Histochemie 2, 260--265 (1961)

Cytogenetics Laboratory, Botany Department, Calcutta University, 35 Ballygunj Circular Road, Calcutta 19, India

A COMPARATIVE STUDY OF T H E EFFECT OF RNA AND DNA TREATMENT ON SOMATIC

NUCLEI I N PLANT CELLS

By

A~u~ KuMa~ S ~ M A and ARORANA SHARMA

With 8 Figures in the Text

(Received March 7, 1967)

Introduction The importance of ribonucleic acid (RNA) and desoxyribonucleic acid (DNA)

in cell metabolism is well-known. Polyploidy, one of the major cellular changes, can be induced through spindle break down. A number of chemicals, from colehicine onwards, are at present known (D'A~ATo 1949; Ls.VAN 1949; EmSTI and DVSTIN 1957), which are capable of causing chromosomal doubling through this process. Spindle disturbance as is well known is caused by viscosity change of the plasma.

I t is obvious that cytoplasmic viscosity is dependent, to a considerable extent, on its nucleic acid content. Evidently, in a cell, if a change in the viscosity occurs, the nucleic acid balance is also upset. I f the idea tha t this viscosity balance is controlled to a large extent by the amount of nucleic acid present, is correct, then artificial feeding of the tissue with nucleic acid should conside- rably affect the viscosity balance of the cytoplasm. On this principle, it was thought worthwhile to find out the effects of RNA and DNA feeding on the tissue. This experiment, it was surmised, would also demonstrate the comparative ability of RNA and DNA in disturbing the balance of viscosity in the plasma.

The purpose of including DNA within the scope of this work was two-fold. In addition to the objective mentioned above, another particular aspect of its property needs serious consideration, namely, the reductional separation of chromosomes in the somatic cells. I t has been established, through the works of I-IUSKI~S and others (tIUSKISS 1947, 1948; WILSO~ and CnEXG 1949), that sodium nucleate t reatment can alter the pat tern of chromosome division in the somatic cells. Instead of equational division, reductional separation has been observed in such cases. These experiments imply that the addition of nucleic acid, given in the form of a salt in the plasma, is responsible for the process. I t is not known, however, as to how the cell will behave if these nucleic acids are added to the tissue in a much purer form. No doubt, their high molecular weight stands, to a large extent, against their penetrability. Even then, the minute quantity, tha t would have access to the cell, should be able to bring about changes in the plasma. This assumption is based on the fact tha t even the addition of water from a different source, may often cause distinct changes in cellular vis- cosity (SKA~MA and SEn 1951a). In view of all the above-mentioned facts, it was thought that the effect of RNA and DNA on reductional separation of

Effect of I~NA and DNA on nuclei 261

chromosomes in the somat ic mer i s t emat i c t issue, if any , should be s tudied. On adu l t cells, the effect of ILNA alone in inducing divis ion has a l r eady been r epor t ed b y SttAI~MA and SE~ (1954b).

The p resen t inves t iga t ion , u n d e r t a k e n wi th the above objects , deals wi th the e//ect o/ R N A and D N A on somatic meristematic cells with special reference to the i r influence on somatic reduction and polyploidy.

Materials and Methods For the present work, in order to study the comparative effects of desoxyribonucleic

acid (DNA) and ribonueleic acid (RNA), solutions of different concentrations were prepared in distilled water.

Two sets of experiments were carried out. In one, only the effects of different concen- trations of the acids were observed. In the other, different concentrations of the acid solutions were mixed in different proportions with Knop's solution. The latter was added to allow a healthy growth of the tissue. A control set of experiment was set up in which the roots were allowed to grow in Knop's medium without undergoing any treatment whatsoever.

The experiments were conducted on roots of Allium cepa. Bulbs were taken with healthy young roots and placed at the mouths of jars containing the solutions, so that the roots were completely submerged. At specified intervals, root-tips were removed from the treated bulb, heated for a few seconds in a mixture (9:1) of 2% aceto-orcein solution and normal hydro- chloric acid and squashed in 1% aceto-orcein. Paraffin blocks following Lewitsky's fixation were also prepared for keeping controls.

The slides were observed at a Leitz Ortholux microscope at a visual magnification of X 12O0.

Observations and Discussion

Two sets of exper imen t s were carr ied out, (i) wi th the acid solut ion alone and (ii) wi th a mix tu re of the acid and K n o p ' s solution, in different propor t ions . The resul ts ob ta ined were recorded (Figs. 1- -8) . No effect was no ted wi th in 1 hour t r e a t m e n t . I t m a y be no ted t h a t in the control set, where the rootswere allow- ed to grow in K n o p ' s med ium, no cytological effect was no ted wi th in 1 hour of t r e a tmen t .

One of the chief effects of t r ea t ing a t issue wi th r ibonueleic acid (RNA) is po lyp lo idy . Chromosome doubl ing has been no ted even af ter three hours t rea t - men t wi th 0.75 % R N A solution. This behav iour is m a i n t a i n e d in different sets of exper imen t s even af ter recovery in K n o p ' s solution. I t has been found t h a t even twen ty - four hours t r e a t m e n t in a mix tu re of 0.5 % t~NA and K n o p ' s solut ion is effective in inducing po lyp lo idy . This fact can be t a k e n to i m p l y t h a t reduc t ion in the concent ra t ion of the nucleic acid solut ion does not, in any way, s t and aga ins t the i r po lyplo id is ing effect. A s imilar effect has been no t iced with three hours t r e a t m e n t wi th a mix tu re of 0.75% I~NA and K n o p ' s solution. Ano the r effect caused b y this m ix tu r e is the fo rmat ion of mul t ipo la r configurat ions wi th in the cell. No d o u b t t h e y represent an in t race l lu la r metabol ic disorder. Bu t i t is also p robab le t h a t such mul t ipo la r configurat ions often help the t issue to rever t back to the no rma l s ta te f rom a high po lyp lo id condit ion, as no ted in the case of g a m m e x a n c (S~A~MA and C~AVDKW~I 1960).

F r o m the occurrence of c-mitosis, no ted in several sets of exper iments , i t is ev iden t t h a t po lyp lo idy here is the resul t of spindle b reakdown. As the l a t t e r is the ou tcome of the change in v iscos i ty of the cy toplasm, i t appears t h a t in- corpora t ion of R N A is capable of causing this v iscos i ty change. This resul t

262 Amr~v KUMA~ SHARMA and A~e~a~ra SI~A~A:

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implies, therefore, that v iscos i ty balance in the cytoplasm is dependent to large extent on its nucleic aeid bManee. As R N A forms one of the main eon- st i tuents of the cytoplasm, its incorporation can easi ly lead to an upset of the normal balance.

Effect of RNA and DNA on nuclei 263

The data obtained with DNA tally with those recorded with RNA, with regard to polyploidy. In these experiments as well, polyploidy has been encoun- tered after t rea tment both in low and high concentrations of DNA solution. When used in a mixture with Knop 's solution, similar induction of polypolidy has been observed. DNA too, therefore, may be taken to cause regular disturb- ance of the viscosity balance of the cell. No doubt, its high molecular weight hinders its rapid penetration, but it is clear that the minimal amount, which finds access to the cells, is sufficient to affect the spindle.

A problem arises as to how DNA is capable of disturbing the physical state of the plasma in a systematic way. So far as the chemical constitution of the cytoplasm is concerned, it is well-known tha t RNA is present in significant amount in the cytoplasm, DNA being practically absent in it. In order to confirm this idea, the author performed Brachet 's pyronin-methyl green staining on the material used in this experiment. Following this test, the cytoplasm took intense stain with pyronin and the chromosomes with methyl green, as expected.

As DNA is not a constituent of the cytoplasm, it is difficult to visualise its role in causing viscosity change in the latter. Polyploidy, recorded after DNA treatments, could have been at tr ibuted to a general metabolic disorder if it did not occur regularly in all series of experiments, including the repeats. Such systematic occurrence suggests that certain regular and organized changes are caused in the cytoplasm, which are responsible for this effect. I t may be assumed that the conversion products of DNA may help directly or indirectly the formation of I%NA precursors, which are ult imately responsible for the disturbance in the viscosity balance of the cytoplasm, resulting in spindle breakdown. I t is difficult to visualise the direct and close interrelationship between DNA and RNA specially in view of the recent works carried out on this aspect (B~AcHET 1957).

Another important effect, noted after t reatment with both I~NA and DNA is the reductional separation of chromosomes in the somatic tissue. Such reduction is frequent not only after t reatments with high concentration of the solutions used, but it is also observed with low concentrations and for low periods. This phenomenon is marked even when a mixture of nucleic acid and Knop 's medium is used as the medium of growth.

The induction of reduction through sodium nucleate t reatment by HVSKINS (1. c.) can be interpreted as due to an upset of the nucleic acid balance within the cell. The similar behaviour noted after DNA treatment, in the present series of experiments, indicates tha t even when nucleic acid is applied in a comparatively pure form, the pat tern of nucleic acid division is altered. Nuclear division is distinctly controlled, to a large extent, by a subtle equilibrium, maintained by the two types of nucleic acids within the cell. The change in this equilibrium in favour of DNA may appear to be the reason for this altered pat tern of nuclear division. But, the observed fact that , even with the addition of I%NA, such reduc- tional separation of chromosomes is possible may indicate tha t the shift in balance in favour of I%NA too is capable of changing the course of nuclear division.

I t seems difficult to reconcile such an assumption, apparently arising out of the observed facts, with our knowledge regarding the two types of nuclear division, mitosis and meiosis. Under normal conditions, reductional separation of chromosomes, as is well known, occurs only during the formation of germ cells,

264 ARUN KUMAR SHARMA and ARCKANA SHAR~A:

being strictly controlled by subtle intra- and extraeellular factors. I t is improb- able that such a regulated well-organized subtle process can be caused by a general shift in balance in favour of both I~NA and DNA. In view of the strictly controlled nature of this type of nuclear division, a shift in balance in one direction only should control its manifestation. In view of this, it is likely that only the incorporation of DNA can change the pattern of nuclear division from equational to reductional type. As t~NA also is capable of inducing similar effect, it can be inferred that RNA, after its entrance into the cell, passes through its usual cycle and after an interval a part or whole of it undergoes conversion directly or in all probability, indirectly into DNA precursors, ultimately causing reductional separation of chromosomes. As none of these effects have been noted before treatment for one hour, sufficient time is thus allowed for its gradual and com- plete conversion and assimilation into the cycle.

An analysis of the causal factors of induced polyploidy and reduction, men- tioned above, reveals that while RNA, immediately after its entrance, causes direct change in the viscosity of the cytoplasm leading to c-mitosis, DNA needs ultimately be converted into t~NA precursors to cause the same effect. For reductional separation of chromosomes, the role of the two nucleic acids is just the reverse. The simultaneous occurrence of the two processes can easily be explained by the fact that none of these effects is visible before one hour of treatment. The time, therefore, is sufficient enough for the complete conversion of one indirectly into the other precursors through the normal process of cell cycle within the cell.

Summary The effect of different concentrations of ribonucleie acid (RNA) and desoxy-

ribonucleie acid (DNA) has been studied on meristematie somatic cells of Allium cepa. The principal effects noted with both are polyploidy and reductional separation of chromosomes.

I t has been assumed that RNA causes viscosity change in the plasma, which is responsible for polyploidy. DNA, though entering in a minute quantity due to its high molecular weight, gets ultimately converted into RNA precursors and finally causes the same effect. Somatic reduction is caused by an increase in the DNA content of the plasma and as such, application of this chemical causes reductional separation of chromosomes. RNA, on the other hand, needs gradual conversion into DNA precursors to cause the effect. The concurrent nature of the two processes allows the simultaneous occurrence of polyploidy and somatic reduction after a brief interval following artificial treatment.

References CARLSON, J. G. : Protoplasmic viscosity changes in different regions of the grasshopper neuro-

blast during mitosis. Biol. Bull 90, 109 (1946). D'AMATO, F. : SuFimpiego del gammesano come agente poliploidizzante. Caryologia (Pisa)

l, 209 (1949). EmsTI, O. J., and P. DUSTIN jr. : Colchicine in agriculture, medicine, biology and chemistry.

Ames, Iowa: Iowa State College Press 1957. HUSKINS, C. L. : The subdivision of chromosomes and their multiplication in nondividing

tissues. Possible interpretation in terms of gene structure and gene action. Amer. Naturalist 81, 401--434 (1947).

Effect of I~NA and DNA on nuclei 265

]:IusKINS, C. L. : Segregation and reduction in somatic tissues. I. Initial observations on All ium cepa. J. Hered. 39, 310--325 (1948).

LEVA~, A.: The influence on chromosomes and mitosis by chemicals, as studied by the All ium test. Proc. 8th Intern~t. Congr. Genetics (Hereditas), p. 325--337, 1949.

SItARMA, A. K., and M. CI-IAUDttURI: An aspect of Gammexane effecton on chromosomes. Curr. Sei. ~8, 498--499 (1959).

- - , and S. SEX: Study of the effect of water on nuclear constituents. Genet. Iberie~ 6, 19--32 (1954a).

- - - - Induction of division though nucleic acid treatment, Caryologia (Pisa) g, 151--159 (1954b).

Wmso?% G. B., and K. C. C~E~5: Segregation and reduction in somatic tissues. II. The separation of homologous chromosomes in Tril l ium species. J. Hered. 40, 1--6 (1949).

Dr. A~uN Ku~A~ SItARMA and Dr. ARCmtNA SHAm~A, Cytogenetics Laboratory, Botany Department, Calcutta University,

35 BMlygunj Circular Road, Calcutta 19, India