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The induction of sports in chrysanthemums by gamma radiation
Radiation
Botany,
1962, pp. 297 to 303.
THE INDUCTION
Pergamon
Press Ltd. Printed
in Great Britain.
OF SPORTS IN CHRYSANTHEMUMS GAMMA RADIATION
BY
Ii. J. M. BOWEN, P. A. CAWSE and M. J. DICK Wantage Research Laboratory (AERE), Wantage, Be&s., Great Britain (Received
4 January
1962)
Abstract-The effect of gamma rays from cobalt-60 on cuttings of seven chrysanthemum varieties has been studied. The LDsg for the cuttings were in the range 3.0-4.3 krad. Irradiation caused plants to flower much later than controls. About 40 per cent of the irradiated plants showed colour sports; the overall percentage of sports and the percentages of different types depended markedly on variety. One sport of one variety appeared to be caused by the breaking of a periclinal chimaera. The remainder were believed to be mutants in the broadest sense of the term. The number of mutant cells was estimated to lie between 2.2 and 27 per lo6 cells per krad. Most sports were produced by irradiating varieties with a chromosome number of 54 and fewest from those with the highest chromosome numbers. A hypothesis is advanced that radiation-induced sports may have more chromosomes than their parent, but rarely have less. RCsum&-L’effet des rayons gamma du cobalt 60 sur des boutures de sept varieds de Chrysanthtmes a CtC Ctudit. La LDsos pour les boutures Ctait de l’ordre de 3,0-4,3 krad. L’irradiation a cause une floraison plus tardive que les controles. Le pourcentage total des variants et les pourcentages de differents types dtpendaient d’une man&e marquee de la variett. 11 est apparu qu’un variant ttait cause par la dissociation d’une chimtre ptricline. On croit que les variants restants sont des mutants au sens le plus large du terme. Le nombre de cellules mutantes a et& estime &re situ& entre 2,2 et 27 pour lo8 cellules et par krad. La majoritt des variants a ete produite en irradiant des varietts a nombre chromosomique 54 et beaucoup moins de varittb a nombres chromosomiques plus ClevCs. On avance une hypothtse suivant laquelle les variants induits par des rayons peuvent avoir plus de chromosomes que leurs parents mais rarement moins. Zusaznznenfassung-Die Wirkung der Gammabestrahlung (Coso) auf Steklinge von sieben Chrysanthemum-varietaten wurde untersucht. Die LDs, fur die Stecklinge lagen zwischen 3,0 und 4,3 Kilorad. Die bestrahlten Pflanzen bliihten vie1 splter ah die Kontrollen. Ungefahr 40 prozent der best&hen Pflanzen hatten Farbenmutationen; der Gesamtprozentsatz der Sports und der Prozentsatz der verschiedenen Typen hing von den Varietaten ab. Eine Mutation in einer Varietat schien durch die Stijrung eines Periklinalchimlrs hervorgerufen worden zu sein. Die Ubrigen sind anscheinend Mutationen im weiten Sinne des Wortes. Das Verhlltnis von normalen zu mutanten Zellen ist schiitzungsweise zwischen 15,000 und 180,000, abhlngig von der Varietat. Es wird die Hypothese vorgeschlagen, dass Bestrahlungsmutante mehr Chromosomen haben konnen als ihre Eltern, aber selten weniger. INTRODUCTION
forms may give rise to bud sports with new colours, which can be propagated by taking vegetative cuttings. Bud sports originate in an apparently random manner, and it may take many years for an important variety to yield its full quota of sports. For example, the pink variety Sweetheart was first raised in 1939 as a
THERE are two ways in which
new varieties of cultivated chrysanthemums may arise. The majority of new forms have been selected from the progeny of crosses between fertile parents, but many of the better forms produced in this way do not readily set seed. Secondly, many 297
298
THE
INDUCTION
OF SPORTS
IN CHRYSANTHEMUMS
seedling from a cross of White Buttercup Excelda. It was six years before any sports noted, but eleven sports were known by The history of sporting in this variety has reconstructed in Table 1, which also the chromosome numbers as measured
with were 1950. been gives by
DOWRICK.
In the same way the variety Loveliness was grown for five years before any other colours appeared, but ten sports appeared in the next decade. The present studies were undertaken to evaluate the potentialities of ionizing radiation in the production of chrysanthemum bud sports. Similar studies have been reported by JANK,@) BREEHAN and SAGAWA(~~) and SHAPIRO and SHOERTJES.‘~~)
Table
Variety
Chromosome
Sweetheart Egerton Sweetheart Red Sweetheart Christine Sweetheart Bronze Sweetheart Salmon Sweetheart Pearl Sweetheart Apricot Sweetheart Peach Sweetheart Orange Sweetheart Deep Red Sweetheart Golden Sweetheart Cream Sweetheart Deep Pink Sweetheart *Chromosome
1. History
Methodr
BY GAMMA
and materials
Rooted
of seven varieties of Chrysan(Sweetheart, Pearl Sweetheart, Salmon Sweetheart, Golden Sweetheart, Orange Sweetheart, Red Sweetheart and Mortar Jewel) were obtained commercially in February 196 1. Two weeks after removal of the terminal shoot they were irradiated with doses of gamma radiation from cobalt-60 ranging from 2.5 to 10 krad, at a dose rate of 0.537 rad/sec. The plants were replanted and raised in a glasshouse until 23rd May, and were then planted in rows in the field. Normal cultivation and disease control practices were carried out on the irradiated plants and on an equal number of unirradiated controls. Mutations were scored in two ways, per themum
of shorting
cuttings
mort$olium
in Sweetheart .
Year Introduced
No.*
Sport from
1939 1945 1945 1946 1946 1946 1957 1949 1949 1949 1950 1950 1961 1961
55 (+54) 55 55 54 (+55) 54 ($53, 55 55 55 55 ($51,
RADIATION
56)
54, 56, 59)
56
Sweetheart Sweetheart Sweetheart Red Sweetheart Sweetheart Sweetheart Sweetheart Sweetheart Sweetheart Red Sweetheart ? Sweetheart ? Red Sweetheart Sweetheart
numbers from Dowrick@) Table
Variety Dose -Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart Mortar Jewel
2. Percentage
survival
in 7 varieties
of irradiated
chrysanthemums
0
2.5 krad
5 krad
7.5 krad
10 krad
98 97 97.5 97.5 98 98.5 99
83.3 76.6 67.2 78.5 90.3 81.0 96.9
11 1 8 11.5 24 19 25.5
1 0 0 0 12.5 0 7
0 0 0.5 0 4 0 0
LD,,
(krad) 3.45 2.99 2.99 3.33 4.19 3.52 4.30
H. J. M. BOWEN,
P. A. CAWSE
bloom and per plant. Each bloom was inspected for mutant streaks on petals, coloured sectors or complete colour changes, recorded, and removed. Scoring was carried out at weekly intervals from mid-August till November, and a few late-flowering plants were lifted and allowed to flower in the glasshouse after frost had destroyed the blossoms left in the field. The pigment distribution in the petals of the original varieties, and in sports arising as a result of irradiation, was studied by stripping off the upper and lower epidermis lay.ers and examining them under the microscope. Petal cross-sections were also examined. No fixative was found which could be used as a permanent mount for these sections. The anthocyanin pigments fade rapidly and dissolve in water, ethanol, glycerine and other mounting fluids tried; Karo syrup preserves the pigment for at least a week.
and M. J. DICK
299
ning of September, the plants treated with 2.5 krad began to flower in mid-August and produced the maximum number of blossoms in mid-October. The few survivors from the higher doses of radiation flowered even later and had to be removed to a glasshouse to avoid the November frosts. The number of blooms per surviving plant did not appear to be much affected by 2.5 krad, but after 5 or more krad the blooms were fewer in number and larger in size. .Number of plants showing sports In many varieties more than half the irradiated plants showed colour sports of some kind, but none of the control plants showed any. Up to four different sports were observed on any one plant; the percentage distribution of multiple sports is given in Table 3. The distribution of the different types of colour sports is shown in Table 4. Other types of sport observed included unusual leaf shapes, chlorophyll deficiencies in leaves and alterations in the type of bloom (single, incurving and small-sized blooms), but these were not scored. Only a small proportion of the Sweetheart sports were propagable, in the sense that a whole branch showed mutants which could be broken up and propagated vegetatively. The majority involved sectors of blooms, individual flowers (petals) or sectors of petals. The percentage of propagable Sweetheart sports is shown in Table 5: all the Mortar Jewel sports were propagable.
Survival Results The percentage survival of the varieties studied is given in Table 2. In each case about 200 cuttings were taken per dose. LD,,s were calculated from these data and are also given in Table 2. Very few surviving plants failed to flower in 1961. Flowering time This was not scored, but it was obvious that in all varieties the irradiated plants tended to flower considerably later than did controls. Thus although the control plants in the Sweetheart varieties were beginning to flower in mid-July and were at their peak at the begin-
Number of blooms showing colour sports Although the proportion of plants
Table 3. Percentage of plants showing one or more sports after 2.5 krad No. of sports
0
1
2
3
4
5743 42.0 41.7 94.9 80.2 46.2 73.3
31.7 55.2 43.3 5.1 16.7 36.3 20.8
6.8 2.8 9.2 0 3.1 16.2 5.9
3.1 0 3.3 0 0 1.3 0
0.6 0 2.5 0 0 0 0
Variety Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart Mortar Jewel
showing
300
THE
INDUCTION
OF SPORTS
IN CHRYSANTHEMUMS
Table 4. Percentage of plants showing dif/ent
BY GAMMA
RADIATION
(@es of sport af%r 2.5 krad
sport
Swt.
Pearl
Salmon
Golden
Orange
Red
Apricot
Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart
57.8 2.1 7.5 2.6 11.5 4.4
7.5 42.0 10.0 0.6 0 33.1
0.6 1.4 41.7 0 0 0
7.5 52.4 16.7 94.9 7.3 24.4
21.1 0 40.0 1.3 80.2 1.9
14.9 0 2.5 0.6 4.7 46.2
6.2 0 1.7 0 0 0
Bronze
Cream
Deep Pink
Deep Red
Egerton
0 0.7 0 0 0 1.3
O-6 0 0 0 0 0
0 0 0 0 0 4.4
0 4.2 1.7 0 0 1.9
Variety 0 0 b 0 0.5 0
Table 4A Sport
Apricot
Bronze
Cream
3.7
10.2
9.1
Yellow
Deep
Pink
Jewel Mortar
Variety Mortar
Jewel
8.6
Table 5. Percentage of plants showing propagable
Swt.
Sport Variety Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart
-100 0.7 2.5 0.6 1.6 2.5 Table
0.5
73.3
sports after 2.5 krad
Pearl
Salmon
Golden
Orange
Red
Apricot
Bronze
Cream
Deep Pink
Deep Red .
Egerton
4.3 100 1o:o 0 0 33.1
0.6 1.4 100 0 0 0
0 6.3 0.8 100 5.7 5.6
2.5 0 2.5 1.3 100 1.3
3.1 0 0 0.6 1.0 66.9
5.0 0 0.8 0 0 0
0 0 0 0 0.5 0
0 0.7 0 0 0 1.3
0.6 0 0 0 0 0
0 0 0 0 0 4.4
0 2.1 0.8 0 0 0.6
6. Percentage
No. of sports
of blooms with one or mere colour sports after
0
2.5 krad
1
2
3
No. of mutants per bloom
7.59 10.34 12.12 1.25 1.42 30.49 2.52
0.26 0.12 1.15 0 0.04 1.02 0
0.09 0 0.28 0 0 0 0
0.0838 0.1058 0.1526 0.0125 0.0150 0.0603 0.0252
Variety Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart Mortar Jewel
92.06 89.54 86.45 98.75 98.54 68.01 97.48
Table 7. Percentage of blooms showing colour sports after 2.5 krad Sport
Swt.
Pearl
Salmon
Golden
Orange
Red
Apricot
Bronze
Cream
Deep Pink
Deep Red
Egerton
92.06 0.36 1.01 0.40 0.60 1.26
0.95 89.54 2.86 0.02 0 26.50
0.06 0.12 86.45 0 0 0
0.93 9.16 3.00 98.75 0.47 3.89
1.59 0 7.37 0.74 98.54 0.20
2.80 0 0.28 0.09 0.33 68.01
1.52 0 0.28 0 0 0
0 0 0 0 0.10 0
0 0.06 0 0 0 0.32
0.53 0 0 0 0 0
0 0 0 0 0 1.16
0 0.88 0.46 0 0 0.14
Variety Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart
H. J. M. BOWEN,
P. A. CAWSE
colour sports was quite high, the proportion of blooms with colour sectors was much lower. Fewer double and triple sports were observed, as shown in Table 6. The percentage of blooms showing different types of colour sports is given in TabIe 7. Pigment distribution According to Dowrick,c3) flower colour in Chrysanthemum is determined by the presence or absence of three different pigments, a soluble anthocyanin, a soluble flavone and an insoluble The pigments isolated from plastid pigment. chrysanthemum petals include the anthocyanin chrysanthemin (3, 5, 7, 3’, 4’ pentahydroxy flavylium chloride) from red varieties,04) and the chemically related flavones apigenin (5, 7,4’ trihydroxy flavone; from a white variety) and luteolin (5, 7, 3’, 4’ tetrahydroxy flavone; from a yellow variety). (*,13) Red and yellow petals contain in addition xanthophyll pigments such as chrysanthemaxanthin.(6) Chrysanthemum petals are approximately five cells thick. The cells in the upper epidermal layer, which are unusually large and swollen, contain soluble pigments in the vacuolar sap, with or without a yellow pigment confined to granules in the cytoplasm. The inner layers of ceils are white or colourless. The lower epidermal layer of cells contain no soluble pigments, but have white or yellow granules in the cytoplasm. Since the i nner layers of cells are more or less transparent, the colour of the whole bloom is determined by the pigments present in both the upper and lower epidermal cells, which are generally different. Differences in the of the pigments can relative proportions markedly alter the colour of the bloom. Table 8 shows the distribution of pigments in the petals of Sweetheart varieties. Dz$iculties in scoring colour sports Although there was no difficulty in scoring propagable colour sports, it was not always possible to distinguish some types of sports which were only present as petal streaks. In particular, Red, Orange and Bronze Sweetheart were difficult to tell apart, and the same might be said of Apricot and Peach Sweetheart. Most colour sports were easy to see, but streaks D
and M. J. DICK
301
of Apricot Sweetheart on Sweetheart, or Egerton Sweetheart on Pearl Sweetheart were hard to see and some may have been missed. DISCUSSION Our survival values agree in order of magnitude with those of other workers, for example JANK.@) Jank obtained a much higher percentage of sports by pruning his plants seven times between irradiation and flowering. It is possible to make a rough estimate of absolute mutation rates induced by irradiation. This can be done as follows. The smallest and commonest mutations observed were petal sectors about 0.5 mm broad. These are assumed to represent the extent of growth of single cells affected by the radiation dose. Since the mean petal width was 7.22 mm, and the mean number of petals per bloom was 158, each bloom could show 2275 different mutant sectors. In the variety Salmon Sweetheart, we see from Table 6 that the average number of mutants per bloom was only 0.1526, so that we can estimate the proportion of cells mutated per krad as about 27 per loo. For Gold Sweetheart, the same calculation gives a figure of 2.2 per 106. In fact the proportion of mutated cells is probably much greater than this, but the majority of them either die or fail to find phenotypic expression through reduced viability. It is not possible to make definite statements about the mode of origin of the sports without detailed cytological and genetical studies. Some hypotheses will be advanced here to account foi the observations on the Sweetheart varieties. The sports may be newly formed periclinal chimaeras, or they may result from the destruction of a pre-existing periclinal chimaera. They may arise from chromosomal abnormalities, for example the addition or deletion of a whoIe chromosome, or of parts of a chromosome, or they may result from point mutations. CUANY@) has reviewed the evidence that shoot apices are formed from at least two layers of cells in most species, with three layers in Solanaceous plants(“) and four in Malus.(7~ Neither shoot apices nor the breeding behaviour of Sweetheart chrysanthemums have been studied, but it is reasonable to assume that (a) the apices have at least two layers of cells and (1,)
302
THE
INDUCTION
OF SPORTS Table
Variety -Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart Apricot Sweetheart Bronze Sweetheart Cream Sweetheart Deep Pink Sweetheart Deep Red Sweetheart Egerton Sweetheart
IN CHRYSANTHEMUMS
8. Distribution
of
BY GAMMA
pigments in jletals oj’sweetheart
varieties
Colour of Soluble pigment in upper epidermal cells
Colour of Granular pigment in upper epidermal cells
pink v. pale pink pale pink none red red pink red none deep pink deep red none
white white white yellow yellow yellow white yellow white white yellow pale yellow
some varieties may be periclinal chimaeras. SHAPIROand BROERTJEGO) report a marked difference of frequency of sports caused by destruction of a periclinal chimaera and those caused by mutational change. The former are frequent and may affect the whole plant, while the latter are less frequent and appear as sectorial chimaeras. We only found examples of sporting affecting whole plants in irradiated Red Sweetheart, where 33.1 per cent of the plants were changed to Pearl Sweetheart. A similar situation has been found by Shapiro and Broertjes for the pair of chrysanthemums Masterpiece and Bronze Masterpiece, and by SAGAWAand MEHLQUIST(~) for the carnations William Sim and White or Pink Sim. We conclude that Red Sweetheart is probably a periclinal chimaera of Pearl Sweetheart, but this must await confirmation by breeding trials. The remainder of the sports are probably genetic in origin. In this connection it must be remembered that the chrysanthemum has the longest known history of any horticultural plant in cultivation, and that its heredity is complex. The chrysanthemum is a hexaploid, with 6n=54, but most of the Sweetheart varieties have an extra chromosome and Golden Sweetheart has two extra (cf. Table 1). The present work suggests that the most sports are produced by irradiating varieties
RADIATION
Colour of Granular pigment in lower epidermal cells white white white pale pale pale pale pale white white pale pale
yellow yellow yellow yellow yellow yellow yellow
with a chromosome number of 54 and fewest from those with the highest .chromosome numbers. In the absence of any information on chromosome number, pink, salmon or red varieties may perhaps give better results than orange or yellow ones.(s) Chromosome deletions and translocations could account for the results, but changes in chromosome number might also be important. The chromosome numbers of the sports we have obtained have not actually been counted, but if they are assumed to be the same as those given by Dowrick@) the data in Table 4 can be rearranged to show how often one variety gives rise to another with a different assumed chromosome number, as in Table 9. Table
9. Frequency
of sporls
chromosome
Parent chromosome 54 54 54 55 55 55 56 56 56
classiJied by ,bresumed number
Assumed Sport no. chromosome no. 54 55 56 54 55 56 54 55 56
change
0/0 plants showing it 0 63.3 16.7 0.6 30.0 22.3 0 5.1 0
in
H. J. M. BOWEN,
P. A. CAWSE
Some points require clarification in Table 9. The figures for 54 + 55 may be misleading in that 40.0 per cent of them refer to Orange Sweetheart sports of Salmon Sweetheart. Some of these may have been Bronze Sweetheart sports and so belong in the 54 + 54 row. Secondly the zero in the 56 + 56 row reflects the fact that only one variety of Sweetheart has 56 chromosomes. The data summarized in Table 9 suggest that radiation induced sports often have more chromosomes than their parent, but very rarely have less. The original variation in chromosome number is presumably brought about by the failure of two chromosomes to separate at mitosis, so that of two daughter cells one has one more and the other one less chromosomes than the parent. If our hypothesis is correct, there must be pronounced diplontic selection against chromosome deficient cells, but not against cells with an extra chromosome. In the case of Sweetheart varieties, this means that Salmon Sweetheart and Bronze Sweetheart are very rare sports, and that Golden Sweetheart can seldom be induced to sport at all. The latter observation is familiar to nurserymen. HORTICULTUIUL APPLICATIONS It is obvious that the technique of irradiation is a useful one for producing bud sports, once the optimum dose has been established. In the present case eleven different propagable sports were produced in one year, one ofwhich (Cream Sweetheart) is certainly an unrecorded type. It took eleven years for a comparable number of sports to occur by natural means, from a far larger population of plants. In the same way Mortar Jewel gave rise to five sports, one of which (Apricot) is probably identical with the parent Mortar Gem, but the others may be new. Moreover, it is unlikely that the sporting potential of an irradiated chrysanthemum plant is exhausted after a single year. BAUER(~)has shown that repeated cutting back of irradiated black currant bushes led to the production of mutants several years after the irradiation took place. Mutant cells are likely to be somewhat less viable than normal cells, and it may take some time for them to build up apical meristems
and M. J. DICK
303
so that the mutant tissue can be identified. It is to be expected that next year’s growths from our chrysanthemum stools will yield more sports, of which a high percentage will be propagable. The ideal method of propagation after irradiation would start from single cells, which would yield non-chimerical sports. Such a method has recently been discovered for S~intpaulia~~~~ and its application to other plants such as Chrysanthemum would greatly simplify the production and classification of radiationinduced sports. Acknowledgtnenti-The assistance of Miss H. DEMAINE in determining the distribution of pigments is gratefully acknowledged. C. BROERTJES of I.T.A.L. Wageningen, Netherlands, S. SHAPIRO of Brookhaven National Laboratory, Upton, N.Y., and J. B. STEVENSON of Colham Green Nurseries, Hillingdon, Middlesex, were most helpful in supplying unpublished information.
REFERENCES BAUER R. (1957) The induction of vegetative mutations in Ribes nigrum. Hereditas 43, 323-337. CUANYR. L. (1960) Nature of somatic mutations induced by radiation in flowering plants. Proceedings of the 2nd Inter-American Symposium {jeaceful aj$lication of nuclear energy. Buenos
on the Aires,
1959, pp. 29-37. Pan American Union, Washington D.C. DOWRICK J. (195 1) Sporting in chrysanthemums. The Chrysanthemum 152-155. (1953) The chromosomes of chrysanthemum. Heredity 7, 59-72. HOWARD H. W. (1959) Experiments with a potato periclinal chimaera. Genetica 30, 278-29 1. 5. JANK H. (1957) Experimental production of mutations in Chrysnnthemum indicum by X-rays. ,&hter
27,223-234.
6. KARRER P. and JUCKER E. (1943) Carotenoids from winter aster blooms. Chrysanthemaxanthin. Helv.
Gem.
Acta. 26, 626-30.
7. PRATT C. (1960) Changes in structure of a periclinal chromosomal chimaera of apple following X-irradiation. Nature, Land. 186, 255256. 8. RAO P. S. (1942) Occurrence of luteolin in the flowers of Chrysanthemum indicum. Proc. Indiatr Acad. Sci. 15A, 123-7.
304
THE
INDUCTION
OF
SPORTS
IN
CHRYSANTHEMUM
9. SAGAWA Y. and MEHLQUIST G. A. L. (1957) The mechanism responsible for some X-ray induced changes in flower colour of the carnation, Dianthus caryofihyllus. Amer. J. Bat. 44, 397403. IO.
SHAPIRO induced .National
S. and BROERTJES C. (1961) sports in floricultural plants. Laboralory Rel,orl BNL 5077.
RadiationBrook-haven
11. SHEEHAN T. J. and SAGAWA Y. (1959) The effects of gamma radiation on Chrysanthemum and Gladiolus. Proc. Flu. Hod. SOG. 72, 388-39 1.
BY
GAMMA
RADIATION
12. SPARROW A. H., SPARROW R. C. and SCHAIRER L. A. (1960) The use of X-rays to induce somatic mutations in Saintpaulia. African Violet Magazine 13, 32-37. 13. WADE M. and HAT-TORI T. (1953) Biogenesis of anthocyans. II. Isolation of apigenin-7-monoglucoside from the petals of the white. chrysanthemum. h4isc. Rep. Res. Inst. nat. Resour. (Japan) no 32, 67-70. 14. WILLSTATTER R. and BOLTON E. K. (1916) Anthocyanins of the winter aster (chrysanthemum). Annalen 413, 136-148.
Botany,
1962, pp. 297 to 303.
THE INDUCTION
Pergamon
Press Ltd. Printed
in Great Britain.
OF SPORTS IN CHRYSANTHEMUMS GAMMA RADIATION
BY
Ii. J. M. BOWEN, P. A. CAWSE and M. J. DICK Wantage Research Laboratory (AERE), Wantage, Be&s., Great Britain (Received
4 January
1962)
Abstract-The effect of gamma rays from cobalt-60 on cuttings of seven chrysanthemum varieties has been studied. The LDsg for the cuttings were in the range 3.0-4.3 krad. Irradiation caused plants to flower much later than controls. About 40 per cent of the irradiated plants showed colour sports; the overall percentage of sports and the percentages of different types depended markedly on variety. One sport of one variety appeared to be caused by the breaking of a periclinal chimaera. The remainder were believed to be mutants in the broadest sense of the term. The number of mutant cells was estimated to lie between 2.2 and 27 per lo6 cells per krad. Most sports were produced by irradiating varieties with a chromosome number of 54 and fewest from those with the highest chromosome numbers. A hypothesis is advanced that radiation-induced sports may have more chromosomes than their parent, but rarely have less. RCsum&-L’effet des rayons gamma du cobalt 60 sur des boutures de sept varieds de Chrysanthtmes a CtC Ctudit. La LDsos pour les boutures Ctait de l’ordre de 3,0-4,3 krad. L’irradiation a cause une floraison plus tardive que les controles. Le pourcentage total des variants et les pourcentages de differents types dtpendaient d’une man&e marquee de la variett. 11 est apparu qu’un variant ttait cause par la dissociation d’une chimtre ptricline. On croit que les variants restants sont des mutants au sens le plus large du terme. Le nombre de cellules mutantes a et& estime &re situ& entre 2,2 et 27 pour lo8 cellules et par krad. La majoritt des variants a ete produite en irradiant des varietts a nombre chromosomique 54 et beaucoup moins de varittb a nombres chromosomiques plus ClevCs. On avance une hypothtse suivant laquelle les variants induits par des rayons peuvent avoir plus de chromosomes que leurs parents mais rarement moins. Zusaznznenfassung-Die Wirkung der Gammabestrahlung (Coso) auf Steklinge von sieben Chrysanthemum-varietaten wurde untersucht. Die LDs, fur die Stecklinge lagen zwischen 3,0 und 4,3 Kilorad. Die bestrahlten Pflanzen bliihten vie1 splter ah die Kontrollen. Ungefahr 40 prozent der best&hen Pflanzen hatten Farbenmutationen; der Gesamtprozentsatz der Sports und der Prozentsatz der verschiedenen Typen hing von den Varietaten ab. Eine Mutation in einer Varietat schien durch die Stijrung eines Periklinalchimlrs hervorgerufen worden zu sein. Die Ubrigen sind anscheinend Mutationen im weiten Sinne des Wortes. Das Verhlltnis von normalen zu mutanten Zellen ist schiitzungsweise zwischen 15,000 und 180,000, abhlngig von der Varietat. Es wird die Hypothese vorgeschlagen, dass Bestrahlungsmutante mehr Chromosomen haben konnen als ihre Eltern, aber selten weniger. INTRODUCTION
forms may give rise to bud sports with new colours, which can be propagated by taking vegetative cuttings. Bud sports originate in an apparently random manner, and it may take many years for an important variety to yield its full quota of sports. For example, the pink variety Sweetheart was first raised in 1939 as a
THERE are two ways in which
new varieties of cultivated chrysanthemums may arise. The majority of new forms have been selected from the progeny of crosses between fertile parents, but many of the better forms produced in this way do not readily set seed. Secondly, many 297
298
THE
INDUCTION
OF SPORTS
IN CHRYSANTHEMUMS
seedling from a cross of White Buttercup Excelda. It was six years before any sports noted, but eleven sports were known by The history of sporting in this variety has reconstructed in Table 1, which also the chromosome numbers as measured
with were 1950. been gives by
DOWRICK.
In the same way the variety Loveliness was grown for five years before any other colours appeared, but ten sports appeared in the next decade. The present studies were undertaken to evaluate the potentialities of ionizing radiation in the production of chrysanthemum bud sports. Similar studies have been reported by JANK,@) BREEHAN and SAGAWA(~~) and SHAPIRO and SHOERTJES.‘~~)
Table
Variety
Chromosome
Sweetheart Egerton Sweetheart Red Sweetheart Christine Sweetheart Bronze Sweetheart Salmon Sweetheart Pearl Sweetheart Apricot Sweetheart Peach Sweetheart Orange Sweetheart Deep Red Sweetheart Golden Sweetheart Cream Sweetheart Deep Pink Sweetheart *Chromosome
1. History
Methodr
BY GAMMA
and materials
Rooted
of seven varieties of Chrysan(Sweetheart, Pearl Sweetheart, Salmon Sweetheart, Golden Sweetheart, Orange Sweetheart, Red Sweetheart and Mortar Jewel) were obtained commercially in February 196 1. Two weeks after removal of the terminal shoot they were irradiated with doses of gamma radiation from cobalt-60 ranging from 2.5 to 10 krad, at a dose rate of 0.537 rad/sec. The plants were replanted and raised in a glasshouse until 23rd May, and were then planted in rows in the field. Normal cultivation and disease control practices were carried out on the irradiated plants and on an equal number of unirradiated controls. Mutations were scored in two ways, per themum
of shorting
cuttings
mort$olium
in Sweetheart .
Year Introduced
No.*
Sport from
1939 1945 1945 1946 1946 1946 1957 1949 1949 1949 1950 1950 1961 1961
55 (+54) 55 55 54 (+55) 54 ($53, 55 55 55 55 ($51,
RADIATION
56)
54, 56, 59)
56
Sweetheart Sweetheart Sweetheart Red Sweetheart Sweetheart Sweetheart Sweetheart Sweetheart Sweetheart Red Sweetheart ? Sweetheart ? Red Sweetheart Sweetheart
numbers from Dowrick@) Table
Variety Dose -Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart Mortar Jewel
2. Percentage
survival
in 7 varieties
of irradiated
chrysanthemums
0
2.5 krad
5 krad
7.5 krad
10 krad
98 97 97.5 97.5 98 98.5 99
83.3 76.6 67.2 78.5 90.3 81.0 96.9
11 1 8 11.5 24 19 25.5
1 0 0 0 12.5 0 7
0 0 0.5 0 4 0 0
LD,,
(krad) 3.45 2.99 2.99 3.33 4.19 3.52 4.30
H. J. M. BOWEN,
P. A. CAWSE
bloom and per plant. Each bloom was inspected for mutant streaks on petals, coloured sectors or complete colour changes, recorded, and removed. Scoring was carried out at weekly intervals from mid-August till November, and a few late-flowering plants were lifted and allowed to flower in the glasshouse after frost had destroyed the blossoms left in the field. The pigment distribution in the petals of the original varieties, and in sports arising as a result of irradiation, was studied by stripping off the upper and lower epidermis lay.ers and examining them under the microscope. Petal cross-sections were also examined. No fixative was found which could be used as a permanent mount for these sections. The anthocyanin pigments fade rapidly and dissolve in water, ethanol, glycerine and other mounting fluids tried; Karo syrup preserves the pigment for at least a week.
and M. J. DICK
299
ning of September, the plants treated with 2.5 krad began to flower in mid-August and produced the maximum number of blossoms in mid-October. The few survivors from the higher doses of radiation flowered even later and had to be removed to a glasshouse to avoid the November frosts. The number of blooms per surviving plant did not appear to be much affected by 2.5 krad, but after 5 or more krad the blooms were fewer in number and larger in size. .Number of plants showing sports In many varieties more than half the irradiated plants showed colour sports of some kind, but none of the control plants showed any. Up to four different sports were observed on any one plant; the percentage distribution of multiple sports is given in Table 3. The distribution of the different types of colour sports is shown in Table 4. Other types of sport observed included unusual leaf shapes, chlorophyll deficiencies in leaves and alterations in the type of bloom (single, incurving and small-sized blooms), but these were not scored. Only a small proportion of the Sweetheart sports were propagable, in the sense that a whole branch showed mutants which could be broken up and propagated vegetatively. The majority involved sectors of blooms, individual flowers (petals) or sectors of petals. The percentage of propagable Sweetheart sports is shown in Table 5: all the Mortar Jewel sports were propagable.
Survival Results The percentage survival of the varieties studied is given in Table 2. In each case about 200 cuttings were taken per dose. LD,,s were calculated from these data and are also given in Table 2. Very few surviving plants failed to flower in 1961. Flowering time This was not scored, but it was obvious that in all varieties the irradiated plants tended to flower considerably later than did controls. Thus although the control plants in the Sweetheart varieties were beginning to flower in mid-July and were at their peak at the begin-
Number of blooms showing colour sports Although the proportion of plants
Table 3. Percentage of plants showing one or more sports after 2.5 krad No. of sports
0
1
2
3
4
5743 42.0 41.7 94.9 80.2 46.2 73.3
31.7 55.2 43.3 5.1 16.7 36.3 20.8
6.8 2.8 9.2 0 3.1 16.2 5.9
3.1 0 3.3 0 0 1.3 0
0.6 0 2.5 0 0 0 0
Variety Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart Mortar Jewel
showing
300
THE
INDUCTION
OF SPORTS
IN CHRYSANTHEMUMS
Table 4. Percentage of plants showing dif/ent
BY GAMMA
RADIATION
(@es of sport af%r 2.5 krad
sport
Swt.
Pearl
Salmon
Golden
Orange
Red
Apricot
Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart
57.8 2.1 7.5 2.6 11.5 4.4
7.5 42.0 10.0 0.6 0 33.1
0.6 1.4 41.7 0 0 0
7.5 52.4 16.7 94.9 7.3 24.4
21.1 0 40.0 1.3 80.2 1.9
14.9 0 2.5 0.6 4.7 46.2
6.2 0 1.7 0 0 0
Bronze
Cream
Deep Pink
Deep Red
Egerton
0 0.7 0 0 0 1.3
O-6 0 0 0 0 0
0 0 0 0 0 4.4
0 4.2 1.7 0 0 1.9
Variety 0 0 b 0 0.5 0
Table 4A Sport
Apricot
Bronze
Cream
3.7
10.2
9.1
Yellow
Deep
Pink
Jewel Mortar
Variety Mortar
Jewel
8.6
Table 5. Percentage of plants showing propagable
Swt.
Sport Variety Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart
-100 0.7 2.5 0.6 1.6 2.5 Table
0.5
73.3
sports after 2.5 krad
Pearl
Salmon
Golden
Orange
Red
Apricot
Bronze
Cream
Deep Pink
Deep Red .
Egerton
4.3 100 1o:o 0 0 33.1
0.6 1.4 100 0 0 0
0 6.3 0.8 100 5.7 5.6
2.5 0 2.5 1.3 100 1.3
3.1 0 0 0.6 1.0 66.9
5.0 0 0.8 0 0 0
0 0 0 0 0.5 0
0 0.7 0 0 0 1.3
0.6 0 0 0 0 0
0 0 0 0 0 4.4
0 2.1 0.8 0 0 0.6
6. Percentage
No. of sports
of blooms with one or mere colour sports after
0
2.5 krad
1
2
3
No. of mutants per bloom
7.59 10.34 12.12 1.25 1.42 30.49 2.52
0.26 0.12 1.15 0 0.04 1.02 0
0.09 0 0.28 0 0 0 0
0.0838 0.1058 0.1526 0.0125 0.0150 0.0603 0.0252
Variety Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart Mortar Jewel
92.06 89.54 86.45 98.75 98.54 68.01 97.48
Table 7. Percentage of blooms showing colour sports after 2.5 krad Sport
Swt.
Pearl
Salmon
Golden
Orange
Red
Apricot
Bronze
Cream
Deep Pink
Deep Red
Egerton
92.06 0.36 1.01 0.40 0.60 1.26
0.95 89.54 2.86 0.02 0 26.50
0.06 0.12 86.45 0 0 0
0.93 9.16 3.00 98.75 0.47 3.89
1.59 0 7.37 0.74 98.54 0.20
2.80 0 0.28 0.09 0.33 68.01
1.52 0 0.28 0 0 0
0 0 0 0 0.10 0
0 0.06 0 0 0 0.32
0.53 0 0 0 0 0
0 0 0 0 0 1.16
0 0.88 0.46 0 0 0.14
Variety Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart
H. J. M. BOWEN,
P. A. CAWSE
colour sports was quite high, the proportion of blooms with colour sectors was much lower. Fewer double and triple sports were observed, as shown in Table 6. The percentage of blooms showing different types of colour sports is given in TabIe 7. Pigment distribution According to Dowrick,c3) flower colour in Chrysanthemum is determined by the presence or absence of three different pigments, a soluble anthocyanin, a soluble flavone and an insoluble The pigments isolated from plastid pigment. chrysanthemum petals include the anthocyanin chrysanthemin (3, 5, 7, 3’, 4’ pentahydroxy flavylium chloride) from red varieties,04) and the chemically related flavones apigenin (5, 7,4’ trihydroxy flavone; from a white variety) and luteolin (5, 7, 3’, 4’ tetrahydroxy flavone; from a yellow variety). (*,13) Red and yellow petals contain in addition xanthophyll pigments such as chrysanthemaxanthin.(6) Chrysanthemum petals are approximately five cells thick. The cells in the upper epidermal layer, which are unusually large and swollen, contain soluble pigments in the vacuolar sap, with or without a yellow pigment confined to granules in the cytoplasm. The inner layers of ceils are white or colourless. The lower epidermal layer of cells contain no soluble pigments, but have white or yellow granules in the cytoplasm. Since the i nner layers of cells are more or less transparent, the colour of the whole bloom is determined by the pigments present in both the upper and lower epidermal cells, which are generally different. Differences in the of the pigments can relative proportions markedly alter the colour of the bloom. Table 8 shows the distribution of pigments in the petals of Sweetheart varieties. Dz$iculties in scoring colour sports Although there was no difficulty in scoring propagable colour sports, it was not always possible to distinguish some types of sports which were only present as petal streaks. In particular, Red, Orange and Bronze Sweetheart were difficult to tell apart, and the same might be said of Apricot and Peach Sweetheart. Most colour sports were easy to see, but streaks D
and M. J. DICK
301
of Apricot Sweetheart on Sweetheart, or Egerton Sweetheart on Pearl Sweetheart were hard to see and some may have been missed. DISCUSSION Our survival values agree in order of magnitude with those of other workers, for example JANK.@) Jank obtained a much higher percentage of sports by pruning his plants seven times between irradiation and flowering. It is possible to make a rough estimate of absolute mutation rates induced by irradiation. This can be done as follows. The smallest and commonest mutations observed were petal sectors about 0.5 mm broad. These are assumed to represent the extent of growth of single cells affected by the radiation dose. Since the mean petal width was 7.22 mm, and the mean number of petals per bloom was 158, each bloom could show 2275 different mutant sectors. In the variety Salmon Sweetheart, we see from Table 6 that the average number of mutants per bloom was only 0.1526, so that we can estimate the proportion of cells mutated per krad as about 27 per loo. For Gold Sweetheart, the same calculation gives a figure of 2.2 per 106. In fact the proportion of mutated cells is probably much greater than this, but the majority of them either die or fail to find phenotypic expression through reduced viability. It is not possible to make definite statements about the mode of origin of the sports without detailed cytological and genetical studies. Some hypotheses will be advanced here to account foi the observations on the Sweetheart varieties. The sports may be newly formed periclinal chimaeras, or they may result from the destruction of a pre-existing periclinal chimaera. They may arise from chromosomal abnormalities, for example the addition or deletion of a whoIe chromosome, or of parts of a chromosome, or they may result from point mutations. CUANY@) has reviewed the evidence that shoot apices are formed from at least two layers of cells in most species, with three layers in Solanaceous plants(“) and four in Malus.(7~ Neither shoot apices nor the breeding behaviour of Sweetheart chrysanthemums have been studied, but it is reasonable to assume that (a) the apices have at least two layers of cells and (1,)
302
THE
INDUCTION
OF SPORTS Table
Variety -Sweetheart Pearl Sweetheart Salmon Sweetheart Golden Sweetheart Orange Sweetheart Red Sweetheart Apricot Sweetheart Bronze Sweetheart Cream Sweetheart Deep Pink Sweetheart Deep Red Sweetheart Egerton Sweetheart
IN CHRYSANTHEMUMS
8. Distribution
of
BY GAMMA
pigments in jletals oj’sweetheart
varieties
Colour of Soluble pigment in upper epidermal cells
Colour of Granular pigment in upper epidermal cells
pink v. pale pink pale pink none red red pink red none deep pink deep red none
white white white yellow yellow yellow white yellow white white yellow pale yellow
some varieties may be periclinal chimaeras. SHAPIROand BROERTJEGO) report a marked difference of frequency of sports caused by destruction of a periclinal chimaera and those caused by mutational change. The former are frequent and may affect the whole plant, while the latter are less frequent and appear as sectorial chimaeras. We only found examples of sporting affecting whole plants in irradiated Red Sweetheart, where 33.1 per cent of the plants were changed to Pearl Sweetheart. A similar situation has been found by Shapiro and Broertjes for the pair of chrysanthemums Masterpiece and Bronze Masterpiece, and by SAGAWAand MEHLQUIST(~) for the carnations William Sim and White or Pink Sim. We conclude that Red Sweetheart is probably a periclinal chimaera of Pearl Sweetheart, but this must await confirmation by breeding trials. The remainder of the sports are probably genetic in origin. In this connection it must be remembered that the chrysanthemum has the longest known history of any horticultural plant in cultivation, and that its heredity is complex. The chrysanthemum is a hexaploid, with 6n=54, but most of the Sweetheart varieties have an extra chromosome and Golden Sweetheart has two extra (cf. Table 1). The present work suggests that the most sports are produced by irradiating varieties
RADIATION
Colour of Granular pigment in lower epidermal cells white white white pale pale pale pale pale white white pale pale
yellow yellow yellow yellow yellow yellow yellow
with a chromosome number of 54 and fewest from those with the highest .chromosome numbers. In the absence of any information on chromosome number, pink, salmon or red varieties may perhaps give better results than orange or yellow ones.(s) Chromosome deletions and translocations could account for the results, but changes in chromosome number might also be important. The chromosome numbers of the sports we have obtained have not actually been counted, but if they are assumed to be the same as those given by Dowrick@) the data in Table 4 can be rearranged to show how often one variety gives rise to another with a different assumed chromosome number, as in Table 9. Table
9. Frequency
of sporls
chromosome
Parent chromosome 54 54 54 55 55 55 56 56 56
classiJied by ,bresumed number
Assumed Sport no. chromosome no. 54 55 56 54 55 56 54 55 56
change
0/0 plants showing it 0 63.3 16.7 0.6 30.0 22.3 0 5.1 0
in
H. J. M. BOWEN,
P. A. CAWSE
Some points require clarification in Table 9. The figures for 54 + 55 may be misleading in that 40.0 per cent of them refer to Orange Sweetheart sports of Salmon Sweetheart. Some of these may have been Bronze Sweetheart sports and so belong in the 54 + 54 row. Secondly the zero in the 56 + 56 row reflects the fact that only one variety of Sweetheart has 56 chromosomes. The data summarized in Table 9 suggest that radiation induced sports often have more chromosomes than their parent, but very rarely have less. The original variation in chromosome number is presumably brought about by the failure of two chromosomes to separate at mitosis, so that of two daughter cells one has one more and the other one less chromosomes than the parent. If our hypothesis is correct, there must be pronounced diplontic selection against chromosome deficient cells, but not against cells with an extra chromosome. In the case of Sweetheart varieties, this means that Salmon Sweetheart and Bronze Sweetheart are very rare sports, and that Golden Sweetheart can seldom be induced to sport at all. The latter observation is familiar to nurserymen. HORTICULTUIUL APPLICATIONS It is obvious that the technique of irradiation is a useful one for producing bud sports, once the optimum dose has been established. In the present case eleven different propagable sports were produced in one year, one ofwhich (Cream Sweetheart) is certainly an unrecorded type. It took eleven years for a comparable number of sports to occur by natural means, from a far larger population of plants. In the same way Mortar Jewel gave rise to five sports, one of which (Apricot) is probably identical with the parent Mortar Gem, but the others may be new. Moreover, it is unlikely that the sporting potential of an irradiated chrysanthemum plant is exhausted after a single year. BAUER(~)has shown that repeated cutting back of irradiated black currant bushes led to the production of mutants several years after the irradiation took place. Mutant cells are likely to be somewhat less viable than normal cells, and it may take some time for them to build up apical meristems
and M. J. DICK
303
so that the mutant tissue can be identified. It is to be expected that next year’s growths from our chrysanthemum stools will yield more sports, of which a high percentage will be propagable. The ideal method of propagation after irradiation would start from single cells, which would yield non-chimerical sports. Such a method has recently been discovered for S~intpaulia~~~~ and its application to other plants such as Chrysanthemum would greatly simplify the production and classification of radiationinduced sports. Acknowledgtnenti-The assistance of Miss H. DEMAINE in determining the distribution of pigments is gratefully acknowledged. C. BROERTJES of I.T.A.L. Wageningen, Netherlands, S. SHAPIRO of Brookhaven National Laboratory, Upton, N.Y., and J. B. STEVENSON of Colham Green Nurseries, Hillingdon, Middlesex, were most helpful in supplying unpublished information.
REFERENCES BAUER R. (1957) The induction of vegetative mutations in Ribes nigrum. Hereditas 43, 323-337. CUANYR. L. (1960) Nature of somatic mutations induced by radiation in flowering plants. Proceedings of the 2nd Inter-American Symposium {jeaceful aj$lication of nuclear energy. Buenos
on the Aires,
1959, pp. 29-37. Pan American Union, Washington D.C. DOWRICK J. (195 1) Sporting in chrysanthemums. The Chrysanthemum 152-155. (1953) The chromosomes of chrysanthemum. Heredity 7, 59-72. HOWARD H. W. (1959) Experiments with a potato periclinal chimaera. Genetica 30, 278-29 1. 5. JANK H. (1957) Experimental production of mutations in Chrysnnthemum indicum by X-rays. ,&hter
27,223-234.
6. KARRER P. and JUCKER E. (1943) Carotenoids from winter aster blooms. Chrysanthemaxanthin. Helv.
Gem.
Acta. 26, 626-30.
7. PRATT C. (1960) Changes in structure of a periclinal chromosomal chimaera of apple following X-irradiation. Nature, Land. 186, 255256. 8. RAO P. S. (1942) Occurrence of luteolin in the flowers of Chrysanthemum indicum. Proc. Indiatr Acad. Sci. 15A, 123-7.
304
THE
INDUCTION
OF
SPORTS
IN
CHRYSANTHEMUM
9. SAGAWA Y. and MEHLQUIST G. A. L. (1957) The mechanism responsible for some X-ray induced changes in flower colour of the carnation, Dianthus caryofihyllus. Amer. J. Bat. 44, 397403. IO.
SHAPIRO induced .National
S. and BROERTJES C. (1961) sports in floricultural plants. Laboralory Rel,orl BNL 5077.
RadiationBrook-haven
11. SHEEHAN T. J. and SAGAWA Y. (1959) The effects of gamma radiation on Chrysanthemum and Gladiolus. Proc. Flu. Hod. SOG. 72, 388-39 1.
BY
GAMMA
RADIATION
12. SPARROW A. H., SPARROW R. C. and SCHAIRER L. A. (1960) The use of X-rays to induce somatic mutations in Saintpaulia. African Violet Magazine 13, 32-37. 13. WADE M. and HAT-TORI T. (1953) Biogenesis of anthocyans. II. Isolation of apigenin-7-monoglucoside from the petals of the white. chrysanthemum. h4isc. Rep. Res. Inst. nat. Resour. (Japan) no 32, 67-70. 14. WILLSTATTER R. and BOLTON E. K. (1916) Anthocyanins of the winter aster (chrysanthemum). Annalen 413, 136-148.