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Chemical mutagenesis in Triticum aestivum
387
MUTATION RESEARCH
CHEMICAL MUTAGENESIS IN T R I T I C U M
AESTIVUM*
H. K. S H A M A R A O * * AND E. R. S E A R S * * *
Field Crops Department, University of Missouri, Columbia, Mo. (U.S.A.) (Received A u g u s t 7th, J964)
SUMMARY
Treatment of common wheat with ethyl methanesulfonate had effects strikingly different from those characteristically obtained in irradiation experiments. Of 558 M x plants, I75 had recognizable mutant sectors, almost all of which were necrotic or chlorophyll-defective stripes in leaves. Such sectors are rarely seen following irradiation. Chromosome aberrations were relatively infrequent. In M~, one family segregated for a viable, simply recessive chlorina mutation, and several segregated lethal lutescent mutants. There were numerous other lethals, including three dwarfs with short, thick leaves. In addition to awn and spike mutants attributable to deficiency or duplication for known genes, there were a compactum-like dominant mutation, a mutant with one or a few central spikelets missing from each spike, and a type with the entire spike aborted except for one to three sterile spikelets. The production of distinctive mutations in common wheat by ethyl methanesulfonate is believed to involve changes in function of genes which are duplicated on other chromosomes of this hexaploid species. The change in function releases the genes from the masking action of their duplicates, while maintenance of the original function by the duplicates keeps the mutations from being automatic lethals.
INTRODUCTION
The effectiveness of numerous chemicals in inducing mutation in both animals and plants is now well established (see review by SMITH24). Many of these chemicals are alkylating agents--that is, organic reagents capable of transferring their alkyl groups to other compounds. One of the most promising of these is ethyl methanesulfonate (EMS), discovered by Kolmark (cited by WESTERCAARD'~°)to be mutagenic in Neurospora and subsequently shown to be mutagenic in bacteriophage 'L bacterialS,~% Drosophila 3, 4, Habrobracon 13, barley 2, 5,9,1l, Arabidopsis 16,19, maize 1", tetraploid wheat', and hexaploid wheat 10, 17, ~.~,27 Abbreviation: EMS, ethyl methanesulfonate. * J o u r n a l Series No. 2773 of the Missouri Agricultural E x p e r i m e n t Station. A p p r o v e d by the Director. * * N o w Scientific Officer, Biology Division, Atomic E n e r g y E s t a b l i s h m e n t , T r o m b a y , Byculla, t3ombay-8, lndia. *** Geneticist, Crops Research Division, Agricultural Research Service, U.S. D e p a r t m e n t of Agriculture. Address: Curtis Hall, University of Missouri, Columbia, Mo.
MutMion Research x (i964) 387 399
388
H. K. SHAMA RAO AND E. R. SEARS
KONZAK el al. n have e m p h a s i z e d t h a t while some chemical m u t a g e n s are fully, radiomimetic, giving essentially the same p a t t e r n of m u t a t i o n s a n d gross c h r o m o s o m a l aberl ations as the ionizing radiations, EMS produces more m u t a t i o n s a n d m a n y fewer chromosomal aberrations. There is a m p l e theoretical s u p p o r t for this tinding in the conclusion of HAWLEY AND BROOKES 12 t h a t EMS reacts with D N A to a l k y l a t e the guanine c o m p o n e n t and cause it to pair with t h y m i n e i n s t e a d of cytosine during D N A duplication. This is said to result in the s u b s t i t u t i o n of an adenine t h y m i n e pair for guanine -cytosine. The a b i l i t y of a m u t a g e n to induce true gene changes r a t h e r t h a n mere deficiencies and d u p l i c a t i o n s is difficult to establish in a higher plant, the only valid criterion n o r m a l l y being the occurrence of b a c k - m u t a t i o n from the induced m u t a n t . In common wheat, however, m u t a t i o n s t h a t are not the result of simple loss or duplication of p a r t s of chromosomes (:an be identified with a reasonable degree of c e r t a i n t y , because the nullisomics and t e t r a s o m i c s for all 21 chromosomes c o n s t i t u t e all the possible deficiencies a n d duplications. H e x a p l o i d w h e a t varieties have the a b i l i t y to tolerate large deficiencies and duplications; in fact, p l a n t s deficient for an entire chromosome are essentially normal, being in most cases difficult to distinguish from t h e i r n o r m a l sibs. Therefore, in con> parison with diploids, r e l a t i v e l y few cells which c a r r y gross a b e r r a t i o n s are e l i m i n a t e d , a n d the a b e r r a t i o n s recovered t e n d to be a true sample of those t)roduced b y the treatment. Monosomics were used in the present e x p e r i m e n t , with the idea t h a t recessive m u t a t i o n s p r o d u c e d on the chromosomes concerned might lead to d e t e c t a b l e m u t a n t sectors in the M1 plants. Offspring from such sectors would include disomics, which would necessarily be h o m o z y g o u s for the m u t a t i o n concerned; a n d in each case it would be k n o w n on which c h r o m o s o m e the m u t a t i o n was located. The monosomics used here were eB (formerly II) a n d 6B (X), each involving a chromosome responsible for the suppression of several abnormalities'a°. Deficiency for the suppressing genes should give rise to m u t a n t s d i s p l a y i n g these abnormalities. B y i r r a d i a t i n g w h e a t m o n o s o m i c s , TSUNEWAKI AND H E Y N E 2~, 2,a were able to d e m o n s t r a t e the presence on these a n d o t h e r chromosomes of d o m i n a n t genes for v i a b i l i t y a n d g r o w t h c h a r a c t e r s as well as for morphological characters. It m i g h t be e x p e c t e d t h a t all recessive m u t a t i o n s in the monosome would show u p in M~, for the d o m i n a n t allele would be missing. However, the critical factor in expression of the m u t a n t m a y be the presence of 2 doses of the recessive, not the absence of the d o m i n a n t . In o t h e r words, the d o m i n a n t allele m a y have little or no suppressing effect on the recessive, and the h e m i z y g o t e m a y be little if a n y different from the heterozygote. This t y p e of gene change is exemplified b y N e a t b y ' s virescent"L a recessive m u t a n t which is not expressed when hemizygous. A p r e l i m i n a r y r e p o r t "a dealt v e r y briefly with M, d a t a a n d o b s e r v a t i o n s on M~ seedlings. MATERIAI.S AND METHODS
Seeds of n o r m a l Chinese spring wheat and of monosomics 2B a n d 6B of this v a r i e t y were first b r o u g h t to a stable moisture c o n t e n t of 12% with 1.5o d e n s i t y sulphuric acid in a dessicator k e p t at 2o °. The chemical t r e a t m e n t s consisted of soaking seeds in EMS solution at 3 o°. I m m e -
Mulation Research I 1I()()4) 387-:;90
CHEMICAL MUTAGENESIS
TABLE
IN
389
T. aeslivum
I
SURVIVAL AND S E E D L I N G HEIGHT AFTER E M S AND X - R A Y TREATMENT IN PILOT EXPERIMENT
3laterial treated
K i n d of treatment
Number of seeds
A t z-leaf stage (iermiMean nation keigkl
.~lt 3-leaf stage (;ermiMean nation keight
Survival after 3 ° days
(%)
(cm)
(°o)
(cm)
o,,)
Mono-2B
Control 0.05 M EMS, 6 h o.o 7 M EMS, 6 h X - r a y s , 24 k R X - r a y s , 48 k R
IO 20 2o 2o 20
loo loo o IOO lOO
7.6 1. 9 -2.2 t. 1
too 1oo IO IOO too
36.8 19. 5 o.I ~ 3.3 8. 4
90 95 o 95 (~5
Mono-6B
Control 0.05 M EMS, 6 h 0.07 M EMS, 6 h X - r a y s , 24 k R X - r a y s , 48 k R
to 20 20 20 20
1oo ioo o 95 5°
6.8 I. 4
IOO ioo 3° IOO 80
36.8 15. 3 2.5 4.7 (>. l
95 9o o 8o o
0.9 0.2
d i a t e l y after t r e a t m e n t , the seeds were t h o r o u g h l y washed with distilled w a t e r at room t e m p e r a t u r e . X - r a y s were a d m i n i s t e r e d at an i n t e n s i t y of 72o R / m i n at 15o kV, 9 mA, w i t h o u t filter. To o b t a i n meiotic p r e p a r a t i o n s , i n d i v i d u a l a n t h e r s fixed for at least two d a y s in 1:3 acetic alcohol were m o r d a n t e d for 3o min in 2°/'0 ferric a c e t a t e a n d soaked overnight in I°,'o a c e t o - c a r m i n e before squashing. The slides were m a d e p e r m a n e n t b y a liquid CO2 technique. PILOT EXPERIMENT
In order to d e t e r m i n e a dose r a t e for X - r a y s a n d EMS which would p r o J u c e a large percentage of defective seedlings at a high s u r v i v a l rate, a pilot e x p e r i m e n t was cond u c t e d which i n v o l v e d 15 different EMS t r e a t m e n t s a n d 2 X - r a y dosages. EMS conc e n t r a t i o n s of o.o5, o.o 7, a n d o.xo M were used for 3, 6, 12, 24, a n d 48 h, and X - r a y t r e a t m e n t s of 24ooo a n d 48ooo R were given. T w e n t y seeds of each monosomic were included in each t r e a t m e n t , a n d IO in each control. The EMS t r e a t m e n t s of 3 h d u r a t i o n caused no m a r k e d change in seedling height or in percentage of g e r m i n a t i o n a n d survival. T r e a t m e n t s for 12 h or longer c o m p l e t e l y i n h i b i t e d g e r m i n a t i o n , as did t h e 6-tl t r e a t m e n t with o . I o M solution. Of the 2 remaining t r e a t m e n t s , b o t h of 6 h (Table I), the one a t o.o 7 p e r m i t t e d some g e r m i n a t i o n b u t allowed no seedlings to survive. The o.o5 M, 6-h t r e a t m e n t , on the other hand, caused a fairly drastic a n d uniform r e d u c t i o n in seedling height, i n d i c a t i n g effectiveness of the t r e a t m e n t , b u t p e r m i t t e d s u r v i v a l n e a r l y equal to t h a t of t h e control. No difference was n o t i c e d in the s e n s i t i v i t y of monosomics 2B a n d 6B to EMS. S e n s i t i v i t y to X - r a y t r e a t m e n t was s t r i k i n g l y g r e a t e r for mono-6B t h a n for mono-2B (Table I). A more drastic reduction in height was noted, s t a r t i n g with the first leaf, a n d s u r v i v a l was less, p a r t i c u l a r l y at 48 k R , where it was zero for 6B a n d 65% for 2B. This g r e a t e r s e n s i t i v i t y of monosomic 6B agrees with the findings of TSUNEWAKI AND HEYNE
2:~.
Mutation Research i ( i 9 6 4 ) 3 8 7 - 3 9 9
"4
"2
Mono-6B
Mono-eB
~oo
5° kR
300
2oo
EMS
3° kR
lo
loo
Control
300
EMS
lo
bet o/ seed~
.Yum-
3° kR
Control
~llaterial Kind uf treated treatment
--
i 1.8
i i 5
Mean heigkt (cm)
AND SFRVIVAL
82, 5
4.0
100,0
92.0
91.o
]-3
90.0
15. 5
68.0
0.0
7.0
8.0
34.3
o.o
1.0
-'3.3
O.O
o.o
J.O
43.7
o.o
11
O. 5
2.7
0.0
o.o
o.o
14.3
o.o
O. 5
,.O
O.O
l .o
o.o
6.3
1o.o
% no/ :e~:mihated
--
35.0
--
--
35.2
Mea~ hdgk! (cm)
AND X-RAYS
88.0
27.3
IOO.O
94.0
92.o
l ~ .o
1 OO.O
,\'O:g~
9.5
03.7
0.0
5.0
8.0
56.3
O.O
• - o
~: - 0 ,o
2,o
0.o
0.0
0.0
o.o
20.0
O.O
)0°o = .
o.o
~.3
o.0
o.o
o.o
7.3
O.O
..... / D
Third-leaf stage (of control) o witk iudicated reduction in keigkl
F O L L O \ V I N G TRI'.'ATMKNTS \VITtt E M S
1;irst-leaf ,;tage ( of conh'ol) % wiih iudica/ed redmTio~t in keigkt ,\'one : 25 % :: 5oo0 7500
IIEIGHT REDI'CTION
TABLE
o
0.5
~.7
o.o
i .o
o.o
5.3
O.O
H(IIzd
93.5
9 7 .0
90.0
95.0
93,o
88.0
IOO.O
(%)
Survival ?o ~wt after ,;'ermi- 3o days
>
> Z
> O
ha
Cla VO ©
aestivum
CHEMICAL MUTAGENESIS IN T. TABLE
391
III
PHENOTYPIC CHANGES OBSERVED IN ~'I 1 GENERATION FOLLO~rING EMS TREATMENT K i n d of change Leaves Albino Yellow
stripe stripe
Pale-green stripes B r o w n stripes Necrotic p a t c h e s Glossy
Notched Punched Spikes Compactoid Subcompactoid Non-squarehead
N u m b e r of p l a n t s affected N u m b e r of affected sectors N u m b e r of p l a n t s studied * l'rogeny about 75°,
Chromosome constitution of affected plants iVIonosomic 6B* Monosomic 2B Disomics
Untreated control
3 46 4 3 36 7 18 13
4 15 I 3 13 7 14 1[
o 4 o [ 8 2 12 o
o o o o o o O o
2 i o
i i 3
o o o
o o o
lO 7 133 286
52 73 18o
16 27 92
o 2o
of selfed m o n o s o m i c 6B. N o t a n a l y z e d cytologically, b u t p r e s u m a b l y comprised of monosomics and 25°o disomics. MAIN EXPERIMENT
On the basis of the results of the pilot experiment, 300 seeds each of monosomics 2B and 6B were subjected to 0.05 M EMS for 6 h at 3 o°. X-ray treatments calculated as 3 o o o o R and 5oooo R (but see below) were given to IOO-seed lots of mono-2B, and 3 o o o o R was given to 2oo seeds of mono-6B.
3I~ survival and growth Following EMS treatment, monosomic 2B showed greater reduction in size than did mono-6B, particularly at the first-leaf stage (Table II). After 30 days, however, this difference had largely disappeared. As in the pilot experiment, survival was excellent--97 % for mono-6B and 88% for mono-2B. After X-ray treatment (Table II), practically no killing or even reduction in height occurred. The sharp contrast with the results of the pilot experiment, the absence of speltoid mutations, and the scarcity of chromosome aberrations indicated that considerablv less than the calculated X-ray doses were actually administered. Therefore, the X-rayed material was not carried into M2. Voluminous data on X-ray experiments with wheat are available in the literature to supplement the information obtained from the small pilot experiment. Mutant seclors in M~
Of the 558 M~ plants grown following EMS treatment, 175 had recognizable mutant sectors(Table III). A number had 2 or more distinctly different sectors, bringing the total number of sectors observed to 233. The most striking phenotypic changes in the EMS-treated generation (Table III) were albino, yellow, and pale-green longitudinal stripes of various sizes in leaves
3lutation Research I ( I 9 6 4 ) 3 8 7 3 9 9
392
14. K . S H A M A
RAO
AND
E.
R. SEARS
(Fig. Ia). In addition, m a n y sectors were observed in which notches developed, if the sector was marginal (Figs. Ib, 3), or holes if the sector was internal (Fig. 3)- These sectors, in which the notches or holes were usually fairly regularly spaced, were designated notched and punched, respectively. Necrotic patches on the leaf-blade were also of c o m m o n occurrence. Monosomic 6B showed slightly, but not significantly, more leaf sectors per plant than monosomic 2B. Necrotic patches were almost twice as numerous in mono-6B, but the increase m a y be attributed to the presence on 6B of a dominant inhibitor of necrosis. Induction of simple deficiency for this gene in a monosomic-6B plant results in a necrotic-patch sector. The 2-fold increase in yellow stripes in mono-6B over mono-2B cannot be so simply explained, as complete deficiency for chromosome (~B does not result in chlorophyll abnormality. However, this increase is well below the I .0 point for statistical significance "1",'\ I ~ I , E
CHROMOSOMAL
Type of sector None Chlorophyll defective Necrotic Brown streaked Yellow, notched, glossy Compactum ? Compactoid Subcompactoid Nomsquarchead Totals
ABERRATIONS
IV
IN ~ I
AFTER
]2~{~ TRI~;ATMENT
Nunzber of plants with indicaled aberralion Ring HelevoRing (,J 4 None c~f morphic heterom,,vphic four bivalenl bii,alent 52 47 o 2 o l o o o
4 o 2 o o o o o o
o o 4 o I o ~ l l
o o 2 o o o o o o
~02
(~
8
2
Spike abnormalities included subcompactoid and compactoid types, most of which are attributable to increased dosage of the vulgare gene Q. One of these, however, has proven due to a simple dominant m u t a t i o n very similar in its effect to that of the gene C which characterizes T r i t i u m aestivum ssp. c o m p a c t u m ~. The spikes listed as non-squarehead in 3 plants of the mono-2B population were lax, like speltoid spikes, but did not have the typical glume characters of speltoids. Since chromosome 2B has an effect on glumes opposite to that of the speltoid chroinosome (5A), it is possible t h a t the spikes were speltoid modified by deficiency for 2B. One plant with a non-squarehead spike had a second spike t h a t was compactoid. This was the spike that segregated compactum-like plants in M~ (ref. 22). The X - r a y t r e a t m e n t did not produce any phenotypic changes during the treated generation. All controls were also normal. Of the I75 EMS-treated plants showing m u t a n t sectors, 62 were examined cytologically (Table IV), and 12 were found to have gross chromosome aberrations (rings of 4 and/or heteromorphic bivalents). Of the 325 plants with no detectable m u t a n t sectors, 5t~ were examined, and 4 had an aberration. Since X - r a y treatments of high intensity result in m u c h higher frequencies of gross aberrations than these (SEARS, unpublished), the conclusion of HESL()T et al. "~, FAHMY AND FAHMYa, and E()NZAK
Mtttattotz Research
I (I9t~1,) " ; 8 7 - 3 ~ q
CHEMICAL MUTAGENESIS
IN
T. aestivum
393
et al? ~ t h a t E M S p r o d u c e s r e l a t i v e l y few c h r o m o s o m e a b e r r a t i o n s is confirmed. U l t r a violet t r e a t m e n t s m a y also induce a b e r r a t i o n s in frequencies g r e a t e r t h a n those found here for EMS (ref. 25). T h e a b e r r a t i o n r a t e for n o r m a l p l a n t s (7.I°~o) was less t h a n half t h a t for p l a n t s with m u t a n t sectors (I9.4°,,~), b u t t h e n u m b e r s are so small t h a t t h e difference is of no s t a t i s t i c a l significance. If t h e r e were a real difference, it would p r e s u m a b l y be a t t r i b u t a b l e to differences in t h e s e v e r i t y of the t r e a t m e n t from seed to seed. Since the sectors o b s e r v e d d i d n o t as a rule involw~ whole tillers, there was little likelihood in a n y p a r t i c u l a r case t h a t the floret s t u d i e d cytologically came from the m u t a n t sector o b s e r v e d in the p l a n t concerned. Also, the a b e r r a t i o n s o b s e r v e d were not of a sort t h a t could h a v e been responsible for the m u t a n t characteristics of the sectors. Deficiencies involving more t h a n 2 homologous or homoeologous loci would be required to p r o d u c e t h e necrotic sectors t h a t chiefly c h a r a c t e r i z e d the p l a n t s in which gross c h r o m o s o m a l a b e r r a t i o n s were detected. H e t e r o m o r p h i c b i v a l e n t s involve deficiency for p a r t or all of only one a r m of one chromosome, a n d rings of 4 p r e s u m a b l y involve no deficiency. Sizeable deficiencies in the m o n o s o m e s could h a v e occurred a n d not h a v e been detected, and, in the case of mono-6B, could have been responsible for necrotic sectors. M s and later generations F o r the M2 generation, seeds were sown from each of the M1 p l a n t s t h a t h a d m u t a n t sectors. In addition, offspring were grown from enough non-sectorial p l a n t s to m a k e a t o t a l of 62 progenies each from mono-6B a n d 2B. Since the M1 p l a n t s from mono-2B h a d been classifiable into monosomics a n d disomics (mainly on the basis of r e d u c e d awn d e v e l o p m e n t in t h e monosomics), the Ms families from the original m o n o - 2 B source were s e p a r a b l e into those from monosomic M1 p l a n t s (46) a n d those from disomics (16). Of the 62 families d e r i v e d from mono-6B, 8 c a m e from M1 p l a n t s t h a t h a d been identified cytologically as disomic a n d 49 from p l a n t s identified as monosomic. The r e m a i n i n g 5 families were from M1 i n d i v i d u a l s not classified as to c h r o m o some constitution. Some families consisted of seeds from a single M~ spike, others of seeds from 2 or more. I n the l a t t e r case, the p l a n t s coming from different spikes were k e p t s e p a r a t e , b u t the d a t a from different spikes of the same p l a n t are c o m b i n e d in Table V. In nc~ case were there more t h a n 2o offspring of a single spike, a n d the most from one p l a n t was 31. The average size of f a m i l y was a b o u t 18. All the families t h a t segregated seedling m u t a n t s , plus a n u m b e r of others containing no seedling m u t a n t s , were raised to m a t u r i t y (Table V). P h e n o t y p i c changes were o b s e r v e d in a t o t a l of 37 families. Since in a n u m b e r of families more t h a n one k i n d of m u t a t i o n was observed, a t o t a l of 63 m u t a n t s were recorded. I n some cases t h e occurrence of more t h a n one m u t a n t in a f a m i l y was a c c o u n t e d for b y d e r i v a t i o n of the f a m i l y from 2 or more spikes of tile Mt p l a n t concerned. I n a n u m b e r of cases, however, more t h a n one m u t a n t was recovered in tile p r o g e n y of a single spike. Chlorophyll defectives a n d necrotic t y p e s were only observed in monosomic 2B. The most frequent t y p e of c h l o r o p h y l l defective was the lethal lutescent (Fig. 2b). I n 2 cases these came from M~ p l a n t s with yellow sectors. The chlorina t y p e (pale yellow-green; Fig. 4 b) affected 5 out of I5 s u r v i v i n g i n d i v i d u a l s in the f a m i l y in which
Mulation Research I (1964 ) 387 399
394
H. K. SHAMA RAO A N D E. R. SEARS TABLE
V
NUMBER OF ~I 2 FAMILIES FROM DIFFERENT SOURCES SEGREGATING SPECIFIED MUTANTS Kind
,~'OID'Cg
of mutant M ono-614
3 I o no- 2 B
D isomic
L u t e s c e n t (lethal) Chlorina Speckled Necrotic (lethal) N e c r o t i c (viable) I)warf (lethal)
o o o o o ;
8 I I r 3 o
o o o o o t
1)warf
o 2
3 5
o 3
3 l o ~ o o o 1 0 o
7 o i l e I ~ o 5 i
o o o o o o o o e o
(bush}',
late)
Other lethals l.ate Missing spikelet Rudimentary spike Increased awns Reduced awns Non-hooded Compactum Speltoid
Semi-speltoid Base-compact N u m b e r of m u t a n t s N u m b e r (if f a m i l i e s sown transplanted affected
x0
,t l
6
54 24 13
40 27 ~8
='4 I I 0
it occurred, indicating this character to be simple recessive to normal green. This was borne out by tile results in subsequent generations. In the family with the speckled mutants there were 3 surviving plants, all of which had light-brown spotted leaves. One of tile necrotic families from monosomic 2B was distinctly different from others in being necrotic only between veins and lethal at an advanced stage of growth. Nine out of 16 plants were affected. Lethal dwarfs of very distinctive type having short, thick, dark-green leaves giving the appearance of young maize seedlings (Fig. 2a) were observed in 3 families. The use of gibberrellic acid to promote the growth of these dwarf seedlings met with no success. Heavy-tillering dwarfs appeared in 3 families from monosomic 2B. All were late in flowering and probably, could be classified as winter types. Among the distinctive spike mutations was one having one or a few spikelets missing from each spike (Fig. 6a c), a character not exhibited by any of the nulli- or tetrasomics. One family segregated plants with rudimentary spikes, the whole spike being reduced to I to 3 sterile spikelets (Fig. 6d). Mutants with both increased and decreased awn development were observed, as well as one lacking tile hooded character that is present in the variety, Chinese spring. These awn effects can all be accounted for by simple deficiency of known genes. The most commonly, occurring spike mutation observed was "semi-speltoid", in which the top half of the spike appeared to be speltoid and the bottom half bore densely arranged spikelets. One mutant very closely resembled the naturally occurring subspecies compactum.
M u t a t i o n lqcscarch r ( z 9 6 4 ) 3 8 7 : 3 ! ) 9
CHEMICAL MUTAGENESIS IN T . a e s t i v u m
395
\ to B
5
ti,, 1. ° Fig. I. L e a v e s from M 1 p l a n t s , s h o w i n g (a) yellow a n d (b) n o t c h e d sectors. Iqg. 2. T w o t y p e s of lethal m u t a n t s in M2, (a) dwarf w i t h short, t h i c k leaves a n d (b) lutescent. (c) N o r m a l seedling of s a m e age. Fig. 3. L e a v e s of a n M 1 p l a n t , w i t h (left) p u n c h e d a n d (right) n o t c h e d sectors. Fig. 4- F i r s t leaves of (a) n o r m a l a n d (b) chlorina M 2 seedlings. Fig. 5. M2 spikes of t h e t y p e s : (a) n o r m a l , (b) s u b c o m p a c t o i d , (c, d) c o m p a c t o i d . Fig. 6. (a c) M u t a n t spikes w i t h m i s s i n g spikelets. (d) R u d i m e n t a r y spike f r o m M~ s e g r e g a n t .
Mutation Research I (1964) 387-399
396
H.K.
S H A M A R A O A N D E . R. S E A R S
It was obtained from a comPact M 1 spike and behaved in M2 to M4 as a simple dominant, like compactum 22. Also observed was a mutant, "base-compact" (Fig. 5d), in which the base of the spike was more compact, and the top less compact, than in compactoid (Fig. 5c) and subcompactoid (Fig. 5b) types. Relatively few cytological observations were made in M2, and these were largely confined to the mutant plants (Table VI). Only among the plants with spike mutations were aberrations found that may have been responsible for the mutant character. In particular the speltoid mutant had a heteromorphic bivalent, and this may have involved deficiency for the vulgare gene 0. Of the semi-speltoid mutants, one had a heteromorphic bivalent, suggesting that at least this one may have been a speltoid with its phenotype modified by changes elsewhere in the genome. TABI.E
V[
CHROMOSOME CONSTITUTION OF MUTANTS I~2XAMINED IN ~'~2
T y p e (,f m u t a n ! -
Number examined
N u m b e r with heteromorphic bivalent
Chlorina Speckled Necrotic (viable) Missing spikelets Increased awns Reduced awns Non-hooded Compactum Speltoid Semi-speltoid Base-compact
l l 3 l 2 2 ~ i l 13 ~
o o 2 o
Totals
27
o o o l i
0
It is doubtful that any mutation-causing deficiencies except relatively large ones would have been detected cytologically in this material, particularly since these would have been homozygous (or hemizygous, if in a monosome). With the large number of chromosomes of different lengths and arm ratios, it is extremely difficult to detect homozygous deficiencies for much less than an entire arm. DISCUSSION
Because the complete series of nullisomics and tetrasomics is available in hexaploid wheat, it is possible to predict the kinds of mutations that will be produced by an agent whose effect is primarily to induce deficiencies and duplications. X-rays and neutrons are evidently such agents, for the mutations they induce in wheat are all, or nearly all, accountable for as deficiencies or duplications. In particular, chlorophyll defects, which are notably absent among the nullisomics and tetrasomics, are also lacking among the radiation-induced mutants. Ethyl methanesulfonate, however, appears to have the ability to induce mutations of kinds not predictable from the aneuploid series nor obtainable by irradiation. Although the modes of inheritance of most of the EMS-induced mutants have not been definitely established, it is certain that at least some of the mutants inwAve single genes. The chlorina mutant, for example, behaves as a simple recessive; the compac-
_lIutaEion Rc,~carch I ( I 9 6 4 ) ] 8 7
39(~
CHEMICAL MUTAGENESIS IN
T. aestivum
397
turn-like m u t a n t is a simple d o m i n a n t ; a n d a n u m b e r of the lethals s e g r e g a t e d in a p p r o x . 1:3 ratio. The m u t a n t with missing spikelets a n d the one with r u d i m e n t a r y spikes affected 3 i n d i v i d u a l s of 2o a n d 2 of 16, respectively. E v e n those m u t a n t s t h a t s e g r e g a t e d in low f r e q u e n c y m a y well have i n v o l v e d single genes, for a m u t a t i o n m a y sometimes affect less t h a n an entire spike. This would p r e s u m a b l y be p a r t i c u l a r l y true of m u t a t i o n s i n d u c e d b y EMS, which, because of the m a n n e r of its action, is believed c a p a b l e of h a v i n g d e l a y e d effects 6. M a n y of the E M S - i n d u c e d m u t a n t s were of t y p e s not recovered following r a d i a t i o n t r e a t m e n t s . The chlorina a n d o t h e r chlorophyll m u t a n t s , the 2 spike m u t a n t s j u s t m e n t i o n e d , a n d v a r i o u s peculiar t y p e s of dwarfs a n d lethals are m u t a n t s such as rarely, if ever, a p p e a r in r a d i a t i o n experiments. This fact, coupled with the evidence from t h e nullisomies a n d t e t r a s o m i c s t h a t these are not deficiencies or duplications, rules o u t the possibility t h a t t h e y are due to simple losses or a d d i t i o n s of c h r o m a t i n . T h a t t h e y m i g h t be due to more c o m p l e x p a t t e r n s of chromosome a b e r r a t i o n , involving homoeologous loci on several chromosomes, is n e g a t e d b y the evidence of the simple i n h e r i t a n c e of the m u t a t i o n s . If the E M S - i n d u c e d m u t a t i o n s are s i m p l y inherited a n d are not due to deficiencies or d u p l i c a t i o n s of genes or chromosome segments, t h e y m u s t be the result of changes within genes. A c t u a l l y such changes are to be a n t i c i p a t e d from w h a t is k n o w n a b o u t the m a n n e r in which EMS reacts with D N A 12 I t could also be a n t i c i p a t e d t h a t a m u t a g e n capable of inducing intragenic changes would be p a r t i c u l a r l y effective in a polyploid. HALDANE 7 a n d HARLAND 8 p o i n t e d out t h a t the d u p l i c a t i o n of genes in polyploids m a k e s m a n y of these genes non-essential a n d therefore a v a i l a b l e for drastic changes t h a t m a y even alter their function. Such changes are usually not t o l e r a t e d in diploids, where almost e v e r y gene has an essent i a l function to perform. H e x a p l o i d wheat, which a p p a r e n t l y has the large m a j o r i t y of its genes present in triplicate, p r e s u m a b l y has a v a s t reservoir of loci t h a t can be m u t a t e d in various w a y s w i t h o u t serious d e t r i m e n t to the plant. An e x a m p l e of the k i n d of locus u n d e r consideration is N e a t b y ' s virescent on chrom o s o m e 3 A. E x p e r i m e n t s have shown 21 t h a t virescent (v) is a m u t a t i o n which has a n t i m o r p h i c action with respect to its n o r m a l allele, V; t h a t is, whereas V p r o m o t e s t h e d e v e l o p m e n t of chlorophyll, v interferes with it. Loss of l T has no effect on chlorop h y l l d e v e l o p m e n t , because it has v i r t u a l d u p l i c a t e s on chromosomes 3 A a n d 3 D. O n l y b y reducing the t o t a l dosage of V a n d its d u p l i c a t e s from 6 to 2 can it be shown t h a t V is essential to n o r m a l chlorophyll d e v e l o p m e n t (SEARS, unpublished). I t seems a reasonable a s s u m p t i o n t h a t at least some of the E M S - i n d u c e d m u t a t i o n s are like v in t h a t t h e y are effective because t h e y are changed in function. T h e i n t r a genic changes i n v o l v e d m a y be s u b s t i t u t i o n s of one nucleotide for another, or t h e y m a y be deletions of nucleotides. The resulting m i n u t e r e a r r a n g e m e n t s or deletions, which do not i n a c t i v a t e the gene b u t only change its function, are more effective t h a n changes of m u c h g r e a t e r physical e x t e n t , such as deletions of an entire chromosome. If it is true t h a t EMS induces intragenic changes p r i m a r i l y , it would be reasonable to e x p e c t a fair p r o p o r t i o n of these to be d o m i n a n t , for it seems u n l i k e l y t h a t all would h a p p e n to have j u s t the right level of a c t i v i t y t h a t 2 doses would be effective whereas one was not. I t m a y therefore be assumed t h a t the m u t a n t sectors o b s e r v e d in t h e M1 p l a n t s were m o s t l y or all due to d o m i n a n t m u t a t i o n s . C y t o p l a s m i c or p l a s t i d effects are not e n t i r e l y r u l e d out as a cause of at least some of the sectors,
3lutation Research I (1964) 387 399
~0~
H. K. SHAMA RAO AND E. R. SEARS
however. A t t e m p t s to recover the presumed dominant m u t a n t s in M~ largely failed, but this m a y be attributed to (x) failure of the sector to extend into the floral organs of the spike, and/or (2) lethality of the m u t a n t when it affects an entire embryo or seedling. As an example of the latter factor, it seems likely that the large majority of the m a n y sectors recordect that involved necrosis would have been lethal at a very early stage, probably in the embryo. T w o of the 8 lethal mutations classified as lutescent in Table V came from M~ plants with yellow sectors. These were presumably instances of recovery of dominant mutations. In the planning of this experiment it was assumed that if mutations could be induced on m o n o s o m e s , these might appear in M~ even though recessive, and thus be i m m e d i a t e l y located as to the chromosome concerned. The results did not bear out this expectation. E x c e p t for certain predictable types of m u t a t i o n e v i d e n t h due to deficiencv for particular loci on the m o n o s o m e , there was no substantial difference between the frequency of M~ mutant sectors in m o n o s o m i c s and disomics. Further consideration shows that m o s t mutations of the type expected following EMS treatment would probably not be expressed when hemizygous. In the disomic 5 alleles of the gene being changed are present, and the recessive mutant in one dose is unable to o v e r c o m e the effect of all 5 alleles but can do so in 2 doses. In the m o n o s o m i c there are still 4 alleles for the mutant gene to overcome, and the probability of its being able to do st) is not great. ACKNO\VLED(;EMENTS
This investigation was carried out while the senior author was an at the University of Missouri under the Point Four Program of the national D e v e l o p m e n t , United States (iovernment. He is grateful for the deputation and to A I D for providing financial assistance. grateful to Dr. C. F. KONZAI¢ for recommending the use of ethyl and for supplying information concerning methods of treatment.
Exchange Visitor Agency for haterto A E E T (India) Both authors are methanesulfonate
I{EFEI~ENCES 1 D'AMA'ro, I;., G. T. SCARASCl,X, 1,. M. MON'rl AND A. BOZZINI, T y p e s and fre:luencies of c h l o r o p h y l l m u t a t i o n s in durum w h e a t i n d u c e d b y r a d i a t i o n s and chemicals. Radiatio~ l~ota~v, 2 (I962) 217 230. EnRW~:Nm~:RC;, l,., C h e m i c a l m u t a g e n e s i s ; b i o c h e m i c a l and chemical p o i n t s of v i e w on m e c h a n i s m s of action. Chemical .,llutagenesis. Erwin-Ba~r-Geddch/nis~,orlesungen I, ~ 9 5 9 . . l ' ) ] t a ~ l d l . D e u t . . I k a d . II'iss. t~erlin, NI. ;led., (I96.3} I24 13(~. :~ FAHMV, (). (;. AND ]¥][. J. FAHMY, Cytogenetic analysis of the a c t i o n of carcinogens and t u m o r inhibitors in Drosophila melam)gaster. X. T h e n a t u r e of the m u t a t i o n s i n d u c e d b y t h e m e s y l o x y esters in relation t o m o l e c u l a r cross-linkage. (;enetics, 46 (1901) 447 458. a FAH.~'IV, (). G. AND ~'I. J. FAHMY, Cytogenetic analysis of t h e acti,'m of carcinogens a n d t u m o u r i n h i b i t o r s in l)roso,t~hila ~nelanocaster. N l . M u t a g e n i c ef~ficiency of the m e s y l o x y esters on sperm in relation t o m o l e c u l a r s t r u c t u r e . Genetics, 40 (~9(~I) ~ l l l l 1-'4. 5 GAUL, H., I ' n g e u O h n l i c h hohe M u t a t i o n s r a t e n bei Gerste nach A n w e n d u n g y o n . / ~ t h v h n e t h a n s u l f o n a t und ROntgenskrahlen. Na/u;'~viss., 49 (I96-') i 2. 6 (;REEN, D. ~I. AND I). I~. IXRIEG, T h e d e l a y e d origin of m u t a n t s i n d u c e d b y exposure of e x t r a c e l l u l a r p h a g e T 4 t o e t h y l m e ' ~ h a n e s u l f o n a t e . Proc. Natl. Acad. Sci. U.S., 47 (1961) o. t 72. 7 HALDANE, J. B. S., 7"he Ca~tse~" (~f Evolution. [ , o n g m a n s , Green and ('o., London, IQ32. s [-IARLAND, S. C., The genetics of cotton. XI. F u r t h e r e x p e r i m e n t s on the inheritance of c h l o r o p h y l l d e f i c i e n c y i n N e w W o r l d c o t t o n . . ] . ( ; e n e t . , 29 (I934) 181 105. 9 HESLOT, H., 1~.. IrERRARY, I~. [,I~.VY AND C. MONaRD, I~echerches sur les substances m u t a g b n e s (halogdno 2 d t h y l ) a m i n e s , d d r i v d s oxygdnds du sulfure de his (chloro-z d t h y l e ) , e s t e r s sulfon i q u e s e t .';ulfuriques. Co~nt~/. Rtl~d. 248 (~959) 729 732.
Mutation Resvar~h I (~964) 387 399'
CHEMICAL MUTAGENESIS IN T . a e s t i v u m
39c~
10 I(ONZAK, C. 1~., Physical and chemical m u t a g e n e s i s in w h e a t breeding. Proc. 2nd Intern. Wheat Genet. Syrup., I963, in the press. Jl KONZAK, C. F., R. A. NILAN, J. R. HARLE AND R. E. HEINER, Control of factors affecting the response of p l a n t s to mutagens. Brookhaven Syrup. Biol., 14 11961 ) 128 i57. 12 LAWLEY, P. D. AND }). BROOKES, Acidic dissociation of 7:9 dialkylguanines and its possible relation to m u t a g e n i c properties of alkylating agents. Nature, 192 11961) lO81 -lO82. 1:~ LOBBECKE, E. A. AND I~. C. VON BORSTEL, Mutational response of H a b r o b r a c o n oocytcs in n l e t a p h a s e and p r o p h a s e to ethyl m e t h a n e s u l f o n a t e and nitrogen m u s t a r d . Genelics, 57 (191)2} 853 864. 14 LOVELESS, A., Increased rate of p l a q u e - t y p e and host-range m u t a t i o n following t r e a t m e n t of bacteriophage in vitro w i t h ethyl m e t h a n e s u l p h o n a t e . Nature, 181 (1958) 1212 1213. 15 LOVELESS, A. AND S. HOV,'ARTH, M u t a t i o n of bacteria at high levels of survival by ethyl m e t h a n e s u l p h o n a t e . Nature, 184 (I959) 1780 ~782. 10 MCI£ELVIE, A. D., Studit's in the induction of m u t a t i o n s in ~4rabidopsis lhaliana (l..) H e y n h . Radiation Bolany, 3 (1963) lO5 123. 17 MAc KEY, J., Chemical induction of m u t a t i o n in c o m m o n wheat. Wheat Inform. Serv., 14 (19()2) 911. is NEUFFER, M. O. AND G. FICSOR, Mutagenic action of ethyl inethanesulfonate in m a i z e Science, 139 11963) 1296-1297. 19 ROBBELEN, G., \Virkungsvergleich zwischen - ~ t h y l m e t h a n s u l f o n a t und R 6 n t g e n b e s t r a h l e n in M u t a t i o n s v e r s u c h mit Arabidopsis thaliana. Naturwiss., 49 (1962) 65. ~0 SEARS, E. R., The aneuploids of c o m m o n wheat. Montana Slate Coll. Agr. Expl. Slat. Bz~ll., 572 (1954) 58 pp. 2~ SEARS, E. R., N e a t b v ' s virescent, a chlorophyll a b e r r a t i o n in c o m m o n wheat. Ge~letics, 44 11959) 534. ~:~ SHAMA I ~ a o , H . lx~. AND ~'I. r . 1)RABHAKARA RAO, EMS-induced compactum-type m u t a t i o n in Triticum aeslivum ssp. wdgare. Crop. Sci., 3, in the press. 2a SHAMA RAO, H. I'L AND E. R. SEARS, EMS-induced m u t a t i o n s in hexaploid wheat. Genetics, 47 (1962) 983 984.. z* SMITH, H. H., Mutagenic specificity and directed m u t a t i o n . Mutation and Plant Breeding. N.4S-NRC, 891 11901 ) 413 436. '25 STEINITZ-SEARS, L. M. AND E . 1~. SEARS, Ultraviolet and X - r a y induced c h r o m o s o m a l aberrations in wheat. Genetics, 42 (1957) 623 630. 2G STRAUSS, B. S., Specilicity of the mutagenic action of the alkylating agents. Natz¢re, I91 11967) 73 ° 731 . 27 SXVAM1NATHAN, ~ . S., \7. L. CHOPRA AND S. BHASKARAN, C h r o m o s o m e a b e r r a t i o n s and t h e frequency and s p e c t r u m of m u t a t i o n s induced b y ethyl m e t h a n e s u l p h o n a t e in barley and wheat. lndian.[.Genel., 22 (1962) 192 2o 7. 28 TSUNEWAKI, 11. AND E. G. HEYNE, Radiological stud), of w h e a t monosomic,;. [. I)ifferential sensitivity of monosomic X and the disomic to X-irradiation. Genetics, 44 (I959) 933 940. 29 TSUNV;WAKL I(. AND E. G. HEYNE, Radiological s t u d y of w h e a t monosomics. II. Differential sensitivity of 16 nlonosomics and the disomic to a single dosage of X-irradiation. Genetics, 44 (1959) 9 4 7 954. 3~ \VESTERGAARD, M., Chemical mutagenesis in relation to the concept of the gone. E.rperientia, 13 11957) 224-234.
Mutation Research i 11964) 387 399
MUTATION RESEARCH
CHEMICAL MUTAGENESIS IN T R I T I C U M
AESTIVUM*
H. K. S H A M A R A O * * AND E. R. S E A R S * * *
Field Crops Department, University of Missouri, Columbia, Mo. (U.S.A.) (Received A u g u s t 7th, J964)
SUMMARY
Treatment of common wheat with ethyl methanesulfonate had effects strikingly different from those characteristically obtained in irradiation experiments. Of 558 M x plants, I75 had recognizable mutant sectors, almost all of which were necrotic or chlorophyll-defective stripes in leaves. Such sectors are rarely seen following irradiation. Chromosome aberrations were relatively infrequent. In M~, one family segregated for a viable, simply recessive chlorina mutation, and several segregated lethal lutescent mutants. There were numerous other lethals, including three dwarfs with short, thick leaves. In addition to awn and spike mutants attributable to deficiency or duplication for known genes, there were a compactum-like dominant mutation, a mutant with one or a few central spikelets missing from each spike, and a type with the entire spike aborted except for one to three sterile spikelets. The production of distinctive mutations in common wheat by ethyl methanesulfonate is believed to involve changes in function of genes which are duplicated on other chromosomes of this hexaploid species. The change in function releases the genes from the masking action of their duplicates, while maintenance of the original function by the duplicates keeps the mutations from being automatic lethals.
INTRODUCTION
The effectiveness of numerous chemicals in inducing mutation in both animals and plants is now well established (see review by SMITH24). Many of these chemicals are alkylating agents--that is, organic reagents capable of transferring their alkyl groups to other compounds. One of the most promising of these is ethyl methanesulfonate (EMS), discovered by Kolmark (cited by WESTERCAARD'~°)to be mutagenic in Neurospora and subsequently shown to be mutagenic in bacteriophage 'L bacterialS,~% Drosophila 3, 4, Habrobracon 13, barley 2, 5,9,1l, Arabidopsis 16,19, maize 1", tetraploid wheat', and hexaploid wheat 10, 17, ~.~,27 Abbreviation: EMS, ethyl methanesulfonate. * J o u r n a l Series No. 2773 of the Missouri Agricultural E x p e r i m e n t Station. A p p r o v e d by the Director. * * N o w Scientific Officer, Biology Division, Atomic E n e r g y E s t a b l i s h m e n t , T r o m b a y , Byculla, t3ombay-8, lndia. *** Geneticist, Crops Research Division, Agricultural Research Service, U.S. D e p a r t m e n t of Agriculture. Address: Curtis Hall, University of Missouri, Columbia, Mo.
MutMion Research x (i964) 387 399
388
H. K. SHAMA RAO AND E. R. SEARS
KONZAK el al. n have e m p h a s i z e d t h a t while some chemical m u t a g e n s are fully, radiomimetic, giving essentially the same p a t t e r n of m u t a t i o n s a n d gross c h r o m o s o m a l aberl ations as the ionizing radiations, EMS produces more m u t a t i o n s a n d m a n y fewer chromosomal aberrations. There is a m p l e theoretical s u p p o r t for this tinding in the conclusion of HAWLEY AND BROOKES 12 t h a t EMS reacts with D N A to a l k y l a t e the guanine c o m p o n e n t and cause it to pair with t h y m i n e i n s t e a d of cytosine during D N A duplication. This is said to result in the s u b s t i t u t i o n of an adenine t h y m i n e pair for guanine -cytosine. The a b i l i t y of a m u t a g e n to induce true gene changes r a t h e r t h a n mere deficiencies and d u p l i c a t i o n s is difficult to establish in a higher plant, the only valid criterion n o r m a l l y being the occurrence of b a c k - m u t a t i o n from the induced m u t a n t . In common wheat, however, m u t a t i o n s t h a t are not the result of simple loss or duplication of p a r t s of chromosomes (:an be identified with a reasonable degree of c e r t a i n t y , because the nullisomics and t e t r a s o m i c s for all 21 chromosomes c o n s t i t u t e all the possible deficiencies a n d duplications. H e x a p l o i d w h e a t varieties have the a b i l i t y to tolerate large deficiencies and duplications; in fact, p l a n t s deficient for an entire chromosome are essentially normal, being in most cases difficult to distinguish from t h e i r n o r m a l sibs. Therefore, in con> parison with diploids, r e l a t i v e l y few cells which c a r r y gross a b e r r a t i o n s are e l i m i n a t e d , a n d the a b e r r a t i o n s recovered t e n d to be a true sample of those t)roduced b y the treatment. Monosomics were used in the present e x p e r i m e n t , with the idea t h a t recessive m u t a t i o n s p r o d u c e d on the chromosomes concerned might lead to d e t e c t a b l e m u t a n t sectors in the M1 plants. Offspring from such sectors would include disomics, which would necessarily be h o m o z y g o u s for the m u t a t i o n concerned; a n d in each case it would be k n o w n on which c h r o m o s o m e the m u t a t i o n was located. The monosomics used here were eB (formerly II) a n d 6B (X), each involving a chromosome responsible for the suppression of several abnormalities'a°. Deficiency for the suppressing genes should give rise to m u t a n t s d i s p l a y i n g these abnormalities. B y i r r a d i a t i n g w h e a t m o n o s o m i c s , TSUNEWAKI AND H E Y N E 2~, 2,a were able to d e m o n s t r a t e the presence on these a n d o t h e r chromosomes of d o m i n a n t genes for v i a b i l i t y a n d g r o w t h c h a r a c t e r s as well as for morphological characters. It m i g h t be e x p e c t e d t h a t all recessive m u t a t i o n s in the monosome would show u p in M~, for the d o m i n a n t allele would be missing. However, the critical factor in expression of the m u t a n t m a y be the presence of 2 doses of the recessive, not the absence of the d o m i n a n t . In o t h e r words, the d o m i n a n t allele m a y have little or no suppressing effect on the recessive, and the h e m i z y g o t e m a y be little if a n y different from the heterozygote. This t y p e of gene change is exemplified b y N e a t b y ' s virescent"L a recessive m u t a n t which is not expressed when hemizygous. A p r e l i m i n a r y r e p o r t "a dealt v e r y briefly with M, d a t a a n d o b s e r v a t i o n s on M~ seedlings. MATERIAI.S AND METHODS
Seeds of n o r m a l Chinese spring wheat and of monosomics 2B a n d 6B of this v a r i e t y were first b r o u g h t to a stable moisture c o n t e n t of 12% with 1.5o d e n s i t y sulphuric acid in a dessicator k e p t at 2o °. The chemical t r e a t m e n t s consisted of soaking seeds in EMS solution at 3 o°. I m m e -
Mulation Research I 1I()()4) 387-:;90
CHEMICAL MUTAGENESIS
TABLE
IN
389
T. aeslivum
I
SURVIVAL AND S E E D L I N G HEIGHT AFTER E M S AND X - R A Y TREATMENT IN PILOT EXPERIMENT
3laterial treated
K i n d of treatment
Number of seeds
A t z-leaf stage (iermiMean nation keigkl
.~lt 3-leaf stage (;ermiMean nation keight
Survival after 3 ° days
(%)
(cm)
(°o)
(cm)
o,,)
Mono-2B
Control 0.05 M EMS, 6 h o.o 7 M EMS, 6 h X - r a y s , 24 k R X - r a y s , 48 k R
IO 20 2o 2o 20
loo loo o IOO lOO
7.6 1. 9 -2.2 t. 1
too 1oo IO IOO too
36.8 19. 5 o.I ~ 3.3 8. 4
90 95 o 95 (~5
Mono-6B
Control 0.05 M EMS, 6 h 0.07 M EMS, 6 h X - r a y s , 24 k R X - r a y s , 48 k R
to 20 20 20 20
1oo ioo o 95 5°
6.8 I. 4
IOO ioo 3° IOO 80
36.8 15. 3 2.5 4.7 (>. l
95 9o o 8o o
0.9 0.2
d i a t e l y after t r e a t m e n t , the seeds were t h o r o u g h l y washed with distilled w a t e r at room t e m p e r a t u r e . X - r a y s were a d m i n i s t e r e d at an i n t e n s i t y of 72o R / m i n at 15o kV, 9 mA, w i t h o u t filter. To o b t a i n meiotic p r e p a r a t i o n s , i n d i v i d u a l a n t h e r s fixed for at least two d a y s in 1:3 acetic alcohol were m o r d a n t e d for 3o min in 2°/'0 ferric a c e t a t e a n d soaked overnight in I°,'o a c e t o - c a r m i n e before squashing. The slides were m a d e p e r m a n e n t b y a liquid CO2 technique. PILOT EXPERIMENT
In order to d e t e r m i n e a dose r a t e for X - r a y s a n d EMS which would p r o J u c e a large percentage of defective seedlings at a high s u r v i v a l rate, a pilot e x p e r i m e n t was cond u c t e d which i n v o l v e d 15 different EMS t r e a t m e n t s a n d 2 X - r a y dosages. EMS conc e n t r a t i o n s of o.o5, o.o 7, a n d o.xo M were used for 3, 6, 12, 24, a n d 48 h, and X - r a y t r e a t m e n t s of 24ooo a n d 48ooo R were given. T w e n t y seeds of each monosomic were included in each t r e a t m e n t , a n d IO in each control. The EMS t r e a t m e n t s of 3 h d u r a t i o n caused no m a r k e d change in seedling height or in percentage of g e r m i n a t i o n a n d survival. T r e a t m e n t s for 12 h or longer c o m p l e t e l y i n h i b i t e d g e r m i n a t i o n , as did t h e 6-tl t r e a t m e n t with o . I o M solution. Of the 2 remaining t r e a t m e n t s , b o t h of 6 h (Table I), the one a t o.o 7 p e r m i t t e d some g e r m i n a t i o n b u t allowed no seedlings to survive. The o.o5 M, 6-h t r e a t m e n t , on the other hand, caused a fairly drastic a n d uniform r e d u c t i o n in seedling height, i n d i c a t i n g effectiveness of the t r e a t m e n t , b u t p e r m i t t e d s u r v i v a l n e a r l y equal to t h a t of t h e control. No difference was n o t i c e d in the s e n s i t i v i t y of monosomics 2B a n d 6B to EMS. S e n s i t i v i t y to X - r a y t r e a t m e n t was s t r i k i n g l y g r e a t e r for mono-6B t h a n for mono-2B (Table I). A more drastic reduction in height was noted, s t a r t i n g with the first leaf, a n d s u r v i v a l was less, p a r t i c u l a r l y at 48 k R , where it was zero for 6B a n d 65% for 2B. This g r e a t e r s e n s i t i v i t y of monosomic 6B agrees with the findings of TSUNEWAKI AND HEYNE
2:~.
Mutation Research i ( i 9 6 4 ) 3 8 7 - 3 9 9
"4
"2
Mono-6B
Mono-eB
~oo
5° kR
300
2oo
EMS
3° kR
lo
loo
Control
300
EMS
lo
bet o/ seed~
.Yum-
3° kR
Control
~llaterial Kind uf treated treatment
--
i 1.8
i i 5
Mean heigkt (cm)
AND SFRVIVAL
82, 5
4.0
100,0
92.0
91.o
]-3
90.0
15. 5
68.0
0.0
7.0
8.0
34.3
o.o
1.0
-'3.3
O.O
o.o
J.O
43.7
o.o
11
O. 5
2.7
0.0
o.o
o.o
14.3
o.o
O. 5
,.O
O.O
l .o
o.o
6.3
1o.o
% no/ :e~:mihated
--
35.0
--
--
35.2
Mea~ hdgk! (cm)
AND X-RAYS
88.0
27.3
IOO.O
94.0
92.o
l ~ .o
1 OO.O
,\'O:g~
9.5
03.7
0.0
5.0
8.0
56.3
O.O
• - o
~: - 0 ,o
2,o
0.o
0.0
0.0
o.o
20.0
O.O
)0°o = .
o.o
~.3
o.0
o.o
o.o
7.3
O.O
..... / D
Third-leaf stage (of control) o witk iudicated reduction in keigkl
F O L L O \ V I N G TRI'.'ATMKNTS \VITtt E M S
1;irst-leaf ,;tage ( of conh'ol) % wiih iudica/ed redmTio~t in keigkt ,\'one : 25 % :: 5oo0 7500
IIEIGHT REDI'CTION
TABLE
o
0.5
~.7
o.o
i .o
o.o
5.3
O.O
H(IIzd
93.5
9 7 .0
90.0
95.0
93,o
88.0
IOO.O
(%)
Survival ?o ~wt after ,;'ermi- 3o days
>
> Z
> O
ha
Cla VO ©
aestivum
CHEMICAL MUTAGENESIS IN T. TABLE
391
III
PHENOTYPIC CHANGES OBSERVED IN ~'I 1 GENERATION FOLLO~rING EMS TREATMENT K i n d of change Leaves Albino Yellow
stripe stripe
Pale-green stripes B r o w n stripes Necrotic p a t c h e s Glossy
Notched Punched Spikes Compactoid Subcompactoid Non-squarehead
N u m b e r of p l a n t s affected N u m b e r of affected sectors N u m b e r of p l a n t s studied * l'rogeny about 75°,
Chromosome constitution of affected plants iVIonosomic 6B* Monosomic 2B Disomics
Untreated control
3 46 4 3 36 7 18 13
4 15 I 3 13 7 14 1[
o 4 o [ 8 2 12 o
o o o o o o O o
2 i o
i i 3
o o o
o o o
lO 7 133 286
52 73 18o
16 27 92
o 2o
of selfed m o n o s o m i c 6B. N o t a n a l y z e d cytologically, b u t p r e s u m a b l y comprised of monosomics and 25°o disomics. MAIN EXPERIMENT
On the basis of the results of the pilot experiment, 300 seeds each of monosomics 2B and 6B were subjected to 0.05 M EMS for 6 h at 3 o°. X-ray treatments calculated as 3 o o o o R and 5oooo R (but see below) were given to IOO-seed lots of mono-2B, and 3 o o o o R was given to 2oo seeds of mono-6B.
3I~ survival and growth Following EMS treatment, monosomic 2B showed greater reduction in size than did mono-6B, particularly at the first-leaf stage (Table II). After 30 days, however, this difference had largely disappeared. As in the pilot experiment, survival was excellent--97 % for mono-6B and 88% for mono-2B. After X-ray treatment (Table II), practically no killing or even reduction in height occurred. The sharp contrast with the results of the pilot experiment, the absence of speltoid mutations, and the scarcity of chromosome aberrations indicated that considerablv less than the calculated X-ray doses were actually administered. Therefore, the X-rayed material was not carried into M2. Voluminous data on X-ray experiments with wheat are available in the literature to supplement the information obtained from the small pilot experiment. Mutant seclors in M~
Of the 558 M~ plants grown following EMS treatment, 175 had recognizable mutant sectors(Table III). A number had 2 or more distinctly different sectors, bringing the total number of sectors observed to 233. The most striking phenotypic changes in the EMS-treated generation (Table III) were albino, yellow, and pale-green longitudinal stripes of various sizes in leaves
3lutation Research I ( I 9 6 4 ) 3 8 7 3 9 9
392
14. K . S H A M A
RAO
AND
E.
R. SEARS
(Fig. Ia). In addition, m a n y sectors were observed in which notches developed, if the sector was marginal (Figs. Ib, 3), or holes if the sector was internal (Fig. 3)- These sectors, in which the notches or holes were usually fairly regularly spaced, were designated notched and punched, respectively. Necrotic patches on the leaf-blade were also of c o m m o n occurrence. Monosomic 6B showed slightly, but not significantly, more leaf sectors per plant than monosomic 2B. Necrotic patches were almost twice as numerous in mono-6B, but the increase m a y be attributed to the presence on 6B of a dominant inhibitor of necrosis. Induction of simple deficiency for this gene in a monosomic-6B plant results in a necrotic-patch sector. The 2-fold increase in yellow stripes in mono-6B over mono-2B cannot be so simply explained, as complete deficiency for chromosome (~B does not result in chlorophyll abnormality. However, this increase is well below the I .0 point for statistical significance "1",'\ I ~ I , E
CHROMOSOMAL
Type of sector None Chlorophyll defective Necrotic Brown streaked Yellow, notched, glossy Compactum ? Compactoid Subcompactoid Nomsquarchead Totals
ABERRATIONS
IV
IN ~ I
AFTER
]2~{~ TRI~;ATMENT
Nunzber of plants with indicaled aberralion Ring HelevoRing (,J 4 None c~f morphic heterom,,vphic four bivalenl bii,alent 52 47 o 2 o l o o o
4 o 2 o o o o o o
o o 4 o I o ~ l l
o o 2 o o o o o o
~02
(~
8
2
Spike abnormalities included subcompactoid and compactoid types, most of which are attributable to increased dosage of the vulgare gene Q. One of these, however, has proven due to a simple dominant m u t a t i o n very similar in its effect to that of the gene C which characterizes T r i t i u m aestivum ssp. c o m p a c t u m ~. The spikes listed as non-squarehead in 3 plants of the mono-2B population were lax, like speltoid spikes, but did not have the typical glume characters of speltoids. Since chromosome 2B has an effect on glumes opposite to that of the speltoid chroinosome (5A), it is possible t h a t the spikes were speltoid modified by deficiency for 2B. One plant with a non-squarehead spike had a second spike t h a t was compactoid. This was the spike that segregated compactum-like plants in M~ (ref. 22). The X - r a y t r e a t m e n t did not produce any phenotypic changes during the treated generation. All controls were also normal. Of the I75 EMS-treated plants showing m u t a n t sectors, 62 were examined cytologically (Table IV), and 12 were found to have gross chromosome aberrations (rings of 4 and/or heteromorphic bivalents). Of the 325 plants with no detectable m u t a n t sectors, 5t~ were examined, and 4 had an aberration. Since X - r a y treatments of high intensity result in m u c h higher frequencies of gross aberrations than these (SEARS, unpublished), the conclusion of HESL()T et al. "~, FAHMY AND FAHMYa, and E()NZAK
Mtttattotz Research
I (I9t~1,) " ; 8 7 - 3 ~ q
CHEMICAL MUTAGENESIS
IN
T. aestivum
393
et al? ~ t h a t E M S p r o d u c e s r e l a t i v e l y few c h r o m o s o m e a b e r r a t i o n s is confirmed. U l t r a violet t r e a t m e n t s m a y also induce a b e r r a t i o n s in frequencies g r e a t e r t h a n those found here for EMS (ref. 25). T h e a b e r r a t i o n r a t e for n o r m a l p l a n t s (7.I°~o) was less t h a n half t h a t for p l a n t s with m u t a n t sectors (I9.4°,,~), b u t t h e n u m b e r s are so small t h a t t h e difference is of no s t a t i s t i c a l significance. If t h e r e were a real difference, it would p r e s u m a b l y be a t t r i b u t a b l e to differences in t h e s e v e r i t y of the t r e a t m e n t from seed to seed. Since the sectors o b s e r v e d d i d n o t as a rule involw~ whole tillers, there was little likelihood in a n y p a r t i c u l a r case t h a t the floret s t u d i e d cytologically came from the m u t a n t sector o b s e r v e d in the p l a n t concerned. Also, the a b e r r a t i o n s o b s e r v e d were not of a sort t h a t could h a v e been responsible for the m u t a n t characteristics of the sectors. Deficiencies involving more t h a n 2 homologous or homoeologous loci would be required to p r o d u c e t h e necrotic sectors t h a t chiefly c h a r a c t e r i z e d the p l a n t s in which gross c h r o m o s o m a l a b e r r a t i o n s were detected. H e t e r o m o r p h i c b i v a l e n t s involve deficiency for p a r t or all of only one a r m of one chromosome, a n d rings of 4 p r e s u m a b l y involve no deficiency. Sizeable deficiencies in the m o n o s o m e s could h a v e occurred a n d not h a v e been detected, and, in the case of mono-6B, could have been responsible for necrotic sectors. M s and later generations F o r the M2 generation, seeds were sown from each of the M1 p l a n t s t h a t h a d m u t a n t sectors. In addition, offspring were grown from enough non-sectorial p l a n t s to m a k e a t o t a l of 62 progenies each from mono-6B a n d 2B. Since the M1 p l a n t s from mono-2B h a d been classifiable into monosomics a n d disomics (mainly on the basis of r e d u c e d awn d e v e l o p m e n t in t h e monosomics), the Ms families from the original m o n o - 2 B source were s e p a r a b l e into those from monosomic M1 p l a n t s (46) a n d those from disomics (16). Of the 62 families d e r i v e d from mono-6B, 8 c a m e from M1 p l a n t s t h a t h a d been identified cytologically as disomic a n d 49 from p l a n t s identified as monosomic. The r e m a i n i n g 5 families were from M1 i n d i v i d u a l s not classified as to c h r o m o some constitution. Some families consisted of seeds from a single M~ spike, others of seeds from 2 or more. I n the l a t t e r case, the p l a n t s coming from different spikes were k e p t s e p a r a t e , b u t the d a t a from different spikes of the same p l a n t are c o m b i n e d in Table V. In nc~ case were there more t h a n 2o offspring of a single spike, a n d the most from one p l a n t was 31. The average size of f a m i l y was a b o u t 18. All the families t h a t segregated seedling m u t a n t s , plus a n u m b e r of others containing no seedling m u t a n t s , were raised to m a t u r i t y (Table V). P h e n o t y p i c changes were o b s e r v e d in a t o t a l of 37 families. Since in a n u m b e r of families more t h a n one k i n d of m u t a t i o n was observed, a t o t a l of 63 m u t a n t s were recorded. I n some cases t h e occurrence of more t h a n one m u t a n t in a f a m i l y was a c c o u n t e d for b y d e r i v a t i o n of the f a m i l y from 2 or more spikes of tile Mt p l a n t concerned. I n a n u m b e r of cases, however, more t h a n one m u t a n t was recovered in tile p r o g e n y of a single spike. Chlorophyll defectives a n d necrotic t y p e s were only observed in monosomic 2B. The most frequent t y p e of c h l o r o p h y l l defective was the lethal lutescent (Fig. 2b). I n 2 cases these came from M~ p l a n t s with yellow sectors. The chlorina t y p e (pale yellow-green; Fig. 4 b) affected 5 out of I5 s u r v i v i n g i n d i v i d u a l s in the f a m i l y in which
Mulation Research I (1964 ) 387 399
394
H. K. SHAMA RAO A N D E. R. SEARS TABLE
V
NUMBER OF ~I 2 FAMILIES FROM DIFFERENT SOURCES SEGREGATING SPECIFIED MUTANTS Kind
,~'OID'Cg
of mutant M ono-614
3 I o no- 2 B
D isomic
L u t e s c e n t (lethal) Chlorina Speckled Necrotic (lethal) N e c r o t i c (viable) I)warf (lethal)
o o o o o ;
8 I I r 3 o
o o o o o t
1)warf
o 2
3 5
o 3
3 l o ~ o o o 1 0 o
7 o i l e I ~ o 5 i
o o o o o o o o e o
(bush}',
late)
Other lethals l.ate Missing spikelet Rudimentary spike Increased awns Reduced awns Non-hooded Compactum Speltoid
Semi-speltoid Base-compact N u m b e r of m u t a n t s N u m b e r (if f a m i l i e s sown transplanted affected
x0
,t l
6
54 24 13
40 27 ~8
='4 I I 0
it occurred, indicating this character to be simple recessive to normal green. This was borne out by tile results in subsequent generations. In the family with the speckled mutants there were 3 surviving plants, all of which had light-brown spotted leaves. One of tile necrotic families from monosomic 2B was distinctly different from others in being necrotic only between veins and lethal at an advanced stage of growth. Nine out of 16 plants were affected. Lethal dwarfs of very distinctive type having short, thick, dark-green leaves giving the appearance of young maize seedlings (Fig. 2a) were observed in 3 families. The use of gibberrellic acid to promote the growth of these dwarf seedlings met with no success. Heavy-tillering dwarfs appeared in 3 families from monosomic 2B. All were late in flowering and probably, could be classified as winter types. Among the distinctive spike mutations was one having one or a few spikelets missing from each spike (Fig. 6a c), a character not exhibited by any of the nulli- or tetrasomics. One family segregated plants with rudimentary spikes, the whole spike being reduced to I to 3 sterile spikelets (Fig. 6d). Mutants with both increased and decreased awn development were observed, as well as one lacking tile hooded character that is present in the variety, Chinese spring. These awn effects can all be accounted for by simple deficiency of known genes. The most commonly, occurring spike mutation observed was "semi-speltoid", in which the top half of the spike appeared to be speltoid and the bottom half bore densely arranged spikelets. One mutant very closely resembled the naturally occurring subspecies compactum.
M u t a t i o n lqcscarch r ( z 9 6 4 ) 3 8 7 : 3 ! ) 9
CHEMICAL MUTAGENESIS IN T . a e s t i v u m
395
\ to B
5
ti,, 1. ° Fig. I. L e a v e s from M 1 p l a n t s , s h o w i n g (a) yellow a n d (b) n o t c h e d sectors. Iqg. 2. T w o t y p e s of lethal m u t a n t s in M2, (a) dwarf w i t h short, t h i c k leaves a n d (b) lutescent. (c) N o r m a l seedling of s a m e age. Fig. 3. L e a v e s of a n M 1 p l a n t , w i t h (left) p u n c h e d a n d (right) n o t c h e d sectors. Fig. 4- F i r s t leaves of (a) n o r m a l a n d (b) chlorina M 2 seedlings. Fig. 5. M2 spikes of t h e t y p e s : (a) n o r m a l , (b) s u b c o m p a c t o i d , (c, d) c o m p a c t o i d . Fig. 6. (a c) M u t a n t spikes w i t h m i s s i n g spikelets. (d) R u d i m e n t a r y spike f r o m M~ s e g r e g a n t .
Mutation Research I (1964) 387-399
396
H.K.
S H A M A R A O A N D E . R. S E A R S
It was obtained from a comPact M 1 spike and behaved in M2 to M4 as a simple dominant, like compactum 22. Also observed was a mutant, "base-compact" (Fig. 5d), in which the base of the spike was more compact, and the top less compact, than in compactoid (Fig. 5c) and subcompactoid (Fig. 5b) types. Relatively few cytological observations were made in M2, and these were largely confined to the mutant plants (Table VI). Only among the plants with spike mutations were aberrations found that may have been responsible for the mutant character. In particular the speltoid mutant had a heteromorphic bivalent, and this may have involved deficiency for the vulgare gene 0. Of the semi-speltoid mutants, one had a heteromorphic bivalent, suggesting that at least this one may have been a speltoid with its phenotype modified by changes elsewhere in the genome. TABI.E
V[
CHROMOSOME CONSTITUTION OF MUTANTS I~2XAMINED IN ~'~2
T y p e (,f m u t a n ! -
Number examined
N u m b e r with heteromorphic bivalent
Chlorina Speckled Necrotic (viable) Missing spikelets Increased awns Reduced awns Non-hooded Compactum Speltoid Semi-speltoid Base-compact
l l 3 l 2 2 ~ i l 13 ~
o o 2 o
Totals
27
o o o l i
0
It is doubtful that any mutation-causing deficiencies except relatively large ones would have been detected cytologically in this material, particularly since these would have been homozygous (or hemizygous, if in a monosome). With the large number of chromosomes of different lengths and arm ratios, it is extremely difficult to detect homozygous deficiencies for much less than an entire arm. DISCUSSION
Because the complete series of nullisomics and tetrasomics is available in hexaploid wheat, it is possible to predict the kinds of mutations that will be produced by an agent whose effect is primarily to induce deficiencies and duplications. X-rays and neutrons are evidently such agents, for the mutations they induce in wheat are all, or nearly all, accountable for as deficiencies or duplications. In particular, chlorophyll defects, which are notably absent among the nullisomics and tetrasomics, are also lacking among the radiation-induced mutants. Ethyl methanesulfonate, however, appears to have the ability to induce mutations of kinds not predictable from the aneuploid series nor obtainable by irradiation. Although the modes of inheritance of most of the EMS-induced mutants have not been definitely established, it is certain that at least some of the mutants inwAve single genes. The chlorina mutant, for example, behaves as a simple recessive; the compac-
_lIutaEion Rc,~carch I ( I 9 6 4 ) ] 8 7
39(~
CHEMICAL MUTAGENESIS IN
T. aestivum
397
turn-like m u t a n t is a simple d o m i n a n t ; a n d a n u m b e r of the lethals s e g r e g a t e d in a p p r o x . 1:3 ratio. The m u t a n t with missing spikelets a n d the one with r u d i m e n t a r y spikes affected 3 i n d i v i d u a l s of 2o a n d 2 of 16, respectively. E v e n those m u t a n t s t h a t s e g r e g a t e d in low f r e q u e n c y m a y well have i n v o l v e d single genes, for a m u t a t i o n m a y sometimes affect less t h a n an entire spike. This would p r e s u m a b l y be p a r t i c u l a r l y true of m u t a t i o n s i n d u c e d b y EMS, which, because of the m a n n e r of its action, is believed c a p a b l e of h a v i n g d e l a y e d effects 6. M a n y of the E M S - i n d u c e d m u t a n t s were of t y p e s not recovered following r a d i a t i o n t r e a t m e n t s . The chlorina a n d o t h e r chlorophyll m u t a n t s , the 2 spike m u t a n t s j u s t m e n t i o n e d , a n d v a r i o u s peculiar t y p e s of dwarfs a n d lethals are m u t a n t s such as rarely, if ever, a p p e a r in r a d i a t i o n experiments. This fact, coupled with the evidence from t h e nullisomies a n d t e t r a s o m i c s t h a t these are not deficiencies or duplications, rules o u t the possibility t h a t t h e y are due to simple losses or a d d i t i o n s of c h r o m a t i n . T h a t t h e y m i g h t be due to more c o m p l e x p a t t e r n s of chromosome a b e r r a t i o n , involving homoeologous loci on several chromosomes, is n e g a t e d b y the evidence of the simple i n h e r i t a n c e of the m u t a t i o n s . If the E M S - i n d u c e d m u t a t i o n s are s i m p l y inherited a n d are not due to deficiencies or d u p l i c a t i o n s of genes or chromosome segments, t h e y m u s t be the result of changes within genes. A c t u a l l y such changes are to be a n t i c i p a t e d from w h a t is k n o w n a b o u t the m a n n e r in which EMS reacts with D N A 12 I t could also be a n t i c i p a t e d t h a t a m u t a g e n capable of inducing intragenic changes would be p a r t i c u l a r l y effective in a polyploid. HALDANE 7 a n d HARLAND 8 p o i n t e d out t h a t the d u p l i c a t i o n of genes in polyploids m a k e s m a n y of these genes non-essential a n d therefore a v a i l a b l e for drastic changes t h a t m a y even alter their function. Such changes are usually not t o l e r a t e d in diploids, where almost e v e r y gene has an essent i a l function to perform. H e x a p l o i d wheat, which a p p a r e n t l y has the large m a j o r i t y of its genes present in triplicate, p r e s u m a b l y has a v a s t reservoir of loci t h a t can be m u t a t e d in various w a y s w i t h o u t serious d e t r i m e n t to the plant. An e x a m p l e of the k i n d of locus u n d e r consideration is N e a t b y ' s virescent on chrom o s o m e 3 A. E x p e r i m e n t s have shown 21 t h a t virescent (v) is a m u t a t i o n which has a n t i m o r p h i c action with respect to its n o r m a l allele, V; t h a t is, whereas V p r o m o t e s t h e d e v e l o p m e n t of chlorophyll, v interferes with it. Loss of l T has no effect on chlorop h y l l d e v e l o p m e n t , because it has v i r t u a l d u p l i c a t e s on chromosomes 3 A a n d 3 D. O n l y b y reducing the t o t a l dosage of V a n d its d u p l i c a t e s from 6 to 2 can it be shown t h a t V is essential to n o r m a l chlorophyll d e v e l o p m e n t (SEARS, unpublished). I t seems a reasonable a s s u m p t i o n t h a t at least some of the E M S - i n d u c e d m u t a t i o n s are like v in t h a t t h e y are effective because t h e y are changed in function. T h e i n t r a genic changes i n v o l v e d m a y be s u b s t i t u t i o n s of one nucleotide for another, or t h e y m a y be deletions of nucleotides. The resulting m i n u t e r e a r r a n g e m e n t s or deletions, which do not i n a c t i v a t e the gene b u t only change its function, are more effective t h a n changes of m u c h g r e a t e r physical e x t e n t , such as deletions of an entire chromosome. If it is true t h a t EMS induces intragenic changes p r i m a r i l y , it would be reasonable to e x p e c t a fair p r o p o r t i o n of these to be d o m i n a n t , for it seems u n l i k e l y t h a t all would h a p p e n to have j u s t the right level of a c t i v i t y t h a t 2 doses would be effective whereas one was not. I t m a y therefore be assumed t h a t the m u t a n t sectors o b s e r v e d in t h e M1 p l a n t s were m o s t l y or all due to d o m i n a n t m u t a t i o n s . C y t o p l a s m i c or p l a s t i d effects are not e n t i r e l y r u l e d out as a cause of at least some of the sectors,
3lutation Research I (1964) 387 399
~0~
H. K. SHAMA RAO AND E. R. SEARS
however. A t t e m p t s to recover the presumed dominant m u t a n t s in M~ largely failed, but this m a y be attributed to (x) failure of the sector to extend into the floral organs of the spike, and/or (2) lethality of the m u t a n t when it affects an entire embryo or seedling. As an example of the latter factor, it seems likely that the large majority of the m a n y sectors recordect that involved necrosis would have been lethal at a very early stage, probably in the embryo. T w o of the 8 lethal mutations classified as lutescent in Table V came from M~ plants with yellow sectors. These were presumably instances of recovery of dominant mutations. In the planning of this experiment it was assumed that if mutations could be induced on m o n o s o m e s , these might appear in M~ even though recessive, and thus be i m m e d i a t e l y located as to the chromosome concerned. The results did not bear out this expectation. E x c e p t for certain predictable types of m u t a t i o n e v i d e n t h due to deficiencv for particular loci on the m o n o s o m e , there was no substantial difference between the frequency of M~ mutant sectors in m o n o s o m i c s and disomics. Further consideration shows that m o s t mutations of the type expected following EMS treatment would probably not be expressed when hemizygous. In the disomic 5 alleles of the gene being changed are present, and the recessive mutant in one dose is unable to o v e r c o m e the effect of all 5 alleles but can do so in 2 doses. In the m o n o s o m i c there are still 4 alleles for the mutant gene to overcome, and the probability of its being able to do st) is not great. ACKNO\VLED(;EMENTS
This investigation was carried out while the senior author was an at the University of Missouri under the Point Four Program of the national D e v e l o p m e n t , United States (iovernment. He is grateful for the deputation and to A I D for providing financial assistance. grateful to Dr. C. F. KONZAI¢ for recommending the use of ethyl and for supplying information concerning methods of treatment.
Exchange Visitor Agency for haterto A E E T (India) Both authors are methanesulfonate
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CHEMICAL MUTAGENESIS IN T . a e s t i v u m
39c~
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Mutation Research i 11964) 387 399