Effect of ionizing radiation on roses: Induction of somatic mutations

Environmental and E~perimental Botany, Vol. 20, pp. 325 to 333

0098-8472/80/10014)325 $02.(10/0

Pergamon Press lad. 1980. Printed in Great Britain

EFFECT OF I O N I Z I N G R A D I A T I O N ON ROSES: I N D U C T I O N OF SOMATIC M U T A T I O N S PUSHPA LATA* National Botanical Research Institute, Lucknow, India

[Received 1 ,\'bvember 1979; received~r publication 22 January 1980) LATA P. Effect of ionizing radiation on roses: induction of somatic mutations'. ENVIRONMENTALAND EXPERIMENTAL BOTANY '~0, 325-333, 1980.--Bud-wood from seven rose cultivars exhibiting five different colors were exposed to 0, 3, 4, and 5 krad of gamma rays. A similar response was observed for all exposed cuhivars; it included dose response reductions in bud-take, number and height of shoots, survival, flowers, petal weight and pollen fertility. The LDs0 for white and mauve-flowered cultivars was found to be lower than the yellow, red and pink-flowered ones; the latter were more prone to mutations. Many phenotypically detectable variations in leaf, flower and growth habit were recorded in irradiated populations. Only three mutations, one in growth habit and two in flower colors, were successfully isolated and propagated. The results suggest that the Floribunda rose, i.e., Pink Parfait was more suitable for induction of mutations as compared with the six Hybrid Teas. INTRODUCTION

ROSE BREEDING through conventional means is h a m p e r e d due to variation in ploidy levels, apomixis, high sterility and seed d o r m a n c y acc o m p a n i e d with poor seed germination. T h e occurrence of spontaneous mutations, and the ease with which roses can be p r o p a g a t e d vegetatively, render them suitable for i m p r o v e m e n t by induced somatic mutations. Several workers have successfully induced growth habit and flower color mutations in roses, ~2) the majority of mutants was neither further evaluated nor multiplied on a large scale and were lost. In the present experiment seven rose cultivars possessing five different colors were subjected to g a m m a irradiation, with the aim to induce mutations and to study the effects of ionizing radiation on various morphological characters. Efforts were also m a d e to isolate and p r o p a g a t e the induced mutations in pure form; the pro-

p a g a t a b l e mutations studies.(12)

were

used

for detailed

MATERIALS AND METHODS

Six vigorous rose cultivars from ' H y b r i d T e a ' (H.T.) group, viz. Caledonia, Virgo (whiteflowered); Prelude (mauve-flowered); Oklahoma, Papa Meilland (red-flowered); Q u e b e c (clear yellow-flowered) and one from the ' F l o r i b u n d a ' (F1.) group, i.e., Pink Parfait (medium to light pink, yellow at base) were used. Four uniform lots of b u d - w o o d each consisting of at least 25 b u d d i n g 'eyes' were prep a r e d from each cultivar. O n e lot served as the control, and the rest were exposed to 3, 4 and 5 krad of g a m m a rays, using a 6°Co source at a rate of 1.8 krad/min. T h e 'eyes' were b u d d e d on the "Edward' stock plants on the treatment day. Suckers were removed from time to time to encourage growth of budded shoot.

*Present address: Department of Plant Biology and Microbiology, Queen Mary College, University of London, London, U.K. EEB ~ 325

326

PUSHPA LATA

Observations on various characters were made daily, fortnightly or at monthly intervals. Parameters used were: time and percentage of bud-take, shoot number and height, survival, flowering period and number of flowers, petal weight and pollen fertility. Petals from at least ten flowers were used for determining the average petal weight. The pollen fertility was determined on the basis of acetocarmine stainability. Abnormalities in leaf, flower or habit of growth were also recorded. The data on budtake and average number and length of shoots were compared after six months of budding since t'urther growth was altered either by transplanting or pruning of plants. All the plants were pruned during mid-October 1970 but observations were continued until the next pruning season. Data were analysed statistically. Significant differences by analysis of variance (F test) were found at the 0.1 to 2.5~{, levels among various treatments. However, the differences between various cultivars were nonsignificant. The t-test was performed to determine the significance of differences between control and 3krad, the control and 4krad, and the control and 5 t~rad exposures. Somatic mutations which were detected as chimaera were encouraged to grow by repeated pruning of normal shoot or tissue. The budding 'eyes' from mutated sectors were used in further propagation and the resultant plants were again screened for the persistence of mutated tissues. Those which produced normal tissue, were discarded. This process was repeated until the pure mutations either in growth habit or inflower color were established in the successively budded plants. RESULTS Bud-take

The union of stock and scion tissues is necessary for the sprouting of budded 'eyes', and is largely dependent on the cambial activity of both. This union appears to have been achieved in the majority of cases by the end of second fortnight (Figs. 1-7). Irradiated buddings yielded a lower percentage of bud-take relative to the controls except in Papa Meilland (Fig. 3).

Shools

More shoots per plant were observed in control plants as compared with the irradiated populations (Table 1). Significant differences were recorded between various treatments; however, the 3krad exposure was the least harmful to the production of shoots. Non-irradiated plants produced taller shoots than the irradiated ones (Table 1). The decline in shoot length was correlated with increase in exposure, the differences were statistically significant. Survival

A statistically significant decrease in the percentage of surviving plants was recorded with increase in exposure (Table 2). The survival of non-irradiated plants was higher than the irradiated ones except for Papa Meilland. The data on survival one year after pruning, i.e., 18 months after budding, were considered suitable tor determining LD~ 0 of perennial roses. Present data suggest that 3 krad of gamma rays were sufficient to produce 5 0 ~ lethality ill Caledonia and Prelude. The LDs0 for Oklahoma, Papa Meilland, Pink Parfait and Qm'l~cc was found m lw 4krad and between 3 ;rod .t krad for Virgo. Growth habit

One 5krad treated plant of Quebec was found to be dwarf [.Figs. 8 and 9) and anolher 4krad treated one showed a climbing tendency (Fig. 10) and malformed leaves. The branch showing the climbing tendency (Fig. 10, arrow) was encouraged to grow further by pruning the upright growing shoots. The budding 'eyes' from the climbing shoot were used for further propagation. In the successive populations onty two plants out of 13, retained the climbing habit of growth (Fig. 11 ).

Leaf One plant each of Oklahoma (4krad), Papa Meilland (3krad), Quebec ( 4 k r a d ) a n d two of Pink Parfait (3krad) produced malformed and smaller leaves, about the size of the normal.

E F F E C T O F I O N I Z I N G R A D I A T I O N ON R O S E S

327

Table 1. Effect of gamma irradiation on growth of cultivated roses Average number of shoots Name of cullivar Caledonia Oklahoma Papa Meilland Pink Parfait Prelude Quebec Virgo t-value

Average length of longest shoot (cm)+SE

Control

3krad

4krad

5krad

Control

3krad

4krad

5krad

2.3 2.2 2.4 3.7 4.6 3.8 3.0

2.3 1.8 2.4 3.5 2.6 2.6 2.2 1.65

1.4 2.1 1.9 2,3 2.5 2.0 1.3 3.16I"

1.0 1.7 3.1 2.2 2.6 2.0 1.3 2.61 *

31+1.9 47_+2.3 40+2.5 42-1-2.9 35_+1.6 48_+1.3 38_+2.0

26-t-2.3 35-t-2.0 34+2.5 39-+1.4 29-+2.0 32_+2.4 28_+2.8 2.30*

16+2.5 32_+2.1 25+2.9 25__3.0 26+2.4 30_+2.3 10--+3.5 4.06I"

22 22_+3.6 29+5.5 27+7.0 25-1-2.3 17_+9.2 9_+4.5 3.47t"

Significant at 1% level of treatments * Significant differences from control at p < 0.05. t Significant differences from control at p <=0.01.

Table 2. Effect of gamma irradiation on survival of cultivated roses (°,,o) Survival Name of cultivar Caledonia Oklahoma Papa Meilland Pink Parfait Prelude Quebec Virgo t-value

After 6 months

After 18 months

Control

3krad

4krad

5krad

Control

3krad

4krad

5krad

88 92 54 96 1O0 100 92

76 63 87 89 76 77 67 1.77

35 64 76 63 59 66 32 3.73t

4 56 30 15 22 11 23 7.49++

72 85 39 88 79 100 73

40 52 40 86 42 77 50 2.10"

4 46 20 44 19 55 8 4.63 +

0 26 0 15 23 15 3 7.80 +

Significant at 0.1 o,~ level for *Significant differences from 1Significant differences from +,Signiticant differences ti'om

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1 7. Percentage of bud-take after different fortnights in rose cultivars. 1, (:ah'dimia: '2. Oklahoma; 3, Papa Meilland; 4, Pink Parfait; 5, Prelude; 6, Quebec; 7, Virgo. l"ms

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Fro. 19. Control (at the top); deep pink {on the left) and light pink (on the right) mutant flowers of Pink Parfait.

EFFECT OF IONIZING RADIATION ON ROSES Leaf abnormalities in two Pink Parfait and one Quebec plant were accompanied with a variation in flower color. These plants later produced deep pink, very light pink and light yellow flowers. Only one Oklahoma plant produced variegated leaves during initial stages of growth.

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produced by the control and 4 and 5krad treated plants were highly significant. Abnormal flowers showing a variation in number, size, shape and color of petals as compared with the normal flowers were observed both in the control and irradiated plants (Table 3, numbers in brackets); the frequency

Ft(;. 18. Effect of gamma irradiation on llowcring period of garden roses.

Flowers A delay of 4--6 weeks in the commencement of flowering was apparent in the 4 and 5krad treated plants (Fig. 18). Long intermittent breaks in the flowering period were commonly observed in the H.T. roses after irradiation with the higher exposures. A continuous flowering was observed in the F1. rose, Pink Parfait. On an average H.T. produced fewer blooms relative to the F1. rose (Table 3). The decrease in blooms correlated with increase in exposure. The differences between the number of flowers

was higher in the latter. Out of 68 abnormal flowers produced in various cultivars, 14 had reduced and 9 had increased numbers of petals (Figs. 12, 14, 15 and 17). Cultivar Oklahoma produced three flowers with 5 petals (Fig, 12), two with 4 petals, one with 10 petals and one with 60 petals (Figs. 14 and 15). A loss in color intensity was observed in two cases each from Oklahoma and Quebec. One 5krad treated plant of Oklahoma produced flowers with fringed petal margins (Fig. 13). A large number (34) of floral abnormalities was

PUSHPA LATA

332

Table 3. Effect of gamma irradiation on flowers of cultivated roses

Name

of cuhivar Caledonia Oklahoma Papa Meilland Pink Parfait Prelude Quebec Virgo t-value

Average petal wt/flower (gm)

No. of flowers produced during 18 months 3krad

4krad

5krad

Control

Irradiated

196(2) 304 (5) 158(1) 1198(11) 290(1) 786 (2) 216 (1) 1.10

48(1) 357 (9) 56(1) 680(21) 71 403 (2) 42 2.12"

12 160 (4) 30 285(2) 44 93 12 2.94~

5.9 9.0 8.3 3.1 3.1 9.0 5.5

5.0 7.1 7.1 3.4 3.0 8.3 5.0 0.58

Control 350 (2) 623 (1) 167 1710 (1) 437 (1) 1375 513

Average pollen fertility (°Jo ) Control Irradiated 19.5 28.5 25.0 31.5 20.0 18.0 23.5

16.0 26.0 22.0 28.5 17.0 18.0 20.0 1.0

Significant at 2.5% level for treatments *Significant differences from control at p < 0.1 tSignificant differences from control at p=<0.02. Number of abnormal flowers in brackets. observed in Pink Parfait including 4 with lighter and 10 with deeper color variations and also one which involved deep color streaks on petals (Fig. 16). One of each of these color mutations, detected on 3krad treated plants, was isolated and propagated in pure form (Fig. 19) by the technique discussed earlier. Only three plants with the flower color mutations were obtained from the 40 buddings carried out during 1971 1972 for isolation of these mutants. Petal weight

The average weight of petals was found to be reduced after irradiation (Table 3) except for Pink Parfait. In the latter case several compact flowers with increased petal numbers (Fig. 17) were also used. Pollen fertility

Irradiation brought about a loss in pollen t~rtility (Table 3).

DISCUSSION

In vegetatively propagated roses several mutation breeders have reported a reduction in bud-take, plant height, survival and flowering after mutagen treatments and with increasing

exposures of ionizing radiation exposures. (5' v, 13) Present results also indicate a similar pattern. Rose cultivar Pink Parfait was found to be a segmental-allotetraploid in a previous study. (~2) Other cultivars were not studied in detail but all were tetraploids. (9'1°'14) It has b e e n ' r e ported by BOROJEVIC and others (1) that genetic differences can induce significant changes in radiosensitivity of individuals; these differences were probably responsible for the variations in LD50 of the roses in the present study. Chlorophyll mutations are known to be restricted in polyploids as compared with diploids. (1) The low frequency of chlorophyll mutations has been reported in roses by several" workers (2'v) and also supported by the present results where one such mutation was observed out of a population of about 500 plants. A loss in pollen fertility is a usual feature of most mutagen treated plants; (4'~1'82) the loss may or may not be counter-balanced by a restoration of female fertility. Such restoration of female fertility was recorded in irradiated plants of Pink Parfait, Quebec and Virgo, in another study. (~°) These results were in line with another report of gamma-induced mutants of the rose cultivar Montezuma. (~ ~) Various genes are known to regulate the growth habit, growth rate and differention of

EFFECT OF IONIZING RADIATION ON ROSES tissuesJ 6~ T h e climbing habit in roses is controlled by a single gene, which is d o m i n a n t over the bush habit. ~8) M a n y climbing roses have arisen as b u d sports or as a result of hybridization(t 1, i5) WYLIE(t5) surveyed 411 sports out of which 140 were c l i m b e r s . . T h e development of such periclinal chimaeras from a normal tissue depends on the cell layers' carrying of m u t a n t genes (2' is) and their participation in future propagations. (2~ Since 11 plants out of 13 failed to show the climbing tendency in successive propagation it can be reasoned that such development failed to occur. A variation in the degree of filling of flowers in the irradiated plants of roses has been reported(2, 5, v) and also confirmed by the present results. T h e doubleness in roses is controlled by several d o m i n a n t genes which are quantitative in expression. (s) T h e complex genetic constitution of garden roses (x4~ is responsible for the high frequency of spontaneous and induced flower color mutations32, 5, 7, 15~ Several m u t a t i o n breeders have reported a proportionately higher n u m b e r of variations in flower color intensities rather than total color changes3 z's'7) These findings are supported by the present results. With roses white is recessive to color, yellow recessive to mauve and red, and pink is dom i n a n t to all colors3 s) The major constituents of flower colors in garden roses are anthocyanins, flavonols and cartenoids, which in a certain proportion gives a specific color or shade33) A fairly high n u m b e r of mutations in red- and pink-flowered roses suggests that anthocyanin rich (3) cultivars are more suitable for inducing flower color mutations. From the present experiment it can be concluded that although g a m m a rays produce some harmful effects there is also a high occurrence of induced mutations in roses. T h e successful isolation and multiplication of one climbing and two flower color mutations indicates the fruitfulness of this technique when applied to garden roses.

Acknowledgements---The author wishes to express her gratitude to Dr. M. N. GEPTA for the guidance in the course of work and the Director, N.. B. R. 1. LU(:KNOWfor providing tacilities.

333

REFERENC, E$ 1. BoRojEvm K., GOTTASCHALKW. and ~|I~.~KEA. (1977) Factors influencing the mutant spectrum and the quality of mutants. Pages 146-150 in Manual on mutation breeding. Tech. Rep. Ser. 119, IAEA, Vienna. 2. BROERTJES C. and VAN HARTEN A. M. (1978) Application of mutation breeding methods in the improvement oat` vegetatively propagated crops. Elsevier, Amsterdam. 296 pp. 3. DE VR~ES D. P., VAN KEtrlen H. A. and DE BRUYN J. W. 0974). Breeding research on rose pigments. I. The occurrence of flavonoids and carotenoids in rose petals. Euphytica 23, 447-457. 4. GAUL H. (1977) Mutagen effects in first generation after seed treatment. Pages 87-98 in Manual on mutation breeding, Tech. Rep. Ser. 119, IAEA, Vienna. 5. GUPTA M. N. and SHUKLA R. (1971) Mutation breeding of garden roses. Effects of gamma irradiation on some scented roses. 57pn. 57. Breed. 21, 129-136. 6. HANSEL H. (1966) Induction of" mutations in barley. Some practical and theoretical results. Pages 117-138 in Mutation in plant breeding, Proc. Panel, IAEA, Vienna. 7. KAICHER U. S. and SWARUP V. (1972) Induced mutations in roses. Indian 57. Genet. Plant. Breed. 32, 257-265. 8. LAMMERTSW. E. (1943) The scientific basis of rose breeding. Am. Rose Annu. 30, 71 74. 9. LATA P. (1972) Hybridization in modern roses. Indian J. Ornament. Hortic. 3, 15 21. 10. LATA P. (1974) Hybridization in modern roses. III. Selfing in control and irradiated plants. Curr. Sci. 43, 690q592. 11. LATA P. (1975) Hybridization in modern roses. IV. Hybrids between control and mutants of cultivar Montezuma. SABRAO jour. 7, 103 108. 12. LATA P. 11975) Ett'ects of ionizing radiation on roses. II. Meiotic studies on rose cultivar Pink Parfait and its gamma ray induced mutants. Cytologia. lO, 633-638. 13. LATA P., GUPTA M. N. and MURTY A. S. (1977) Effects of ionizing radiation on Rosa dama.scena Mill. New Bot. 4, 23-27. 14. SHAHARE M. L. and SHASTRY S. V. S. 11963) Meiosis in garden roses. Chromosoma 13, 702-724. 15. WVLm A. P. (1954) The history of garden roses. I1..7. Roy. Hortic. Soc. B0, 8--24.