- Email: [email protected]
Genetic effects of chronic gamma irradiation in durum wheat
IZadialiutL
&Amy,
1968,
GENETIC
Vol.
8, pp. 49 Lo 58. Pcrgamon
EFFECTS B. DONINI
Laboratory
Press. l’rinlcd
in Great
Brit&.
OF CHRONIC GAMMA IN DURUM WHEAT” and G. T. S$LUUSCIA
IRRADIATION
MUGNOZZA
for the Applications of Nuclear Energy in Agriculture, for Nuclear Research, Rome, Italy
National
Committee
and F. D’AMATO Institute
of Genetics, (Received
The
University,
20 March
Pisa,
Italy
1967)
Abstract-Three
cultivars of durunt wheat, Cappelli, Aziziah and Russello, and a radiation induced stable mutant of Cappelli, brachytic, were subjected to chronic y-irradiation from @%o for their whole lift cycle (from the 3-leaf stage to harvest). Radiation exposures of 5, 10, 15, 20, 25 and 30 R/20-hr day were applied. Mature spikes were harvested from the irradiated plants keeping spikes produced by the main culms (‘main spikes’) separate from those produced by tillers (‘secondary spikes’). Both spikelet fertility and germination of caryopses decreased with increasing exposure rate. In the first generation from irradiated plants (yz of previous workers), no case of chlorophyll mutation was found. In the second generation (ya of previous workers), chlorophyll mutations were ascertained in two categories of plants: (a) plants heterozygous for the mutation (all spikes segregating for the mutation) and (6) plants chimeric for the mutation (segregation for mutants occurring in one or two-but not allspikes). The two types of mutations occurred with a frequency of 20 and 80 per cent respectively. This shows that, in durutn wheat plants irradiated for their whole life cycle, the majority of recoverable mutations are induced in the post-fertilization diplophase: i.e. during development and maturation of the embryos. Seventy two out of 78 of the mutations occurring in chimeric plants of Cappelli and Aziziah were found in the progeny of the ‘main’ spikes of irradiated plants. The possible reasons for this striking difference between ‘main’ and ‘secondary’ spikes in undergoing mutation during embryogenesis are discussed. A great variation in response to genetic effects of radiation among the four types of durum wheat tested was ascertained. It was ascribed to different characteristics of their life cycle rather than to differences in radiation sensitivity.
R&us&---Trois cultivars de blt duntnz, Cappelli, Aziziah et Russell0 ainsi qu’un mutant stable de Cappelli, brachylique induit par les radiations ont ttt soumis a une irradiation chronique par les rayons gamma du soCo pendant toute leur vie (de la 3e feuille a la rtcolte). Les doses de rayonnement ttaient de 5, 10, 15,20,25, et 30 R/ZOhr de jour. Les tpis matures, provenant de plantes irradites, ont ttt recoltts en separant les tpis prod&s par les chaumes principaw (‘epis principaux’) de ceux produits par les talles (‘tpis secondaires’) . La fertilite des Cpillets et la germination des caryopses decroit avec la dose. Au tours de la premiere g&&ration provenant de plantes irradiees (yz de chercheurs anttrieurs) on n’a rencontrt aucune mutation. En seconde generation (yB de chercheurs anttrieurs) des mutations chlorophylhennes apparaissent avec certitude dans deux categories de plantes. (a) les plantes *Contribution Nucleari della
No. 147 from the Laboratorio Casaccia, S. Maria di Galeria,
per le Applicazioni Rome, Italy. 49
in Agricoltura
de1 C.N.E.N.,
Centro
Studi
50
13. DONINI,
G. T. SCARASCIA
MUGNOZZA
and
F. D’AMATO
Mtirozygoles
pour une mutations (tous les i-pis qui &g&gent pour la mutation) ct (6) les plantes qui ont des chimtres pour la mutation (stgrbgation pour dcs mutants survenant dans un ou deux Cpis, mais pas dans tous). Les deux types de mutations sont produits h des frCCeci montre que dans les bits durum irradiCs pendant quences de 20 et 8O”/b respectivement. toute la vie, la majoritt des mutations qui peuvent apparaitre sont induites pendant la diplophase apris la fkcondation c’est&dire pendant le d&eloppement et la maturation des embryons. Parmi 78 mutations provenant de plantes avec chimeres chez Cappelli et Aziziah, on en a rencontrC 72 dans la descendance d’epis “principaux” provenant de plantes irradiCes. On discute les raisons possibles de cette importante diffkrence cntre Cpis ‘principaux’ et ‘secondaires’ du point de vue de la production de mutations au tours de l’embryogtntse. On a confirm6 I’existence d’une grande variation dans la rGponse aux radiations pour les quatre types de durum en ce qui concerne les efFets gtnCtiques. Cette grande variation a &t attribuCe g diffkrents caracteres du cycle biologique plut6t qu’a dcs dX&ences de radiosensibilitt?. Zusammenfassung-Drei Ziichtungen der Weizenart durum, Cappelli Aziziah und Russello, und eine strahleninduzierte stabile Mutante von Cappelli, braclylic, wurden wghrend ihres ganzen Lebenszyklus (vom 3-Blatt-Stadium bis zur Ernte) einer chronischen Gammastrahlung aus einer 60Co Strahlenquelle ausgesetzt. Es wurden Strahlendosen von 5, 10, 15,20, 25 und 30 R/20-Std.-Tag angewendet. Von den bestrahlten Pflanzen wurden reife .&en geerntet, wobei man die Ahren, die man von den Haupttrieben erhielt (‘Haupt-Ahrenj von denen der Wurzelschijsslinge (‘sekundire iihren’) getrennt hielt. Mit zunehmender Strahlendosis nahm die Fruchtbarkeit der iihrchen sowie Keimung der Karyopsen ab. In der ersten Generation der bestrahlten Pflanzen (yz friiherer Arbeiten) wurde kein Fall von Chlorophyllmutation festgestellt. In der zweiten Generation (ys friiherer Arbeiten) wurden Chlorophyllmutationen bei 2 Gruppen festgestellt: (a) Pflanzen, heterozygot fiir die Mutation (bei allen Ahren segregiert die Mutation) und (6) Pflanzen, bei denen die Mutation in Form einer Chimare vorgliegt (Segregation der Mutanten in einer oder zwei-aber nicht allen-Ahren). Die zwei Mutationsformen traten mit einer Hgufigkeit von 20 und 80 Prozent respektive auf. Dies zeigt, dass in der Weizenart durum, die wlhrend des ganzen Lebenszyklus bestrahlt wurde, die Mehrzahl der restituierbaren Mutationen in der Diplophase nach der Befruchtung induziert wird: d.h. wihrend der Entwicklung und Reifung der Embryonen. Bei Cappelli und Aziziah wurden 72 von 78 der Mutationen der ChimPrcn in den Nachkommen der ‘Haupt’-P;hren bestrahlter Pflanzen gefunden. Die m6glichen Griinde fiir diesen auffallenden Unterschied zwischen ‘Haupt’und ‘Sekundlr’Pihren bei der Mutation wlhrend der Embryogencse werden diskutiert. Bei den untersuchten 4 Typen der Weizenart durum wurde eine grosse Variation im Hinblick auf die genetische Wirksamkeit der Strahlung festgestellt. Sie wurde mehr den verschiedenen Merkmalen ihres Lebens zyklus zugeschrieben als den Unterschieden in der Strahlenempfindlichkeit.
INTRODUCTION IN A PREVIOUS paper, ce) data on the response to chronic y-irradiation ( 60Co source) of several cultivars of durum and bread wheats were reported. Among five cultivars of durum wheat tested, the cultivar Aziziah appeared the most radiation sensitive and the cultivar Cappelli the most radioresistant, both in terms of spikelet fertility and seed germination in the irradiated plants. Since the chronic radiation exposures used, 52, 72 and 148 R/20-hr day induced a strong decrease in spikelet fertility, especially in the
most radiation sensitive cultivars of durum wheat, any analysis of genetic effects in that material would have been inadequate. New experiments on chronic y-irradiation in durum wheat were designed for genetic analyses. In the present paper, the results of these experiments are presented. MATERIALS AND METHODS Three cultivars of durum wheat, Cappelli, Aziziah and Russello, and a radiation induced stable mutant of Cappelli, h-achytic, were used. Seedlings at the 3-leaf stage were transplanted
GENETIC
EFFECTS
OF DfJHUA4
in the y-radiation field of the CNEN(“) at distances from the G°Co source corresponding to exposuresof 5, 10, 15, 20, 25 and 30 R/20-111day. For each wheat type and radiation exposure 25 seedlings were planted and the plants were irradiated for their whole life cycle up to harvest (114 days). Control material was grown in the y-field in a shielded sector. Mature spikes were harvested from each irradiated plant, keeping spikes produced by the main culms separate from those produced by tillers; they are referred to in the Tables and text as ‘main’ and ‘secondary’ spikes, respectively. For each plant progeny, seeds were sown in the greenhouseas spike progenies, either ‘main’ or ‘secondary’, and scored for possibly occurring chlorophyll mutations; this material is referred to in Table 3 as ‘first generation from chronically irradiated plants’. When the plants reached the 3-leaf stage they were transplanted in the field and grown to maturity. Plants were harvested separately and spike-progenies from all spikes in each plant were grown in the greenhousefor analysis of chlorophyll mutations. On account of the labour involved, only a part of the harvested material was analysed: this ‘second generation’, therefore, consisted of the progeny of G-10 chronically irradiated plants in each variety and radiation exposure. ‘First’ and ‘second generation’ from chronically irradiated plants were regarded as more appropriate terms than yr and y,.(‘y12) As already realized by NYBOM et a1.,(12) in experiments of chronic irradiation for the whole-life cycle, the plants harvested from the gamma Table
1. S/it&let
JertiliQ,
in /ter certl of control,
5R/day
51
field correspond in part, not totally, to the X,-generation after treatment of seeds with acute irradiation (see Discussion). RESULTS
At 5 R/day, there was indication, in some materials, of an increased spikelet fertility as compared to control; at higher exposure rates, spikelet fertility progressively decreased with increasing exposure rate (Table 1). The adverse effect of chronic irradiation at exposure rate of 20 R/day and higher was also manifested in the reduced germination of caryopses collected from irradiated plants: in Table 2, germination is expressedas percentage of seedlings produced by the caryopses. In both Tables 1 and 2, the effects of chronic irradiation are reported separate1y for ‘main’ and ‘secondary’ spikes. No case of chlorophyll mutation was found in the progeny (first and second generation) of control plants, grown in the shielded area of the y-field. This conforms with the very low spontaneous chlorophyll mutation rate of durum wheat cultivars ascertained previously.@) The material analysed in the ‘first generation’ from chronically irradiated plants is listed in Table 3; in this material, tillering was as expected from spaced durum wheat plants. In Table 3, by number of plants irradiated is meant the number of plants harvested from the y-field. The great variation in these numbers is due to unfavourable environmental conditions occurring after the transplantation of seedlings in the y-field. In the material
of tnain (M) and secondary wheal plants
15R/day
1OR/day
WHEAT
(S)
20R/day
spikes of chronically
25R/day
irradialed
durum
SOR/day
MateriaI M
s
A4
s
M
S
M
S
M
S
M
S
Cappelli Aziziah Russcllo
96.73
75.00
83.33
67.50
72.82
52.50
64.13
60.62
51.81
56.25
39.85
43.75
107.22 115.20
119.31 92.08
80.22 97.60
79.95 66.90
64.25 87.60
60.22 53.95
40.68 7040
52.27 50.35
36.88 54*80
50.00 48.20
28.13 38.00
34.09 28.77
brachytic Cappe"i
106.63
131.66
92.53
87.50
86.30
75.83
71.37
54.16
62.24
55.00
48.54
36.66
B. DONINI,
52 Table
G. T. SCARASCIA
MUGNOZZA
and F. D’AMATO
2. Percentage of seedlings jvoduced by caryopses collected from main (M) secondary (S) spikes of control and chronically irradiated durum wheat blants
Control
5R/day
1OR/day
and
15R/day
Material M Cappelli Aziziah Russell0 Cappelli brachytic
s
M
s
95.84 98.60 97.42 98.96 94.85 96.80 93.98 97.72 94.42 95.20 94.61 95.64 93.70 89.25 93.90 86.82
M
s
M
s
95.80 95.33 90.30 92.74
98.63 95.70 93.33 87.68
94.26 92.26 89.76 89.95
91.90 94.64 96.91 84.90
20R/day
25R/day
30R/day
Material M Cappelli Aziziah Russell0 Cappelli brachytic
listed in Table 3, no case of chlorophyll mutation was found. Screening of chlorophyll mutations in the ‘second generation’ was limited to only part of the plants grown in the ‘first generation’ (see Since all spikes present Material and Methods. in each plant (2-5) were analysed, plants showing mutants could be classified in two categories. (u) Plants in which segregation occurred for mutants of same phenotype in each spike, are regarded as heterozygous for the mutation (type A of NYBOM et al. ;(12) (b) Plants in which mutants occurred in only one or two spikes, while the remaining spikes did not contain mutations ; these plants are regarded as chimeric for the mutation (type B of NYBOM
et a1.(12)
Since, as can be seen in Table 5, the number of seedlings in a durum wheat spike is sufficiently high, there is good probability for the occurrence of at least one mutant in mutated spikes.@151 In Tables 4 and 5, the number and frequency of plants ascertained as heterozygous and chimeric for chlorophyll mutations in the ‘second generation’ from irradiated plants are
S
M
92.82 94.32 78.57 85.00 75.00 79-18 79.84 87.87 69.96 82.85 86.84 83.13
S
M
S
80.19 67.09 69.43 70.00 55.86 63.93 84.00 48.04 76.00 80.14 64.67 61.80
reported. In Table 5, the frequency of mutated seedlings is also shown. It is apparent that both the frequency of heterozygous plants and the frequency of chimeric plants and mutated spikes were highest at radiation exposures of 20-30 R/day. The frequencies of mutated seedlings (Table 5) were generally related to the frequencies of mutated spikes. In some radiation exposures (e.g. Cappelli, 25 R/day; Aziziah, 25 and 30 R/day; Cappelli brachytic, 20 R/day) a ‘segregation ratio’ of mutants in mutated spikes clearly greater than the overall ‘segregation ratio’ of the experiments was observed. This is to be ascribed to a greater average size of the mutated sector in the spikes.(3~7~8) The analysis of all ‘segregation ratios’ in the experiments did not reveal any relation of mutated sector size in the spike to radiation exposure. In Table 6, the frequencies of chimeric plants found in Cappelli and Aziziah have been reported separately for progenies originating from the ‘main’ and the ‘secondary’ spikes of the irradiated individuals. It is seen that 72 out of 78 mutations occurring in chimeric plants in the ‘second generation’ were born by the main spikes of the irradiated plants.
GENETIC Table
3. Material
analysed
in the Jirst
EFFECTS
generation
OF ZIURUM
from chronically secondarv sbikesl
No. spikes analysed Material Cappelli
R/day 5
10 15 20 25 30
Aziziah
5
10 15 20 25 30
Russell0
5
10 15 20 25 30
Cappelli brachytic
5
10 15 20 25 30
No. plants irradiated ---.
WHEAT
irradiated
&rum
53
No. of seedlings analysed
A4
S
668 145 504 521 501 975
51.38 28.56 37.32 33.08 23.47
14.84 10.35 10.50 12.70 12.84
13.91
11.89
301 307 106 12 206 78
17.20 13.31 16.14
10.37
444
41.56 27,37
S
M
s
12 10 14 14 16 16
39 16 37 35 48 59
45 14 48 41 39 82
2004 457 1381 1158 1127 821
6 12 8 4 12
30 38 28 4 38 55
29
516
40 15 3 35 22
500 452 15 467 361
12 11 12 7 15 16 16 14 12 7
9 7
Mean No. seedlings per spike
M
10
(M and S main and
wheat plants
--
79 56 35 14 88 70 80 46 31 15 34 48
DISCUSSION In these experiments, plants were irradiated for their whole-life cycle, from 3-leaf stage to harvest. Under these conditions, irradiation of distinct developmental stages should have resulted in the induction of mutation in cells of both the haplophase and the diplophase. These may belong to the following three categories. (u) Mutations induced in somatic (diploid) cells of the vegetative phase before meiosis. If a cell heterozygous for the mutation occurred in the layer of the shoot apex responsible for the
26 8 26
3284 1533 324 206
--__
3.75 12.28 6.56
25 26
1360 561
56 210 58 203 168
26 18
29
3450 1468 784
501 243 270
8 25 18
319
107
31.91 25.29 21.26
840 773
225 135
24.70 16.10
9
9.05 14.71 15.45 8.01 43.12
6.67 7.06 4.00 5.88 3.54 17.07 7.00 8.07 644 8.12 646
19.26 13.50 9.31 13.37 9.00 7950
organization of the sporogenous tissue, L-2, 3,(‘s) zygotes, and seeds, heterozygous and homozygous for the mutation, would be formed. Manifestation of the homozygous individuals (mutants) in the next generation (our ‘first generation’ from irradiated plants or ys of previous workers@Js)) would strictly depend on spike progeny size, mutated sector size and ‘segregation ratio’ of the mutation.(s~7~s) Plants originating from seeds heterozygous for the mutation will segregate for the mutants in all of their spikes (our ‘second generation’ or y3. W))
54 Table
B. DONINI, 4.
Plants
ascertained
G. T. SCARASCIA
as heterozygous
for
durum wheat plants.
MUGNOZZA
and F. D’AMATO
chlorophyll mutations in the second generation All spikes (2-5) in each plant were analysed
Cappelli
R/day 5 10 15 20 25 30
No. plants irradiated 6 9 10 6 9 10
540 360 750 370 403 388
1 1 2 2 3 2
0.18 0.27 0.26 0.54 0.74 0.51
__--.
No. plants No. plants irradiated analysed 6 10 8 4 9 10
Russell0
Way
chronically
irradiated
Aziziah Heterozygous plants No. %
No. plants analysed
No. plants No. plants irradiated analysed
from
204 226 187 15 171 136 Cappelli
Heterozygous plants ~-No. %
No. plants irradiated
No. plants analysed
Heterozygous plants %~No. 0 1 0 0 1 1
0 0.44 0 0 0.58 0.73
brachytic Heterozygous plants No. l
%
___~~.
5 10 15 20 25 30
6 10 8 6 9 10
357 454 109 64 158 116
0 1 0 0 2 2
0 0.22 0 0 1.26 1.72
6 10 8 6 9 10
326 432 263 159 211 211
0 1 0 0 1 2
0 0.23 0 0 0.47 0.94
In barley plants irradiated for the whole life cycle only a few instancesof mutations were observed in y2. This was taken as an indication that most of the mutations were induced at a stage later than the separation of the male and female organs of the flowers.(12) The possibility of diplontic selection was not discussedby the authors. In a total of 115 mutations ascertained in our experiments, 23 occurred in hetorozygous plants (type A of NYBOM et aZ.).P2) The frequency of plants heterozygous for mutation in our material (20 per cent) compares very well with that of 20.7 per cent found in the during development (diplontic selection(3~7~*)) irradiation of barley from germination to or happened to involve only a reduced sector maturity (18 mutations of type A and 69 of in the spike. type B).t12) Therefore, the data obtained in both barley and durum wheat, a largely diploidized species in the genetic system of its *Fusion of gametes bearing one and same mutation chlorophyll apparatus,(“) show very clearly is disregarded, due to the low probability of its occurrence. that, in experiments of chronic irradiation for
(6) Mutation induced in gametes or zygotes, resulting in seeds heterozygous for the mutation.* In experiments of the type performed by us, heterozygous plants cannot be distinguished as to mode of origin, either (a) or (b). (c) Mutation induced during embryo development and maturation.(lO) This will lead to chimeric plants which will segregate for mutants in one or some-but not in allspikes in the ‘second generation’. Since no mutants were recovered in the ‘first generation’ of chronically irradiated plants it must be assumed that mutations induced in vegetative cells were either selected against
5. Plants
as chimeric
for chlorofihyll
6 9 10 6 9 10 6 10 8 4 9 10 6 10 8 6 9 10 6 10 8 6 9 10
5 10 15 20 25 30 5 10 15 20 25 30 5 10 15 20 25 30 5 10 15 20 25 30
Aziziah
Russell0
Cappelli brachytic
326 432 263 159 211 211
357 454 109 64 158 116
204 226 187 15 171 136
540 360 750 370 403 388
No. plants No. plants R/day irradiated anaiysed
ascertained
Cappelli
Material
Table
3 6 17 11 13 14
No.
0.46 0.38 0.63 1.89 o-94
0.00
1.26 0.00
0.00 044 0.00 0.00
0.88 1.60 6-66 3.40 1.47
o-00
0.55 1.66 2.26 2.97 3.22 3.61
0, ,0
plants
791 1020 596 402 509 485
873 1106 324 192 482 354
582 620 526 42 498 405
1408 946 2062 947 1065 1128
No. soikes A analysed
3 1 10 2
0 2
3 6 19 12 19 14
No.
0.00
0.19 O-16 0.24 0.78 0.61
0.00
0.18 0.00 0.00 0.41 0.00
0.00
0.32 0.57 2.38 2.00 0.49
wheat plants.
45,010 50,707 32,285 18,854 23,332 15,916
30,66 1 26,640 8,919 5,443 12,724 9,072
24,513 22,233 20,293 1,096 15,865 12,163
61,761 45,151 92,903 41,633 44,845 46,208
No. seedlines analysed
durum
0.21 0.63 0.92 1.26 1.78 1.24
%
spikes
irradiated
Mutated
in the second generation from chronically each plant were analysed
Chimer+
mutations
0 3 2 13 23 16
0 13 0 0 3 0
0 20 9 2 74 26
I1 27 50 30 94 39
NO.
Mutated
0.00 0.059 0.061 0.689 0.985 1.005
0.00 0.48 0.00 0.00 0.23 0.00
1.25 04 1.82 4.66 2-13
0.18 0.60 0.54 0.72 2.09 0.84
0, /o
seedling
All spikes (2-5)
in
56
B. DONINJ,
Table
G. T. SCARASCIA
6. Plunts ascertained as chimeric for wheat plants. Plants derivedfrom
MUGNOZZA
and F. D’AMATO
chloro~lyll mutations in the second generation front chronically irradiated the main (M) and the secondary (S) spikes of irradiated individuals Chimeric
No plants Material
R/clay
Cappelli
5 10 15 20 25 30
Aziziah
5 10 15 20 25 30
No. plants irradiated
analysed
M
S
6 9 10 6 9 10
416 328 596 287 273 191
124 32 154 83 130 197
6 10 8 4 9 10
180 197 186 15 152 127
24 29 1 0 19 9
Mutated
plants S
M No.
No. spikes analysed
%
No.
%
M
3 6 17 10 12 11
O-72 l-82 2.85 3.48 4.39 5.76
0 0 0 1 1 3
0.00 0.00 0.00 l-20 0.77 1.52
1070 863 1640 742 735 560
338 83 422 205 330 568
0 2 3 1 5 2
0.00 1.01 1.61 6.66 3.28 1.57
0 0 0 0 1 0
0.00 0.00 0.00 0.00 5.26 o-00
528 542 523 42 447. 383
54 78 3 0 51 22
spikes
M
Mutated No. seedlings analysed
S
durum
s
seedlings
M
S -
Material
R/day
No.
y.
No.
y.
M
s
No.
y.
No.
%
15,357 3,605 19,112 8,777 14,814 26,560
11 27 50 27 78 21
0.24 0.65 0.67 O-82 2.59 1.07
0 0 0 3 16 18
0.00 0.00 o-00 0.34 1.08 0.67
0 20 9 2 66 26
0.00 1.03 0.44 1.82 4.55 2.20
0 0 0 0 8 0
0.00 0.00 0.00 0.00 5.86 0.00
~____ Cappelli
5 10 15 20 25 30
3 6 19 11 17 11
0.28 0.69 1.15 1.48 2.31 1.96
0 0 0 1 2 3
0.00 0.00 0.00 0.48 0.60 0.52
46,404 41,546 73,791 32,856 30,031 19,648
Aziziah
5 10 15 20 25 30
0 2 3 1 8 2
0.00 0.36 0.57 2.38 l-78 0.52
0 0 0 0 2 0
0.00 0.00 0.00 0.00 3.92 0.00
22,776 19,370 20,230 1,096 14,501 11,813
the whole-life cycle, the majority of recoverable mutations are mutations induced in the postfertilization diplophase, i.e. during embryo development and maturation. This conclusion is further strengthened by the analyses on the relative proportions of type A and B mutations induced in barley by y-irradiation in different periods of the cycle life: from germination to
1,737 2,863 63 0 1,364 350
heading, from flowering to maturity and from heading to maturity.(12) In the experiments of NYBOM et a1.W and in ours, the mutation rates recorded after chronic exposures were lower than, or did not exceed, those obtainable in the same materials with acute irradiation of seeds. These results are at variance with those of MIKAELSEN(*~)
GENETIC
EFFECTS
and SWAMINATHAN who reported, in barley and bread wheat respectively, that the mutation rates were higher after chronic than after acute radiation exposures. An interesting result of our experiment lies in the demonstration that 92 per cent (72 out of 78) of the mutations occurring in chimeric plants of Cappelli and Aziziah (Table 6) were borne by the ‘main’ spikes of the irradiated plants. The causes for this striking difference between ‘main’ and ‘secondary’ spikes in undergoing mutation during embryogenesis are not known. The folIowing possibilities may be suggested. (a) Embryogenesis in the ‘main’ spikes may take longer than in the ‘secondary’, resulting in an accumulated radiation exposure greatel in the former than in the latter. (b) Cell division in embryos developing in ‘main’ spikes may run with a mitotic cycle time longer than in embryos of ‘secondary’ be, at any daily spikes ; the result would exposure rate, an increased radiation effect(15) on dividing cells in the first category of embryos. (c) The number of cells constituting the apical primordia of ‘primary’ spikes in embryos is obviously greater than that of ‘secondary’ spikes, thereby increasing probability for the occurrence of mutations. The great variation in response to genetic effects of radiation among the four types of durum wheat tested cannot be explained. Previous studies with acute(‘e5) and chronic(“) irradiations have shown that Aziziah is more radiosensitive than Cappelli and Russell0 when effects on growth, survival at maturity and spike fertility are considered;(lp51s) however, no clear differences between Aziziah and Cappelli and among Aziziah, Cappelli and Russell0 are found when chromosome damage(s) and chlorophyll mutation rates(b) are analysed after acute irradiation of dry seeds. In our opinion, the differences in the frequency of mutations induced by chronic radiation exposure among the four durum wheat types must be searched for in the characteristics of their life cycle,(s) rather than in differences in radiation sensitivity. Acknr&e&ement-The
authors
wish
to express
their
OF
DURUM
57
WHEAT
appreciation 10 Mr. BEINAT for their very
M. BELLATALLA efficient technical
and Mr. assistance.
V.
REFERENCES I. AVANZI chimici italiane 353-364.
S. (1961) Effetti di radiazioni e mutageni sulla crescita e sopravvivenza in varietj. Afti Assoc. &ret. f&al. 6, di grano duro.
S.. BRUNORI A. and GIORGI B. (1966) 2. AVANZI Radiation response of dry seeds in two varieties of Triticum durum. n4utation Res. 3, 426-437. 3. D’AMATO F. (1965) Chimera formation in mutagen treated seeds and diplontic selection, pp. 303-3 16. In The use of induced mutations in plant breeding, Radiation Botagl Suppl. 5, Pergamon Press, Oxford. 4. D’AMATO F., SCARASCIA G. T., BELLIAZZI U., BASSANI A., CAMBI S., CEVOLO~TO P., GIACALONE F. and TAGLIATI S. (1962) The gamma radiation field of the “Comitato Nazionale per 1’Energia Nucleare”, Rome. Radiation Botatp 1,243-246. 5. D’AMATO F., SCARASCIA G. T., MONTI L. M. and BOZZINI A. (1962) Types and frequencies of chlorophyll mutations in durwn wheat induced by radiations and chemicals. Radiation Bolany 2, 217-239. 6. DONINI B., SCARAXIA MUGNOZZA G. T. and D’AMATO F. (1964) Effects of chronic gamma irradiation in durum and bread wheats. Radiation Botarp 4, 387-393. 7. GAUL H. (1959) Uber Chimlrenbildung Gerstenpflanzen nach Riintgenbestrahlung Samen. Flora 147, 207-241.
in von
8. GAUL H. (1961) Studies on diplontic selection after X-irradiation of barley seeds, pp. 117-136. In Effecti of Ionizing Radiations on Seeds, IAEA, Vienna. T. and INOSHITA I. (1965) Effects of 9. KAWAI gamma irradiation on growing rice plants. Irradiations at four main developmental stages. Radiation Botany 5, 233-255. 10.
MERICLE Mutation Radiation
L. W. and MERICLE induction by proembryo Botagf 1, 195-202.
II.
MIKAELSEN K. (1956) of chronic gamma Intern. Cottf. Peacejkl Nations 7, 34--39.
12. NYBOM N., EURENI~ERO
R.
P. (1962) irradiation.
Studies on genetic effects radiation in plants. Proc. Use.r AI. Energy, United
GUSTAFSSON I>. (1956)
A., The
GRANI-IALL I. and genetic effects of
58
B. DONINI,
G. T. SCARASCIA
chronic gamma irradiation on barley. Hereditas 4!&74-84. 13. POPHAM R. A. (1963) Developmental studies of flowering. Brookhaven Symp. Biol. 16, 138-156. 14. SWAMINATHAN M. S. (1965) A comparison of mutation induction in diploids and polyploids, pp. 619-641. In The use of induced mutations in
MUGNOZZA
and
F. D’AMATO
plant breeding. Radiation Botany Suppl. 5, Pergamon Press, Oxford. 15. VAN’T HOF J. and SPARROW A. H. (1963) The effect of mitotic cycle duration on chromosome breakage in meristematic cells of Pisum sativum. Proc. Natl. Acad. Sci., US. 50, 855-860.
&Amy,
1968,
GENETIC
Vol.
8, pp. 49 Lo 58. Pcrgamon
EFFECTS B. DONINI
Laboratory
Press. l’rinlcd
in Great
Brit&.
OF CHRONIC GAMMA IN DURUM WHEAT” and G. T. S$LUUSCIA
IRRADIATION
MUGNOZZA
for the Applications of Nuclear Energy in Agriculture, for Nuclear Research, Rome, Italy
National
Committee
and F. D’AMATO Institute
of Genetics, (Received
The
University,
20 March
Pisa,
Italy
1967)
Abstract-Three
cultivars of durunt wheat, Cappelli, Aziziah and Russello, and a radiation induced stable mutant of Cappelli, brachytic, were subjected to chronic y-irradiation from @%o for their whole lift cycle (from the 3-leaf stage to harvest). Radiation exposures of 5, 10, 15, 20, 25 and 30 R/20-hr day were applied. Mature spikes were harvested from the irradiated plants keeping spikes produced by the main culms (‘main spikes’) separate from those produced by tillers (‘secondary spikes’). Both spikelet fertility and germination of caryopses decreased with increasing exposure rate. In the first generation from irradiated plants (yz of previous workers), no case of chlorophyll mutation was found. In the second generation (ya of previous workers), chlorophyll mutations were ascertained in two categories of plants: (a) plants heterozygous for the mutation (all spikes segregating for the mutation) and (6) plants chimeric for the mutation (segregation for mutants occurring in one or two-but not allspikes). The two types of mutations occurred with a frequency of 20 and 80 per cent respectively. This shows that, in durutn wheat plants irradiated for their whole life cycle, the majority of recoverable mutations are induced in the post-fertilization diplophase: i.e. during development and maturation of the embryos. Seventy two out of 78 of the mutations occurring in chimeric plants of Cappelli and Aziziah were found in the progeny of the ‘main’ spikes of irradiated plants. The possible reasons for this striking difference between ‘main’ and ‘secondary’ spikes in undergoing mutation during embryogenesis are discussed. A great variation in response to genetic effects of radiation among the four types of durum wheat tested was ascertained. It was ascribed to different characteristics of their life cycle rather than to differences in radiation sensitivity.
R&us&---Trois cultivars de blt duntnz, Cappelli, Aziziah et Russell0 ainsi qu’un mutant stable de Cappelli, brachylique induit par les radiations ont ttt soumis a une irradiation chronique par les rayons gamma du soCo pendant toute leur vie (de la 3e feuille a la rtcolte). Les doses de rayonnement ttaient de 5, 10, 15,20,25, et 30 R/ZOhr de jour. Les tpis matures, provenant de plantes irradites, ont ttt recoltts en separant les tpis prod&s par les chaumes principaw (‘epis principaux’) de ceux produits par les talles (‘tpis secondaires’) . La fertilite des Cpillets et la germination des caryopses decroit avec la dose. Au tours de la premiere g&&ration provenant de plantes irradiees (yz de chercheurs anttrieurs) on n’a rencontrt aucune mutation. En seconde generation (yB de chercheurs anttrieurs) des mutations chlorophylhennes apparaissent avec certitude dans deux categories de plantes. (a) les plantes *Contribution Nucleari della
No. 147 from the Laboratorio Casaccia, S. Maria di Galeria,
per le Applicazioni Rome, Italy. 49
in Agricoltura
de1 C.N.E.N.,
Centro
Studi
50
13. DONINI,
G. T. SCARASCIA
MUGNOZZA
and
F. D’AMATO
Mtirozygoles
pour une mutations (tous les i-pis qui &g&gent pour la mutation) ct (6) les plantes qui ont des chimtres pour la mutation (stgrbgation pour dcs mutants survenant dans un ou deux Cpis, mais pas dans tous). Les deux types de mutations sont produits h des frCCeci montre que dans les bits durum irradiCs pendant quences de 20 et 8O”/b respectivement. toute la vie, la majoritt des mutations qui peuvent apparaitre sont induites pendant la diplophase apris la fkcondation c’est&dire pendant le d&eloppement et la maturation des embryons. Parmi 78 mutations provenant de plantes avec chimeres chez Cappelli et Aziziah, on en a rencontrC 72 dans la descendance d’epis “principaux” provenant de plantes irradiCes. On discute les raisons possibles de cette importante diffkrence cntre Cpis ‘principaux’ et ‘secondaires’ du point de vue de la production de mutations au tours de l’embryogtntse. On a confirm6 I’existence d’une grande variation dans la rGponse aux radiations pour les quatre types de durum en ce qui concerne les efFets gtnCtiques. Cette grande variation a &t attribuCe g diffkrents caracteres du cycle biologique plut6t qu’a dcs dX&ences de radiosensibilitt?. Zusammenfassung-Drei Ziichtungen der Weizenart durum, Cappelli Aziziah und Russello, und eine strahleninduzierte stabile Mutante von Cappelli, braclylic, wurden wghrend ihres ganzen Lebenszyklus (vom 3-Blatt-Stadium bis zur Ernte) einer chronischen Gammastrahlung aus einer 60Co Strahlenquelle ausgesetzt. Es wurden Strahlendosen von 5, 10, 15,20, 25 und 30 R/20-Std.-Tag angewendet. Von den bestrahlten Pflanzen wurden reife .&en geerntet, wobei man die Ahren, die man von den Haupttrieben erhielt (‘Haupt-Ahrenj von denen der Wurzelschijsslinge (‘sekundire iihren’) getrennt hielt. Mit zunehmender Strahlendosis nahm die Fruchtbarkeit der iihrchen sowie Keimung der Karyopsen ab. In der ersten Generation der bestrahlten Pflanzen (yz friiherer Arbeiten) wurde kein Fall von Chlorophyllmutation festgestellt. In der zweiten Generation (ys friiherer Arbeiten) wurden Chlorophyllmutationen bei 2 Gruppen festgestellt: (a) Pflanzen, heterozygot fiir die Mutation (bei allen Ahren segregiert die Mutation) und (6) Pflanzen, bei denen die Mutation in Form einer Chimare vorgliegt (Segregation der Mutanten in einer oder zwei-aber nicht allen-Ahren). Die zwei Mutationsformen traten mit einer Hgufigkeit von 20 und 80 Prozent respektive auf. Dies zeigt, dass in der Weizenart durum, die wlhrend des ganzen Lebenszyklus bestrahlt wurde, die Mehrzahl der restituierbaren Mutationen in der Diplophase nach der Befruchtung induziert wird: d.h. wihrend der Entwicklung und Reifung der Embryonen. Bei Cappelli und Aziziah wurden 72 von 78 der Mutationen der ChimPrcn in den Nachkommen der ‘Haupt’-P;hren bestrahlter Pflanzen gefunden. Die m6glichen Griinde fiir diesen auffallenden Unterschied zwischen ‘Haupt’und ‘Sekundlr’Pihren bei der Mutation wlhrend der Embryogencse werden diskutiert. Bei den untersuchten 4 Typen der Weizenart durum wurde eine grosse Variation im Hinblick auf die genetische Wirksamkeit der Strahlung festgestellt. Sie wurde mehr den verschiedenen Merkmalen ihres Lebens zyklus zugeschrieben als den Unterschieden in der Strahlenempfindlichkeit.
INTRODUCTION IN A PREVIOUS paper, ce) data on the response to chronic y-irradiation ( 60Co source) of several cultivars of durum and bread wheats were reported. Among five cultivars of durum wheat tested, the cultivar Aziziah appeared the most radiation sensitive and the cultivar Cappelli the most radioresistant, both in terms of spikelet fertility and seed germination in the irradiated plants. Since the chronic radiation exposures used, 52, 72 and 148 R/20-hr day induced a strong decrease in spikelet fertility, especially in the
most radiation sensitive cultivars of durum wheat, any analysis of genetic effects in that material would have been inadequate. New experiments on chronic y-irradiation in durum wheat were designed for genetic analyses. In the present paper, the results of these experiments are presented. MATERIALS AND METHODS Three cultivars of durum wheat, Cappelli, Aziziah and Russello, and a radiation induced stable mutant of Cappelli, h-achytic, were used. Seedlings at the 3-leaf stage were transplanted
GENETIC
EFFECTS
OF DfJHUA4
in the y-radiation field of the CNEN(“) at distances from the G°Co source corresponding to exposuresof 5, 10, 15, 20, 25 and 30 R/20-111day. For each wheat type and radiation exposure 25 seedlings were planted and the plants were irradiated for their whole life cycle up to harvest (114 days). Control material was grown in the y-field in a shielded sector. Mature spikes were harvested from each irradiated plant, keeping spikes produced by the main culms separate from those produced by tillers; they are referred to in the Tables and text as ‘main’ and ‘secondary’ spikes, respectively. For each plant progeny, seeds were sown in the greenhouseas spike progenies, either ‘main’ or ‘secondary’, and scored for possibly occurring chlorophyll mutations; this material is referred to in Table 3 as ‘first generation from chronically irradiated plants’. When the plants reached the 3-leaf stage they were transplanted in the field and grown to maturity. Plants were harvested separately and spike-progenies from all spikes in each plant were grown in the greenhousefor analysis of chlorophyll mutations. On account of the labour involved, only a part of the harvested material was analysed: this ‘second generation’, therefore, consisted of the progeny of G-10 chronically irradiated plants in each variety and radiation exposure. ‘First’ and ‘second generation’ from chronically irradiated plants were regarded as more appropriate terms than yr and y,.(‘y12) As already realized by NYBOM et a1.,(12) in experiments of chronic irradiation for the whole-life cycle, the plants harvested from the gamma Table
1. S/it&let
JertiliQ,
in /ter certl of control,
5R/day
51
field correspond in part, not totally, to the X,-generation after treatment of seeds with acute irradiation (see Discussion). RESULTS
At 5 R/day, there was indication, in some materials, of an increased spikelet fertility as compared to control; at higher exposure rates, spikelet fertility progressively decreased with increasing exposure rate (Table 1). The adverse effect of chronic irradiation at exposure rate of 20 R/day and higher was also manifested in the reduced germination of caryopses collected from irradiated plants: in Table 2, germination is expressedas percentage of seedlings produced by the caryopses. In both Tables 1 and 2, the effects of chronic irradiation are reported separate1y for ‘main’ and ‘secondary’ spikes. No case of chlorophyll mutation was found in the progeny (first and second generation) of control plants, grown in the shielded area of the y-field. This conforms with the very low spontaneous chlorophyll mutation rate of durum wheat cultivars ascertained previously.@) The material analysed in the ‘first generation’ from chronically irradiated plants is listed in Table 3; in this material, tillering was as expected from spaced durum wheat plants. In Table 3, by number of plants irradiated is meant the number of plants harvested from the y-field. The great variation in these numbers is due to unfavourable environmental conditions occurring after the transplantation of seedlings in the y-field. In the material
of tnain (M) and secondary wheal plants
15R/day
1OR/day
WHEAT
(S)
20R/day
spikes of chronically
25R/day
irradialed
durum
SOR/day
MateriaI M
s
A4
s
M
S
M
S
M
S
M
S
Cappelli Aziziah Russcllo
96.73
75.00
83.33
67.50
72.82
52.50
64.13
60.62
51.81
56.25
39.85
43.75
107.22 115.20
119.31 92.08
80.22 97.60
79.95 66.90
64.25 87.60
60.22 53.95
40.68 7040
52.27 50.35
36.88 54*80
50.00 48.20
28.13 38.00
34.09 28.77
brachytic Cappe"i
106.63
131.66
92.53
87.50
86.30
75.83
71.37
54.16
62.24
55.00
48.54
36.66
B. DONINI,
52 Table
G. T. SCARASCIA
MUGNOZZA
and F. D’AMATO
2. Percentage of seedlings jvoduced by caryopses collected from main (M) secondary (S) spikes of control and chronically irradiated durum wheat blants
Control
5R/day
1OR/day
and
15R/day
Material M Cappelli Aziziah Russell0 Cappelli brachytic
s
M
s
95.84 98.60 97.42 98.96 94.85 96.80 93.98 97.72 94.42 95.20 94.61 95.64 93.70 89.25 93.90 86.82
M
s
M
s
95.80 95.33 90.30 92.74
98.63 95.70 93.33 87.68
94.26 92.26 89.76 89.95
91.90 94.64 96.91 84.90
20R/day
25R/day
30R/day
Material M Cappelli Aziziah Russell0 Cappelli brachytic
listed in Table 3, no case of chlorophyll mutation was found. Screening of chlorophyll mutations in the ‘second generation’ was limited to only part of the plants grown in the ‘first generation’ (see Since all spikes present Material and Methods. in each plant (2-5) were analysed, plants showing mutants could be classified in two categories. (u) Plants in which segregation occurred for mutants of same phenotype in each spike, are regarded as heterozygous for the mutation (type A of NYBOM et al. ;(12) (b) Plants in which mutants occurred in only one or two spikes, while the remaining spikes did not contain mutations ; these plants are regarded as chimeric for the mutation (type B of NYBOM
et a1.(12)
Since, as can be seen in Table 5, the number of seedlings in a durum wheat spike is sufficiently high, there is good probability for the occurrence of at least one mutant in mutated spikes.@151 In Tables 4 and 5, the number and frequency of plants ascertained as heterozygous and chimeric for chlorophyll mutations in the ‘second generation’ from irradiated plants are
S
M
92.82 94.32 78.57 85.00 75.00 79-18 79.84 87.87 69.96 82.85 86.84 83.13
S
M
S
80.19 67.09 69.43 70.00 55.86 63.93 84.00 48.04 76.00 80.14 64.67 61.80
reported. In Table 5, the frequency of mutated seedlings is also shown. It is apparent that both the frequency of heterozygous plants and the frequency of chimeric plants and mutated spikes were highest at radiation exposures of 20-30 R/day. The frequencies of mutated seedlings (Table 5) were generally related to the frequencies of mutated spikes. In some radiation exposures (e.g. Cappelli, 25 R/day; Aziziah, 25 and 30 R/day; Cappelli brachytic, 20 R/day) a ‘segregation ratio’ of mutants in mutated spikes clearly greater than the overall ‘segregation ratio’ of the experiments was observed. This is to be ascribed to a greater average size of the mutated sector in the spikes.(3~7~8) The analysis of all ‘segregation ratios’ in the experiments did not reveal any relation of mutated sector size in the spike to radiation exposure. In Table 6, the frequencies of chimeric plants found in Cappelli and Aziziah have been reported separately for progenies originating from the ‘main’ and the ‘secondary’ spikes of the irradiated individuals. It is seen that 72 out of 78 mutations occurring in chimeric plants in the ‘second generation’ were born by the main spikes of the irradiated plants.
GENETIC Table
3. Material
analysed
in the Jirst
EFFECTS
generation
OF ZIURUM
from chronically secondarv sbikesl
No. spikes analysed Material Cappelli
R/day 5
10 15 20 25 30
Aziziah
5
10 15 20 25 30
Russell0
5
10 15 20 25 30
Cappelli brachytic
5
10 15 20 25 30
No. plants irradiated ---.
WHEAT
irradiated
&rum
53
No. of seedlings analysed
A4
S
668 145 504 521 501 975
51.38 28.56 37.32 33.08 23.47
14.84 10.35 10.50 12.70 12.84
13.91
11.89
301 307 106 12 206 78
17.20 13.31 16.14
10.37
444
41.56 27,37
S
M
s
12 10 14 14 16 16
39 16 37 35 48 59
45 14 48 41 39 82
2004 457 1381 1158 1127 821
6 12 8 4 12
30 38 28 4 38 55
29
516
40 15 3 35 22
500 452 15 467 361
12 11 12 7 15 16 16 14 12 7
9 7
Mean No. seedlings per spike
M
10
(M and S main and
wheat plants
--
79 56 35 14 88 70 80 46 31 15 34 48
DISCUSSION In these experiments, plants were irradiated for their whole-life cycle, from 3-leaf stage to harvest. Under these conditions, irradiation of distinct developmental stages should have resulted in the induction of mutation in cells of both the haplophase and the diplophase. These may belong to the following three categories. (u) Mutations induced in somatic (diploid) cells of the vegetative phase before meiosis. If a cell heterozygous for the mutation occurred in the layer of the shoot apex responsible for the
26 8 26
3284 1533 324 206
--__
3.75 12.28 6.56
25 26
1360 561
56 210 58 203 168
26 18
29
3450 1468 784
501 243 270
8 25 18
319
107
31.91 25.29 21.26
840 773
225 135
24.70 16.10
9
9.05 14.71 15.45 8.01 43.12
6.67 7.06 4.00 5.88 3.54 17.07 7.00 8.07 644 8.12 646
19.26 13.50 9.31 13.37 9.00 7950
organization of the sporogenous tissue, L-2, 3,(‘s) zygotes, and seeds, heterozygous and homozygous for the mutation, would be formed. Manifestation of the homozygous individuals (mutants) in the next generation (our ‘first generation’ from irradiated plants or ys of previous workers@Js)) would strictly depend on spike progeny size, mutated sector size and ‘segregation ratio’ of the mutation.(s~7~s) Plants originating from seeds heterozygous for the mutation will segregate for the mutants in all of their spikes (our ‘second generation’ or y3. W))
54 Table
B. DONINI, 4.
Plants
ascertained
G. T. SCARASCIA
as heterozygous
for
durum wheat plants.
MUGNOZZA
and F. D’AMATO
chlorophyll mutations in the second generation All spikes (2-5) in each plant were analysed
Cappelli
R/day 5 10 15 20 25 30
No. plants irradiated 6 9 10 6 9 10
540 360 750 370 403 388
1 1 2 2 3 2
0.18 0.27 0.26 0.54 0.74 0.51
__--.
No. plants No. plants irradiated analysed 6 10 8 4 9 10
Russell0
Way
chronically
irradiated
Aziziah Heterozygous plants No. %
No. plants analysed
No. plants No. plants irradiated analysed
from
204 226 187 15 171 136 Cappelli
Heterozygous plants ~-No. %
No. plants irradiated
No. plants analysed
Heterozygous plants %~No. 0 1 0 0 1 1
0 0.44 0 0 0.58 0.73
brachytic Heterozygous plants No. l
%
___~~.
5 10 15 20 25 30
6 10 8 6 9 10
357 454 109 64 158 116
0 1 0 0 2 2
0 0.22 0 0 1.26 1.72
6 10 8 6 9 10
326 432 263 159 211 211
0 1 0 0 1 2
0 0.23 0 0 0.47 0.94
In barley plants irradiated for the whole life cycle only a few instancesof mutations were observed in y2. This was taken as an indication that most of the mutations were induced at a stage later than the separation of the male and female organs of the flowers.(12) The possibility of diplontic selection was not discussedby the authors. In a total of 115 mutations ascertained in our experiments, 23 occurred in hetorozygous plants (type A of NYBOM et aZ.).P2) The frequency of plants heterozygous for mutation in our material (20 per cent) compares very well with that of 20.7 per cent found in the during development (diplontic selection(3~7~*)) irradiation of barley from germination to or happened to involve only a reduced sector maturity (18 mutations of type A and 69 of in the spike. type B).t12) Therefore, the data obtained in both barley and durum wheat, a largely diploidized species in the genetic system of its *Fusion of gametes bearing one and same mutation chlorophyll apparatus,(“) show very clearly is disregarded, due to the low probability of its occurrence. that, in experiments of chronic irradiation for
(6) Mutation induced in gametes or zygotes, resulting in seeds heterozygous for the mutation.* In experiments of the type performed by us, heterozygous plants cannot be distinguished as to mode of origin, either (a) or (b). (c) Mutation induced during embryo development and maturation.(lO) This will lead to chimeric plants which will segregate for mutants in one or some-but not in allspikes in the ‘second generation’. Since no mutants were recovered in the ‘first generation’ of chronically irradiated plants it must be assumed that mutations induced in vegetative cells were either selected against
5. Plants
as chimeric
for chlorofihyll
6 9 10 6 9 10 6 10 8 4 9 10 6 10 8 6 9 10 6 10 8 6 9 10
5 10 15 20 25 30 5 10 15 20 25 30 5 10 15 20 25 30 5 10 15 20 25 30
Aziziah
Russell0
Cappelli brachytic
326 432 263 159 211 211
357 454 109 64 158 116
204 226 187 15 171 136
540 360 750 370 403 388
No. plants No. plants R/day irradiated anaiysed
ascertained
Cappelli
Material
Table
3 6 17 11 13 14
No.
0.46 0.38 0.63 1.89 o-94
0.00
1.26 0.00
0.00 044 0.00 0.00
0.88 1.60 6-66 3.40 1.47
o-00
0.55 1.66 2.26 2.97 3.22 3.61
0, ,0
plants
791 1020 596 402 509 485
873 1106 324 192 482 354
582 620 526 42 498 405
1408 946 2062 947 1065 1128
No. soikes A analysed
3 1 10 2
0 2
3 6 19 12 19 14
No.
0.00
0.19 O-16 0.24 0.78 0.61
0.00
0.18 0.00 0.00 0.41 0.00
0.00
0.32 0.57 2.38 2.00 0.49
wheat plants.
45,010 50,707 32,285 18,854 23,332 15,916
30,66 1 26,640 8,919 5,443 12,724 9,072
24,513 22,233 20,293 1,096 15,865 12,163
61,761 45,151 92,903 41,633 44,845 46,208
No. seedlines analysed
durum
0.21 0.63 0.92 1.26 1.78 1.24
%
spikes
irradiated
Mutated
in the second generation from chronically each plant were analysed
Chimer+
mutations
0 3 2 13 23 16
0 13 0 0 3 0
0 20 9 2 74 26
I1 27 50 30 94 39
NO.
Mutated
0.00 0.059 0.061 0.689 0.985 1.005
0.00 0.48 0.00 0.00 0.23 0.00
1.25 04 1.82 4.66 2-13
0.18 0.60 0.54 0.72 2.09 0.84
0, /o
seedling
All spikes (2-5)
in
56
B. DONINJ,
Table
G. T. SCARASCIA
6. Plunts ascertained as chimeric for wheat plants. Plants derivedfrom
MUGNOZZA
and F. D’AMATO
chloro~lyll mutations in the second generation front chronically irradiated the main (M) and the secondary (S) spikes of irradiated individuals Chimeric
No plants Material
R/clay
Cappelli
5 10 15 20 25 30
Aziziah
5 10 15 20 25 30
No. plants irradiated
analysed
M
S
6 9 10 6 9 10
416 328 596 287 273 191
124 32 154 83 130 197
6 10 8 4 9 10
180 197 186 15 152 127
24 29 1 0 19 9
Mutated
plants S
M No.
No. spikes analysed
%
No.
%
M
3 6 17 10 12 11
O-72 l-82 2.85 3.48 4.39 5.76
0 0 0 1 1 3
0.00 0.00 0.00 l-20 0.77 1.52
1070 863 1640 742 735 560
338 83 422 205 330 568
0 2 3 1 5 2
0.00 1.01 1.61 6.66 3.28 1.57
0 0 0 0 1 0
0.00 0.00 0.00 0.00 5.26 o-00
528 542 523 42 447. 383
54 78 3 0 51 22
spikes
M
Mutated No. seedlings analysed
S
durum
s
seedlings
M
S -
Material
R/day
No.
y.
No.
y.
M
s
No.
y.
No.
%
15,357 3,605 19,112 8,777 14,814 26,560
11 27 50 27 78 21
0.24 0.65 0.67 O-82 2.59 1.07
0 0 0 3 16 18
0.00 0.00 o-00 0.34 1.08 0.67
0 20 9 2 66 26
0.00 1.03 0.44 1.82 4.55 2.20
0 0 0 0 8 0
0.00 0.00 0.00 0.00 5.86 0.00
~____ Cappelli
5 10 15 20 25 30
3 6 19 11 17 11
0.28 0.69 1.15 1.48 2.31 1.96
0 0 0 1 2 3
0.00 0.00 0.00 0.48 0.60 0.52
46,404 41,546 73,791 32,856 30,031 19,648
Aziziah
5 10 15 20 25 30
0 2 3 1 8 2
0.00 0.36 0.57 2.38 l-78 0.52
0 0 0 0 2 0
0.00 0.00 0.00 0.00 3.92 0.00
22,776 19,370 20,230 1,096 14,501 11,813
the whole-life cycle, the majority of recoverable mutations are mutations induced in the postfertilization diplophase, i.e. during embryo development and maturation. This conclusion is further strengthened by the analyses on the relative proportions of type A and B mutations induced in barley by y-irradiation in different periods of the cycle life: from germination to
1,737 2,863 63 0 1,364 350
heading, from flowering to maturity and from heading to maturity.(12) In the experiments of NYBOM et a1.W and in ours, the mutation rates recorded after chronic exposures were lower than, or did not exceed, those obtainable in the same materials with acute irradiation of seeds. These results are at variance with those of MIKAELSEN(*~)
GENETIC
EFFECTS
and SWAMINATHAN who reported, in barley and bread wheat respectively, that the mutation rates were higher after chronic than after acute radiation exposures. An interesting result of our experiment lies in the demonstration that 92 per cent (72 out of 78) of the mutations occurring in chimeric plants of Cappelli and Aziziah (Table 6) were borne by the ‘main’ spikes of the irradiated plants. The causes for this striking difference between ‘main’ and ‘secondary’ spikes in undergoing mutation during embryogenesis are not known. The folIowing possibilities may be suggested. (a) Embryogenesis in the ‘main’ spikes may take longer than in the ‘secondary’, resulting in an accumulated radiation exposure greatel in the former than in the latter. (b) Cell division in embryos developing in ‘main’ spikes may run with a mitotic cycle time longer than in embryos of ‘secondary’ be, at any daily spikes ; the result would exposure rate, an increased radiation effect(15) on dividing cells in the first category of embryos. (c) The number of cells constituting the apical primordia of ‘primary’ spikes in embryos is obviously greater than that of ‘secondary’ spikes, thereby increasing probability for the occurrence of mutations. The great variation in response to genetic effects of radiation among the four types of durum wheat tested cannot be explained. Previous studies with acute(‘e5) and chronic(“) irradiations have shown that Aziziah is more radiosensitive than Cappelli and Russell0 when effects on growth, survival at maturity and spike fertility are considered;(lp51s) however, no clear differences between Aziziah and Cappelli and among Aziziah, Cappelli and Russell0 are found when chromosome damage(s) and chlorophyll mutation rates(b) are analysed after acute irradiation of dry seeds. In our opinion, the differences in the frequency of mutations induced by chronic radiation exposure among the four durum wheat types must be searched for in the characteristics of their life cycle,(s) rather than in differences in radiation sensitivity. Acknr&e&ement-The
authors
wish
to express
their
OF
DURUM
57
WHEAT
appreciation 10 Mr. BEINAT for their very
M. BELLATALLA efficient technical
and Mr. assistance.
V.
REFERENCES I. AVANZI chimici italiane 353-364.
S. (1961) Effetti di radiazioni e mutageni sulla crescita e sopravvivenza in varietj. Afti Assoc. &ret. f&al. 6, di grano duro.
S.. BRUNORI A. and GIORGI B. (1966) 2. AVANZI Radiation response of dry seeds in two varieties of Triticum durum. n4utation Res. 3, 426-437. 3. D’AMATO F. (1965) Chimera formation in mutagen treated seeds and diplontic selection, pp. 303-3 16. In The use of induced mutations in plant breeding, Radiation Botagl Suppl. 5, Pergamon Press, Oxford. 4. D’AMATO F., SCARASCIA G. T., BELLIAZZI U., BASSANI A., CAMBI S., CEVOLO~TO P., GIACALONE F. and TAGLIATI S. (1962) The gamma radiation field of the “Comitato Nazionale per 1’Energia Nucleare”, Rome. Radiation Botatp 1,243-246. 5. D’AMATO F., SCARASCIA G. T., MONTI L. M. and BOZZINI A. (1962) Types and frequencies of chlorophyll mutations in durwn wheat induced by radiations and chemicals. Radiation Bolany 2, 217-239. 6. DONINI B., SCARAXIA MUGNOZZA G. T. and D’AMATO F. (1964) Effects of chronic gamma irradiation in durum and bread wheats. Radiation Botarp 4, 387-393. 7. GAUL H. (1959) Uber Chimlrenbildung Gerstenpflanzen nach Riintgenbestrahlung Samen. Flora 147, 207-241.
in von
8. GAUL H. (1961) Studies on diplontic selection after X-irradiation of barley seeds, pp. 117-136. In Effecti of Ionizing Radiations on Seeds, IAEA, Vienna. T. and INOSHITA I. (1965) Effects of 9. KAWAI gamma irradiation on growing rice plants. Irradiations at four main developmental stages. Radiation Botany 5, 233-255. 10.
MERICLE Mutation Radiation
L. W. and MERICLE induction by proembryo Botagf 1, 195-202.
II.
MIKAELSEN K. (1956) of chronic gamma Intern. Cottf. Peacejkl Nations 7, 34--39.
12. NYBOM N., EURENI~ERO
R.
P. (1962) irradiation.
Studies on genetic effects radiation in plants. Proc. Use.r AI. Energy, United
GUSTAFSSON I>. (1956)
A., The
GRANI-IALL I. and genetic effects of
58
B. DONINI,
G. T. SCARASCIA
chronic gamma irradiation on barley. Hereditas 4!&74-84. 13. POPHAM R. A. (1963) Developmental studies of flowering. Brookhaven Symp. Biol. 16, 138-156. 14. SWAMINATHAN M. S. (1965) A comparison of mutation induction in diploids and polyploids, pp. 619-641. In The use of induced mutations in
MUGNOZZA
and
F. D’AMATO
plant breeding. Radiation Botany Suppl. 5, Pergamon Press, Oxford. 15. VAN’T HOF J. and SPARROW A. H. (1963) The effect of mitotic cycle duration on chromosome breakage in meristematic cells of Pisum sativum. Proc. Natl. Acad. Sci., US. 50, 855-860.