- Email: [email protected]
Embryo and endosperm response to ageing in Triticum Durum seeds as revealed by chromosomal damage in the root meristem
Mutation Research Elsevier Publishing Company, A m s t e r d a m Printed in The Netherlands
349
EMBRYO AND E N D O S P E R M R E S P O N S E TO A G E I N G IN T R I T I C U M DURUM SEEDS AS R E V E A L E D BY CHROMOSOMAL DAMAGE IN T H E ROOT M E R I S T E M G A B R I E L L A CORSI AND SILVANA AVANZI
Institute of Botany, University of Pisa (Italy) (Received F e b r u a r y 6th, 1969)
SUMMARY
A cytological analysis was carried out on root tips of seedlings raised from intact aged seeds (1963 crop), intact young seeds (1966 crop) and from both aged and young embryos transplanted onto either aged or young endosperms. The collected data indicate that the incidence of chromosomal aberrations detectable in an aged embryo is independent of its transplantation onto either aged or young endosperms. This implies that young endosperms do not induce any recovery from chromosomal damage in aged seeds. However, the possibility is not excluded that products responsible for chromosome aberrations might be formed in the endosperm and transferred to the embryo under some experimental conditions.
INTRODUCTION
The incidence of chromosomal damage in plants increases with the age of seeds from which they are grown4,% 1°. Several investigators indicated that chromosomal damage in aged seeds results from a chemical action by metabolites and/or waste products accumulating in seeds during ageing. In fact, it has been found that extracts of whole aged seeds of several species are able to break chromosomes in young seeds of the same or other species 5. However, it is not known in which part of the seed these products accumulate, and regarding cereal seeds, the question m a y be raised of whether the embryo is directly affected by the ageing process or the nuclear damage detectable in the embryo results from endosperm ageing. This question is not only a part of the general problem of ageing but also refers to the more particular problem of embryo-endosperm relations in seeds. NUTI RONCHI AND MARTINI 9, using the embryo transplantation technique, found that in Triticum vulgare the endosperm of old seeds inhibited the onset of mitosis in embryos of young seeds and the growth of both root and coleoptile in seedlings raised from them. These data have been confirmed b y more recent experiments. FLORIS' investigations e have shown that when young embryos are transplanted onto old instead of onto young endosperms, germination and seedling growth are clearly decreased. Mutation Res., 7 (1969) 349-355
(;AI¢I{IF.I.I.A('()RSI, AII.V\NA .\V:\NZI
35 °
However, no d a t a are a v a i l a b l e on the chromosome d a m a g e in either aged elnbrvos t r a n s p l a n t e d onto e n d o s p e r m s of y o u n g seeds or e m b r y o s excised fr(~m your~g seeds a n d t r a n s f e r r e d onto aged endosperms. In the p r e s e n t paper, the results of this t y p e of investigation are reported. MATERIALS AND METHODS Seeds (caryopses) of d u r u m wheat, I963 ("aged") a n d 1966 ( " y o u n g " ) crops, were used for the p r e s e n t investigation. In a p r e l i m i n a r y e x p e r i m e n t carried out in A p r i l 1967, t h e caryopses were s o a k e d in deionized w a t e r at 2o ° in the d a r k . A f t e r 24 h, t h e following e m b r y o t r a n s p l a n t a t i o n s were m a d e using the procedure described b y MELETTI ~. (;r) 1966 h o m o t r a n s p l a n t s (embryos excised from y o u n g seeds onto e n d o s p e r m s of y o u n g seeds: EMy/ENy) ; (2) 1963 h o m o t r a n s p l a n t s (embryos excised from aged seeds onto e n d o s p e r m s of aged seeds : EM~/EN~) ; (3) E M a / E N y h e t e r o t r a n s p l a n t s (embryos excised from aged seeds on endos p e r m s of y o u n g seeds); (4) E M y / E N a h e t e r o t r a n s p l a n t s (embryos excised from y o u n g seeds on endos p e r m s of aged seeds). I n a second e x p e r i m e n t carried out d u r i n g J u l y - A u g u s t 1967, aged a n d y o u n g seeds were s o a k e d either at 2o ° or at 3 °. T h e same 4 t y p e s of e m b r y o t r a n s p l a n t a t i o n s as in the first e x p e r i m e n t were m a d e in all possible combinations. T r a n s p l a n t e d 40
{ 3o
<
20
ta
14
16
18
20
22
2 4 T I M E (h)
Fig. I. Percentage of aberrant anaphases in intact seeds (1963 and 1966 crops), young seed homotransplants (EM~/ENy), aged seed homotransplants (EMa/ENa) and transplants EMa/ENy and EMy/ENa (aged embryo onto young endosperm and young embryo onto aged endosperm) of durum wheat. On the abscissa: hours of germination after 24 h of presoaking. • , aged intact seeds; • - - - , EMa/ENa; • . . . . . . . , EMa/ENy; , young intact seeds; - - , EMy/ENy; . . . . . , EMy/ENa. caryopses of t h e first a n d second e x p e r i m e n t s t o g e t h e r w i t h i n t a c t seeds soaked u n d e r t h e same conditions were f u r t h e r i m b i b e d in P e t r i dishes at 24 ° in the dark. F o r t h e cytological s t u d y t h e c e n t r a l seminal roots of seedlings were collected e v e r y 2 h, s t a r t i n g from t h e I 4 t h h of i m b i b i t i o n . A t each collection time, 25 roots were fixed in L a Cour's 2BD, a n d t h e i r tips were p r e p a r e d as F e u l g e n squashes. A s e p a r a t e slide was m a d e for each r o o t t i p ; t h e slides were m o u n t e d in C a n a d a balsam. Chromosome b r e a k a g e was a n a l y z e d at a n a p h a s e , a n d all a n a p h a s e s p r e s e n t in each slide
J~lutation Ties., 7 (1969) 349 355
RESPONSE TO AGEING IN T r i t i c u m durum SEEDS
351
7O
~ 60
~'~
~so (3
~
'~,..
"'" "~ "" ,, ", \ ",
40
O Ur~ 30
/"
\ ~'~.
20
/"
,/ ~'-~_. . . . . .
,
/
"'.
.-e/'" ,
/
~~
o
14
16
18
20
22
24
TIME(h)
Fig. 2. Percentage of c h r o m a t i d breaks (B') in intact seeds of b o t h 1963 and 1966 crops, y o u n g seed h o m o t r a n s p l a n t s (EMy/ENy), aged seed h o m o t r a n s p l a n t s (EMa/ENa) and t r a n s p l a n t s EMa/ E N y a n d E M y / E N a (aged e m b r y o onto y o u n g e n d o s p e r m and y o u n g e m b r y o onto aged endosperm) of d u r u m wheat. On the abscissa: h o u r s of germination after 24 h of presoaking. • , aged i n t a c t seeds; • . . . . , E M a / E N a ; • . . . . . . . , EMa/ENy; - - , y o u n g intact seeds; . . . . , EMy/ ENy; . . . . . . . , EMy/ENa.
were scored. In some preliminary investigations the time of the onset of the first mitosis in the meristem was detected; thereafter the analysis was limited to the five collection times following the mitotic initiation. Nuclear damage was expressed as percent of aberrant anaphases, chromatid breaks (B') and chromosome breaks (B"). RESULTS
The nuclear damage observed in the first experiment is illustrated in Figs. i, 2 and 3- In Fig. i, it is seen that chromosome damage, expressed as percentage of
v
15
~o o*
16
18
20
".
22
24 TIME(h)
Fig. 3. Percentage of c h r o m o s o m e breaks (B") in intact seeds of b o t h 1963 and 1966 crops, y o u n g seed h o n l o t r a n s p l a n t s (EMy/ENy), aged seed h o m o t r a n s p l a n t s (EMa/ENa) and t r a n s p l a n t s EMa/EN~ a n d E M y / E N a (aged e m b r y o onto y o u n g e n d o s p e r m and y o u n g e m b r y o onto aged endosperm) of d u r u m wheat. On the abscissa: h o u r s of germination after 24 h of presoaking. @--, aged i n t a c t seeds; • . . . . , EM~/ENa; • . . . . . . . , E M a / E N y ; - - , y o u n g intact seeds; , E M y / E N y ; . . . . . . . , EMy/ENa.
~rutation Res., 7 (1969) 349-355
352
(iAI~,RII.;LLA CORSI, SlI_VAN\ AVANZI
aberrant anaphases, is clearly higher when aged embryos are involved than when aged endosperms are used. This fact is even more apparent, if the percentage of chromatid breaks is determined (lqg. 2). In fact, the roots grown from EM~/ENr transplants show chromosomal damage as high as that shown bv roots of intact aged seeds and of EM~/EN~ homotransplants; the roots grown from intact young seeds, EMy/ENy homotransplants and EMy/EN~ heterotransplants, are clearly less damaged. The aberrations are mostly of the chromatid type. Anaphases with B" aberrations which were formed in the resting embryo before chromosome duplication are much less frequent; they never exceed 25/o o/ of aberrant anaphases (Table 1).
TABLE
I
ANAPHASES
SHOWING
CHROMOSOME-TYPE
PHASES IN INTACT AND TRANSPLANTED
DAMAGE
(g")
IN
PERCENT
OF
TOTAL
ABERRANT
ANA-
SEEDS
E M a a n d E N a : e m b r y o a n d e n d o s p e r m , r e s p e c t i v e l y , f r o m a g e d seeds; E M y a n d E N y : e m b r y o a n d e n d o s p e r m , r e s p e c t i v e l y , f r o m y o u n g seeds.
Seed or transplant
I n t a c t y o u n g seeds I n t a c t a g e d seeds EMy/ENy EMa/ENa EMy/ENa EMa/ENy
Hours after embryo transplantation or after end of seed soaking (no transplantation) ±4 z6 28 20 o o o o o o
9.o o o o o o
o i 2.o 2o.o 7.1 o 6.0
24.6 7.0
17.6 20.0
24 15. 3 23.0
o
2 i.o
14.2
16.6 4.5 25.o
22.2 ti, t ] 2. 7
25.o 4-5 5.6
Table I I gives a survey of the results obtained in the second experiment. In this table, the anaphases scored within the 12 h following the onset of mitosis are pooled, these being cells that divide for the first time during germination. I t is seen that at both soaking temperatures (2o and 3°), the chromosome damage in aged embryos is higher than that induced b y aged endosperms in embryos of young seeds. Anaphases showing B'-type aberrations constitute the predominant fraction of aberrant anaphases in the second experiment as well (Table III). DISCUSSION
The above results seem to indicate that the chromosomal damage induced by ageing in the embryo is not the result of the senescence of the endosperm. In fact, an embryo from an aged seed (1963) shows a comparable incidence of chromosomal aberrations independent of its transplantation onto either aged or young endosperms (Figs. I, 2 and 3). This also implies that young endosperms do not induce any recovery from chromosomal damage in aged seeds. However, a young embryo seems to respond with low chrornosomal damage to transplantation onto aged endosperms (Figs. I and 2); the damage is at any rate much lower than that occurring in aged embryos. FLORISs has shown that soaking the seed prior to transplantation at 3 ° instead of at 2o ° decreased manifold the germination and growth of young embryos transplanted onto aged endosperms. This observation led to the suspicion that the subMutation Res., 7 (I969) 3 4 9 - 3 5 5
,,al
cj~
..q
II
Groups~ I Number of anaphases scored
20.2
51.3 12.2 32"7 39-5 13. 4
25.2 7-4 19,7 21. 4 8,2
B" per zoo anaphases
12, 4
Aberrant anaphases (~o)
4.0 0. 7 5.4 2.6 0. 9
o
B" per zoo anaphases
347 1296 227 428 1252
841
11 Number of anaphases scored
26.8 6.2 20.7 20.3 6. 7
lO.8
Aberrant anaphases (%)
53.6 io.2 37-4 34.3 ii.t
17.8
t3" per ioo anaphases
4.6 0. 9 5.7 4 .6 1. 5
o.8
lY" per zoo anaphases
1139 616 939 lO2O
III Number ofanaphases scored
5,5 13.7 I4. I 8.8
Aberrant anaphases (%)
8,4 23.0 25.4 13. 9
t3" per ~oo anaphases
o-3 1. 9 1.9 o.i
B" per ioo anaphases
994 288 395 469
I V Number ofanaphases scored
6. 9 20. 5 20.2 6.8
Aberrant anaphases (°o)
12. 5 37.5 36.7 ii.o
B" per IOO anaphases
0.6 4.5 3.5 0.8
B" per IOO anaphase~
III
a Transplants
Groups a
99.2 9o.o 93.6 81.4 9o. 7 91.6
(%J
1 -Ariaphases with B '
o 6.8 3.8 14.4 7.2 6.9
(,<>)
dnaphases with B" o~ o.8 3.2 2.6 4.2 2.1 I. 5
(%)
Anaphases with t~" I B" 93.4 85.3 87.2 80.8 83.9 88.0
(%)
1l Anaphases with B '
m a d e i n 4 d i f f e r e n t g r o u p s , a s i n T a b l e 1 i.
Intact young seeds Intact aged seeds EMy/ENy EMa/ENa EM~/ENy EM~,/EN~
Seed or transplant
5.4 7.9 9.4 14. 9 I2.6 8.3
(%)
Anaphases with B" 1.2 6.8 2.4 4-3 3.5 3.7
(%)
93.6 90.0 89-4 97-7
(%)
IlI A n a p h a s e s Ariawith phases B" ~ B" with B"
4,7 8.0 6.1 2.3
(%)
1.7 2.0 4-5 o
(%)
AnaAnaphases phases with with B" B" ! B"
91.5 86.6 88.7 90.6
(%)
IV dnaphases with B '
5.7 II.8 6.3 6. 3
(%)
Ariaphases with B"
2.8 1.6 5 .o 3.1
(%)
Anaphases with B" F B"
RELATIVE PERCENTAGES OF ANAPHASES KVITft g ~ ABERRATIONS, g " ABERRATIONS AND g z t t~" ABERRATIONS IN THE FIRST MITOTIC CYCLE OF THE ROOT MERISTEM
TABLE
a T r a n s p l a n t s m a d e in 4 d i f f e r e n t g r o u p s : I, e m b r y o s a n d e n d o s p e r m s f r o m s e e d s s o a k e d a t 2o':; I I , e m b r y o . ~ a n d e n d o s p e r m s f r o m s e e d s s o a k e d a t 3°; 111, e m b r y o s f r o m s e e d s s o a k e d a t 2 0 ° a n d e n d o s p e r n a s f r o m s e e d s s o a k e d a t 3°; I V , e m b r y o s f r o l n s e e d s s o a k e d a t 3 ° a n d e n d o s p e r m s f r o m s e e d s s o a k e d a t 2 o °.
Intact y o u n g s e e d s lOO8 Intact aged seeds 748 Emy/ENy lO3O EMa/ENa 492 EMa/ENy 653 EMy/F,N a 869
Seed or transplant
PERCENTAGE OF ABERRANT ANAPHASES OF CHROMATID (B') AND CIIROMOSOME (1~~') BREAKS IN INTACT AND TRANSPLANTED SEEDS
TABLE
~J oq
©
0
.JS~
(;A|-H~II':LLA ('()I,:SI, S l I . \ A2~,\ AV.XYZI
stances resp(msib/e for tlm negative ei]ects on the en/brv~) \w,r~~ lea(l~ed h()m tl,' endosperm by soaking at 2(; and not, ~w t() a lesser extent, by snaking at 3 - TII(' results of our second experiment seen| to rule (rot this possibility, since at b()th presoaking temperatures (2o and 3"), the EM~/EN:. transplants wen, damaged t~ the same extent as both intact aged seeds and EM:,/EN~ transplants (Table l I, groups I and II). It should also be noted that aged endosperms did n()t induce any clear increase in chromosomal dalnage in the young embryo (Table II). In this sense the results of the second experiment arc even more definitive than the results (~f the first one. Student's I test was used for the statistical analysis which indicated that the difference was significant only when aged embryos were compared with young embryos, independently of their transplant onto aged or young endosperms. It therefore seems, that the chromosomal damage detectable in the ro()t meristem of aged embryos under our experimental conditions is not the result of a senescence of the endosperm. However, the possibility, that products responsible for chromosome aberrations are formed in the endosperm and then transferred t() the embryo cannot be excluded by these experiments. In fact, the seeds empl(ued in the present investigation are some years younger than the caryopses emplob-ed by I~'LORIS% ~ which might account for the discrepancy between FLORIS' results and our own. It should also be noted that simply the process of transplanting seems to decrease the damage detectable in the intact aged seeds (see EM,,/EN~ in comparison with intact aged seeds in Table II). This fact probably indicates that some substances are lost during the transplanting procedure. The problems raised by the present investigation will be studied in experiments combining embryo transplantation and labeling with metabolic precursors. Another finding of our investigation is that chromosomal damage is predominantly of the chromatid type. The fact that both types of aberrations, B' and B", are present, is in line with the discovery that in the radicle meristem of dry (resting) seeds of Triticum durum there occur nuclei with 2C and with 4C DNA contents~, 3. Other investigators~,% ~°, however, detected mainly or exclusively, chromosome-type aberrations in aged seeds. A possible explanation of this disagreement is that previous data may have been collected when the first mitotic cycle was over or nearly complete, while our study of chromosome breakage at different times disclosed the response of both G,~ and G~ cells. Nevertheless the high incidence of B' aberrations requires an explanation. Table I I shows the relative percentages of anaphases with the three types of aberrations: B', B" and B ' + B " . The relatively high frequency of anaphases with B ' + B " aberrations probably indicates that some mutagenie agent(s) is (are) also working during the germination process; i.e. the high incidence of chromatid-type damage might be a specific response to mutagen(s) of some phases (DNA-synthetic phase, post-synthetic phase or both) of the metabolically active mitotic cycle. The possibility that the incidence of B'-type damage may decrease in the course of seed ageing cannot be excluded. In fact, soon after the harvest, during the dormancy period of Triticum durum, germinated seeds show exclusively chromatid-type damage in the root tip. A study is now in progress to investigate qualitatively and quantitatively how chromosomal damage evolves over the whole life span of Triticum durum seeds. 3lutation lees., 7 (t969) 349 355
RESPONSE TO AGEING IN T r i t i c u m
durum
SEEDS
355
ACKNOWLEDGEMENTS
We are grateful to Dr. C. 17LORIS for suggestions and advice and to Prof. P. MELETTI for discussion and criticism. The technical assistance of Mrs. V. SBRANA, R. BERTINI and G. CIONINI is also acknowledged. REFERENCES I AVANZI, S., A. M. INNOCENTI AND A. M. TAGLIASACCHI, Conlportanlento alla germinazione e mutabilitk cronaosomica s p o n t a n e a in Triticum d u r u m Desf. d u r a n t e i primi due anni di vita del seine, Giorn. Botan. Ital., lO2 (i968) 381-395 . 2 AVANZI, S., A. BRUNORr, ~'. D'AMATO, ~r. NUTI RONCHI AND G. T. SCARASCIA ~.IUGNOZZA, Occurrence of 2C ((;1) and 4C (G~) nuclei in the radicle nleristems of d r y seeds in Triticum d u r u m Desf. I t s implications in studies on c h r o m o s o m e breakage and on developmental processes, Caryologia, 16 (i963) 553 558. 3 AVANZI, S., A. BRUNORI AND g. GIORGI, Radiation response of dry seeds in two varieties of Triticum durum, Mutation Res., 3 (1966) 426-437 . 4 D'AMATO, F., Di alcuni aspetti fisiologici e genetici dell'invecchiamento dei semi. Contributo al p r o b l e m a della senescenza e della mutabilit~ s p o n t a n e a nei vegetali, Caryologia, 6 (1954) 217 240. 5 D'AMATO, F., AND O. HOFFMANN-OSTENIIOF, Metabolism and s p o n t a n e o u s m u t a t i o n s in plants, A d v a n . Genet., 8 (1956) 1-28. 6 FLORIS, C., The possible role of the e n d o s p e r m in the aging of the e m b r y o in the w h e a t seed, Giorn. Botan. Ital., 73 (1966) 349-35 o. 7 FLORIS, C., C o m p o r t a m e n t o di enlbrioni ed endospermi provenienti da semi di Triticunl di et~ crescente, Giorn. Botan. Ital., lO2 (1968) 559-560. 8 MELETTI, P., Revisione critica delle tecniche di ineesto fra graminacee con particolare riguardo ai metodi di t r a p i a n t o embrionale, Nuovo Giorn. Botan. Ital., 66 (1959) 43S-45 o. 9 N u r i RONCHI, V., AND G. MARTINI, GerminabilitS., sviluppo delle plantule e frequenza di aberrazioni cromosomiche in r a p p o r t o alla etk del seine di frumento, Caryologia, 15 (1962) 293-3o2. io SAX, K., Aspects of aging in plants, A n n . Rev. Plant Physiol., 13 (1962) 489-506.
Mutation Res., 7 (1969) 349-355
349
EMBRYO AND E N D O S P E R M R E S P O N S E TO A G E I N G IN T R I T I C U M DURUM SEEDS AS R E V E A L E D BY CHROMOSOMAL DAMAGE IN T H E ROOT M E R I S T E M G A B R I E L L A CORSI AND SILVANA AVANZI
Institute of Botany, University of Pisa (Italy) (Received F e b r u a r y 6th, 1969)
SUMMARY
A cytological analysis was carried out on root tips of seedlings raised from intact aged seeds (1963 crop), intact young seeds (1966 crop) and from both aged and young embryos transplanted onto either aged or young endosperms. The collected data indicate that the incidence of chromosomal aberrations detectable in an aged embryo is independent of its transplantation onto either aged or young endosperms. This implies that young endosperms do not induce any recovery from chromosomal damage in aged seeds. However, the possibility is not excluded that products responsible for chromosome aberrations might be formed in the endosperm and transferred to the embryo under some experimental conditions.
INTRODUCTION
The incidence of chromosomal damage in plants increases with the age of seeds from which they are grown4,% 1°. Several investigators indicated that chromosomal damage in aged seeds results from a chemical action by metabolites and/or waste products accumulating in seeds during ageing. In fact, it has been found that extracts of whole aged seeds of several species are able to break chromosomes in young seeds of the same or other species 5. However, it is not known in which part of the seed these products accumulate, and regarding cereal seeds, the question m a y be raised of whether the embryo is directly affected by the ageing process or the nuclear damage detectable in the embryo results from endosperm ageing. This question is not only a part of the general problem of ageing but also refers to the more particular problem of embryo-endosperm relations in seeds. NUTI RONCHI AND MARTINI 9, using the embryo transplantation technique, found that in Triticum vulgare the endosperm of old seeds inhibited the onset of mitosis in embryos of young seeds and the growth of both root and coleoptile in seedlings raised from them. These data have been confirmed b y more recent experiments. FLORIS' investigations e have shown that when young embryos are transplanted onto old instead of onto young endosperms, germination and seedling growth are clearly decreased. Mutation Res., 7 (1969) 349-355
(;AI¢I{IF.I.I.A('()RSI, AII.V\NA .\V:\NZI
35 °
However, no d a t a are a v a i l a b l e on the chromosome d a m a g e in either aged elnbrvos t r a n s p l a n t e d onto e n d o s p e r m s of y o u n g seeds or e m b r y o s excised fr(~m your~g seeds a n d t r a n s f e r r e d onto aged endosperms. In the p r e s e n t paper, the results of this t y p e of investigation are reported. MATERIALS AND METHODS Seeds (caryopses) of d u r u m wheat, I963 ("aged") a n d 1966 ( " y o u n g " ) crops, were used for the p r e s e n t investigation. In a p r e l i m i n a r y e x p e r i m e n t carried out in A p r i l 1967, t h e caryopses were s o a k e d in deionized w a t e r at 2o ° in the d a r k . A f t e r 24 h, t h e following e m b r y o t r a n s p l a n t a t i o n s were m a d e using the procedure described b y MELETTI ~. (;r) 1966 h o m o t r a n s p l a n t s (embryos excised from y o u n g seeds onto e n d o s p e r m s of y o u n g seeds: EMy/ENy) ; (2) 1963 h o m o t r a n s p l a n t s (embryos excised from aged seeds onto e n d o s p e r m s of aged seeds : EM~/EN~) ; (3) E M a / E N y h e t e r o t r a n s p l a n t s (embryos excised from aged seeds on endos p e r m s of y o u n g seeds); (4) E M y / E N a h e t e r o t r a n s p l a n t s (embryos excised from y o u n g seeds on endos p e r m s of aged seeds). I n a second e x p e r i m e n t carried out d u r i n g J u l y - A u g u s t 1967, aged a n d y o u n g seeds were s o a k e d either at 2o ° or at 3 °. T h e same 4 t y p e s of e m b r y o t r a n s p l a n t a t i o n s as in the first e x p e r i m e n t were m a d e in all possible combinations. T r a n s p l a n t e d 40
{ 3o
<
20
ta
14
16
18
20
22
2 4 T I M E (h)
Fig. I. Percentage of aberrant anaphases in intact seeds (1963 and 1966 crops), young seed homotransplants (EM~/ENy), aged seed homotransplants (EMa/ENa) and transplants EMa/ENy and EMy/ENa (aged embryo onto young endosperm and young embryo onto aged endosperm) of durum wheat. On the abscissa: hours of germination after 24 h of presoaking. • , aged intact seeds; • - - - , EMa/ENa; • . . . . . . . , EMa/ENy; , young intact seeds; - - , EMy/ENy; . . . . . , EMy/ENa. caryopses of t h e first a n d second e x p e r i m e n t s t o g e t h e r w i t h i n t a c t seeds soaked u n d e r t h e same conditions were f u r t h e r i m b i b e d in P e t r i dishes at 24 ° in the dark. F o r t h e cytological s t u d y t h e c e n t r a l seminal roots of seedlings were collected e v e r y 2 h, s t a r t i n g from t h e I 4 t h h of i m b i b i t i o n . A t each collection time, 25 roots were fixed in L a Cour's 2BD, a n d t h e i r tips were p r e p a r e d as F e u l g e n squashes. A s e p a r a t e slide was m a d e for each r o o t t i p ; t h e slides were m o u n t e d in C a n a d a balsam. Chromosome b r e a k a g e was a n a l y z e d at a n a p h a s e , a n d all a n a p h a s e s p r e s e n t in each slide
J~lutation Ties., 7 (1969) 349 355
RESPONSE TO AGEING IN T r i t i c u m durum SEEDS
351
7O
~ 60
~'~
~so (3
~
'~,..
"'" "~ "" ,, ", \ ",
40
O Ur~ 30
/"
\ ~'~.
20
/"
,/ ~'-~_. . . . . .
,
/
"'.
.-e/'" ,
/
~~
o
14
16
18
20
22
24
TIME(h)
Fig. 2. Percentage of c h r o m a t i d breaks (B') in intact seeds of b o t h 1963 and 1966 crops, y o u n g seed h o m o t r a n s p l a n t s (EMy/ENy), aged seed h o m o t r a n s p l a n t s (EMa/ENa) and t r a n s p l a n t s EMa/ E N y a n d E M y / E N a (aged e m b r y o onto y o u n g e n d o s p e r m and y o u n g e m b r y o onto aged endosperm) of d u r u m wheat. On the abscissa: h o u r s of germination after 24 h of presoaking. • , aged i n t a c t seeds; • . . . . , E M a / E N a ; • . . . . . . . , EMa/ENy; - - , y o u n g intact seeds; . . . . , EMy/ ENy; . . . . . . . , EMy/ENa.
were scored. In some preliminary investigations the time of the onset of the first mitosis in the meristem was detected; thereafter the analysis was limited to the five collection times following the mitotic initiation. Nuclear damage was expressed as percent of aberrant anaphases, chromatid breaks (B') and chromosome breaks (B"). RESULTS
The nuclear damage observed in the first experiment is illustrated in Figs. i, 2 and 3- In Fig. i, it is seen that chromosome damage, expressed as percentage of
v
15
~o o*
16
18
20
".
22
24 TIME(h)
Fig. 3. Percentage of c h r o m o s o m e breaks (B") in intact seeds of b o t h 1963 and 1966 crops, y o u n g seed h o n l o t r a n s p l a n t s (EMy/ENy), aged seed h o m o t r a n s p l a n t s (EMa/ENa) and t r a n s p l a n t s EMa/EN~ a n d E M y / E N a (aged e m b r y o onto y o u n g e n d o s p e r m and y o u n g e m b r y o onto aged endosperm) of d u r u m wheat. On the abscissa: h o u r s of germination after 24 h of presoaking. @--, aged i n t a c t seeds; • . . . . , EM~/ENa; • . . . . . . . , E M a / E N y ; - - , y o u n g intact seeds; , E M y / E N y ; . . . . . . . , EMy/ENa.
~rutation Res., 7 (1969) 349-355
352
(iAI~,RII.;LLA CORSI, SlI_VAN\ AVANZI
aberrant anaphases, is clearly higher when aged embryos are involved than when aged endosperms are used. This fact is even more apparent, if the percentage of chromatid breaks is determined (lqg. 2). In fact, the roots grown from EM~/ENr transplants show chromosomal damage as high as that shown bv roots of intact aged seeds and of EM~/EN~ homotransplants; the roots grown from intact young seeds, EMy/ENy homotransplants and EMy/EN~ heterotransplants, are clearly less damaged. The aberrations are mostly of the chromatid type. Anaphases with B" aberrations which were formed in the resting embryo before chromosome duplication are much less frequent; they never exceed 25/o o/ of aberrant anaphases (Table 1).
TABLE
I
ANAPHASES
SHOWING
CHROMOSOME-TYPE
PHASES IN INTACT AND TRANSPLANTED
DAMAGE
(g")
IN
PERCENT
OF
TOTAL
ABERRANT
ANA-
SEEDS
E M a a n d E N a : e m b r y o a n d e n d o s p e r m , r e s p e c t i v e l y , f r o m a g e d seeds; E M y a n d E N y : e m b r y o a n d e n d o s p e r m , r e s p e c t i v e l y , f r o m y o u n g seeds.
Seed or transplant
I n t a c t y o u n g seeds I n t a c t a g e d seeds EMy/ENy EMa/ENa EMy/ENa EMa/ENy
Hours after embryo transplantation or after end of seed soaking (no transplantation) ±4 z6 28 20 o o o o o o
9.o o o o o o
o i 2.o 2o.o 7.1 o 6.0
24.6 7.0
17.6 20.0
24 15. 3 23.0
o
2 i.o
14.2
16.6 4.5 25.o
22.2 ti, t ] 2. 7
25.o 4-5 5.6
Table I I gives a survey of the results obtained in the second experiment. In this table, the anaphases scored within the 12 h following the onset of mitosis are pooled, these being cells that divide for the first time during germination. I t is seen that at both soaking temperatures (2o and 3°), the chromosome damage in aged embryos is higher than that induced b y aged endosperms in embryos of young seeds. Anaphases showing B'-type aberrations constitute the predominant fraction of aberrant anaphases in the second experiment as well (Table III). DISCUSSION
The above results seem to indicate that the chromosomal damage induced by ageing in the embryo is not the result of the senescence of the endosperm. In fact, an embryo from an aged seed (1963) shows a comparable incidence of chromosomal aberrations independent of its transplantation onto either aged or young endosperms (Figs. I, 2 and 3). This also implies that young endosperms do not induce any recovery from chromosomal damage in aged seeds. However, a young embryo seems to respond with low chrornosomal damage to transplantation onto aged endosperms (Figs. I and 2); the damage is at any rate much lower than that occurring in aged embryos. FLORISs has shown that soaking the seed prior to transplantation at 3 ° instead of at 2o ° decreased manifold the germination and growth of young embryos transplanted onto aged endosperms. This observation led to the suspicion that the subMutation Res., 7 (I969) 3 4 9 - 3 5 5
,,al
cj~
..q
II
Groups~ I Number of anaphases scored
20.2
51.3 12.2 32"7 39-5 13. 4
25.2 7-4 19,7 21. 4 8,2
B" per zoo anaphases
12, 4
Aberrant anaphases (~o)
4.0 0. 7 5.4 2.6 0. 9
o
B" per zoo anaphases
347 1296 227 428 1252
841
11 Number of anaphases scored
26.8 6.2 20.7 20.3 6. 7
lO.8
Aberrant anaphases (%)
53.6 io.2 37-4 34.3 ii.t
17.8
t3" per ioo anaphases
4.6 0. 9 5.7 4 .6 1. 5
o.8
lY" per zoo anaphases
1139 616 939 lO2O
III Number ofanaphases scored
5,5 13.7 I4. I 8.8
Aberrant anaphases (%)
8,4 23.0 25.4 13. 9
t3" per ~oo anaphases
o-3 1. 9 1.9 o.i
B" per ioo anaphases
994 288 395 469
I V Number ofanaphases scored
6. 9 20. 5 20.2 6.8
Aberrant anaphases (°o)
12. 5 37.5 36.7 ii.o
B" per IOO anaphases
0.6 4.5 3.5 0.8
B" per IOO anaphase~
III
a Transplants
Groups a
99.2 9o.o 93.6 81.4 9o. 7 91.6
(%J
1 -Ariaphases with B '
o 6.8 3.8 14.4 7.2 6.9
(,<>)
dnaphases with B" o~ o.8 3.2 2.6 4.2 2.1 I. 5
(%)
Anaphases with t~" I B" 93.4 85.3 87.2 80.8 83.9 88.0
(%)
1l Anaphases with B '
m a d e i n 4 d i f f e r e n t g r o u p s , a s i n T a b l e 1 i.
Intact young seeds Intact aged seeds EMy/ENy EMa/ENa EM~/ENy EM~,/EN~
Seed or transplant
5.4 7.9 9.4 14. 9 I2.6 8.3
(%)
Anaphases with B" 1.2 6.8 2.4 4-3 3.5 3.7
(%)
93.6 90.0 89-4 97-7
(%)
IlI A n a p h a s e s Ariawith phases B" ~ B" with B"
4,7 8.0 6.1 2.3
(%)
1.7 2.0 4-5 o
(%)
AnaAnaphases phases with with B" B" ! B"
91.5 86.6 88.7 90.6
(%)
IV dnaphases with B '
5.7 II.8 6.3 6. 3
(%)
Ariaphases with B"
2.8 1.6 5 .o 3.1
(%)
Anaphases with B" F B"
RELATIVE PERCENTAGES OF ANAPHASES KVITft g ~ ABERRATIONS, g " ABERRATIONS AND g z t t~" ABERRATIONS IN THE FIRST MITOTIC CYCLE OF THE ROOT MERISTEM
TABLE
a T r a n s p l a n t s m a d e in 4 d i f f e r e n t g r o u p s : I, e m b r y o s a n d e n d o s p e r m s f r o m s e e d s s o a k e d a t 2o':; I I , e m b r y o . ~ a n d e n d o s p e r m s f r o m s e e d s s o a k e d a t 3°; 111, e m b r y o s f r o m s e e d s s o a k e d a t 2 0 ° a n d e n d o s p e r n a s f r o m s e e d s s o a k e d a t 3°; I V , e m b r y o s f r o l n s e e d s s o a k e d a t 3 ° a n d e n d o s p e r m s f r o m s e e d s s o a k e d a t 2 o °.
Intact y o u n g s e e d s lOO8 Intact aged seeds 748 Emy/ENy lO3O EMa/ENa 492 EMa/ENy 653 EMy/F,N a 869
Seed or transplant
PERCENTAGE OF ABERRANT ANAPHASES OF CHROMATID (B') AND CIIROMOSOME (1~~') BREAKS IN INTACT AND TRANSPLANTED SEEDS
TABLE
~J oq
©
0
.JS~
(;A|-H~II':LLA ('()I,:SI, S l I . \ A2~,\ AV.XYZI
stances resp(msib/e for tlm negative ei]ects on the en/brv~) \w,r~~ lea(l~ed h()m tl,' endosperm by soaking at 2(; and not, ~w t() a lesser extent, by snaking at 3 - TII(' results of our second experiment seen| to rule (rot this possibility, since at b()th presoaking temperatures (2o and 3"), the EM~/EN:. transplants wen, damaged t~ the same extent as both intact aged seeds and EM:,/EN~ transplants (Table l I, groups I and II). It should also be noted that aged endosperms did n()t induce any clear increase in chromosomal dalnage in the young embryo (Table II). In this sense the results of the second experiment arc even more definitive than the results (~f the first one. Student's I test was used for the statistical analysis which indicated that the difference was significant only when aged embryos were compared with young embryos, independently of their transplant onto aged or young endosperms. It therefore seems, that the chromosomal damage detectable in the ro()t meristem of aged embryos under our experimental conditions is not the result of a senescence of the endosperm. However, the possibility, that products responsible for chromosome aberrations are formed in the endosperm and then transferred t() the embryo cannot be excluded by these experiments. In fact, the seeds empl(ued in the present investigation are some years younger than the caryopses emplob-ed by I~'LORIS% ~ which might account for the discrepancy between FLORIS' results and our own. It should also be noted that simply the process of transplanting seems to decrease the damage detectable in the intact aged seeds (see EM,,/EN~ in comparison with intact aged seeds in Table II). This fact probably indicates that some substances are lost during the transplanting procedure. The problems raised by the present investigation will be studied in experiments combining embryo transplantation and labeling with metabolic precursors. Another finding of our investigation is that chromosomal damage is predominantly of the chromatid type. The fact that both types of aberrations, B' and B", are present, is in line with the discovery that in the radicle meristem of dry (resting) seeds of Triticum durum there occur nuclei with 2C and with 4C DNA contents~, 3. Other investigators~,% ~°, however, detected mainly or exclusively, chromosome-type aberrations in aged seeds. A possible explanation of this disagreement is that previous data may have been collected when the first mitotic cycle was over or nearly complete, while our study of chromosome breakage at different times disclosed the response of both G,~ and G~ cells. Nevertheless the high incidence of B' aberrations requires an explanation. Table I I shows the relative percentages of anaphases with the three types of aberrations: B', B" and B ' + B " . The relatively high frequency of anaphases with B ' + B " aberrations probably indicates that some mutagenie agent(s) is (are) also working during the germination process; i.e. the high incidence of chromatid-type damage might be a specific response to mutagen(s) of some phases (DNA-synthetic phase, post-synthetic phase or both) of the metabolically active mitotic cycle. The possibility that the incidence of B'-type damage may decrease in the course of seed ageing cannot be excluded. In fact, soon after the harvest, during the dormancy period of Triticum durum, germinated seeds show exclusively chromatid-type damage in the root tip. A study is now in progress to investigate qualitatively and quantitatively how chromosomal damage evolves over the whole life span of Triticum durum seeds. 3lutation lees., 7 (t969) 349 355
RESPONSE TO AGEING IN T r i t i c u m
durum
SEEDS
355
ACKNOWLEDGEMENTS
We are grateful to Dr. C. 17LORIS for suggestions and advice and to Prof. P. MELETTI for discussion and criticism. The technical assistance of Mrs. V. SBRANA, R. BERTINI and G. CIONINI is also acknowledged. REFERENCES I AVANZI, S., A. M. INNOCENTI AND A. M. TAGLIASACCHI, Conlportanlento alla germinazione e mutabilitk cronaosomica s p o n t a n e a in Triticum d u r u m Desf. d u r a n t e i primi due anni di vita del seine, Giorn. Botan. Ital., lO2 (i968) 381-395 . 2 AVANZI, S., A. BRUNORr, ~'. D'AMATO, ~r. NUTI RONCHI AND G. T. SCARASCIA ~.IUGNOZZA, Occurrence of 2C ((;1) and 4C (G~) nuclei in the radicle nleristems of d r y seeds in Triticum d u r u m Desf. I t s implications in studies on c h r o m o s o m e breakage and on developmental processes, Caryologia, 16 (i963) 553 558. 3 AVANZI, S., A. BRUNORI AND g. GIORGI, Radiation response of dry seeds in two varieties of Triticum durum, Mutation Res., 3 (1966) 426-437 . 4 D'AMATO, F., Di alcuni aspetti fisiologici e genetici dell'invecchiamento dei semi. Contributo al p r o b l e m a della senescenza e della mutabilit~ s p o n t a n e a nei vegetali, Caryologia, 6 (1954) 217 240. 5 D'AMATO, F., AND O. HOFFMANN-OSTENIIOF, Metabolism and s p o n t a n e o u s m u t a t i o n s in plants, A d v a n . Genet., 8 (1956) 1-28. 6 FLORIS, C., The possible role of the e n d o s p e r m in the aging of the e m b r y o in the w h e a t seed, Giorn. Botan. Ital., 73 (1966) 349-35 o. 7 FLORIS, C., C o m p o r t a m e n t o di enlbrioni ed endospermi provenienti da semi di Triticunl di et~ crescente, Giorn. Botan. Ital., lO2 (1968) 559-560. 8 MELETTI, P., Revisione critica delle tecniche di ineesto fra graminacee con particolare riguardo ai metodi di t r a p i a n t o embrionale, Nuovo Giorn. Botan. Ital., 66 (1959) 43S-45 o. 9 N u r i RONCHI, V., AND G. MARTINI, GerminabilitS., sviluppo delle plantule e frequenza di aberrazioni cromosomiche in r a p p o r t o alla etk del seine di frumento, Caryologia, 15 (1962) 293-3o2. io SAX, K., Aspects of aging in plants, A n n . Rev. Plant Physiol., 13 (1962) 489-506.
Mutation Res., 7 (1969) 349-355