Cytogenetic abnormalities in cotton (Gossypium hirsutum L.) cell cultures1

RUZHONG LI AND DAVID M. STELLY~ Department of Soil and Crop Sciences, Texas A & M University, College Station, TX 77843-2474, U.S.A.

AND

NORMA L. TROLINDER Agricultural Service, United States Department of Agriculture, Cropping System Research Luboratory, Route 3 Box 215,

Lubbock, TX 79401, U. S. A.

Corresponding Editor: R. L. Phillips

Received October 17, 1988

Accepted June 15, 1989

LI, R., STELLY, D. M. and TROLINDER, N. L. 1989. Cytogenetic abnormalities in cotton (Gossypium hirsutum L.) cell cultures. Genome, 32: 1 128 - 1 134.

High frequencies of somaclonal phenotypic and cytogenetic variation have been observed previously among regenerants from cotton (Gossypium hirsutum L., 2n = 4x = 52). In this study we endeavored to determine if cytogenetic abnormalities would be detectable in cotton cell cultures and if so, whether or not the observed abnormalities would parallel those expected on the basis of previous cytogenetic analyses of cotton somaclones. Paired samples from suspension cultures established from 21-month-old 'Coker 312' and 8-month-old 'Coker 315' calli were pretreated or not pretreated with colchicine to detect cytogenetic abnormalities at metaphase or anaphase- telophase, respectively. Cell cultures established from both calli were found to vary in chromosome number. Hypoaneuploidy was common, but hyperaneuploidy and polyploidy were rare. Modal chromosome numbers for the 'Coker 312' and 'Coker 315' cultures were 46 and 50, respectively. Bridges at anaphase and telophase were frequent in the 'Coker 312' cultures but rare in the 'Coker 315'cultures. Cytogenetic differences between the cultures could be due to effects of culture age, genotype, their interaction, or other factors. Very small chromosomes, presumably centric fragments, as well as ring chromosomes and putative bridges between metaphase chromosomes occurred at low frequencies. The prevalence of hypoaneuploidy and rarity of hyperaneuploidy and polyploidy in cultures paralleled previous results on cotton somaclones, indicating that cytogenetic abnormalities arising in vitro probably contribute significantly to cotton somaclonal variation. The occurrence of hypoaneuploidy and bridges, including multiple bridges within single cells, is concordant with the hypothesis that breakage - fusion - bridge cycles may accumulate during in vitro culture of cotton.

Key words: cotton, Gossypium, tissue culture, cytogenetics.

LI, R., STELLY, D. M. et TROLINDER, N. L. 1989. Cytogenetic abnormalities in cotton (Gossypium hirsutum L.) cell cultures. Genome, 32 : 1128 - 1134.

Des frkquences klevCes de variations cytogknktiques et phknotypiques ont kt6 observkes antkrieurement parmi des rkgknkrants de coton (Gossypium hirsutum L., 2n = 4x = 52). Dans cette Ctude, nous avons visC B dkterminer si des anomalies cytogknktiques pourraient etre dkcelkes dans des cultures de cellules de coton et, dans l'affirmative, si les anomalies observkes pourraient ou non correspondre aux anomalies attendues, d'aprks les analyses cytogknktiques antkrieures de somaclones de coton. Des Cchantillons en double de suspensions de cultures de cals de (Coker 315, ktablies depuis 21 mois et de cals de (Coker 312, Ctablies depuis 8 mois ont CtC prktraitks ou non B la colchicine pour dkceler les anomalies cytogknCtiques A la mCtaphase ou B l'anaphase- tklophase respectivement. Les cultures des deux cals ont prksentC des variations dans le nombre de chromosomes. L'hypoaneuploidie s'est avCrke etre presque gknkrale, tandis que l'hyperaneuploi'die et la polyploidie ont kt6 rares. Le nombre chromosomique modal pour les cultures de (Coker 3 15, et (Coker 3 12, a Ctk respectivement de 46 et de 50. Les ponts B l'anaphase et B la tklophase ont kt6 fkquents dans les cultures de (Coker 312,, mais rares dans celles de (Coker 315, . Les diffkrences cytogCnktiques entre les cultures pourraient relever des effets de l'iige des cultures, des gknotypes, des interactions entre ces deux facteurs ou meme d'autres facteurs. De tds petits chromosomes, pksumkment des fragments centriques aussi bien que des chromosomes en anneaux, et des ponts possibles entre les chromosomes des mktaphases ont kt6 prksents B de faibles frkquences. La prkvalence de l'hypoaneuploi'die et la raretk de l'hyperaneuploidie et de la polyploidie dans les cultures ont 6tk B l'kgal des rksultats antkrieurs sur les somaclones de coton, ce qui indique que les anomalies cyto- gknCtiques survenant in vitro contribuent, probablement de f a~on significative, B la variation somaclonale chez le coton. L'occurrence de l'hypoaneuploidie et de ponts, incluant les ponts multiples dans les cellules individuelles, concorde avec l'hypothkse que les cycles de bris et de fusion de ponts peuvent s'accumuler au cours de la culture du coton in vitro.

Mots c l b : coton, Gossypium, culture de tissus, cytogknetique. [Traduit par la revue]

Introduction explant tissue, culture age, and genotype have been reported to

Plant chromosomal variation induced by cell and tissue cul- affect the occurrence of cytological instability (Torrey 196 l ;

ture has been well documented (Sunderland 1977; Bayliss Shimada 1971 ; McCoy et al. 1982). Durations of callus culture

1980; Bhojwani and Razdan 1983). Media components, (Torrey 1967; Novak 1981) and suspension culture (Singh and Harvey 1975) affect the range of cytological abnormalities. ow ever, instances of cytolo~ical stability-after long-term cul-

lcontribution TA-24018 from the Texas Agricultural Experiment ture have been reported (cooper et ale 1964; ~or s tog et al. Station, College Station, TX, U.S .A. 1969; Edallo et al. 1981 ; McCoy and Phillips 1982; Rhodes

2Author to whom reprint requests should be sent. et al. 1986).

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TABLE 1. Variation in chromosome number in Gossypium hirsutum suspension cell cultures

No. of 'Coker 3 12' 'Coker 3 15 ' chromosomes

per cell No. of cells Frequency (%) No. of cells Frequency (%)

Total

NOTE: F, chromosome fragment; B, metaphase bridge. *Value in parentheses is the actual number of chromosc

Cotton has been one of severa1,crops that has proved difficult to regenerate after tissue culture. Very little work has been reported on the chromosomal instability of cotton cultures or cytogenetic abnormalities of somaclones. Recently Bajaj and Gill (1985) reported on chromosomal variation in calli raised from embryos, ovules, anthers and hypocotyl segments of two diploid (2n = 2.x = 26) cotton species, Gossypium arboreum and G. herbaceum. The calli varied in chromosome number from 13, putatively haploid, to 78 putatively hexaploid. Trolinder and Goodin (1987) recently reported that G. hir- sutum 'Coker 3 12' was more easily and expeditiously regener- ated from cultures than were other genotypes. However, they observed a high frequency of infertile plants among regener- ants. Stelly et al. (1985, 1989) found most G. hirsutum somaclones to be both phenotypically and cytogenetically aberrant, as well as partially or completely sterile. Hypoaneu- ploidy was common, whereas hyperaneuploidy and polyploidy were rare and not observed, respectively. High frequencies of tertiary monosomy were observed among the somaclones, leading to the hypothesis that breakage -fusion -bridge (BFB) cycles may accumulate during cotton cell culture and resolve themselves by secondary events involving fusions of nonhomo- logous BFB chromatids to form tertiary chromatids (Stelly et al. 1989).

We are not aware of any reports on the cytogenetic behavior of G. hirsutum cell cultures. The present study was undertaken to determine if detectable chromosomal variation occurs in G. hirsutum cell cultures' and, if so, to determine which types of cytogenetic anomalies might be most common. We are also interested in knowing whether the types and frequencies of abnormalities would parallel expectations based on the previ- ous analyses of somaclones (Stelly et al. 1989).

Materials and methods Calli of G. hirsutum 'Coker 3 12' and 'Coker 3 15' (2n = 4x = 52)

were initiated from hypocotyl segments of the respective varieties. The medium (MS) used for induction and long-tern culture was that of Murashige and Skoog (1962), supplemented with 0.1 mg/L 2,4-D

lmes observed.

3 0 ~

NO. OF CHROMOSOMES

FIG. 1. Frequencies of chromosome numbers in cells from callus- derived suspension cultures of cotton (Gossypium hirsutum L.) varieties 'Coker 3 12' (*) and 'Coker 3 15' (+).

(2,4-dichlorophenoxyacetic acid), 0.1 mg/L kinetin, 100 mg/L myoinositol, B5 vitamins (Gamborg et al. 1968), and 30 g/L glucose. The medium was solidified with 1.6 g/L Gelrite and 0.75 g/L MgCl,. The pH of the medium was adjusted to 5.8. For suspension cell cul- ture the same medium was used without Gelrite and MgCl,. The calli were subcultured at 1-month intervals, whereas the suspension cul- tures were subcultured at 10- to 12-day intervals. The callus cultures were maintained under a photoperiod regime of 16 h light : 8 h dark- ness, at 60-90 pE rn-, S-' and 28 f 2°C.

Cell samples were collected for cytological observation on the 2nd or 3rd day after transfers. Paired samples, pretreated and not, were used for cytological analysis. One subsample was collected without pretreatment before fixation for examination of mitotic abnormalities. The other subsample was incubated for 4-4.5 h under normal culture conditions after the addition of sufficient 1 % colchicine solution in MS medium to give a final colchicine concentration of 0.075 -0.1 %. This sample was used subsequently for determining the numbers of chromosomes. All samples were fixed in 95 % ethyl alcohol -glacial acetic acid (3: 1) for 24 h and then transferred to 70% ethyl alcohol for storage. The cell samples were hydrolyzed 10- 12 min with 6 N HC1 at room temperature. After rinsing twice with distilled H,O, a small piece of cellular material was transferred to a slide and stained with toluidine blue 0 (Hsu and Stewart 1982). After heating several times,

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GENOME VOL. 32, 1989

samples were squashed. For each sample, 20-50 slides were exam- ined. Only relatively well spread, intact metaphase cells were counted.

Dispersion statistics were calculated for the frequency data on normal mitotic divisions, assuming binomial distribution for abnor- mal versus normal mitotic divisions. Two-tailed t-tests were used to evaluate the probability of obtaining the observed frequencies under the null hypothesis, i.e., equality of the samples from the older 'Coker 312' culture versus the younger 'Coker 315' culture.

Results and discussion Chromosome numbers of the 'Coker 3 12' and 'Coker 3 15'

cultures were tremendously variable (Fig. 1)' with a range of 2 1 - 100 chromosomes per cell. Hypoaneuploidy (Figs. 2A and 2B) was far more common than hyperaneuploidy (Fig. 2C, Table 1). Only 1 of 80 cells from 'Coker 3 12'cultures and 7 of 109 cells from the 'Coker 3 15' cultures had the normal number of chromosomes. It is possible that even these few cells classi- fied as normal carried undetected abnormalities.

Hypoaneuploidy involved ,the loss of one to several chromo- somes. The range of chromosome numbers was wider for the 'Coker 3 12' culture than for the 'Coker 3 15' culture. From 39 to 49 chromosomes were present in most 'Coker 312' cells. Whereas from 48 to 50 chromosomes were present in most 'Coker 315' cells. However, both cultures exhibited a marked propensity to lose chromosomes. Results on chromosome numbers in these cultures paralleled those observed among somaclones from 'Acala SJ-2' and 'Acala SJ-5', which also exhibited very high frequencies of hypoaneuploidy and very low frequencies of euploidy , hyperaneuploidy , and polyploidy (Stelly et al. 1989).

The overall frequency of metaphase cells with more than 80 chromosomes (Fig. 2C) was about 11 % , whereas only about 4% of the cells had more than 100 chromosomes. Actual fre- quencies of cells with high numbers of chromosomes might have been higher than observed, because we selectively ana- lyzed well-spread cells, which may have inadvertently led to preferential exclusion of cells with many chromosomes. It is not clear why cells with between 52 and 80 chromosomes were not observed. The cells with very high chromosome numbers were typically clustered on slides, indicating that such cells reproduce in vitro . This observation supports the conclusion by Bayliss (1975) that "both polyploid and structurally altered complements were being produced continuously, rather than originating from the explant used to initiate the culture line."

Chromosome structural changes also occurred in the cotton cultures. Centric fragments (Fig. 2D) were especially common in the 'Coker 315' cell line (Table 1). As many as six frag- ments were observed in a single cell. Centric fragments of roughly similar size are frequently recovered among mono- somic progeny from certain monosomic maternal parents in G. hirsutum (D. M. Stelly , unpublished). A few ring chromo- somes and putatively dicentric chromosomes were found in the 'Coker 312' cultures. Stickiness between chromosomes was not observed, but we observed rare metaphase figures from 'Coker 315' cultures that seemed to be bridges between chro- matid arms of two different chromosomes (Fig. 2E), i.e.,

--

FIG. 2. Mitotic metaphase abnormalities in cells from callus-derived suspension cultures of cotton (Gossypium hirsutum L.). (A) 'Coker 3 15'' 2n = 50, including one abnormally small chromosome (arrow). (B) 'Coker 315" 2n = 50. (C) 'Coker 3 12'' aneupolyploid cell, ca. 2n = 98. (D) 'Coker 3 15'' 2n = 47 + 2 centric fragments (arrowheads). (E) 'Coker 3 15'' metaphase with putative interchromosomal bridges. (F) Single bridge at late anaphase in 'Coker 3 12'.

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NOTES 1133

between nonsister and perhaps nonhomologous chromatids. Such connections might arise in the metaphase after an inter- chromosomal fusion event, i.e., following an anaphase with a double bridge -breakage event.

Samples not pretreated with colchicine before fixation were examined for abnormalities at metaphase through interphase. The frequencies of bridges in anaphase and telophase cells from 'Coker 312' and 'Coker 315' were very high and low, respectively (Table 2). From 0 to 6 bridges were found in single cells (Figs. 2F and 3A-3D). Bridges were also observed between daughter interphase nuclei, some of which were very fine and most easily detected by projections from daughter nuclei and (or) pieces of attenuated chromatin posi- tioned between the nuclei (Fig. 3D). An unequal mitotic divi- sion (Fig. 3E) was observed in a 'Coker 315' cell, possibly explaining the origin of rare cells with very low numbers of chromosomes, e.g., one cell was observed with only 2 1 chro- mosomes. Such cells may not reproduce and hence remain scarce. Chromosomes that lagged or were excluded from daughter nuclei were occasionally noted in cultures from both 'Coker 312' and 'Coker 315' (Fig. 3F).

The overall relative frequencies of normal anaphase and telophase figures in the 'Coker 3 12' (66 %) versus 'Coker 3 15' (91.7 %) cultures were statistically different (P < 0.00 1). Effects of genotype, culture age, their interaction, or other fac- tors could account for this and other cytogenetic differences between the varietal cultures.

Our results demonstrate that mitotic abnormalities and hypo- aneuploidy occurred frequently in these two cultures. Com-

that BFB cycles may indeed accumulate in specific cell lines. The common occurrences of bridges and hypoaneuploidy reported herein are congruent with the hypothesis that the BFB cycles accumulate in vitro, but evidence for the fusion of non- homologous BFB chromatids during culture remains more cir- cumstantial. The observation of a few ring chromosomes suggests that fusion events do occur during cotton cell culture.

Improper replication of heterochromatic segments, whether centromeric and (or) telomeric, could lead to numerous types of cytogenetic abnormalities (McCoy and Phillips 1982; Murata and Orton 1984), including the formation of BFB cycles and a diverse array of secondary cytogenetic aberra- tions. All resultant cell lines would be deficient for at least some chromatin (Johnson et al. 1987) and some expectedly would be deficient in chromosome number, although not nec- essarily deficient for whole chromosomes (Murata and Orton 1984; Stelly et al. 1989). The cytogenetic, genetic, and pheno- typic arrays of subsequently formed somaclones could be diverse.

In summary, we feel that strong cytogenetic parallelisms were evident between results obtained with cotton somaclones and cotton cell cultures. The results do no prove but are con- gruent with the proposed involvement of BFB cycles during cell culture of G. hirsutum in vitro and the formation of tertiary monosomes observed among somaclones. If cytogenetic aber- rations can b.e reduced in vitro, it may be possible to reduce levels of somaclonal variation in cotton.

Acknowledgements bined with phenotypic (stelly et al. 1985, 1989; Trolinder and R. ~i thanks D ~ . W. ~l~~~~ for his instruction and Goodin 1987) and c~togenetic On G. hirsutum use of tissue culture facilities, and Dr. Stuart Lyda and Mr. somaclones, the present results suggest that Jesus C. Mabellos for occasionally supply callus cultures that ties are prevalent in G. hirsutum cultures and lead to high fre- were usd for the development of cytological techniques. The quencies of h ~ ~ o a ~ ~ u ~ ~ o ~ ~ ~ in culture and regeneFn!s. research was made possible by support from the Food and At least some of the extreme phenotypic variation and sterility ~ ~ r i ~ ~ l ~ ~ ~ ~ organization (CpR/85/()49/ 310 1 12). among somaclones must result from the in vitro cytogenetic instability.

Murata and Orton (1984) observed that in celery cultures, 17 % of the cells formed anaphase bridges, and 44 % possessed one or more dicentric or multicentric chromosomes. Their overall results indicated the BFB cycles and chromosome fus- sions may generate many of the cytogenetic abnormalities that arise during culture. In the present study 'Coker 3 12' cotton cultures exhibited a high cellular frequency (3 1.2 %) of bridges at anaphase and telophase and a moderate cellular frequency (8.75 %) of lagging chromosomes and fragments. We have not seen reports of such a high frequency of bridges in cultures of other crop plants. To explain the frequent occurrence of ter- tiary monosomy among cotton somaclones it has been hypoth- esized (Stelly et al. 1989) that chromatid BFB cycles can accumulate per cell line during cotton tissue culture, and that two or more affected chromatids may fuse to form a dicentric chromatid and (or) chromosome, which might function directly as a tertiary monosome with monocentric activity or undergo further loss of intercentromeric chromatin to yield a function- ally monocentric tertiary monosome. The observation of as many as six bridges in a 'Coker 312' anaphase cell suggests

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FIG. 3. Late anaphase and early telophase mitotic stages in cells from callus-derived suspension cultures of cotton (Gossypium hirsutum L.). (A) 'Coker 312', two bridges. (B) 'Coker 3 12', four bridges. (C) 'Coker 3 12', six bridges. (D) 'Coker 3 12', chromatin body at center of very fine bridge (not visible in photograph). (E) 'Coker 315', unequal mitotic products with isolated chromosome. (F) 'Coker 315', isolated chromosome.

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