Mitotic events and other disorders induced in cowpeas, Vigna unguiculata (L.) Walp. by ionizing radiation

Mitotic events and other disorders induced in cowpeas, Vigna unguiculata (L.) Walp. by ionizing radiation

Radiation Botany, 1971, Vol. 11, pp. 375 to 381. Pergamon Press. Printed in Great Britain. M I T O T I C EVENTS AND O T H E R DISORDERS INDUCED IN C ...

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Radiation Botany, 1971, Vol. 11, pp. 375 to 381. Pergamon Press. Printed in Great Britain.

M I T O T I C EVENTS AND O T H E R DISORDERS INDUCED IN C O W P E A S , VIGNA UNGUICULATA (L.) WALP. BY I O N I Z I N G R A D I A T I O N OLATUNDE A. OJOMO Research Division, Ministry of Agriculture and Natural Resources, Ibadan, Nigeria and H. R. CI-H'IEDA

Department of Agronomy, Faculty of Agriculture, University of Ibadan, Ibadan, Nigeria (Revised: manuscrlpt received24 February 197 I)

OJOMO O. A. and CHHEDAH. R. Mitotic events and other disorders induced in cowpeas, Vigna unguiculata (L.) Walp. by ionizing radiation. RADIATION BOTANY 11, 375--381, 1971.Studies of cytological, some morphological and other disorders were made in the MI generation of cowpeas following exposures to X-rays and thermal neutrons. Though the incidence of chromosome aberrations was very high at the first mitotic stage, seed germination was apparen fly unaffected, but seedling survival diminished as level of treatment increased. The frequency of cells showing chromosome disorders decreased as growth advanced, the ceils having been apparently eliminated by diplontic selection. These events appear to be related to the appearance of leaf spots, alteration in the leaflet number of normally trifoliate leaves, and gross leaf distortions at early growth and their disappearance from subsequent growth. INTRODUCTION

published data show that ionizing radiatious produce several primary effects in the M I generation of plants grown from the treated seeds. Thus SJODIN,(s) BLIXT el a/.,(z) WELI.~NSIEK,(ls) SPEC~AN(ix) have noticed primary effects such as chlorophyll---deficient spots which occur only on the first leaves. Other effects such as leaf distortions and changed leaf digits were observed. T h e plants however, recovered to normal growth. SPARROW(10)attributed these changes to radiation induced physiological changes but that they m a y as well be related to chromosome changes. VIG (14) studied mitotic events in relation to the occurrence of leaf spots in T219 m u t a n t strain of Glycine max and found a correlation between them. H e attributed the incidence of spots to loss of one or more chromosomes besides other events. T h e preliminary work on mutagenic treatments of cowpeas in Nigeria indicate that the 375

crop shows a strong phenotypic buffering against radiation, and not m a n y permanent primary effects are visually discernable. This p a p e r reports the study of chromosome disturbances and their correlation with some primary changes produced in cowpeas after ionizing radiation. Not much information is available on mutation work in this crop.

MATERIALS A N D M E T H O D S

T w o varieties of cowpeas, Local brown, a native brown and large seeded variety with 1000 kernel weight of 200 g and Westbred, an exotic but adapted white small seeded variety with 1000 kernel weight of 100 g were used. Neutron and X - r a y irradiation were used. In 1969, the varieties were irradiated at the Brookhaven National Laboratories in New York, using X - r a y exposures ranging from 7"5 kR to 17-5 kR and thermal neutron radiation ranging

376

OLATUNDE A. OJOMO and H. R. CH'HEDA

from 6.15 × 10X2n/cm2/sec to 3.86 × 1013n/cm~/ see. There was no lethal exposure within the X - r a y range but the highest neutron exposure was lethal. In 1970, seeds were irradiated only with X-rays to include a lethal exposure. The exposure range of 10-30 kR was used and the treatment was made at the Institute of Genetics, University of Lund, Sweden. 20 and 30 kR of X - r a y proved sub-lethal and lethal, respectively. Cytological observations were made only of X-ray treated materials, at the Cytogenetic Division in Svalof, Sweden. Seeds were germinated in Petri-dishes and some of their root tips excised early in the morning after three days, in order to catch the first mitotic divisions. Some seeds of Local brown treated with 20 kR X - r a y were allowed to germinate for 10-15 days before removing their root tips. All the root tips were fixed in Carnoy's solution for 5-6 hr and washed with tap water. They were later hydrolysed for 6 rain in N H C L before being squashed in a drop of Lactopropionic-orcein (DYER(S)). Records were made of all abnormal types observed and frequency of the different configurations were estimated. For observations of morphological changes and other disorders, seeds were planted in boxes containing sand-earth mixtures. About 80-100 seedlings which emerged and looked apparently vigorous per treatment were transplanted into pots where they were allowed to grow to maturity. The number of seedlings which emerged relative to the number of seeds planted and the n u m b e r of seedlings which survived relative to the number transplanted were observed. Chlorophyll-deficient spots were noted on leaves of seedlings, and they were scored on a 5-grade scale, from 0 to 4. Plants with normal leaves showing no spots were assigned to grade 0 and those plants with deformed leaves on which the spots had become diffuse were assigned to grade 4. Grades 1-3 were intermediate between the above extremes. Plants in which the first trifoliate leaves showed leaflet number more or less than 3 were also observed. A systematic record was made of the incidence as more and more leaves were formed. Two sets of data are presented, one set for

neutron treatments recorded in 1969 and the second set for X - r a y treatments made in 1970. RESULTS

The results of seed germination and seedling survival following the respective treatments are presented in Table 1. Seed germination was based on the n u m b e r of seeds which emerged from the soil relative to seeds sown and seedling survival was based on the numbers of plants which became established out of a number of seedlings that were transplanted from those which emerged. Seed germination, on the average, was not drastically affected by the treatments when compared with the control. However, significant differences between control and high doses of irradiation occurred for n u m b e r of plants which survived. The lower exposures did not produce any effects. The results of cytological investigations of first mitotic division are presented in Table 2. T h e extremely small chromosome size prevented detailed observations of chromosome morphology, though some chromosome abnormalities which occurred during mitosis in treated root tips were easily discernable. Similarly it was not also possible to discern if two or more events occur per cell. The most common chromosome abnormality was bridges, followed by fragments and/or laggards. The last two, where they did not occur alongside of a bridge, were included as a group because of the difficulty in differentiating between them. Some evidence of sticky chromosomes and also some reductional groupings were observed, though infrequently. T h e y were classified as "others" in Table 2. Comparative figures of chromosome abnormalities that were observed for the variety Local brown at the sub-lethal X - r a y exposure level of 20 k R at 3, 10 and 15 days after sowing the treated seeds are presented in Table 3. As expected, the frequency of chromosome abnormalities decreased with increasing n u m b e r of cell divisions. T h e value of 30 per cent at first mitosis had dropped to 10 and 5 and at 1 and 2 weeks of growth, respectively. Bridge conformations appeared to disappear more rapidly than the other types of chromosome disorders.

I O N I Z I N G R A D I A T I O N ON COWPEAS

377

Table 1. The effects of ionizing radiation on seedgermination and seedling survival in cowpeas Local brown

Westbred

%

%

%

Germ*

Survived

%

Germ

Survived

X-ray (1970) 1 10kR

2 3 4 5 6

15kR 20kR 25kR 30kR Control

88.8 82.8 74.1 88.8 78.4 87-0

100.0 100.0 43.3 25.0 0.0 100.0

78-4 72.4 74.1 81.0 75-0 82.0

I00.0 100-0 66.0 50.0 0.0 I00.0

1 2 3 4 5 6

Neutron (1969) 6-15 × 101~n/cm 2 8.76 × 10X2n/cm2 1.02 × 10XSn/cm2 1.22 x 1013n/cm 2 3"86 × 1013n/cm 2 Control

83.0 67.4 62.2 59.3 63.0 83.0

100.0 100.0 92.0 56.2 0.0 I00-0

74.1 65.9 62.8 60.8 63.2 81.0

100.0 100.0 100.0 66-3 0'0 98.3

*Number of seeds planted -- 200. 1"Number of seedlings transplanted = 80.

Table 2. Frequenciesof different chromosomeconfigurations in cowpeastreatedwith X-ray Dose

Number anaphase cells

Number normal

Bridges

Westbred

Control 15kR 20kR 25kR 30kR

50 25 31 88 56

50 20 14 60 33

0 3 4 14 9

0 2 2 11 12

Local brown

Control 15kR 20kR 25 kR 30kR

100 56 47 109 74

100 46 33 64 44

0 7 10 25 20

0 1 3 14 8

Variety

Fragments and Others Laggards

Total abnormal

% abnormal

0 0 1 3 2

0 5 7 28 23

0"0 20.0 30.0 31.8 41.8

0 2 1 3 2

0 10 14 42 30

0.0 18.6 29.1 38.5 40.5

Table 3. Percentageof mitotio cells of cowbea, Var. Local brown showing chromosomeaberrations at differentstages of germinationfollowing 20 kR of X-ray radiation Days

Anaphase

Normal

Bridges

Fragments

Others

Total abnormal

% abnormal

3 10 15

47 59 60

33 53 57

10 4 1

3 2 2

1 ---

14 6 3

29.1 10.1 5-0

O L A T U N D E A. O J O M O and H. R. C H H E D A

378

T h e n u m b e r of plants w h i c h o c c u r in the various grades o f c h l o r o p h y l l - - d e f i c i e n t spots, hereafter referred to as l e a f spots are presented in T a b l e 4. T h e leaf spots were observed o n l y in the first simple leaves a n d less frequently in the first trifoliate l e a f a n d not at all in subsequent leaves. All the plants in the control h a d a g r a d e o f 0, whilst n o n e of the t r e a t e d plants o c c u r r e d in this grade. F u r t h e r , the f r e q u e n c y of plants in the higher grades increased as t r e a t m e n t levels increased. This t r e n d was observed for b o t h o f the varieties used. Different kinds of changes in leaves a n d p l a n t forms h a v e b e e n r e p o r t e d in o t h e r crops following ionizing r a d i a t i o n . I n this study, m o r p h o l o g i c a l l y visible changes were restricted

to a l t e r a t i o n in the n u m b e r a n d shape o f leaflets w h i c h occur in the e a r l y trifoliate leaves. T h e n u m b e r a n d progress o f o c c u r r e n c e starting with the first trifoliate l e a f a r e s u m m a r i z e d in T a b l e 5. A t all levels o f t r e a t m e n t , plants occur w h i c h b e a r c h a n g e d leaflet n u m b e r , thus on the first few trifoliate leaves, one, two, four o r five leaflets a p p e a r instead o f three (Fig. 1). T h e n u m b e r o f plants showing the t r a i t on the second a n d t h i r d trifoliate leaves decreased, a n d no plants showed it on the fourth a n d s u b s e q u e n t trifoliate leaves. I t was n o t e d t h a t none o f the p l a n t s w h i c h d i d n o t show a l t e r e d l e a f d i g i t n u m b e r at the first trifoliate leaf stage showed it a t a l a t e r state. Similarly, n e a r l y all plants showed serious

Table 4. Frequencydistributionofplants within differentgrades of leaf s~Ootsin cowpeasfollowing ionizing radiation Number of plants

0

1

2

3

4

40 40 40 40 40

0 0 0 0 40

15 0 0 1 0

25 39 26 I 0

0 1 8 8 0

0 0 6 0 0

1.6 2.0 2.5 2.7 0.0

40 40 40 I0 40

0 0 0 0 40

32 29 0 0 0

7 11 31 3 0

1 0 9 34 0

0 0 0 3 0

1.2 1.7 2.2 2-8 0.0

Neutron (1969) Westbred 6"15 × 1012n/cm s 8.76 X 10t2n/cm a 1.02 × 10tSn/cm 2 1.22 × 10XSn/cm2 Control

40 40 40 40 40

0 0 0 40 40

38 38 27 10 0

2 1 12 27 0

0 1

0 0

1

0

3 0

0 0

I'0 1.1 1.4 1.9 0.0

Local brown 6-15 × lOtSn/cm a 8.76 × lOtan/cm 2 1.02 x lOXSn/cm~ 1.22 × 10Xan/cm ~ Control

40 40 40 40 40

0 1 0 0 40

40 27 25 23 0

0

0

0

12 15 17 0

0 0 0 0

0 0 0 0

Dose

X-ray (1970) Westbred 10kR 15 kR 20 kR 25kR Control Local brown 10 kR 15 kR 20 kR 25 kR Control

Number of plants in grades

-

Mean

1.0 1.2 1.4 1.4 0.0

4-

51.

1 2

5

FIG. 1. Alteration in leaflet n u m b e r of compound leaves following ionizing radiation. I. Normal leaf with three leaflets. 2. Leafshowing only two leaflets. 3a, b. Leaves showing four leaflets. 4. Compound leafwith 1 leaflet. 5. Leaf showing five leaflets.

R.B.f.p. 378

IONIZING RADIATION ON COWPEAS Table 5. Frequency distribution of leaflet changes in cowpeas following ionizing radiation Dose X-ray 1970 Westbred 10 kR 15 kR 20kR 25 kR Control

Number of plants

Trifoliates 1st

2nd

3rd

80 80 I0 18 80

2 4

0 1

0 0

2 4 0

0 I 0

0 0 0

80 80 80 36 80

4 6 15 10 0

0 1 6 5 0

0 0 9 3 0

Neutron (1969) Westbred 6.15 × 10X2n/cm2 8.76 × 10X2n/cm2 1.02 × 10XSn/cmz 1'22 × 10aSn/cm2 Control

80 80 80 80 80

5 5 5 4 0

1 0 0 2 0

0 0 0 0 0

Local brown 6.15 × 10XZn/cmz 8.76 × 10a2n/cm 2 1.02 × 10XSn/cm2 Control

80 80 80 80

8

2

0

9 10 0

0 0 0

0 0 0

Local brown 10 kR 15 kR 20 kR 25 kR Control

distortions of the leaflets of early-formed compound leaves. In nearly all cases these plants recovered and eventually produced normal leaves later on. DISCUSSION

T h e effects of X - r a y and thermal neutron treatments of Local brown and Westbred varieties of cowpeas are presented for the plant population directly raised from the treated seed (M1). Though data presented were for two different treatments in two different years, the same trends were observed. Seed germination, as defined by the ability of seeds to produce plumule and radicle under o p t i m u m growth conditions, did not appear to be affected by the different

379

types and levels of radiation. T h e n u m b e r of seedlings which survived after being transplanted appeared to be inversely proportional to exposure. More plants survived from seeds that were treated with a relatively low radiation exposure than at high exposures. Similarly, observations were recorded by SJODm(9) and MmKE. (7) T h e y advanced a physiological reason for their observation. The first phase of germination is the swelling of ceils by hydration followed by enzymatic activation and metabolism. The materials and energy necessary for this initial growth are already available in the seed and so the young embryo has no need to form new substances, but only to activate those already stored in the cotyledons. This stage of germination is apparently unaffected by radiation, therefore, damage to the embryo which might arise from ionizing radiation become apparent at later stages ofontogenesis. T h e incidence of chlorophyll deficient spots on leaves of M 1 generation of legumes after mutagenie treatment of seeds has been commonly observed and reported by various workers, e.g. BLrXT et aL,(9) W~'-LLENSIEK,(13)SPECKMAN,(11) Mmr.a~ (7) and SJODIN.(9) The observations recorded in cowpeas are similar. T h e spots appeared only on the primary leaves and disappeared gradually from the compound leaves formed later on. Alteration in the n u m b e r of leaflets produced by the first to third trifoliate leaves followed the same pattern. As recorded in this study, the n u m b e r of leaves showing the phenomenon decreased from the first trifoliate leaf till the third and did not occur in the forth and subsequent leaves. It is conceivable, therefore, that both events, leaf-spots and changed leaf digits arise from the same cause. Vio04) related mitotic events with the occurrence of leaf spots in Glydne max. He suggested that the incidence might be related to chromosome disorders which appeared at early mitosis and disappeared with increasing n u m b e r of cell divisions. I n cowpeas, all the doses of X-ray radiation used on the two varieties, produced fairly high levels of chromosome abnormality, which ranged from about 18 per cent at the lowest exposure of 10 k R to 41 per cent at 30 kR. As plants continued to grow, the frequency of

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OLATUNDE A. OJOMO and

ceils carrying chromosome abnormalities decreased. It would appear that such cells were unable to reproduce themselves relative to normal cells and consequently became eliminated. This phenomenon has been observed in other crops by SWAMINATHAN,(12) MAGKEY,(6) Vio(14) and GAUL,(4) and has been variously termed diplontic selection, intra-somatic cell selection or competitive elimination of cells containing lethal or sub-lethal levels of chromosome disorders. The working hypothesis for diplontic selection was developed by GAUL(a) from preliminary studies in barley. H e proposed that at the time of irradiation of dry seeds, the primary cells which latter produce the first-formed tiller buds were already more or less differentiated in the embryo. As such, there is probably only a little chance of cell competition and selection at this stage. The morphological effects and other disorders due to the chromosome aberrations are therefore, morpholigically expressed. However, with successive growth in the areas of the embryo where new ceils are developed, much greater intercellular competition is likely in which the cells carrying chromosomal disorders above a certain limit are disadvantaged. The visible morphological changes due to these cell types are subsequently eliminated. From the work of Kosra and SMITH,(s) ANDERSONand ROBERTSON, (1) y o n WETTSTEIN (15) and NELSON,(s) colour changes in leaves of mutants occur from results of blocks at some intermediate stages in chlorophyll synthesis, an essentially physiological activity. The incidence of leaf spots following ionizing radiation in legumes might represent localized blocks in the pathway of chlorophyll synthesis. I t seems, therefore, plausible to suggest that the occurrence of leaf spots and changed digits at early ontogenesis and their disappearance at later stages might be related to physiological disorders due to the high level of chromosome abnormalities which also occur at early ontogenesis and become drastically reduced as growth progresses. SLmilar suggestions were advanced by SPARROW(10) whilst working with ionizing radiation in Tradescantia paludosa. He observed that

H. R. CHHEDA

dosages which caused marked morphological changes also caused a considerable amount of chromosome aberrations. However, m a n y plants which showed serious morphological disorders recovered to normal growth patterns. He thus suggested that the morphological changes were due to radiation induced physiological changes and possibly to chromosomal aberrations. Though cytological investigations were made only for X - r a y treated seeds, disappearance of leaf spotting and leaflet abnormalities were observed to follow the same trend in the progeny of neutron treated seeds. T h e incidence would appear also to be due to the proposed diplontic selection of invisible cells.

Acknowledgements--I express sincere appreciations to the Brookhaven National Laboratories, New York and UDDA LtrsDQULST,Swedish Seed Association, Svalof, for their help in irradiating the seeds used in this exercise, to Doctors O. Nw~xmTI, J. ~SJoDINand S. ELLESTROMfor their help in the cytological investigations and to the Swedish International Development Authority for providing funds for part of the studies.

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As,mm~soN I. C. and R.OBERTSOND. S. (1960) The role of carotinoids in protecting chlorophyll from photo-destruction. Plant Physiol. 35, 531-534. BLIXTS., GELIN O., MOSSBERGR., AHNSTRONG., EHRENBERGI. and LOFGRENB. A. (1964) Studies of induced mutations in peas. IX. Induction of leaf spots. Agr. Hort. Genetica22, 186-194. DYER A. F. (1963) The use of lacto-propionic orcein in rapid squash methods for chromosome preparations. Stain Technol. 38, 85-90. GAUL H. (1961) Studies of diplontic selection after X-irradiation of harley seeds. In, Effects of ionizing radiation of dry seeds, pp. 117-138. Proc. Syrup. Karlsruhe, 1960. Kosva V. M. and SMrrHJ. H. C. (1951) Chlorophyll formation in a mutant white seedllngs-3. Arch. Bioehem. Biophys. 34~ 189-195. M_AcKeYJ. (1961) Methods of utilizing induced mutation in crop improvement. In, Mutation and plant breeding,pp. 336-364. WAS-NRC Publ. 891. MmKz A. (1961) Comparison of the effects of X-ray and thermal neutron on viability and growth of sweet clover (Melilotus alba) after irradation of dry seeds, pp. 403-410. In, Effects of ionizing radiation on seeds. Proc. Syrup. Karlsruhe, 1960.

I O N I Z I N G R A D I A T I O N ON COWPEAS 8. NELSON O. E. (1967) Biochemical genetics of higher plants. Ann. Rev. Genet. 1, 245-268. 9. SjODINJ. (1962) Some observations in XI and X2 of Viola faba L., after treatment with different mutagens. Hereditas 18, 565-586. 10. SPARROW A. H. (1951) Some cytological and morphological changes induced in plants by ionizing radiation. Science 114, 48. 11. SPECKMANNG . J . (1964) The mutagenic effect of treatment with EMS at different temperatures in P. sativum. Euphytica 13, 337-344. 12. SwAMrNATX-tANM. S. (1961) Effect of diplontic selection on the frequency and spectrum of

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mutations induced in polyploids following seed irradiation, pp. 279-288. In, Effects of ionizing radiation on seeds. Proc. Symp. Karlsruhe, 1960. 13. WELLENSmK S.J. (1959) Neutronic mutations in peas. Euphytica 8, 209-215. 14. Vio. B. K. (1969) Relationship between mitotic events and leaf spotting in Glycine max. Can. 07. Genet. Gytol. 11, 147-152. 15. YON WET'rSTEIN D. (1961) Nuclear and cytoplasmic factor in development of chloroplast structure and function. Can. 07. Botany 39~ 15371545.