Fagopyrum 8 (1988): 20-22
Chromosome counts and karyotype analysis in buckwheat
(Fagopyrum escu/en/um Moench.)
Tornaz Slnkovic , Borut Bohanec
VTOZD za agronom ljo , Blotehn l§ka fakulteta , Unlverza E. Kardelja v Ljubljani , Ljubljana,
Jugoslavlja
Key words: buckwheat, chromosome analysis, karyotype
Abstract
Cytological methods for the study of buckwheat chromosome number were developed.
Roots grown in ylyo and In Yltro have been analysed; also In vitro grown roots have high
mitotic Index. Estimation of karyotype picture of common buckwheat Fagopyrllm esclI/entllm
Moench was presented.
"traduction
Cytogenetical analysis Is a very Important step In' the study of different methods of In Yltro
growth of buckwheat tissues . It has become especilally Important lor the study of the
somaclonal variation and for the study of anther derived plants. Buckwheat Is a plant with
relatively small chromosomes what makes cytogenetical analyses more complicated . Karyotype
analysis of buckwheat were studied by Mansurova and Soboleva (1968) and Gohil and Rathar
(1983). Our attempt was to establish a repeatable method for chromosome studies In different
plant materials (from In vivo and in Yitro). Beside that we also made efforts for Identification of
chromosome pairs with proposal for karyotype picture of buckwheat.
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Picture 1. Karyogram of common buckwheat cv.
Darja . Bar represents 4 prn.
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21
Material and methods
Plant material tested was root tips taken from seedlings or adventive roots from in vitro
grown shoots. At 10 AM optimal mitotic index was achieved . Root tips from termir'QI ell.J latera!
roots were pretreated with 3 different cytostatycs which are most frequently used in
cytogenetical observations of plant material: colchicine (0.02 % vs), 1-monobromonaphtalene
(saturated suspension) and 8- hldroxyquinoline (45 mg/100ml). Material was exposed for 24
hours at 4 °c or for 4 hours at room temperature (18°C). After pretreatment. material was
fixed in freshly prepared acetic alcohol fixative (Farmer's fixative) for at least 24 hours.
Substitution of acetic acid with propionic acid was also tested.
Different hydrolysis treatments have been tested where duration and concentr ..iions of HCI
varied from 3-12 minutes and concentrations from 1N-4N (60:t1 °C). Following Feulgen staining
was modified after Goltenboth,1978. PH was increased to 3.6. Freshly prepared Feulgen stain
was preferably used. Feulgen staining was combined with aceto-carmine stain (Krikorian et al.
1983) to intensify the colour of small and gentle chromosomes of buckwheat. Aceto- orcein
staining was less efficient. For karyotype studies permanent preparations were essential.
Permanent preparation were made by C02 freezing following mounting in euparal. Method for
karyotype analysis was based on measurements of 3 different cells with 'ideally' contracted
and well spread chromosomes. All chromosome arms were measured 5 times using a Vernier
ruler (to 0.1 rnm). Photomicrographs were taken with a Zeiss Photo microscope equiped with a
~reen filter. Negative mater ial was Agfa Orto 25. Measurements were made on prints (mag.
3500 x).
Results and discussion
A. Staining technique
From different procedures studied following staining procedure was found to be optimal: there was no big difference between in vivo and in vitro grown roots, sometimes in vitro grown
roots have higher mitotic index.
- 8-hidroxyquinoline has caused sufficient metaphase arresting and overcontraction of
chromosomes was avoided. Treatment for 4 hours at room temperature was superior to long
term treatment at lower temperature.
- We found no differences between two fixatleves, so Farmer's fixative was more routinely
used.
- Hot hydrolysis was found to be of crucial importance; 3 minutes in 3 N HCI was optimal.
- Feuigen preparation after Goltenboth (1978) modified with increased pH to 3.6 was optimal.
B. Karyotype analysis
The chromosome number in Fagopyrum escu/entum Moench is 2n=2x=16. By our
observations, chromosomes can be devided in two groups- 4 metacentrics and 4
submetacentrics. By chromosome lenght 3 chromosomes can be easily determined (1, 4 and 8)
while chromosomes 2- 3 and 5- 6- 7 are of similar size and have the same centromere
position. In some preparations we have noticed some constrictions on different chromosomes;
by our present knOWledge we can not determine wheat her this constrictions represent satelite
or heterochromatine regions on chromosomes. Results of karyotype determination is presented
in the Table 1 and in Picture 1.
22
Table 1: Karyotype characteristics 01 Fagopyrum esculenfum Moench. cv. Darja
L.
No.
3
4
3 .822
3 .200
2 .982
2 .769
5
6
3 .415
3 .138
7
8
2 .422
1
2
2 .899
R.L.
A.R.
Type
15 .51
12 .98
12 .10
11.24
13 .86
12 .73
11 .76.
9 .83
1 .435
1 .450
1 .627
1 .186
1 . 001
1 .028
1.00 1
1 .003
SM
SM
SM
SM
M
M
M
M
L.: lenght in prn
RL.: relative lenght expressed as % of the total chromosome lenght
A.R arm ratio-Ienght of long arm devlded by that of short arm of the chromosome
Type: M rnetacentrlc chromosome, SM submetacentric chromosome
References
Mansurova V.V., Soboleva N.A. ef sf. 1968. In Genetlka, selekcija i semenovodstvo
grechihi. Alekseeva E.S. and Pausheva P" 208 pp . Kiev 1988
Gohil RN.. Rathar G.M., Tah ir I., Farooq S. 1983. Comparative cytology, growth and grain
composition 01 west Hima layan buckwheats. In Proc. 2nd Inti. Symp . Buckwheat,
Miyazaki, p. 87-107
Goltenboth F. 1978. Chromosomen -praktikum. Georg Thieme Verlag , Stuttgart, pp. 212
Krikorian A.D., O'Connor S.A., Fitter M.S. 1983. Chromosome number v.ariation and
karyotype stability in cultures and culture-derived plants, In Handbook 01 plant cell
culture . edited by D.A. Evans. W.R. Sharp, P.V. Ammirato and Y. Yamada,
Macmillan Publishing Co , Collier Macmillan Publishers. New York , London: 541-581.