BIOLOGIA PLANTARUM (PRAHA)

21 (4) : 253--258, 1979

Induction of Polyploidy and C-Tumours after Treating �9 411ium c e p a Root Tips with the Herbicide "Treflan"

A. KABARITY and A. NA~S

Department of Botany and Microbiology, Kuwait University*

Abstract. Treflan has the ability to induce polyploidy in Allium cepa root tips. The frequency of polyploidy was reduced after allowing the roots to recover, which indicates tha t the process of polyploidy is a reversible one, if we apply Treflan for a short time (4 h). As soon as the chemical is removed from the ceils, they go on with their normal way in division. I t was found tha t the concentration 350 • 10 -5 mi Treflan per 100 ml wa~er is an effective concentration in producing polyploidy in the roots of AUium cepa.

Treflan induced C-tumours in root tips of Allium cepa. Two types of enlargement were noticed, complete swelling along the majori ty of root tips and complete swelling with a certain enlargement immediately after 2 mm terminal tips. The histological examination of these zones revealed that swellings were due to cell enlargement both in meristematic and in tumour zones. This conclusion was evidenced by the results obtained from the nucleoplasmic index.

Trifluralin is a toluidene herbicide (a, a, a-trifluro 2,6-dinitro N, N-dipropyl p-toluidine) (HoLLIST and For 1971). It is purchased as a liquid formulation of 44.5% active ingredient manufactured by Elanco Company under the trade name Treflan. It is an effective chemical for selective weed control as a premergence soil incorporated treatment in many crops including cotton, peanuts, soybeans, sunflowers and others.

KABARITY and NAHAS (1978) and DELCOURT and DEYSSON (1976) found that the main cytological effects of Treflan upon mitosis of Allium cepa root tips is stathmokinetic. Treflan inhibits the spindle mechanisms at prophase, delays chromosomal separation and also inhibits daughter nuclei formation. Therefore, the ability of Treflan to induce polyploidy and C-tumours was investigated to demonstrate the mode of action of this herbicide upon plant cells.

M A T E R I A L AND M E T H O D S

Bulbs of Allium cepa were allowed %o germinate in %ap water at 20 ~ After the roots grew 2--4 cm, they were placed in solutions which contained the test substances for the time given in the text at 20 ~ The root tips were then fixed in Cornoy 3 : 1 for 24 h. The paraffin method was used for preparing root tips which had produced C-turnouts. The chromosomes were

Received iVovemb6r 6, 1978; accep~d January 9, 1979 * Addresa: P. O. Box 5969, Kuwait.

253

2 5 4 A. K A B A R I T Y , A. NAHAS

TABLE 1

Frequency of polyploid cells in the tumour zone in AUiu~n cepa roots after t r ea tment with Treflan for 4 h

Concentration R e c o v e r y t i m e • 10 -5 ml (100 ml) -1 0 h 1 h 2 h 4 h 20 h

700 3.3 0.236 0.615 0.719 300 9.7 0.982 1.255 2.074 0.526 170 5.09 O. 133 2.463 1.204 0.837

9 5.4 0.347 0.952 2.431 1.682 0 0 0.0 0.0 0.0 0.0

4660 to 6263 cells scored for each t rea tment .

stained by leucobasic fuchsin whereas the cytoplasm was stained with 1% light green.

Two parameters were determined from the zones of both treated and untreated roots, namely the meristematic zone and the turnout zone; the nucleoplasmic index (NP) and the number of cell layers from the centre to the epidermal layer which were counted in ten different sections taken at random from each zone of the four roots. The nucleoplasmie index was determined by using the following formula:

V n N P =

Vc -- Vn

R E S U L T S

1. Induction of Polyploidy after Treating ,411ium cepa Root Tips with Treflan for 4---96 h

In the present work Treflan was investigated for the induction of polyploidy in the following concentrations: 700, 350, 170, 90 • 10-5 ml per 100 ml H20.

Cytological investigations indicated that the control roots possessed normal mitotic phases, and all their cells in the meristematic region had the diploid chromosome number. The treated roots on the other hand showed a certain degree of polyploidy. The polyploidy level varied from one root to another. The majority, however, had cells containing the tetraploid chromosome (Table 1). This indicates that Treflan has the ability to induce polyploidy.

Table 1 represents the frequency of roots having polyploid cells after the treatment with different concentrations of Treflan. The maximum rate of polyploidy {9.7%) appeared when Allium cepa root tips were treated with the concentration 350 • 10 -5 ml Treflan per 100 ml HzO. Meanwhile the percentage of polyploid cells reached 3.3 in the concentration 700 • 10 -5 ml Treflan per 100 ml H20. The number of cells which were found as polyploid cells was 4- the same after the treatment with 170 and 90 • 10-5 ml Treflan per 100 ml H20. It must be mentioned here that the value of polyploid cells given in Table 1 is that of cells which indicate obviously 32 chromosomes during division.

INDUCTION OF POLYPLOIDY AND C-TUMOURS 2 5 5

2. Frequency of P o l y p l o k l y in ,411ium cepa Cells after 4 h Treatment and F i x e d 1 h Later

After one hour recovery of the root cells previously t reated with different concentrations of Treflan for 4 hours, the frequency of polyploidy varied according to the concentrations applied.

Table 1 shows tha t the highest number of polyploid cells appears after treating Allium cepa root cells with 350x 10-5 ml Treflan for 4 hours and fixed one hour later, whereas the concentrations 700, 170 and 90• 10 -2 ml Treflan per 100 ml H20 show lower frequency of polyploidy which are ~ the same.

I t was noticed that the frequency of polyploidy was reduced after allowing the roots to recover for one hour before fixation. This indicates . that the process of polyploidy is a reversible one, if the application of Treflan was only for 4 hours. As soon as the chemical was removed from the cells, they returned to their normal way of division.

3. Frequency of Polyploidy in Allium cepa Root Cells after Treatment with Treflan for 4 h a n d F ixed 2 h Later

In general the tendency of the decrease of polyploid cells continues after allowing the roots to recover for 2 hours. Table 1 shows tha t the highest percentage of polyploidy (2.46) was reached in the concentration 170 x 10- 5 ml Treflan per 100 ml H20. The other concentrations used in this t rea tment showed a fluctuation in the value of polyploid cells produced.

4. Frequency of Polyploidy in ,4ilium cepa Root Cells after 4 h T r e a t m e n t w i t h Treflan and Fixed 4 h Later

Table 1 indicates tha t there is a jump in the number of polyploid cells after treating All ium cepa root tips with 350 • 10 -5 ml Treflan per 100 ml H20, and allowing the roots to recover for 4 hours before fixation. The other concentrations used in this experiment showed a considerably low frequency of polyploidy ranging from 0.7 to 2.43~o after treating All ium cepa roots with 700• 10-5 and 90X 10 -5 ml Treflan per 100 ml H20 and allowing the roots to recover for 4 hours before fixation. I t must be mentioned tha t the concentration of 350 • 10-a ml Treflan per 100 ml H20 which caused the highest value of polyploidy in this investigation caused the same effect after 4 hours of t rea tment and, therefore, may be considered as an effective con- centration in producing polyploidy in the roots of All ium cepa.

5. Frequency of Polyploidy i n ,4lliurn cepa Root Cells after 4 h Treatment with T r e f l a n and Fixed 20 h Later

The result of the above mentioned t rea tment is represented in Table l, which indicates tha t the frequency of polyploid cells is very low. The highest value reached was only 1.68 ~o after t rea tment with concentration 90 • 10 .2 ml Treflan per 100 ml H20 and allowing the roots to recover for 20 hours before fixation.

6. Induction o f C-Tumours after Treating Allium cepa Roots with Treflan

Nature of C-tumours induced by Treflan:

1) M o r p h o l o g i c a l c h a n g e s in t h e r o o t s : Treflan reduced the rate of root growth so much tha t they appeared stunt and thick compared with

256 A. KABARITY, A. NAHAS

Fig. 1. Diagrammatic representation of different layers (A) and zones (B) examined in Alliutn cepa root tips.

the untreated roots, which appeared long and thin (Fig. 1). Swelling in the roots appeared after 3- -4 days of t reatment . Two types of enlargement were noticed:

a -- Complete swelling along the majori ty of the root tips. b -- Complete swelling bu t with a certain enlargement immediately after

2 mm terminal tips.

2) H i s t o l o g y o f t h e r o o t : Microscopic observation on transverse sections of the root tips which had been t reated showed tha t the enlargement included both the meristematic and the tumour zones (Figs. 2, 3). However, the main swelling area involved the elongation zone of the root. The increase of the radial diameter was found to be due to the abnormal cell enlargement of the cortex area. This enlargement was quite evident from the determination of the surface area of sections of the different zones. For example, the meristematic zone of the t reated roots had an average area of 0.45 mm2 compared with the untreated roots of the same zone which had an area of 0.09 mm 2. This area had increased more than five times in the tumour zone. The diameter of the t reated area reached 1.31 mm 2 as compared with the control value of 0.38 mm 2 (Table 2).

The increase in surface section area which causes the abnormal tumour swelling of the root was found to be due to cell enlargement of the cortex (II layer) and pith (III layer) layers rather than to an increase in cell number. This was proved by the determination of the number of cell layers from the centre of the section to the peripheral layer.

Table 2 shows that the number of cell rows in the t reated roots is more than in untreated ones by almost one or two layers. Such difference remains the same in both the meristematic and tumour zones. Thus it could be concluded that the abnormal complete swelling along the majori ty of the root was due to cell enlargement both in meristematic and in tumour zones. The cell enlargement of the cortex and pith areas involved only the cell volume whereas the nucleus volume remained constant in cells of the t reated and untreated roots and in both zones. This conclusion was evidenced by the results obtained from the nucleoplasmic index.

C-tumours were formed in root tips of Allium cepa due to the treatment with Treflan. I t was found in this investigation tha t the main reason for these swellings in the roots is due to the abnormal enlargement of the region

INDUCTION OF POLYPLOIDY AND C-TUMOURS 257

TABU 2 The nucleoplasmic index (NP), the mean area of sections, the number of layers, the volume of cells (Vc), and the volume of nuclei (Vn) in the t rea ted and unt rea ted root tips of Allium c e ~ root cells with 350• 10 -5 ml Treflan per 100 ml H20 for 96 h

A: Meristematic zone

ZNucleoplasmio Vc (Volume of cell) Vn (Volume of index N P M 2 nucleus) M 2 Diameter Number

of of

I I I I I I I I I I U I I I I I I Section layers layer layer layer layer layer layer layer layer layer [mm 2]

Control 0.56 0.13 0.42 192 494 233 69 55 69 0.09 15

Trea tment 0.38 0.13 0.15 398 950 823 110 120 110 0.45 17

B: Tumour zone and the corresponding zone in the untreated roots

Nueleoplasmic Vc (Volume of cell) Vn (Volume of Diameter Number index NP M 2 nucleus) M 2 of of

I I I I I I I I I I I I I I I I I I Section layers layer layer layer layer layer layer layer layer layer [mm 2]

Control 0.36 0.13 0.27 206 809 261 55 96 55 0.38 20

Trea tment 0.031 0.021 0.028 2689 4665 3430 82 96 96 1.31 21

between the meristematic area and the differentiated cells of a root. Nor- mally cells elongate linearly to the axis of the root. They seem to show a polarity in this respect. When Treflan is present, an enlargement of the cell takes place in all directions. That is, an iso-diamctric expansion occurs, rather than a polarized elongation. Cells of the cortex and pith become inflated. This leads to a swelling at a particular place along the root. Sections of treated and untreated roots within five or six layers of cells show where the change occurs and reveal particularly the difference in the shape of individual cells. These comparative studies confirm the opinion that the direction of growth is altered when Treflan is present. This reaction was also found after the t reatment with colchicine ( E m s ~ and Dvs r r s 1955). Grovcth promoting substances, such as naphthalene acetic acid and indolebutyric acid induce C-tumours (LEVA~ 1942, BEROER and WXTKUS 1949).

RAS~ (1964) stated that there was no evidence ofnon-chromosomal DNA in tumour cells. DNA synthesis extends over 50--60% of the interphase period in both normal and tumorous bean tissues.

Not all compounds that create tumours arrest mitosis. In fact, certain phytohormones that do not stop mitosis may induce root tip enlargements. The idea of an autonomy of C-mitosis and C-tumours gains support from these general observations with several chemicals (L:~vA~ and 0ST~R- OR~N 1943).

Generally, favourable conditions for growth increase the promotion of a tumour from a specific t reatment (L~.vAN and STv.INOO~.R 1947). The range in concentration is fairly broad, but there are limits marked by minimum

2 5 8 A. K A B A R I T Y , A. N A H A S

and maximum concentrations. The formation of C-~umours within certain limits is proportional to concentration. Finally, the thresholds for C-mitosis and C-tumours are close to each other with some indication that the threshold for the latter process is lower than that for C-mitosis (Lv, vA~ and 0STaR- ORION 1943).

As soon as the independence of C-mitosis and C-tumour was suspected, a specific experiment was designed to test autonomy (LV.vA~ 1942). Root primordia of Allium fistulosum were subjected to intense X-ray treatment. Consequently, the mitotic capacity of meristematic cells was destroyed. Following X-irradiation, bulbs were placed over colehicine, and typical C-tumours formed with no evidence for several days in these roots. Therefore enlargement occurs without a simultaneous division of cells. Polyploidy following a C-mitosis is not necessary for tumour formation (L~vAN 1942). Swelling at the hypocotyl where seedlings were soaked in colchicine gave the first evidence that tumours were in no way related to C-mitosis or induced polyploidy. Although cells in the hypocotyl are not meristematic, they are capable of elongating or expanding. Colchicine causes an iso-diametric expansion of cells much the same as among cortical cells in roots (HAwxEs 1942).

The tumour formation is proportional to concentration within certain limits (GERE~LINO 1939). Different species show different degrees of response to the same concentration. Another factor is the specific moment when seedlings are placed in colchicine (Wv, YLA~D 1948). If the seedling has not yet elongated, there is swelling throughout the entire hypocotyl. But the seedling that has already elongated to about 2"3 mm before treatment begins, practically shows swelling at the hypoeotyl (R~ES 1950). All these points fall in line within the proposition that tumour formation is basically a growth response to the chemical used.

R E F E R E N C E S

BEI~GER, C., WITKUS, E.: Further s tudies of the cyto logica l effects of combined t rea tments w i th eolchicine and naphtha lene acetic acid. - - Amer . J . Bet. 36 : 794--795, 1949.

DELCOURT, A., DEYSSON, (~.: Effects de la Trifluraline sur los Meristemes Radicula ires d'Alliu~n sa~iv'um L. -- Cytologia 41 : 75--84, 1976.

ExosTI, O. J . , DusTn~, P.: Colchieine in Agriculture , Medicine, B io logy and Chemistry . - - I o w a State Coll. Press, Amos, I o w a 1955.

G~.m~ML~G, G.: Troubles morpho log iqucs determines par la eolchieine chez v~g~tattx. - - Assoc. France Avanc . Sci. 63 : 595--607, 1939.

HAWKES, J . : Some effects of the drug eolchieine on cell divis ion. -- J . Goner. 44 : 11--22, 1942. HOZ~IST, R. I . , FoY, G. I . : Trifluralin interact ion w i t h soil const i tuents . - - W e e d Sci. 19: 11,

1971. KABARITY, A., NAHAS, A.: Cytological s tudies of the effect of some herbicides on plants . --

Manuscript 1979. L:~VAN, A. : The macroscopic colehicine effect -- a harmonic action. Observat ions on the n a p h t h a -

lene series. - - Heredi tas 29 : 361--443, 1942. LEVAN, A., 0STERGREI~, ~. : The m e c h a n i s m of c-mitot ic action. Observat ions on the n a p h t h a -

lene series. - - Heredi tas 29 : 381--443, 1943. L~.VA~, A., STEINGSE~, E. : The resistance of co lch icum and b u l b o c o d i u m to the c -mitot ic act ion

o f colehicine. - - Heredi tas 33 : 552--660, 1947. RASCH, E. M.: D N A synthes i s in p lant t u m o r cells. -- Exp. Cell Res. 36 : 445--486, 1964. REES, G.: Beitr~ge zur W i r k u n g des Colehicine bei der S a men b eh a n d lu n g . - - P l a n t a 38 : 324

to 376, 1950. WEY!~WD, H. : The act ion of chemicals on plants and its s ignificance in medic ine . -- Krebs-

forschung 56 : 148--164, 1948.

FiAgur~ at the end of the i s ~ .

A. K A B A R I T u A. NAHAS I N D U C T I O N OF P O L Y P L O I D Y AND C-TUMOURS

Fig. 2. Transverse section in A l l i u m cepa root tips in meris temat ie zone. a -- control; b -- af ter t r e a t m e n t wi th 350• 10 -s ml Treflan per 100 ml H20 for 96 h.

A. K A B A R 1 T Y , A. N A H A S I N D U C T I O N OF P O L Y P L O I D Y AND C-TUMOURS

Fig. 3. a - - Transverse sect ion in u n t r e a t e d root t ips of A l l i u m cepa in a zone cor responding t u r n o u t zone in t r e a t e d roots (control). b -- Transverse sect ion in t u m o u r zone a f te r t r e a t m e n t w i t h 350• 10 -5 ml Treflan per 100 ml H 2 0 for 96 h.