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Developmental Biology

Combining Histochemical Staining and Image Analysis to Quantify Starch in the Ovary Primordia of Sweet Cherry during Winter Dormancy

Published: March 20th, 2019



1Instituto Agroalimentario de Aragón - IA2 (CITA-Universidad de Zaragoza), Centro de Investigación y Tecnología Agroalimentaria de Aragón

We present a methodology to quantify the starch content in the ovary primordia in sweet cherry (Prunus avium L.) during winter dormancy by using an image analysis system combined with histochemical techniques.

Changes in starch in small structures are associated with key events during several plant developmental processes, including the reproductive phase from pollination to fertilization and the onset of fruiting. However, variations in starch during flower differentiation are not completely known, mainly due to the difficulty of quantifying the starch content in the particularly small structures of the flower primordia. Here, we describe a method for the quantification of starch in the ovary primordia of sweet cherry (Prunus avium L.) by using an image analysis system attached to the microscope, which allows relating the changes in starch content with the different phases of dormancy from autumn to spring. For this purpose, the dormancy status of flower buds is determined by evaluating the bud growth of shoots transferred to controlled conditions at different moments in winter time. For the quantification of starch in the ovary primordia, flower buds are sequentially collected, fixed, embedded in paraffin wax, sectioned, and stained with I2Kl (potassium iodide-iodine). Preparations are observed under the microscope and analyzed by an image analyzer that clearly distinguishes starch from the background. Starch content values are obtained by measuring the optical density of the image that corresponds to the stained starch, considering the sum of the optical density of each pixel as an estimation of the starch content of the frame studied.

Temperate woody perennials adapt to the seasons by modulating their growth and development. While they develop during spring and summer, they stop growing during the autumn to go dormant in winter1. Although dormancy allows them to survive at low winter temperatures, chilling is a prerequisite for a proper budburst in spring2. The important implications of dormancy in temperate fruit production and forestry have led to diverse efforts to determine and predict the dormancy period3. In fruit tree species, empirical experiments transferring shoots to forcing conditions and statistical predictions bas....

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1. Dormancy Determination and Plant Material Collection

  1. Sample the flower buds in the field. Dormancy studies are long-term experiments and require adult trees big enough to collect buds and shoots all winter without compromising the trees’ development during the next spring. Special orchard management could be required depending on the training system; thus, pruning may be less severe than for fruit production purposes.
    1. Each week, from the start of autumn until the onset of bud break, colle.......

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Dormancy studies require the determination of the moment when the chilling requirements are fulfilled. Despite the lack of phenological changes during winter under field conditions (Figure 1A), cherry trees do not recover the capacity of growth in suitable conditions until they pass a certain period under low temperatures. The regular transference of shoots to a controlled conditions chamber (Figure 1B) during winter time allowed.......

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Dormancy in woody perennials presents clear implications in fruit production and forestry in a changing climate, although the biological process behind dormancy remains unclear. Dormancy studies can be approached from different points of view, but the research looking for a biological marker for winter dormancy has intensified over the last years. However, most attempts to find an unequivocal indicator showing when a bud has broken dormancy have been unsuccessful3. The methodology described herein.......

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The authors gratefully thank Maria Herrero and Eliseo Rivas for their helpful discussion and advice. This work was supported by the Ministerio de Economía y Competitividad — European Regional Development Fund, European Union [grant number BES- 2010-037992 to E. F.]; the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria [grant numbers RFP2015-00015-00, RTA2014-00085-00, RTA2017-00003-00]; and the Gobierno de Aragón — European Social Fund, European Union [Grupo Consolidado A12-17R].


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Name Company Catalog Number Comments
Precision scale Sartorius CP225D
Stereoscopic microscope Leica Microsystems MZ-16
Drying-stove Memmert U15
Paraffin Embedding station Leica Microsystems EG1140H
Rotatory microtome Reichert-Jung 1130/Biocut
Microtome blade Feather S35 Stainless steel
Bright field microscope Leica Microsystems DM2500
Digital Camera Leica Microsystems DC-300
Image Analysis System Leica Microsystems Quantiment Q550

  1. Kurokura, T., Mimida, N., Battey, N. H., Hytönen, T. The regulation of seasonal flowering in the Rosaceae. Journal of Experimental Botany. 64 (14), 4131-4141 (2013).
  2. Rohde, A., Bhalerao, R. P. Plant dormancy in the perennial context. Trends in Plant Science. 12 (5), 217-223 (2007).
  3. Fadón, E., Rodrigo, J. Unveiling winter dormancy through empirical experiments. Environmental and Experimental Botany. 152, 28-36 (2018).
  4. Fadón, E., Rodrigo, J., Herrero, M. Is there a specific stage to rest? Morphological changes in flower primordia in relation to endodormancy in sweet cherry (Prunus avium L.). Trees - Structure and Function. , (2018).
  5. Fadón, E., Herrero, M., Rodrigo, J. Dormant flower buds actively accumulate starch over winter in sweet cherry. Frontiers in Plant Science. 9 (171), (2018).
  6. Loescher, W. H., Mccamant, T., Keller, J. D. Carbohydrate reserves, translocation and storage in woody plant roots. HortScience. 25 (3), 274-281 (1990).
  7. Hedhly, A., et al. Starch turnover and metabolism during flower and early embryo development. Plant Physiology. , (2016).
  8. Rodrigo, J., Hormaza, J. I., Herrero, M. Ovary starch reserves and flower development in apricot (Prunus armeniaca). Physiologia Plantarum. 108 (1), 35-41 (2000).
  9. Julian, C., Rodrigo, J., Herrero, M. Stamen development and winter dormancy in apricot (Prunus armeniaca). Annals of Botany. 108 (4), 617-625 (2011).
  10. Alcaraz, M. L., Hormaza, J. I., Rodrigo, J. Pistil starch reserves at anthesis correlate with final flower fate in avocado (Persea americana). PLoS One. 8 (10), e78467 (2013).
  11. Smith, A. M., Zeeman, S. C. Quantification of starch in plant tissues. Nature Protocols. 1 (3), 1342-1345 (2006).
  12. Eliceiri, K. W., et al. Biological Imaging Software Tools. Nature Methods. 9 (7), (2013).
  13. Rodrigo, J., Herrero, M. Influence of intraovular reserves on ovule fate in apricot (Prunus armeniaca L.). Sexual Plant Reproduction. 11, 86-93 (1998).
  14. Zhou, J., Spallek, T., Faulkner, C., Robatzek, S. CalloseMeasurer: A novel software solution to measure callose deposition and recognise spreading callose patterns. Plant Methods. 8 (1), (2012).
  15. Faulkner, C., et al. An automated quantitative image analysis tool for the identification of microtubule patterns in plants. Traffic. 18 (10), 683-693 (2017).
  16. Kuhn, B. F. Determination of starch in ovules of the sour cherry cv. "Stevnsbaer.&#34. European Journal of Horticultural Science. 71 (3), 120-124 (2006).
  17. Johansen, D. A. . Plant microtechnique. , (1940).
  18. Ruzin, S. E. . Plant microtechnique and microscopy. , (1999).
  19. Rodrigo, J., Rivas, E., Herrero, M. Starch determination in plant tissues using a computerized image analysis system. Physiologia Plantarum. 99 (1), 105-110 (1997).
  20. Alcaraz, M. L., Hormaza, J. I., Rodrigo, J. Ovary starch reserves and pistil development in avocado (Persea americana). Physiologia Plantarum. 140 (4), 395-404 (2010).
  21. Suarez, C., Castro, A. J., Rapoport, H. F., Rodriguez-García, M. I. Morphological, histological and ultrastructural changes in the olive pistil during flowering. Sexual Plant Reproduction. 25, 133-146 (2012).
  22. Lang, G. A., Early, J. D., Martin, G. C., Darnell, R. L. Endodormancy, paradormancy, and ecodormancy - Physiological terminology and classification for dormancy research. HortScience. 22 (3), 371-377 (1987).
  23. Hedhly, A., Vogler, H., Eichenberger, C., Grossniklaus, U. Whole-mount clearing and staining of arabidopsis flower organs and siliques. Journal of Visualized Experiments. 2018 (134), 1-10 (2018).
  24. Kaufmann, H., Blanke, M. Changes in carbohydrate levels and relative water content (RWC) to distinguish dormancy phases in sweet cherry. Journal of Plant Physiology. 218 (July), 1-5 (2017).
  25. Herrero, M., Dickinson, H. G. Pollen-pistil incompatibility in Petunia hybrida: changes in the pistil following compatible and incompatible intraspecific crosses. Journal of Cell Science. 36, 1-18 (1979).
  26. Carpenter, A. E., et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biology. 7 (10), R100 (2006).

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