The genetical control of radiosensitivity—II growth measurements in Lycopersicum and Melandrium

Radiation

THE

Botany,

1962,

pp.

277

to 295.

Pergamon

Press

Ltd.

Printed

in Great

Britain.

GENETICAL CONTROL OF RADIOSENSITIVITY-II GROWTH MEASUREMENTS IN LYCOPERSICUM AND MELANDRIUM D. R. DAVIES Wantage

Research Laboratory AERE, (Received 14 February 1962)

Berks.,

U.K.

Abstract-A

genetical control of radiation response has been detected in Lycopersicum and Melandrium species. The nature of this control was investigated by analyses of the progenies of a diallel cross of six genotypes of Lycopersicurn and of five genotypes of Melandrium. Seeds and seedlings were irradiated and growth measurements taken at various stages of development. There were numerous difficulties associated with the utilization of growth data for the assessment of comparative radiosensitivity-the most important being the strong positive correlation between control values and the degree of growth depression. A metric of sensitivity had to be calculated which removed this correlation. Evidence of both additive and dominance variation was found and also of pronounced reciprocal differences. The genetical control varied according to the stage irradiated and the stage measured. The significance of this variation and of the reciprocal differences is considered, and the value of growth data for radiobiological investigations discussed.

RCsum&--Un controle gtnetique de la reponse aux radiations a tte detect6 dans des esptces de Lycopersicum et de Melandrium. La nature de ce controle a ete etudiee par analyse de la progeniture provcnant de croisements dialltles de six genotypes de Lycopersicum et de cinq genotypes de Melandrium. Des grains et des plantules ont et& irradiees et la croissance mesurte a divers stades du developpement. 11 y eut de nombreuses difficult& resultant de l’utilisation des don&es sur la croissance dans l’evaluation de la radio-sensibilite comparee. La plus importante etait due a l’existence de la trPs forte correlation positive entre les valeurs du controle et le degre de depression de la croissance. Une metrique de la sensibilite a du etre calculte afin d’ecarter cette correlation. Une variation additive et de dominance ainsi que des differences reciproques prononcees ont ete mises en evidence. Le controle gtnttique variait d’aprts le stade irradie et le stade mesure. La signification de cette variation ainsi que des differences reciproques a tte considerte et la valeur des don&es sur la croissance dans ces investigations radiobiologiques a ete discutte. Zusammenfassung-In Lycopersicumund Melandriumarten wurde genetische Regulierung der Bestrahlungsreaktion gefunden. Die Eigenart dieser Regulierung wurde durch die Analyse der Nachkommenschaft einer Diallelkreuzung von seche Lycopersicumgenotypen und ftinf Melandriumgenotypen untersucht. Samen und Keimlinge wurden bestrahlt und Wachstumsmessungen an verschiedenen Entwicklungsstadien vorgenommen. Zahlreiche Schwierigkeiten zeigten sich in der Ausniitzung der Wachstumsdaten fiir die Bewertung der vergleichenden Strahlungsempfindlichkeit-die Wichtigste davon war die starke positive Korrelation zwischen den Werten der Kontrollen und dem Grad der Wachstumunterdruckung. Eine Empfindlichkeitmetrik musste berechnet werden urn diese Korrelation auszuschalten. Nachweis fur additative sowohl als dominante Variation wurde gefunden, sowohl wie such bedeutende reziproke Unterschiede. Dir genetische Regulierung war verschieden, und hing vom Stadium bei Bestrahlung und Stadium zur Zeit der Messung ab. Die Bedeutung dieser Variation und der der reziproken Unterschiede wird erwogen und der Wert der Wachstumdaten fiir strahlenbiologische Arbeiten wird besprochen. 277

278

THE

GENETICAL

CONTROL

INTRODUCTION factors are known to be important in determining the radiosensitivity of higher organisms, and the particular importance of some nuclear factors in this context has been indicated recently.c5) It has been shown that variations in nuclear volume and D.N.A. content, chromosome size and number, nucleolar and heterochromatin content, and centromere number and position, account to a large extent for the range of radiation tolerances found in various species and genera of higher organisms. That the particular genetic constitution of an organism has an influence on its radiosensitivity has also been recognized, but few attempts have been made to analyse critically the role of this genetic factor.@*rf In an earlier study(‘) the nature of this genetic control was investigated by analyses of three seedling characters in the control and irradiated progenies of a diallel cross of six Lycopersicum varieties. These analyses showed evidence of additivity and dominance for radiosensitivity and pronounced consistent and inconsistent maternal effects were also observed. The main aims of the present experiments were firstly, to confirm that the radiosensitivity of a plant is to some extent genetically controlled. Secondly, to compare the relative radiation response of genotypes irradiated as dry systems (seeds) and as actively metabolizing ones (seedlings). Thirdly, to obtain further information regarding the reciprocal differences observed in the earlier experiments. Lastly to clarify the role of cellular elimination in affecting the ultimate radiation response of a plant.

NUMEROUSbiological

MATERIALS AND METHODS Two separate experiments were undertaken. For the comparison of seed and seedling irradiation Lycopersicum esculentum was used, and for the comparison of developmental stages, the two dioecious species Melandrium album and M. rubrum were used. The same six Lycopersicum varieties were used as in a previous experiment, ‘-LI Harbinger; L2 Early Market; L3 Essex Wonder; L4 Ailsa Craig; L5 Hundredfold; L6 Carter’s Sunrise. These were crossed in all possible combinations including reciprocals, and the parental and F, seeds equilibrated over

OF RADIOSENSITIVITY-II Calcium Chloride for seven days and then divided into three groups. (a) Irradiated (17286 rad Coso gamma rays, at 1116 rad/min) and then immediately soaked in nitrogenated water for 25 min. (b) Non-irradiated, soaked as in (a). (c) Soaked as in (a) and irradiated (2000 rad Coso gamma rays, at 1600 rad/hr) at the 3-4 leaf stage (18 days growth). All seeds were planted singly in paper pots, and the plants grown initially in a greenhouse. There were seven plants per sub-plot, three sub-plots (a, b, c above) per plot, thirty-six plots (genotypes) per block and four blocks. The same design was used in the greenhouse and field. Characters scored were height at the 48th day, fresh weight at the 110th day after sowing, and flowering time. Five forms of Melandrium were used and crossed-Ml M. rubrum (ex Pembrokeshire, M. rubrum (ex Cardigapshire, U.K.) ; M2 U.K.); M3 M. album (ex U.S.S.R. Seed collection); M4 M. album (ex Cardiganshire, U.K.); M5 M. album (ex Poznam University, Poland). Seeds were placed over Calcium Chloride for seven days then exposed to 2000 rad of Coso gamma rays at 2000 rad/hr. They were then germinated in petri dishes, transferred to boxes in the greenhouse and later planted in the field. There, as in the greenhouse they were grown in a randomized design of three blocks, each block having twenty-five plots (genotypes) and each plot being composed of two sub-plots one of thirty irradiated and the other of thirty control seeds. Characters scored were-summed cotyledon lengths at the 25th day, summed leaf lengths at the 35th day, weight and stem number at the 162nd day after sowing, and flowering time. RESULTS The Metric of Radiosensitivity There were marked differences between genotypes in the extent to which growth was depressed after irradiation. The importance of this genetic factor was shown in the data from two genotypes which had similar control weights. One was reduced after irradiation to 2 1a7 per cent and the other to 74.5 per cent of its control value. However, the magnitude of this depression

D. R. DAVIES

in growth after irradiation could not be used as a measure of radiosensitivity. It was found that the amount by which the growth of any genotype was depressed, was directly correlated with the size of the unirradiated or control form of that genotype. Genotypes which produced large plants when unirradiated consistently showed a greater depression of growth than those which produced small plants when unirradiated (Fig. 1). It was necessary therefore to determine whether there were genetic differences in sensitivity which were separable from differences in growth. The correlation of growth depression and control growth was removed by fitting a regression line to the observations relating the differences between control and irradiated sub-plot means and the control

279

value, for each block independently. The deviation of the actual observation from the expected value on the regression line was then taken as. the metric of radiosensitivity. A positive value, i.e. a greater difference between control and irradiated plot means than would have been predicted from the regression line, indicated a more sensitive, and a negative value a more resistant genotype. Occasionally irradiated plot means were larger than the control, or in the case of flowering time smaller than the control. This meant that the expected value on the regression line was negative. To correct for this, the deviation (the metric of sensitivity) was always multiplied by the sign of the expected value and in this way it was ensured that a sensitive genotype had a positive value and a

0

0

. l

0

l

100

600

CONTROL FIG.

loou

Boo

WEIGHT,

G

1. An example of the relation between control weight values and growth in Qml,ersicum. Each point represents a single genotype.

depression

THE

280

GENETICAL

CONTROL

resistant one a negative value. It must be emphasized that the metric used could only give a relative measurement of response for those particular genotypes used in a given experiment. It may have minimized true differences and in using it we ignored the fact that large plants may have been genuinely more sensitive than small ones. At least it was shown that the large plants did not give rise to smaller irradiated plants than did the small control plants. However, the main aim of the experiment was the assessment of parent-offspring relationships and the metric used should be satisfactory for this purpose although it only gave relative values. Induced

Variation

Exposure of any living organisms to a mutagen usually results in an increased variation within the immediate or later generations of the treated population. Due to the correlation between the reduction in growth and control values referred to earlier, the first generation progenies in these experiments generally showed a decreased variation, both within and between genotypes (Table 1). Data for flowering time demonstrated this very well, though there was an increase in the mean after irradiation. Numerous reports of radiation induced stimulation of growth exist but in only a few instances is convincing evidence provided. The only statistically significant stimulation induced in the present experiment was for flowering time. Two genotypes of Lycopersicum, Ll x L4 and L2 x L3, flowered 5.6 and 5.2 days earlier Table

1. Within

OF RADIOSENSITIVITY-II (mean of four blocks, P=O*O2 to 0.01 and 0.05 to 0.02 respectively) and one genotype of Melandrium, M3 xM5, flowered 10.3 days earlier than the control (mean of three blocks, P=O-02 to O-01). None of these genotypes was markedly different in terms of any of the other characters measured and they were not at the extreme ranges of expression of any of the characters. The

filol variation

Control Plant

--

Melandrium

control

for

all genotypes

Seed irradiated

Seedling irradiated

Character Mean

Mean Square

Mean

Mean Square

Mean

Mean Square

time

15.42 763 48.80

5.27 36616 35.07

11.03 521 67.41

5.08 25592 32.22

13.59 604 55.39

3.85 21385 32.97

Cotyledon First leaf Weight Tiller No. Flowering time

21.83 51.96 256.41 6.57 75.89

11.57 216.50 7835 3.22 148.12

18.37 37.65 244.48 6,72 80.87

-Lycopcrsicum

genetic

Analyses of variance of the progenies of the two diallel crosses were made in order to detect genetic variation of the following kind: (a) variation between the mean effects of each parental line. (b) dominance. This can be subdivided, a significant b, value indicating unidirectional dominance and a significant b, value indicating asymmetry of gene distribution. (c) consistent maternal effect (average rociprocal difference related to a given line). (d) inconsistent maternal effect (reciprocal difference related to a particular cross). For a detailed account of this analysis the reader is referred to a paper by HAYMAN. Other analyses involved comparisons of the variances of array means ( Vr) and covariances within arrays of family means on to the nonrecurrent parent means ( Wr), (array= progenies of a common parent). Comparisons could also be made of Wr and the covariance within arrays of family means on to the mean of all offspring of that array ( Wlr). In all these calculations the mean values of

Height Weight Flowering

5.78 104.77

5898 2.98 80.82

D. R. DAVIES the reciprocal families were used initially. If the Wr/Vr and WY/ Wr relationships are represented graphically certain genetic information can be drawn from the distribution of the points representing each array on the graph.‘BglO) Lycopersicum

The calculated values of the metrics of radiosensitivity after seed irradiation are given in Table 2. The first statistic in each sector refers to the value from height, the second from weight, and the third from flowering time. The results of the analyses of variance for radiosensitivity after seed irradiation are given in Table 5. The genetic control ofradiosensitivity was clearly demonstrated in the data from height and weight measurements. There were significant differences between the mean effects of each parental line, and evidence of dominance, which was definitely ambidirectional in the case of weight values and a tendency in this Table

2. Mean

values of the calculated

mekics

281

direction also for height values. Pronounced reciprocal differences were found, their extent being well illustrated in the extreme cross of L2 and L3. The hybrid with L2 as maternal parent was the most radioresistant in the whole experiment and the reciprocal was the most radiosensitive (Table 2). Radiosensitivity data from flowering times gave no evidence of differences between parental lines, some evidence of dominance, this time towards resistance, and again pronounced reciprocal differences. Joint regression tests of the WT/ Vr and W’r/ WT relationships were significant for the weight and height data, only after the removal of array Ll, in other words parent Ll showed genetic interaction with the other genotypes. A significant regression line through points representing mean Wr/Vr or Wlr/ Wr values for all blocks could only be drawn in the case of W’r/ Wr from height data (Table 12). From that graph it was seen that there was no significant interaction

of radioseruilivily for Lycopersicum

genolylles, after seed irradiation

6

Ll

L2

L3

L4

L5

L6

Ll

0.22 14.85 - 1.8

2.31 144.24 2.20

- 1.26 20.54 -0.82

-0.19 - 37.93 -0.85

0.20 60.49 -2.22

-2.13 - 103.54 -3.66

L2

0.78 170.24 4.23

- 1.29 -25.71 -2.40

-3.63 - 193.55 -6.32

-2.11 44X2 -1.72

-3.91 - 189.42 -1.31

0.45 - 34.04 -3.05

L3

1.94 61.33 2.35

4.88 299.77 13.39

0.70 -2.64 - 1.65

3.06 57.61 4.50

-3.16 - 73.39 -543

- 1.08 - 103.92 -4.34

L4

0.06 - 13.23 0.23

0.56 3.55 -1.02

-0.14 - 38.58 -0.49

4.23 196.31 3.61

-0.03 - 57.42 - 1.98

- 1S6 1744 - 1.08

L5

2.75 192.01 4.15

-0.85 30.39 1.41

1.04 - 66.95 1.17

1.84 33.11 3.34

- 1.88 - 89.28 -3.34

-1.91 - 94.97 0.41

L6

- 1.09 -90.13 - 1.84

0.67 -2.36 - 1.85

0.64 34.02 - 1.65

2.H 134.94 3.98

i., T --

-2.14 -150.11 -2.32

-048 -47.94 -1.24

*The first statistic in each sector refers to data from height, flowering time.

the second from weight

and the third from

THE

282

GENETICAL

CONTROL

OF RADIOSENSITIVITY-II

remaining after the removal of array L.1 (slope of the regressiou line did not deviate significantly from 0.5) and no overdominance (intercept of the line on the H’r axis did not differ significantly from 0). Genotypes L4 (sensitive) and L5 (resistant) had most of recessive alleles. In the case of the other measurements the nonsignificant Wrl VY and W%/ Wr regressions could be due to interaction or maternal effects. An attempt was made to eliminate the possible upsetting influence of the maternal effects on the WrI Vr and W’rl Wr estimates by analysing the data from rows (common female parent) and columns (common male parent) separately. No improvement in the relationships was found. After seedling irradiation there was evidence of genetic differences in radiosensitivity only from the flowering time data (Tables 3 and 5). The absence of differences in the other characters could have been attributable to too low a level Table

3. A4ean ualues of the calculated

melrics

of‘ radiosensitiuily

of damage to allow the expression of genetic differences in radiosensitivity. This seems unlikely, however, in view of the fact that some genotypes were more badly damaged after seedling than after seed irradiation. Significant Wrl VY and W’rl Wr joint regression lines were obtained from the flowering time data (Table 12). These indicated the presence of some degree of dominance but not of overdominance or interaction. Genotype L6, the most radiosensitive, had most recessive alleles. The highly significant maternal effect observed from the height data after seedling irradiation was of particular interest and will be considered later. A joint regression test of the metrics of radiosensitivity showed no correlation between seed and seedling radiosensitivity. When the three control characters were analysed no evidence could be found of genetical differences between the parents and only some . for Lycopersicum

genobpes,

aflier

seedling irradiation

8

Ll

L2

L3

Ll

0.63 47.28 -2.62

2.22 52.52 1.22

- 0.46 73.33 - 1.33

- 0.74 - 54.97 -0.81

0.80 65.07 -1.61

L2

-0.24 8.65 - 1.04

-0.24 - 53.42 0.82

-1-30 - 37.09 -6.31

- 0.20 33.96 2.96

- 1.64 51.73 0.68

-0.42 29.52 -0.87

L3

-044 - 39.67 -2.31

0.22 -7.91 2.52

0.37 -27.96 -0.92

1.23 32.63 1.71

-1.34 - 108.83 - 1.59

1.06 16.19 2.07

L4

-0.64 -4.94 -1.31

1.55 0.91 0.20

0.28 11.43 -3.75

0.85 2-l-67 1.81

- 0.05 - 34.93 -0.93

0.59 83.03 4.14

L5

0.15 20.86 -3.15

0.75 1.66 - 1.78

0.21 28.07 0.79

1.90 -52.64 3.66

-0.57 - 22.84 1.75

0.82 -73.33 2.04

L6

- 0.46 -5.63 -2.47

046 - 17.23 -1.17

-0.04 -7.69 0.48

- 1.99 - 66.70 1.38

-0.68 26.04 -2.24

148 68.57 6.52

2

L4

L5

*The first statistic in each sector refers to data from height, the second from weight flowering time.

L6 -0.17 -11.68 -3.14

and the third from

283

D. R. DAVIES evidence of dominance for flowering time (Tables 4 and 5). It is quite clear therefore that different gene systems were involved in the control of radiosensitivity and growth, and it was very likely that different systems controlled sensitivity after seed and seedling irradiation. This conclusion could be verified by a modificationt2) of an analysis of variance devised by ALLARD. This is concerned with the relative magnitudes of Wr and 1-r under different conditions, and will test the significance of variation due to dominance (D) to arrays (A) to blocks (B) to the difference between rows and columns (R) (common female and common male parent respectively) and to the difference between seed radiosensitivity, seedling radiosensitivity and control growth (S). A significant dominance component can be expected, except when dominance is complete and the array component detects differences in the proportion of dominant and recessive alleles between parents. A significant DxA usually indicates non-allelic interTable

Ll

-

-k. A4ean clalues

L2

action. This analysis for flowering time data is given in Table 6. There was evidence of dominance, and the significant A x D x R interaction indicated the influence of the maternal effects on the estimates of nuclear gene interactions.@) The significant A x D x S component showed that a different genetic control existed for the three parameters measured (tlvo of radiosensitivity and one control). .\Ielandrium

The results of the analyses of variance estimating the genetic variation for radiosensitivity are given in Table 11. In the irradiated series (seed irradiation only, Tables 7 and 8) genetic variation in radiation response was demonstrated from cotyledon, first leaf, and flowering time measurements but not from fresh weight data and tiller numbers. There was no correlation between radiosensitivity at early and late growth stages. From cotyledon measurements there was evidence of significant differences in

for the control characters L3

c?

of Lycopersicum

genolyles

L4

L5

L6

-

Ll

15.35 733.63 7.86

13.04 688.57 14.43

15.36 686.97 11.80

14.33 653.43 16.09

17.29 774.35 7.19

15.52 837.01 9.10

L2

15.71 763.32 12.02

13.93 770.95 13.59

15.23 705.66 14.11

15.84 695.69 11.36

15.42 753.93 12.01

16.67 79744 12.24

L3

16.18 780.06 9.17

12.27 674.17 10.76

15.02 773.80 19.41

16.95 853.66 13.11

16.59 798.22 10.96

15.34 799.20 10.01

L4

14.81 769.58 11.92

14.05 751.58 13.04

15.78 788.84 12.50

12.85 760.30 13.45

16.88 867.74 7.96

16.88 819.50 8.86

L5

16.39 653.00 9.66

15.40 827.59 13.08

15.90 78448 11.31

15.98 749.82 15.10

14.89 815.63 10.97

14.80 698.06 16.32

L6

15.17 768.44 15.45

15.81 794.85 9.98

15.94 738.75 17.13

15.84 632.57 16.25

15.61 830.12 13.59

15.70 894.67 7.39

---

0 --

*The first statistic in each sector refers to height, the second to weight

and the third to flowering

time.

6.79 18.41 5.00 6.62 6.87 6.42 2.75 112.27~~~ 5.49

5 1 5 9 15 5 10 3 105

143

a b, bs bs b

Total

Figures quoted are Mean Squares. *Zero value is due to the fact that xxx P<.OOl. xx P=O.Ol to 0.001. x P=O.O5 to 0.01.

: B Bt

Control

N

Item

Table 5. Analysk

deviations

15.47 15.36 16.41 12.27 13.86 29.07 17.92 3.67 *

xx x xx xx xxx xxx

from

a regression

1.23 3.61 1.95 4.19 3.41 12.05 xxx 1.20 2.65

line were

12988

36456 40480 63588 46188 51608 59944 40996 xxx xxx xxx xxx xx

x

used as the metric,

19653 22550 3655 16513 12629 23337 14536 142735 xxx 19471

Control

Seed irradiation

Seedling irradiation

Seed irradiation

seed and seedling irradiation Weight

aftir

Height

of variance of radiosensitivity

32.55 0.22 58.42 x 24.06 33.92 61.30 x 21.68 323.92 xxx 23.51

Control

20.29 1.64 63.85 28.08 38.24 106.83 60.76 15.93

Seed irradiation

and these must add up to zero.

6640

5363 820 7861 6554 6608 5739 13071

Seedling irradiation

Flowering

and of control values in Lycopersicum

x xxx xxx

x

time

54.91 x 52.83 13.72 14.66 16.89 29.34 22.47 18.61

Seedling irradiation

Z

&

E 2 < 3

T u 8 ii

g

$

g

Z

r 8

P

2

8

Z

D. R. DAVIES Table 6. Analysis glowering times,

of variance of Wr and Vr for radiosensitioity determinedfrom after seed and seedling irradiation and for the control character (LYCOPERSICUM) Item

Arrays (A) Blocks (B) Dominance (D) Rows and Columns (R) Radiosensitivity and Control AxBxS AxDxR AxDxS BxDxS BxRxS Others non-significant Residual Total

parental lines. the radiosensitivity of The components of variation (c) and (d) did not necessarily indicate maternal effects in dioecious species such as these. The reciprocal differences in radiosensitivity among male and female progenies were the same, however, indicating that sex linked factors did not contribute to the reciprocal differences. From first leaf measurements there was again a significant (a) component and also evidence of dominance, this time, at least in the males, with a tendency towards radioresistance. Sex differences in radiosensitivity were not significant. The estimates of the genetic control of radiosensitivity from flowering dates were different (Table 11). In the males, but not in the females, there were parental differences and dominance, again towards resistance.. The Wrj Vr and Wlr/ Wr regressions for radiosensitivity from cotyledon data are given in Fig. 2. In no instance was there overdominance, and the only evidence of gene interaction was in the w’r/ WY graph from cotyledon measurements in males (deviation of slope from O-5 significant at the 0.02 to 0.01 level). The two remaining significant Wrl Vr and Wlr/ Wr values (from first leaf data) are given in Table 12-in C

285

N

(S)

Mean Square 5 3 1 1 2

307.43 364.15 22717.00 xxx 1516.90 312.23

30 5 10 6 6

223.96 403.68 370.52 58048 511.05

188

194.90

30

131.72

x x xx xxx xx

287

these there was again no interaction or overdominance. The genetic control of the growth data themselves (Tables 9 and 10) was of interest only in so far as it could be compared with that for radiosensitivity. Comparison of the genetic variation Table 11, and of the Wr/ Vr and Wlr/ Wr regression lines (Table 12) and the distribution of array points showed clearly that there was a different genetic control of growth and radiosensitivity. DISCUSSION Radiation induced growth reduction in plants is generally considered to be primarily due to nuclear damage, and is a parameter which has been extensively used in radiobiological investigations. However in the present experiments it was observed that the extent of this radiation induced growth depression was directly correlated with control growth. values. Genotypes which were potentially larger consistently showed a greater depression than those which were smaller. Thus growth reduction could not be used as a metric of radiosensitivity. This correlation is one which has been generally ignored in the past, but unless the metric used

THE

286

Table

7. Mean

GENETICAL

values of the calculated

CONTROL

metrics

OF RADIOSENSITIVITY-II

of radiosensitivib

for

male

Melandrium

genoppes,

af&er seed irradiation

d Ml

M2

M3

M4

M5

Ml

2.61 9.36 - 34.2 1 -0.28 2.36

1.21 8.94 24.8 1 0.60 3.89

-0.01 -0.24 36.76 1.oo 0.81

-0.75 -5.84 2.32 0.04 - 3.64

1.86 2.89 -23.93 0.80 2.91

M2

2.81 3.46 13.89 -0.20 -2.21

0.56 10.85 10.93 0.90 4.15

-0.01 - 13.16 5.33 - 1.05 -7.61

-4.24 - 1.87 52.23 0.63 0.86

0.79 - 1.03 - 37.50 -0.28 -3.75

M3

0.54 5.42 -2.56 0.77 7.85

1.05 2.31 35.56 0.11 -0.25

- 0.47 4.64 -4.12 0.01 8.67

- 0.93 - 7.55 - 19.95 0.09 0.96

-2.17 - 7.55 - 7.29 - 0.65 - 14.76

M4

-0.11 5.50 -67.82 0.23 6.30

- 1.44 - 1.15 - 1.86 0.31 -0.04

-2.22 -2.42 -49.15 -0.22 5.78

-2.06 -4.22 18.25 - 0.66 1.26

-2.50 -9.09 - 15.75 0.94 -0.08

M5

1.62 0.47 -30.17 0.09 0.37

1.76 -0.49 -21.11 -0.88 -0.29

1.42 - 1.92 13.58 -0.17 1.51

- 0.45 -4.72 0.87 -0.21 0.23

1.12 0.85 - 35.25 -1.13 -3.69

0

The first statistic in each sector refers to data from cotyledons, the sicond from first leaves, the third from weight, the fourth from tiller numbers and the last from flowering time.

D. R. DAVIES

Table

8. Mean

values

of the calculated

Ml

M 1

M2 p----

M4

1.77 i i .a2 53.2 1 0.15 0.91

1.59 10.34 2764 -0-17 0.63

0.77 - 0.38 83.66 1.07 1.99

- 1-21

1.74 1.64 27.32 - 0.62 - 1.53

2.11 14.92 -1.17 o-15 4.47

- 15.36 3-20 -0.60 - 7-97

3.70 -41.22 0.77 6.02

- 65.48

0.54 4.67

--

M5

Melandrium

female

M3

-0.14 4.43 M4

for

M2

-0.78

M3

metrics of radiosensilivily after seed irradiation

1 -74 4.51 58.94 - 0.77 5.83

-0.09

- -

0.14 1.24

0.82 7.36 -43.28 0.34 1.29

- 0.60 5.40

0.86 2.73 la.27 0.54 -1.14

- 3.22 -5.76 - 49.95 0.26 4.33

1 .a6 o-94 -9.16 -0.95 4.72

1.20 -4.25 11.68 -0.25 - l-96

0.81

genotypes,

M5

3.28 a.84 - 0.08

3.31 4.25 -71.65

-2-99

0.32 - 0.07

-4.78 -5.84 - 17.60 0.29 -2.63

-0-34 -3.51 -30-71 o-09 - 4.24

- 1.22 -9.09 - 30.73 -0.51 - 1.22

- 1.45 - 10.76 4744 0.13 - 10.16

-3-39 - 12.89 - 37-84

- i .a3 - 10.33 -2.93 -0.52 -44-95

- 0.30 -5.13 - 0.98

-6-10 21.06 0.31 i -38

-

1.52 3.83 - 54.36 - 1,25 0.25

The first statistic in each sector refers to data from cotyledons, the second from first leaves, the third from weight, the fourth from tiller numbers and the last from flowering time.

THE

288

GENETICAL

CONTROL

OF

RADIOSENSITIVITY-II

Table 9. Mean values for the control characters of male Melandrium

Ml

M2

M3

gerwtyjes

M4

M5

Ml

20.93 4640 260.00 746 87.49

19.68 .48.77 297.49 7.49 85.14

22.08 53.25 285.00 6.35 82.88

20.13 54.78 235.33 7.43 79-88

22.09 50.74 233.15 6.94 72.11

M2

24.70 59.96 324.92 7.83 83.28

21.96 53.03 218.69 7.67 81.22

26.6 I 67.26 293-88 5.68 51.05

23-28 59.03 239.78 5.66 59.55

25.92 60.63 2 16.76 5.43 55.62

M3

18.40 53.64 236.20 7.01 73.82

15.75 46.04 296.47 8.33 83.65

14.69 31.96 170-98 4.89 85.14

17.25 38.75 241.83 6.05 83.38

15.90 33.25 208.22 5.45 78.11

M4

26-97 85.59 294.41 7.22 74.26

24.12 5448 290.39 7.98 82.18

25.86 58-96 335-89 6.05 74.66

25.66 54.56 290.02 6.33 84.26

25.92 46-29 244.9 1 5-39 71.38

M5

21-19 49-63 275.02 7-40 77.68

19.16 48.35 264.81 8.25 78.65

22-95 54-35 233.18 4.92 66.25

22.98 39.00 278.86 6-06 79.4 1

21.67 50.08 154.67 4-91 64-2 1

0

The weight,

first statistic in each sector the fourth to tiller numbers

refers and

to cotyledons, the the last to flowering

second to first times.

leaves,

the

third

to

D. R. DAVIES

Table

10. Mean

values_lbr

the control

characters

289

o/female

Melandrium

genoppeJ

d

Ml

M2

M3

M4

M5

20.60 47.26 340.72 7-54 88.58

20.69 49.14 317.61 7.95 89.28

23.05 60.26 250.7 1 5.97 77.69

21.91 64-17 303.60 7.43 80.18

23.92 57.90 263.72 6-18 61.22

23.94

56.5 1 342.35 7-85 83-82

23.65 52.87 244.97 7.03 81.77

27-2 1 6144 328.43 5.81 47.93

23.20 58.0 1 271.74 5.89 57.07

27-40 65-40 2’37.09 5.41 45-63

M3

18.95 50.38 338.95 7.33 73.86

16.17 50.34 315-74 8.10 80.29

15.15 31.99 252.34 5.69 84.04

17.55 39.94 331.34 6-95 87.89

18.05 42-89 282-50 5.85 69-19

M4

27.02 75.80 377.48 7.24 74.19

26-2 1 54-l 1 359.34 8-46 80.60

24.86 55.98 409*09 6.08 74.68

25.38 51.33 375-52 7-13 86-42

28-25 44-94 295.03 5.11 7.1.10

M5

21.58 52.02 301.22 6-92 72.09

20.36 49.86 292.13 8.21 72.73

22.33 51.03 268.06 5.73 68.33

22.68 45.6 1 358.32 6-51 78-32

21.95 43.60 209.96 5.10 63.09

Ml

M2 --

r:----

The first statistic in each sector refers to cotyledons, the second to first leaves, the third weight, the fourth to tiller numbers and the last to flowering times.

to

-

Male

First leaf

67.47

63~61 70.93 85.61

482.43 422q38 228.69 281.16 544 274.29 120.70 5.13 xxx 816.13 107.15 x 77-47 xxx 3150~82 xxx 2.96 200.07

81.10 xxx 24.43 xx 2.20 4.23

2.90

1~81 x 8-35 8.27 x

43.17 xxx 283.98 x 3.46 387.52 x 1.37 35.06 1.83 21.67

Female

length

402.26 944.53 x 85.86 30644 282.02 313.13 47.40 2428*08 xxx 226.26

57.56

88.90 x 194.05 64.05

590.92 xxx 323.55 x 128.47 10.31

12437 xxx 26414 xxx 3274 3460 x 5681 xxx 6323 1670 xx 3260 1367

2187

2591 3542 712

3994

3588 1431 220

Male

Female

22530 xxx 9103 x 2826 3044 3563 8426 3460 xx 12765 xx 1953

208- 1

3371 3119 8859 xx

4348

0.65 6.42 1.19 xxx 244 0*75

12.14 xxx 1.87 0.25 0.72

0.76

1.58 0.63 0.75

1.23

1.16 2.09 1.30 x

Male

and of control values in

5331 2725 1075

Weight

after seed irradiation

Female

length

of radiosmsitivi~

20.66

59.07 xx 6449 93.75 xx

50.15

81.60 x 81.59 x 64.61

Male

741.87 xxx 862.82 xx 50.41 361.57 xx 287.23 xx 872.00 10865 xxx 628.92 xxx 101.48

36.32

56.60 x 123.73 22.67

53.54

39.15 29-93 67.11

Female

Flowering time

8.21 xxx 396.37 xx o-75 392.25 x 0.48 85.84 0.23 149.21 0.38 148.17 8.14 xxx 696.32 0.80 179.26 xxx 1.75 854.84 xxx I ~08 78.97

0.75

O-97 0.40 1.06

1.10

I *32 0.61 1.72

Female

Tiller no.

Melandrium

Figures quoted are Mean Squares. *Zero value is due to the fact that deviations from a regression line were used as the metric, and these must add up to zero. xxx P<.OOl. xx P=O.Ol to 0.001. x P=OaO5 to 0.01.

74

10 64 2 48

a bl b2 b3 b zl B Bt

Total

3.95 136.77 4.65 xxx 77.18 xxx 3.20

4 1 4 5

Total

zb $ u

74.17 xxx 13.69 x 3,74 2.18

74

B Bt -* 1.36

482

i

2a83 x 4.35 5.08 xx

35.65 xxx 2.32 1.43 4.06

Male

.--

11. Analysis o/variance

Cotyledon

.Table

d

a bl b2 b3 10 4 6

Item

b

N 4 1 4 5

-...

_

85 -2 .”

‘2 ‘Z

x .z

-.._

=I

2

2

iz 3 ii 5 2

$

E

3

8

F

3 +I i;

Control Radiosensitivity

First

Flowering time

Flowering

Weight

Height

Control Radiosensitivity

Control Radiosensitivity

Tiller

no.

Control Radiosensitivity

Weight

leaf

Control Radiosensitivity

Cotyledon

1

-

-

Seedling

radiosensitivity

Seed radiosensitivity

I .084 + 4.304

-

-

Control

0.832 f0.23

-

-

Seedling

0.286*0.027

-

-

-

Seed radiosensitivity radiosensitivity

-

-

-

Control

-

-

137.52

-

Wr

0.443kO.134

-

b W’r/

28.28

-

radiosensitivity

c

-

-

1.141

4.455

1.762 ,L 0.870

-

Seedling

-

-

Seed radiosensitivity

b Wr/Vr

Lycopersicum

-

0.53410.155

-

-

7.182 & 1.406

-

-

-

-

-

c

-

10.331+

26.798+

lines in

-

0.973 kO.272

-

-

-

0.35 110.054

-

384*252

k3.240

0.061 kO.295

-0.761

-

0.943;0.120

b Wr/Vr

regression

-

W’r/Wr

0.864&0.041

and

0.631 f0.565

0.280&0.176

c

of Wr/Vr Melandrium

Control

-

-

0.478hO.037

2.831 kO.356 -

-

0.346*0.038

0.552kO.041

-

0.670 *0.033

0.392 kO.007

b W’,/Wr

-

--1838*2525

-

-

-

time

13.985

c

d

12. Slopes and intercepti

6.030 *2.398

28.40

-

1.021 *to.064

-

0.939+0.268

-

-

0.730*0.137

0.976&0.151

b Wr/Vr

Table

b W’r/

Wr

&I.178

1.151*0.464

-

-

-

-

-

-

-

c

-

0.274t0.036

-

0.595+0.046

-

0.418,tO.O63

0.372 $10.055

-

0.499&0*103

0.412&0.006

-0.761

0

-

2.555

0.134

34.64 -

f

--8.341-+0.24

-

--103+436

5.70

33.72 1 k22.854

1.042f

0.665*

c

: F; v,

7

P

292

THE

GENETICAL

CONTROL

OF

TY cf

RADlOSENSlTlVl

40

RADIOSENSITIVITY-II.

80

40

Vr Wr

Wr RADKXENSITIVITY

Wr

CONTROL

40

$?

9

80

40

VT

FIG.

2. Wr/Vr

and

80

W17/Vr graphs from the The array points represent

diallel analyses mean values

of cotyledon for all blocks.

80

data

Wr

in Melandrium.

D. R. DAVIES in any comparison of varieties, species or genera which do not have the same control value, takes account of the correlation, an incorrect assessment of comparative radiosensitivity is obtained. Instead of differences in sensitivity to primary nuclear damage differences in growth will be measured. The correlations are not removed by expressing growth of irradiated plants as a fraction of control growth for example, but they can be removed in the way described earlier. Such correlations may be due to the different growth rates of the various genotypes; since growth is exponential a given initial radiation induced delay in growth will result in a more marked depression in a faster than in a slower growing genotype. There is no information available on the relative delay in genetically divergent forms. Other difficulties can arise in the interpretation and utilization of some growth data since a reduction in growth can be due to various factors, one example being an upset hormonal metabolism. Another factor which appeared of importance in the present experiments was related to the growth and competition of cells in irradiated meristems. Following irradiation the meristems are depopulated due to cell differentiation and death, and later repopulated by a mixture of normal and mutant cells-the latter usually having a slower growth rate. Cellular competition may result in a swamping and an elimination of the mutant cells. The extent of this elimination is directly related to growing conditions-the better the conditions the greater the advantage normal cells have.@) Since optimum environmental conditions for growth will be specific for each genotype, the rate of elimination in one given environment will vary among genotypes. Again, elimination rates are related to meristematic organization,(@ and possibly to the general level of damage. The importance of some of these factors may not be very great when the radiosensitivity of closely related genotypes, such as those used in the present experiment, are compared in terms of growth parameters, but in comparisons of widely divergent species and genera they can be particularly important. It was shown earlier that comparisons of irradiated plants over a fixed unit of time are invalid unless the forms

293

compared have a similar control value, or the correlation with control growth is removed. Similarly comparisons of divergent forms over a fixed unit of control growth will not give valid measures of chromosomal damage unless the rate of cell elimination is similar, and from the evidence of the present experiments the role of this process must not be underestimated. The metric of radiosensitivity used, though only a relative one, was adequate for the main purpose of the experiment-the assessment of parent-offspring relationships. Clear confirmation was obtained of earlier evidence(7*2) that the radiation response of an organism is a heritable character. The genetical control was quite different from that concerned with the growth of the plant. There was evidence of dominance, mostly ambidirectional but in some. instances towards greater radioresistance. Ambidirectional dominance was detected in a previous experiment@) using the same genotypes of Lycopersicum and scoring three seedling characters. Its possible significance in relation to the evolution of the character “radiosensitivity”, has been discussed previously.@) Lawrence from a study of the genetic control of radiation induced chromosome breakage in rye has shown dominance to be exclusively in the direction of increased sensitivity in that species.ou Reciprocal differences were observed in all instances after seed irradiation of Lycopersicum and also possibly, as explained earlier, in Melandrium. This could have been due to the influence of the endosperm on embryo development immediately post-irradiation. The fact that maternal effects were detected even when mature plant characters were scored did not disprove this concept since the initiating event which resulted in radiation damage at all stages, had occurred in the embryo. However, the evidence of maternal effects even after seedling irradiation indicated that true cytopiasmic effects, and not necessarily endosperm influences, could have been involved in the production of these pronounced reciprocal differences. READ has reviewed the evidence for attributing radiation induced growth reduction in the roots of Vicia faba to chromosome damage and there is a considerable amount of evidence which indicates that delay in division and cell death in other

294

THE

GENETICAL

CONTROL

organisms are primarily attributable to nuclear damage. The cytoplasm is inherently resistant to radiation. Nevertheless it might be expected, and it has been established, that the cytoplasm plays a role in the development of nuclear damage. Duryee’s experiments on nuclear transplantation in amphibian eggs showed that the development of lesions in irradiated nuclei was dependent on the presence of an irradiated cytoplasm.(*) ORD and DANIELLI(~~) have shown similarly that irradiated amoeba nuclei transferred into a normal cytoplasm developed normally. A similar dependence on the cytoplasm for the development of nuclear damage was shown in NAKAO’S results.02) It is quite likely therefore that the different cytoplasmic constitution of the cells in the reciprocal crosses in the present experiments had a differential effect on the development or repair of nuclear damage and gave rise to the pronounced reciprocal differences in radiosensitivity. The different genetical control of sensitivity observed in Melandrium when measurements were made at different growth stages, was not unexpected when one considers the changes that occur in an irradiated meristem. Radiosensitivity values obtained from very early postirradiation growth stages, more accurately measure the relative sensitivities of genotypes to primary damage, i.e. chromosome aberration production. At the later growth stages the meristematic population will be different due to the processes of cell elimination referred to earlier. Hence measurements at later stages will indicate the relative sensitivities of different genotypes to aberration production as well as indicating the relative amounts of mutant cell elimination. There was no correlation between sensitivities determined from cotyledons or first leaves and from mature weights. In fact there were no genetic differences between the genotypes. at the mature stages. This differential response it must be emphasized was not related to the different size of the plants at the different stages as the growth correlation had been removed. In Lycopersicum on the other hand, genetic differences were observed at the mature stages and this may have been due to the greater amount of damage induced, or to a less efficient elimination of mutant cells.

OF RADIOSENSITIVITY-II The different responses obtained after seed and seedling irradiation may indicate the inherently different response of a resting and an actively metabolizing system, or be related to the different meristematic activity and organization within the two tissues. The results certainly show that extrapolation of results from growing plants to resting seeds or vice versa need not necessarily be accurate. The inherent resistance to chromosome aberration production as well as the ability to eliminate mutant cells from the meristem are two important factors in determining the survival and recovery of an acutely irradiated plant, and their relative roles as well as the role of other factors have to be carefully considered if data from growth measurements are utilized in radiobiological experiments. At present there is no information available that would permit identification of the basic differences which underlie the heritable ‘variations observed in the genotypes examined. Nevertheless the effect of this genetic factor has been shown to be of such a magnitude that it must be taken into account in any consideration of factors which influence the radiosensitivity of higher organisms. Acknowledgments-The assistance of Mr. B. E. COOPER and Mr. .T. HOBSON of the Computer Section, Harwell, is gratefully acknowledged. REFERENCES 1. ALLARD R. W. (1956) The analysis of geneticenvironmental interactions by means of diallel crosses. Genetics 41, 305-318. 2. DAVIES D. R. (1962) The genetical control of radiosensitivity. I. Seedling characters in tomato. Heredity. In press. 3. DAVIES D. R. (unpub. observations.) 4. DURYEE W. R. (1949) The nature of radiation injury to amphibian cell nuclei. 3. Natl. Cancer Inst. 10, 735-758. 5. EVANS H. J. and SPARROW A. H. (1961) Nuclear factors affecting radiosensitivity. II. Dependence on nuclear and chromosome structure and organization. In a symposium on fundamental aspects of radiosensitivity. Brookhaven Symposium in Biology, 13.

D. Ii. 6. GAUL H. Studies on diplontic selection after X-irradiation of barely seeds. Symfxwium on the effects of ionizing radiation on see& and &heir significance for crol improvement. Sponsored by I.A.E.A. and F.A.O. Karlsruhe, Germany (1960). 7. GRAHN D. (1958) The genetic factor in acute and chronic radiation toxicity. Proceedings of the International Conferences on the Peaceful Uses of Atomic Energy (2nd. Con& Geneva 1958, United Nations) 22, 394-399. 8. HAYMAN B. I. (1954) The analysis of variance of diallel tables. Biometrics 10, 235-24-k. 9. HAYMAN B. I. (1958) The theory and analyses of diallel crosses-II. Genetics 43, 63-85.

DAVIES

295

10. JINKS J. L. and HAYMAN analysis of diallel crosses. News Letter 27, 48-54. 11. LAWRENCE

C. W.

(unpub.

B. I. (1953) Maize Genetics

The Coop.

observations).

12. NAKAO Y. (1953) Action of irradiated on untreated chromosomes of the Nature, Land. 172, 625-626.

cytoplasm silkworm.

13. ORD M. J. and DANIELLI J. F. (1956) The site of damage in amoeba exposed to X-rays. Quart. J. Micr. Sn’. 97, 29-37. 14. READ J. (1959) Radiation biology of Vicia faba in relation to the general jwoblem. Blackwell Scientific Publications, Oxford. 270 p.