International Journal of Agriculture and Crop Sciences.
Available online at www.ijagcs.com
IJACS/2013/5-10/1125-1127
ISSN 2227-670X ©2013 IJACS Journal
Optimised techniques for mitotic studies in sweet
cherry (P. avium L.)
Tahereh Javanmard1, Zabihollah Zamani2, Seyed Mahmoud Ghaffari3, Mansour Omidi4
1. M. Sc of Horticultural Sciences, Graduated at University of Tehran, Tehran, Iran
2. Dept. of Horticultural Sciences, College of Agricultural Sciences & Engineering, Campus of Agriculture and
Natural Resources, University of Tehran, Karaj, Iran.
3. Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
4. Department of Agronomy and Plants Breeding, College of Agriculture and Natural Resources, University of
Tehran, Karaj, Iran.
Corresponding author: [email protected]
ABSTRACT: Sweet cherry (Prunus avium L.) is one of the Prunoideae subfamily of Rosaceae, which is
characterized by small chromosomes and the preparation its cells for studying of mitotic chromosomes is
difficult. Different methods at each stage of slide preparation, from collecting root tips to chromosome
staining, were applied to determine the best method, for studying its chromosomes and karyotype
preparation. Results showed root tips which were collected from germinating seeds were better than
those from young seedlings. Optimized technique consisted of placing samples in 0.002 molar 8-hydroxy
quinoline for 4 hours at 4 °C. After washing by distilled water, they were fixed in pinar solution (6 ethanol:
3 chloroform: 2 propionic acid) for 48-72 hours at 4 °C and stored in fresh pinar at -18 °C. Chromosomes
then were hydrolyzed by 1N HCl at 60 °C and stained with feulgen and squashed in 2% aceto-carmine.
This improved protocol allowed the identification of chromosomes in mitosis division.
Key words: karyotype; metaphase; staining; mitosis
INTRODUCTION
Cherries belong to the family of Rosaceae, subfamily Prunoideae, to the genus Prunus, subgenus Cerasus
(Linnaeus, 1753; Ohba, 1992). Developements in fruit breeding and progress in molecular biology requires enough
knowledge on the genetics and the genomes and more information about the karyotype (Schuster, 1996). The
precise cytogenetic description of fruit species, especially the genus Prunus is difficult because the mitotic
chromosomes in Prunus are characterized by small size and the detection of these chromosomes is difficult
(Lespinasse et al., 1986; Salesses Bonherta, 1993; Schuster, 1996; Bouvier et al., 2000; Martinez-Gomez et al.,
2005). In prunus species, slide preparation for mitotic studies are reported for apricot (P. armeniaca L.), peach (P.
persica L.) and almond (P. dulcis Mill.) (Medeira & Warden, 1986; Warden and Medeira, 1986; Gostjeva,
2001).Cytogenetical study in P. aviumis restricted to detection of chromosome numbers in mitotic studies (Schuster
& Ahne, 1999). In sweet cherry, there are few reports about slide preparation for mitotic studies and chromosome
counting. In reported publications, results have shown poor quality in terms of observation and counting of
chromosomes (Singh et al., 1984; Soodan et al., 1988). So, it seems essential to have an improved technique for
slide preparation and chromosome staining for counting chromosomes and karyotype analysis in sweet cherry and
other relative species.
Materials and methods
1. For study of sweet cherry mitotic chromosomes, root tips were obtained by two methods.
– Young seedlings were placed in perforated pots and 1-1.5 cm protruding meristemic root tips were cut.
– Meristemic root tips (1 cm in length) were cut from germinating seeds.
2. Pretreatment
Samples were treated by two different solutions:
Intl J Agri Crop Sci. Vol., 5 (10), 1125-1127, 201
– 0.002 molar 8-hydroxy quinoline for 4 hours at room temperature.
– 0.2% colchicines for 4 hours at 4°C.
3. Fixation
Two fixative solutions were used for incubation of root tips in them.
– Karnoy I solution: (3 vol absolute ethanol: 1 vol acetic acid) used for 24 hours at room temperature.
– Pinar solution: (6vol absolute ethanol: 3 volchloroform: 2 vol propionic acid) used for 48-72 hours at 4 °C.
For preserving root tips for long time, after fixation, samples were washed by distilled water, rot tips which
were fixed by karnoy I solution, were preserved in 70% ethanol at 4 °C and the ones which were fixed in
pinar, were preserved at -18 °C in fresh pinar solution.
4. Hydrolysis
For hydrolyzing, 1N HCl was used in different times and temperatures.
– Samples were immersed into HCl for and inserted in water bath for 10 and 15 minutes at 60 °C.
– Root tips were immersed into HCl at room temperature for 15 and 20 minutes.
5. Staining
For chromosome staining, two different staining solutions and immersing durations were applied:
– Aceto- orcein 2% for 48 hours at room temperature.
– Feulgen for 3 hours in darkness at room temperature and then root tips were squashed using acetocarmine 2%.
6. Microscopy
Chromosomes were studied under oil immersion by a phase contrast microscope at a magnification of 1000x.
RESULTS
Root tips from germinated seeds were superior to root tips from seedlings and had more mitotic division. Root
tips were cut when 1 cm long and immediately were placed in pretreatment solutions for 4 hours. Both of these
solutions had qualified results but 8-hydroxy qinoline solution was somehow better. After washing samples by
distilled water, they were placed in fixative solutions, which preserving in pinar solution (Fasihi&Ghaffari, 2001) for
48-72 hours at 4 °C produced a much better condition in the observation of the chromosomes. Root tips were
hydrolyzed in 1 N HCl at 60 °C for 10 minutes, whereas 15 minutes resulted to cell break down, and hydrolyzing
without water bath at 60 °C resulted to fade and pale chromosomes. Samples were stained with feulgen reaction
for 3 hours in darkness and by squashing in 2% aceto-carmine had better results compared to acto-orcein.
For studying mitosis stages and chromosome counting, this optimized protocol allowed the qualified
observation of chromosomes in meristemic cells from sweet cherry root tips. As it is shown in figure 1, sweet cherry
is a diploid species of Prunus genus with 2n=16 chromosomes in somatic cells, in accordance with previous
reports (Kobel, 1927; Darlington, 1928; Crane, & Lawrence, 1929; Raptopoulus, 1941; Schuster & Ahne, 1996).
Chromosome complement of this species had 4 metacentric and 4 submetacentric chromosomes.
Root tips should be white and at active growing stage for successful study of mitotic division, while in brown
root tips mitotic division is decreased (Martinez-Gomez et al., 2005). According to pleasant work, by modifying the
methods reported before, the optimized methods for each stage of slide preparation for mitotic studies and
chromosome counting in sweet cherry (P. avium) has been introduced.
Intl J Agri Crop Sci. Vol., 5 (10), 1125-1127, 201
Figure 1. mitotic chromosomes in sweet cherry (metaphase stage).
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