 2000). These plants present ecology importance for being Material and Methods resource sources for the wildlife. They serve as shelter and In vitro establishment: Neoregelia concentrica (Vellozo) also hold fleshy fruits, nectar and water (stored between the L.B. Smith fruits were collected from adult plants grown in leaves) (Balke et al ., 2008). Bromeliads also have green house and the seeds were extracted mechanically. commercial value as an ornamental plant, due to the beauty They were submitted to disinfestation in ethanol 70% for of its leaves and flowers (Vesco et al ., 2011). For this one minute and sodium hypochlorite (50% commercial reason, illegal gathering has been carried out in natural solution and 2.5 activated chlorine) for 10 minutes. environments aiming income supplement. It is threatening Subsequently, the seeds were washed 3 times in autoclaved some species with extinction (Negrelle et al ., 2012). distilled water, to remove the disinfesting solution excess Therefore, the mass multiplication of bromeliads and inoculated in test tubes containing 10ml of MS medium rises as an economical and ecologic viable option. (Murashige & Skoog, 1962) with half of the original However, propagation through side shoots results in a concentration, supplemented with 30gL -1 sucrose and limited number of plants, and they may be contaminated solidified with 7gL -1 agar. The medium pH was adjusted to by Fusarium sp (Feuser et al ., 2003). According to Guerra 5.8 before autoclaving at 120ºC for 20 minutes. After & Vesco (2010), the employment of plant tissue culture inoculation in horizontal laminar flow cabinet the plant techniques is potentially important in a wide propagation material was kept in growth room at 27±2 oC with a 16-hour of species of this family, as verified by Silveira et al ., photoperiod, under fluorescent lamps providing 25.2µmol (2009) with Neoglaziovia variegata (Arr. Cam.) Mez, m-2s-1 photosynthetic photon flux. Santos et al ., (2010) with Acanthostachys strobilacea (Schult. f.) Klotzsch, Huang et al ., (2011) with Guzmania , Shoot multiplication: Obtained plants, (180-day old), were Vesco et al ., (2011) with Billbergia zebrina (Herbert) inoculated in test tubes containing 10 mL stationary liquid Lindley and Silva et al., (2012) with Nidularium MS medium supplemented with 10mgL -1 citric acid, 30gL -1 innocentii Lem. and Nidularium procerum Lindm. sucrose and plant growth regulator 6-benzilaminopurine Among the factors able to influence In vitro (BAP) or kinetin (KIN) with concentrations 0.0, 5.0, 10.0 propagation, cytokinins are considered the most important and 15.0µM. The medium pH was adjusted to 5.8 before factor, as they affect shoot induction, besides the autoclaving at 120ºC for 20 minutes. After inoculation in significant effects related to the histological modification horizontal laminar flow cabinet the plant material was kept in in plant tissues (Magyar-Tábori et al ., 2010). Although a growth room at 27±2 oC with a 16-hour photoperiod, under these modifications may be observed in different plant fluorescent lamps providing 25.2µmol m -2 s-1 photosynthetic organs, leaf is the most important, since the structural photon flux. 2180 JOÃO PAULO RODRIGUES MARTINS ET AL., The experimental design was completely randomized in (%) presented quadratic-growing model with the factorial arrangement (two cytokinin types x four concentration raise, independent on the cytokinin type concentrations), with four repetitions, each parcel composed employed, acting positively in the break of apical by five test tubes. In total 20 explants per treatment. dominance and in the induction of adventitious shoots (Fig. The evaluation was performed at 60-day 1). The employment of cytokinins in the In vitro shoot subcultivation. The analyzed phytotechnical features were: induction is well documented, as in the papers of Pompelli Budding (%), shoot average number, longest-leaf average & Guerra, 2005; Silva et al ., 2009; Silveira et al ., 2009; length (cm), fresh- and dry-mass (mg) of the budding. To Jafari et al ., 2011; Khan et al ., 2011 and Wu et al., 2012. obtain dry mass, the plant material was previously kept in However, in concentrations above ideal, it may occur an forced ventilation oven at 65ºC until stabilization. All inhibitory effect, decreasing the organogenic-response obtained data were submitted to analysis of variance and frequency (Arumugam et al ., 2009), which agrees with the the averages were compared through Tukey’s test at 5% results obtained, in which concentrations above 11.68 µM probability and regression analysis. of the studied cytokinins were harmful to the budding frequency in explants of N. concentrica (Fig. 1). Anatomy analysis: To perform anatomic characterization, When BAP was used in the medium, compared to tree plants from each treatment were sampled randomly at KIN, it was observed a larger budding in the explants of 60-day growth and fixed in a FAA (formaldehyde, acetic N. concentrica (Table 1). The lower In vitro acid and ethanol 50%, proportion 0.5:0.5:9, v/v) for 48 multiplication rates of N. concentrica in the media where hours, followed by conservation in ethanol 70% (Johansen, it was employed KIN, may be related to the activity of 1940). enzymatic systems in the cleavage of furfuryl group. An Cross-sections were performed in the median region example is the cytokinins oxidase/dehydrogenase which of the third completely expanded leaf in the rosette central catalyzes the cytokinin degradation (Schmülling et al ., region with aid of a manual microtome. Those were 2003; Frébort et al ., 2011) decreasing or inhibiting the cleared with sodium hypochlorite 3% (v/v) and organogenesis response. posteriorly stained with an astra blue and safranin The average shoot number presented a significant solution, using glycerin 50% to assemble the sliders. interaction for each of the cytokinin types and The cross-sections were observed in an optical concentrations. The highest shoot number was presented microscopy coupled to a digital camera to capture images. by the explants grown in BAP supplemented media, when The photomicrographs were used to measure the anatomy compared with KIN (Table 2). Silva et al ., (2009) and characteristics using the software UTHSCSA-Imagetool ® Silveira et al ., (2009) verified a higher shoot number in calibrated with microscopy ruler. Vriesea scalaris E. Morren and N. variegata respectively, The experimental design was completely randomized when BAP was employed at the media, if compared to in factorial arrangement (two cytokinin types x four KIN, likewise what we verified with N. concentrica. concentrations), with three repetitions per treatment. It was However, this is not common to all plant species, as analyzed thickness of the following anatomic characteristic: observed in Artemisia amygdalina Decne (Rasool et al., adaxial and abaxial epidermis (µm), water-storage 2013), in which the use isolated BAP was not efficient in parenchyma (µm) and chlorophyll parenchyma (µm). the formation of new shoots. All the data were submitted to analysis of variance The lowest values were observed in the exogenous-