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Feb 2, 2017 - A Modified Chromosome Squash Technique to Determine Mitotic Activity in Primed. Mericarps. Author(s): Michael W. Olszewski, Wallace G. Pill ...
Society of Commercial Seed Technologists (SCST) Association of Official Seed Analysts

A Modified Chromosome Squash Technique to Determine Mitotic Activity in Primed Mericarps Author(s): Michael W. Olszewski, Wallace G. Pill and Thompson D. Pizzolato Source: Seed Technology, Vol. 27, No. 1 (2005), pp. 101-103 Published by: Association of Official Seed Analysts and the Society of Commercial Seed Technologists (SCST) Stable URL: http://www.jstor.org/stable/23433221 Accessed: 02-02-2017 15:33 UTC REFERENCES Linked references are available on JSTOR for this article: http://www.jstor.org/stable/23433221?seq=1&cid=pdf-reference#references_tab_contents You may need to log in to JSTOR to access the linked references. JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected].

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A Modified Chromosome Squash Technique to Determine Mitotic Activity in Primed Mericarps Michael W. Olszewski, Wallace G. Pill* and Thompson D. Pizzolato ABSTRACT

Mericarps of 'Moss Curled' parsley (Petroselinum crispum Mill. Nym. ex A.W. Hill) were hydroprimed (1 or 3 days in water at 20 °C) or osmotically primed in polyethylene glycol (-1.0 MPa for 7 or 21 days at 20 °C). Extracted

whole embryos then were subjected to a modified chromosome squash tech nique which sequentially involved fixing, staining, tissue squashing, and chromosomal viewing. The technique quantified relative embryonic mitoti activity. The number of cells per embryo with late anaphase activity was greatest for 3 day hydroprimed mericarps (15), then 7 or 21 day osmotic primed mericarps (3), with 1 day hydroprimed mericarps showing no mitotic activity.

EXPERIMENTAL TECHNIQUES A simple modified chromosome squash technique to determine relative mitotic activity in embryos extracted from primed mericarps of'Moss Curled' parsley (.Petroselinum crispum (Mill.) Nym. ex A.W. Hill is described. Mericarps were hydroprimed (1 or 3 d in water at 20 °C) or osmotically primed in polyethyl

ene glycol (-1.0 MPa for 7 or 21 d at 20 °C). These treatments were imposed by placing mericarps in 125 x 80 x 20 mm transparent polystyrene boxes con

taining two layers of germination blotter (Germination Blotter No. 385; Seedburo, Chicago, IL) moistened with 15 ml of either deionized water or 284

g-H polyethylene glycol 6000 (Michel and Kaufman, 1973). These treatments were replicated twice, with 4 mericarps examined in each replicate. After priming, the distal 0.5 mm of pericarp tissue was removed and the intact embryo dislodged from the depleted layer (endosperm) cavity by applying a slight force to the mericarp just proximal to the embryo position. Chromosome squashes were performed using modified techniques of Berlyn

and Miksche (1976) and Jensen (1962). The extracted embryos were fixed in 4 ml ethanol: glacial acetic acid (3:1 by vol.; 100% conc.) with 0.1 g saffranin, and incubated at 60 °C for 3 h. Subsequently, embryos were transferred to 1 N hydrochloric acid (HCl) at 60 °C for 10 min. The embryos then were placed on a microscope slide and flooded with filtered carmine solution (0.5 g carmine,

55 ml deionized water, plus 45 ml glacial acetic acid). The slide was passed over an ethanol flame and excess carmine solution was removed by blotting with Whatman #1 filter paper. The procedure (flooding, flaming, carmine removal) was repeated two more times to remove HCl. The embryo was then

teased apart with a dissecting needle, and incubated for 9-12 h in carmine solution. To prevent evaporation of carmine solution, slides were contained in Michael W. Olszewski, Wallace G. Pill* and Thompson D. Pizzolato, Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19717-1303. *Corresponding author. Received May 25, 2004.

101

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102 Vol. 27, no. 1,2005

lidded polystyrene boxes with double layered water. After incubation, excess carmine solut placed over the teased apart embryo, and vert

disperse the tissue into an approximate single ce

a Wild M20 phase contrast microscope with an 400X. The selective staining permitted viewin (Fig. 1). The number of cells per embryo exhibit for eight embryos of each priming treatment. T late anaphase cell was traced using a drawing tub RESULTS AND DISCUSSION

High relative mitotic activity was noted for 3 d hydroprimed embryos mean ± standard error), while 1 d hydroprimed embryos had no mitoti

ity. Relative mitotic activities of the 7 and 21 d primed embryos w

and 3 ± 1, respectively. Mericarp imbibition was lower with osmotic pr

than with 3 d hydropriming (Olszewski, 2004) resulting in lower mitotic activity. Many ultrastructural, physiological and metabolic occur during priming that lead to improved germination performan

cially under adverse seed bed conditions (Pill, 1995). Late anaph

selected because of its ease of recognition, although any other mito

Figure 1. The chromosome squash technique was used to identify late an cells (arrows) as a measure of relative mitotic activity per embryo. The e intact embryo was fixed, stained, and squashed into a single cell layer, late anaphase cells viewed through a phase contrast microscope and coun

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Seed Technology / Seed Tech Notes 103

could be used to assess relative mitotic activity. Mericarp

seeded dispersal units of the schizocarp fruits of the Apiaceae, nique could be applied to embryos of other plant families. Alth

mosome squash technique has not been reported previousl primed seed, it provided a simple and fairly rapid way to assess

activity at selected times during the seed priming process. REFERENCES

Berlyn, G. P. and J. P. Miksche. 1976. Botanical Microtechnique and Cytochemistr Iowa State Univ. Press, Ames, IA. Jensen, W. A. 1962. Botanical Histochemistry. Freeman, San Francisco, CA.

Michel, B. E. and M. R. Kaufmann. 1973. The osmotic potential of polyethylene glyc

6000. Plant Physiol. 51:914-916.

Olszewski, M. W. 2004. The effects of priming on the anatomy, pathology and germ nation of parsley mericarps. Ph.D. Diss., Univ. of Delaware, Newark, DE.

Pill, W. G. 1995. Low water potential and presowing germination treatments to impro

seed quality, p. 319-359. In Basra, A.S. (ed.) Seed quality: basic mechanisms and agricultural implications. Haworth Press, Binghampton, NY.

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