Published September, 1980
1MutagenicEffects of SodiumAzide in Rice
~’
M. Afsar Awan, C. F. Konzak, J. N. Rutger, and R. A. Nilan
ABSTRACT
Seeds of rice (Oryza sativa L. spp. japonica) cultlvar
’MS’ were presoaked in distilled water and treated for 2
or 3 hours with 0, 0.12, 0.50, 0.75, 1.0, 1.25, 1.50, and
1.75 mMsodium azide solutions prepared in 0.1 M phos-
phate buffer (pH 3). After treatment the seeds were
rinsed for 1 hour in 15 C tap water. Seeds receiving a
2-hour azide treatment were redried in a fume hood at
room temperature for 24, 48, 72, and 96 hours prior to
planting to develop treatment procedures suitable for
transporting dry, treated seeds rather than growing seed-
lings. Although the scope of this study did not permit
field evaluation of the redried treated seeds, it was found
that seeds treated for 2 hours with I mMazide and re-
dried for 9~ hours had more injury than seeds treated
for 3 hours with I mMazide without redrying. Thus,
a 1 mMazide treatment for 2 hours with redrying may be
near the maximum permissible for sufficient field vi-
ability of the M1. Seeds with a 3-hour azide treatment
were planted immediately after rinsing. The seedlings
were transported by air to the California test site and
transplanted for the mutation study.
C~teria used to assess the biological effects of azide
on rice were germination, seedling height, and seed ste-
rility in the Mz generation, and chlorophyll-deficient
seedlings and viable mutations in the Me generation.
In general, an .increase in azide concentration, along
with an increase in the post-treatment redrying period,
resulted in a decrease in M1 germination and seedling
height. Azide treatment also induced sterility, the high.
est concentration producing maximum(62.2%) seed ste-
rility. The same treatment induced chlorophyll mutations
in 98.5% of the M1 panicle progenies and in 14% of the
M~seedlings. The v,ridis type of mutation was most fre-
quent.
The highest frequency of viable mutations score~ in
the adult plant stage was 4.64% on an Mz plant basis.
All azide concentrations were mutagenic. Short-culm
and dwarf mutant types were more frequent than the
early-flowering mutant types in the azide-induced muta-
tion spectrum. Azide should prove useful as a mutagen
for the genetics and practical breeding of rice.
Additional index words: Viable mutations, Chloro-
phyll mutations, Sterility, Seedling growth, Azide, Oryza
sativa.
INDUCTION o[ useful mutations in
sativa L.) has gained wide interest
rice (Oryza
in the last
decades due to an increasing awareness of the poten-
tial of this method for creating genetic variability,
Various attempts have been made to determine the
most effective and efficient mutagens and treatments
l Scientific Paper No. 5288. Cooperative investigations among
College of Agric. Res. Ctr., Pullman, WA,Project No. 1568; SEA,
USDA; and Unix’. of California, Davis. Supported in part by
a Training Fellowship from the Int. Atomic Energy Agency,
Vienna, via the U.S. Natl. Sci. Foundation, Natl. Res. Court,
and U.S. Dep. of Energy Contract No. DE-AMO6-76RLO-2221.
DOE/RL/02221-50. Received 20 Feb. 1979.
~Former I.A.E.A. research fellow, Dep. of Agronomy and Soils
(now senior scientific officer, Nuclear Institute for Agric. and
Biology, Faisalabad, Pakistan); professor of agronomy and ge-
netics, Dep. of Agronomy and Soils and Program in Genetics,
W. ashington State Unix’., Pullman, WA99164; research geneti-
cist, SEA, USDA,Davis, CA 95616; and professor of agronomy
and genetics, Dep. of Agronomy and Soils, and Program in Ge-
netics, Washington State Unix’., Pullman, WA99194.
663
for the induction of desirable traits in a crop cultivar.
As a result, an extensive array of highly effective mu-
tagenic agents are nowavailable.
Azide has been shown to be a potent chemical mu-
tagen in both higher and lower organisms (Sideris and
Nilan, 1970; Konzaket al., 1972; Nilan et al., 1973;
Kleinho[s et al., 1974). The potentially high muta-
genicity of azide was disclosed by Spence (1965) while
studying the repair mechanism of radiation-induced
damage in barley (Hordeum sp.) seeds. He observed
that the azide-treated population used as a control
p.roduced a significanfnumber of chlorophyll-deft-
c~ent mutations. By adjusting the pH of the azide
solutions, as well as modifying the metabolic state of
the treated seeds, Nilan and Sander (1974) induced
mutation frequencies approximately equal to the high-
est frequency obtained by ethyl methanesulphonate.
In barley, azide appears to be the most efficient and
effective of all mutagens tested for the induction of
chlorophyll-deficient, morphological, and certain bio-
chemical mutations (Konzak et al., 1965; Kleinhofs et
al., 1974; Konzak et al., 1975; Niknejad, 1976; Nilan
et al., 1976; Walther, 1976). Nilan et al. (1976)
ported that azide treatment produces some delay in the
germination of treated seeds, and low levels of p.hy-
siological damage as measured by M~ seed germina-
tion, seedling growth and plant survival at azide con-
centrations producing sufficient progeny for mutation
studies.
In recent studies, Owais et al., (1978) have demon-
strated a highly mutagenic substance present in azide-
treated barley embryos which is not azide. The active
substance is believed to be a metabolic derivative
of azide.
Very little information is yet available about azide
mutagenicity in rice. Mustafa (1976) conducted pre-
liminary studies on the mutagenic and other effects
of azide in rice and observed a high frequency of
chlorophyll-deficient mutations in Mz. The research
described here indicates that azide is a potent mutagen
for inducing chlorophyll-deficient, as well as viable
physiological and morphological, mutations in rice.
MATERIALS AND METHODS
Seeds of the japonica rice cultivar "M5’ were used in this study.
M5is a tall (120 cm) cultivar that is widely grown in California
(Carnahan et al., 1975).
About 1,000 uniform seeds per treatment were presoaked in
flowing tap water for 20 hours at 25 C, then exposed to sodium
azide solutions. The azide treatment solutions were prepared
in a fume hood to exhaust any evolved hydrazoic acid. These
solutions were made by adding aliquots of a freshly prepared
1 M aqueous stock solution of practical grade (commercial)
sodium azide salt to 0.1 Mpotassium phosphate buffer previously
adjusted to pH 3.0 with concentrated phosphoric acid. The
following combinations of treatment periods and sodium azide
concentrations were used:
a) 3-hour treatments with buffered sodium azide solutions at
0, 0.12, 0.25, 0.50, 0.75, 1.0, 1.25, 1.50, or 1.75 mMconcen-
trations.
b) 2-hour treatments with buffered sodium azide solutions at
0, 0.12, 0.25, 0.50, 0.75, or 1.0 mMconcentrations.
664 CROP SCIENCE, VOL. 20, SEPTEMBER-OCTOBER 1980

Table 1. Relative mean germination values and their standard deviations for the Mi rice seeds treated with sodium azide.

Azide concentrations (raM)

Treatment Redrying
time- period 0.0 0.12 0.25 0.50 0,75 1.0 1.25 1.50 1.75
hours hours
3 0 100±1.1 99.1±1.2 97.8±1.3 96.4~1.5 92.7±1.6 90.0±1.8 88.6~1.0 86.5±0.8 84.4±0.9
2 24 100±1.1 96.5±1.2 92.5±0.7 81.5±1.7 75.0±1.7 72.9~2.5
2 48 100±1.2 94.9±0.9 89.4±1.6 76.3±1.6 72.7±1.6 68.9~1.0
2 72 100±0.9 92.8±1.4 89.3±1.3 77.1±2.1 73.1±1.7 69.0~1.4
2 96 100±1.3 91.9±1.4 87.9±1.4 75.0±1.8 72.8±1.6 70.2~0.9

Table 2, Average height Imm)of 6-day old M5seedlings from seed treated 2 hours with sodium azide and redried.
Azide concentrations (mM)
Treatment Pc~lrying
time period 0.0 0.12 0.25 0.50 0.75 1.0
~urs
2 24 49.2±0.9 46.7~2.1 42.6±1.4 41.8±0.8 36.3~1.2 35.2~1.8
2 48 46.6~1.1 42.9~1.2 42.8±2.1 39.8±0.8 38.6±1.9 34.4±1.8
2 72 44.3±2.0 41.8~1.4 39.4±1.4 36.1±1.8 35.4~0.8 30.5±0.8
2 96 39.7±0.4 37.5±1.0 35.5±1.5 31.8±1.3 30.6±0.4 21.2±0.7

Table 3, Panicle stel~ility of MI rice plants following a 3-hour Table 4. Frequencies of chlorophyll mutations in Mz progenies
azide treatment. following 3-hour azide treatments of rice cultivar M5.
Azide concentration Sterility Based on M 1 pan/des Basedon Mz seedlings
Azide
mM % concen- No. No. No.. No.
tration analyzed mutated Percent analyzed mutated Percent
0.00 17.0
0.12 21.2 rnM --no,-- % no. %
0.25 27.0
0.50 30.5 0.00 164 0 0 15,549 0 0
0.75 no data 0.12 134 43 30.1 5,405 113 2.1
1..00 35.6 0.25 136 59 43.4 4,192 163 3.9
1.25 41.6 0.50 274 187 68.3 18,869 777 4.1
1.50 49.3 0.75 152 118 77.6 15,537 688 4.4
1.75 62.2 1.00 144 112 77.8 17,172 902 5.3
1.25 210 165 78.6 22,690 1,730 7.6
L.S.D. 0.05 = 7,05 1.50 152 124 81.5 17,068 1,983 11.6
1.75 70 69 98.5 10,003 1,403 14.1
Presoaked seeds were placed in 250 ml Erlenmeyer flasks and
covered with 200 ml of treatment solution. The flasks were 30 C. Chlorophyll-deficient mutants were scored when the seed-
maintained at 25 C in a water bath. During all presoaks and lings were 21 days old. The mutants were classified into eight
azide treatments the seeds were slowly and continuously bub- classes: I. albino (white); II. xantha (yellow); III. viridis
bled with air, After treatment, the seeds were washed in flow- (light green); IV. alboviridis (upper portion white, lower por-
ing tap water (ca 15 C) for 1 hour. tion green); V. viridoalbina (upper portion green, lower portion
Fifty seeds per treatment from each of the 3-hour treatments white); VI. xanthaviridis (upper portion yellow, lower portion
were planted in the laboratory to determine the per cent ger- green); VII. viridoxantha (upper portion green, lower por-
mination and seedling l~:eight according to methods described by tion yellow); and VIII. striata (chlorophyll deficiency in stripes).
Myhi11 and Konzak (1967). The remaining seeds were sown For the analysis of viable physiological and morphological
plastic pots in the greenhouse to produce transplantable seed- mutants, a minimum of 15 seeds per M~ plant per azide treat-
lings for the field.experiment. ment was collected from the panicles. These seeds were planted
Seeds from the 2-hour treatments were redried for 24, 48, 72, in plant rows (7.5 × 15 cm apart) at the Rice Research Facil-
and 96 hours at room temperature (ca 23 C) in a fume hood, ity, Univ. of California, Davis, during June, 1977. At maturity,
then planted in the lal:~oratory to determine only the per cent the Ma plants were classified for several qualitative, viable mu-
germination and seedling height. This was done to establish tants. The mutant classes were: I. early (flowering at least
a basis for estimating whether azide-treated seeds would retain 1 week earlier than the control); II. late (flowering 1 week
sufficient viability after redrying for convenience in handling more later than the control); III. short culm (15 to ~0%shorter
when transporting to another location. than the control); and IV. dwarf (80 to 90 cm). All variants
Seedling height was determined as the mean height of 6- were termed viable mutants, although we have not yet confirmed
day-old seedlings. Increase in seedling height during each 24- their breeding behavior. Short-culm and dwarf mutants were
hour period also was recorded up to 6 days after germination. progeny tested in 1978. Because of extreme phenotypic vari-
ability in the experimental field in 1978, only the true-breeding
Thirty-day-old seedlings from the 3-hourtreatment were trans- short or dwarf lines (both called short in 1978) were recorded.
planted to the field, in rows 7.5 × 15 cm apart, in a randomized
complete block experiment with 3 replications at Davis, Cali- Relative mean values were determined by the formula treat-
fornia on 8 June 1976. ment mean/mean of control × 100, with standard errors calcu-
lated according to Steel and Torrie (1960).
At maturity, three panicles per Mx plant per treatment were
harvested and stored separately in envelopes. Induced sterility
was determined as the l:,roportion of undeveloped seeds on the RESULTS
panicles from Mx plants relative to those from plants of the
untreated control. The percent induced sterility was analysed M~ Germination
by analysis of variance.
The Mt germination percentage was reduced by ~-
Chlorophyll mutation analyses were carried out in an air-sup-
ported plastic greenhouse. Unthreshed panicles from individual hour treatments with increasing concentration of azide
Ma plants were sown in a sand bed at a temperature of 25 to (Table 1). Redrying of 2-hour azide-treated seeds for
 AWAN ET AL.: SODIUM AZIDE EFFECTS IN RICE 665
Table 5. Spectrum of M, chlorophyll mutations from &hourazide-treated rice seeds of cultivar M5.
Total no. of
Xantha- Virido-
Azide Albo~ Virido- mutant
Xantha xantha
concentration Albina Viridis viridis viridis albina Striata seedlings
mM no. % no. % no. % no. % no. % no. % no. % no. %
0.00 .............. 0
0.12 25 22.1 48 42,5 40 35.4 ........ 113
0,25 43 26.4 68 41,7 52 31.9 ........ 163
0.50 247 31.8 317 40.8 213 27.4 .......... 777
0.75 239 34.8 203 29.5 246 35.8 .......... 688
1.00 291 32.3 351 38.9 108 12.0 26 2.9 9 1.6 83 9.2 34 3.8 - - 902
1.25 348 20.1 157 8.5 509 29.4 440 25.4 19 1.1 208 12.0 - 49 2.8 1,730
1.50 387 19.5 190 9.6 483 24.4 829 41.8 3 0.2 77 3,9 - - 14 0.7 1,983
1.75 361 25.7 161 11.5 542 38.6 317 22.6 .... 22 1.6 1,403

_.9O
~
100 100
.-~90
~ ~ 80
o
.o---
o 70

~oncentrauon mM v 60
0.12
" 50 /4odium azide
a: 40
0.25
0.50
0.75
.~50
.-q...-- / ConcentrationrnM

1.00 ~ ~Y e--0.25
-- 30 1.25 ~ 30 / ...-’" 0--0.50
.-.o-" ~--0.75
1.50 ..... -o-- .... o--1.00
® 20 1.75
1
10
I I I I I
I I I I
2 3 ~ 5 6
3 4 5 6 Daysof Growth
Days of Growth
Fig. 2. Seedling height of M5treated 2 hours with sodium aflde
Fig. 1. Seedling height of M5treated 3 hours with sodium az~de. followed by 96 hours of redrying.

24 to 96 hours resulted in further decreases in germina- MtPanicle Sterility

tion in somecases.
Seedling Height Reduction
The average seedling height decreased with increas-
ing mutagen concentrations in both the 3-hour and
2-hour azide treatments (Table 2). Meanvalues
seedling height for the g-hour azide treatments (Fig.
1) showthat all except the two lowest azide concen-
trations had a profoundinhibitory effect on the growth
of I-day-old seedlings. However,except at the highest
concentration, the differences in seedling growth
amongthe azide-treatment concentrations narrowed
between 3 and 6 days, indicating the temporal and
largely physiological nature of the growthinhibition.
Followingthe 2-hour azide ffeatment, redrying periods
of 24 to 96 hours decreased seedling growth (Table
2). Maximum growth depression occurred for the 96-
hour redrying period. The seedling height showed a
largely linear relationship with azide concentrations
at or below0.75 mM(Fig. 2). The curvilinear growth
response of the 1.0 mMtreatment, however, demon-
strates the strong, early inhibitory effects followedby
recovery from the azide-induced injury.
A markedincrease in the M1panicle sterility oc-
curred with increasing azide concentrations (Table
3). MIpanicles of the untreated control had only 17%
infertile florets but, at the highest azide concentration
(1.75 raM), 62%of the M~panicle florets were sterile
comparedto 21’%sterility in the lowest 3-hour azide
concentration (0.12 raM).
Chlorophyll Mutations
Three-hourtreatments with all azide concentrations
used in this study proved effective for inducing
chlorophyll mutations (Table 4). The frequency
chlorophyll mutations was calculated on an M1pani-
cle basis as well as on an M2seedling basis. Usingboth
criteria, the highest M~mutation frequency based on
M, panicles and M2seedlings (98.5 and 14.1%, re-
spectively), was induced by the highest concentration
of azide. However,each increase in the azide treat-
ment concentration caused an increase in mutation
frequency.
The spectrum of induced cholorophyll mutations
from the 3-hour azide treatment (Table 5) indicates
that, with the lower azide concentrations (0.12 to 0.75
666 CROP SCIENCE,
Table 6. Frequency and spectrum of viable mutations in the Mz generation 3 azide-treated
breeding short mutants in the M~generation.
Azide No. Total
concentration analyzed frequency
m~ no. % no.
0.00 2,360 0 0
0.12 618 1.94
0.25 623 2.08
0.50 4,250 1.29
0.75 1,216 2.30
1.00 L340 2.39
1.25 2~268 4.10
1.50 2,039 4.27
1.75 925 4.64
Includes both "short" and"dwarf" from the Msgeneration.
raM), there was a relatively narrow mutation spectrum,
comprised of albina, xantha, and viridis only. Xantha
mutants occurred in a relatively high frequency ex-
cept in the 0.75 n~Mazide treatment, in which the
xantha frequency was lowest. Higher concentrations
of azide (1.0 to 1.75 raM) induced a comparatively
wider spectrum of chlorophyll mutations.
Amongthe total of 7,759 mutant seedlings observed,
there were 28%viridis, 25%albina, and 21%xantha-
viridis.
V:iable Mutations
The viable mutation frequencies (Table 6), cal-
culated on an Msplant basis, show that all concentra-
tions of azide used in this study induced viable muta-
tions. The highest mutation frequency (4.64%) was
observed in progenies from the highest treatment con-
centration (1.75 raM) while the lowest mutation fre-
quency of 1.29% wa~ induced by the 0.50 mMconcen-
tration. No mutants were found in the control popu-
lations. It was observed that, above 0,50 raM, the
mutation frequency increased with increasing azide
concentration. In all the treatments, this trend was
similar to that for chlorophyll mutation frequencies,
although the latter were higher compared to the
readily observable viable mutation frequencies.
The spectrum of vilable mutations (Table 6) reveals
that short-culm mutants were the most frequent in
most of the treatments. The next most frequent were
dwarf, late- and early-flowering variants. The ma-
jority of dwarf mutants were of late-flowering habit.
Amongthe 354 mutant plants, 49.2% were short-culm,
28.5% dwarf, 9.6% early- and 12.7% late-flowering
types. Progeny tests of the combined short-culm and
dwarf M2plants revealed that 21 of these mutants were
true breeding for reduced height (Table 6). Additional
M~selections appeared to be segregating for reduced
height in the M~, suggesting the induction of dominant
mutations, but this cannot be confirmed until addi-
tional progeny tests are conducted.
DISCUSSION
Various physical and chemical mutagens have
been known to affect the germinability of seed. Re-
suits of this study indicate that the range of azide
concentrations and redrying periods used in this in-
VOL. 20, SEPTEMBER-OCTOBER 1980
rice seeds, from hour and numberof true-
M~plants
Mstrue-breeding
Short Early Late short mutant
culm Dwarf flowering flowering lines~
0 0 0 0
7 2 3 3
6 1 2 4 5
21 12 10 12 5
18 4 2 4 2
6 4 4 10 1
45 35 6 7 1
55 23 6 3 4
16 22 2 2 0
vestigation strongly affected seed germination. Nik-
nejad (1976) reported a reduction in germination
barley seeds treated with azide and stored for different
durations. Walther (1976) obtained similar results.
The magnitude of germination reduction with azide
treatments in this study is in agreement with results
of other investigators (Levy and Ashri, 1973; Gichner
et al., 1975). However, the recent finding of a mu-
tagenic azide derivative in barley embryos suggests
that the observed germination inhibition may have a
more complex basis (Owais et al., 1978).
Ma seedling growth is widely used as an index in
determining the biological effects of various physical
and chemical mutagens (Konzak et al., 1972). Muta-
gens differ in their mechanism and mode of action in
the biological system. Hence, the extent of reduction
in growth is related to the mechanism of action for a
given mutagen. As a respiratory inhibitor, azide may
inhibit an energy supply system (Yonetani and Ray,
1965; Vigers and Ziegler, 1968), resulting in the inhibi-
tion of mitosis which can be associated with seedling
growth depression. Physiological conditions of the
seed at the treatment time greatly influence the mag-
nitude of growth depression (Nilan et al., 1976; Klein-
hofs et al., 1978).
In the present study, reduction in seedling height
occurred with each corresponding increase in azide
concentration. Seedling growth retardation was more
pronounced in the 2-hour azide and redrying treatment
than compared in the 3-hour treatment without re-
drying. Preliminary studies had shown that the 2-
hour treatment without redrying had little measur-
able effect on 6-day seedling growth. Results from
this study indicate that the 2-hour 1 mMazide treat-
ment with redrying may be the most severe treatment
applicable to rice seeds intended for dry transport to
the field in order to obtain M1plants.
The relatively slow growth of the first leaf to the
sixth day after azide treatment might be considered
to result from mitotic delay due to the inhibition of
physiological processes. Walther (1976) observed
similar reduction in the growth of the first leaf in
azide-treated barley after 15 days. However, in this
study the direct relationship between seedling growth
reduction, and azide treatments combined with realty-
ing suggests that azide is biologically effective in rice.
These findings also confirm the earlier results of Si-
deris et al. (1969) and Niknejad (1976).
 AWAN ET AL.: SODIUM AZIDE EFFECTS IN RICE 667

Panicle fertility may be influenced by a number of ful types of mutants such as short-culm, dwarf, and
environmental factors such as temperature, humidity, early-flowering were induced in rice by azide treat-
and wind velocity during flowering, and certain of ment. The frequencies of viable mutations on an M2
these factors may cause reduced panicle fertility as plant basis were lower than the frequencies of chloro-
shown by the controls. However, mutagens generally phyll mutations. This is probably due in part to the
reduce the reproductive capacity of the plants and in- fact that only distinct viable mutations were scored,
crease the number of sterile florets much more than but it is likely also that more genes are involved in
the environmental effects. In this study, induced floret chlorophyll synthesis than in the types of morphologi-
sterility increased with increasing concentrations of cal variants scored. In practice, it is likely that, ex-
azide. This result confirms earlier results of Mustafa cept for easily detectable mutations, many morphologi-
(1976), who found that azide treatments induced high cal and physiological alterations are left undetected.
levels of sterility in Mt rice plants. The induction of Hence, it is difficult to make a strict comparison of
MX sterility in barley by azide has been reported by the frequencies of viable vs. chlorophyll mutations.
Kleinhofs et al., (1974); Konzak et al. (1975); and The occurrence of short-culm, dwarf and early mu-
Niknejad (1976). Since azide does not induce chromo- tations at a relatively high frequency in the mutation
somal abberations in barley (Niknejad, 1976; Walther, spectrum suggests that azide mutagenicity can be ex-
1976), its effect on sterility has been attributed to gene ploited toward the development of short-strawed, non-
mutations expressed as gametic or zygotic lethals (Mi- lodging, and earlier cultivars of rice.
lan et al., 1976).
The induction of 62.2% sterility with the 1.75 mM ACKNOWLEDGMENTS
azide treatment compared to 17% sterility in the un- The advice and counsel of Dr. R. F. Line during the course
treated control indicates the high mutagenic potency of this study, the support of the Pakistan Atomic Energy Com-
mission, and the encouragement and support of the Directors
of azide in rice. of the Nuclear Institute for Agriculture and Biology, Faisalabad,
The induction of chlorophyll-deficient mutations Pakistan, are gratefully acknowledged.
is directly related to the mutagenic efficiency of a mu-
tagen (Konzak et al., 1965). The most effective azide
treatment resulted in chlorophyll mutations in a very
high frequency (98.5%) of the M! panicle progenies.
The maximum frequency of mutations on an M2 seed-
ling basis was about 14%. Nilan et al. (1976) have
shown that azide treatment induced a maximum fre-
quency of 64% chlorophyll mutations on an MI spike
basis in barley seeds presoaked for 16 hours.
The spectrum of chlorophyll mutations determined
from 7,759 mutant seedlings revealed that viridis types
were predominant, followed by albina, xanthaviridis,
xantha, viridoalbina, striata, viridoxantha, and albo-
viridis. Nilan et al. (1976) reported a similar spectrum
of azide-induced mutations in barley. Mustafa (1976)
observed much lower frequencies of tigrina (horizon-
tally banded) and xantha mutations as compared to
striata in M2 rice seedings of the cultivar 'Dawn.'
The high frequency of mutations observed in this
study with 20 hours of presoaking suggests that muta-
tion frequencies also in rice are enhanced by treat-
ments of pregerminated seeds. Possibly this could be
due to the optimum production and activity of the
azide metabolite at some stage in the germinating em-
bryo. This optimal activity may occur during the pe-
riod of maximum inhibition of seedling growth fol-
lowing the treatment.
Variable frequencies of azide-induced chlorophyll-
deficient mutations have been reported with different
treatment conditions (Sideris and Nilan, 1970; Klein-
hofs et al., 1974; Gichner et al., 1975). It can be in-
ferred from these findings that lower concentrations
of azide may be effective for inducing mutations if the
appropriate pregermination treatments are employed.
Induced viable mutations may provide a valuable
source of genetic variability to plant breeders. Sev-
eral physical and chemical mutagens have been used
to induce desirable agronomic traits in crop plants.
Azide has been reported to be the most effective and
efficient of all mutagens for barley (Nilan et al., 1977;
Niknejad, 1976). In the present study, potentially use-
668 CROP SCIENCE, VOL. 20, SEPTEMBER-OCTOBER 1980