PlantCell Reports
Plant Cell Reports (1986) 5: 280- 283
© Springer-Verlag 1986
Sister chromatid exchanges in garlic (Allium sativum L.) callus cells J. D o l e ~ e l 1 and F. J. N o v f i k 2 1 Czechoslovak Academy of Sciences, Institute of Experimental Botany, Sokolovskfi 6, CS-77200 Olomouc, Czechoslovakia 2 International Atomic Energy Agency, P.O. Box 100, A-1400 Vienna, Austria Received January 16, 1986 / Revised version received April 21, 1986 - Communicated by A. R. Gould
ABSTRACT A technique is described for differential staining of sister chromatids and the study of sister chromatid exchanges (SCEs) in garlic (Allium sativum L.) callus cells. BrdU incorporation into newly synthesized DNA was ensured by culturing calli on medium containing i00 pM BrdU + 0.01 ~ M FudR + 1 p M Urd. SCEs were visualized by FPG staining technique and their frequency was analysed. Mean frequency of SCEs in callus cells was higher than that in meristem root-tip cells. Using the same staining method, cell cycle time of callus cells was analysed. It was found that it ranges from 48 to 132 hrs. The method described represents a new approach in the study of instability of plant cells cultured in vitro.
genetic
ABBREVIATIONS BrdU = 5-bromo-2'-deoxyuridine; 2,4-D = 2,4-dichlorophenoxyaeetie acid; FPG = fluorescent-plus-Giemsa; FudR = 5-fluoro-2'-deoxyuridine; SCE = sister chromatid exchange; SSC = 0.15 M NaCI + 0.015 M Na-citrate; T = thymidine-containing strand of the DNA duplex; B =5-bromo-2'-deoxyuridine-containing strand of the DNA duplex; Urd = uridine. INTRODUCTION Somaclonal variation as a novel variability is now considered an plant
improvement
(Larkin
and
source of genetic important tool for Scowcroft
1983), suggesting its mutagenic effect. The SCE test has been widely used for the detection of mutagen activity because of its superior sensitivity and ease of scoring (Perry 1983a). It is important that
excellent
correlation
has
been
found
between
chemicals that induce SCEs and those causing mutations (Abe and Sasaki 1982). Todate, no report has been made on studies of the frequency of SCEs in plant cells cultured in vitro. In this communication, a method is described for sister chromatid diffrentiation in garlic (Allium sativum L.) calius cells. Evaluation was made on the frequency of SCEs in callus cells and this was compared with that in meristem root-tip cells. MATERIALS AND METHODS Sister chromatid exchange reflects an interchange between DNA molecules at homologous loci within a replicating chromosome. The detection of SOEs in cytological preparations has been greatly simplified by BrdU-dye technique (Perry and Wolf 1974). Following this method, chromosomes in the second mitosis after BrdU incorporation display unifilarly substituted chromatids dark and bifilarly substituted chromatids light. The technique involves staining with Hoechst 33258 fluorescent dye, irradiation with UV light and hot saline treatment followed by Giemsa staining.
1981).
Recently, regenerants have been obtained carrying single gene mutations (Evans and Sharp 1983). This will make it possible to use tissue culture as a tool to introduce variation not only into vegetatively propagated plants, but also into virtually all seed propagated species (Evans et al. 1984). Contrary to the above mentioned achievements, our knowledge on the nature of somaclonal variation remains rather limited (Wenzel 1983). Some variation can be explained due to expression of mutant cells existing in the explants (Barbier and Dulieu 1983). It
Offprintreques~ to: J. Dole~el
is apparent, however, that genetic changes are also induced during in vitro culture (LSrz and Scowcroft
Garlic
callus
culture
(line
H-7),
used
in
our
experiments was originally obtained from leaf explants of Allium sativum L. cv. Bzeneck~ pali6~k (Dole~el 1982). The culture was maintained in dark on the medium BDS (Dunstan and Short 1977) supplemented with i ~M 2,4-D + 5 ~ M kinetin at 25°C. Transfers to fresh medium were made at four weeks intervals. At the beginning of the experiment, the culture was in its 49th passage (approx. 4 years old). The culture consisted of mainly diploid cells (71%) with lower frequency of tetraploid (23%) and highly polyploid and aneuploid (6%) cells.
281 To incorporate BrdU,
the
culture was
transferred to
inferior following more conventional RNase treatment.
the fresh medium containing i00 ~ M BrdU + 0.01 p M FudR
Thus,
+i p M
frequency
Urd.
containing
Further BrdU
transfers
were
made
at
to
the
72
hours
Samples of calli were taken after 48, 144,
168,
fresh
media
intervals.
72,
96,
120,
for
the
preparation
analysis,
substituted
by
HCI
of
the
slides
RNase
treatment
for
the
SCE
treatment
in
FPG
was
staining
technique.
192, 216, 240, 264, 288 and 312 hours. The
calli were pretreated with 2.5 mM colchicine and fixed
The staining patterns of chromosomes in garlic callus
in fresh 3:1 fixative.
cells depended on the length of the culture containing
medium.
After
48
and
The fixed callus pieces were treated with 1% pectinase
differentiation of SCEs was observed,
in 0.i M citrate buffer,
being
pH 4.7 at 37 ° C for 60 min,
and in 0.25% cellulase under
the same
15 min.
in 45% acetic
Squashes were
made
conditions for acid and
stained
chromosomes
observed after
coverslips were removed by the dry-ice method (Conger
low,
not
and Fairchild 1953).
Their
frequency
Some preparations were incubated
with 0.01% RNase at 37°C for 60 min. stained with i0 ~ M min.
After a brief wash
mounted
with
fluorescent
a
coverslips were SSC
at
drop
sun
50°C
of
lamp 60
in 0.5 x SSC, 0.5
x
for
removed
for
All slides were
Hoechst 33258 in 0.5 x SSC for 30 SSC
75
and
min.
slides were
and
exposed
min.
to
Thereafter,
slides
incubated
Those
slides
in 1 x
not
being
incubated with RNase were treated with 5N HCI at 20°C for
15
water.
min
and
washed
in
two changes
of
times, only
homogenously.
showing
SCE
differentiation
higher with
were
with
were
only
Their frequency was rather metaphases
after
but never exceeded 20%. cells
no
the chromosomes
Metaphases
exceeding 10% of all
the
on BrdUhours
differentiation
96 hours. was
72
longer
After 144 hours not
chromosomes
observed
observed. incubation
but
showing
SCE
cells
with
also
chromosomes showing iso-nonstaining regions (Fig. ib). After
216
hours,
only
few
metaphases
showed
SCE
differentiation and finally after 264 hours of culture on
BrdU-containing medium,
only cells
showing
iso-
nonstaining regions were observed.
distilled
Finally, all preparations were stained with 3%
Giemsa in 0.067 M phosphate buffer, pH 6.7 for i0 min, air dried and mounted with Euparal. For the
evaluation of
SCE frequency in callus cells,
samples of calli were taken after 120, 192 hours
of
Slides were
culture
on
the medium
prepared using
144,
168 and
containing BrdU.
the FPG method comprising
HCI treatment as described above. The
SCE
was
established
frequency
in garlic
using
the
garlic (Dole{el et al.
meristem root-tip FPG
1986a).
method
cells
modified
In the present
for
work,
however, the incorporation of BrdU was made during two consecutive rounds of DNA replication. Briefly, garlic cloves
(of
the
same
genotype
as
used
for
callus
a
culture initiation) were grown in the dark at 25°C on beakers containing 250 ml Hoagland's solution (diluted ten times)
which was
renewed every
24 hours
and was
continuously aerated by bubbling air at the rate of i0 ml/min. When the roots reached 15 to 20 mm length, the solution was changed for the same Hoagland's solution but containing also i00 ~ M BrdU + 0.01 ~ M FudR + item Urd.
This solution was renewed every 12 hours.
After
50 hours, the roots were pretreated, fixed and stained using the same method as used for calli. After scoring 200 to 300 chromosomes in each sample, mean
SCE
values
calculated. t-test.
and
The means
their
standard
errors
were
were compared using Student's
RESULTS Very good resolution of
SCEs
in garlic
callus cells
was obtained if the chromosomes substituted with BrdU for
two
rounds
of
DNA
replication
were
according to the procedure described (Fig. hydrolysis
i
(Gonzalez-Gll
and
Navarette
processed la).
Acid
1982)
was
necessary in the present experiment to achieve distinct staining patterns. The resolution of SCEs was
Fig.
i
Metaphase
callus cells
chromosomes
of
Allium
sativum L.
(a) substituted with BrdU for two rounds
of replication,
(b) substituted with BrdU
for three
rounds of replication, approximately three out of four chromatids are deeply stained. Bar = i0
~m
282 Since
it
was
difficult
to
find
complete
metaphase
The incorporation
of BrdU
during different
phases or
cells with sister chromatid differentiation, SCE frequency per chromosome was calculated evaluating individual chromosomes. The results of the study of
during different numbers of DNA replication cycles results in discernible staining patterns of
SCE frequency in meristem root-tip cells (control) and that in callus cells after different periods of culture on BrdU-eontaining medium are summarized in
implies that this technique can be used for the study
Tabl@ I. For all sampling times, mean frequency of SCEs per chromosome was higher than that of control. For the sampling times of 120, 144 and 192 hours, this difference was statistically significant at P = 0.05 level. Mean frequency of SCE per chromosome (calculated using data from all sampling times) in callus cells was 7.47, that is, 16% higher than that of meristem root-tip cells (6.42). On the other hand, the differences in the mean SCE frequencies per chromosome in callus cells after different length of culture on BrdU-medium were not statistically significant at P = 0.2 level. Table i. Comparison of mean frequencies of SCEs per chromosome in garlic callus cells with those of meristem root-tip cells
Tissue
Culture on BrdU-medium (hrs)
Number of chromosomes analysed
50
310
6.42 +/- 0.29
callus
120
285
7.55 +/- 0.28 *
callus
144
291
7.42 +/- 0.21 *
callus
168
274
T.ll +/- 0.28
callus
192
220
7.78 +/- 0.38 *
meristem
*)
Number of SCEs per chromosome +/-S.E.
statistically significant from control root-tip cells) at P = 0.05 level
(meristem
DISCUSSION In the present paper, a method for differential staining of sister chromatids in garlic (Allium sativum L.) callus cells is described. To simplify the handling of in vitro material, differential labelling of sister chromatids has been achieved by continuous cultivation on BrdU-containing medium. Thus chromosomes showing SCEs had TB-BB constitution. The advantage of simplified labelYing procedure was, however, somehow counteracted by the fact that the chromosomes with TB-BB constitution gave inferior resolution of SCEs using FPG technique in comparison with chromosomes with TT-TB constitution. However, it was possible to greatly improve the resolution of SCEs when conventional RNase treatment was substituted by the treatment with 5N HCI as reported by Gonz~lez-Gil and Navarette (1982). This enabled study of the staining patterns of chromosomes and SCE frequency in garlic callus cells.
chromosomes
after
staining
with
FPG
method.
This
of DNA replication patterns of chromosomes and/or cell cycle kinetics (Schubert and DSbel 1983). In our experiment,
chromosomes
showing
SCEs
were
observed
only after 96 hours on Brd-containing medium and then up to 264 hours. time
of
garlic
According to this, callus
cells
ranges
the from
cell cycle 48
to
132
hours. The first metaphases with chromosomes showing iso-nonstaining regions were observed after 144 hours, thus confirming the shortest cell cycle time of 48 hours, detected in our experiment. This result indicates that cell cycle time of garlic callus cells is significantly longer than that of meristem root-tip cells (22 hours, Dole{el et al. 1986a). This is in agreement with other reports on cell cycle kinetics in cultured plant cells (cf. Gould 1984). As the present study was directed towards the visualization and analysis of SCEs, the labelling method chosen did not allow detailed analysis of the cell cycle kinetics. It was not possible to distinguish longer cycle time arising from prolongation of certain cell cycle compartments from delayed cell divisions. However, the labelling method using combined incorporation of BrdU and thymidine allows this problem to be analysed if different timings of incorporation of analogs and fixation are used (CortSs and Gonz~lez-Gil 1982). For the analysis of SCE frequency in garlic callus cells, four sampling times were chosen with respect to the frequency of metaphases showing SCE differentiation. For all of them, the frequency of SCEs per chromosome observed was higher in comparison with that of meristem root-tip cells. This result is interesting as the current knowledge evidently shows that the increase in SCE frequency may be due to mutagen treatment of cells (Carrano and Thompson 1982). In connection with genetic instability of cultured plant cells cultured in vitro, the effect of growth regulators (for example 2,4-D) is often considered. Although our earlier results did not confirm mutagenic activity of tissue culture media and/or its components (Dole~el and Novlk 1984a,b), in a recent study we have observed the increase of SCE frequency in garlic meristem root-tip cells after treatment with low doses of 2,4-D (Dole~el et al.
1986a). On the other hand, there is a great number of other factors which may influence SCE frequency. OXygen • I tension (GutiSrrez and Lopez-Saez 1982) or balance in DNA-precursor pools for DNA synthesis (Perry 1983b) are two of them. It is evident that such conditions can cause the increase in SCE frequency as compared to the cells of the intact meristem. It was observed that the prolongation of S-phase and decrease in the rate of replication-fork movement caused an increase in SCE
283 frequency that
the
(Guti~rrez et al. 1981). cell
cycle
of
garlic
In view of the fact callus
cells
is
considerably longer as compared to root meristem cells, it may be supposed that the increased SCE frequency is related to the longer cell cycle time. The ever-increasing interest in somaclonal variability and its application to crop improvement evokes a more profound insight into the sources of variability of cultured cells (Evans et al. 1984). We expect the SCE test will make it possible to achieve other important results in this sphere of investigation. Garlic tissue culture was shown to be genetically unstable and numerous somaclones were obtained differing from one another in one or more phenotypic traits (Nov~k 1980, 1984). It is possible now to test the impact of culture medium composition independent on in vitro conditions by the same method and using the same material, because a method has been also developed for differential staining of sister chromatids in meristem root-tip cells of garlic (Dole~el et al. 1986a). Moreover, there is a possibility to divide a tissue culture cell population into subpopulations according to the cell cycle length and study them independently. Research is in progress to use the system described to study the SCE frequency in garlic tissue culture including the factors that influence it.
Conger AD, Fairchild LM (1953) Stain Technol. 28: 281-283. Cortes F, Gonz~les-Gil G (1982) Cytologia 4 7 : 4 8 1 - 4 8 7 Dole~el J (1982) Ph. D. Thesis, Inst. Exptl. Botany, Prague Dole~el J, Nov~k FJ (1984a) Biol. Plant. 2 6 : 2 9 3 - 2 9 4 Dole~el J, Nov~k FJ (1984b) Z. Pflanzenphysiol. 114: 51-58 Dole~el J, C~hal~kov~ J, Nov~k FJ (1986a) Caryologia in press Dole~el J, Lucretti S, Nov~k FJ (1986b) Environm. Exp. Bot. - in press Dunstan DI, Short KC (1977) Physiol. Plant. 4 1 : 7 0 - 7 2 Evans DA, Sharp WR (1983) Science 2 2 1 : 9 4 9 - 9 5 1 Evans DA, Sharp WR, Medina-Filho HP (1984) Amer. J. Bot.
71:759-774
Gonzalez-Gil G, Navarette MH (1982) Chromosoma 86: 375-382 Gould AR (1984) CRC Critical Reviews in Plant Sciences i: 3 1 5 - 3 4 4 . Guti~rrez C, L6pez-S~ez JF (1982) Mutat. Res. 103: 295-302 Guti~rrez C, Schvartzman JB, L~pez-S~ez JF (1981) Exp. Cell Res. 1 3 4 : 7 3 - 7 9 Larkin PJ, Scowcroft WR (1981) Theor. Appl. Genet. 60: 197-214 Lbrz
AKNOWLEDGEMENTS The authors wish to thank Mrs. Antonietta Moras for typing the manuscript. J.D. received a one year research fellowship from Comitato Nazionale per la Ricerea e per io Sviluppo dell'Energia Nucleare e delle Energie Alternative, Roma, Italy. REFERENCES Abe S, Sasaki M (1982) In: Sandberg AA (ed) Sister chromatid exchange, Alan R Liss, New York, pp 461-514. Barbier M, Dulieu H (1983) Plant Sci. Lett. 29: 201206 Carrano AV, Thompson LH (1982) Cytogenet. Cell Genet. 33:57-61
H, Scowcroft WR (1983) In: Potrykus I et al. (eds) Protoplasts (Poster Proceedings), Birkhauser, Basel Boston Stuttgart, pp 152-153 Nov~k FJ (1980) Z. Pflanzenzuchtg. 8 4 : 2 4 0 - 2 6 0 Nov~k FJ (1984) In: Proc. 3rd Eucarpia Allium Symposium, Inst. Hort. Plant Breeding, Wageningen, pp. 39-43. Perry PE (1983a) In: Castellani A (ed) The use of human cells for the evaluation of risk from physical and chemical agents, Plenum Publ. Corp., New York, pp 49-61 Perry PE (1983b) Mutat. Res. 1 0 9 : 2 1 9 - 2 2 9 Perry PE, Wolf S (1974) Nature 2 5 1 : 1 5 6 - 1 5 8 Schubert I, DUbel P (1983) In: Chaloupka
J et al.
(eds) Progress in cell cycle controls, Acad. Sci. Prague, pp 188-189 Wenzel F (1983) Progress in Botany 45: 174-188.
Czech.
Plant Cell Reports (1986) 5: 280- 283
© Springer-Verlag 1986
Sister chromatid exchanges in garlic (Allium sativum L.) callus cells J. D o l e ~ e l 1 and F. J. N o v f i k 2 1 Czechoslovak Academy of Sciences, Institute of Experimental Botany, Sokolovskfi 6, CS-77200 Olomouc, Czechoslovakia 2 International Atomic Energy Agency, P.O. Box 100, A-1400 Vienna, Austria Received January 16, 1986 / Revised version received April 21, 1986 - Communicated by A. R. Gould
ABSTRACT A technique is described for differential staining of sister chromatids and the study of sister chromatid exchanges (SCEs) in garlic (Allium sativum L.) callus cells. BrdU incorporation into newly synthesized DNA was ensured by culturing calli on medium containing i00 pM BrdU + 0.01 ~ M FudR + 1 p M Urd. SCEs were visualized by FPG staining technique and their frequency was analysed. Mean frequency of SCEs in callus cells was higher than that in meristem root-tip cells. Using the same staining method, cell cycle time of callus cells was analysed. It was found that it ranges from 48 to 132 hrs. The method described represents a new approach in the study of instability of plant cells cultured in vitro.
genetic
ABBREVIATIONS BrdU = 5-bromo-2'-deoxyuridine; 2,4-D = 2,4-dichlorophenoxyaeetie acid; FPG = fluorescent-plus-Giemsa; FudR = 5-fluoro-2'-deoxyuridine; SCE = sister chromatid exchange; SSC = 0.15 M NaCI + 0.015 M Na-citrate; T = thymidine-containing strand of the DNA duplex; B =5-bromo-2'-deoxyuridine-containing strand of the DNA duplex; Urd = uridine. INTRODUCTION Somaclonal variation as a novel variability is now considered an plant
improvement
(Larkin
and
source of genetic important tool for Scowcroft
1983), suggesting its mutagenic effect. The SCE test has been widely used for the detection of mutagen activity because of its superior sensitivity and ease of scoring (Perry 1983a). It is important that
excellent
correlation
has
been
found
between
chemicals that induce SCEs and those causing mutations (Abe and Sasaki 1982). Todate, no report has been made on studies of the frequency of SCEs in plant cells cultured in vitro. In this communication, a method is described for sister chromatid diffrentiation in garlic (Allium sativum L.) calius cells. Evaluation was made on the frequency of SCEs in callus cells and this was compared with that in meristem root-tip cells. MATERIALS AND METHODS Sister chromatid exchange reflects an interchange between DNA molecules at homologous loci within a replicating chromosome. The detection of SOEs in cytological preparations has been greatly simplified by BrdU-dye technique (Perry and Wolf 1974). Following this method, chromosomes in the second mitosis after BrdU incorporation display unifilarly substituted chromatids dark and bifilarly substituted chromatids light. The technique involves staining with Hoechst 33258 fluorescent dye, irradiation with UV light and hot saline treatment followed by Giemsa staining.
1981).
Recently, regenerants have been obtained carrying single gene mutations (Evans and Sharp 1983). This will make it possible to use tissue culture as a tool to introduce variation not only into vegetatively propagated plants, but also into virtually all seed propagated species (Evans et al. 1984). Contrary to the above mentioned achievements, our knowledge on the nature of somaclonal variation remains rather limited (Wenzel 1983). Some variation can be explained due to expression of mutant cells existing in the explants (Barbier and Dulieu 1983). It
Offprintreques~ to: J. Dole~el
is apparent, however, that genetic changes are also induced during in vitro culture (LSrz and Scowcroft
Garlic
callus
culture
(line
H-7),
used
in
our
experiments was originally obtained from leaf explants of Allium sativum L. cv. Bzeneck~ pali6~k (Dole~el 1982). The culture was maintained in dark on the medium BDS (Dunstan and Short 1977) supplemented with i ~M 2,4-D + 5 ~ M kinetin at 25°C. Transfers to fresh medium were made at four weeks intervals. At the beginning of the experiment, the culture was in its 49th passage (approx. 4 years old). The culture consisted of mainly diploid cells (71%) with lower frequency of tetraploid (23%) and highly polyploid and aneuploid (6%) cells.
281 To incorporate BrdU,
the
culture was
transferred to
inferior following more conventional RNase treatment.
the fresh medium containing i00 ~ M BrdU + 0.01 p M FudR
Thus,
+i p M
frequency
Urd.
containing
Further BrdU
transfers
were
made
at
to
the
72
hours
Samples of calli were taken after 48, 144,
168,
fresh
media
intervals.
72,
96,
120,
for
the
preparation
analysis,
substituted
by
HCI
of
the
slides
RNase
treatment
for
the
SCE
treatment
in
FPG
was
staining
technique.
192, 216, 240, 264, 288 and 312 hours. The
calli were pretreated with 2.5 mM colchicine and fixed
The staining patterns of chromosomes in garlic callus
in fresh 3:1 fixative.
cells depended on the length of the culture containing
medium.
After
48
and
The fixed callus pieces were treated with 1% pectinase
differentiation of SCEs was observed,
in 0.i M citrate buffer,
being
pH 4.7 at 37 ° C for 60 min,
and in 0.25% cellulase under
the same
15 min.
in 45% acetic
Squashes were
made
conditions for acid and
stained
chromosomes
observed after
coverslips were removed by the dry-ice method (Conger
low,
not
and Fairchild 1953).
Their
frequency
Some preparations were incubated
with 0.01% RNase at 37°C for 60 min. stained with i0 ~ M min.
After a brief wash
mounted
with
fluorescent
a
coverslips were SSC
at
drop
sun
50°C
of
lamp 60
in 0.5 x SSC, 0.5
x
for
removed
for
All slides were
Hoechst 33258 in 0.5 x SSC for 30 SSC
75
and
min.
slides were
and
exposed
min.
to
Thereafter,
slides
incubated
Those
slides
in 1 x
not
being
incubated with RNase were treated with 5N HCI at 20°C for
15
water.
min
and
washed
in
two changes
of
times, only
homogenously.
showing
SCE
differentiation
higher with
were
with
were
only
Their frequency was rather metaphases
after
but never exceeded 20%. cells
no
the chromosomes
Metaphases
exceeding 10% of all
the
on BrdUhours
differentiation
96 hours. was
72
longer
After 144 hours not
chromosomes
observed
observed. incubation
but
showing
SCE
cells
with
also
chromosomes showing iso-nonstaining regions (Fig. ib). After
216
hours,
only
few
metaphases
showed
SCE
differentiation and finally after 264 hours of culture on
BrdU-containing medium,
only cells
showing
iso-
nonstaining regions were observed.
distilled
Finally, all preparations were stained with 3%
Giemsa in 0.067 M phosphate buffer, pH 6.7 for i0 min, air dried and mounted with Euparal. For the
evaluation of
SCE frequency in callus cells,
samples of calli were taken after 120, 192 hours
of
Slides were
culture
on
the medium
prepared using
144,
168 and
containing BrdU.
the FPG method comprising
HCI treatment as described above. The
SCE
was
established
frequency
in garlic
using
the
garlic (Dole{el et al.
meristem root-tip FPG
1986a).
method
cells
modified
In the present
for
work,
however, the incorporation of BrdU was made during two consecutive rounds of DNA replication. Briefly, garlic cloves
(of
the
same
genotype
as
used
for
callus
a
culture initiation) were grown in the dark at 25°C on beakers containing 250 ml Hoagland's solution (diluted ten times)
which was
renewed every
24 hours
and was
continuously aerated by bubbling air at the rate of i0 ml/min. When the roots reached 15 to 20 mm length, the solution was changed for the same Hoagland's solution but containing also i00 ~ M BrdU + 0.01 ~ M FudR + item Urd.
This solution was renewed every 12 hours.
After
50 hours, the roots were pretreated, fixed and stained using the same method as used for calli. After scoring 200 to 300 chromosomes in each sample, mean
SCE
values
calculated. t-test.
and
The means
their
standard
errors
were
were compared using Student's
RESULTS Very good resolution of
SCEs
in garlic
callus cells
was obtained if the chromosomes substituted with BrdU for
two
rounds
of
DNA
replication
were
according to the procedure described (Fig. hydrolysis
i
(Gonzalez-Gll
and
Navarette
processed la).
Acid
1982)
was
necessary in the present experiment to achieve distinct staining patterns. The resolution of SCEs was
Fig.
i
Metaphase
callus cells
chromosomes
of
Allium
sativum L.
(a) substituted with BrdU for two rounds
of replication,
(b) substituted with BrdU
for three
rounds of replication, approximately three out of four chromatids are deeply stained. Bar = i0
~m
282 Since
it
was
difficult
to
find
complete
metaphase
The incorporation
of BrdU
during different
phases or
cells with sister chromatid differentiation, SCE frequency per chromosome was calculated evaluating individual chromosomes. The results of the study of
during different numbers of DNA replication cycles results in discernible staining patterns of
SCE frequency in meristem root-tip cells (control) and that in callus cells after different periods of culture on BrdU-eontaining medium are summarized in
implies that this technique can be used for the study
Tabl@ I. For all sampling times, mean frequency of SCEs per chromosome was higher than that of control. For the sampling times of 120, 144 and 192 hours, this difference was statistically significant at P = 0.05 level. Mean frequency of SCE per chromosome (calculated using data from all sampling times) in callus cells was 7.47, that is, 16% higher than that of meristem root-tip cells (6.42). On the other hand, the differences in the mean SCE frequencies per chromosome in callus cells after different length of culture on BrdU-medium were not statistically significant at P = 0.2 level. Table i. Comparison of mean frequencies of SCEs per chromosome in garlic callus cells with those of meristem root-tip cells
Tissue
Culture on BrdU-medium (hrs)
Number of chromosomes analysed
50
310
6.42 +/- 0.29
callus
120
285
7.55 +/- 0.28 *
callus
144
291
7.42 +/- 0.21 *
callus
168
274
T.ll +/- 0.28
callus
192
220
7.78 +/- 0.38 *
meristem
*)
Number of SCEs per chromosome +/-S.E.
statistically significant from control root-tip cells) at P = 0.05 level
(meristem
DISCUSSION In the present paper, a method for differential staining of sister chromatids in garlic (Allium sativum L.) callus cells is described. To simplify the handling of in vitro material, differential labelling of sister chromatids has been achieved by continuous cultivation on BrdU-containing medium. Thus chromosomes showing SCEs had TB-BB constitution. The advantage of simplified labelYing procedure was, however, somehow counteracted by the fact that the chromosomes with TB-BB constitution gave inferior resolution of SCEs using FPG technique in comparison with chromosomes with TT-TB constitution. However, it was possible to greatly improve the resolution of SCEs when conventional RNase treatment was substituted by the treatment with 5N HCI as reported by Gonz~lez-Gil and Navarette (1982). This enabled study of the staining patterns of chromosomes and SCE frequency in garlic callus cells.
chromosomes
after
staining
with
FPG
method.
This
of DNA replication patterns of chromosomes and/or cell cycle kinetics (Schubert and DSbel 1983). In our experiment,
chromosomes
showing
SCEs
were
observed
only after 96 hours on Brd-containing medium and then up to 264 hours. time
of
garlic
According to this, callus
cells
ranges
the from
cell cycle 48
to
132
hours. The first metaphases with chromosomes showing iso-nonstaining regions were observed after 144 hours, thus confirming the shortest cell cycle time of 48 hours, detected in our experiment. This result indicates that cell cycle time of garlic callus cells is significantly longer than that of meristem root-tip cells (22 hours, Dole{el et al. 1986a). This is in agreement with other reports on cell cycle kinetics in cultured plant cells (cf. Gould 1984). As the present study was directed towards the visualization and analysis of SCEs, the labelling method chosen did not allow detailed analysis of the cell cycle kinetics. It was not possible to distinguish longer cycle time arising from prolongation of certain cell cycle compartments from delayed cell divisions. However, the labelling method using combined incorporation of BrdU and thymidine allows this problem to be analysed if different timings of incorporation of analogs and fixation are used (CortSs and Gonz~lez-Gil 1982). For the analysis of SCE frequency in garlic callus cells, four sampling times were chosen with respect to the frequency of metaphases showing SCE differentiation. For all of them, the frequency of SCEs per chromosome observed was higher in comparison with that of meristem root-tip cells. This result is interesting as the current knowledge evidently shows that the increase in SCE frequency may be due to mutagen treatment of cells (Carrano and Thompson 1982). In connection with genetic instability of cultured plant cells cultured in vitro, the effect of growth regulators (for example 2,4-D) is often considered. Although our earlier results did not confirm mutagenic activity of tissue culture media and/or its components (Dole~el and Novlk 1984a,b), in a recent study we have observed the increase of SCE frequency in garlic meristem root-tip cells after treatment with low doses of 2,4-D (Dole~el et al.
1986a). On the other hand, there is a great number of other factors which may influence SCE frequency. OXygen • I tension (GutiSrrez and Lopez-Saez 1982) or balance in DNA-precursor pools for DNA synthesis (Perry 1983b) are two of them. It is evident that such conditions can cause the increase in SCE frequency as compared to the cells of the intact meristem. It was observed that the prolongation of S-phase and decrease in the rate of replication-fork movement caused an increase in SCE
283 frequency that
the
(Guti~rrez et al. 1981). cell
cycle
of
garlic
In view of the fact callus
cells
is
considerably longer as compared to root meristem cells, it may be supposed that the increased SCE frequency is related to the longer cell cycle time. The ever-increasing interest in somaclonal variability and its application to crop improvement evokes a more profound insight into the sources of variability of cultured cells (Evans et al. 1984). We expect the SCE test will make it possible to achieve other important results in this sphere of investigation. Garlic tissue culture was shown to be genetically unstable and numerous somaclones were obtained differing from one another in one or more phenotypic traits (Nov~k 1980, 1984). It is possible now to test the impact of culture medium composition independent on in vitro conditions by the same method and using the same material, because a method has been also developed for differential staining of sister chromatids in meristem root-tip cells of garlic (Dole~el et al. 1986a). Moreover, there is a possibility to divide a tissue culture cell population into subpopulations according to the cell cycle length and study them independently. Research is in progress to use the system described to study the SCE frequency in garlic tissue culture including the factors that influence it.
Conger AD, Fairchild LM (1953) Stain Technol. 28: 281-283. Cortes F, Gonz~les-Gil G (1982) Cytologia 4 7 : 4 8 1 - 4 8 7 Dole~el J (1982) Ph. D. Thesis, Inst. Exptl. Botany, Prague Dole~el J, Nov~k FJ (1984a) Biol. Plant. 2 6 : 2 9 3 - 2 9 4 Dole~el J, Nov~k FJ (1984b) Z. Pflanzenphysiol. 114: 51-58 Dole~el J, C~hal~kov~ J, Nov~k FJ (1986a) Caryologia in press Dole~el J, Lucretti S, Nov~k FJ (1986b) Environm. Exp. Bot. - in press Dunstan DI, Short KC (1977) Physiol. Plant. 4 1 : 7 0 - 7 2 Evans DA, Sharp WR (1983) Science 2 2 1 : 9 4 9 - 9 5 1 Evans DA, Sharp WR, Medina-Filho HP (1984) Amer. J. Bot.
71:759-774
Gonzalez-Gil G, Navarette MH (1982) Chromosoma 86: 375-382 Gould AR (1984) CRC Critical Reviews in Plant Sciences i: 3 1 5 - 3 4 4 . Guti~rrez C, L6pez-S~ez JF (1982) Mutat. Res. 103: 295-302 Guti~rrez C, Schvartzman JB, L~pez-S~ez JF (1981) Exp. Cell Res. 1 3 4 : 7 3 - 7 9 Larkin PJ, Scowcroft WR (1981) Theor. Appl. Genet. 60: 197-214 Lbrz
AKNOWLEDGEMENTS The authors wish to thank Mrs. Antonietta Moras for typing the manuscript. J.D. received a one year research fellowship from Comitato Nazionale per la Ricerea e per io Sviluppo dell'Energia Nucleare e delle Energie Alternative, Roma, Italy. REFERENCES Abe S, Sasaki M (1982) In: Sandberg AA (ed) Sister chromatid exchange, Alan R Liss, New York, pp 461-514. Barbier M, Dulieu H (1983) Plant Sci. Lett. 29: 201206 Carrano AV, Thompson LH (1982) Cytogenet. Cell Genet. 33:57-61
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