A S T U D Y O N T H E C O M P A R A T I V E E F F E C T O F C H E M I C A L S O N C H R O M O S O M E ' S O F R O O T S ,

P O L L E N M O T H E R C E L L S A N D P O L L E N G R A I N S

BY ARUN KUMAR S ~ M A AND SANTOSH KUMAR SARKAR (Cytogenetics Laboratory, Botany Department, Calcutta University, Calcutta-19)

Originally Received August 22, 1956

Received in revised form March 16, 1957 (Communicated by Dr. I. Banerji, F.A.SC.)

INTRODUCTION

EXTENSIVE investigations have been carried out on chromosome breakages induced by chemicals (Levan and Tjio, 1948; Carr, 1950, etc.). Levan who has contributed much to our knowledge in this direction considers their action to be sub-narcotic in character. Apart from their practical utility, sllch studies are likely to throw some light on fundamental problems also. The reaction of different chromosome segments to a particular chemical gives an indication of the differences in the same chromosome. Localised breakages occurring in the region of the secondary constrictions in Vicia faba are considered to signify that they differ from the other chromosomal regions.

In spite of the large amount of work on induction of chromosome breakages by chemicals, little attention has been paid to the response of the chromosomes from the germinal and somatic tissues of a plant to the same chemical. It is known that a good fixative for the root tips, e.g., Lewitsky's fluid, is often unsuitable for a study of the meiotic stages. Mitotically dividing cells from different tissues show often differences in response to the same fixative. Such records would imply that there is considerable varia- tion in the chemical make-up of the chromosomes themselves.

Mitotic chromosomes from soma have been the favourite material for an evaluation of the effects induced by various chemicals. Pollen grains have not interested investigators in this line, nor has there been any attempt at comparison of the effect of the same chemical on somatic and meioitc cells. Their differential responses, if any, would give an indication of the qualitative and quantitative differences in the make-up of the chromosomes as also the suitability or otherwise of particular types of cells for such investigations. 288

Effect of Chemicals on Chromosomes of Roots, etc. 289

The present investigation was undertaken to evaluate the effect of three different chemicals on mitotic and meiotic cells of Nothoscordum fragrans Kunth.

MATERIALS AND METHODS 1. Materials

The species utilized in the present investigation is Nothoscordum fra- grans Kunth. It was grown in sawdust and garden soil mixture ( l : 1) in flower pots and also in soil at the University College Compound at Bally- gunge, Calcutta.

Root-tips, flower-buds and mature pollen grains of this species were utilized for the investigation.

2. Chemicals studied for their comparative effects on different mitotically and meiotically dividing cells

(a) Aesculine,

(b) Paradichlorobenzene, and

(c) Resorcinol.

Solutions were prepared in distilled water. Aesculine and Paradichloro- benzene were used as saturated solutions. Resorcinol was applied in 0.1 tool. solution.

3. Method of application of chemicals

(a) In root-tips.--The root-tips were treated separately in different chemical solutions at a temperature of 10-14 ° C. and the period of treatment was varied in each experiment.

(b) For the study of Meiosis in P.M.C.'s in flower-buds and in mature flowers for pollen mitosis.--O) By injection: The solution (1 c.c.) was taken in a glass syringe and injected into the inflorescence axis. It was noted that more than 1 c.c. of the solution could not be injected.

(ii) By dipping the inflorescence tip in the solution.--The tip of the in- florescence while still attached to the plant was kept immersed for differing

.periods in a solution of the chemical in a petri dish either at the room tem- perature (26 ° C.) or at slightly lower temperatures ranging from 10-14 ° C.

In addition to this, experiments were also carried out in which inflores- cence tips with buds and mature flowers were treated after removal at a temperature of 10-14 ° C. for varying periods in the solution.

290 ARIm KUMAR SHARMA AND SANTOSH KUMAR SARKAR

4. Method of staining (a) Root-tips.--After treatment, the root-tips were fixed and stained in

Aceto orcein (2%): Hydrochloric acid (N) mixture (9: 1) by slight heating and smeared in Aceto orcein solution (1%) to study the effect of different chemicals used.

(b) Flower-buds and mature flowers.--After treatment, the anthers of suitable size were taken and smeared in Aceto carmine (1%) to study the effect of the chemical on meiotic chromosomes.

In order to evaluate the effect of the chemical on chromosomes of pollen grains, anthers of suitable size were smeared and fixed in Nawaschin's fluid as usual. Staining was carried out with the Crystal Violet technique.

Temporary smear preparations of dividing pollen grains with Aceto- carmine (1%) were also used.

All the diagrams were drawn with a x 12 eyepiece and an 1.3 apo- chromatic objective with a condenser 1.4 N.A. at a table magnification of approximately, x 2,000.

OBSERVATIOIgS

Effect on mitotically dividing cells (a) On root-tips.IIn the smear preparations, divisional figures and

specially the metaphase and anaphase stages were carefully examined to study the effect of the chemical on the chromosomes. Large number of dividing cells were observed, thereby indicating no antimitotic action at the initial stage. Considerable erosion and fragmentation of chromosomes occurring at random were noted both in metaphase and in anaphase. In comparatively lower concentrations, the number of fragments was found to be two to three and in higher concentrations the number was six to ten. Cells with a higher number of fragments could obviously divide no further. It was observed that comparatively longer chromosomes were fragmented more than the smaller ones (Plate XXX, Fig. 1).

(b) On pollen grains.--After treatment, pollen grain chromosomes were studied. Diplochromosome formation was detected, but in no case was fragmentation of chromosomes observed even after prolonged treatment (Plate XXX, Fig. 2).

Effect on meiotic cells After chemical treatment of meiotic cells, irregularities in division,

such as lagging, non-disjunction and stickiness were detected, but in no case

Effect of Chemicals on Chromosomes of Roots, etc. 291

was fragmentation of chromosomes observed even after prolonged treat- ment (Text-Figs. 1 and 2).

_. < Z > ." __Z~"- 0 o

TtxT-FIGs. 1 and 2. Meiotic plates of N. fragrans Kunth. showing non-disjunction and agging of chromosomes after prolonged (3 hours) treatment in Resorcinol (0.1 ml.).

The results obtained are summarised in Tables I and II.

TABLE I

Effect noted after short period of treatment

Root-tips Flower-buds

Chemicals Time of No. of frag- used treat- ments in Meiotic cells Pollen grains

ment Meta- Ana- phase phase

Aesculine 1 hr. 2-3 1-3 Only stickiness of chro- No irregularity in mosomes noted chromosomes

noted

Paradichlo- 3½ hrs. 1-2 1-2 Do. Do. robenzene

Resorcinol 1 hr. 2-3 2-3 Do. Do.

From the tables it is apparent that root-tip chromosomes undergo fragmentation, whereas pollen grain chromosomes do not show any frag- ments. Meiotic cells too do not reveal the presence of fragments. By increasing the period of treatment, the number of fragments goes on increasing in root-tip nuclei, but still pollen grain chromosomes do not undergo any fragmentation.

DISCUSSION

It has been emphasised in the text that not all the mitotic cells behave in a similar way. Their different behaviour is apparent even after fixation

292 ARUN KUMAR SHARMA AND SANTOSI-I KUMAR SARKAR

TABLE II

Effect noted after prolonged treatment

Root-tips Flower-buds

Chemicals Time of No. of frag- used treat- ments in Meiotic cells Pollen grains

ment Meta- Aria- phase phase

Aesculine 3 hrs. 6-10 6-8 Lagging, non-disjunc- tion and stickiness of chromosomes but no fragments noted

No fragmentation, but diplochromo- somes in few cases

Paradichlo- 6 hrs. 5-8 5-8 Do. Do. robenzene

Resorcinol 3 hrs. 7-10 6-10 Do. Do.

in the same chemical. The present investigation brings out clearly the differential response of chromosomes of pollen grains and root-tips to specific chemicals.

With all the chemicals, root-tip chromosomes undergo heavy fragmenta- tion, whereas the mitotically dividing chromosomes of pollen grains remain almost unaffected. It is a positive indication of the differential set-up of the chromosomes of these two types of cells. At the same time erosion and stickiness in root tips are not uncommon. Evidently all these are brought about by a disturbance of the nucleic acid metabolism of the cell. The number of fragments goes on increasing with increase in the period of treatment.

Pollen grain chromosomes rarely show any response. Only in certain cases were diplochromosomes noted. This indicates a slight disturbance in the spindle mechanism and a failure of chromosome separation.

It seems likely that the metabolism of the chromosomes of pollen grains and root-tips, may to some extent be different. The protein and nucleic acid synthesis of the chromosomes may be somewhat different in the two. Such a suggestion does not naturally involve the assumption of fundamental chemical differences in chromosome structure.

Arun Kumar Sharma and Santosh Kumar Sarkar

Proc. Ind. Acad. Sci., B, Vol. XLV, Pl. XXX

FIG. 1.

FIG. 2.

Photomicrograph of somatic metaphase plate of Nothoscordum fragrans Kunth. when treated in Resorclnol (0-1 mol.) for I hour showing fragmentation of chromosomes.

Photomicrograph of pollen grain metaphase of N. fragrans Kunth. showing 10 chromo- somes and no fragment after prolonged (3 hours) treatment in Resorcinol (0- I mol.).

Effect of Chemicals on Chromosomes of Roots, etc. 293

As regards meiotic chromosomes, irregularities not involving fragmenta- tion have been recorded. Such irregularities may arise out of a disbalance of the nucleic acid cycle and a disturbance of the spindle mechanism. The absence of fragmentation in meiotic cells may possibly be due to differences in their metabolic set-up as compared to the mitotic ones as also the physical state of the chromosomes and the bivalents themselves.

SUMMARY

1. A comparative study of the action of Aesculine, Paradichloro- benzene and Resorcinol on the root-tip, pollen grain and pollen mother cell chromosomes of Nothoscordum fragrans Kunth. reveals considerable erosion and fragmentation at meta- and anaphase in root-tips.

2. The division is mitotic in pollen grains but yet prolonged treat- ment resulted only in diplochromosome formation and not chromosome fragmentation. Fragmentation was also not observed in meiotic cells. Chemical treatment produced only irregularities in division such as lagging, non-disjunction and stickiness.

3. The marked differences in response to chemicals between the root- tip cells on the one hand and the pollen grains and P.M.C.'s on the other is suggestive only of differences in chromosome metabolism and not the ultimate structure of the chromosomes.

Carr, J. G.

Levan, A. and Tjio, J.H. ..

REFERENCES

.. "Chemically induced mutations--Biochemical aspects of Genetics," British Biochemical Society Symposium No. 4, 1950.

"Chromosome fragmentation induced by phenols," Hereditas, 1948, 34, 250-52.

.. "Induction of chromosome fragmentation by phenols," Ibid., 1948, 34, 453-84.