Modifications in Leaf Anatomy of Banana Plants Cultivar Maçã Subjected to Different Silicon Sources In Vitro

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1 Modifications in Leaf Anatomy of Banana Plants Cultivar Maçã Subjected to Different Silicon Sources In Vitro J. Magno Queiroz Luz 1, S. Abreu Asmar 1, M. Pasqual 2, A. Gomes de Araujo 2, L.A..S. Pio 2 and R. Ferreira Resende 1 1 Universidade Federal de Uberlândia, Avenida Amazonas s/n, Umuarama , Uberlândia, MG, Brazil 2 Universidade Federal de Lavras, Campus UFLA, , Lavras, MG, Brazil Keywords: Musa spp., silicon, anatomical characteristics Abstract In vitro plant culture under conventional heterotrophic conditions promotes the development of certain undesirable morpho-physiological characteristics, such as reduced epicuticular wax deposition and mesophyll differentiation, rudimentary vascular bundles and little control of stomata opening and closing. Silicon is a beneficial element for plants and affects anatomical characteristics of leaf surface. This study analyzed morphological differences in banana plant cultivar Maçã plants as a result of the use of silicon added to the medium for in vitro cultivation. Shoots of banana plants cultivar Maçã established in vitro were inoculated on MS, supplemented with 30 g L -1 sucrose, 1 mg L -1 NAA (naphthaleneacetic acid) and solidified with 1.8 g L -1 Phytagel TM. Three sources of silicate were added to the MS medium, sodium silicate, potassium silicate or calcium silicate at 1 g L -1, and MS medium without silicate, as the control treatment. The experimental design was completely randomized with five replications. After 45 days, anatomical characteristics and photosynthetic and transpiration rates were evaluated. The addition of calcium silicate resulted in greater thickness of upper and lower epidermis, mesophyll, palisade parenchyma and increased photosynthetic rate. The use of silicon improved micropropagated anatomy of banana plant cultivar Maçã leaves. INTRODUCTION Over the past 30 years, micropropagation growth of shoot tips and meristems has been the basis of mass propagation of banana seedlings (Gübbük and Pekmezci, 2004). Tissue culture provides large-scale disease free production of plants to supply banana producers. Despite its benefits for seedling production, in vitro cultivation, under conventional heterotrophic conditions promotes the development of certain undesirable morpho-physiological characteristics, such as reduced epicuticular wax deposition and mesophyll differentiation, rudimentary vascular bundles and little control of stomata opening and closing (Romano and Martins-Loução, 2003). The composition of the culture medium plays an important role on cell and tissue growth responses. Silicon is considered as a beneficial element for plants and, according to Epstein (1999), plants grown in silicon enriched environment differ from those grown in the absence of this element, especially in chemical composition, cell mechanical strength, leaf surface characteristics, tolerance to biotic and abiotic stresses and resistance to pests and diseases. Therefore, this study analyzed the morphological differences in leaf anatomy of plants as a result of the use of silicon added to the culture medium for in vitro cultivation of banana plant cultivar Maçã. MATERIAL AND METHODS Shoots of banana plant cultivar Apple already established in vitro were inoculated in MS medium (Murashige and Skoog, 1962), supplemented with 30 g L -1 sucrose, 1 mg L -1 NAA (naphthaleneacetic acid) and solidified with 1.8 g L -1 Phytagel TM. Three silicate sources were tested in the MS medium, sodium silicate (Na 2 SiO 3 ), potassium silicate (K 2 SiO 3 ) or calcium silicate (CaSiO 3 ) at a dosage of 1 g L -1, and MS Proc. 7 th IS on In Vitro Culture and Horticultural Breeding Ed.: D. Geelen Acta Hort. 961, ISHS

2 medium without silicate, as the control treatment. The ph was adjusted to 5.8 before autoclaving at 121 C and 1.2 atm for 20 min. Subsequently, in laminar flow hood, the explants were inoculated in 20-ml flasks containing 30 ml of MS culture medium modified according to the treatment. The flasks were sealed with polypropylene caps and parafilm. After inoculation, the flasks were kept in conventional growth room at 25±2 C, with 16 hours lighting, with an intensity of 52.5 W m -2 s -1, supplied by white fluorescent lamps. After 45 days, the treatments were evaluated for leaf anatomy and photosynthetic and transpiration rates. The anatomical studies were done using the middle third of the second fully expanded leaf collected from five different plants per treatment, previously fixed in 70% FAA (formaldehyde - acetic acid - ethyl alcohol 70%) (Johansen, 1940) for 72 hours and then preserved in ethanol 70% (v v -1 ). The cross sections were obtained in a table microtome type LPC, after clarification in sodium hypochlorite (1-1.25% active chlorine), triple-rinsed in distilled water, and staining in Safrablau solution (0.1% astra blue and 1% safranin) and subsequently mounted on semipermanent slides with water glycerol (Kraus and Arduin, 1997). The slides were observed and photographed under a light microscope, Olympus BX 60, coupled to a Canon A630 digital camera. The images were analyzed with the image analysis software UTHSCSA ImageTool, with the measurement of five fields per repetition for each variable analyzed. The thickness of the abaxial surface epidermis (EAB), adaxial epidermis (EAD), abaxial hypodermis (WCH), adaxial hypodermis (HAD), mesophyll (MF), palisade parenchyma (PP), spongy parenchyma (PE) were measured and the ratio of the palisade and spongy parenchyma (PP/PE) was calculated. Plant transpiration and photosynthesis rates were evaluated by analysis of infrared gas exchange (IRGA) model LI To evaluate these variables, fully expanded leaves were selected in seven plants per treatment, from 10 hours, and the flux density of photosynthetic photon was fixed in the chamber of the device to 100 μmol m -2 s -1. The experimental design was completely randomized (CRD) with five repetitions, and 15 fields for the cross sections. The data were analyzed using the statistical program SISVAR 4.3 (FERREIRA, 2000) and the averages were compared by the Scott-Knott test at 5% probability. RESULTS AND DISCUSSION The banana plant is a species listed as bifacial or dorsiventral with the palisade parenchyma facing the adaxial epidermis and immediately below the adaxial hypodermis, while the spongy one is directed to the abaxial epidermis, which was confirmed in this study (Fig. 1). The palisade parenchyma cells are typically elongated, arranged in rows, with one or more cell layers, while the spongy parenchyma cells are not well defined (Costa et al., 2009). The characteristics evaluated related to tissue thickness showed significant effects for all parameters except for the spongy parenchyma (Table 1). Greater thickness was observed in the adaxial and abaxial epidermis surfaces in seedlings grown in the presence of calcium silicate. All treatments containing silicon showed greater thickness of the adaxial hypodermis surface. Greater thickness was obtained in treatments with calcium silicate and in the control for the abaxial surface (Table 1). Epstein (1999) reported that, when accumulated by plants, silicon provides anatomical changes in their tissues, such as the appearance of thicker epidermis cells, due to the deposition of silicon. Increased thickening of the palisade parenchyma was observed using calcium silicate. According to Alquini et al. (2006), the palisade and spongy parenchyma are photosynthetically important due to the presence of chloroplasts which convert light into chemical energy and store it in the form of carbohydrates. Thus, the lack of differentiation may result in low photosynthetic efficiency and, consequently, problems during the acclimatization phase. Results of this study confirm those of other authors, such as Sandoval et al. (1994) and Costa et al. (2009), which showed that micropropagated plants have thin epidermis, 240

3 cells with irregular size and sinuosity, hypodermis composed of large cells, lack of differentiation between the palisade and spongy parenchyma and a thin cuticle layer. There were no significant differences in transpiration rate among the treatments. However, in seedlings grown in the presence of calcium silicate, an increase in photosynthetic rate was observed (Table 2). Photosynthesis can vary according to plant growth environment and the two major environmental limitations to photosynthesis rate are CO 2 availability and radiation (Zhou and Han, 2005). Since the amount of radiation was fixed in the IRGA chamber (100 µmol m -2 s -1 ), the increase in photosynthesis rate observed in calcium silicate grown seedlings may be related to morphological adaptations that promote CO 2 capture and incident radiation exploitation by the leaves. Thus, the addition of calcium silicate may have promoted an increase in photosynthetic rate by thickening the palisade parenchyma (Table 1), which allowed greater use of incident radiation. These results demonstrate that the addition of calcium silicate is important to improve internal structures of the leaves banana plants cultivar Maçã. Transpiration did not change in the presence of silicon, probably due to high relative humidity inside the flasks. CONCLUSION The addition of calcium silicate provided greater thickness of upper and lower epidermis, mesophyll, and palisade parenchyma, and increased the photosynthesis rate. The use of silicon favored micropropagated banana plant Maçã leaf anatomy. ACKNOWLEDGEMENTS This work was sponsored by the FAPEMIG and CAPES (Brazil). Literature Cited Alquini, Y. et al Epidermis. p In: B. Appezzato-da-Glória and S.M. Carmello-Guerreiro (eds.), Anatomia vegetal. Viçosa, MG: UFV. Costa, F.H.S. et al Anatomical changes in micropropagation banana plants in response to acclimatization. Ciência Rural, Santa Maria 39(2): , mar./abr. Epstein, E Silicon. Annual Review of Plant Physiology and Plant Biology, Palo Alto 50: Ferreira, D.F SISVAR 4.3: System analysis of variance for balanced data, statistical analysis and program design of experiments. Lavras: UFLA/DEX, Software. Gübbük, H. and Pekmezci, M In vitro propagation of some new banana types (Musa spp.). Turkish Journal of Agriculture and Forestry, Istanbul 28(5): Johansen, D.A Plant microtechnique. New York: McGraw Hill. 523p. Kraus, J.E. and Arduin, M Basic manual of methods in plant morphology. Rio de Janeiro: UFRRJ. 198p. Murashige, T. and Skoog, F A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, Copenhagen 15(3): Romano, A. and Martins-Loução, M.A Water loss and morphological modifications in leaves during acclimatization of Cork Oak micropropagated plantlets. Acta Hort. 616: Sandoval, J.A., Müller, L.E. and Weberling, F Foliar morphology and anatomy of Musa cv. Grande Naine (AAA) plants grown in vitro and during hardening as compared to field-grown plants. Fruits, Les Ulis 49(1): Zhou, Y.M. and Han, S.J Photosynthetic response and stomatal behaviour of Pinus koraiensis during the fourth year of exposure to elevated CO 2 concentration. Photosynthetica, Amsterdam 43(3):

4 242 Tables Table 1. Modification in leaf tissue thickness of banana plants cultivar Maçã cultivated in vitro for 45 days in culture medium containing different sources of silicon. Sources of silicon Abaxial epidermis Adaxial epidermis Abaxial hypodermis Adaxial hypodermis Mesophyll Palisade parenchyma (PP) Spongy parenchyma (PE) Control 18,70b 20,74b 095,08a 099,19b 340,31b 72,62b 95,69a 0,78c MS+Na 2 SiO 3 20,08b 21,45b 084,45b 131,03a 357,62b 66,91c 91,75a 0,75c MS+K 2 SiO 3 20,11b 22,45b 084,23b 136,65a 359,14b 73,78b 87,37a 0,89b MS+CaSiO 3 25,38a 28,55a 103,29a 135,26a 400,57a 93,30a 94,21a 1,03a Averages followed by same letter in columns do not differ by the Scott-Knott test at 5% probability. PP/PE Table 2. Photosynthetic (A) and transpiratory (E) rate in leaves of banana plant cultivar Maçã cultivated in vitro for 45 days in culture medium containing different sources of silicon. Sources of silicon A (μmol m -2 s -1 ) E (mmol m -2 s -1 ) Control 0,35 b 0,27 a MS+Na 2 SiO 3 0,95 b 0,16 a MS+K 2 SiO 3 0,23 b 0,46 a MS+CaSiO 3 1,56 a 0,40 a Averages followed by same letter in columns do not differ by the Scott-Knott test at 5% probability. 242

5 Figures Fig. 1. Microscopy of cross sections of banana plant cultivar Maçã leaves cultivated in vitro with different sources of silicon. (A) MS, (B) Na 2 SiO 3, (C) K 2 SiO 3, (D) CaSiO 3. ead = adaxial epidermis; eab = abaxial epidermis; had = adaxial hypodermis; hab = abaxial hypodermis; pp = palisade parenchyma; pe = spongy parenchyma. Bars = 100 µm. 243

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Study of leaves structures that determine the resistance to dryness at succulent plants

Study of leaves structures that determine the resistance to dryness at succulent plants Cristescu Mihaela 1*, Anton Doina 1, Mandă Manuela 1, Nicu Carmen 1 1 University of Craiova, Faculty of Horticulture