BIOLOGIA PLANTARTJM (PRAHA) 11 (6):432--441, 1969 Cytogenetic Studies on the Effect of Chronic Gamma Irradiation on Viciafaba SOHEIR iV[. Ai~ER, I. u MOSTAFA and EvoN MIKHAEL* National :Research Centre, Dokki, Cairo, and Radiobiology Department, Atomic Energy Establishment, Cairo, U.A.R.** 1~eceived December 13, 1968 Abstract. Vieia faba seeds (cv. Giza 1) were planted in the Inshas gamma radiation field where they were chronically irradiated during the whole life of the plant. The percentage of the induced abnormal P.M.Cs, as well as the frequency of abnormal P.M.Cs in the different meiotic stages were proportional with the given doses. The main types of chromosome aber- rations were anaphase and telophase bridges, fragmentation and lagging chromosomes. The nearest plants to the source showed an inhibition of shoot growth, flower and seed sterility and irregular branching. At the dosage levels used irradiation had no effect on pollen fertility. Seeds of the 1st filial generation were used for both mitotic and meiotic studies. The percent- age of the mitotic abnormalities was proportional with the doses. The most dominant type of anomaly was the presence of micronuclei in the different stages of mitosis and in the resting cells. Irradiation affected also ether types of anomalies e.g. lagging chromosomes, fragments, bridges.., etc. Meiosis, and pollen fertility (2nd generation) were normal~ Much research has been done concerning the effect of ionizing radiation on the plant. The aim was the determination of radiosensitivity and study of the genetic, morphogenetic and cytological effects. In this connection the work of: SPARROW and CHRISTE~SE~ (1953), SPARROW and SI~GLETO~ (1953), NYBOM (1956), ~qYBOM et al. (1956), SPAn~ow and Bo~D (1956), SPARROW et al. (1961), SPARROW and MIxSC~E (1961) and YAMAKAWAand SPARROW (1965, 1966) may be mentioned. The present work was undertaken in order to follow the changes that might occur after chronic irradiation of Vicia faba plants. The effect to be considered included meiotic irregularities, pollen fertility, morphological responses of the irradiated plants and irregularities in root-mitosis of seeds produced by the irradiated plants. * This is a part of the M.Sc. Thesis submitted by Miss Even Mikhael. ** Address: Laboratory of Genetics and Cytology, ~ational Research Centre, Dokki, Cairo, U.A.R. 432
Transcript
BIOLOGIA PLANTARTJM (PRAHA)
11 (6 ) :432- -441 , 1969
Cytogenet ic Studies on the Effect o f Chronic G a m m a Irradiat ion on Viciafaba
SOHEIR iV[. Ai~ER, I . u MOSTAFA a n d E v o N M I K H A E L *
National :Research Centre, Dokki, Cairo, and Radiobiology Depar tment , Atomic Energy Establ ishment , Cairo, U.A.R.**
1~eceived December 13, 1968
Abstract . Vieia faba seeds (cv. Giza 1) were planted in the Inshas gamma radia t ion field where they were chronically irradiated during the whole life of the plant. The percentage of the induced abnormal P.M.Cs, as well as the frequency of abnormal P.M.Cs in the different meiotic stages were proportional with the given doses. The main types of chromosome aber- ra t ions were anaphase and telophase bridges, f ragmentat ion and lagging chromosomes. The nearest plants to the source showed an inhibit ion of shoot growth, flower and seed sterility and irregular branching. At the dosage levels used irradiation had no effect on pollen fertility.
Seeds of the 1st filial generation were used for bo th mitotic and meiotic studies. The percent- age of the mitotic abnormalit ies was proportional with the doses. The most dominant type of anomaly was the presence of micronuclei in the different stages of mitosis and in the resting cells. I r radia t ion affected also e ther types of anomalies e.g. lagging chromosomes, fragments, br idges. . , etc.
Meiosis, and pollen fertility (2nd generation) were normal~
Much research has been done concerning the effect of ionizing radiation on the plant. The aim was the determination of radiosensitivity and study of the genetic, morphogenetic and cytological effects. In this connection the work of: SPARROW and CHRISTE~SE~ (1953), SPARROW and SI~GLETO~ (1953), NYBOM (1956), ~qYBOM et al. (1956), SPAn~ow and Bo~D (1956), SPARROW et al. (1961), SPARROW and MIxSC~E (1961) and YAMAKAWA and SPARROW (1965, 1966) may be mentioned.
The present work was undertaken in order to follow the changes that might occur after chronic irradiation of Vicia faba plants. The effect to be considered included meiotic irregularities, pollen fertility, morphological responses of the irradiated plants and irregularities in root-mitosis of seeds produced by the irradiated plants.
* This is a par t of the M.Sc. Thesis submi t ted by Miss Even Mikhael. ** Address: Labora tory of Genetics and Cytology, ~a t iona l Research Centre, Dokki, Cairo,
U.A.R.
432
GAMMA I R R A D I A T I O N ON VICIA F A B A 4 3 3
Materials and Methods
Material Cultivation
Air dry seeds of Vicia faba (cv. Giza 1) were sown in a sector from the irradiated field of the Co ~~ unit at Nuclear Energy Establishment at Ins- h~s. The sector consisted of 20 rows; the 1st was 5 meters from the source and the distance between each two successive rows was one meter. 40 Seeds (30 cm apart) were sown in each row, except for the ]st 5 rows where the sector was narrow and 25 seeds were sown in each row.
Samplln9 and Abnormal i t ies Evalut lon
Flower buds were collected after 50 days from sowing, from 10- -15 plants /row. Flower buds were collected from 5 plants only from the 6th row, because of the weak development of the flo- wer buds, and the small number of plants cultivated. Flower buds were fixed in Carnoy's fluid, and exami- ned using the acetocarmine smear met- hod. Abnormalities were counted in the 1st and 2rid meta-, ana- and te- lophases in the pollen mother cells. Stain ability of the pollen grains in acetocarmine was used as the index for determining pollen sterility.
Plan~s which remained ~fter collec- tion of the flower buds (about 20 plants /row) were left in the field for the pro- duction of seeds.
Mitotic and Meiotic Studies
A part of the seeds was used for mitotic studies. Observations were made from permanent root-tip squash preparations stained after Feulgen technique. Irradiation doses received by the flower buds and the seeds were calculated, using Kinsm~n law.
The other part of seeds was used for meiotic studies (2nd filial generation).
Abbreviation used: P.M.C. - - pol len m o t h e r cell
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434 SOHEIR M. A~IER, I. Y. ~IOSTAFA, EVON MIKttAEL
Seeds of 5 different plants (from each row) were sown in rows. 15 Seeds from each plant were sown 30 cm apart in one row.
M e i o s i s
Results and Discussion
The percentage of the induced abnormal pollen mother cells was pro- portional with the given doses of radiation (Table 6, compare Table 7 for the irradiation dose per day).
TABLE 2
ANALYSIS OF TEE PErCEnTAGE of the types of the occurring abnormal i t ies in the 1st meiotic division of P. M. Cs of chronically i r radiated Vicia faba plants
1 Metaphase Percentage of the different type i of abnormali t ies
Abnormal P.M.Cs in the different meiotic stages tended to increase in frequency as the dose increased (Table 1). A relatively high percentage of abnormal P.M.Cs persisted till the 2nd telophase stage. As is evident from Table 1, the frequency of abnormal P.M.Cs in the 2rid telophase stage is higher than that in the 1st telophase stage. SPARROW et al. (1952) reported that cells which are irradiated while undergoing division do not display the full extent of chromosomal damage until they have passed through inter- phase and have divided again.
T y p e s o f t h e I n d u e e d A b n o r m a l i t i e s
The percentage of the observed bridges in the 2nd anaphase and 2nd telo- phase stages (e.g. Figs 1, 2) did not exceed those observed in the 1st ann- phase and 1st telophase stages (e.g. Fig. 1 and Tables 2 and 3).
I t seems that most of the observed bridges in the 2nd anaphase stage are st icky bridges, since a high percentage of sticky bridges was observed in the 2nd metaphase stage (about 70~o , by 2075 r e.g. Fig. 6 and Table 3).
All the used doses of Co 6~ Gamma-radiat ion affected a considerable per- centage of fragments in the different meiotic stages (Figs. 1, 3 and Tables 2 and 3).
G A M M A I R R A D I A T I O N O N V I C I A .FABA 435
The occurrence of lagging chro- -~ mosomes was a common phenome- non in the different meiotic stages (e.g. Figs. 2, 4, 6 and Tables 2, 3). .e Most of the observed lagging chro- mosomes were single (e.g. Figs. 2, 4 .~ and 6). Sometimes more than one -~ lagging chromosome was present in the cell (e.g. Fig. 5). The per- o centage of lagging chromosomes was proportional with the doses (392--718 r), whereas the doses (1058--2075 r) affected only a low ~. percentage of lagging chromosomes ~" (Table 4).
I t is worth mentioning tha t when :~ the percentage of the induced types of abnormalities was calculated as percentage of the observed abnormal .~ P.M.Cs, no relation could be traced between the percentage of the in- duced abnormalities and the em- ployed doses (Table 4). When the proportion of abnormalities per .~ cell was calculated (Table 5), an .~ increasing proportion of bridges and fragments with increasing dose of accumulated radiation was ob- served.
Beside the above mentioned types of anomalies Co ~ Gamma-ra- diation affected the following ano- malies, however, in a low percentage.
Stickiness was observed in the different meiotic stages (e.g. Fig. 7).
Unequal distribution of the chro- ~ mosomes to the opposite poles at the 1st anaphase and 2nd metap- hase stages, and disturbed 2nd meta- and anaphases (e.g. Fig. 8) were classified under disturbed type o in Tables 3 and 4.
All the employed doses induced micronucleated cells (e.g. Fig. 3 and ~ Tables 2, 3 and 4). Some doses indu- o ced multinucleated cells (Tables 3 ~ ~% and 4); the largest number of nuclei observed was 5. ~ ~ ~
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TABLE 4
PERCENTAGE Of t h e abnormal i t i e s occurr ing in t h e meios is of chronica l ly i r r ad ia ted Vicia faba *lants
P e r cen t age of t he di f ferent t y p e s of abnorma l i t i e s Dose
(r)
392 471 575 718
1058 1440 2075
br id . m i e r o n u - m u l t i n u - s t ick, brid. f rag. lag. dis t .
Chronic irradiation of Vicia faba plants with Gamma-rays (392--2075 r) did not reduce pollen fertility (Table 6, compare Table 7 for the irradiation dose per day). This indicates tha t cells containing aberrations did not con- t inue to divide or were out-grown by the normal cells (SAx 1950). The
TABLE 5
PROPORTIOI~ o f t h e di f ferent t y p e s o f abnorma l i t i e s pe r cell in t h e meios is o f chronica l ly i r rad ia ted Vicia faba p lan t s
presented results are in line with those of SAx (1950) on Tradescantia palu- dosa, and NYBo~ (1956) on several plant species. The authors reported tha t chronic exposure to Gamma-rays did not seriously reduce pollen fertility or seed set.
Morphological Responses and Seed Product ion
Inhibition of shoot growth was observed of Vicia faba plants given doses of 1440--2075 r of Gamma-rays. Such doses caused also a high degree of
G A M M A I R R A D I A T I O N O N V I C I A F A B A 437
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branching, retardation of flowering and later complete seed sterility (Table 6, compare Table 7 for the irradiation dose per day). When such plants were analysed for meiotic irregularities a high degree of chromosome aberrations was observed (Table 6).
SrARROW et al. (1956) reported on the irregular branching and flowering of an amphidiploid tobacco hybrid (Nicotiana glauca X langsdorffii), after exposure to chronic Gamma radiation.
TAB~.E 7
ACCUMULATED DOSE a n d average dose ( r ) /day rec ieved b y va r ious p l a n t s (rows) t h r o u g h t he whole life o f t he p l an t
Dose received b y p l an t s a t the t i m e of
D i s t ance f r o m t h e Collection of F .B s* M e a s u r e m e n t of p l a n t source h e i g h t a n d b r anch ing** H a r v e s t i n g o f seeds*** in (m)
No F . B s 4682 3871 3249 2773 2390 2086 1830 1620 1383 1297 1169 1062
968 885
Aver . dose ( r ) /day
73 60 51 Did no t 43 p roduce 37 seeds 33 29 25 22 2O 18 17 15 14
* No of days ** No of d a y s
*** No of d a y s
o f exposure till collect ion o f F . B s 27 d a y s of exposure till m e a s u r e m e n t o f p l a n t he igh t s a n d b r a n c h i n g 56 days of exposure till h a r v e s t i n g of seeds 64 days
The present results are in accordance with those of READ (1959) who suggested that the severity of growth inhibition can be correlated with the amount of chromosome damage. Seed sterility of Vicia faba plants ir- radiated with Gamma-rays (for doses compare Tables 6 and 7) may be due to ovule sterility as EWALD and I~)DRIGUEZ (1957) suggested in their ex- periments on Hordeum distichum.
GAMMA I R R A D I A T I O N ON V I C I A ~ A B A 439
Delayed Effect
Seeds produced by chronically irradiated plants were used for both mitotic and meio- tic studies.
Mitosis
Effect on tiae mitotic index and the mitotic stages
Mitotic index of Vicia faba was not affec- ted by any of the applied doses (Table 8). No distinct relation could be traced bet- ween the doses and the percentage of the mitotic stages (Table 8). Percentage and types of the induced ano- malies
The percentage of the induced anomalies in all the mitotic stages was increased by increasing the dose (Table 8). Mieronuclei comprised the most dominent type of ano- malies affected by Gamma-irradiation. Mic- ronuclei were observed in both resting and dividing cells where their percentage was high (Table 9).
Bridges were observed also in a rela- t ively high percentage (Table 9). Lagging chromosomes were observed in the me- taphase stage and later stages. Both frag- ments and disturbed type (disturbed meta- and anaphases e.g. Fig. 9) were caused by the different doses, but, in a low percen- tage (Table 9). Tripolar anaphases (e.g. Fig. 10) were observed only after treat- ment with the highest dose. Such pheno- menon was only met with in few cells. Multinucleated cells (e.g. Figs l l , 12) were met with after t reatments with the diffe- rent doses. Table 10, represents the propor- tion of micronuclei, lagging chromosomes and bridges per cell.
Meiosis and Pollen Fertility
As is evident from Table 6, plants origi- nated from chronically irradiated seeds (F 2 -- generation of irradiated plants) pro- ved to be normal.
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TABLE 9
PEECENTAGE of the different types of abnormali t ies occurring in root-mitosis of seeds produced b y chronically irradiated Vicia faba plants
Percentage of the different types of abnormali t ies
PROPORTIO~ of the different types of abnormali t ies per cell in root-mitosis of seeds produced b y chronically irradiated Vicia faba plants
Dose Micronuclei Lagging* Bridge**
(r) No. I Per cell No. Per cell No.
885 1062 1297 1620
No of divid. cells
1675 1154 1252 1419
f
45 0.027 77 0-067
118 0.094 129 0-091
2 0.001 20 0-017 24 0.019 63 i 0-044
I Per cell
33 0.020 21 0.018 61 0.048 51 0.036
* Include lag. and frag. ** Inc lude brid. and micro. -k brid. and lag.
R e f e r e n c e s
FAVRET, E. A., RODRIGUEZ, A. A.: Induc t ion of muta t ions in barley seeds by acute t reatmenbs by g a m m a radiat ions f rom Cobalt 60. -- Rev. Inves t . Agric. 11 : 313--316, 1957.
NYBOM, N.: Some fur ther exper iments on chronic g a m m a irradiat ion of plants . -- Bot. Notiser 1 0 9 : 1 - - i 1 , 1959.
N~ZBOM, N., GUSTAFS~0N, A., GRANHALL, I., EHRENBERG, L.: The genetic effects of chronic g a m m a irradiat ion in barley. -- Heredi tas 42 : 74--84, 1956.
READ, J . : Radia t ion biology of Vicia faba in relat ion to the general problem. -- Blackwell Scientific Publication, p. 270, 1959.
SAX, K.: The cytological effects of low intensi ty radiation. -- Science 1 1 2 : 332--33, 1950. SPARROW, A. H., MOSES, M. J . , STEELE, R.: A cytological and cytochemical approach to an
unders tand ing of radia t ion damage in dividing cells. -- Brit . J . Radiol. 25 : 282--188, 1952. SPARROW, A. t I . , C~RZSTENSEN, E.: Tolerance of certain higher p lants to chronic exposure to
gamma-rad ia t ion f rom Co6% -- Science l l B : 697--98, 1953. SPARROW, A. H. , SZNGLETON, W. R.: The use of radiocobalt as a source of g a m m a rays and some
effects of chronic irradiat ion on growing plants . -- Amer. Nat . 87 : 29--47, 1953. SPARROW, A. H. , BOND, V.: Some eytogenetic and morphogenet ic effects of ionizing radiat ion on
plants . -- In : Proceedings of the Conference on Radioact ive Iso topes in Agriculture. -- U.S. Govt. Pr int ing Office, pp. 125-- 139, 1956.
SPARROW, A. H., GUNCKEL, J. E., SCHAIRER, L. A., HAGEN, G- L.: Tumor formation and other morphogenctic responses in an amphidiploid tobacco hybrid exposed to chronic gamma irradiation. -- Amcr. J . Bot. 43 : 377--388, 1956.
GAMMA IRRADIATION ON V I C I A F A B A 4 4 1
SPARROW, A. H., CUANY, R. L., ~IKSCHE, J-. P., SCHAIRER, L. A.: Some factors affecting the responses of plaats to acute and chronic radiation exposures. -- IAEA, Vienna, 1961.
SPARI~OW, A. H., lVilI~SCHE, J. P.: Correlation of nuclear volume and DNA content with higher plant tolerance to chronic radiation. -- Science 134:282--83, 1961.
YAMAKAWA, K., SPARROW, A. H.: Correlation of interphase chromosome volume and reduction of viable seed set by chronic irradiation of 21 cultivated plants during reproductive stages. -- Rad. Bet. 5 : 557--565, 1965.
YA~AKAWX, K., SPARROW, A. H.: The correlation of interphase chromosome volume with pollen abortion induced by chronic gamma-irradiation. -- Rad. Bet. 6 : 21--38, 1966.
S. ]VI. A~EI~, I. Y. MOSTAFA and EvoI~ IVIII(~AEL, Ng~rodnl v59zkumnel centrum, Dokki, Kah~ra a Odd~tlenl radiobiologie, Atomov~ agentura Kah39ra, SAR: Cytogenetiek~ studium vlivu ehroniek6ho 9area z~en i na Vicia f aba . -- Biol. Plant. 11 : 432--441, 1969.
Semena Vicla faba byla vyseta na gama polo a rostliny byly chronicky oza~ovs po eelou vegetaSni dobu. Procento indukovan3~ch abnorms PMC a frekvence ab- norm~lnfch PMC v rflzn3~ch meiotick3~ch stadiich byly primo tlm~rn6 pou~it6 dosi. NejSast~j~ilmi typy chromosoms aberaci byly mflstky v anaf~zi a teIof~i, zi, fragmenty, lagging ehromo- somy. Rostliny, kter6 byly nejbli~e zdroji zh~eni vykazovaly inhibici odno~tovgmi, sterilitu kv~tfl a semen a nepravideln6 v~tven/. Pou~it6 dose z~en l neovlivfiovaly fertilitu pylu. Semena pry6 filis generate byla pou~ita pro studium mitosy a rostliny FI pro studium meiose. Pro- eento meiotick39ch abnormalit bylo primo dm~rn6 dosi. NejSast~jw typem anoms byla pH- tomnost mikrojader v rfizn:~ch stadileh mitosy i v interf~zi. Zs indukovalo i dalw typy anomhlil, jako lagging chromosomy, fragmenty, mflstky atd. Meiose i fertility pylu v generaei, ns po oza~ov~nf, byly normhlnf.
The plates will be found at the end o] the issue.
SOHEIR M. AMER, I. Y. MOSTAFA, EVON M I K H A E L GAMMA I R R A D I A T I O N ON VIC, I A F A B A
Plate 1. (Figs. 1--4): Fig. 1. A group of P.M.Cs showing 1st anaphaso with bridge and frag- men t (left), 2nd telophase with bridge (middle) and 2nd anaphase with fragment. Fig. 2. A group of P.M.Cs in the 2nd anaphase stage showing lagging chromosomes and a bridge- f ragment configuration. Fig. 3. 2nd metaphase wi th f ragment (left P.M.C.) and 2nd telo- phase with mieronucleus (right P.M.C.). Fig. 4. 1st Anaphase with lagging chromosome. (Dose 1440 r)
SOHEIR M. AMER, I. Y. MOSTAFA, EVON MIK H A E L GAMMA I R R A D I A T I O N ON V I C I A F A B A
Plate l l . (Figs. 5--8) : Fig. 5. 2nd Metaphase with 2 lagging chromosomes. Fig. 6. 2nd Meta- phase with sticky bridge and lagging chromosome. Fig. 7. Sticky 1st metaphase. Fig. 8. Dis turbed 2rid anaphase. (Dose 1440 r)
SOHEIR M. AMER, I. Y. MOSTAFA, EVON MIK H A E L GAMMA IRRADIATION ON V I C I A F A B A
Plate III . (Figs. 9--12): Fig. 9. Disturbed metaphase, Fig. 10. micronucleated cell, and tripolar anaphase, "Fig. 11 binucleated cell (left) and trinucleated cell (right), and Fig. 12. two mul- tinucleated cells; as occurring in root--mitosis of chronically irradiated Vicia faba seeds. (Dose 1620 r)
