Pergamon Adv. Space Res. Vol. 14, No. 10, pp. (10)363-(10)372, 1994

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MUTAGENIC EFFECTS OF HEAVY ION RADIATION IN PLANTS

M. Mei,* H. Deng,* Y. Lu,* C. Zhuang,* Z. Liu,** Q. Qiu,** Y. Qiu* and T. C. Yang***

* South China Agricultural University, Guangzhou, P. R. China ** Zhongshan University, Guangzhou, P. R. China *** NASA Johnson Space Center, SD4, Houston, TX 77058, U.S.A.

ABSTRACT

Genetic and developmental effects of heavy ions in maize and rice were investigated. Heavy particles with various charges and energies were accelerated at the BEVALAC. The frequency of occurence of white-yellow stripes on leaves of plants developed from irradiated maize seeds increased linearly with dose, and high-LET heavy charged particles, e.g., neon, argon, and iron, were 2-12 times as effective as gamma rays in inducing this type of mutation. The effectiveness of high-LET heavy ion in (1) inhibiting rice seedling growth, (2) reducing plant fertility, (3) induc- ing chromosome aberration and micronuclei in root tip cells and pollen mother cells of the first generation plants developed from exposed seeds, and (4) inducing mutation in the second generation, were greater than that of low-LET gamma rays. All effects observed were dose-de- pendent; however, there appeared to be an optimal range of doses for inducing certain types of mutation, for example, for argon ions (400 MeV/u) at 90-100 Gy, several valuable mutant lines with favorable characters, such as semidwarf, early maturity and high yield ability, were obtained. Experimental results suggest that the potential application of heavy ions in crop improvement is promising. RFLP analysis of two semidwarf mutants induced by argon particles revealed that large DNA alterations might be involved in these mutants.

INTRODUCTION

There were many studies on the biological effects of ionizing radiation in plants in the past. Most of the experiments were done with low-LET radiation, some with neutrons, and only a few with heavy ions. Earlier studies with ~ seeds indicated that low energy heavy ions were effective in producing various bioeffects, e.g., growth inhibition and tumor and mutation inducation/1/. Recently, the effect of medium energy carbon ions on germination of four kinds plant seeds was examined in China/2/. Data obtained from other studies also showed that heavy ions were efficient in inducing cell inactivation, somatic mutation, and neoplastic transfor- mation/3,4,5,6/. The high-energy heavy-ion beams generated by accelerators have large pen- etration distances and thus are suitable for irradiating large plant seeds.

For the understanding of the mechanisms of radiation effects as well as exploring the potential use of heavy-ion radiation in crop improvement, we investigated the bioeffects of heavy ions ac- celerated at BEVALAC and found that heavy ions can be more effective than low-LET photons in inducing developmental and mutagenic effects in maize and rice seeds.

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(10)364 M. IVlei etal.

METHODOLOGY

Plant Materials

Maize and Rice seeds

A special genetic stock of Zea m~.=, heterozygous for LWl/IW 1 alleles, from Ratio Seeds

Company (Indiana, USA) was used for all the experiments. These seeds contain a recessive lemon white gene (Iw I ) and a dominant gene (Lw I ) in the first chromosome. A deletion or dam- age of Lw I in embryonic cells for leave development will cause white-yellow stripes on plant

leaves. Seeds of Bianpizhan, an ~ rice ( ~ Z a ~ variety, originated in South China with stable inheritance, were used for investigating the developmental and mutagenic effects in M 1 generation and mutation in M 2 generation. For mutation breeding study, another Indicava-

riety, Xiangzhan, with pleasant aroma but poor yield was used. The water content of the seeds used for irradiation was 14.3%.

Irradiation

For heavy ion irradiation, seeds were exposed at the plateau region of the Bragg curve to mini- mize the dose component from secondary particles. Four different heavy charged particle beams generated by the BEVALAC at the Lawrence Berkeley Laboratory were used. Table 1 shows the physical parameters of these heavy-ion beams. The detailed irradiation conditions were described in an earlier report/71. Seeds were sowed 2-3 months after irradiation for germi- nation.

Table 1 Physical Parameters of Various Radiations Studies in Rice Experiments

Lead Residual Track Average Initial Foil Range in LET

Radiation Energy (cm) Water (cm) (keV/t~m)

60Co 1.17 MeV -- -- 0.27 40Ar 570 MeV/u 0.32 13.80 90.00

400 MeV/u 0.23 5.90 117.00

56Fe 600 MeV/u 0.40 8.39 190.00

Gamma ray irradiation was performed with a 60Co source at Radiation Center of Guangdong, South China Agricultural University. Seeds were irradiated 1-2 days before planting. Both gamma-ray and heavy-ion exposures were conducted at room temperature.

Observation of Bioloaical Effects in M I ~

For measuring the mutation frequency in maize, total number of leaves and the number of leaves with white-yellow stripes in each treatment were counted and used to calculate the fre- quency of occurence of stripes in plants developed from irradiated seeds. The effects on growth and development of the third leaf were also investigated in one experiment to determine the relationship between growth retardation and mutation. Inhibition of seedling growth and

Effects of Heavy Ions in Plants (10)365

M 1 plant fertility of rice were used to estimate the radiation damages in M 1 generation. For de-

termining seedling growth inhibition, the height of 20 seedlings at the 7th day after germination were measured for each dose.

The average seed fertility of 20 randomly chosen M 1 plants in each treatment was computed to determine the M 1 fertility. The percentage of reduction in seedling growth or fertility in plants from irradiated seeds, compared to that from non-irradiated seeds, were calculated. For cyto- logical analysis, root tip cells from the seedling with root about 1-cm in length or pollen mother cells in meiotic stage were used. Root tips were fixed in Camoy's solution, hydrolyzed in 1 molar hydrochloric acid, stained with standard Schiff procedure, and then used for preparing slides. For studying pollen mother cells, anthers were fixed with modified Carnoy's method, stained with Carbon fuchsin, and then squashed to make slides. The number of cells for esti- mating frequency of micronuclei formation or chromosome aberration for each dose was about 2,000. These cells were obtained from ten plants or seedlings. The data so obtained, except those from seedling growth inhibition, were fitted by using least square method and the equa- tion Y= aD to generate the dose-response curves, where D is the radiation dose and a the con-

stant. The standard errors were calculated from the formula S.E. = (pq/N) 1/2, where p is the percentage of observed event, q = 1- p, and N is the number of samples observed.

Scorina of Mutation in M 2 ~

The seedlings of M 1 generation were planted in the field at the rate of one plant per hill, and M 1 plants were harvested on plant basis. M 2 progenies then were raised as separated rows.

Mutants with obvious variation in plant height, growth-stage duration, grain shape, and sheath color were scored in the field. The quantitative characters of panicles were also examined in 150 randomly chosen plants for each treatment. The standard for selecting mutants with trans- gressive variation in characters of panicle length, effective panicles per._plant, spikelet per pani- cle, grain per panicle, and grain weight was according to Lu/8/, X< or X>X ~. 1.960".

RFLP Analysis of Mutants

For a better understanding of the molecular nature of heavy-ion-induced mutation, two semid- wad mutants induced by argon particles were chosen for restriction-fragment-length-polymor- phism (RFLP) analysis. Results were compared with their original varieties, Bianpizhan and Xiangzhan. Plant DNA was extracted from fresh-frczen leaf tissue, digested with five restriction enzymes found to be most efficient in detecting polymorphism in rice: EcoRV, Xbal, EcoRI, Hindlll, and Dra1191. DNA extraction, enzyme digestion, electrophoresis, and Southern analysis were performed according to McCouch et a1110/. Rice random genomic clones, which were classified as single copy and mapped on genetic map 110/, were selected as probes for DNA molecular hybridization. These probes were kindly provided by Dr. Tanksley, Comell University, distributed throughout 12 chromosomes of rice.

RESULTS

Somatic Mutation Induced bv Various Radiations in Maize

Quantitative studies with neon, argon, iron particles and gamma rays to determine the RBEs for mutation induction were also done/12/. The mutation frequency, calculated from the number of leaves having white-yellow stripes, increased linearly with dose for all radiations studied

(10)366 M. Meiet al.

(Figure 1). The RBE for neon, argon, and iron ions were 2.02, 8.31, and 12.45 respectively. By using the mutation of alcohol dehydrogenase 1 (Adh 1) of maize pollen grain as endpoint, Freeling reported that the overall RBE of neon ion to X ray was 5 1131, a value higher than that from this study. The cause of this difference might be related to different materials and end- points used. In some plants developed from seeds exposed to argon ion beam, white stripes extending from stem to leaf sheath, to leaf and even to staminate flower, were found. White stripe also occured in some of the inbred plants in the second generation. These results indi- cate that heavy ions produced critical damages in the apical area of seed embryos, where the germ cells were located. These mutated germ cells might be involved in the formation of stripes in the second generation plants.

Effects on Growth Inhibition and Chromosome Aberration in Rice

The biological effects in rice plants developed from seeds exposed to argon, iron particles and gamma rays were compared recently 1141. Figure 2 shows the dose-response curves for seedling height reduction, which possessed shoulders for gamma rays and were exponential for iron particles. For argon ions, the shoulder of the dose-response curve was much smaller than that of the curve for gamma rays. The final slopes calculated from these curves are

10.9xl 0 "3, 3.4xl 0 "3, and 1.67x10 -3 for iron, argon ions and gamma rays respectively. Seed fertility of M 1 plants decreased with increased dose for heavy ions and gamma rays, as shown in Figure 3. For 400 MeV/u argon ions and iron particles, the dose-response curves were expo- nential; the others had shoulders. The final slopes calculated from the curves for iron, argon

(570 MeV/u)ions and gamma rays were 15.6x10 "3, 4.0x10 "3, and 1.9x10 "3 respectively. These values are higher than that from curves shown in Figure 2 for the same radiations. Evidently, plant fertility was more sensitive than seedling height to radiation and can be used to measure radiation injury in the second generation (M 1).

! I / 0 . . . . . . . . I

o.12 [ / J. ,,., .......

o o°.1°!/ / 0.06

0.04

0 .02

50 100 150

oOSE (Gy)

B w

o ~

Figure 1. Dose responses of white-yellow stripes formation in plant leaves after maize seeds exposed to four types of radiation.

Figure 2. Seedling height reduction measured at the 7 day after seed germination. Rice seeds were irradiated with three types of radiation.

Radiation also caused an increase in micronuclei formation and chromosome aberration, includ- ing chromosome bridges and fragments, in root tip cells. A linear relationship was found be- tween the frequency of micronuclei or chromosome aberrations and radiation exposure, as shown in Figure 4A and B. The RBE for all these bioeffects, summarized in Table 2, appeared to increase with an increase of LET between 90 and 190 keV/l~m, which is in agreement with re- suits from maize study. Different values, however, were obtained for various endpoints used.

Effects of Heavy Ions in Plants (10)367

RBE for micronudei formation for argon and iron ions, for example, were higher than those for growth inhibition. This difference may be due to the fact that heavy charged particles caused more damages in chromosomes which resulted in the formation of more micronuclei and that one micronuleus was lethal to plant cells. Figure 5 shows the frequency of chromosome aber- rations in meiotic pollen mother cells induced by irradiating seeds with 570 MeV/u argon ions or gamma rays, which are higher than that observed in root tip cells. For argon ions, the RBE for chromosome aberration from meiotic mother cells, however, was about the same as that for plant fertility (Table 2). There may be a dose relationship between the damage of heavy ions to pollen mother cells in meiotic stage and the seed fertility of M 1 plants.

lO0 90 80

"70

60

20

, , A s o c = ~ . ~

o 4 o k (sTo Mov/u)

I i r /~ 100 200 3O0 0

ooeE (ev)

3.0

| 2.0

i 1.0

(A)

100 200 ]OO ~O0 ~ D / t

I 1,o

e~n

Figure 3. Dose response curves for M 1 plant fertility after gamma rays, argon, or iron particle irradiation of rice seeds.

Figure 4. Induction of micronuclei formation (A) and chromosome aberration (B) in root tip cells of irradiated rice seeds.

TABLE 2 RBE of Various Bioeffects of Rice Seeds Exposed to Three Types of Heavy Ions

Radiation

60Co

40At

60 Fe

RBE

Micro- Chromosome Semi- nuclei ~ dwarf

LET Growth Forma- Root Tip Pollen Muta- keV/um ~ Ferti l i lv tion Cel Mother Cell tion

027 1.0 1.0 1.0 1.0 1.0 1.0

90 2.0 22 4.3 3.3 2.3 2.3

117 - - - - 3 . 8 8 - - - - - -

190 6.5 7.44 113 42 -- 8.4

*RBEs were determined at 50% growth inhibition or 50% fertility leverls. D50.G.I. and D50.F of

gamma rays were 290 Gy and 255 Gy respectively.

Mutation Induced in the Second Generation of Rice

In order to compare the effectiveness of heavy ions with gamma rays in inducing mutation, the frequencies of semidwarf mutation were estimated and plotted as a function of dose (Figure 6).

(10)368 M. Meiet al.

All curves could be fitted with a linear relationship for the doses used in this study. The iron beam was the most effective in inducing this type of mutation. It was, however, also effective in producing severe inhibition of plant development. As a result, a much lower number of plants could be screened in the second generation. For this reason, more efforts were made to deter- mine the spectrum of mutation from the seeds irradiated with 400 MeV/u argon ions/15/. Changes in panicle length, effective panicle per plant, spikelet per panicle, grain per panicle, grain weight, grain shape, sheath color, plant height, and duration of growth stage were exam- ined; and, mutants with characters of semidwart, earlier maturity, and other alterations were se- lected in the second generation plants. Mutation frequency, shown in Table 3A and B, in- creased with doses in the lower dose region, around 90 Gy or lower, but decreased at higher doses, e.g., higher than 120 Gyo There thus appeared to be an optimal range of doses for some types of mutation. More recently, several breeding materials selected from mutants in- duced by 400 MeV/u argon ions at dose of 100 Gy have been obtained in our laboratory. These materials, namely Xiang-Ar-series, not only retain the pleasant aroma as original variety (Xiangzhan) but also show desirable plant height, maturity and disease resistance. Semidwarf, eady maturity, resistant to bacterial leaf blight, and high yielding ability were obtained 116/. This result demonstrats the potential use of heavy ions in crop improvement.

0 ~,~" i ~ M~.,~,)

~ 5 ° C

3.0

2.0

1.0

0 ~*, ar~ u=.u

I 5°©

1 3°0

2°0 ~

1°

i i i i 100 2o0 300 t~oo 500 100 200 3(30 400 500

oose (ey) DOSE (Oy)

Figure 5. Chromosome aberration induced by exposueing rice seeds to argon ions or gamma rays in meiotic pollen mother cells.

Figure 6. Dose responses of semidwarf mutat- ion frequency in M 2 generation of rice seeds exposed to gamma rays or heavy ions.

Molecular Alteration of Semidwarf Mutants Induced by Aroon Ions

Because no specific genes or DNA fragments goveming character of dwarf in rice have been cloned, random genomic clones, which had been assigned to respective chromosomes/10/, were chosen as probes to detect alteration of restriction pattern in semidwarf mutants, Ar-10 and Xiang-Ar-1. Over 100 genomic clones were tested, and identical patterns were observed for DNA from Ar-10 and Bianpizhan, or Xiang-Ar-1 and Xiangzhan for most probes. Only a few clones, such as RG620, mapped on chromosome 4, exhibited very different patterns between Ar-10 and Bianpizhan, as shown in Figure 7. The polymorphism of restriction fragment pattern between each pair of mutant and its original was found on more than one lane for each probe used, i.e., two or more restriction enzymes could generate RFLP with a given probe. More than one restriction sites, thus, were affected. Previous analysis of mutants indicated that only large changes of DNA, e.g., deletion or insertion, could generate this type of RFLP/10/. Therefore, mutant Ar-10 most likely have undergone a large genetic change, rather than base substitution or point mutation. This observation is in consistent with those previously reported for mutation induced by ionizing radiation in human and hamster cells 117, 18, 19/.

Effects of Heavy Ions in Plants (l 0)369

TABLE 3A. Mutation Spectra and Frequency in M 2 Generation from Irradiated Rice Seeds.

Freouen~ P/=) Dose Nurnberof Panicle Effective Panicle Sp~elet/ Grain/ 1000 Grain

_ Plar Baz a_ Total"

30 150 0.00 0.00 0.00 0.63 2.00 2.00 60 150 0.00 2.67 0.00 0.00 2.00 4.00 90 150 0.67 3.33 2.67 4.67 4.00 12.00

120 150 0.00 2.67 0.00 0.00 0.67 3.33 150 42 0.00 0.00 0.00 0.00 0.00 0.00

*Total Mutation Frequency - (No. Plants with Mutated Character(s))/Total No. Plants Examined x 100

TABE 3B. Mutation Spectra and Frequency in M 2 Generation from Irradiated Rice Seeds.

Mutalbn F re0tR¢l (%) D_0.SP, blg._c LP_lams SemUwa Totar

30 3120 1.67 1.15 1.19 1.57 2.02 60 1470 4.16 3.13 2.43 3.12 4.56 90 1390 4.10 5.90 3.47 3.47 6.83

120 630 3.52 4.29 1.77 2.86 4.92 150 140 1.43 0.00 1.43 1.43 1.43

*Total Mutation Frequency - (No. Plants with Mutated Character(s))/Total No. Plants Examined x 100

DISCUSSION

An early study on growth inhibition and formation of visible white-yellow stripes on leaves of maize plants from seeds irradiated by 220 MeV/u oxygen ions or gamma rays showed that the effects on growth and stripe formation on the third leaf increased with dose for beth radiations /11/. The efficiency, however, was different. For oxygen particles, a dose of 8.2 Gy caused a 50% decrease of leaf area, and 3.0 Gy produced stripes in 50°/= of plants. For gamma rays, the doses required to produce these effects were 76 and 55 Gy respectively. These results sug- gested that oxygen ion was about 2.7 times more effective in inducing mutation than in retard- ing plant growth, and only about 1.37 times for gamma rays. Oxygen ions were thus more ef- fective than gamma rays in producing mutation for a given effect on leaf growth.

Experimental data presented here also demonstrated that energetic high-LET heavy ions can be much more effective than photons in causing various biological effects in plant. Table 2 shows the summary of RBEs for all biological effects observed in rice in this study. Because only heavy ions with LETs up to 190 keV/l~m were used, this table shows the RBE-LET relation- ship in the range of 0.27-190 keV/l~m. Our studies show that Iron beam with LET about 190 keV/l~m was the most effective radiation examined. The results reported by Hirono et al with Arabidopsis 111, however, indicated that the RBE of heavy ions reached a maximum at 72-174 keV/gm for growth inhibition, and at 70 keVIgm for somatic mutation. The possible cause for this difference may be related to the track structure of heavy ions, since only low energy particle beams (10 MeV/u) were used in their study.

Several investigators have studied the relationship between RBE and LET for various end- points, e.g., cell inactivation/3, 4/, HGPT mutation/5/, neoplastic transformation/6/, and proline prototrophs induction/20/in mammalian cells, and have reached same conclusion, i.e., the

(10)370 M. Meiet al.

value of RBE increases with LET up to about 100-200 keV/pm. Interestingly, present studies with plants showed similar LET dependence, suggesting that similar molecular damages may be responsible for various biological effects in both systems.

From the dose-response curves for various bioeffects, effective cross sections for seedling growth inhibition, plant fertility reduction, chromosome aberrations, micronuclei formation, and semidwarf mutation induction were calculated and plotted as a function of LET (Figure 9). The calculation of cross section of fertility and growth inhibition was done according to Hironro et al /1/, cross section = 161JDo; and others were determined as 16L ~, (Good head,/21/), where L is

LET in keV/um, D o the dose required to reduce fertility or seedling height to 37% of control in

cGy, and Zthe slope of dose-effect curves. The cross sections rise steeply with LET up to 190 keV/pm for all effects studied. The cross section of iron particles with LET of 190 keV/pm for growth inhibition and fertility reduction is about thousand times as great as those of low-LET gamma rays. Because high-energy heavy ions were used, their long-ranged delta rays may cause an overestimation of target size. This steep increase of cross section with LET, neverth- less, is similar to that reported previously for ArabidoDsis /1/ and mammalian cells/21/. The cross sections of growth inhibition, shown in Figure 9, agree with those reported by Hirono/1/. For

ArabidoDsis the cross section reaches a value of 0.1 pm 2 at LET approximately 200 keV/pm, as

compared to 10 -4 pm 2 for low-LET photons. The cross sections for chromosome aberrations or mutation induction are 10-100 times smaller than that for growth inhibition, suggesting that nuclear area or genes involved in these effects may be limited.

• • 6 • 5 • • HindH

~ D

0 4 i~ 3 a I I

t " D

i 0 I

0

EcoRV

F~ure 7. Southem blot analysis of DNA from semidwarf mutant, Ar-10 (lane 1,3, and 5) and original variety, Blanpizhan (Lane 2, 4, and 6).

/ /

10.3

I 10.4

O

10-5

10-6 Z~F~mty &G~owlh kC~llon C~ze M~X~on eu=,,~, ~%) OI~F. IC.A.

10 1OO 1OOO LET (keV/pM 2)

Figure 8. Effective cross sections as a function of LET for maize mutation (white-yellow stipe formation), growth inhibition, fertility, micronuclei formation (M.F.), chromosome aberration (C.A.), and semidwarf mutation in second generation.

Other investigators have also demonstrated that high-LET heavy ions could effectively induce chromosome deletion and rearrangement and cause large DNA deletion /18, 22, 23/.

Effects of Heavy Ions in Plaats (10)371

Cytogenetlo analysis of radialton-induced TG r mutants of cultured human fibroblasts by Cox and Messon 122/in the 70s led to the speculation that the majority of mutants arised by changes in chromosome structure rather than by true gene mutation. Recently, Thacker/18/ and Kronenberg and Little 119/have examined the characterization of structure alteration at the DNA level in TK or HPRT mutants of cultured mammalian cells induced by densely ionizing radiation with molecular biological technique, and found that high proportion of gene deletions or rear- rangements occured in these mutants. They concluded that the mutation induced by ionizing radiation in these loci likely involves large-scale genetic changes. Our results of RFLP analysis also indicated that semidwarf mutation may be resulted from large genetic damage in DNA struc- ture, e.g., deletion. This type of DNA changes should give stable mutation and should be use- ful for identifying gene(s) essential in controlling important characters in plants.

Experimental results presented in this report showed that high-LET heavy particles can induce mutation more effectively than photons, produce more character changes in a single plant, and suggested that the potential use of heavy ions in crop Improvement is highly promising. The successful selection of Xiang-Ar-series, which are valuable breeding materials, strongly sup- ports this suggestion. Studies on potential effects of heavy ions in plants, however, are only at the begining stage and much more remain to be explored in the near future.

ACKNOWLEDGEMENTS

This project was supported by the National Natural Science Foundation of China. Part of the RFLP analysis was supported by the Rockefeller Foundation. We wish to acknowledge the heavy ion beam time kindly provided by the BEVALAC of Lawrence Berkeley Laboratory and the valuable assistance from Q. Zuo, W. Huang, and Z. Mei in performing these experiments.

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