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Endoreduplication in Floral Structure, Vegetative and Fruits of Red Pitaya with White Pulp

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Endoreduplication in Floral Structure, Vegetative and Fruits of Red Pitaya with White Pulp 931 Original Article ENDOREDUPLICATION IN FLORAL STRUCTURE, VEGETATIVE AND FRUITS OF RED PITAYA WITH WHITE PULP ENDORREDUPLICAÇÃO EM ESTRUTURA FLORAL, VEGETATIVA E FRUTOS DE PITAYA VERMELHA DE POLPA BRANCA Thatiane Padilha de MENEZES 1; Leila Aparecida Salles PIO 2; José Darlan RAMOS 3; Moacir PASQUAL 4; Tesfahum Alemu SETOTAW5 1. Doutora em Agronomia, Universidade Federal de Lavras – UFLA, Lavras, MG, Brasil. [email protected]; 2. Professora, Doutora em Agronomia, Universidade Federal de Lavras – UFLA, Lavras, MG, Brasil; 3. Professor, Doutor em Agronomia, Universidade Federal de Lavras – UFLA, Lavras, MG, Brasil; 4. Professor, Doutor em Agronomia, Universidade Federal de Lavras – UFLA, Lavras, MG, Brasil. [email protected]; 5. Doutor em Agonomia, Universidade Federal de Lavras – UFLA, Lavras, MG, Brasil. ABSTRACT: Endoreduplication is the change of cellular cycle that result DNA duplication without cell division and could result endopolyploid cells. This phenomenon is common in plants and animals and considered as evaluative strategy. Although endoreduplication reported in various plant species, the information about these phenomena in red pitaya is rare. Therefore, this work was done with the objective of studying the endoreduplication in Hylocereus undatus Haw. using flow cytometry analysis. In this study were used the tissue from the flower structure, fruits, roots, cladode, and thorns of the pitaya plant.To determine the DNA content approximately 50 mg the sample of each treatment with Pisum sativum (the internal standard reference) were grind in plate of petri dishes contained 1 mL of cold Marie buffer to release the nucleus.The nuclear suspension was filtered through 50 µm mesh. The nucleuses were colored with 25 µL of 1 mg/L mL of propidium iodide. For each treatment, three samples of 10 thousand nucleuses were analyzed in cytometry Facscalibur (Becton Dickinson). The content of nuclear DNA (pg) was estimated as the ratio between fluorescence intensity of the G1 nucleus of the standard and the G1nucleus of the sample multiplied by the quantity of DNA of the internal reference. In conclusion, the endoreduplication occurred in all part of the plants analyzed except in the aculeus and the roots. The analysis evidenced different index of DNA content in the tissues analyzed being observed up to four different ploidy levels. The phenomena of endoreduplication occurs in all parts of the plant analyzed except in aculeus and roots. KEYWORDS: Flow cytometry. DNA content. Endopolyploidy. Hylocereus undatus Haw. INTRODUCTION Consequently the edoreduplicated cells are produced and this is one form of somatic polyploidy Some decades ago, pitaya is cactaceae (NAGL, 1976; D’AMATO, 1984). The increase in native to Tropical and subtropical America is the ploidy level can increase the metabolism and unknown. But recently, itoccupied an increasing genetic redundancy that improve the productivity importance in exotic fruit market of Europe due to and quality of the plant (COMAI, 2005). It also its exotism and organoleptic characteristics. The accelerates the metabolism and improves the interest for the plant is recent and required more physiological functionsof the plant (BARROW, studies involving the agronomic, genetics and 2006). technology (MARQUES et al., 2011; BASTOS et This phenomenon is found in abundance al., 2006). among angiosperms that grows in adverse It is known that during the development of environmental conditions suggested as an evaluative some plants the normal cellular cycle can be advantage (BARROW; MEISTER, 2003).It’s also substituted by the altered cellular cycle where did reported in yeast and animals andbelieved to be the not occur mitoses (CHEVALIER et al., 2011). This part of normal standard development or a response altered cellular cycle is denominated as endocycle for physiological or genetic stimulus(EDGAR; or endoreduplication which occur the DNA ORR-WEAVER, 2001). It can also be associated duplication without separation of chromosomes and with the increase in the size of the nucleus, cells, cytokinesis (de VEYLDER et al., 2011). rate of metabolism, and cellular differentiation in The endoreduplication can be initiated in variety of plants (SABELLI; LARKINS, 2008). various phases of plant development (de VEYLDER The endoreduplication can be proved using et al., 2011), thatcan result the variation of DNA the flow cytometry technique that measures the content within the organism (COMAI, 2005). physical and/or chemical characteristics of a single Received: 10/02/16 Biosci. J., Uberlândia, v. 32, n. 4, p. 931-939, July/Aug. 2016 Accepted: 02/05/16 932 Endoreduplication in floral structure… MENEZES, T. P. et al. particle. In this technique, the measurements are suspension was aspirated through two gas chambers done when the number of particles in suspension with the help of pipette of Pasteur and filtered using passed individually through the focus of light the 50 µm mesh. The nucleuses were colored with generating pulses of reflected light and the the addition of 25 µL of 1 mg mL -1 solution of fluorescence that are collected are converted in to propidium iodide for each sample. The samples pulses of electric current by optical sensor. This were analyzed immediately after the preparation. principle can measure for example the DNA content For each sample, 10 thousand nucleuses were of the cell (DOLEZEL; BARTOS, 2005). analyzed using the logarithmic scale. The analysis The DNA content estimation has a number was realized using cytometer Facscalibur (Becton of application from basic research to plant breeding Dickinson) and the histogram is obtained with the including estimating the genome size, evaluation of software Cell Quest and analyzed statistically with ploidy, detection of mixed ploidy and aneuploidy, the software WinMDI 2.8. evaluation of cellular cycle, study the chromosome The nuclear DNA content (pg) of the plants elimination, cell separation, chromosome or was estimated using the ratio of fluorescence cense organelles, among others (DOLEZEL; BARTOS, intensity of G1 nucleus of the standard reference (P. 2005). sativum ) and the G1 nucleus of the sample Due to the importance of edoreduplication multiplied by the DNA content of the standard in plants and the absence of information about this reference (9.09 pg). The results obtained were phenomenon in pitaya fruit Brazilian , this work was submitted for analysis of variance (ANOVA) and done to study the occurrence of these phenomena in the means were clustered using the Scott-Knott test, various parts of red pitaya plant through flow at 5% error probability by the software Sisvar cytometry analysis with the quantification of the (FERREIRA, 2011). DNA content of these structures. RESULTS AND DISCUSSION MATERIAL AND METHODS The quantification of nuclear DNA through The experiment was realized in pitaya plant flow cytometer in a species is possible by with four years old maintained by a structure type of comparing the pick formed by nucleus at G1 trellis installed in experimental site of the interface stage of cellular cycle of the standard Horticulture Research of the Department of reference (the material where its DNA content Agriculture, Federal University of Lavras, Brazil. known) to the G1 pick of the sample to be analyzed. Pitaya plants were used with five years of age in 3 x In the present study the samples in majority of the 3 spacing, supported perpendicularly to the ground treatments presented more than one pick. Due to this in eucalyptus stakes up to 1.80 m. reason, pea ( Pisum sativum ) was used as a standard The flow cytometer analysis was realized in reference since its pick did not overlays to the picks floral structure of pitaya (sepals - the internal produced by the samples. segment of perianth and petals - the external The coefficient of variation was below 2% segment of perianth, stigma, anther, fillet, the bract for all the histograms that show high level of of the pericarp, ovaries, ovary wall, ovules and reliability of the results obtained in our study. pollen. The flowers completely opened with 32 cm Some flower tissues (stigma, ovules, ovary length were used for analysis. In addition, tissues wall, pollen), mature fruits (bract of epicarp, and such as bract of epicarp, epicarp, mesocarp, seeds) and immature (tissue of mesocarp, bract of endocarpoof the mature fruit (approximately 100 g) epicarp, mesocarp, and endocarp) did not show and immature (approximately 30 g), full seeds of picks in the histogram obtained therefore it is not mature fruits, thorns, cactus stems and roots were possible to quantify the DNA of these materials. In subjected for analysis. Three samples of different contras in other treatments we obtained histograms tissue taken from three different plants in each with good quality. treatment were analyzed to verify the presence of The treatments showed significant endoreduplication. difference at 5 % probability. The analysis of flow To determine the DNA, content cytometer indicated the existence of various level of approximately 50 mg of the tissue sample ploidy in different structure of the plant that corresponding to each treatment together with evidenced the phenomena of endoreduplication. Pisum sativum (the internal standard reference) were However, in aculeus and roots the histograms grinded in plate of petridish contain 1 mL of cold presented only one value for the DNA content Marie buffer to release the nucleus. The nucleus represented by one pick in the histogram. The rest of Biosci. J., Uberlândia,
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