Microsc. Microanal. Page 1 of 7 doi:10.

1017/S1431927610000279

Microscopy Microanalysis
AND MICROSCOPY SOCIETY OF AMERICA 2010

Influence of Blue Light on the Leaf Morphoanatomy of In Vitro Kalanchoe pinnata ~Lamarck! Persoon ~Crassulaceae!
Marcos Vinicius Leal-Costa,1,4, * Luana Beatriz dos Santos Nascimento,1 Nattacha dos Santos Moreira,1 Fernanda Reinert,1,4 Snia Soares Costa,3 Celso Luiz Salgueiro Lage,2,4 and Eliana Schwartz Tavares 1
Universidade Federal do Rio de Janeiro, Centro de Cincias da Sade, Instituto de Biologia, Departamento de Botnica, sala A1-104, Avenida Carlos Chagas Filho, Cidade Universitria, 21.941-902, Rio de Janeiro, Brazil 2 Universidade Federal do Rio de Janeiro, Instituto de Biofsica Carlos Chagas Filho, Rio de Janeiro, Brazil 3 Universidade Federal do Rio de Janeiro, Ncleo de Pesquisas de Produtos Naturais, Rio de Janeiro, Brazil 4 Universidade Federal do Rio de Janeiro, Programa de ps-graduao em Biotecnologia Vegetal, Rio de Janeiro, Brazil
1

Abstract: Kalanchoe pinnata ~Lamarck! Persoon ~Crassulaceae! ~air plant, miracle leaf! is popularly used to treat gastrointestinal disorders and wounds. Recently, the species was tested to treat cutaneous leishmaniasis with successful results. This medicinal activity was associated with the phenolic fraction of the plant. Blue light induces biosynthesis of phenolic compounds and many changes in anatomical characteristics. We studied the effects of supplementary blue light on the leaf morphology of in vitro K. pinnata. Plants cultured under white light ~W plants! only and white light plus blue light ~WB plants! show petioles with plain-convex section, amphistomatic leaf blades with simple epidermis, homogeneous mesophyll with densely packed cells, and a single collateral vascular bundle in the midrib. W plants have longer branches, a larger number of nodes per branch, and smaller leaves, whereas WB plant leaves have a thicker upper epidermis and mesophyll. Leaf fresh weight and leaf dry weight were similar in both treatments. Phenolic idioblasts were observed in the plants supplemented with blue light, suggesting that blue light plays an important role in the biosynthesis of phenolic compounds in K. pinnata. Key words: blue light, Crassulaceae, in vitro culture of medicinal plants, plant development, Kalanchoe pinnata, leaf anatomy

I NTR ODUCTION
Blue light regulates several aspects of plant functioning, from stomatal movement to hypocotyl elongation ~Taiz & Zeiger, 2009!. Blue light enrichment in the incident light may influence some characteristics such as plant height, number of nodes per branch, and biomass accumulation ~Yorio et al., 2001; Glowacka, 2004; Sarala et al., 2007; Wozny & Jerzy, 2007; Kurilcik et al., 2008; Poudel et al., 2008!. Besides morphogenetic events, blue light also stimulates the biosynthesis of phenolic compounds in plants ~Caldwell et al., 1983; Maffei et al., 1999; Wade et al., 2001; Rao & Ravishankar, 2002; Meng et al., 2004; Saleh, 2007!. Many of these compounds are active substances of medicinal interest ~Jansen et al., 2008!. Some studies have included Crassulacean species as medicinal plants in ethnobotanical checklists ~Medeiros et al.,
Received July 9, 2009; accepted March 4, 2010 *Corresponding author. E-mail: mlealcosta@gmail.com

2004; Lans, 2006; Taufner et al., 2006!. Kalanchoe pinnata ~Lamarck! Persoon, popularly used to treat gastrointestinal disorders and wounds, was recently tested as a treatment for cutaneous leishmaniasis ~da-Silva et al., 1999; Muzitano et al., 2006a, 2006b!, with positive results. The disease is characterized by skin lesions that do not heal. K. pinnata activity in the treatment of leishmaniasis was attributed to a flavonoidic fraction of its aqueous extract ~da-Silva et al., 1999; Muzitano et al., 2006a, 2006b!. Synthesis of flavonoids, which are phenolic compounds, can be stimulated by blue light exposure ~Meng et al., 2004!. Flavonoids are known to protect plants against light induced damage ~Shirley, 1996; Jaakola et al., 2004!. Vigorous branching, thick leaves, and dense vascularization are some characteristics linked to light-induced damage avoidance ~Larcher, 2000!. Additionally, some anatomical modifications, such as increased leaf thickness and reduction in epidermal cell length and xylem vessel diameter ~Schuerger et al., 1997; Rapparini et al., 1999!, are the result of light quality. Light quality also influences biosynthesis of secondary metabolites ~Namdeo, 2007; Jansen et al., 2008;

Marcos Vinicius Leal-Costa et al.

Risnen et al., 2008!. In vitro plant culturing allows assessment, in controlled conditions, of the influence of light on plant development and secondary metabolite production ~Rao & Ravishankar, 2002; Fila et al., 2006; Kurilcik et al., 2008; Poudel et al., 2008!. We addressed the effects of supplementary blue light on leaf morphology and phenolic inclusions in K. pinnata using optical microscopy and a microchemical test in in vitro culturing of plants.

M ATERIALS
Plant Material

AND

M ETHODS

The plants used in this work were monoclonal. The matrix plant was obtained from the internal garden of Ncleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, where several specimens are grown under natural conditions. A voucher specimen was deposited on the Jardim Botnico do Rio de Janeiro under number RB292.697.

Establishment of In Vitro Culture

Leaf borders of the matrix plant were cut into sections of 1 cm 2 and surface-sterilized under aseptic conditions ~commercial detergent in aqueous solution for 15 min, 5 min in sodium hypochlorite solution, washed in 70% ethanol solution, and between each step, explants were rinsed with distilled water!. Sterilized explants were transferred to autoclaved glass flasks containing MS medium ~Murashige & Skoog, 1962! without growth regulators and supplemented with 0.6 mM myo-inositol, 2.43 mM pyridoxine, 4.1 mM nicotinic acid, 1.48 mM thiamine, and 30 g L 1 sucrose. The medium was jellified with agar ~8 g L 1 ! at pH 5.7 adjusted before autoclaving. In vitro plants were cultured under 12 mmol m 2 s 1 ~1.6 W m 2 ! of photosynthetic active radiation ~PAR!. Light was supplied by white fluorescent lamps ~Sylvania 20 WF20T12!. The spectral curves of the lamps can be viewed in Figure 1. There was no significant difference in the photosynthetic light quantity/quality measured outside and inside the flasks. Ultraviolet wavelengths were mainly blocked by glass. The growth room temperature and photoperiod were 24 6 28C and 16 h, respectively. Subsequent subcultures were done using nodal segments.
Figure 1. Spectral power distribution of the ~A! white and ~B! blue fluorescent lamps.

room temperature was 24 6 28C and photoperiod was 16 h. The PAR levels of both treatments ~white lamps and white lamps plus blue lamps! were similar ~12 mmol m 2 s 1 !. The fluence of both treatments was 1.6 W m 2. After four months, five flasks per treatment ~25 plants! were randomly taken for plant growth analysis, and the remaining three flasks ~15 plants! were used for analysis of leaf morphology. With four months of growth, plants reached their maximum growth without signs of senescence and/or nutritional limitation of the medium.

Morphological Parameters
Plant development was evaluated according to the following parameters: leaf fresh weight, leaf dry weight, length of the longest branch, and number of nodes of the longest branch. For dry weight determination, plants were dehydrated at 608C for 24 h. Anatomical analyses were performed on fully developed leaves from both treatments. They were fixed in 37% formaldehyde, glacial acetic acid, and 70% ethanol solution ~Johansen, 1940! and kept in 70% ethanol solution. Whole leaves were embedded with a Leica Historesin Embedding Kit. Sections 10 mm thick were obtained using a rotary microtome ~American Optical! and stained with toluidine blue ~OBrien et al., 1965!. Sections were taken from the middle third of the leaf blade and the petiole. Toluidine

In Vitro Culture under Different Light Qualities

In vitro plants after the fourth subculture were used to study the effect of blue light on the leaf internal morphology. Two light treatments were used: white lamps ~W plants! and white lamps plus blue lamps ~WB plants!. Fluorescent lamps were used for both treatments ~Sylvania 20 W day light, Sylvania 20 W blue light F20T12! ~Fig. 1!. Eight flasks with five plantlets were used per treatment. The growth

Blue Light and In Vitro K. pinnata Morphoanatomy

Figure 3. Drawings of K. pinnata epidermis. ~A! Adaxial side; ~B! abaxial side. WB plant. Scale bar, 50 mm.

Figure 2. Transverse sections of K. pinnata petiole. ~A,B! Control plants; ~C,D! plants cultured under supplementary blue light. avb, accessory vascular bundle; e, epidermis; p, parenchyma tissue; vb, main vascular bundle. Scale bars: A,C 100 mm; B,D 50 mm.

blue was used to identify phenolic idioblastic cells ~Ramalingan & Ravindranath, 1970!. The following anatomical parameters were measured: leaf thickness, epidermal thickness, mesophyll thickness, and number of mesophyll cell layers.

between treatments ~Table 1!. WB plants have larger chloroplasts with conspicuous starch grains when compared to W plants. There is a single collateral vascular bundle in the midrib ~Fig. 5! in W and WB plants. W plants have longer branches and a larger number of nodes ~Table 2; Fig. 6A!. However, leaves are smaller in W plants than in WB plants and are rolled up ~Fig. 6B!. Leaf fresh weight and leaf dry weight were similar for W plants and WB plants ~Table 2!. Toluidine blue indicated phenolic indioblastic cells associated with the vascular cells in parenchyma tissue in petioles and leaf blades of WB plants only ~Fig. 7B,D!.

Statistical Analysis
Statistical analysis was conducted with GraphPad Instat 3.0 for Windows . Data were analyzed with Students t-test ~ p 0.05!.

D ISCUSSION
Reports of blue light effects on plant development are widespread in the scientific literature ~Spalding & Folta, 2005!. Blue light effect on leaf thickness is species dependent. The thickness of pepper leaves increases more under blue light in combination with red light than under red light alone ~Schuerger et al., 1997!; whereas addition of blue light decreases leaf thickness in peach ~Rapparini et al.,

R ESULTS
Petioles are plain-convex ~flat upper surface and rounded lower surface! in cross section under the two light treatments. Epidermal cells are rectangular and elliptic with convex external periclinal walls ~parallel to the leaf surface! ~Fig. 2!. Stomata are rarely found. The petiole is filled with chlorophyll and ground parenchyma. In the center, embedded in the ground parenchyma, one collateral vascular bundle is present ~Fig. 2A,C!. Near to the adaxial side are two accessory collateral vascular bundles ~Fig. 2B,D!. Plants grown under white light ~W plants! and plants supplemented with blue light ~WB plants! have amphistomatic leaves with similar epidermis. In frontal view epidermal cells have sinuous anticlinal walls on both sides of the leaf ~Fig. 3!. The epidermis is simple with rectangular and eliptic cells in cross section ~Fig. 4!. W plants have thinner leaves than WB plants ~Table 1!. The mesophyll is homogeneous and densely packed in both W and WB plants, although cells are visibly larger in WB plants ~Fig. 4B!. The number of mesophyll cell layers did not differ significantly

Table 1. Anatomical Parameters of Kalanchoe pinnata ~Lamarck! Persoon Cultured under White Light ~W Plants! and White Light plus Blue Light ~WB Plants!. W Plants Epidermal thickness ~adaxial side! ~ mm! Epidermal thickness ~abaxial side! ~ mm! Mesophyll thickness ~ mm! Leaf thickness ~ mm! Number of mesophyll cell layers
a

WB Plants
a

23.91 6 1.40

31.71 6 1.62 a 17.68 6 2.30 233.95 6 9.78 a 273.90 6 9.95 a 5.80 6 0.15

17.35 6 1.64 154.32 6 7.44 a 196.00 6 8.47 a 5.45 6 0.15

Indicates statistical difference. T test, p , 0.5, n

15.

Marcos Vinicius Leal-Costa et al.

Figure 5. Transverse sections of K. pinnata leaf blade in the midrib. ~A! Control plant; ~B! plant cultured under supplementary blue light. e, Epidermis; m, mesophyll; vb, vascular bundle. Scale bars: A,B 100 mm.

1999!. Thick leaves, homogeneous and densely packed mesophyll, and large vacuoles are characteristic of succulent leaves and are often observed in crassulacean acid metabolism ~CAM! species ~Nelson & Sage, 2008!. These traits are of great importance in preventing CO 2 leakage during the day when malate is decarboxylated and CO2 is refixed via the Calvin-Benson cycle. Reduced CO2 conductance in CAM

Table 2. Morphological Parameters of Kalanchoe pinnata ~Lamarck! Persoon Cultured under White Light ~W Plants! and White Light plus Blue Light ~WB Plants!. W Plants Leaf fresh weight ~mg! Leaf dry weight ~mg! Length of the higher branch ~cm! Number of nodes
a

WB Plants 82.20 6 14 9.84 6 1.73 0.71 6 0.10 a 3.88 6 1.33 a

104.82 6 10.51 10.04 6 0.71 2.10 6 0.27 a 6.30 6 1.87 a

25.

Figure 4. Transverse sections of K. pinnata leaf blade. ~A! Control plant; ~B! plant cultured under supplementary blue light. e, Epidermis; m, mesophyll. Scale bars: A,B 50 mm.

Indicates statistical difference. T test, p , 0.5, n

Blue Light and In Vitro K. pinnata Morphoanatomy

Figure 6. Plants cultured under ~A! white light and ~B! white light plus blue light. Observe the rolled leaf margins in panel A. Need to show these at the same magnification for comparison.

Figure 7. ~Color online! Detail of transections of K. pinnata showing a vascular bundle in the ~A,B! petiole and ~C,D! midrib. ~A,C! Control plant; ~B,D! plant cultured under supplementary blue light. id, Phenolic idioblasts; ph, phloem; x, xylem. Scale bars: AD 50 mm.

plants also increases the efficiency to reuse of CO 2 from respiration. Limited efflux of internally generated CO 2 is particularly important to CAM function during stressful situations, enhancing photosynthetic efficiency ~Nelson et al., 2005!. Thicker WB leaves could represent an advantage under water stress, favoring the process of transplantation to ex vitro culturing. Large chloroplasts with large starch grains are characteristic of plants under high light ~Meier & Lichtenthaler, 1981; Oguchi et al., 2003!. Light also influences chloroplast

position and development ~Tholen et al., 2008!. Removing blue wavelength from spectral radiation determines some shade-type features, such as the position of chloroplasts, perpendicular to the main direction of light ~Tholen et al., 2008!. Blue light alone and blue light associated to red light increased starch biosynthesis in Doritaenopsis plants in comparison to white light ~Shin et al., 2008!. WB plants also exhibited chloroplasts with more starch content. Blue light inhibits dry weight accumulation on spinach, radish, and lettuce ~Yorio et al., 2001!. Tomato plants under

Marcos Vinicius Leal-Costa et al.

blue light exhibit smaller shoots with reduction in the number of nodes, internode length, and fresh weight reduction ~Glowacka, 2004!. Pines and narcissi are shorter under blue light than plants under white light ~Sarala et al., 2007; Wozny & Jerzy, 2007!. Blue light also reduces shoot height in in vitro grapes without reduction in the number of nodes ~Poudel et al., 2008!. In vitro Chrysanthemum plants also exhibit reduced dry and fresh weight and smaller internodes linked to blue light exposure ~Kurilcik et al., 2008!. In this work, WB plants were smaller and had fewer nodes than W plants. Nevertheless, their biomass accumulation, as a measure of dry weight, was equivalent. This is probably a result of larger leaves. Blue light often improves synthesis of phenolic compounds, including flavonoids, in plants. Vigna sinensis and Phaseolus vulgaris have increased phenolic content promoted by blue light exposure ~El-Khawas & Khatab, 2007!. Anthocyanin accumulation is induced by blue light in Arabdopisis thaliana ~Cominelli et al., 2008!. Phenolic idioblasts are observed in K. daigremontiana, Sedum dendroideum ~Balsamo & Uribe, 1988; Duarte & Zaneti, 2002!. Densely filled cells, probably of phenolic content, are viewed in K. daigremontiana and K. pinnata ~Kondo et al., 1998!. These reports and the greenish reaction observed in WB plants lead us to believe that the inclusions in the idioblasts of WB plants are phenolic compounds.

R EFER ENCES
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C ONCLUSIONS
Supplementary blue light under the intensity tested here did not affect plant growth. However, significant morphological effects were observed under supplementary blue light. Although W plants were larger than WB plants, they had smaller and thinner leaves. Shorter plants under supplementary blue light are a desirable character that permits a controlled culture in reduced spaces, including greenhouse culture. Additionally, accumulation of phenolic compounds, visible in the idioblasts using toluidine blue, was only found in WB plants. Overall, in vitro culture of K. pinnata under supplementary blue light was improved, especially as a source of phenolic compounds. Further tests on a larger scale are desirable if commercial in vitro culturing is intended for the production of phytotherapeutic medicines.

A CKNOWLEDGMENTS
This work was supported by Fundao de Amparo Pesquisa do Estado do Rio de Janeiro-FAPERJ and Coordenao de Aperfeioamento de Pessoal de Nvel Superior-CAPES.

Blue Light and In Vitro K. pinnata Morphoanatomy

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