GALLIC ACID: ITS IMPORTANCE IN CYTOCHEMICAL STUDIES

by

A. K. SHARMA and DEEPESH DE

Cytogenetics Laboratory, Department of Botany, Calcutta University, Calcutta, India.

(With Plate X)

Received 231h July 1954.

INTRODUCTION

In recent years practically speaking most of the groups of chemi-cals have been demonstrated to possess the chromosome-affecting property (Carr, 1950; D' AMATO) 1950: LEVAN and TJIOJ 1948; LEVAN) 1949). The types of changes that are generally brought about, of course, differ in certain degrees. Importance of such studies are far-reaching, and immense practical scope has been indicated.

The pyrogallol derivative- gallic acid- is one, which has been little investigated from this aspect so far. As its related compounds show well-marked chromosome affecting property, it was thought that an -investigation in this direction might prove fruitful here too. In addition to bringing out its subnarcoti?iing property, a study of its importance in karyotype analysis was thought highly desirable. This forms a part of the programme of work taken up in this laboratory involving rese­arches on fixing property of mutagenic chemicals.

Fortunately, on investigation, as the text would reveal, it has heeu shown to be endowed with the capability of« so-called>> mutagenesis. 'l'he tf'rm «;mutagenic», however, has been used here in a wider sense, not strictly implying gene mutation, but also the gross structural <"hangf's, of chromosomes. The possibility of its use in the study of chromo~ome morphology has further been elucidated. Furthermore, certain interesting facts regarding the manifestation of narcotic and lethal properties have been brought about, indicating the further possi­bilities of research in this aspect.

MATERIALS INVESTIGATED

The materials for the investigation of the present work were obtained from the bulbs of A..lli-um cepa Linn.

180] [Caryologia, Vol. VI, n. 2-3, 1954

GALLIC ACID: ITS IMPOR'l'A.NOE IN OYTOOHEMIOAL STUDIES 181

PREPARATION OF SOLUTION

Gallic acid is sparingly soluble in water. It has the chemical formula CiH4 0 2 (OH) 3 , the molecular weight being 129. 6.45 gms. Chemically pure white crystals of Gallic acid were dissolved in 1000 c. c. and thus 0.05 M solution was obtained. This solution was used as the stock solotion and solutions of different strength were obtained from this solution. Treatment of the stock solution in Hot Bath for about half an hour is necessary for complete dissolution of the crystals.

METHODS FOR DIRECT TREATl\lENTR WITHOUT RECOVERY

Healthy root tips were taken from the bulbs and in no case tips more than an inch long were selected.

The experiments were performed either with tips cut or intact in bulb. The cut ones hardly exceeded half an inch in length. In case of intact treatments in the solution of gallic acid, the bulbs were set as sueh on the mouth of a glass vase containing the solution of desired strength. The wall of the vase was covered with a black carbon paper so as to prevent access of light to the root-tips.

RECOVERY EXPERIMENTS

Several cases of fragmentation and erosion and also of subnar­cotic effects were obtained in the somatic cells at specific treatments in definite concentration for a particular period. The time, concentra­tion of the solution and the temperature bringing about fragmentation were carefully noted and plants were subjected to such conditions before undertaking any recovery experiment.

The bulbs with treated root-tips were transferred to glass vases containing physiological culture solution, viz., the Knop's solution. These glass vases were also covered with black paper. The root-tips were kept in this solution till their complete recovery.

Composition of Knop's solution Ca(N03 ) 2

KH2P04

MgS04

KCl FeP04

Distilled water

))

))

))

))

))

))

3 ~ms. 1 g-m. 1 gm. 1 gm. trace 6000 c.c.

All the chemicals used were chemically pure.

182 SHARMA and DEEPESH

In order to test the period required for complete recovery, the tips were constantly observed at intervals of 24 hours from the initial starting of recovery experiment. On continuous observation as soon as the plant was noted to have completely recovered, the experiment was not further prolonged. In case of necessity of prolonging the recovery experiment for a considerable period, continuous change of Knop's solution was given, in order to avoid any physiological disba.lanee causing disturbance in the metabolism of somatic cells.

TECHNIQUE

Subsequent to treatment or treatment followed by recovery the materials were transferred directly to a mixture of 2% orcein in 4r,% acetic acid and (N) HCl in the proportion of 9:1 respectively. The tube containing the mixture and the inaterial was gently heated over a bunsen flame for 5-10 seconds. If necessary, the process was ~ repeated 3-4 times. The contents were then poured in a watch glass and allowed to settle for a few minutes. After some time, the root­tips were transferred to a drop of 1% orcein in 45% acetic acid on a dry slide. Only the growing tip was taken, the non-meristematic regions being rejected. The materials being sufficiently softened could be smeared by applying unifor:m pressure over the cover glass. For further spreading of the metaphase plates the slide was passed over the flame for a second and again pressed. The process improves the spreading. A filter paper was next put on the slide and with !'luffieient pressure, superficial orcein was carefully wiped off. Slides were then sealed as usual.

Precautions :

1) Over-heating of root-tips in 2% orcein mixture was avoided. This precaution has been found to be absolutely necessary in view of recent findings from our laboratory (SHARMA & RoY, 1954), whereby orcein has been noted to cause fragmentation on heating.

2) After heating, the root-tips were allowed to remain in 2% orcein-NHCl mixture for a minute or two, so that all the tissues might take up stain uniformly. On immediate smearing after heating, the epidermal tissue was found to stain brightly, leaving the inner ones nearly unstained.

GALLIO AOID: ITS IMPORTANCE IN OYTOOHEMIOAL STUDIES 183

3) Uniform pressure throughout the cover glass has been found to be essential.

4) Too much pressure was avoided in order to prevent disruption of cells and irregular scattering of chromosomes.

Photo-micrographs were taken of suitable preparations from tempo­rary mounts generally at a table magnification of X ± 900 approx. imately using U.C.E. compensating eye piece of X 20 and X 10 and 1.3 apochromatic objective with an aplanatic condenser of 1.4 N .A.

MODE OF REACTION OF THE CHEMICAL

It is reported that Oxyquinoline, Coumarin &c. (SHARMA & BHAT'IA­CHARJEEJ 1952; SHARMA & BALJ 1954) can behave as pre-fixatives themsel­ves. when root-tips are treated with a solution of particular strength under rigid time control. So far as this character of Oxyquinoline is con­cerned, gallic acid is parallel to Oxyquinoline as gallic acid also acts as a good pre-fixing agent. As has already been mentioned, no detailed post-fixation becomes necessary before staining of materials treated in gallic acid. The chromosome structure becomes absolutely clear due to differential contraction and swelling of chromosome arms resulting out of hydration and dehydration of chromosome segments. A scatte­ring of the chromosomes is affected by pressure over a background of plasma with increased viscosity.

OBSERVATIONS

TABLE I

Ta.b"te sh.Qtotng concentrations requifi"OO!. to bring about fra.gf!Umtatfon

at different temperatures and period

Condition of Concentration Period Temperature

Treatment

Cut -OOlM 3 hrs. Room temp. Intact .oolM 3 hrs. Cold Cut .005M 15m1ns. Cold Cut . 005 M 2-45 mins . Cold Intact .005M 3 hrs. Room. temp.

184 SHARMA and DEEPESH

Inferences deduced from the Table I : It is apparent that a very low concentration of the solution of the

chemical is necessary for bringing about effects of the subnarcotic level and these concentrations vary from .001 M to .005 M. It is rather interesting to note here that the cut and the intact root-tips behave differently in details. In order to bring about subnarcotic effects cut tips require only 15 minutes, whereas the intact tips need 3 hrs- a longer period, under same concentration and temperature. Thus the in the cut tips the effects is more pronounced than in the intact ones.

So far as the treatment period is concerned, it can be easily inferred from the above table that in higher concentration .005 M, the period of treatment is different in cut and intact tips. But in lower concentration .001 M, the period is independent of the condition of the root-tip­cut or intact.

The last point to be noted is that cold temperature (10°-14° C) is an essential requisite for bringing about frag.mentation of intact tips, but different is the case with the cut ones.

TABLE II

Table showing different degrees ot effect in concentration wMch> il8 known

to be opPimum at different periods

Concentration Period Temperature Effect

.0005M 1% hrs. Cold Spindle mechanism affected, slight stickiness .

. 0005 M 3 hrs. Cold Perfect chromosome structure

.0005 M 4% hrs. Cold Do

Inferences deduced from the Table II: From the above table it is detected that there is a specific concentr­

ation zone of .0005 M below the mutagenic zone of .001 M to .005 M, the treatment in which brings about clarification of chromosome struc­tures in detail. This is an indication of the presence of a concentration zone of mutagenic chemicals whose activity threshold lies below sub­narcotic thr.eshold of Levan.

The karyotypes are brought out distinctly with even minute details being visible. The effect of the chemical on chromosome structure at this concentration is considered to be « optimum )). In case of the other chemicals tested so far, this optimum acthity similarly has got

GALLIO ACID: ITS IMPORTA.KOE IN OYTOOHEJIIOAL STUDIES 185

a. lower and upper threshold. But so far as gallic add is concerned, this zone is extremely limited in its application and the effect is only revealed at a specific concentration of .0005 l\f. In such specific level, the time range is remarkably wide, varying between 11;2 to 4I;2 hour!'. Similar to the concentration the temperature required is extreme!~·

rigid, being strictly within 10°-14° C during the operation.

TABLE III

Results of Recovery Experiments

Concentration Period Temperature Effect

.OOlM :J hrs. treatment fol- 100-140 c Complete recovery lowed by 24 hrs. recovery

.005 M 3 hrs. treatment fol- Do. lowed by recovery of:

1. 24 hrs. Fragments persisted 2. 48 hrs. Fragmentation fre-

quency increased

3. 72 hrs. Fragmentation fre-quency quite high possibly resulting in lethality

Inferences deduced from the Table III : Subnarcotic effect has been brought about in concentrations between

001 M to .005 M. Recovery experiments show that the former concentra­tion is actually subnarcotic in effect, and the fragments produced -gradually disintegrate in subsequent division. But the latter concen­tration, though apparently subnarcotic in effect, gradually brings about lethality as a delayed effect. The delay necessary for lethality is unlike the effect of lethal concentration of Levan where the result is immediate.

DISCUSSION

Levan has classified the chemical effects on chromosomes and other cell structures under the categories of lethal, narcotic and the sub­narcotic ones. The latter two concentrations as he suggests are rever­sible, _so that during the operation, though the cell is intoxicated to some extent, death is never caused. The result is the manifestations of several cytological peculiarities as regards the chromosomes, which

186 SHARMA and DEEPESH

could be noticed after a certain period of recovery. Lethal chemicals, however, cause changes which are naturally irreversible.

A particular chemieal can behave according to him as narcotic, subnarcotic as well as lethal in specific concentration ranges. The threshold zones too, in most of these cases, have been worked out.

In any case the lower threshold concentration bringing about just the subnarcotic effect on the cell structure has been considered as the viRible minimal effect. Below this zone, it is implied from his statements, the cells remain unaffected or at least no visible change in the cell structure is apparent.

It has been emphasized in a series of publications from this laborat­ory (SHARMA & BALJ 1953; SHARMA & BHATTACHARYYA, 1954), that most of the chemicals, if not all, bringing about so-called radio-mimetic effect ca.n safely be applied for the study of chromosome analysis. The eoncentrations required for the elucidation of chromosome structure lie always below the subnarcotic zone of Levan. After treatment for a limited and controlled period in the .required concentration, direct squashing of the root-tips in orcein following hydrolysis, brings about the details of chromosome structure. The plasma becomes highly af­fec·ted, the viscosity being altered. At the same time, considerable ehanges in the chromosome structure appears mainly through physical means. The constriction regions comes out highly clarified due to differential contraction of different chromosome segments. This also leads to the straightening of the chromosome arms, thus facilitating the study of chromosome morphology, specially in long chromosomed ones, where under normal :fixation the arms remain highly foreshortened. The perfection in preparation is further attained by the scattering of the chromosomes caused as a result of pressure over the solidified plasma.

As far as gallic acid is concerned, it has been pointed out that an extremely specific concentration, viz., .0005M is required for the clari:fie­ation of chromosome structure. In this respect the chemical behave~ differently from the others for the same, tried so far. In cases of oxy­quinoline, coumarin etc. concentrations can be varied within a certain limit with the consequent increase or decrease of the time of treatment, for the purpose of karyotype analysis. In their cases, the period of treatment has been recorded as extremely limited, the lowering or in­crease of which yield no effect or drastic results respectively. By the term «drastic», the clumping of chromosomes, fragmentation or pyc­notic appearance of the same is meant. On the other hand, in case of

GALLIO ACID: ITS IMPORTANCE IN CYTOCHEMIOAL STUDIES 187

gallic acid, the concentration remaining fixed, the time period can be varied even within the range of 1 ¥:! hours (3 to 41,6 hours) without causing a,ny ;marked change in appearance. This possibly suggests that the ehemical is unable to accumulate within the cell and as such the prolonged treatment causes no drastic effect whatsoever. Contrary might be the case with coumarin, aesculin and such other chemicals.

Another important fact that has emerged out fro;m the report pre­sented involves fragmentation of chromosomes. It is generally regarded that concentrations bringing about subnarcotic effect do not cause lethality to the plant. On the basis of this assumption fragmentation, tranl-lloeation ete. are generally characterized under reversible changes. It ha~ been brought out here that both concentrations .001 and .005M arf> able to induee such cytological effects. But the behaviour of the cell is different following recoYery experiment!>. Keeping for a period of ~4 hours of the treated bulbs in Knop's solution results in complete recovery of the plant with practically eliminations ·of most of the fragments. 'Vhereas in the other concentration, the same type of reco­very for 24, 48 and 72 hours gradually leads to increase in number of fragments in the cells ultimately eausing lethality. It suggests that irrespective of the fact that fragmentation is a subnarcotic phenomenon, it at the same time may give an indication of the initiation of the lethality of the tiRsue. Lethality, therefore, may not universally be brought about immediately, manifested in peculiarities noted by Levan, but may be delayed in its appearance yielding at its initial stage certain cytological phenomenon, classified under Levan's subnarcotic reactions.

Another point worth discussing about is the result obtained in treatments of excised and intact root-tips. The root-tips treated intact in bulb require more period of treatment for the manifestation of mutagenic effect. Further in room temperature the effect is practically absent. In higher concentrations the reverse is the case with excised ones. Even in low period of treatment, the fragmentation can be brought about. The temperature required is not specific, higher and lower ones both being ineffective. In lower concentration, no doubt, the two types of root-tips behave similarly.

The results may be interpreted if one take_s into consideration the physiological set-up of the excised and intact root-tips. It may be suggested that under intact natural condition all the physiological set up being normally maintained, the roots possess a high degree of resistivity to the chemical treatment. The resistance may go even so

188 SHARMA and DEEPESH

far as to require temperature different from its normal environment, for its break. As such the chemical is only effective under prolong!:'d treatment and at specified temperature. On the other hand, the excised ones being consequently physiologically unbalanced lose their natural resistivity and as such become susceptible to ehemiical treatments. Therefore, extremely short treatment can cause fragmentation. The temperature required too, as such, is not specific. As a treatment in lower concentration of the chemical is extremely limited in its effeet, no difference could be noticed in the behaviour of excised and intact ones.

LI'fERATrRE CITED

1. CARR .J. G., 1950. - Chemically inducerl mutation. Biochemical aspects of f/('1Wfic.~.

(British Biochemical Society, Symposia N. 4) 25-31.

2. D'AMATO F., 1950. - Notes on the chromosome breaks ·inrluc,ed by pure gama.u•uc.

Caryologia, 2: 361-364.

3. LEVAN A., 1949. - The •influence on chromosomes and mitosi.~ of che111icals as stmliictl

by Allium test. Proc. 8th Int. Cong. Genet. - Hereditas Suppl. Vol. 325-337. 4. LEVAN A. & 'i.'JIO .J. H., 1948. - Induction of chromosome fmgmentation by pll~no/.~.

Hereditas, 3.~: 453-489.

5. SHARMA A. K. & BHATTACHARJEJE, DIPTI, l!l-:i2. - Permanent mounts of cht·om0801/lC.~

after B-Oxy quinoline and squashing. Stain Tech., 27: 201-203.

6. SHARMA A. K. & BAL A. K., 1953. - Ooumal'in in chromosome analysis. Stain Tech,

28: 255-257.

7. SHARMA A. K. & BHATrACHARYYA N. K., 1954. - An 'inr:esti{lation on the possiiJilitif's

of the u.~e of phenols i·n chromosome analy.~i.Y. Proc. 41st Ind. Sc. Gong. Seetion VI,

148-149.

8. SHARMA A. K. & MIRA RoY, 1954. - The scope of orceln stainill[l in the Mutl.lf of the

effects of chemicals on chromo.~omc.~. Chromosoma (In Press).

EXPLANATION OF PLATE X.

Fig. 1. -Optimum effect bringing about details of the karyotype in metaphase following

treatment in .0005 M for 3 hrs.

Figs. 2-4.. - Prophase, metaphase and anaphase stages respectiYely showing heavy

fragments following treatment in .005 M for :: hrs.

CARYOLOGIA Vol. VI, Plate X.

A. K. SHARMA and DEEPESH DE.- Gallic acid: its importance in cytochemical studies.

GALLIO ACID: ITS IMPORTANCE IN CYTOCHEMIOAL STUDIES 189

SUMMARY

1. Allium test has been performed with gallic acid as the testing chemical.

2. Both excised and intact tips have been subjected to treatments, the time and concentrations to bring about. fragmentations have been found to be different in certain

aspects in the two. The significance of the behaviour has been discussed. 3. A certain concentration has been worked out to bring about the chromosome

details, thus facilitating the study of chromosome morphology.

4. Evidences brought forward show that fragmentations do not only indicate the subnarcotized conditions of the root, but at the same time the initiation of lethality.

RIASSUNTO

:El stato sperimentato l'acido gallico nella sua azione sui cromosomi delle radici di

Allium. 11 tempo e le concentrazioni per indurre frammentazioni cromosomiche sono

diverse a seconda che le radici siano tagliate o intatte, essendo in quest'ultimo caso piu

resistent! per l'integritA delle condizioni fisiologiche generali. Certe concentrazloni pos­

sono facilitare lo studio della morfologia cromosomica. La frammentazione del cromosomi

e indice non soltanto delle condizioni di subnarcotizzazione delle radici, rna nello stesso

tempo anche dell'inizio di una condizione di letalita.