Root tips of Vicia faba for the study of the induction of chromosomal aberrations

Mutation Research, 31 (1975) 4Ol-412 © Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands

4Ol

ROOT TIPS OF VICIA FABA FOR T H E STUDY OF T H E INDUCTION OF CHROMOSOMAL ABERRATIONS

t3. A. KIHLMAN Department of Genetics and Plant Breeding, Agricultural College, S-75o o7 Uppsala (Sweden) (Received May I2th, 1975) (Accepted June 4th, 1975)

INTRODUCTION

One of the oldest, simplest and least expensive methods for studying the induction of chromosomal aberrations utilizes plant root tips as experimental material. The merits of this material were already realized by radiobiologists in the I93O'S. The root tip technique is particularly well suited for the study of chemically induced aberrations. However, the method is no exception to the rule that reliable and valuable results can only be obtained when the experiments are correctly and carefully carried out. This requires a good knowledge of the material and its reactions towards different types of treatment.

THE MATERIAL

Root tips from several plant species have been used for the study of induced chromosomal aberrations. However, most common as experimental material are probably root tips of Vicia faba (horse bean, broad bean, field bean) or of various Allium species (e.g. the common onion, Allium cepa, the tree onion, Allium proliferum, and the Welsh leek Alliumfistulosum). Because of their low chromosome numbers and large chromosomes, all these are favourable cytological materials, but they also have the advantage of being available all the year round and easily handled and cultivated. In this paper only one material will be described, i.e. root tips of the field bean, Vicia faba, var. minor. For information about the cultivation and handling of Allium root tips, the reader may consult ref. 2. Vicia faba is a popular material which has been used extensively, not only in cytological, but also in physiological and radiobiological studies. An additional advantage, of course, is that its physiological properties and reactions towards external agents are so well known. As shown in Fig. I, a root tip cell of Viciafaba contains 5 pairs of nearly equally long chromosomes with sub-terminal centromeres (S-chromosomes) and one pair with median centromere (M-chromosome), the diploid chromosome number (2n) thus being Abbreviations: maleic hydrazide, 1.2-dihydro-3.6-pyridazinedione ; tepa, Tris(i-aziridinyl)-phosphine oxide; thiotepa, Tris(I aziridinyl)-phosphine sulphide.

402

B.A. KIHLMAN

Fig. i. The chromosome complement of the field bean, Vicia faba, var minor (2n = t:). lrron~ I¢IHLMANL(By courtesy of Plenum Press, New York.) I2. The M-chromosome contains a secondary (nucleolar) constriction which separates a large satellite from the rest of the chromosome. The ratio of the length of the Mchromosome to the m e a n length of the S-chromosomes is approx. ~.3 : I. If chemically induced aberrations were r a n d o m l y d i s t r i b u t e d between the chromosomes, 2. 3 times as m a n y aberrations should occur in S- as in M-chromosomes. However, since aberrations induced b y chemical substances are seldom r a n d o m l y distributed, the S/M a b e r r a t i o n ratio usually diverges considerably from 2.3"L

Fig. 2. Chromatid exchange localized in the nucleolar constriction after treatment with 8-ethoxycaffeine. From KIHLMAN1. (13y courtesy of Prentice-Hall, Englewood Cliffs.)

THE ROOT TIP TECHNIQUE

403

After treatment with certain chemicals, chromosomal aberrations m a y be localized almost exclusively in some particular region of one of the chromosome pairs t (Fig. 2). At 20 ° the mean duration of the mitotic cycle in lateral roots of the field bean has been estimated to be 19. 7 h. The interphase time was 17. 7 h, 8.1 of which were spent in S (the period of D N A synthesis), 4.1 in G1 (the period between telophase and S), and 5.5 in G~ (the period between S and prophase). Mitosis itself lasted 2 h. METHOD FOR GROWING ROOTS FROM SEEDS

Soaking Seeds are soaked for 6-12 h in tap water. Before soaking, seeds m a y be disinfected b y short immersion (3-1o rain) in a decanted 5% calcium hypochlorite solution. However, as a rule, disinfection is not necessary.

Germination After soaking, the seeds are allowed to germinate in moist Vermiculite or in Perlite (Deutsch. Perlite AB, Dortmund) for 4 days at 20 °. At the end of this 4-day period, the seedlings have grown 3-5 cm long p r i m a r y roots and are ready to be transferred to a water tank. However, before this is done, the seed coat should be removed and the shoot cut off. If lateral roots are to be used in the experiment, the tip of the p r i m a r y root should be cut off as well, since the removal of the primary root m e d s t e m stimulates the growth of the lateral roots.

Growth period in water The water in the t a n k should be fresh, well aerated and of constant temperature

(e.g. 20 °) during the growth period. These requirements can usually be met b y growing the roots in thermo-regulated, running tap water. When tap water cannot be used (for instance when it is too heavily chlorinated), Hoaglands salt solution is recommended. This solution has the following composition: 0.005 M KNO~, 0.005 M Ca(N03)~, 1201 100

~ 8o

~_6o m ~ 40

20

8

12 16 20 24 28 32 Time between treatment and fixation, hours

36

40

4/,

Fig. 3. Frequencies of chromosomal aberrations obtained at various times after treatments with 8-ethoxycaffeine and tepa. A, i h IO-2 M8-ethoxycaffeine, 20°; striped area indicates the proportion of "sub-chromatid" exchanges. B, i 3/4 h zo-~ M8-ethoxycaffeine, z7 °. C, 2 h 7-5" lO'-4M tepa.

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t~. A. KIHLMAN

o.oo 5 M MgSO4, o.ooi M KH2PO4, and o.5% iron tartrate (i nil/l). Primary roots can be used after 241l in the water tank. To obtain lateral roots of suitable length (I 2 cm), the seedlings usually have to be grown for 4 days. TREATMENT OF ROOT TIPS WITH CHEMICALS

When chemical substances are being tested for their chromosome-breaking activity, it should be realized that the effect is dependent not only on the structure and dose of the chemical, but also on factors such as the stage of the cell cycle at the time of treatment and the pH, temperature and oxygen tension of the treatment solution. Thus, for instance, there are substances in plant materials that act mainly or

A

B

1

Fig. 4. " S u b - c h r o m a t i d " (A) and c h r o m a t i d (B) exchanges in m e t a p h a s e after exposure of cells to 8-ethoxycaffeine during p r o p h a s e and late interphase, respectively. F r o m KIHLMAN AXD KRO.NB O R G a.

405

THE ROOT TIP TECHNIQUE

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Fig. 5. C h r o m a t i d - t y p e a b e r r a t i o n s i n d u c e d b y t h e t r i f u n c t i o n a l a l k y l a t i n g a g e n t tepa. A, C h r o m atid e x c h a n g e (from STURELIDS); B, c h r o m a t i d b r e a k a n d i s o c h r o m a t i d b r e a k (arrows) w i t h p r o x i m a l a n d distal sister union.

exclusively during late interphase and mitosis. Methylated oxypurines, such as caffeine, 8-methoxycaffeine, 8-ethoxycaffeine, and 1,3,7,9-tetramethyluric acid are substances of this type 1. These agents produce "sub-chromatid" exchanges in prophase and chromatid aberrations in G~, but very few, if any, aberrations at other stages of the cell cycle (Figs. 3A, B; 4 A, B). Other substances appear to be active during all stages of the cell cycle, but since the formation of the aberrations is dependent on TABLE I T Y P E S AND FREQUENCIES OF CHROMOSOMAL ABERRATIONS OBTAINED AT 5 AND 18 h AFTER I - h TREATMENTS WITH BLEOMYCIN.

Concentration of Bleomycin (mg[l)

2

2. 5

Time Abnormal between metaphases treatment (%) and fixation (h)

Aberrations per Ioo cells Breaks Exchanges Chrom- Iso"SubChromatid chrom- chrom- arid atid arid"

Chromosome

5

43

4

4

58

12

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18

15

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5

3

Total aberrations per IOO cells 78 19

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B. 3_. KIHLMAN

D N A synthesis, only cells exposed during or before S contain a b e r r a t i o n s in the first mitosis after t r e a t m e n t . Such S - d e p e n d e n t effects are p r o d u c e d b y a l k y l a t i n g agents (Fig. 3C). T h e a b e r r a t i o n s p r o d u c e d b y these agents are exclusively of t h e c h r o m a t i d t y p e (Fig. 5A, B). F i n a l l y , there are substances such as t h e antibiotics P h l e o m y c i n a n d B l e o m y c i n t h a t are active d u r i n g all stages of the cell cycle, a p p a r e n t l y independ e n t l y of D N A synthesis, a n d t h a t produce"sub-chromatid-", c h r o m a t i d - , a n d chromos o m e - t y p e a b e r r a t i o n s (Table I). The effect of substances with acid or basic p r o p e r t i e s is influenced b y the p H of t h e t r e a t m e n t solution. Thus, t h e herbicide maleic h y d r a z i d e has a 6-fold stronger effect at p H 4.7 t h a n at p H 7-3 (Fig. 6). O O

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TemperQture, °C

Fig. 6. The influence of tile pH of the treatment solution on the frequency of aberrations obtained after treatments with the herbicide maleic hydrazide, l:rom KIHLMANL(By courtesy of Plenum Press, New York.) Fig. 7- The influence of treatment temperature oil the frequency of aberrations obtained after treatments with 8-methoxycaffeine. From I~IHLMANAND I ~ R O N B O R G a. Most chemical substances are more active at high t h a n at low t e m p e r a t u r e s . M e t h y l a t e d o x y p u r i n e s are exceptions to tiffs rule. These substances a p p e a r to be most active at t e m p e r a t u r e s b e t w e e n IO ° a n d I5 °, a n d p r a c t i c a l l y inactive at t e m p e r a t u r e s a b o v e 25 ° (Fig. 7). The f r e q u e n c y of c h r o m o s o m a l a b e r r a t i o n s p r o d u c e d b y chemical substances m a y also be d e p e n d e n t on t h e o x y g e n tension during t r e a t m e n t . As a rule, a reduction of the o x y g e n tension results in a lower f r e q u e n c y of a b e r r a t i o n s (Fig. 8).

407

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IO0/O

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Concentration of oxygen in the gas phase (%)

Fig. 8. The influence of oxygen concentration on the frequencies of aberrations obtained after treatments with potassium cyanide (A), methylphenylnitrosamine (B), 8-ethoxycaffeine (C), and maleic hydrazide (D). From KIHLMANx. (By courtesy of Prentice-Hall, Englewood Cliffs.)

All these modifying factors should be considered when the experiment is planned. I should like to suggest the following procedure.

Preparation and volume of treatment solution. Size of experiment Freshly prepared solutions should always be used, since aqueous solutions of chemical substances are often unstable. For the same reasons the solutions should not be exposed to strong light or high temperatures. If the substance does not dissolve easily in cold water, the water should be heated to the required temperature before the substance is added. As soon as the substance has dissolved, the solution should be chilled to room temperature. The treatments may be carried out in vials or tubes containing at least 25 ml of solution. Primary roots of three seedlings or lateral roots of one seedling can be treated in such vials. The number of seedlings to be used per treatment and fixation time depends on the type of root. In experiments with lateral roots, at least two seedlings should be used and in experiments with primary roots, at least six.

Treatment conditions Treatment as well as recovery should take place in the dark. Well aerated, buffered solutions should be used and the temperature should be kept constant, since it is important that the treatments are carried out under controlled conditions.

Concentration of the substance to be tested The chemical substances should be tested over a wide range of concentrations.

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B . A . KIHLMAN

The upper limit of concentration is set by the water solubility of the substance as well as by its toxicity. The lethal concentration, if any, is of course dependent on the duration of the treatment. Plainly, there is also an upper limit of concentration worth testing. If the purpose is to find out whether the substance is a genetic risk, there is little point in testing concentrations more than Ioo times higher than those likely to occur in the environment of man (after allowing for concentration along food chains,

etc.). Duration of the treatment period It is advisable to keep the period of treatment as short as possible. If the treatment period is short, it is easier to keep the treatment conditions constant. It is also easier to determine the stage(s) of the mitotic cycle during which the aberrations aTe produced. However, since some substances are active only when given over an extended period, 24-11 treatments should be included in the test. It should then be borne in mind that cells fixed after such long periods of treatment represent a very heterogeneous population with respect to the stage of the cell cycle at the tinle of treatment.

Fixation time Since different chemical substances may produce aberrations at different stages of the cell cycle and affect the duration of the mitotic cycle differently, several fixation times should be used. When aberrations are produced in G2 or mitosis, they appear in metaphase-anaphase from a few minutes to about 8 h after the beginning of the treatment. Methylated oxypurines produce this type of effect (Fig. 3 A, B). On the other hand, when roots are exposed to alkylating agents, tile appearance of aberrations in metaphase is delayed and the peak frequency of aberrations m a y not be obtained until the second mitosis after treatment, i.e. at about 4 ° h after the beginning of treatment (Fig. 3C). It is, therefore, advisable that the test should include at least one experiment in which roots are fixed at short intervals and over a period long enough to cover at least one, and preferably two, whole mitotic cycles.

Colchicine treatment When aberrations are to be scored in metaphase, the roots are, as a rule, treated with colchicine before fixation. Colchicine prevents the formation of a functional mitotic spindle and thereby the arrangement of chromosomes on the metaphase plate. Furthermore, the splitting of the chromosomes into daughter chromosomes is delayed by colchicine, and, as a result, there is an accumulation of cells at a stage (c-metaphase) corresponding to metaphase in untreated cells. The analysis of the aberations is much facilitated by these effects. A suitable treatment is to expose the roots for 2 3 h at 19-2o ° to an aerated solution containing o.o2-o.o5°(, colchicine. FIXATION AND STAINING OF ROOT TIPS

Fixatio~ Roots are conveniently fixed in a mixture containing 3 parts of IOO% alcohol and • part of glacial acetic acid. The alcohol can be either ethanol or methanol, the latter usually giving better results. The fixative should be freshly prepared and cool

THE ROOT TIP TECHNIQUE

409

(4-1o°). Although fixation is completed within 15-2o min, the preparation of permanent slides is facilitated b y leaving the roots in the fixative overnight at 4 °. If kept in a freezer, roots can be stored in the fixative for several days, or even weeks.

Staining and preparation of slides For preparation of slides, the Feulgen squash technique is recommended. A description of this well-known technique m a y seem superfluous but is nevertheless given here. From the fixative the roots are transferred to distilled water and left there for a few minutes. They are then hydrolysed in I M hydrochloric acid (HC1) at 60 ° for 6- 7 rain. After hydrolysis, the roots are transferred to leukobasic fuchsin, i.e., the Feulgen stain. Leucobasic fuchsin is prepared in the following way. One gram basic fuchsin (a suitable stain is "Diamantfuchsin ffir die Bakteriologie", Art. 1358, E. Merck AG, Darmstadt) is dissolved in 200 ml of distilled water that has been heated to boiling point. After the solution has been cooled to 5 o°, it is filtered into a dark bottle. To the filtered solution is added 20 ml of I M HC1 and, when the temperature of the solution is 25 ° or less, 1.2 g potassium pyrosulphite (potassium metabisulphite), K2S~O5. The solution is allowed to bleach at room temperature for 24 h in the dark. The bottle should be tightly stoppered. The decolorized stain is stored in the dark at 4 °. When staining is complete, which at 20 ° takes 45-60 min, the roots are transferred onto clean slides. The number of roots to be used for each slide depends on the type of root. The meristem from one primary root or three lateral roots usually gives a suitable number of cells. On the slide, the dark-stained tips are separated from the rest of the roots, crushed in a drop of 45% acetic acid with the flat end of an aluminium rod and squashed under a cover slip. The pressure is applied under several thicknesses of blotting paper. Sideways movements of cover slip must be avoided. Preparations are made permanent according to the dry-ice procedure. The slides are placed on a block of dry ice and left there until they are thoroughly frozen which takes a couple of minutes. The cover slip is then popped off b y slipping a scalpel under one of its corners. The material sticks to the slide, which is quickly passed to lOO% ethanol (two changes). From the second change of ethanol the preparations can be mounted in Euparal, using a clean cover slip. If Canada balsam is used as a mounting medium, the slide is passed from ethanol to xylene (two changes) before mounting. SCORING OF SLIDES

Chromosomal aberrations m a y be scored both in anaphase and in metaphase. However, scoring at metaphase is usually preferable, since only at this stage is it possible to distinguish the various types of aberration and to make a detailed analysis of the effect. As a rule, anaphase scoring should be used only in preliminary experiments to find out whether a chemical is active. The more rapid and simple anaphase method m a y also be preferable when the purpose of the experiment is to obtain an approximate idea of the concentrations which should be used or of the stage of the mitotic cycle during which the substance is most active. When anaphases are scored, the effect can usually be given only as the percentage of abnormal anaphases or as fragments and bridges per (lOO) anaphase(s). However, there is one type of aberration that can be scored more easily at anaphase than at metaphase, and that is the so-called

410

1~. A. KIHLMAN

Fig. 9. Side-arm bridges at anaphase after treatment with 8-ethoxycaffeine during early prophase.

"sub-chromatid" exchange (Fig. 4A). During anaphase such exchanges give rise to characteristic side-ann bridges or "pseudochiasmata" (Fig. 9). Metaphase scoring makes it possible to distinguish not only between "subchromatid-", chromatid- and chromosonm-type aberrations, but also between the various types of breaks and exchanges that are produced by chemical substances. These aberrations have been described and schematically and photomicrographically illustrated in an earlier publication". A "sub-chromatid" exchange is shown in Fig. 4A, chromatid exchanges in Figs 2, 4 B and 5A, whereas Fig. 5B shows one chromatid break and one isochronmtid break with sister chromatid union. It is recommended that slides are masked and coded before analysis. The number of cells that should be analysed for each treatment and fixation time depends on the magnitude of the effect ; the lower the frequency of aberrations, the more cells should be analysed. When the treatment produces more than 3o aberrations per IOO cells, it is usually sufficient to analyse lOO-2OO cells. The cells of untreated lateral roots contain less than IC}i~aberrations. Therefore, any marked increase above r%, based on an analysis of 3oo cells or more, indicates that the chemical substance in question produces chromosonml aberrations. CONCLUSIONS

Advantages and disadvantages of Vicia root tips as a test material Advantages. The material is easily available all the year round ; it is inexpensive and easy to grow and handle. The method does not require sterile conditions or any expensive material or equipment. The root meristem contains a high proportion of cells in mitosis. The chromosome number is low (2n = 12) and tile chromosome very large, which makes scoring easy and accurate. An idea of the size of the Vicia chromosomes may be obtained by

THE ROOT TIP TECHNIQUE

411

t

Fig. I o. H i g h f r e q u e n c y of sister c h r o m a t i d e x c h a n g e s o b t a i n e d after t r e a t m e n t s w i t h t h i o t e p a a n d d e m o n s t r a t e d b y a modified F P G t e c h n i q u e 4.

comparing the DNA content and chromosome number of a Vicia cell with those of Chinese hamster or human cells, two other materials commonly used for studying the production of chromosomal aberrations. A diploid root tip cell of Vicia contains about 46 pg of DNA, which is distributed among 12 chromosomes, a Chinese hamster cell 8.3 pg of DNA distributed among 22 chromosomes, and a human cell 7.3 Pg of DNA distributed among 46 chromosomes. The size of the chromosomes also makes Vicia root tips a favourable material for the study of the effects of chemical substances on the frequency of sister chromatid exchanges 4. This phenomenon is of interest in connection with chemical mutagenesis, since like chromosomal aberrations, it appears to be an indication of damage to the genetic material. A further advantage of root tips as a test material is that they can be exposed to known concentrations of chemical substances under controlled environmental conditions. Metabolic disturbances can be measured by biocbemical methods or by autoradiography. Finally, in contrast with artificial systems such as animal and plant cells cultured in vitro, root-tip cells have a very low spontaneous aberration frequency and a stable chromosome number. They also react more normally to treatment. Cells in tissue culture are very sensitive to changes in their environment and may react by fragmentation of chromosomes to treatments that have no effect on cells grown under normal conditions in vivo. Disadvantages. The cell wall constitutes an obstacle to both biochemical and cytological studies on plant root tips. Thus, for squash preparations to be made and good separation of the cells in the root to be obtained, the pectic substances in the middle lamella of the cell wall have to be dissolved by hydrolysis with acids or enzymes. These procedures may change the morphology of the chromosomes and the

4 I2

B. A. KIHLMAN

c o m p o s i t i o n of t h e i r c h e m i c a l c o n s t i t u e n t s , t h e r e b y m a k i n g c e r t a i n t y p e s of c y t o l o g i cal a n a l y s i s (e.g. a n a l y s e s of t h e effects of c h e m i c a l s on t h e f r e q u e n c y of sister c h r o m a t i d e x c h a n g e s 4) difficult, if n o t i m p o s s i b l e . A n o t h e r d i s a d v a n t a g e of t h e m a t e r i a l is t h a t t h e r o o t t i p e o n t a i n s d i f f e r e n t t y p e s of cells w h i c h h a v e m i t o t i c cycles of d i f f e r e n t d u r a t i o n and, p e r h a p s , different sensitivities to chemical treatment. T h e i m p o s s i b i l i t y (|f o b t a i n i n g m o r e t h a n a v e r y m o d e r a t e s v n e h r o n v of t h e cell d i v i s i o n s in t h e r o o t m e r i s t e m is a f u r t h e r d i s a d v a n t a g e . F i n a l l y , as a m e t h o d for d e t e c t i n g c h e m i c a l s u b s t a n c e s w h i c h m a y be a g e n e t i c risk t o m a n , t h e r o o t t i p t e c h n i q u e suffers f r o m t h e serious d i s a d v a n t a g e of n o t t a k i n g i n t o a c c o u n t m a m m a l i a n m e t a b o l i s m . T h e r e f o r e , for t h i s p a r t i c u l a r p u r p o s e it should be u s e d o n l y in c o n j u n c t i o n with, a n d as a e o m p l e m e n t to, t e s t s w i t h m a m m a l i a n s y s t e m s iTz r i v e . R l,iFE l{ EN C b-% 1 KIHLMAN, F,. A., ..tclionx ~/ Chemicals on D i w d i n g Cells, Prentice-Hall, l,;nglcwood Cliffs, N.J..

[966, pp. 20o. 2 K I H L M A N , 1~. :\., Root tips for studying the effects of chemicals on chromosomes, in A. ItOt.LAENDt~:R ( e d . ) , Chelnical 3IutageT2s, Vol. 2, Plenum, New York, ~97 I, pp. 489 5 t43 KIHLMAN, I~. A. AND D. I~RON|3ORG, Catteine, caffeine derix ati\'es and c h r o m o s o m a l abcr|-atio|ts,

V. The influence of temperature and concentration on the induced aberration frequent;" in l'iciafaba, Hcreditas, 71 (|972 ) lot l,S. 4 KIHLMAN, B. A., AND I). KRONBO|¢(;,Sister chromatid exchltllges in Iicia /kcba, I. I)emonstration by a modified fluorescent plus (;iemsa (I:.I)G) technique, Chromosoma, 5| (1975) ~ 1o 5 STURELID, S., ('hromosome-breakinv capacity of tepa and analogues in I'icia /aba and Chinese h a m s t e r cells, Hercditas, ()S ( I(}71 ) 255 27(I.