Vol. XXIX Issue 2
Article 4

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"><!-- [et_pb_line_break_holder] --><html xmlns="http://www.w3.org/1999/xhtml"><!-- [et_pb_line_break_holder] --><head><!-- [et_pb_line_break_holder] --><meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" /><!-- [et_pb_line_break_holder] --><title>Documento sin título</title><!-- [et_pb_line_break_holder] --></head><!-- [et_pb_line_break_holder] --><!-- [et_pb_line_break_holder] --><body><!-- [et_pb_line_break_holder] --><p align="right"><font size="3" face="Arial, Helvetica, sans-serif"><strong>ARTÍCULOS ORIGINALES</strong></font></p><!-- [et_pb_line_break_holder] --><p><font size="4" face="Arial, Helvetica, sans-serif"><strong>Pollen viability and meiotic irregularities in a collection</strong> <!-- [et_pb_line_break_holder] --> <strong>of <em>Stevia rebaudiana </em>(Bertoni) Bertoni cultivated in</strong> <!-- [et_pb_line_break_holder] --> <strong>Tucumán, Argentina</strong></font></p><!-- [et_pb_line_break_holder] --><p><i><font size="3" face="Arial, Helvetica, sans-serif"><strong>Viabilidad de polen e irregularidades meióticas en una</strong> <strong>colección de Stevia rebaudiana (Bertoni) Bertoni cultivada</strong> <strong>en Tucumán, Argentina</strong></font></i></p><!-- [et_pb_line_break_holder] --><p> </p><!-- [et_pb_line_break_holder] --><p><b><font size="3" face="Arial, Helvetica, sans-serif">Budeguer C.J.<SUP>1*</sup>, Camadro E.L.<SUP>2</sup>, Erazzú L.E.<SUP>3</sup></font></b></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><SUP>1</sup> Cátedra de Genética, Facultad de Agronomía y Zootecnia, Universidad Nacional de Tucumán, Argentina;<br /><!-- [et_pb_line_break_holder] --> <SUP>2</sup> Unidad Integrada EEA “Domingo Pasquale” Instituto Nacional de Tecnología Agropecuaria (INTA)-Facultad de Cs. Agrarias, <!-- [et_pb_line_break_holder] --> Universidad Nacional de Mar del Plata y CONICET, Argentina;<br /><!-- [et_pb_line_break_holder] --> <SUP>3</sup> Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agrícola Famaillá, Tucumán, Argentina.<br /><!-- [et_pb_line_break_holder] --> * Autor correspondiente: <a href="mailto:carlosjbudeguer@gmail.com">carlosjbudeguer@gmail.com</a></font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><b>Fecha de recepción</b>: 01/11/2018<br /><!-- [et_pb_line_break_holder] --> <b>Fecha de aceptación de versión final</b>: 03/12/2018</font></p><!-- [et_pb_line_break_holder] --><hr /><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><b>ABSTRACT</b></font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><em>Stevia rebaudiana </em>(Bertoni) Bertoni (2n=2x=22) (Asteraceae family) is a species of economic value due to the presence of steviol glycosides<!-- [et_pb_line_break_holder] --> in leaves -mainly stevioside and rebaudioside A- which are non-caloric sugars. In 2013, a collection of plants (genotypes) from four contrasting<!-- [et_pb_line_break_holder] --> environments was established in Tucuman, Argentina, for evaluation under local conditions and, eventually, breeding purposes. As a first step, pollen<!-- [et_pb_line_break_holder] --> viability and meiosis was studied in a sample of 56 plants. The percentage of pollen viability varied from medium (69.4%) to high (99.6%) in 52<!-- [et_pb_line_break_holder] --> of them, but from low (36.5%) to relatively low (51.5%) in the remaining four. The latter four plants also presented pollen grains of heterogeneous<!-- [et_pb_line_break_holder] --> size, which were classified as n (normal, the most frequent size in the sample), <n and >n. Abnormalities were observed in meiosis and at the tetrad<!-- [et_pb_line_break_holder] --> stage. Pollen viability appears not to be a problem for planning crossing experiments within the collection. Moreover, the observation of parallel<!-- [et_pb_line_break_holder] --> spindles at Anaphase II and dyads and triads at the tetrad stage entails the possibility of eventually exploring ploidy manipulations in breeding.</font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><b>Key words</b>: <em>Stevia</em>; Pollen viability; Meiosis; Sporads</font>.</p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><b>RESUMEN</b></font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><em>Stevia rebaudiana </em>(Bertoni) Bertoni (2n=2x=22) (familia Asteraceae) es una especie de valor economico debido a la presencia de glucosidos de<!-- [et_pb_line_break_holder] --> esteviol en sus hojas, principalmente esteviosido y rebaudiosido A, que son azucares no caloricos. En 2013, se establecio una coleccion de plantas<!-- [et_pb_line_break_holder] --> (genotipos) provenientes de cuatro ambientes contrastantes en Tucuman, Argentina, para su evaluacion en condiciones locales y, eventualmente,<!-- [et_pb_line_break_holder] --> con fines de mejoramiento genetico. Como primer paso, se estudio la viabilidad del polen y la meiosis en una muestra de 56 plantas. El porcentaje<!-- [et_pb_line_break_holder] --> de viabilidad del polen fue de medio (69,4%) a alto (99,6%) en 52 de ellas, pero de bajo (36,5%) a relativamente bajo (51,2%) en las cuatro<!-- [et_pb_line_break_holder] --> restantes. Las ultimas cuatro plantas tambien presentaron granos de polen de tamano heterogeneo, que se clasificaron como n (normal, el tamano<!-- [et_pb_line_break_holder] --> mas frecuente en la muestra), <n y >n. Se observaron anomalias en la meiosis y en el estadio de tetrada. La viabilidad del polen no parece ser un<!-- [et_pb_line_break_holder] --> problema para planificar cruzamientos experimentales en la coleccion. Ademas, la observacion de husos paralelos en Anafase II de diadas y triadas<!-- [et_pb_line_break_holder] --> en la etapa de tetrada conlleva la posibilidad de explorar, eventualmente, manipulaciones de ploidia en el mejoramiento de esta especie.</font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><b>Palabras clave</b>: <em>Stevia</em>; Viabilidad de polen; Meiosis; Esporada</font>.</p><!-- [et_pb_line_break_holder] --><hr /><!-- [et_pb_line_break_holder] --><p> </p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><b>INTRODUCTION</b></font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><em>Stevia rebaudiana </em>(Bertoni) Bertoni (2n=2x=22) belongs<!-- [et_pb_line_break_holder] --> to the Asteraceae family. This species, native to the<!-- [et_pb_line_break_holder] --> Amambay region of Northeastern Paraguay (Soejarto,<!-- [et_pb_line_break_holder] --> 2002; Brandle and Telmer, 2007), was introduced for<!-- [et_pb_line_break_holder] --> cultivation in Argentina by the end of the XXth century.<!-- [et_pb_line_break_holder] --> Its leaves accumulate steviol glycosides, mainly stevioside<!-- [et_pb_line_break_holder] --> and rebaudioside A, which are non-caloric natural sugars<!-- [et_pb_line_break_holder] --> widely used in food products and as dietary supplements<!-- [et_pb_line_break_holder] --> in many countries (Kinghorn and Soejarto, 1985; Brandle<!-- [et_pb_line_break_holder] --> and Rosa, 1992; Brandle and Telmer, 2007). Several of the<!-- [et_pb_line_break_holder] --> widely cultivated lines of <em>S. rebaudiana </em>were experimentally<!-- [et_pb_line_break_holder] --> obtained at the University of New Mexico, USA, in<!-- [et_pb_line_break_holder] --> a program designed to develop polyploids richer in<!-- [et_pb_line_break_holder] --> stevioside than their diploid counterparts (Valois, 1992). In<!-- [et_pb_line_break_holder] --> Brazil, triploid and tetraploid lines from those experiments<!-- [et_pb_line_break_holder] --> were initially cultivated at the CENARGEN/EMBRAPA<!-- [et_pb_line_break_holder] --> germplasm bank before the transference of part of the<!-- [et_pb_line_break_holder] --> collection to CPQBA/UNICAMP (Oliveira <em>et al.</em>,<!-- [et_pb_line_break_holder] --> 2004). In Argentina studies on glycoside content, plant<!-- [et_pb_line_break_holder] --> morphology and reproductive biology in different lines or<!-- [et_pb_line_break_holder] --> varieties were carried out by Kolb <em>et al. </em>(2001), Kryvenki<!-- [et_pb_line_break_holder] --> <em>et al. </em>(2007), Taiariol and Molina (2010), Guerrero <em>et al.</em><!-- [et_pb_line_break_holder] --> (2015) and Caponio <em>et al. </em>(2016).<!-- [et_pb_line_break_holder] --> <em><br /><!-- [et_pb_line_break_holder] --> Stevia rebaudiana </em>has a complex reproductive system,<!-- [et_pb_line_break_holder] --> which is not completely understood. Its flowers are<!-- [et_pb_line_break_holder] --> hermaphrodite, and also protandry has been reported,<!-- [et_pb_line_break_holder] --> thus, cross-pollination appears to be the prevalent mode<!-- [et_pb_line_break_holder] --> of sexual reproduction (Yadav <em>et al.</em>, 2014; Caponio <em>et</em><!-- [et_pb_line_break_holder] --> <em>al.</em>, 2016; Benelli <em>et al.</em>, 2017). In addition, a sporophytic<!-- [et_pb_line_break_holder] --> homomorphic autoincompatibility system would further<!-- [et_pb_line_break_holder] --> promote cross-pollination (Monteiro, 1980; Brandle<!-- [et_pb_line_break_holder] --> <em>et al.</em>, 1998; Yadav <em>et al.</em>, 2014; Caponio <em>et al.</em>, 2016).<!-- [et_pb_line_break_holder] --> For commercial production, however, it is reproduced<!-- [et_pb_line_break_holder] --> agamically via stem cuttings (Kryvenki <em>et al.</em>, 2007; Tamura<!-- [et_pb_line_break_holder] --> <em>et al.</em>, 1984a; b) or micropropagation (Miyagawa <em>et al.</em>, 1986;<!-- [et_pb_line_break_holder] --> Swanson <em>et al.</em>, 1992; Akita <em>et al.</em>, 1994; Constantinovici<!-- [et_pb_line_break_holder] --> and Cachita, 1997) due the poor and highly variable<!-- [et_pb_line_break_holder] --> percentage of viable seed production (Monteiro, 1980;<!-- [et_pb_line_break_holder] --> Duke, 1993; Carneiro <em>et al.</em>, 1997).<!-- [et_pb_line_break_holder] --> Besides the described sexual and agamic reproduction,<!-- [et_pb_line_break_holder] --> in embryological studies Monteiro (1980) observed that<!-- [et_pb_line_break_holder] --> <em>S. rebaudiana </em>can also reproduce apomictically. Along<!-- [et_pb_line_break_holder] --> with agamospermy, this author reported the production<!-- [et_pb_line_break_holder] --> of viable pollen in sexual plants and sterile pollen in the<!-- [et_pb_line_break_holder] --> apomictics, as well as relatively low (65%) pollen viability<!-- [et_pb_line_break_holder] --> in three batches of 10 plants each, introduced in Brazil<!-- [et_pb_line_break_holder] --> from Paraguay in the 1960 and 1970 decades. Furthermore,<!-- [et_pb_line_break_holder] --> Oliveira <em>et al. </em>(2004) only observed sterile pollen in diploid,<!-- [et_pb_line_break_holder] --> triploid and tetraploid lines obtained from CENARGEN/<!-- [et_pb_line_break_holder] --> EMBRAPA, in Brasilia. Caponio <em>et al. </em>(2016), however,<!-- [et_pb_line_break_holder] --> reported pollen viability values between 94.8 and 97.9%<!-- [et_pb_line_break_holder] --> in six plants from two geographic provinces of Argentina:<!-- [et_pb_line_break_holder] --> Misiones (four ecotypes) and Entre Rios (two ecotypes).<br /><!-- [et_pb_line_break_holder] --> Pollen viability can be used as an indicator of<!-- [et_pb_line_break_holder] --> regularities/irregularities in the nuclear division processes<!-- [et_pb_line_break_holder] --> during male sporogenesis and/or gametogenesis. Low<!-- [et_pb_line_break_holder] --> pollen viability in higher plants might have a genetic<!-- [et_pb_line_break_holder] --> and/or an environmental base (Larrosa <em>et al.</em>, 2012; Farco<!-- [et_pb_line_break_holder] --> and Dematteis, 2014). Thus, meiotic analyses can provide<!-- [et_pb_line_break_holder] --> relevant information for both basic and applied studies<!-- [et_pb_line_break_holder] --> (De Souza <em>et al.</em>, 2003), particularly for parental selection<!-- [et_pb_line_break_holder] --> in breeding. In <em>S. rebaudiana</em>, Raina <em>et al. </em>(2013) observed<!-- [et_pb_line_break_holder] --> mostly normal chromosome behavior in two morphotypes<!-- [et_pb_line_break_holder] --> with low pollen viability, but they also observed a few<!-- [et_pb_line_break_holder] --> univalents at diplotene and diakinesis in some meiocytes,<!-- [et_pb_line_break_holder] --> which revealed synaptic problems underlying the sterility.<br /><!-- [et_pb_line_break_holder] --> In 2013, a collection of <em>Stevia rebaudiana </em>was established<!-- [et_pb_line_break_holder] --> in Tucuman, Argentina, to evaluate germplasm from<!-- [et_pb_line_break_holder] --> four contrasting environments for adaptation to local<!-- [et_pb_line_break_holder] --> conditions and, eventually, breeding purposes. The aim<!-- [et_pb_line_break_holder] --> of this communication is to report the results of pollen<!-- [et_pb_line_break_holder] --> viability and meiotic studies carried out in a sample of<!-- [et_pb_line_break_holder] --> plants of this collection.</font></p><!-- [et_pb_line_break_holder] --><p><b><font size="3" face="Arial, Helvetica, sans-serif">MATERIALS AND METHODS</font></b></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif">In November 2013, 120 plants from four origins (30<!-- [et_pb_line_break_holder] --> plants/origin) were established in the experimental field at<!-- [et_pb_line_break_holder] --> <em>Estación Experimental Agropecuaria Famaillá, INTA </em>(27o 00´ <!-- [et_pb_line_break_holder] --> 49” S - 65o 22´ 32” W), in Tucuman province, Argentina.<!-- [et_pb_line_break_holder] --> This locality has an average annual temperature of 20o<!-- [et_pb_line_break_holder] --> C and an average annual precipitation of 965 mm. From<!-- [et_pb_line_break_holder] --> this collection, 56 plants from the “criolla” landrace were<!-- [et_pb_line_break_holder] --> included in the study, which were assigned the codes<!-- [et_pb_line_break_holder] --> T (Tucuman), J (Jujuy), M (Misiones) and F (Formosa),<!-- [et_pb_line_break_holder] --> according to their provenance, followed by consecutive<!-- [et_pb_line_break_holder] --> numbers (Table 1). Plants were placed in the field, in<!-- [et_pb_line_break_holder] --> furrows at a distance of 1 m between furrows and 0.4<!-- [et_pb_line_break_holder] --> m between plants; irrigation was provided by dripping<!-- [et_pb_line_break_holder] --> as needed. The field grown plants were vegetatively<!-- [et_pb_line_break_holder] --> propagated in September-October 2014 by stem cuttings,<!-- [et_pb_line_break_holder] --> and the derived plantlets were cultivated in 2 l pots in<!-- [et_pb_line_break_holder] --> the open, also with irrigation by dripping. Pollen viability<!-- [et_pb_line_break_holder] --> was determined in 56 well-grown plants. Pollen samples<!-- [et_pb_line_break_holder] --> were obtained from mature anthers prior to anthesis, from<!-- [et_pb_line_break_holder] --> florets of 14 of the pot-grown plants per origin (56 plants<!-- [et_pb_line_break_holder] --> in total) in February 2015. From each plant, four florets<!-- [et_pb_line_break_holder] --> were analyzed, and significant differences between means<!-- [et_pb_line_break_holder] --> were detected by Fisher´s Least Significant Difference<!-- [et_pb_line_break_holder] --> (LSD) test at the 5% level with Infostat 2018 (Di Rienzo<!-- [et_pb_line_break_holder] --> <em>et al</em>., 2018).<!-- [et_pb_line_break_holder] --> </font></p><!-- [et_pb_line_break_holder] --><p><a name="tab1" id="tab1"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b>Table 1</b>. Geographical provenance of the <em>Stevia rebaudiana </em>plants collected in Argentina.</font><br /><!-- [et_pb_line_break_holder] --> <img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a05tab1.jpg" width="561" height="233" /></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif">Fresh pollen from five anthers per floret were placed<!-- [et_pb_line_break_holder] --> on glass slides and stained with a 0.5% acetocarmine<!-- [et_pb_line_break_holder] --> solution (0.5 g carmine, 45 ml glacial acetic acid and<!-- [et_pb_line_break_holder] --> 55 ml distilled water, 50 ml glycerol) (Marks, 1954), for<!-- [et_pb_line_break_holder] --> observation under an optical microscope at 125x. On<!-- [et_pb_line_break_holder] --> average, 300 pollen grains per plant were observed. Pollen<!-- [et_pb_line_break_holder] --> grains that were fully stained with well-defined contours<!-- [et_pb_line_break_holder] --> were considered viable, whereas those that were poorly (or<!-- [et_pb_line_break_holder] --> not) stained were considered sterile. In plants with pollen<!-- [et_pb_line_break_holder] --> of heterogeneous size, 200 pollen grains per plant were<!-- [et_pb_line_break_holder] --> measured with a micrometric eyepiece and, according<!-- [et_pb_line_break_holder] --> to diameter, were classified as n (normal, most frequent<!-- [et_pb_line_break_holder] --> diameter in the sample), <n and >n. Following Larrosa <em>et al</em>. (2012) and based on the volume of a sphere, pollen<!-- [et_pb_line_break_holder] --> grains with diameters 1.26 times larger than the average<!-- [et_pb_line_break_holder] --> pollen diameter of each sample were considered to be<!-- [et_pb_line_break_holder] --> chromosomically unreduced (2n), and those with diameters<!-- [et_pb_line_break_holder] --> 1.59 times larger were considered to be chromosomically<!-- [et_pb_line_break_holder] --> double unreduced (4n).<!-- [et_pb_line_break_holder] --> <br /><!-- [et_pb_line_break_holder] --> Meiosis was studied in plants with low pollen viability<!-- [et_pb_line_break_holder] --> (<52%) and heterogeneity in pollen size. To that end,<!-- [et_pb_line_break_holder] --> immature flower buds at various developmental stages<!-- [et_pb_line_break_holder] --> were fixed in absolute ethanol: glacial acetic acid (3:1, v/v)<!-- [et_pb_line_break_holder] --> for 24 hs, and stored in 70% ethanol until use (Mok and<!-- [et_pb_line_break_holder] --> Peloquin, 1975). Meiocytes were removed from anthers<!-- [et_pb_line_break_holder] --> placed on a drop of 0.5% acetocarmine (0.5 g carmine, 45<!-- [et_pb_line_break_holder] --> ml glacial acetic acid and 55 ml distilled water) for staining,<!-- [et_pb_line_break_holder] --> and observed with an optical microscope, starting from the<!-- [et_pb_line_break_holder] --> most advanced stages and working backwards.</font></p><!-- [et_pb_line_break_holder] --><p><b><font size="3" face="Arial, Helvetica, sans-serif">RESULTS</font></b></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif">Fifty-two out of the 56 analyzed plants had medium<!-- [et_pb_line_break_holder] --> to high pollen viability (69.4 to 99.6%), whereas the<!-- [et_pb_line_break_holder] --> remaining plants had low viability (36.5 to 51.5%) (<a href="#tab2">Table<!-- [et_pb_line_break_holder] --> 2</a>). Plants with low to relatively low pollen viability (M10.8,<!-- [et_pb_line_break_holder] --> M11.7, M11.11 and F12.1) also produced pollen grains of<!-- [et_pb_line_break_holder] --> heterogeneous size (<a href="#tab3">Table 3</a>, <a href="#fig1">Figure 1A, B</a>).</font></p><!-- [et_pb_line_break_holder] --><p><a name="tab2" id="tab2"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b>Table 2</b>. Percentage of pollen viability in individual plants of <em>Stevia rebaudiana.</em></font><br /><!-- [et_pb_line_break_holder] --> <img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a05tab2.jpg" width="494" height="394" /></p><!-- [et_pb_line_break_holder] --><p><a name="tab3" id="tab3"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b>Table 3</b>. Percentage of pollen according to size and sporad types in plants of <em>Stevia rebaudiana </em>with low<!-- [et_pb_line_break_holder] -->pollen viability.</font><br /><!-- [et_pb_line_break_holder] --><img src=https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a05tab3.jpg" width="500" height="219" /></p><!-- [et_pb_line_break_holder] --><p><a name="fig1" id="fig1"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a05fig1.jpg" width="503" height="182" /><br /><!-- [et_pb_line_break_holder] --> Figure 1</b>. Variability in pollen grain viability and size: viable (stained), inviable (non-stained) (A); n, <n, >n, (B).<!-- [et_pb_line_break_holder] -->Bar=20μ.</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"> Pollen grains were classified as n (mean diameter<!-- [et_pb_line_break_holder] --> =20.37 μ, the most common in the sample), >n ( =33.83<!-- [et_pb_line_break_holder] --> μ) and <n ( =11.55 μ) (<a href="#tab4">Table 4</a>). The mean diameter<!-- [et_pb_line_break_holder] --> of the largest-sized pollen was 1.66 times that of the n<!-- [et_pb_line_break_holder] --> pollen, and the mean diameter of the smallest-sized pollen<!-- [et_pb_line_break_holder] --> was 0.57 times the diameter of the n pollen. Plant M10.8<!-- [et_pb_line_break_holder] --> had the highest percentage of <n pollen (24.0%), whereas<!-- [et_pb_line_break_holder] --> M11.11 and M11.7 had the highest percentages of >n<!-- [et_pb_line_break_holder] --> pollen (12.0% and 11.9%, respectively).<!-- [et_pb_line_break_holder] --> </font></p><!-- [et_pb_line_break_holder] --><p><a name="tab4" id="tab4"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b>Table 4</b>. Mean, minimum and maximum pollen diameter (μ) and<!-- [et_pb_line_break_holder] -->standard deviations in <em>Stevia rebaudiana.</em></font><br /><!-- [et_pb_line_break_holder] --><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a05tab4.jpg" width="347" height="209" /></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif">All plants produced normal tetrads; nevertheless,<!-- [et_pb_line_break_holder] --> abnormalities were observed in the four plants with low<!-- [et_pb_line_break_holder] --> pollen viability (<a href="#tab3">Table 3</a>). These abnormalities included<!-- [et_pb_line_break_holder] --> monads in M10.8 (2.6%) and M11.7 (1%) (<a href="#fig2">Figure 2A</a>);<!-- [et_pb_line_break_holder] --> dyads in M11.7 (20.8%), M11.11 (2%), and F12.1 (1.4%)<!-- [et_pb_line_break_holder] --> (<a href="#fig2">Figure 2B</a>); and triads in M11.7 (0.9%), M11.11 (16%),<!-- [et_pb_line_break_holder] --> and F12.1 (12.7%) (<a href="#fig2">Figure 2C</a>). Moreover, pentads were<!-- [et_pb_line_break_holder] --> observed in M11.7 (0.9%) and M10.8 (0.5%).<!-- [et_pb_line_break_holder] --> </font></p><!-- [et_pb_line_break_holder] --><p><a name="fig2" id="fig2"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a05fig2.jpg" width="569" height="124" /><br /><!-- [et_pb_line_break_holder] -->Figure 2</b>. Normal and abnormal sporads in <em>Stevia rebaudiana</em>: monad (A), dyad (B), triad (C), normal tetrad (D). Bar=20μ.</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif">The number of cells of the previously mentioned<!-- [et_pb_line_break_holder] --> four plants (M10.8, M11.7, M11.11 and F12.1) analyzed<!-- [et_pb_line_break_holder] --> at various meiotic stages and the irregularities observed<!-- [et_pb_line_break_holder] --> are presented in <a href="#tab5">Table 5</a>. In Metaphase I, out of plate<!-- [et_pb_line_break_holder] --> chromosomes (<a href="#fig3">Figure 3A</a>) were observed in 18.5% of<!-- [et_pb_line_break_holder] --> the cells of M10.8, whereas in the other three plants, this<!-- [et_pb_line_break_holder] --> percentage varied from 2.4 to 6.2%. In Anaphase I, laggards<!-- [et_pb_line_break_holder] --> (<a href="#fig3">Figure 3B</a>) were observed in percentages varying from<!-- [et_pb_line_break_holder] --> 2.7 to 7.9. Metaphases II (not included in the table) were<!-- [et_pb_line_break_holder] --> normal, except for the presence of parallel spindles (<a href="#fig3">Figure<!-- [et_pb_line_break_holder] --> 3C</a>) in F12.1. In Telophase II, 2.8 to 6.7% of the cells<!-- [et_pb_line_break_holder] --> presented chromosomes grouped in three poles instead of<!-- [et_pb_line_break_holder] --> four (<a href="#fig3">Figure 3D</a>).</font></p><!-- [et_pb_line_break_holder] --><p><a name="tab5" id="tab5"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b>Table 5</b>. Percentage of meiotic abnormalities in plants of <em>Stevia rebaudiana </em>with low<!-- [et_pb_line_break_holder] -->pollen viability.</font><br /><!-- [et_pb_line_break_holder] --><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a05tab5.jpg" width="392" height="454" /></p><!-- [et_pb_line_break_holder] --><p><a name="fig3" id="fig3"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a05fig3.jpg" width="568" height="134" /><br /><!-- [et_pb_line_break_holder] --> Figure 3</b>. Meiotic abnormalities in <em>S. rebaudiana</em>: out of plate chromosomes at Metaphase I (A), laggards at Anaphase I (B),<!-- [et_pb_line_break_holder] -->parallel spindles (der.) at Metaphase II (C), chromosomes grouped in three poles in Telophase II (D). Bar=20μ.</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><b>DISCUSSION</b> </font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif">Most plants analyzed in this study (92.9%) had more than<!-- [et_pb_line_break_holder] --> 85.0% pollen viability, thus, this factor is not expected<!-- [et_pb_line_break_holder] --> to be a problem in planning crossing experiments with<!-- [et_pb_line_break_holder] --> the <em>Stevia rebaudiana </em>collection established in Tucuman.<!-- [et_pb_line_break_holder] --> However, abnormalities were detected in meiosis and at<!-- [et_pb_line_break_holder] --> the tetrad stage in the other 8.0%, which could account<!-- [et_pb_line_break_holder] --> for their low pollen viability.<!-- [et_pb_line_break_holder] --> Following Larrosa <em>et al. </em>(2012), 2n and 4n pollen grains<!-- [et_pb_line_break_holder] --> were identified, along with n, <n and other >n pollen<!-- [et_pb_line_break_holder] --> grains. The observation of monads, dyads and triads at<!-- [et_pb_line_break_holder] --> the tetrad stage gave further support to this classification.<!-- [et_pb_line_break_holder] --> Although there are reports on 2n pollen production in the<!-- [et_pb_line_break_holder] --> Asteraceae family (<em>i.e.</em>, Noyes and Allison, 2005; Echeverria<!-- [et_pb_line_break_holder] --> and Camadro, 2017), this is the first report on 2n and 4n<!-- [et_pb_line_break_holder] --> pollen production in <em>S. rebaudiana</em>.<!-- [et_pb_line_break_holder] --> <br /><!-- [et_pb_line_break_holder] --> In Dicotyledons, the first meiotic division is not<!-- [et_pb_line_break_holder] --> followed by cytokinesis; rather, two spindles are formed<!-- [et_pb_line_break_holder] --> in the second division, which are oriented such that<!-- [et_pb_line_break_holder] --> their poles define a tetrahedron; finally, simultaneous<!-- [et_pb_line_break_holder] --> cytokinesis occurs yielding a tetrad of four n microspores.<!-- [et_pb_line_break_holder] --> Abnormalities in spindle orientation and/or cytokinesis<!-- [et_pb_line_break_holder] --> due to the action of meiotic mutants can lead to the<!-- [et_pb_line_break_holder] --> formation of meiocytes with different numbers of<!-- [et_pb_line_break_holder] --> microspores at the tetrad stage (Mok and Peloquin, 1975).<!-- [et_pb_line_break_holder] --> In the present study, the presence of monads, dyads, triads,<!-- [et_pb_line_break_holder] --> and parallel spindles in plants M10.8, M11.7, M11.11 and<!-- [et_pb_line_break_holder] --> F12.1 allows the presumption of the presence of meiotic<!-- [et_pb_line_break_holder] --> mutants. Similarly, the production of <n and other > n<!-- [et_pb_line_break_holder] --> pollen grains could be due to the action of other types of<!-- [et_pb_line_break_holder] --> mutants.<!-- [et_pb_line_break_holder] --> <br /><!-- [et_pb_line_break_holder] --> In summary, pollen viability was high in most of plants<!-- [et_pb_line_break_holder] --> of the <em>S. rebaudiana </em>collection sample grown in Tucuman,<!-- [et_pb_line_break_holder] --> but low pollen viability was also detected, which was<!-- [et_pb_line_break_holder] --> underlined by meiotic irregularities. Some of these<!-- [et_pb_line_break_holder] --> irregularities leading to 2n and 4n pollen formation are,<!-- [et_pb_line_break_holder] --> presumably, under control of meiotic mutants. <!-- [et_pb_line_break_holder] --> Thus, for<!-- [et_pb_line_break_holder] --> breeding purposes, it is advisable to previously screen the<!-- [et_pb_line_break_holder] --> collection for selecting fertile male parents. Moreover, it<!-- [et_pb_line_break_holder] --> is feasible to explore heterotic responses for the traits of<!-- [et_pb_line_break_holder] --> interest at higher ploidy levels by sexual polyploidization<!-- [et_pb_line_break_holder] --> via 2n pollen.</font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><b>BIBLIOGRAPHY</b></font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif">1. Akita M., Shigeoka T., Koizumi Y., Kawamura M. (1994)<!-- [et_pb_line_break_holder] --> Mass propagation of shoots of <em>Stevia rebaudiana </em>using<!-- [et_pb_line_break_holder] --> large scale bioreactor. Plant Cell Rep. Jpn. 13: 180-183.</font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"> 2. Benelli G., Canale A., Romano D., Flamini G., Tavarini S.,<!-- [et_pb_line_break_holder] --> Martini A., Angelini L.G. 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