Plant Cell Reports
Plant Cell Reports (1986) 5:43-46
© Springer-Verlag 1986
Variability of DNA content and karyotype in cell cultures of an interdihaploid Solanum tuberosum L. P. Pijnacker, J. H. M. Hermelink, and M.A. Ferwcrda Department of Genetics, University of Groningen, Centre of Biological Sciences, P.O. Box 14, NL-9750 AA Haren, The Netherlands Received October 16, 1985 / Revised version received December 6, 1985 - Communicated by I. Potrykus
ABSTRACT Karyological and DNA cytophotometric observations were carried out to follow nuclear events in cell cultures of an interdihaploid potato, genotype H2578, 2n=2x=24. The percentage of polyploid cells increased in leaf calluses, but decreased and then remained at a constant level when these cells were cultured in suspension. These suspension cultures did not survive, maybe because they did not become tetraploid. Another suspension culture proliferated with hypotetraploid cells only. Unlike the DNA contents of their interphase nuclei, the karyotypes varied considerably because of numerical and structural chromosome mutations. INTRODUCTION Plant cells grown in vitro, whether as callus or as suspension, do not usually demonstrate a stable chromosome complement (Bayliss 1980, Karp and Bright 1985). Numerical and structural changes, of the chromosomes are common features. These changes, however, are undesirable for somatic cell genetic studies. Cell cultures of the potato (Solanum tuberosum, 2n= 4x=48) also do not escape chromosomal instability. Changes in chromosome number and/or nuclear-DNA content of callus cells of haploid, diploid and tetraploid plants have been described by Jacobsen (1981), Khvilkovskaya (1982), Jacobsen et al. (1983), Sree Ramulu et al. (1984) and Tempelaar et al. (1985). Nuclei with a higher DNA content were also observed in cell colonies which developed from isolated protoplasts of tetraploid plants in liquid medium during two weeks (Sree Ramulu et al. 1984, Carlberg et al. 1984). Moreover, chromosomal instability can be inferred from somaclonal variation among regenerants which passed a callus phase (Karp and Bright, 1985). The present investigation was undertaken to determine the progress of instability in, as well as the relationship between, DNA contents and karyotypes of cells from suspension cultures which originated from leaf calluses of an interdihaploid potato. MATERIALS
AND
METHODS
Material. Interdihaploid Solanum tuberosum plants with genotype H2578 (2n=2x=24), originating from a tetraploid clone (cf. Jacobsen et al. 1983), were used. Leaf Callus and Cell Suspension Cultures. Calluses were derived from leaves and cell suspensions were initiated from these calluses according to de Vries and Bokelmann (1985). The calluses were subcultured
OfJ?rint requests to: L. P. P~nacker
4 weeks after initiation and after 60 days about 5 calluses were taken to initiate a suspension culture. In the first experiment 2 cell suspensions were started at the same time, subcultured twice a week and investigated over a period of about 90 days until the death of these lines. Thereafter another cell suspension was used, which had originated in the same way and had been in culture for 410 days at that time; it was investigated at days 410 and 424. Cytology. For slide preparations, callus, with or without pretreatment with 8-hydroxyquinoline or ~bromonaphtalene, was processed either as root tips according to P~nacker and Ferwerda (1984) or, if the callus was very loose, as cell suspensions. Slides of cell suspensions were made as follows, according to modified procedures of Mouras et al. (1978) and Murata (1983): 20 h after subculturing colchicine (0.05%) was added to the suspension cultures for 3-5 h. After centrifugation at lOOg for 5 min, the medium was discarded, the cells suspended in 60% acetic acid at 4°C and allowed to stand for 15 min. The acetic acid was removed after centrifugation and the cells resnspended in fresh, cold (4°C) fixative (1:3 glacial acetic a c i d - 100% ethanol) for a minimum of 2 h. To remove the cell walls, 5 ml of cells were, after centrifugation and washing in distilled water (twice), incubated in 15% (v/v) pectinase (Sigma P5146)/1.5% (w/v) cellulase RIO (Yakult) in citrate buffer pH 4.8 at 37°C for 3-4 h. They were then centrifuged and rinsed again in distilled water (twice). A drop of the pellet of concentrated cells was transferred to a clean slide and processed as described for root tips. In later experiments better slides were obtained when the cells were brought again from the distilled water to cold fixative. Then, after centrifugation, the final amount of suspension was made up to about 1 ml and, using a Pasteur pipette, 3-4 drops of the suspension were dropped on a slide which was wetted with cold fixative immediately before use, and allowed to air dry. This procedure was carried out on the 410and 424-days-old cell suspensions. For the study of anaphases and the determination of mitotic indices, colchicine was omitted and the cells were fixed 24 h after subculturing. For DNAcytophotometry the colchicine step was also omitted and the cells fixed 72 h after subculturing. The slides were either Giemsa stained according to P~nacker and Ferwerda (1984) or Feulgen stained as follows: the slides were postfixed in ethanol 100% : formaldehyde 40% : glacial acetic acid (85:10:5) for 30 min at room temperature, rinsed in distilled water, hydrolysed in 5N HCI for 20 min at room temp-
44 erature and stained by the Feulgen reaction for 2 h at 4°C in the dark. The slides were then rinsed in running tap water for I0 min, rinsed in distilled water, air-dried and, via xylene, mounted in DePeX. Cytophotometrical D N A - measurements were carried out on the Feulgen stained interphase nuclei with the equipment described by Tempelaar (1980). Interphases, prophases and telophases of root tip cells of H~578 plants were taken as reference. The mean and standard error of a peak of a frequency distribution were calculated by calculating first the mean of all the values of that peak distribution and then by taking only those values which lie within that mean ± 10%.
--_ 2'C
.
|.ml.d.._
4'C
Z
AND
CALLUSES DAY 60 _.j_ . . . . . 16C
CELL SUSPENSION 2'C
4'C
8'C
DAY 39
N=199 "~ , 24C 2L.-7& C
]
N=226
16C
2/*C 27-76C
20
10 ~"
CELL SUSPENSION 2'C
z.'C
8'C
2C
N=200 24C
CELL SUSPENSION .11
DAY 77
16C
10
DISCUSSION
The leaves which were the source material of the callus cultures, were investigated cytophotometrically by Jacobsen et al. (1983) and Tempelaar et al. (1985). They found that 76% of the nuclei had a 2C value (corresponding to the diploid number of 24 chromosomes in G1 phase), 19% had 4C, and 5% had a value higher than 4C. In the present investigation the nuclear-DNA content of the cells of two 60-daysold calluses was established. The data of the 2 calluses did not differ and were pooled (Fig.l). The frequency distribution reveals that main peaks were not found near the 2C and 4C values and that 64% of the nuclei had values higher than 4C (range up to 74C). Supposing that all the 4C nuclei of the leaves were in G1 phase and that these nuclei appeared as 8C nuclei in G2 phase in callus, only 24% (19% + 5%) of the callus nuclei would have values higher than 4C. Consequently, the variation in the DNA contents had increased considerably mainly through polyploidization under the in vitro conditions. This variation was even higher than was established before in younger and older calluses of H2578 by Jacobsen et al. (1983) and Tempelaar et al. (1985). The cytophotometrical analyses of the 2 cell suspensions originating from the 60-days-old calluses were similar and therefore the data of only one suspension are presented (Fig.l). This suspension showed a progressive decrease in the DNA amounts with the time: after 39 days main peaks were found at the 2C, 4C and 8C levels and 57% of the nuclei had DNA contents higher than 4C, after 77 days only 18% of the nuclei had values higher than 4C and cells with 4C nuclei outnumbered those with 2C 1.5 times. The abundance of these cells could have been caused by a synchronously dividing part of the population of diploid cells which were in G2 phase at the moment of fixation. However, the mitotic indices (see below) do not point to that synchrony, which could mean that the 4C cells were mainly tetraploid cells in G1 phase. During leaf development the metaphases (N=50) showed the normal karyotype with 24 chromosomes (Fig.3). The chromosome counts, carried out on 2 calluses and the 2 derived cell suspensions, revealed that up to 11% of the diploid metaphases were aneuploid for 1 or 2 chromosomes. The frequency of aneuploidy increased at higher ploidies, particularly when the chromosome numbers were higher than 4x. The data of the 2 calluses were pooled because they were almost similar; the same applies to the 2 suspension cultures (Fig.2). 31% of the metaphases of the calluses was diploid, hyper/hypodiploidy for 1 or 2 chromosomes inclusive. This percentage of 'diploidy' increased to about 80% in both suspension cultures during the first 39 days of culture while the metaphases with chromosome numbers higher than 5x disappeared progressively. A rather constant 4:1 ratio of metaphases with (about) 24 and (about) 48 chromdsomes was reached and maintained. The course of development of the cell suspensions towards the diploid-tetraploid level as shown by cytophotometry, is thus reflected in the chromosome
.
8'C
10
___
RESULTS
.__
DAY410
N=230
, //-2~C
......
.L r8c ~6C DNA CONTENT PER NUCLEUS
4~
278
Fig.l. Cell cultures of interdihaploid potato genotype H2578. Frequency distribution of DNA content per nucleus in interphase cells of 2 calluses after 60 days of culture, of a cell suspension derived from such calluses at days 39 and 77 of culture growth, and of another cell suspension at day 410. DNA contents in arbitrary units ( m ± s . e . ) are at day 3 9 : 1 7 . 1 ± 0 . 2 = 2 C , 3 5 . 0 ± 0 . 3 = 4 C ; at day 77: 1 7 . 5 ± 0 . I = 2 C , 3 5 . 5 ± 0 . 2 = 4 C ; at day 410: 2 9 . 7 ± 0 . 3 = 3 . 4 C , 6 0 . 2 ± 0 . 3 =6.8C. G1 cells of leaves contained 1 7 . 2 ± 0 . 2 = 2 C and G2 cells 3 5 . 3 ± 0 . 2 = 4 C .
n u m b e r of metopheses
55
n u m b e r of onephoses
6
80 137 &2 100
154 100
88
93 100
50
50 50
58
56 60
50 50
50
% 6O V1 I/} O
40
r,-
=
/ x
O
~t "- = 0 1:3 "-.. fI r_ O OJ 4 - o
E
20 0
.
0
.
.
20
.
.
.
.
40
x.... -x "'"-Q
~ .... n
60
80
days F i g . 2 . Cell c u l t u r e s of i n t e r d i h a p l o i d p o t a t o genot y p e H~578. T o t a l numbers of m e t a p h a s e s and a n a p h a s e s and p e r c e n t a g e s o f a b e r r a n t m e t a p h a s e s and a n a p h a s e s o f 2 c a l l u s e s , d e t e r m i n e d a f t e r 60 d a y s o f c u l t u r e and o f 2 c e l l s u s p e n s i o n s , d e r i v e d from s u c h 6 0 - d a y s o l d c a l l u s e s , p r e s e n t e d o v e r a c u l t u r e p e r i o d of 0 - 9 4 d a y s . Data s u s p e n s i o n s a t day 0 = d a t a c a l l u s e s a t day 60; t h e d a t a a t day 9/4 c o n c e r n one c e l l s u s p e n s i o n . • = m e t a p h a s e s w i t h chromosome numbers from 3x t o 16x and o = m e t a p h a s e s w i t h chromosome numbers from 5x t o 16x, a n e u p l o i d numbers i n c l u s i v e . D i p l o i d m e t a p h a s e s (2x=24) i n c l u d e m e t a p h a s e s h y p e r / h y p o p l o i d f o r 1-2 chromosomes; 3x m e t a p h a s e s h a r d l y o c c u r r e d . x = a n a phases with bridges.
counts. The mitotic indices of the callus cultures were ~0.1% and of the suspension cultures between 0.i and 9.2%. Anaphase bridges were absent in the mitotic cells of the calluses and of the suspensions until
45 Table i. Analyses of 100 karyotypes of 410- and 424-days-old cell suspensions of interdihaploid potato H~578. No. of chrom- No. of metaosomesa/plate phase plates
36 37 38 39 40 41 42 43 44 45 40.6±i •9 e
1 1 12 16 22 23 9 8 3 5 I00
No. of tr~ns- No. of plates No. of dicen- No. of plates No. o~ fraglocations~/ with translo- tricsC/plate with dicenments~/plate plate cations trics 0 2 0-2 0-2 0-2 0-i 0-i 0-i 0-2 0-2 0.5±0.7
0 1 4 5 7 9 7 2 1 3 39
1 2 0-4 0-5 1-4 0-4 0-2 0-2 0-2 1-2 1.6±1.0
1 1 ii 15 22 19 8 5 1 5 88
1 2 0-3 0-3 0-3 0-3 0-i I-4 1-2 0-2 1.2±1.0
No. of plates with fragments 1 1 9 12 13 16 8 8 3 3 74
a) Fragments not included, b) Translocated monocentric chromosomes recognizable as chromosomes longer than the longest chromosome of the normal karyotype, c) Dicentric chromosomes consisting of 2 dicentric chromatids. d) Chromosome segments without a primary constriction being smaller than the smallest chromosome, e) Mean ± standard deviation. day 59 of culture, with an exception at day 39 (Fig.2). The percentage of anaphases with bridges then increased in diploid as well as in polyploid cells and reached a level of 32% within 3 weeks. These bridges resulted from dicentric chromosomes and were not due to stickiness. Chromosome breakage thus began after the chromosome numbers apparently had stabilized. The cause of these chromosome as well as genome mutations is not understood. One of the 2 cel! suspensions did no longer show mitotic divisions after 84 days of culture and the other one after 94 days; they both ceased to grow and could not be investigated any longer. The other cell suspension,inventarized when 410 and 424 days old, grew vigorously (mitotic indices ii-16%). The frequency distributions of the DNA contents of the 410- and 424-days-old cells were similar. The DNA values of the interphase nuclei appeared in 2 peaks at the 3.4C and 6.8C levels, which contents were significantly (P
Plant Cell Reports (1986) 5:43-46
© Springer-Verlag 1986
Variability of DNA content and karyotype in cell cultures of an interdihaploid Solanum tuberosum L. P. Pijnacker, J. H. M. Hermelink, and M.A. Ferwcrda Department of Genetics, University of Groningen, Centre of Biological Sciences, P.O. Box 14, NL-9750 AA Haren, The Netherlands Received October 16, 1985 / Revised version received December 6, 1985 - Communicated by I. Potrykus
ABSTRACT Karyological and DNA cytophotometric observations were carried out to follow nuclear events in cell cultures of an interdihaploid potato, genotype H2578, 2n=2x=24. The percentage of polyploid cells increased in leaf calluses, but decreased and then remained at a constant level when these cells were cultured in suspension. These suspension cultures did not survive, maybe because they did not become tetraploid. Another suspension culture proliferated with hypotetraploid cells only. Unlike the DNA contents of their interphase nuclei, the karyotypes varied considerably because of numerical and structural chromosome mutations. INTRODUCTION Plant cells grown in vitro, whether as callus or as suspension, do not usually demonstrate a stable chromosome complement (Bayliss 1980, Karp and Bright 1985). Numerical and structural changes, of the chromosomes are common features. These changes, however, are undesirable for somatic cell genetic studies. Cell cultures of the potato (Solanum tuberosum, 2n= 4x=48) also do not escape chromosomal instability. Changes in chromosome number and/or nuclear-DNA content of callus cells of haploid, diploid and tetraploid plants have been described by Jacobsen (1981), Khvilkovskaya (1982), Jacobsen et al. (1983), Sree Ramulu et al. (1984) and Tempelaar et al. (1985). Nuclei with a higher DNA content were also observed in cell colonies which developed from isolated protoplasts of tetraploid plants in liquid medium during two weeks (Sree Ramulu et al. 1984, Carlberg et al. 1984). Moreover, chromosomal instability can be inferred from somaclonal variation among regenerants which passed a callus phase (Karp and Bright, 1985). The present investigation was undertaken to determine the progress of instability in, as well as the relationship between, DNA contents and karyotypes of cells from suspension cultures which originated from leaf calluses of an interdihaploid potato. MATERIALS
AND
METHODS
Material. Interdihaploid Solanum tuberosum plants with genotype H2578 (2n=2x=24), originating from a tetraploid clone (cf. Jacobsen et al. 1983), were used. Leaf Callus and Cell Suspension Cultures. Calluses were derived from leaves and cell suspensions were initiated from these calluses according to de Vries and Bokelmann (1985). The calluses were subcultured
OfJ?rint requests to: L. P. P~nacker
4 weeks after initiation and after 60 days about 5 calluses were taken to initiate a suspension culture. In the first experiment 2 cell suspensions were started at the same time, subcultured twice a week and investigated over a period of about 90 days until the death of these lines. Thereafter another cell suspension was used, which had originated in the same way and had been in culture for 410 days at that time; it was investigated at days 410 and 424. Cytology. For slide preparations, callus, with or without pretreatment with 8-hydroxyquinoline or ~bromonaphtalene, was processed either as root tips according to P~nacker and Ferwerda (1984) or, if the callus was very loose, as cell suspensions. Slides of cell suspensions were made as follows, according to modified procedures of Mouras et al. (1978) and Murata (1983): 20 h after subculturing colchicine (0.05%) was added to the suspension cultures for 3-5 h. After centrifugation at lOOg for 5 min, the medium was discarded, the cells suspended in 60% acetic acid at 4°C and allowed to stand for 15 min. The acetic acid was removed after centrifugation and the cells resnspended in fresh, cold (4°C) fixative (1:3 glacial acetic a c i d - 100% ethanol) for a minimum of 2 h. To remove the cell walls, 5 ml of cells were, after centrifugation and washing in distilled water (twice), incubated in 15% (v/v) pectinase (Sigma P5146)/1.5% (w/v) cellulase RIO (Yakult) in citrate buffer pH 4.8 at 37°C for 3-4 h. They were then centrifuged and rinsed again in distilled water (twice). A drop of the pellet of concentrated cells was transferred to a clean slide and processed as described for root tips. In later experiments better slides were obtained when the cells were brought again from the distilled water to cold fixative. Then, after centrifugation, the final amount of suspension was made up to about 1 ml and, using a Pasteur pipette, 3-4 drops of the suspension were dropped on a slide which was wetted with cold fixative immediately before use, and allowed to air dry. This procedure was carried out on the 410and 424-days-old cell suspensions. For the study of anaphases and the determination of mitotic indices, colchicine was omitted and the cells were fixed 24 h after subculturing. For DNAcytophotometry the colchicine step was also omitted and the cells fixed 72 h after subculturing. The slides were either Giemsa stained according to P~nacker and Ferwerda (1984) or Feulgen stained as follows: the slides were postfixed in ethanol 100% : formaldehyde 40% : glacial acetic acid (85:10:5) for 30 min at room temperature, rinsed in distilled water, hydrolysed in 5N HCI for 20 min at room temp-
44 erature and stained by the Feulgen reaction for 2 h at 4°C in the dark. The slides were then rinsed in running tap water for I0 min, rinsed in distilled water, air-dried and, via xylene, mounted in DePeX. Cytophotometrical D N A - measurements were carried out on the Feulgen stained interphase nuclei with the equipment described by Tempelaar (1980). Interphases, prophases and telophases of root tip cells of H~578 plants were taken as reference. The mean and standard error of a peak of a frequency distribution were calculated by calculating first the mean of all the values of that peak distribution and then by taking only those values which lie within that mean ± 10%.
--_ 2'C
.
|.ml.d.._
4'C
Z
AND
CALLUSES DAY 60 _.j_ . . . . . 16C
CELL SUSPENSION 2'C
4'C
8'C
DAY 39
N=199 "~ , 24C 2L.-7& C
]
N=226
16C
2/*C 27-76C
20
10 ~"
CELL SUSPENSION 2'C
z.'C
8'C
2C
N=200 24C
CELL SUSPENSION .11
DAY 77
16C
10
DISCUSSION
The leaves which were the source material of the callus cultures, were investigated cytophotometrically by Jacobsen et al. (1983) and Tempelaar et al. (1985). They found that 76% of the nuclei had a 2C value (corresponding to the diploid number of 24 chromosomes in G1 phase), 19% had 4C, and 5% had a value higher than 4C. In the present investigation the nuclear-DNA content of the cells of two 60-daysold calluses was established. The data of the 2 calluses did not differ and were pooled (Fig.l). The frequency distribution reveals that main peaks were not found near the 2C and 4C values and that 64% of the nuclei had values higher than 4C (range up to 74C). Supposing that all the 4C nuclei of the leaves were in G1 phase and that these nuclei appeared as 8C nuclei in G2 phase in callus, only 24% (19% + 5%) of the callus nuclei would have values higher than 4C. Consequently, the variation in the DNA contents had increased considerably mainly through polyploidization under the in vitro conditions. This variation was even higher than was established before in younger and older calluses of H2578 by Jacobsen et al. (1983) and Tempelaar et al. (1985). The cytophotometrical analyses of the 2 cell suspensions originating from the 60-days-old calluses were similar and therefore the data of only one suspension are presented (Fig.l). This suspension showed a progressive decrease in the DNA amounts with the time: after 39 days main peaks were found at the 2C, 4C and 8C levels and 57% of the nuclei had DNA contents higher than 4C, after 77 days only 18% of the nuclei had values higher than 4C and cells with 4C nuclei outnumbered those with 2C 1.5 times. The abundance of these cells could have been caused by a synchronously dividing part of the population of diploid cells which were in G2 phase at the moment of fixation. However, the mitotic indices (see below) do not point to that synchrony, which could mean that the 4C cells were mainly tetraploid cells in G1 phase. During leaf development the metaphases (N=50) showed the normal karyotype with 24 chromosomes (Fig.3). The chromosome counts, carried out on 2 calluses and the 2 derived cell suspensions, revealed that up to 11% of the diploid metaphases were aneuploid for 1 or 2 chromosomes. The frequency of aneuploidy increased at higher ploidies, particularly when the chromosome numbers were higher than 4x. The data of the 2 calluses were pooled because they were almost similar; the same applies to the 2 suspension cultures (Fig.2). 31% of the metaphases of the calluses was diploid, hyper/hypodiploidy for 1 or 2 chromosomes inclusive. This percentage of 'diploidy' increased to about 80% in both suspension cultures during the first 39 days of culture while the metaphases with chromosome numbers higher than 5x disappeared progressively. A rather constant 4:1 ratio of metaphases with (about) 24 and (about) 48 chromdsomes was reached and maintained. The course of development of the cell suspensions towards the diploid-tetraploid level as shown by cytophotometry, is thus reflected in the chromosome
.
8'C
10
___
RESULTS
.__
DAY410
N=230
, //-2~C
......
.L r8c ~6C DNA CONTENT PER NUCLEUS
4~
278
Fig.l. Cell cultures of interdihaploid potato genotype H2578. Frequency distribution of DNA content per nucleus in interphase cells of 2 calluses after 60 days of culture, of a cell suspension derived from such calluses at days 39 and 77 of culture growth, and of another cell suspension at day 410. DNA contents in arbitrary units ( m ± s . e . ) are at day 3 9 : 1 7 . 1 ± 0 . 2 = 2 C , 3 5 . 0 ± 0 . 3 = 4 C ; at day 77: 1 7 . 5 ± 0 . I = 2 C , 3 5 . 5 ± 0 . 2 = 4 C ; at day 410: 2 9 . 7 ± 0 . 3 = 3 . 4 C , 6 0 . 2 ± 0 . 3 =6.8C. G1 cells of leaves contained 1 7 . 2 ± 0 . 2 = 2 C and G2 cells 3 5 . 3 ± 0 . 2 = 4 C .
n u m b e r of metopheses
55
n u m b e r of onephoses
6
80 137 &2 100
154 100
88
93 100
50
50 50
58
56 60
50 50
50
% 6O V1 I/} O
40
r,-
=
/ x
O
~t "- = 0 1:3 "-.. fI r_ O OJ 4 - o
E
20 0
.
0
.
.
20
.
.
.
.
40
x.... -x "'"-Q
~ .... n
60
80
days F i g . 2 . Cell c u l t u r e s of i n t e r d i h a p l o i d p o t a t o genot y p e H~578. T o t a l numbers of m e t a p h a s e s and a n a p h a s e s and p e r c e n t a g e s o f a b e r r a n t m e t a p h a s e s and a n a p h a s e s o f 2 c a l l u s e s , d e t e r m i n e d a f t e r 60 d a y s o f c u l t u r e and o f 2 c e l l s u s p e n s i o n s , d e r i v e d from s u c h 6 0 - d a y s o l d c a l l u s e s , p r e s e n t e d o v e r a c u l t u r e p e r i o d of 0 - 9 4 d a y s . Data s u s p e n s i o n s a t day 0 = d a t a c a l l u s e s a t day 60; t h e d a t a a t day 9/4 c o n c e r n one c e l l s u s p e n s i o n . • = m e t a p h a s e s w i t h chromosome numbers from 3x t o 16x and o = m e t a p h a s e s w i t h chromosome numbers from 5x t o 16x, a n e u p l o i d numbers i n c l u s i v e . D i p l o i d m e t a p h a s e s (2x=24) i n c l u d e m e t a p h a s e s h y p e r / h y p o p l o i d f o r 1-2 chromosomes; 3x m e t a p h a s e s h a r d l y o c c u r r e d . x = a n a phases with bridges.
counts. The mitotic indices of the callus cultures were ~0.1% and of the suspension cultures between 0.i and 9.2%. Anaphase bridges were absent in the mitotic cells of the calluses and of the suspensions until
45 Table i. Analyses of 100 karyotypes of 410- and 424-days-old cell suspensions of interdihaploid potato H~578. No. of chrom- No. of metaosomesa/plate phase plates
36 37 38 39 40 41 42 43 44 45 40.6±i •9 e
1 1 12 16 22 23 9 8 3 5 I00
No. of tr~ns- No. of plates No. of dicen- No. of plates No. o~ fraglocations~/ with translo- tricsC/plate with dicenments~/plate plate cations trics 0 2 0-2 0-2 0-2 0-i 0-i 0-i 0-2 0-2 0.5±0.7
0 1 4 5 7 9 7 2 1 3 39
1 2 0-4 0-5 1-4 0-4 0-2 0-2 0-2 1-2 1.6±1.0
1 1 ii 15 22 19 8 5 1 5 88
1 2 0-3 0-3 0-3 0-3 0-i I-4 1-2 0-2 1.2±1.0
No. of plates with fragments 1 1 9 12 13 16 8 8 3 3 74
a) Fragments not included, b) Translocated monocentric chromosomes recognizable as chromosomes longer than the longest chromosome of the normal karyotype, c) Dicentric chromosomes consisting of 2 dicentric chromatids. d) Chromosome segments without a primary constriction being smaller than the smallest chromosome, e) Mean ± standard deviation. day 59 of culture, with an exception at day 39 (Fig.2). The percentage of anaphases with bridges then increased in diploid as well as in polyploid cells and reached a level of 32% within 3 weeks. These bridges resulted from dicentric chromosomes and were not due to stickiness. Chromosome breakage thus began after the chromosome numbers apparently had stabilized. The cause of these chromosome as well as genome mutations is not understood. One of the 2 cel! suspensions did no longer show mitotic divisions after 84 days of culture and the other one after 94 days; they both ceased to grow and could not be investigated any longer. The other cell suspension,inventarized when 410 and 424 days old, grew vigorously (mitotic indices ii-16%). The frequency distributions of the DNA contents of the 410- and 424-days-old cells were similar. The DNA values of the interphase nuclei appeared in 2 peaks at the 3.4C and 6.8C levels, which contents were significantly (P
