Radiation damage in shoot apical meristems of Antirrhinum majus and somatic mutations in regenerated buds

Radiation Botany, 1971, Vol. 1I, pp. 157 to 169. Pergamon Press. Printed in Great Britain.

R A D I A T I O N DAMAGE IN S H O O T APICAL MERISTEMS OF ANTIRRHINUM MAJUS AND SOMATIC M U T A T I O N S IN REGENERATED BUDS F. SEKIGUP_~II, K. YAMAKAWA* mad H. Y ~ G U C H I Laboratory of Radiation Genetics, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113 and *Institute of Radiation Breeding, Ohmiya, Ibaraki 319-22, Japan

(Revised manuscript received 12 March 1970) S~.gxoucm F., YAmmC.AWAK. and YAMAGUCHIH. Radiation damage in shoot apical meristems 0fAntirrhinum majns and somatic mutations in regeneratedbuds. RADIATIONBOTANY11, 157-169, 1971.--To obtain information on recovery from radiation damage and the behavior of mutated cells in shoot apical meristems actively growing plants of Antirrhinum majus, heterozygous for flower color, were irradiated for ten days with 50-6000 R]20 hr-day with °°Co gamma rays and the effects investigated in detail by macro- and microscopic observations. Induced somatic mutations appearing in the petals were scored. In irradiated plants, all the various developing buds were somewhat damaged, though apparent differential radiosensitivity appeared among them. We inferred that the active meristem was relatively radiosensitive, and it was less capable of recovery from radiation damage than dormant ones. Anatomical studies revealed that sublethal irradiation results in development of lateral buds, which are normally inactive due to apical dominance, or brought about the formation of abnormal buds, as a result of stunting of the lateral shoot apex. These regenerated buds were of adventitious origin from viable cells in the radioresistant peripheral (or flank) regions of the meristem. Results from scoring the induced somatic mutations suggested that meristem regeneration produced primarily large mutated sectors without chimaeric structure. Morphogenesis of irradiated apical meristems is discussed on the basis of these results. INTRODUG'TION either adjacent to or below the damaged apical INCREASING attention has been given to the meristem. (4,s,ta,ts,~ 7/~,26,,~9,s2-s4) histology of radiation damage and to recovery A great deal of work has also been done with in shoot apical meristems. Recendy, PRATTCa4) radiation-induced somatic mutations in leaves summarized her work and that of others with or stems (19,48) and flowers (t, n, v,15,2s,39,41) of plants herbaceous and woody species, and described heterozygous for several genes. These mutational active meristems as more radiosensitive than events are correlated with the number of cells in inactive ones. There are differences among the organ primordia and with the number of species in which the radiation-induced damage cells in the meristem at the time of irradiation. is either localized in the cells of the tunica or SPARROW et a/.(41) demonstrated that a bud corpus, scattered, and distributed uniformly primordium consisting of a few cells produced throughout the meristem. After recovery from larger mutant sectors than buds compose d of radiation damage, a regenerated meristem in many cells at the time of irradiation. Thus, the carnations was formed from a part of the un- effects of ionizing radiation on apical meristems damaged apical region.(sn) In most cases, how- provide a very interestingsubject for morphoever, new buds are initiated in axillary regions logical studies,(5,a, a4,4a) particularly as an aid to 157

158

F. SEKIGUCHI, K. YAMAKAWA and H. YAMAGUCHI

understanding the composition and function of the derivatives of the meristem, although few studies have actually traced the derivatives of meristem cells into mature tissues.(38) In this paper, sensitivities of the various developing buds of Antirrhinum majus to g a m m a irradiation were compared by macro- or microscopic observations. Also, the radiation-induced somatic mutations affecting petal color in the flowers of a regenerated shoot were investigated. A part of this paper was reported by YA~AKAWA a n d SEKIOUCHI(4s) at the International Symposium on the Present State of Mutation Breeding (15-16 August 1968) held at Mito, Japan. /VIATERIALS A N D M E T H O D S

F 1 hybrids of Antirrhinum majus, heterozygous for flower color, were cultured from seed obtained from the Sakata Seed Co. Ltd. (Yokohama, Japan) in 1965. This F I hybrid was derived from a cross of white x pink with the phenotype of pink. The experiments were carried out at the Institute of Radiation Breeding, Ohmiya, Ibaraki, Japan. The first part is a macro- or microscopic study of the radiation-induced damage and recovery in the various shoot apical meristems. The second part concerns scoring of the somatic mutations which appeared on the flowers of shoot regenerated after irradiation. (A) Macro- and microscopic investigations Thirty three-month-old plants were irradiated with each exposure in the g a m m a field (S°Co, 3004 Ci), the Institute of Radiation Breeding, Japan.(2.) At the start of irradiation, the plants had various developing buds as illustrated schematically in Fig. 1. The growth of Antirrhinum majus is characterized by apical dominance, with apices of the lateral shoot axes remaining dormant in the vegetative phase as long as the apex of parent shoot axis is not inhibited. Radiosensitivity of the various buds was assessed by evaluating the degree of growth inhibition and the capacity to recover from radiation damage. A group of plants were given total exposures ranging from 0-5 to 60 kR with exposure rates of 50-6000 R/20 hr-day, for 10 days beginning 3 June 1966. Another group of

S V

112|11 777

FIo. I. A schematic representation showing positions of the various shoot apical meristems studied in Antirrhinum majus at the start of irradiation. I, inflorescence axis (reproductive phase); I1, upper part of the axis including inflorescence apex and developing floral buds; I2, developed floral buds. II, Iateralshoot axis (vegetative phase) ; II1, upper part of the axis including shoot apex, younger leaves, and developing axillary buds; II~, developed'axillary buds; II~, accessory bud.

plants were exposed to 15 and 30 kR in order to investigate histologically the pattern of radiation damage and recovery in the vegetative shoot apices after sublethal irradiation. The irradiated plants were pruned to the fifth node from the base. A lateral shoot axis, as well as an accessory bud, subtending a single leaf remained; the others had been totally trimmed off. Collections from two plants were made on each of eight sampling days: 0, 3, 5, 7, 14, 21, 28 and 35 days after irradiation. Every lateral shoot axis of the plant was separated under the dissecting microscope into several parts: shoot apex or developing axillary buds, developed axillary buds, and accessory buds. T h e y were killed and fixed in Craf I I I , dehydrated in a tertiary-butyl alcohol series and embedded in paraffin. Materials were sectioned longitudinally at 10-12 ~t, and were stained with safranin and fast green. Macroscopic observation of irradiated plants was continued from the start of irradiation to death. (B) Scoringprocedure of induced somatic mutations To obtain information about induced somatic mutations after sublethal irradiation, fifty plants were exposed with either 15 or 30 kR, for 10 days from 4 to 15 July 1967. The exposure rate was

RADIATION DAMAGE IN SHOOT APICAL MERISTEMS OF ANTIRRHIWUM 1500 and 3000 R/20-hr day, respectively. Induced somatic mutations were scored on the flowers of regenerated shoots from 15 September to 5 November of the same season. Since the scoring of somatic mutations was done by naked eye, only the mutant sectors composed of 80 cells or more could be detected. The mutated sectors were classified by assuming that each spot or streak had the size composed of the n u m b e r of cells listed in Table 1. The mutation rate was estimated for each of the 13 sizes by the formula, M R = ( N o ) (m)/5×105, where M R is the mutation rate, (No) is the estimated n u m b e r of cells per mutation sector, and (m) is the n u m b e r of mutated sectors in a flower. A value of 5 × 105, the average n u m b e r of cells constituting the petal area of a flower in an F 1 hybrid (pink), was obtained as follows: The n u m b e r of cells per m m ~ was 410-4- 8.7 I, and the surface petal area was 12-244-0.53 cm 2 with an average of 20 flowers. Consequently, the mean number of cells making up the surface area of a petal was estimated as about 5 × 105. In this experiment, no apparent difference in development between non-mutated and mutant tissues was noticed.

RESULTS

(A) Differential radiosensitivity among the various

shoot apical meristems in a plant (1) Macroscopic observations on growth inhibition by gamma irradiation Gross differences in radiosensitivity among the various apices (see Fig. 1) were readily apparent at 20 days after irradiation (Fig. 2). At 4-0 kR (400 R/day), flower primordia which must have differentiated on the inflorescence axis became degenerated, though the growth of active terminal buds of the inflorescence axis continued and produced full-grown flowers again. Apparently the developing flower primordia (in the 11 bud of Fig. 1) were more radiosensitive than fully- or half-grown ones (Is). Thus, floral organs which were fully developed at the time of irradiation grew as usual even after 7-5 kR at 750 R/day. Growth of a terminal bud of the inflorescence axis terminated with exposures greater than 7.5 kR. As a result of inhibition of growth of the inflorescence axis, the dormant apical meristems

159

of lateral shoot axes were able to continue their growth, which was more vigorous at exposures of 7.5 and 15 kR than at 1-0 or 4.0 kR. At 30 kR (3000 R/day), dormant apical buds of the lateral shoot axes did not develop immediately, but only after 45 days. These vigorously growing lateral shoot axes did not originate from the intact apices present at the time of irradiation, but those exposed to 15 and 30 kR were adventitious, as was ascertained by microscopic observation. At 45 and 60 kR, death of the plant occurred. Thus, it is obvious that the flower primordia were several times more radiosensitive than vegetative buds, as has been previously reported. (21,ss,47,49~ On the other hand, recovery of growing tissues was essentially complete. Table 2 illustrates the length of the inflorescence axis, main shoot axis, and plant height involving the growth of lateral shoot axes, as well as n u m b e r of lateral shoot axes per mature plant (an average of 10 plants) after short-term irradiation. When the length of inflorescence axis was disregarded, length of the remaining shoot axis was almost the same regardless of exposure. It corresponds to plant size measured at the time of irradiation. At 4.0 kR growth of a terminal bud of the inflorescence axis was only slightly less than at 0-5 kR or less than in the non-irradiated control. Plants irradiated with 7.5 and 15 kR developed lateral shoot axes and maintained the total height even after growth of a terminal bud of the inflorescence axis was inhibited (see Fig. 2). Lateral shoot axes which were slightly damaged at 4.0 kR developed somewhat earlier than those of non-irradiated controls. With exposures of 0.5, 1.0 and 2.0 kR almost the same amount of growth of terminal buds of the lateral shoots was maintained as was the case in non-irradiated control plants. (2) Microscopic investigations on recoveryfrom

the damage in the vegetative apical meristems after sublethal irradiation (i) Usual morphology of the lateral shoot ax.es. The lateral shoot axes existing in the leaf axils were investigated in longitudinal section. Longitudinal sections of apices included in this axis, at the time of irradiation, are presented in Fig. 3. All apices are domes and in a vegetative state.

1/4096=0.00025 1/2048=0.00049 1/1024=0.00098 1/512 =0.00195 1/256 =0-00390 1/128 =0-00780 1/64 =0.01560 1/32 =0.03100 1116 =0.06300 I/8 =0.12500 1]4 =0-25000 1/2 =0.50000 1 = 1.00000

125 245 490 975 1950 3906 7813 15,625 31,250 62,500 125,000 250,000 500,000

Calculated mean no. of cells/ mutation sector ( 80170) ( 175340) ( 345680) ( 7001350) ( 14002700) ( 27505400) ( 550011,000) ( 11,50022,000) ( 22,50044,000) ( 44,50088,000) ( 90,000- 175,000) (180,000- 350,000) (355,000-1,000,000)

Range (No. of cells observed)

N o = S × (5 × IOs)

0.005 0-021 0-043

0.515 0.687 1.375 2.750 55-000

22

33 22 22 11 55

MRt

22 44

No. of mutations per flower, m

15 kR

Exposure

11

11 II 33 I1 22

54 54 22

No. of mutations per flower, m

30 kR

2.750

0.043 0.086 0.515 0.343 1.375

0.013 0.026 0-021

MR t

and (5 × 105) = a v . no. of cells per total area in a flower (410 8.71[mm9).

t M R (mutation r a t e ) = (No) (m)[5 × 105. (aV0) =estimated no. of cells per mutation sector, (m) = n o . of mutation sectors in a flower,

* Average total petal (flower) area = 12.24 0.53 cm z. S-1 = 12.24 cm 9, S-2 -t2 = 0-003 cm 9.

2-xl= 2 -11 = 2-10= 2 -9 -2 -9 = 2 -7 = 2 -6 = 2 -5 = 2 -4 = 2 -8 = 2 -9 = 2-t = 1 =

Size of mutated sector as fraction of total flower area*

S= 1/12.24 cm 9

Table 1. The number of mutations per flower and the estimated mutation ratefor various sizes of sector after sublethal irradiation

RADIATION DAMAGE IN SHOOT APICAL MERISTEMS OF A N T I R R H I N U M

161

Table 2. Length of main axis and inflorescence axis, plant height after development of lateral shoot, and number of the lateral shoots in plants of Antirrhinum majus after a short-term (ten days) irradiation (average of 10 plants) Exposure, R/day

0 50 100 200 400 750 1500 3000

Total exposure, kR 0 0.5 1 2 4 7-5 15 30

Length Of main axis, cm 78-7 72-4 74.9 80.6 73-1 44.6 44-1 39.7

inflorescence axis, cm 35"2 27.6 24.6 32.8 26.4 3"9 5.4 2-9

Plant height, cm

No. of developed lateral shoots

93.6 87-1 90-7 92.9 96" 1 96.9 95.9 73-9

13.1 15.7 13.9 10"7 12"1 8-1 12-7" 10-3"

* No. of developed 'adventitious' shoots. Lateral shoot axes with 5-6 pairs of leaves include the various apical meristems of terminal buds (A), of developing (By or developed (C) axillary buds, and of accessory buds (D). The larger shoot apices (A, C and D) are organized into two tunica layers and a corpus, but the pith rib meristem is not identifiable. Smaller apex (By lack this organization. Leaves are arranged in the decussate phyllotaxis, although it becomes alternate just below the inflorescence--see WARDLAW.Cso) (ii) Radiation damage and recovery in plants exposed to 15 kR (1500 R/day). Radiation damage and recovery in plants exposed to 15 kR (1500 R/day) was investigated in longitudinal sections of vegetative lateral shoot axes as a function of postirradiation time (Fig. 4). Figures 4 A - C illustrate radiation damage to the dormant apical meristem of a terminal bud of lateral shoot axis (II 1 of Fig. 1). First, at the end of the irradiation, damaged cells were observed in the second tunica layer and the corpus of the apex, and did not stain with safranin or fast green (Fig. 4A). This results from the lack of the nuclei and cytoplasms. It suggests that an inner part of the meristem is more radiosensitive than an outer region (the first tunica layer and the peripheral portion) in Antirrhinum majus plants. At 3 day., after irradiation, it is possible to note a depressiolz in the first tunica layer and a flattening of the apex (Fig. 4By. At 5 days after irradiation, the

shoot apex stopped growing, but seemed to show a little elongation in cells of the peripheral portion (Fig. 4C). Figures 4 D - F show radiation damage and recovery of an apical meristem of the developing axillary bud (in the Ill). Although this bud did not initially seem to be affected by irradiation, vacuolated cells were observed below the first tunica layer at 3 days after irradiation (Fig. 4 D). At 7 days after irradiation, disturbance of layering was observed in the central region of the second tunica layer (Fig. 4 E). At 14 days after irradiation, resumption of organization and growth of the meristem became apparent, but it seems still to have retained some degree of radiation damage (Fig. 4 F). Such regenerated meristems are expected to produce young leaves. Both dormant apical meristems of developed axillary buds (II~) of the lateral shoot axis and of accessory bud (118) did not show conspicuously their meristematic activity. As a result of stunting of the inflorescence axis, the several adventitious shoots just below it began to grow (Fig. 5). These vigorously growing lateral shoots do not originate from the dormant apical meristems o f l I 1 bud but from the repaired developing axillary buds existing adjacent to the apex, as shown in Figs. 4 D-F. In this paper, therefore, it is termed a 'false-lateral' shoot. Similar phenomenon is observed following decapitation.

162

F. SEKIGUCHI, K. YAMAKAWA and H. YAMAGUCHI

(iii) Radiation damage and recovery in plants exposed to 30 kR (3000 R/day). As with the 15 kR experiment, radiation damage and recovery in plants exposed to 30 kR (3000 R/day) were investigated in longitudinal sections of vegetative lateral shoot axes. Radiation damage, observed in the inner region of every apical meristem, was more severe than that seen after 15 kR. It results probably from the strong inhibition of cytological and physiological processes after irradiation.(24) Therefore, the 30 kRirradiated plants show some differences in the pattern of recovery from damage, as demonstrated in Fig. 6. Primary damage in the apical meristem of a terminal bud of lateral shoot axis (II1) , observed at the end of irradiation, was the presence of collapsed cells in the second tunica layer and the upper region of the corpus (Fig. 6 A). The majority of the effects on cells of the inner portion become evident during irradiation with 3000 R/day. At 5 days after irradiation, the damaged apex ceases growth and elongation was never observed in the peripheral region as with 15 kR (Fig. 6 B). Differentiation of leaf primordia on the flanks of the apical meristem stopped completely. Apical meristems of the developing axillary buds were inhibited also. Recovery phenomenon of apical meristems exposed to 30 kR, however, occurred in the developed axillary buds (II2) of lateral shoot axis and accessory bud (II3). Figures 6 C - E illustrate a pattern of recovery from radiation damage in dormant apical meristem of IIa buds. Damage seen at the end of irradiation was the loss of the nucleus and cytoplasm in cells of the second tunica layer and the corpus (Fig. 6 C). This damaged meristem stopped growing. At 14 days irradiation, a flattened tip became evident and the pith closely approached cells of the first tunica layer and the peripheral region (Fig. 6 D). Later, a phenomenon of much interest, namely evidence of the occurrence of cell division in cells of the peripheral region, was observed. Consequently, a regenerated meristem differentiated from cells of the periphery of the original dormant apical meristem of I I , buds at 28 days after irradiation (Fig. 6 E), followed by young leaf differentiation. Although the abnormal appearance of the meri-

stem still remained, a bud was noted at 35 days after irradiation. This is termed a 'false-axillary' shoot. Recovery meristems were formed also in dormant apical meristem of I I 3 bud, as shown in Figs. 6 F-J. Radiation damage was not evident immediately after 30 kR. At 7 days after irradiation, however, necrosis of cells was perceived below the first tunica layer (Fig. 6 F). At 14 days, the apex was inhibited and it began to collapse in the areas of necrosis (Fig. 6 G). At 21 days after irradiation, two regenerated meristems developed in peripheral regions (Fig. 6 H), showing dichotomy as observed by HAccms and REICHERT.(16) However, recovery from radiation damage was not complete. The regenerated meristems increased in the number of cells up to 28 days after irradiation (Fig. 6 I). At 35 days (the last sampling time), hyperplasia, ~nlargement of several parts due to numerical increase of the cells, was found (Fig. 6 J). Later, such regenerated meristems can form new buds up to 60 days after irradiation and produce vigorous shoots. As seen in Fig. 7, the plant at about 60 days after 30 kR shows stimulation in growth of one accessory bud (as a consequence of radiation damage of axillary buds), but the other has not developed. Several swollen nodes seen at the base of a grown-up shoot are probably derived from the several protuberances observed in Fig. 6 J. Shoots do not develop from the intact dormant apical meristem of I I 8 buds, but from ones of adventitious origin. They are termed a 'falseaccessory' shoot. Completely normal-looking shoots have sprouted in the nodes of 'falseaccessory' shoot. (B) Petal color mutation in the regenerated shoost after sublethal irradiation of 15 and 30 kR Induced somatic mutation was scored by using an F 1 hybrid (pink) plant, heterozygous for flower color (dominant pink vs. recessive white), expecting that white sectors in a flower of original pink color could appear as a consequence of mutation or would appear as a consequence of the loss of the dominant locus by chromosome deletion. Two total exposures of 15 and 30 kR were administered over a ten-day period. These exposures, which were sufficient to kill the apical meristem of terminal buds of

RADIATION DAMAGE IN SHOOT APICAL MERISTEMS OF AWTIRRHINUM

proportions of chimaeric flower, full flower and entire shoot mutations of the total color mutations, calculated from Table 1. Both full flower and entire shoot mutations are probably the derivatives of one mutated initial cell while chimaeric flowers are traced to more than one initials. At 15 kR, full flower and entire shoot mutations formed 15.0 and 83.5 per cent of the total mutations, respectively. More conspicuous was the case of 30 kR, where 98.0 per cent of the total was of the entire shoot type. It is reasonable to infer that the difference in sector size between 15 and 30 kR is correlated with the difference in their recovery from radiation damage. In both cases, however, the mutations resulting in chimaeric flowers were so few that they were disregarded. The 'false-axillary' or 'false-accessory' shoots produced after 30 kR must originate from fewer cells than 'false-lateral' shoots formed after 15 kR. Sublethal irradiations cause regenerated shoots to develop from a few cells in a less differentiated region, and hence enlargement of the mutant sector results. Consequently, this effect has been termed 'internal disbudding by radiation' (see YAMAKAWAand SEKIGUCHI(4S)).

the inflorescence axis or/and of the lateral shoot axes, caused two different patterns of recovery: 15 kR produced 'false-lateral' shoots and 30 kR induced 'false-axillary' and/or 'false-accessory' shoots. Therefore, it is of interest to obtain information on the size of mutant sectors in flowers of these regenerated shoots. After irradiation, every flower in each plant was examined in detail for size of the mutant sector. At 15 kR, the recovered shoots flowered at an average of 43 days after irradiation. There were 124 inflorescences scored from 31 plants with an average of 4.0 shoots per plant. At 30 kR, the shoots flowered at an average 60 days after irradiation and 103 inflorescences from 39 plants were scored (2.64 shoots per plant). Petal color mutations observed resulted in changes of the original pink to deep pink, light pink or white. Most of the observed changes were light pink c o l o r mutants. In non-irradiated control plants, no apparent color mutants were found. The inferred number of mutations per flower and the estimated mutation frequency for each sector size of 13 types are recorded in Table 1. Frequencies of entire shoot mutations when exposed to 15 and 30 kR were 3.07 and 2"37 per cent, respectively. The reason that mutation frequency after 30 kR was lower than after 15 kR is probably due to the severe growth inhibition with the higher exposure. Figure 8 shows the

0 °~

DISCUSSION

(A) Differences of radiosensitivity among the various

developing buds of irradiated plants It is well known that radiobiological effects on m

I00 15kR

30

kR

E O

"

50

tO .u O IX

o

U

10

ft.

Size ~/~

of

mutation

C h;,rno e r i,c [ - ~ ' 7 - ~ F u I I,

ftower

163

[

J flower

sector I I

Entire shoot

Fxo. 8. Proportions of chimaeric flower, full flower and entire shoot mutations to total mutations. Data from Table 1.

F. SEKIGUCHI, K. YAMAKAWA and H. YAMAGUCHI

164

plants depends on the exposure rate, total exposure, species or variety used, growth stage, and physiological condition of the plant at the time of irradiation. 04) Thus, causes of differences in radiosensitivity among developing buds of a plant are very obscure. In Antirrhinum majus, the buds contain cells of different ages (the most juvenile cells are those of the meristem). A flower primordium was more radiosensitive than the active terminal bud of an inflorescence axis, as blind flower primordia were noticed after irradiation of 4.0 kR (Fig. 2). And also, the full-grown flower at the time of irradiation opened even after 7.5 kR. It was more radioresistant than the developing flower primordium or the active terminal bud of an inflorescence axis. Such a difference in radiosensitivity is related to differences in cell cycle between the meristems. Since the active apical meristem of an inflorescence axis which consisted of young cells is more resistant to radiation than the developing flower primordia, it seems to have relatively shorter cell cycles. These problems will be discussed in detail in a later paper on effects of chronic irradiation with lower exposure rates. In vegetative growth, active apical meristems were more radiosensitive than dormant meristems, being inhibited in their growth by apical dominance. This finding seems to be in line with the well-known fact that dividing cells are generally more susceptible to radiation than non-dividing cells.Csv,4s) Furthermore, it is sug-

gested that radiation damage is related to the degree of differentiation (or age) of the meristems in a shoot, and it was found that the apical meristem of lateral shoot axis was indeed more inhibited by radiation than dormant apical meristems of the axillary buds or of the accessory bud which differentiated from the former.CS~) In the case of reproductive growth, on the other hand, the response to radiation is complicated by involving apical dominance and the age of the meristems in a shoot. However, reproductive growth is several times more radiosensitive than the vegetative one, as reported by m a n y workers.C~x,ss,4v,4s) Since radiosensitivity of cells fluctuates during growth, we need to clarify the characteristics of the growth cycle of proliferating cells, as well as of the transition of the apical meristem from a vegetative to reproductive stage. Radiosensitivity of various developin~ buds in Antirrhinum majus is summarized in Table 3. In conclusion, (i) the developing flower primordium is the most radiosensitive, but the full-grown flower is more resistant than an active terminal bud of the inflorescence axis. (ii) In vegetative shoot, the organized terminal meristem of the axis is more radiosensitive and less capable of recovery from radiation-induced damage than the less-organized terminal meristems of buds which differentiated from the former and are dormant.

Table 3. Radiosensitivity among the various developing buds of a plant, Antirrhinum majus Exposure, R/day

Total exposure, kR

Kind of damaged meristem

400 750 1500

4 7.5 15

Floral bud Apex of inflorescence axis Apex of lateral shoot axis

3000

30

Developed axillary bud and accessory bud

4500

45

All

* See Section A 2-ii. t See Section A 2-iii.

Phenomena exhibited Blind bud Growth of a lateral bud Growth of a 'false-lateral' shoot* Growth of a 'false-axiUary' and/or a 'false-accessory' shoot t Death of plant

RADIATION DAMAGE IN SHOOT APICAL MERISTEMS OF AWTIRRHIWUM (B) Radiation damage and recovery Ionizing radiation causes several different morphological effects in the process of shoot growth, such as stunting or dwarfing, misshapen organs and mottled flowers or leaves, as reported earlier by GUNCKEL and SPARROW.(14) After irradiation, meristematic damage and recovery are revealed, in general, by changes in flower and leaf development due to an effect on organogenesis of the apical meristem. Many workers have reported macroscopically the phenomena of recovery.(1~,14,x7,~5,82,84) In our experiment also, recovery was disclosed by the renewal of flower formation at low exposure and by the vigorous development of lateral shoots at high exposure (see Fig. 2). The former can be brought about by repair of the active apical meristem of a terminal bud of the inflorescence axis per se and the latter by recovery of the plant per se. Responses of the apical meristem to sublethal irradiation also have been studied histologically in seeds,(~) seedlings,(1°,18,4~) or growing plants (6,~s,ne) of herbaceous species and in dormant( 12, l~,sl-s4) or actively growing shoots(9,n,26,~9,35) of woody species. Damaged cells recognized in many plants have possessed the following characteristics: absence of mitosis, a high degree of vacuolation, hypertrophy, absence of or decrease in stainability of the nucleus, deep-staining or thickening of the primary cell wall, and collapsed cells.(s4) Localized damage is in the outer layer(9,29, se) or in the deeper region of the meristem. (6,12,17,23,31--35) In Antirrhinum majus plants, the nucleus and the cytoplasm in cells of the second tunica layer and the corpus disappeared during or soon after irradiation (Figs. 4 A or D, and Figs. 6 A or C). Subsequently, these cells collapsed. However, cells of the peripheral region (or flank) still remained without showing any damage. This indicates that the cells of the central zone are more radiosensitive than those of the peripheral zone in the apical meristem. The pattern of recovery from radiation damage in Antirrhinum majus plants does not disagree with that described by several authors.( 9,11,18,¢~5,28,29, s~-s4) The first visible, cytological alteration was a flattening of the superficial layer in the apical meristem of a terminal bud, subsequently leading to progressive disorganization in the apical meri-

165

stem, and the development of the dormant apical meristems of axillary and/or accessory buds. In most cases, the cells of the more radioresistant peripheral region (or flank) began to divide and played an important part in the formation of a new functioning apex during the process of recovery. Plants irradiated with 15 kR (1500 R/day) formed 'false-lateral' shoots for a renewal -:'he growth at the upper part of lateral shoot axis, though having conspicuous damage such as disturbance of layering in the meristem (see Figs. 4 D-F, and Fig. 5). In plants irradiated with 30 kR (3000 R/day), 'false-axillary' shoots and/or 'false-accessory' shoots, which originated adventitiously from the radioresistant peripheral region, developed considerably later (see Figs. 6 C-E, Figs. 6 F-J, and Fig. 7). The regenerated shoots did not originate from the intact buds but from abnormal proliferation of the damaged meristem.(~5) This might also result from occasional regeneration of the less severely injured cells throughout the various developing buds, as MIr,SCHE et al.(29) observed in Taxus media irradiated chronically with gamma rays. In addition to differences in radiosensitivity among the various developing meristems of a plant as discussed in the preceding section, the histological studies confirmed the fact that there is a relatively radiosensitive portion in every meristem. As shown in Fig. 3, a dormant apical meristem of a terminal bud of the lateral shoot axis has the largest number of cells, whereas dormant apical meristems of axillary buds of lateral shoot axis and of accessory bud have a small number of cells. Thus, it is concluded that the capability of recovery is determined by the size of the radiosensitive portion, as well as by the relative amount of metabolic activity in the meristem. SPARROW et al.(*°) and TAYLOR(45) demonstrated that differential radiosensitivity between actively growing and dormant plants or between terminal and lateral buds was related to the size of the interphase nuclei of shoot apical meristem cells. Therefore, accurate measurement of nuclear volume should be made in the shoot apices of the material used in the present experiments. Dormant apical meristems of a few damaged axillary or accessory buds resumed their growth and replaced the destroyed apical meristem of

166

F. SEKIGUCHI, K. YAMAKAWA and H. YAMAGUCHI

the terminal bud of a lateral axis. This finding indicates that the ultimate survival of the less organized or dormant buds depends primarily on their high capacity for recovery, and not merely on the degree of incipient damage produced. Several explanations for the difference between the repair capacity of highly organized and less organized meristems seem possible: (i) Differentiated cells may be more resist~rtt than undifferentiated proliferating cells, as MERICLE and M~.RICLE(z~) demonstrated with X-rayed barley embryos. This concept is in complete agreement with Bergonie-Tribondeau's law, in which the radiosensitivity of a cell varies in proportion to its proliferating activity and inversely proportional to degree of differentiation. It has been suggested that part, if not most, of the differences in radiosensitivity between an actively dividing and an essentially non-dividing meristematic cell population reside in their different average nuclear volumes.C4°, 46) (ii)Some of the differentiated cells in the peripheral region resume their proliferating activity or capacity as a result of the complete destruction of proliferating cells in the tunica layer and the corpus. This might be called a type of dedifferentiation. (iii) Resumption of proliferating activity occurs more easily in the cells of the less organized apices than those of more highly organized apices. (iv) Furthermore, a retardation of plant growth with ionizing radiation is considered to be caused by either (a) inactivation of auxin, (b) inhibition of auxin transport, or (c) damage to the auxin-synthesizing system of the plant. GORDON(as) has already reported that the major effect of radiation, at least on auxin, is a more or less permanent inhibition of auxin formation. The development of dormant apical meristem of lateral buds in irradiated plants is not due to a stimulation of growth by irradiation, but is due to the destruction of apical dominance. (C) Mutation sectors in the regeneratedshoot after

sublethal irradiation A study of the gamma-ray induction of somatic mutations in the petals of Antirrhinum majus, heterozygous for flower color, was undertaken to clarify certain essential details of the radiobiological responses.(~, sg) As shown in Table 1 and Fig. 8, irradiation of 15 and 30 kR produced

mostly entire or whole flower mutations. Chimaeric flowers were produced in such low frequency that they were disregarded. This result indicates that the regenerated shoot did not originate only from one or a very few cells, but also that the effect of cell competition was not thought to be an important factor in ontogeny. This is supported by the histological study, showing that the regenerated meristem originated adventitiously from the viable cells of the peripheral region (or flank) after sublethal irradiation. Lower doses, however, produce very few entire flower mutations.(~) Segregation ratios for induced mutations (double flower, male sterile, and the alteration of leaf shape or phyUotaxy) in M 1 capsule progenies fits very closely to the expected Mendelian ratio of 3:1 (unpublished data). Segregation ratios close to the expected 25 per cent in the progeny ofLycopersicon esculentum seedling~ irradiated with 10 kR (2 kR/day), were reported previously by YAMAKAWAand SEKIGUGHI.(48) In tomato, it is thought also that regeneration of the apical meristem leads to enlargement of the mutated sector. Thus, we conclude that sublethal irradiation of growing plants over a short period at very high exposure rates is highly effective in producing large mutant sectors, due to the effect of 'internal disbudding by radiation'. Changes of a pre-existing periclinal chimaeric condition following irradiation of buds have been shown in carnation, Car) in potato, OS) and in apple.(a2) SAOAWA and MEHL~UI$T(s6)observed that the radiation-induced destruction of the outer cell layers of the shoot apex of carnation was followed by regeneration of a new apex from the more deep-seated cells. Our results are similar to their observation and it can, therefore, be concluded that the genetic constitution of the germ cells, which are produced from the regenerated shoots, is that of the surviving cells of the radioresistant region in the apex. The enlargement of mutant sectors by means of meristem regeneration following initial radiation damage has now been shown in two species, Antirrhinum majus and Lycopersicon esculentum. This method was also applied to vegetatively propagated Chrysanthemum morifolium plants. We were able to find entire shoot mutations in the recovered shoots when the young plant was

RADIATION DAMAGE IN SHOOT APICAL MERISTEMS OF A.~fTIRRHIJVUM irradiated with total exposures of 5, 10 and 20 kR, at exposure rates of 0.1, 1.0 and 2.0 kR/day, respectively.(2°) In Chrysanthemum cuttings, BowEs et al.(3) have also obtained sports by g a m m a irradiation. TANAKAand SEKIGUCHI(44) chronically irradiated actively growing rice plants with exposure rates of 300-600 R/day, then the plant was cut off just above the ground. Most of the mutated panicles obtained from developing buds in this plant were not chimaeric in nature, as indicated by the scoring of recessive (chlorophyll) mutant segregation. This suggested that sublethal exposures could be one of the possible means for obtaining the enlargement of mutant sectors. This might be a type of internal disbudding, although a histological study of development of the axillary buds in the irradiated plant after it has been cut back seems highly desirable. To obtain complete mutant non-chimaeric shoots after irradiation of vegetatively propagated plants, BAUER,(S) NAKAjIMA(s0) and ZWINTZCHER(51) emphasized the importance of treating a bud primordium when it consists of only a few, undifferentiated cells. In order to sprout such a bud primordium, they also stressed the necessity of cutting back the shoot growing from an irradiated primary bud and thus forcing secondary or adventitious buds. Although this cutting back method has been used widely at present, it is troublesome to cut back repeatedly. By using our internal dishudding method, on the other hand, non-chimaeric sports can be identified in both seed and vegetatively propagated plants following irradiation. These findings might also provide useful information on the number of cells contributing to the formation of a given organ and on the behaviour of mutated ceils entering somatic competition during plant development.

Acknowledgements--The authors are grateful to exdirector Dr. K. Knw.~A, director Dr. T. TAa~UNO, and the members of the Institute of Radiation Breeding for their valuable suggestions and encouragement during the course of these investigations. Thanks are due to Prof. K. SArro, Utsunomiya University, Prof. T. MATSUOand Dr. N. HA~, University of Tokyo, for valuable suggestions and criticisms in preparing the manuscript.

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