Somatic embryogenesis in Alnus glutinosa (L.) Gaertn

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<ul><li><p>ORIGINAL PAPER</p><p>Somatic embryogenesis in Alnus glutinosa (L.) Gaertn</p><p>Elena Corredoira Silvia Valladares </p><p>Ma Teresa Martnez Ana Ma Vieitez </p><p>Ma Carmen San Jose</p><p>Received: 11 March 2013 / Revised: 4 July 2013 / Accepted: 9 July 2013</p><p> Springer-Verlag Berlin Heidelberg 2013</p><p>Abstract Induction of somatic embryos and plant</p><p>regeneration was demonstrated for the first time in Alnus</p><p>glutinosa. Somatic embryos were initiated from zygotic</p><p>embryos collected 13 weeks post-anthesis (WPA), i.e.,</p><p>when they were at globular or early cotyledonary stage and</p><p>were 0.51 mm in length. Induction frequency (16.6 %)</p><p>and the mean number of somatic embryos (4.5 embryos/</p><p>explant) were highest after culture of zygotic embryos,</p><p>collected at 3 WPA, on Murashige and Skoog medium</p><p>(MS) supplemented with 0.9-lM 2,4-dichlorophenoxy-acetic acid and 2.22-lM benzyladenine (BA). Noembryogenic induction was observed on medium with BA</p><p>alone. Initial somatic embryos differentiated indirectly</p><p>from callus tissue formed at the surface of the zygotic</p><p>embryos. Embryogenic competence was maintained by</p><p>secondary embryogenesis, which was affected by explant</p><p>type, plant growth regulators and genotype. Secondary</p><p>embryogenesis was induced by culture of small groups of</p><p>whole somatic embryos or isolated cotyledon explants on</p><p>medium consisting of MS medium (half-strength ma-</p><p>cronutrients) supplemented with 0.44-lM BA. Histologicalstudy of isolated cotyledon explants revealed that</p><p>secondary embryos developed directly from differentiated</p><p>embryogenic tissue on the surface of cotyledons. Somatic</p><p>embryos at successive stages of development, including</p><p>cotyledonary-stage embryos with shoot and root meris-</p><p>tems, were evident. For plantlet conversion, somatic</p><p>embryos were transferred to maturation medium supple-</p><p>mented with 3 % maltose, followed by 6 weeks of culture</p><p>in Woody Plant Medium supplemented with 0.44-lM BAand 0.46-lM Zeatin (Z). This novel protocol appearspromising for mass propagation, conservation and genetic</p><p>transformation of black alder.</p><p>Keywords Black alder Cones Histology Secondaryembryogenesis Somatic embryos Zygotic embryos</p><p>Introduction</p><p>Alders grow throughout the northern hemisphere in</p><p>woodland and riparian habitats. These species prefer cool</p><p>climates, although they can also withstand warmer tem-</p><p>peratures when water is adequate (Prada and Arizpe 2008).</p><p>The genus is thought to comprise approximately 30 spe-</p><p>cies, which are characterized by wind pollination, seed</p><p>dispersal by wind and water, rapid colonization of bare</p><p>ground and a relatively short life span. The male and</p><p>female flowers occur on separate catkins. The female</p><p>flowers are reddish-purple in color and develop into hard</p><p>cones containing seeds. Although alders are usually not the</p><p>focus of forest protection concerns in Europe, because they</p><p>are of minor importance to the forest economy, the eco-</p><p>logical value of the genus and its high value for land rec-</p><p>lamation and reforestation are well recognized (Cech</p><p>1998). Alders are capable of fixing atmospheric nitrogen</p><p>through symbiotic association with the actinomycete</p><p>Communicated by J. Carlson.</p><p>S. Valladares and E. Corredoira contributed equally to this work.</p><p>E. Corredoira (&amp;) S. Valladares M. T. Martnez A. M. Vieitez M. C. San JoseDepartment of Plant Physiology, Instituto de Investigaciones</p><p>Agrobiologicas de Galicia (CSIC), Apartado 122, 15780</p><p>Santiago de Compostela, Spain</p><p>e-mail: elenac@iiag.csic.es</p><p>123</p><p>Trees</p><p>DOI 10.1007/s00468-013-0907-8</p></li><li><p>Frankia alni (Oliveira et al. 2005), which forms nodules on</p><p>the tree roots. Alnus glutinosa (L.) Gaertn, also called</p><p>common alder, black alder or European black alder, is the</p><p>most common tree species in riparian forests. In addition to</p><p>the above-mentioned characteristics, black alder has a use</p><p>in flood control, stabilization of riverbanks and in func-</p><p>tioning of the river ecosystems (Claessens et al. 2010).</p><p>Black alder is also a host to a wide variety of moss and</p><p>lichen.</p><p>Unfortunately, alder populations are threatened by lethal</p><p>alder blight disease, which is caused by the pathogenic</p><p>fungus Phytophthora alni (Brasier et al. 2004).The disease</p><p>has been particularly destructive in the UK, but is also</p><p>found in Austria, Belgium, France, Germany, and Spain,</p><p>where it threatens natural riparian populations of alder</p><p>(Brasier et al. 1995; Cech 1998; Cavelier et al. 1999;</p><p>Streito et al. 2002; Jung and Blaschke 2004; Solla et al.</p><p>2010). Various attempts have been made to control the</p><p>disease through international research programmes, none</p><p>of which has yet succeeded in significantly halting its</p><p>spread or reducing its impact (Gibbs et al. 2003; Webber</p><p>et al. 2004). Genetic engineering may prove useful for</p><p>controlling alder blight disease. Regeneration of plants</p><p>from genetically transformed cells is a key step in devel-</p><p>oping a protocol for the genetic transformation of alder.</p><p>Somatic embryogenesis is an ideal regeneration system</p><p>for genetic transformation. This propagation technique is</p><p>defined as a process in which a bipolar structure, resem-</p><p>bling a zygotic embryo, develops from a somatic cell</p><p>without vascular connection to the original tissue (von</p><p>Arnold et al. 2002). Somatic embryogenesis, in which the</p><p>frequency of chimeras is low and the regeneration rate is</p><p>high, is more attractive than organogenesis as a plant</p><p>regeneration system (Gaj 2001). In addition, somatic</p><p>embryogenesis is considered the best in vitro propagation</p><p>method for most tree species (Merkle and Nairn 2005).</p><p>This technique could make a substantial contribution to the</p><p>conservation of alder species, not only as a means of pro-</p><p>ducing transgenic trees with resistance genes, but also for</p><p>mass propagation of the resistant genotypes produced by</p><p>traditional breeding programmes. However, as far as we</p><p>know, no studies have addressed the capacity of black alder</p><p>tissue to form somatic embryos. Alder species have only</p><p>been micropropagated by multiplication and rooting of</p><p>axillary shoots (Garton et al. 1981; Perinet and Lalonde</p><p>1983; Tremblay et al. 1986; Perinet and Tremblay 1987;</p><p>Barghchi 1988; Welander et al. 1989; Lall et al. 2005; San-</p><p>Jose et al. 2012, 2013).</p><p>The main objective of this study was, therefore, to</p><p>induce somatic embryogenesis from zygotic embryos (ZEs)</p><p>in Alnus glutinosa and to define a protocol for maintenance</p><p>of embryogenic lines and plantlet regeneration.</p><p>Materials and methods</p><p>Collection and sterilization of plant material</p><p>In the first experiment, cones were collected from three A.</p><p>glutinosa trees, of age 2025 years, during August and</p><p>September 2011 (Table 1). The trees, which were desig-</p><p>nated Sarela 1, Sarela 2 and Sar 1, were growing in Garelas</p><p>park (Sarela 1 and Sarela 2) and Branas del Sar park (Sar 1)</p><p>in Santiago de Compostela (Spain). For the second exper-</p><p>iment, carried out in 2012, cones were collected from Sa-</p><p>rela 2 at weekly intervals during August and September,</p><p>i.e., approximately 16 weeks post-anthesis (WPA).</p><p>Anthesis is defined as the time at which approximately half</p><p>of the tree is in bloom.</p><p>The cones were washed in running tap water for 30 min</p><p>and surface sterilized by immersion for 1 min in 70 %</p><p>ethanol followed by immersion for 10 min in a 0.5 %</p><p>solution of free chlorine (Millipore chlorine tablets) plus</p><p>1 % Tween80. The cones were then rinsed twice with</p><p>sterile distilled water. After the sterilization process, the</p><p>seeds were removed from the cones, and intact zygotic</p><p>embryos were excised from seeds under a stereo micro-</p><p>scope in a flow laminar chamber. Isolated zygotic embryos</p><p>were then used as initial explants and were placed in</p><p>individual 20 9 150 mm culture tubes (one zygotic</p><p>embryo 9 tube) containing 16 ml of embryo induction</p><p>medium. A similar sterilization protocol has been used to</p><p>sterilize shoot tips and nodal segments of Paulownia</p><p>tomentosa (Corredoira et al. 2008).</p><p>Induction of somatic embryogenesis</p><p>In the first set of experiments, the explants were initially</p><p>cultured on induction medium consisting of Murashige and</p><p>Skoog medium (MS; Murashige and Skoog 1962) supple-</p><p>mented with 0.5 g/l casein hydrolyzate, 3 % sucrose, 0.7 %</p><p>agar (Difco Bacto, BD, USA), and 2.22-lM benzyladenine(BA; Sigma-Aldrich, St. Louis, MO, USA) in combination</p><p>with 0.9, 2.26, 4.52 or 9.05 lM 2,4-dichlorophenoxyaceticacid (2,4-D; Duchefa, The Netherlands). Before sterilizing</p><p>the medium, by autoclaving at 121 C for 20 min, the pHwas adjusted to 5.7. For each tree, seeds were collected at</p><p>23 WPA, and 48 zygotic embryos were cultured for each</p><p>concentration of 2,4-D, so that there were 192 explants per</p><p>tree. In the second set of experiments, zygotic embryos</p><p>were collected from the Sarela 2 tree at 16 WPA and</p><p>cultured on MS plus 2.22-lM BA, with or without 2,4-D(0.9 lM). Twenty-four explants were used for each com-bination of induction medium and collection date, thus</p><p>providing 288 explants in total. In addition, on each col-</p><p>lection date, another set of 12 seeds and 12 zygotic</p><p>Trees</p><p>123</p></li><li><p>embryos was used to evaluate the mean length of seeds and</p><p>zygotic embryos.</p><p>After 4 weeks of culture on induction medium, the</p><p>explants were transferred to 300 ml jars (4 or 5 explants</p><p>per jar) containing 50 ml of expression medium consisting</p><p>of MS salts supplemented with 0.5 g/l casein hydrolyzate,</p><p>3 % sucrose, 0.7 % Difco bacto agar, and 0.44-lM BA.The explants were transferred monthly to fresh medium</p><p>and were examined periodically to record the number of</p><p>explants exhibiting callus formation and somatic embryos.</p><p>Initial explants were maintained in darkness at 25 C for4 weeks and were then subjected to a 16-h photoperiod (30</p><p>lmol m-2 s-1 from cool-white fluorescent lamps) with25 C light and 20 C dark temperatures. These conditionswere applied at all subsequent stages of the embryo pro-</p><p>liferation, maturation, and germination experiments.</p><p>Maintenance of embryogenic cultures</p><p>Isolated somatic embryos and embryogenic groups were</p><p>excised from the original explants and subcultured sepa-</p><p>rately on MS medium supplemented with 0.44-lM BA plus0.54 lM a-naphthaleneacetic acid (NAA, Duchefa, TheNetherlands). On this medium, embryo proliferation by</p><p>secondary embryogenesis was low. Therefore, the capacity</p><p>for secondary embryogenesis was investigated by subcul-</p><p>turing embryo clumps of 13 whole somatic primary</p><p>embryos at globular to early cotyledonary stage (embryo-</p><p>genic line S1-1) on five proliferation media consisting of</p><p>MS medium (half-strength macronutrients) supplemented</p><p>with 3-mM glutamine, 3 % sucrose, 0.6 % agar (A-1296,</p><p>Sigma-Aldrich, St. Louis, MO, USA) and different com-</p><p>binations of plant growth regulators (PGRs) (Table 3).</p><p>Similar proliferation media have been used to maintain</p><p>different embryogenic lines of Quercus robur (Mallon</p><p>et al. 2012).</p><p>In a further experiment, the embryo proliferation</p><p>capacity of cotyledon explants isolated from somatic</p><p>embryos was evaluated using the same proliferation media,</p><p>except the medium supplemented with 0.54-lM NAA(Table 4).</p><p>To investigate the influence of genotype, groups of 13</p><p>whole somatic embryos at globular-early cotyledonary</p><p>stage, derived from three different embryogenic lines</p><p>named S1-1 (derived from one seed of tree Sarela 1) and</p><p>S2-1 and S2-2 (derived from two seeds of tree Sarela 2)</p><p>were cultured on proliferation medium supplemented with</p><p>0.44-lM BA.In each experiment, three replicate Petri dishes with six</p><p>explants per dish (18 explants in total) were used. Each</p><p>Petri dish was considered as a single replicate in a com-</p><p>pletely randomized block design, and the experiment was</p><p>repeated twice. In embryo proliferation experiments, the</p><p>number of explants forming secondary embryos, the</p><p>number of embryos per embryogenic explant, and the</p><p>multiplication coefficient (MC) were recorded after</p><p>6 weeks of culture. The multiplication coefficient was</p><p>defined as the product of the proportion of explants pro-</p><p>ducing secondary embryos and the mean number of</p><p>embryos per embryogenic explant. In the genotype</p><p>Table 1 Effect of 2,4-D concentration, tree genotype, and size of zygotic embryo on the induction of somatic embryogenesis in Alnus glutinosa</p><p>Tree Collection datea (WPA) Length of zygotic embryo (mm)b Surviving explants with callus (%)c Embryogenesis induction (%)</p><p>2,4-D (lM)</p><p>0.90 2.26 4.52 9.05</p><p>Sarela 1 2 0.8 0.2 50.0 4.2 </p><p>Sarela 2 2 0.6 0.1 44.3 10.0 6.6 </p><p>Sar 1 2 0.9 0.2 45.8 </p><p>Sar 1 3 1.3 0.2 45.8 </p><p>Source of variation df v2 P</p><p>2,4-D concentration (A) 3 8.177 B0.05</p><p>Tree genotype (B) 2 7.873 B0.05</p><p>AXB 9 6.980 ns</p><p>For each tree and collection date, 192 explants were cultured</p><p>Influence of tree genotype and 2,4-D concentration on embryogenic induction was evaluated by the v2 test; ns not significant</p><p>, no responsea Weeks post-anthesisb Values are means standard error of 12 zygotic embryosc After 4 weeks of culture</p><p>Trees</p><p>123</p></li><li><p>experiment, for each embryogenic line the total number of</p><p>somatic embryos per embryogenic explant and the number</p><p>of cotyledonary somatic embryos per embryogenic explant</p><p>were recorded after 6 weeks of culture.</p><p>Histological analysis</p><p>A histological study was performed during secondary</p><p>embryo proliferation. Samples were fixed in a mixture of</p><p>formalin, glacial acetic acid and 50 % ethanol [1:1:18 (v/v/</p><p>v)], dehydrated through a graded n-butanol series and</p><p>embedded in paraffin. Sections (8 lm) were cut on a Re-ichert-Jung rotary microtome and were later stained with</p><p>periodic acid-Schiff (PAS)naphthol blue-black, which is</p><p>commonly used to reveal total insoluble polysaccharides</p><p>and total protein content of the cells (O0Brien and McCully1981). The stained sections were mounted with Euckit,</p><p>and photomicrographs were taken with an Olympus DP71</p><p>digital camera fitted to a Nikon-FXA microscope.</p><p>Macroscopic features were observed in a stereo micro-</p><p>scope (Olympus SZX9) and photographed with an Olym-</p><p>pus DP10 digital camera.</p><p>Embryo maturation and germination</p><p>Somatic embryos, developed to the cotyledonary stage,</p><p>were carefully separated from the proliferation embryo-</p><p>genic explants and cultured on MS medium (half-strength</p><p>macronutrients) supplemented with either 6 % sucrose or</p><p>3 % maltose. Selection of these media was based on the</p><p>findings of previous studies on somatic embryo maturation</p><p>in other woody species (Iraqui and Tremblay 2001; Cor-</p><p>redoira et al. 2006a). After 5 weeks, somatic embryos were</p><p>transferred to glass jars (300 ml) with 25 ml of germination</p><p>medium consisting of Woody Plant Medium (WPM; Lloyd</p><p>and McCown 1981) supplemented with 2 % glucose, 0.6 %</p><p>agar, 0.44-lM BA and 0.46-lM Zeatin trans isomer (Z;</p><p>Duchefa, The Netherlands). This germination medium was</p><p>selected as the most efficient for the proliferation of axillary</p><p>shoot cultures in Alnus glutinosa (San Jose et al. 2013).</p><p>Twenty-five somatic embryos (five embryos per jar) were</p><p>used for each maturation treatment, and the experiment was</p><p>repeated twice. The numbers of somatic embryos that</p><p>developed roots only and those that successfully converted</p><p>into plantlets were recorded after 6 weeks of culture.</p><p>Statistical analysis</p><p>The influence of the 2,4-D concentration and tree origin o...</p></li></ul>

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