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Induced plasmon mutations affecting the growth habit of peanuts, A. hypogaea L.
347
Mutation Research, 51 ( 1 9 7 8 ) 3 4 7 - - 3 6 0 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
INDUCED PLASMON MUTATIONS A F F E C T I N G THE GROWTH HABIT OF PEANUTS, A. bypogaea L.
A. L E V Y * a n d A. A S H R I
The Hebrew University, Faculty of Agriculture, Rehovot (Israel) 21 N o v e m b e r 1977) ( A c c e p t e d 23 March 1 9 7 8 )
(Received
Summary The effectiveness of the acridines ethidium bromide (EB) and acriflavine in inducing plasmon mutations was compared with the alkylating agents ethyl methanesulphonate (EMS) and diethyl sulphate and to 7-rays. The growth habit (trailing versus bunch) of peanuts (A. hypogaea), controlled by geniccytoplasmic interactions, was utilized. Breeding tests distinguishing nuclear from plasmon mutations were developed and are described in detail. Plasmon mutations were induced, b u t there were differences in mutation yields between the cultivars and the mutagens. In the trailing line, T B R [ V 4 ] , 135 independent bunch mutations (in 1804 M2 families) were recovered: 28 bred true while 97 continued to segregate into M3 and M4. Of the 28, 14 were nuclear from an Hb to an hb allele while 14 were in the plasmon. Of the latter, 6 were induced by EMS, 7 by 7-rays and 1 by acriflavine. Somatic segregation of heteroplasmons, i.e. more plasmon mutations, could be responsible for many of the mutations that continued to segregate, b u t in some cases chromosomal aberrations might be involved. In the bunch cultivars there were 32 independent trailing mutations (in 3895 M2 families), one bred true for trailing, while the others continued to segregate into M3 and M4. Plasmon mutations could n o t be ascertained because of the continuing segregations, b u t these mutations manifested sorting o u t of heteroplasmons.
* Present address: Medicinal and Spice Crops Division, Agricultural Research Organization, Volcani Center, Bet-Dagan (Israel). Cultivars: DA, Dixie Anak; V4, Virginia Bet-Dagan No. 4; $9, Sepharadi No. 9; TBR[V4], True Breeding Runner [V4]. Mutagens: Act, acriflavine; DES, diethyl sulphate; EB, ethidium bromide; EMS, e t h y l m e t h a n e sulphonate. Plasmons: [O], "others"; [V4], "V4".
Abbreviations.
6
3 4
1804
7-26
Total
--
--
Act 2
Total
--
--
--
0.8
0.8 1.5
0.2
--
.
2.8
--
.
14
1 9
--
2
1
1
.
.
w
0.8
0.3 3.4
--
1.2
0.5
0.3
.
7
97
51 28
6
I
4
5.4
13.6 10.5
1.3
0.6
2.1 1.9
179
38 141
3716
532 216
755
77
197
511 1428
--
---
--1
--
--
--
1
--
Number
--
---
0.03
---
--
--
--
0.07
--
%
True breeding in M 3
2
1 1
29
11 3
1
1
7 3
3
Number
1.1
2.6 0.7
0.8
2.0 1.4
0.1
1.3
1.5
0.9 0.5
%
Segregating in M 3
MUTAGENICTREAT-
a DES, 1 1 4 . 8 m M ( 1 5 m l / l ) , 30 rain; EMS, 38.7 m M (4 m U D , 24 h; E B 1 = 1.26 m M ( 5 0 0 m g / l ) , 2 4 h; E B 2 = 1.26 m M , 72 h; A c r 1 = 0 . 3 8 m M ( i 0 0 m g / l ) , 48 h; A c t 2 = 0.11 m M ( 3 0 mg/1), 13 d a y s ; 7 - 1 4 = g a m m a i r r a d i a t i o n , 1 4 k~ad; 7 - 2 6 = g a m m a i r r a d i a t i o n , 26 krad.
--
EBI
Developing embryos
14
1
--
--
--
375 266
1
171
--
333 211
%
Number
%
Number
%
Number
Nuclear
Cytoplasmic
No.
Segregating in M 3
BY DIFFERENT
Trailing mutations
True breeding in M 3
INDUCED
No.
MUTATIONS
M2 family
AND TRAILING
Bunch mutations
OF BUNCH
M2 family
M2 FAMILIES)
Bunch cultivars
(% O F T O T A L
Trailing TBR V4
448
7-14
Act
EB2
EB1
EMS
DES
Mature seeds
Treatmerit
NATURE AND FREQUENCY MENTS a
TABLE 1
00
co
349 Introduction
Natural plasmon mutations and divergence have taken part in the evolutionary process [17,32,34]. Studies on the induction of plasmon mutations were undertaken fairly recently, mostly in microorganisms [17,24,30,33] and a few in higher plants dealing with plastid mutations [15,37] or cytoplasmic male sterility [ 16,25]. Such mutations affect the fertility and/or the viability of the plants, thus making genetic analysis of the mutants difficult. EB and acriflavine, reported to be highly effective in inducing plasmon mutations in yeast [35], Chlamydomonas [1] and Trypanosoma [36], have n o t been studied in higher plants. Hence, a study was initiated in 1971 with the bunch versus trailing growth habit of peanuts which is controlled by geniccytoplasmic interactions [2,3], to evaluate the potency of 7-rays, the acridines EB and acriflavine and the alkylating agents EMS and DES in inducing plasmon mutations. This system has the advantages of clear phenotypic differences, cleistogamy and full fertility of the plants. The initiation of the research was stimulated by the natural plasmon variation found in this system [4]. Recently, EB treatments using the procedures developed in the present study [27] were reported to induce cytoplasmic male sterility mutations in P e n n i s e t u m american u m [10]. Apart from the economically important cytoplasmic male sterility used in several crop plants for the production of hybrid cultivars, other agronomic characters affecting yield and resistance to pathogens are cytoplasmically inherited [22,39]. Studies on plasmon-induced mutations in higher plants could, therefore, have important consequences in breeding and genetic vulnerability, as well as theoretical implications for the role of the plasmon in evolution and divergence. Materials and methods Fully mature seeds were irradiated with 7-rays at doses of 14 and 26 krad from a 6°Co source at the Soreq Nuclear Research Centre at a dose rate of 27 krad/h. The acridines EB and acriflavine, and the alkylating agents EMS and DES, were applied to mature seeds or developing embryos attached to the plants. The concentrations were determined after preliminary physiological sensitivity trials [5,26]. The seeds were pre-soaked in deionized water for 1 h at room temperature. During the treatments (Table 1) air was bubbled into the solutions. After the treatments, the seeds were rinsed for 30 min in running tap water, sown immediately and watered well. The developing embryos, at different stages of development, were treated as described earlier [5,27]. 5 pure-line, homozygous cultivars, which represented different botanical types of peanuts [19] according to their branching and reproductive patterns, were treated with the mutagens. The bunch cultivars were V4, DA, $9 and Congo. V4 has the [V4] plasmon and the nuclear genotype HblHblhb2hb:hbshbs [3]; DA, $9 and Congo have the [O] plasmon and the nuclear genotype hblhblHb2Hb2hbshbs [3]. $9 and V4 also served as testers for the growth habit mutations (see Figs. 1--4 below). The trailing line used was TBR[V4] which has the [V4] plasmon and is HblHblHb2Hb2hbshbs [3]. The M~, M2 and where necessary, Ms and M4 plants of the different muta-
350 genic treatments, were studied several times during the growing season for their growth-habit mutations, namely bunch mutants in the trailing cultivar and trailing ones in the bunch cultivars. (For details see Levy and Ashri [27].) The mutants were screened by their pod and seed characteristics to be true to type and n o t chance outcrosses. Border rows and seed-increase plots of the different cultivars grown for several years served as controls for spontaneous mutations.
Results and discussion
Genetic tests distinguishing cytoplasmic from nuclear mutations Breeding tests based on the genetic models proposed by Ashri [2,3], were (I) C y t o p l a s m i c m u t a t i o n s : [ V 4 ] -~ [ m l]
? V4, bunch [ V 4 ] H b i H b 1h b 2 h b 2 h b s h b 5
TBR[V4] (trailing) [V4] Hb 1HblHb2Hb2hb5hb 5
X
Bunch mutant [ml] Hb 1Hbl Hb2Hb2hbshb 5
F 1 trailing [ V 4 ] H b l I - I b l I - I b 2 h b 2 h b s h b 5
®,
F 2 43-I V 4 ] H b l H b l H b 2 - h b s h b 5 I[V4]
HblHblhb2hb2hbshb
trailing 5 bunch
R e c i p r o c a l cross:
F 1 b u n c h [ m 1] H b l H b l H b 2 h b 2 h b s h b ×~
5
F 2 [ml] trailing: bunch ? C o n c l u s i o n . R e c i p r o c a l d i f f e r e n c e s a r e e x p e c t e d in t h e F 1 . F 2 s e g r e g a t i o n will d e p e n d u p o n t h e i n t e r a c t i o n o f t h e m u t a n t c y t o p l a s m a n d t h e d i f f e r e n t loci.
(II) N u c l e a r m u t a t i o n (a) H b 2 ~ h b 2
TBR[V4] trailing [ V 4 ] H b i H b I H b 2 H b 2 h b 5hb 5 Bunch mutant IV4] Hb iHblhb2hb2hbshb 5
Q V4 bunch [V4] Hb iHb lhb2hb2hb 5hb 5
F 1 bunch IV4] HblHblhb2hb2hbshb5
®~
F 2 all b u n c h I V 4 ] I-Ibl H b l h b 2 h b 2 h b s h b 5 R e c i p r o c a l croas. T h e s a m e F I a n d F 2 r e s u l t s will b e o b t a i n e d . C o n c l u s i o n . N o r e c i p r o c a l d i f f e r e n c e s a r e e x p e c t e d in F 1 a n d F 2 ; b u n c h r e c i p r o c a l F I ' s i n d i c a t e recessive m u t a t i o n f r o m H b 2 t o h b 2.
(b) H b I -+ h b I
TBR[V4] trailing
9 V4 bunch [V4] HblHblhb2hb2hbshb5
X
Bunch mutant [V4] hblhblHb2Hb2hbshb5
F I trailing I V 4 ] H b l h b l H b 2 h b 2 h b s h b
®¢
S
F 2 I-~[V4] I-Ibl-Hb2-hbshb $ trailing i-~ all r e m a i n i n g g e n o t y p e s ; b u n c h R e c i p r o c a l cross. T h e s a m e F I a n d F 2 r e s u l t s will b e o b t a i n e d C o n c l ~ l o n . N o r e c i p r o c a l d i f f e r e n c e s a r e e x p e c t e d in F 1 a n d F 2 ; t r a i l i n g r e c i p r o c a l F l ' s i n d i c a t e recessive m u t a t i o n f r o m H b I t o h b I .
Fig. 1. B r e e d i n g t e s t s d i s t i n g u i s h i n g c y t o p l a s m i c f r o m n u c l e a r m u t a t i o n s t h a t c h a n g e g r o w t h h a b i t f r o m t r a i l i n g t o b u n c h . V 4 w a s u s e d as a t e s t e r ( V 4 p l a s m o n ) .
351 (I) C y t o p l a s m i c m u t a t i o n s : [V4] -+ [ m l ] TBR[V 4] Wailing [V4] Hb i H b iHb21-Ib2hb 5hb 5 $9, bunch [O] h b l h b l H b 2 H b 2 h b s h b 5
X
Bunch mutant [ml] HblHblHb2Hb2hbshb S
F 1 trailing [O] H b l h b l H b 2 H b 2 h b s h b 5 (II) Nuclear m u t a t i o n (a) Hb I --~hb I
TBR [V4] trailing 9 S9,bunch [O] h b l h b l H b 2 H b 2 h b s h b 5
X
Bunch mutant [V4] hb l h b l H b 2 H b 2 h b s h b 5
F 1 b u n c h [O] h b l h b l H b 2 H b 2 h b s h b 5
®~
F 2 all b u n c h [O] h b l h b l H b 2 H b 2 h b s h b 5 (b) Hb 2 --~ hb 2 TBR [V4] trailing 9 $9, b u n c h [O] b b l b b l H b 2 H b 2 h b s h b 5
X
Bunch mutant IV4] H b l H b l h b 2 h b 2 h b s h b $
F 1 b u n c h [O] H b l h b l H b 2 h b 2 h b 5 h b 5
®~
F 2 5 Wailing: 11 b u n c h
Conclusion. Trailing F 1 h y b r i d s indicate a p l a s m o n m u t a t i o n w h e r e a s b u n c h F 1 's indicate recessive m u t a t i o n in Hb I or Hb 2 loci. T h e l o c u s i n v o l v e d can be identified b y the presence or absence o f segregation in t h e F 2 .
Fig. 2. Breeding tests distinguishing c y t o p l a s m i c f r o m nuclear m u t a t i o n s that change g r o w t h habit f r o m trailing to b u n c h w h e n $9 is the female parent tester (O p l a s m o n ) .
developed to distinguish nuclear mutations from cytoplasmic ones according to the growth habit of the F1 hybrids and sometimes F2 progenies (Figs. 1--4). When bunch mutations from the trailing cultivar are crossed reciprocally to V4, the nature of the mutations can be determined by the F~ hybrid phenotypes (Fig. 1). This is also true when $9 is the female parent tester (Fig. 2). It should be noted that when V4 is used as a tester reciprocally the plasmon is studied, whereas with $9 as a female tester, only the nuclear genotype is checked. For trailing mutations, $9 as a female parent tester facilitates a rapid and sharp determination of the nature of the mutations (Fig. 4); cytoplasmic mutations produce bunch F~ hybrids whereas nuclear ones yield trailing F~'s. Therefore, reciprocal crosses are not necessary. If V4 is the tester, no phenotypic F~ differences are expected, and F2 segregations are required to distinguish between nuclear and cytoplasmic mutations (Fig. 3). Since the genetic system controlling growth habit in peanuts may be more complex and additional loci with different interactions might be involved [3,21], the identification o f the mutations should be based essentially on the FI results, especially on reciprocal crosses between the mutants and V4.
Nature of bunch mutations from the trailing cultivar No growth habit mutations were detected in the M1. From the different
352
(I) C y t o p l a s m i c m u t a t i o n : [ O ] -+ [ m 2 ]
9 V4, bunch [O] HblHblhb2hb2hbshb
5
X
Bunch cultivar [O] hbl hbl Hb2 Hb2hbshb5 t Trailing mutant [ m 2] h b l h b l H b 2 H b 2 h b s h b 5
F 1 trailing [ V 4 ] H b l h b l H b 2 h b 2 h b s h b 5
®~
F2 1~ I V 4 ] H b l - H b 2 - h b s h b 5 t r a i l i n g ~6all remaining genotypes; bunch R e c i p r o c a l cross
F 1 trailing [ m 2 ] H b l h b l H b 2 h b 2 h b s h b
®~
5
F2 1 [ m 2 ] h b l h b l h b 2 h b 2 h b s h b 5 ' b u n c h i5 I--6 all r e m a i n i n g g e n o t y p e s ; t r a i l i n g T h e F 2 s e g r a t i o n o f 1 : 1 5 is e x p e c t e d if H b 1 a n d H b 2 b e h a v e as d u p l i c a t e l o c i in t h e [ m 2 ] p l a s m o n . However, other interactions and therefore different segregations can be obtained. C o n c l u s i o n . N o r e c i p r o c a l d i f f e r e n c e s a r e e x p e c t e d in F 1. S u c h d i f f e r e n c e s m i g h t a p p e a r in F 2.
(II) N u c l e a r m u t a t i o n (a) h b 1 "-~ H b I Bunch cultivar [O] hblhbl Hb2Hb2hbshbs V4, bunch [V4] HblHblhb2hb2hbshb
5
X
Trailing mutant [O] HblHblHb2Hb2hbshb5
F 1 trailing [ V 4 ] H b l I - I b l H b 2 h b 2 h b s h b 5
@;
F 2 ~ [V4] HblHblHb2-hbshbs, I IV4] Itblltblhb2hb2hbshbs,
trailing bunch
R e c i p r o c a l cross. T h e s a m e F 1 a n d F 2 r e s u l t s will b e o b t a i n e d . (b) h b S -~ H b $ Bunch cultivar
V4, bunch [V4] HblHblhb2hb2hbshbs
×
Trailing mutant [O] hblhblHb2Hb2Hb$Hb5
F 1 trailing [ V 4 ] H b l h b l I - l " b 2 h b 2 H b s b b 5
®~ F2
b u n c h , all g e n o t y p e s h o m o z y g o u s recessive f o r t w o l o c i o r m o r e . 27 3-'2 t r a i l i n g , all r e m a i n i n g g e n o t y p e s .
R e c i p r o c a l cross F 1 trailing [ O ] H b l h b l H b 2 h b 2 H b $ h b 5
®~ F2
b u n c h , g e n o t y p c s h a v i n g t w o d o m i n a n t alleles o r less in a n y c o m b i n a t i o n . 21 t r a i l i n g , all r e m a i n i n g g e n o t y p e s . ~-~
C o n c l u s i o n . N o r e c i p r o c a l d i f f e r e n c e s are e x p e c t e d in F 1. D i f f e r e n c e s in F 2 s e g r e g a t i o n s c a n b e u s e d f o r t h e i d e n t i f i c a t i o n o f t h e locUS i n v o l v e d .
Fig. 3 . B~eedlng t e s t s d i s t i n g u i s h i n g c y t o p l a s m i c f r o m n u c l e a r m u t a t i o n s t h a t c h a n g e g r o w t h h a b i t in c u l t i v a r s $ 9 , C o n g o a n d D A f r o m b t m e h t o t r a i l i n g . V 4 w a s u s e d as a t e s t e r .
353
(I) C y t o p l a s m i c m u t a t i o n : [O] --~[m2] B u n c h cultivar
[O] h b l h b l H b 2 H b 2 h b s h b 5 9 $9, b u n c h [O] b b l h b l H b 2 H b 2 h b s h b 5
Trailing m u t a n t
X
[m2] h b l h b l H b 2 H b 2 h b s h b s
F 1 b u n c h [O] h b l h b l H b 2 H b 2 h b s h b 5 (II) N u c l e s x m u t a t i o n (a) hb 1 --~ Hb 1 B u n c h cultivar
9 $9, b u n c h [O] hb l h b l Hb2 H b 2 h b s h b 5
Trailing m u t a n t
× [O] Hbl Hbl Hb2Hb2hb5hb5 ¢ F 1 trailing [O] H b l h b l H b 2 H b 2 h b s h b 5
(b) hb 5 ~ I-Ib5 B u n c h cultivar
¢ 9 $9, b u n c h [O] h b l h b l H b 2 H b 2 h b s h b 5
Trailing m u t a n t
X
[O] h b l h b l H b 2 H b 2 H b s H b s
F 1 trailing [O] h b l h b l H b 2 H b 2 H b s h b 5
C o n c l u s i o n . B u n c h F 1 h y b r i d s i n d i c a t e a p l a s m o n m u t a t i o n w h e r e a s trailing F 1 's indicate a d o m i n a n t m u t a t i o n in h b I or h b 5. T h e l o c i i n v o l v e d can b e distinguished b y a n o t h e r t e s t e r having t h e G p l a s m o n
[3]. Fig. 4. B r e e d i n g t e s t s d i s t i n g u i s h i n g c y t o p l a s m i c f r o m n u c l e a r m u t a t i o n s w h i c h c h a n g e t h e g r o w t h h a b i t f r o m t h e b u n c h cultivars $ 9 , C o n g o and D A t o trailing w h e n $ 9 is t h e f e m a l e p a r e n t tester.
mutagenic treatments of T B R [ V 4 ] , 137 independent bunch mutations were obtained in the M2 progeny rows. All the bunch mutations, except two that were sterile and 10 that were nearly sterile, were progeny tested in the M3:28 gave uniform bunch progenies while 97 segregated for bunch versus trailing in different ratios (see below}. M3 plants from the 28 true-breeding bunch mutations were crossed reciprocally to V4 and/or to $9 as the female parent. According to the phenotypes of their F1 hybrids with the testers, 14 of them are plasmon mutations (Table 2): 10 mutations crossed to V4 gave reciprocal differences in the F1; since the original cultivar TBR[V4] contained the [V4] plasmon, no reciprocal differences were expected if the mutations did not affect the plasmon (Fig. 1). 4 additional uniform bunch mutations were crossed only to $9 as the female parent, and all the F~'s from these crosses were trailing as expected for plasmon mutations (Fig. 2, i). 3 mutations crossed to V4 were also crossed with S9 as the female parent (Table 2): in two of them, the F~ 's were trailing as expected for a plasmon mutation (Fig. 2, I); the Fl's of the third mutation were bunch, perhaps owing to a change in the genetic background of this mutation [18,21]. F2 populations of the crosses between the above 10 bunch mutations and V4 were studied for growth habit. Only two F2 populations segregated at the ratio of 3 trailing: 1 bunch expected with a plasmon mutation only (Fig. 1, I}. In the F2 populations of the other mutations, different ratios of trailing versus bunch plants were obtained (Table 2}. Such deviations from the expected ratio could
354
TABLE 2 THE ORIGIN OF BUNCH CYTOPLASMIC MUTATIONS FROM TBR[V4] INDUCED BY MATURESEED TREATMENTS AND THE BEHAVIOUR OF THEIR F 1 AND F 2 PROGENIES FROM CROSSES W I T H V 4 A N D / O R $9 Mutation No. 1973
Mutagen a
Tester
F! hybrids, number b and phenotypes with m u t a n t as
F 2 s e g r e g a t i o n b w i t h t h e m u t a n t as male
female
B male
female
T
T
26 200
94 137
5219 5318 5388
7-14 7-26 EMS
$9 $9 $9
3 T 6 T 1 T
. . .
. . .
. . .
5390 5415 5260
EMS Act 1 7-14
$9 V4 V4
1 T 4 T 4 T
. 1 B 3 B
.
.
5260 5261 5261
7-14 7-14 7-14
S9 V4 $9
6B 6 T 1 T
. 4 B .
5279 5308 5332
7-26 7-26 ")'-26
V4 V4 V4
2 T 4 T 5 T
1 B 3 B 4 B
142 246 282
111 232 308
-162 202
-103 162
5381 5394 c
EMS EMS
V4 V4
6 T 2 T
3 B 5 B
345 115
565 370
134 128
141 78
202 68
181 55
5394
EMS
$9
3 T
.
5404 5405 c
EMS EMS
V4 V4
4 T 1 T
5 B 1 B
. .
.
. . .
B
. . .
.
.
22 121
216 501
. 73
. 145
.
.
.
98
.
. 98 28
. 95
. 134 55
a See Table I for details of treatments. b B, B u n c h ; T, T r a i l i n g . c F i t n e s s o f F 2 s e g r e g a t i o n f o r 3 t r a i l i n g : l b u n c h w i t h t h e m u t a n t as m a l e p a r e n t : 5 3 9 4 , P ( 1 d f ) = 0 . 4 0 - 0 . 5 0 ; 5 4 0 5 , P ( 1 d r ) = 0.05---0.10.
result from varying intensities of haplontic selection or from additional changes in the genetic background of the mutants as found for other mutations in peanuts [ 1 8 , 2 1 ] . In this regard, it should be noted that many loci might be involved in the hormonal control of growth habit in peanuts [ 3 , 4 0 ] . 14 other true-breeding independent mutations gave, in reciprocal crosses with V4 and/or $9 as female parent, only bunch FI plants and are therefore nuclear mutations (Fig. 1, II; Fig. 2, II). 8 of them gave no reciprocal F, differences when crossed to V4; hence the mutation was from Hb~ to hb2 and their genotype is the same as V4. This assessment was reinforced by the lack of segregation for growth habit in two F2 populations obtained from these crosses: all the progenies were bunch. Moreover, 5 of the 8 mutations above were also crossed to $9 as the female parent, and all their F, 's were bunch as expected for a nuclear mutation (Fig. 2, II). The other 6 mutations were crossed only to $9 as female parent. They behaved as nuclear mutations because their F,'s were all bunch (Fig. 2, II), but the Hb locus or loci involved could n o t be determined w i t h o u t further test crosses. The other 97 independent bunch mutations in Ms progeny tests gave a wide range of segregations: from 1 to 90% of the plants were bunch in the different
355
u~
20
z
o F--
4c o o z
6 4
0
4~-~
,I - '10
%. BUNCH
2~-~0 ~-40 4t-50 54-60 6~-70 T~-80 8t-90
PLANTS
IN M3
POPULATION
Fig. 5. F r e q u e n c y d i s t r i b u t i o n o f t h e i n d e p e n d e n t s e g r e g a t i n g b u n c h m u t a t i o n s a c c o r d i n g t o t h e p r o p o r t i o n s o f b u n c h p l a n t s in t h e M 3 p r o g e n y r o w s . ( O n e m u t a n t M 2 I~lant per m u t a t i o n w a s t e s t e d in M 3.)
progeny rows (Fig. 5). 85 bunch plants from different segregating M3 families were progeny tested in M4:79 continued to segregate, usually with a preponderance of trailing progenies, while 6 gave bunch progenies only. 26 M3 trailing offspring of different bunch M2 plants were progeny tested in M4:23 gave uniform trailing M4 rows, while 3 segregated with a preponderance of trailing plants. The segregations of the bunch mutants and the M4 results of the above trailing-progeny tests cannot be interpreted as nuclear mutations or outcrosses in view of earlier findings showing that the bunch growth habit is recessive to
'10 z 0
23 LL 0 0 Z
t-t0 %
tt-20 TRAILING
24-30
31-40
PLANTS
41-50 IN
M3
51-60
I
~-70
71-80
81- 9 0
POPULATION
Fig. 6. F r e q u e n c y d i s t r i b u t i o n o f t h e i n d e p e n d e n t segregating trailing m u t a t i o n s a c c o r d i n g t o t h e p r o p o r t i o n s o f trailing plants in t h e M 3 p r o g e n y r o w s . ( O n e m u t a n t M 2 p l a n t per m u t a t i o n w a s t e s t e d in M3. )
356 the trailing one [ 2,21]. Neither can these results be interpreted by nuclear gene models in which dominance of the bunch growth habit [23] or other genie interactions [12] is assumed. The segregations of the offspring of the bunch mutants in the M3 (Fig. 5) and M4 can be interpreted in terms of somatic segregations [15,24]. Accordingly, in the segregating bunch mutations, the initial m u t a n t cells were heteroplasmic and, through sorting o u t of the m u t a t e d particles, bunch plants were recovered in M2. Bunch or trailing plants could be homoplasmic or heteroplasmic, depending on the proportions of the particles in the "mixed cells", as in plastid mutations [9,15,20]. Thus, different segregation ratios of bunch versus trailing offspring could then be obtained as in M3 and M4, depending u p o n the ratios of the particle types and their relative multiplication rates. Somatic segregation can also explain the behaviour of the FI hybrids between V4 and bunch or trailing M3 progenies from the 14 segregating bunch mutations, especially the reciprocal F1 differences and sibs' (from the same pods) segregations (Table 3). Some of the progenies of the segregating bunch mutants were semisterile. Therefore, the possible presence of chromosomal aberrations was checked in the PMC's of such mutants. Indeed, observations of c h r o m o s o m e stickiness at Mx and AI, asynchronized m o v e m e n t of the chromosomes at AI, and hexades with t w o micronuclei at TII suggest that induced chromosomal aberrations led to some of the growth habit changes. The aberrations were difficult to analyze because of the small size and large number of chromosomes (2n = 40) in cultivated peanuts.
TABLE 3 GROWTH HABIT LINES WITH V4
OF
FI'S
FROM
Mutation No. 1972
Growth habit of the mutant a
3577-18 b 3583-9 3618-1
CROSSES
OF
MUTANTS
SEGREGATING
F 1 hybrids, number and phenotypes male
female
B
1B, 2T
1B
T B
1T 5T
2B 2B
3608-1 3657-11 3789-11
B T T
1B IT IT
-2B --
3899-24 b 3929-15 3949-5 b
T T T
----
1T, 1B 2B 1T, 1B
3950-8 3966-5 3946-18
T T T
1T -1T
3B 1B 2B
4693-19 5042-18
B B
-4T
2B 1B
a B , B u n c h ; T, T r a i l i n g . b N o t e s e g r e g a t i o n o f sibs.
FROM
BUNCH
a w i t h m u t a n t as
M3
357
Nature of trailing mutations from the bunch cultivars 32 independent trailing mutations were obtained in the M2 from the different bunch cultivars. All but one of them segregated in the M3 for buhch versus trailing, usually with more bunch progenies (Fig. 6). In 6 trailing mutations, segregation for pod or shoot traits characteristic of other cultivars was observed in the M3; therefore, cross pollination was suspected, and studies with them were discontinued. The continued segregation of the trailing mutants made it difficult to determine their genotype by appropriate test crosses (Figs. 3, 4). However, 13 M3 trailing plants from different mutations were crossed reciprocally to the testers, and their FI and F2 behaviour is summarized in Table 4. The M3 segregations and F1 results of most of the trailing mutations can be interpreted by a nuclear mutation at one of the Hb loci or by appropriate cross-pollination of the M~ plants. However, some of the trailing-mutant results are compatible with somatic segregation, as discussed for the bunch mutants above. Since most of the trailing M2 mutants segregated in the M3, they could have been heteroplasmic or heterozygous for Hb~ or Hbs. In the latter, the segregation expected in M3 was 3 trailing: 1 bunch, but most M3 progeny rows gave different ratios (Fig. 6). Such results could be obtained from sorting out of plasmon mutations, or from changes in the nuclear genetic background [18,21]. It was not possible to eliminate one of the above alternatives with the
TABLE 4 T H E O R I G I N OF T R A I L I N G M U T A T I O N S AND T H E B E H A V I O U R OF T H E I R F 1 A N D F 2 PROGEN I E S F R O M C R O S S E S W I T H V 4 A N D / O R $9 Mutation No. 1973
5146 5590 c 5644
Origin cultivar
V4 V4 $9
Mutagenic treatment a
Act 2 EMS EB1
Tester
V4 $9 $9
F I hybrids, number b and p h e n o t y p e s w i t h m u t a n t as
F 2 segregation b with m u t a n t as
male
female
B
1B 2B 3T
2B 3T .
. 109 .
male
.
female T .
B . 77
. 107
106
89 118
156 148
95 100
249
344
0
70
25
311
0
.
.
5532
Congo
EMS
$9
2B
IT, I B
.
.
.
.
5518 5548
Congo Congo
7-26 7-26
$9 $9
2T, 1 B --
2T, 2 B 2T
. .
. .
. .
. .
5556 5559
Congo Congo
Act 1 Acr 1
V4 V4
5T 2T
5B 4B
176 128
5559
Congo
Act 1
$9
7T
2B
5427 5428
DA DA'
7-14 7-14
$9 $9
6B 2B
. 6B
147
5429
DA
7"14
V4
IT
1B
104
5442
DA
7-14
$9
--
2B
.
5443
DA
7-14
$9
1 T , 2B
2B
225 d
.
. .
. .
. .
.
106 .
T
.
0
.
a S e e Table 1 for d e t a i l s o f t r e a t m e n t s . A c t 2, t r e a t m e n t o f d e v e l o p i n g e m b r y o s w i t h acriflavine 0 . 1 1 m M for 1 3 d a y s . All o t h e r t r e a t m e n t s w e r e o n m a t u r e seeds. b B, B u n c h ; T, Trailing. c M u t a t i o n t h a t b r e d true in t h e M 3. All o t h e r trailing m u t a t i o n s s e g r e g a t e d in t h e M 3. d S e g r e g a t i o n o f a b u n c h F 1 plant.
358 data furnished by the F1 hybrids of the trailing mutants and the known testers. The reciprocal differences between the F1 hybrids of the true breeding mutation No. 5590 (Table 4) cannot be attributed to a nuclear mutation and may again indicate a plasmon mutation.
Concluding remarks Most of the true-breeding plasmon mutations were recovered in the trailing cultivar TBR[V4] after v-ray and EMS treatments of mature seeds (Table 1). The EB treatments proved highly effective in inducing various shoot mutations [27]; however, all the true-breeding growth-habit mutations obtained from the EB treatments were nuclear mutations (Table 1). In pearl millet (P. americanum), EB induces a high frequency of cytoplasmic male sterile mutations [10]; and in microorganisms, EB and acriflavine are highly efficient in the induction of cytoplasmic mutations [1,29,35]. Therefore it was surprising that in the growth habit of peanuts, even severe treatments with these two mutagens were less efficient than EMS and v-rays in inducing such mutations. The effectiveness of EMS and v-rays is in agreement with other studies reporting the induction of cytoplasmic male sterility in barley by EMS and X-rays [16], in sorghum by alkylating agents [28], in sugar beet with v-rays [25] and plastid mutations by EMS in tobacco [15]. The differences in the frequency of plasmon mutations between the mutagens may result from their different modes of action [6,14] or from different intensities of diplontic selection [13,24]. EB has been reported to have a great affinity for circular DNA [31,38]. In view of its low efficiency in inducing plasmon mutations in the growth habit of peanuts, it is tempting to suggest that the cytoplasmic DNA component affecting this system might not be circular. From the treatments of developing embryos, small M1 and M2 populations were obtained. Therefore, it is difficult to compare treatment of mature seeds with treatment of embryos for the induction of plasmon mutations. On the other hand, the differences between the cultivars in the frequency of such mutations underline the genotypic influence on plasmon mutations which is known for nuclear mutations [7,8].
Acknowledgements This research was financed in part by a grant to A.A. made by the U.S. Department of Agriculture Research Service, authorized by P.L. 480, Project No. A10-CR-77, Grant No. FG-Is-281. The report is based in part on the dissertation of A.L. submitted to the Hebrew University in partial fulfilment of the requirements for the Ph.D. degree. We acknowledge with thanks the technical assistance of Y. Berg, A. Cohen, M. Kochavi and R. Offenbach in various phases of this research. References 1 Alexander, N.J., N.W. Gilham and J.E. B o y n t o n , The m i t o c h o n d r i a l g e n o m e o f C h l a m y d o m o n a s : i n d u c t i o n o f m i n u t e c o l o n y m u t a t i o n s b y acriflavine and their inheritance, Mol. Gen. Genet., 130 (1974) 275--290.
359 2 Ashri, A., Genic-cytoplasmic i n t e r a c t i o n s affecting growt h h a b i t in peanuts, A. hypogaea, II. A revised model, Genetics, 60 (1968) 807--810. 8 Ashri, A., Plasmon divergence in p e a n u t s (Arachis hypogaea): A t hi rd plammon and locus affecting growth habit, Theor. Appl. Genet., 48 (1976) 17--21. 4 Ashri, A., Frequencies of p l a s m o n t y p e s in a germ plasm sample of peanuts, Arachis hypogaea, Euphytiea, 25 (1976) 777--785. 5 Ashri, A., and A. Levy, Sensitivity of d e v e l o p m e n t a l stages of p e a n u t (A. hypogaea) e m b r y o s and ovaries to several chemical m u t a g e n t r e a t m e n t s , Radiat. Bot., 14 (1974) 223--228. 6 Auerbach, C., and B.J. Kilbey, M u t a t i o n in eukaryotes, Annu. Rev. Genet., 5 (1971) 163--218. 7 Bllxt, S., M u t a t i o n genetics in Pisum, Agric. Hort. Genet., 30 (1972) 1--293. 8 Borojevic, K., Factors influencing the m u t a n t spectrum and the quality of m u t a n t s : differences depending on the genotype, in: Manual on Mutation Breeding, IAEA, Vienna, 1970, pp. 125--126. 9 Burk, L.G., R.N. Stewart and H. Dermen, Histogenesis and genetics of a plastid controlled c hl orophyl l variegation in tobacco, Am. J. Bot., 51 (1964) 718--723. 10 Burton, G.W., and W.W. Hanna, E t h i d i u m b r o m i d e induc e d male sterility in pearl millet, Crop Sci., 16 (1976) 731--732. 11 Clemen t Jr., W.M., Plasmon m u t a t i o n s in cytoplasmic male sterile pearl millet, Pennisetum typhoides, Genetics, 79 (1975) 583--588. 12 Coffelt, T.A., Inheritance of growth h a b i t in an infraspecific cross p o p u l a t i o n in peanuts, J, Heredity, 65 (1 974) 160--162. 13 D ' A m a t o , F., Chimera f o r m a t i o n in m u t a g e n treated seeds and diplontic selection, in: The Use of I n d u c e d Mutations in Plant Breeding, Radiat. Bot., Suppl. 5 (1965) 303---315. 14 Drake, J.W., Mutagcnic mechanisms, Annu. Rev. Genet., 36 (1969) 247--268. 15 Dulieu, H., Sur les diff6rents t y p e s de m u t a t i o n s extranucl6aires induites par le m 6 t h a n e sulfonate d' ~th yle chez Nicotiana tabacum L., M u t a t i o n Res., 4 (1967) 177--189. 16 Favret, E.A., and G.S. Ryan, Two cytoplasmic male sterile m u t a n t s i nduc e d by X-rays and EMS, Barley Newsletter, 8 (1964) 42. 17 Gilham, N.W., Genetic analysis of the chloroplast and m i t o c h o n d r i a l genomes, Annu. Rev. Genet., 8 (1974) 347--391. 18 Gregory, W.C. (Ed.), A r a d i a t i o n breeding e x p e r i m e n t wit h peanuts, Radiat. Bot., 8 (1968) 81--147. 19 Gregory, W.C., M.P. Gregory, A. Krapovickas, B.W. Smith and J.A. Yarbrough, Structures a nd genetic resottrces of peanuts, in: C.T. Wilson (Ed.), P e a n u t s - Culture and Uses, Am. Peanut Res. Educ. Assoc., O k l a h o m a State Univ., Stillwater, Okla., 1973, pp. 47--133. 20 Hagemann, R., and M. Scholze, S t r u k t u r u n d F u n k t i o n der genetisehen I n f o r m a t i o n in den Plastiden, VII. Vererbung u n d E n t m i s c h u n g genetisch unterschiedlicher Plastidensorten bei Pelargonium zonale, /kit. Biol. Zbl., 93 (1974) 625--648. 21 Hammons, R.O., Genetics of Arachis hypogaea, in: C.T. Wilson (Ed.), Peanuts -- Culture and Uses, Am. Peanut Res. Educ. Assoc., O k l a h o m a State Univ., Stillwater, Okla., 1973, pp. 135--173. 22 Harvey, P.H., C.S. Levings III and E.A. Wernsman, The role of e x t r a c h r o m o s o m a l inheritance in p l a n t breeding, Adv. Agron., 24 (1972) 1--27. 23 Hassan, M.A., and D.P. Srivastava, Inheritance of grow t h h a b i t in g r o u n d n u t (Arachis hypogaea L.), J. Indian Bot. Soc., 45 (1966) 293--295. 24 Jinks, J.L., E x t r a c h r o m o s o m a l Inheritance, Prentice-Hall, Englewood Cliffs, N.J., 1964, p. 178. 25 Kinoshita, T., Genetical studies of cytoplasmic male sterility induced by gamma irradiation in sugar beets, Jap. J. Breed., 26 (1976) 256--265. 26 Levy, A., and A. Ashri, Differential physiological sensitivity of p e a n u t varieties to seed t r e a t m e n t w i t h the m u t a g e n s e t h i d i u m bromide, MNNG and sodium azide, Radiat. Bot., 13 (1973) 369--373. 27 Levy, A., and A. Ashri, E t h i d i u m b r o m i d e - - a n efficient m u t a g e n in higher Plants, Mut a t i on Res., 28 (1975) 397--404. 28 Malinovsky, B.N., N.N. Zoz and A.I. Kitaev, I n d u c t i o n of c yt opl a s mi c male sterility in sorghum by chemical mutagcns, Genetika, 9 (1973) 19--27. (Russian, English abstract) 29 Nagley, P., and A.W. IAnnane, Biogenesis of m i t o c h o n d r i a , XXI. Studies on the nature of the mitocho ndrial genome in yeast: the degenerative effects of e t hl di um bromi de on m i t o c h o n d r i a l genetic i n f o r m a t i o n in a respiratory c o m p e t e n t strain, J. Mol. Biol., 66 (1972) 181--193. 30 Preer Jr., J.R., E x t r a c h r o m o s o m a l inheritance: heredit a ry s y m b i o n t s , m i t o c h o n d r i a , chloroplasts, Ann. Rev. Genet., 5 (1971) 364--406. 31 Richardson, J.P., and S.R. Parker, Effect of e t h i d i u m bromi de on transcription of linear and circular DNA templates, J. Mol. Biol., 78 (1973) 715--720. 32 Sager, R., Evolution of preferential transmission m e c h a n i s m s in c yt opl a s mi c genetic systems, in H.H. Smith (Ed.), E v o l u t i o n of Genetic Systems, 1972, pp. 495---502. 33 Sager, R., Cytoplasmic Genes and Organelles, Academic Press, New York, 1972, p. 177. 34 Singh, A., and J.R. Laughnan, Instability of S male sterile c y t o p l a s m in maize, Genetics, 71 (1972) 607--620. 35 Slonimski, P.P., G. Perrodin and J.H, Croft, E t h i d i u m b r o m i d e i nduc e d m u t a t i o n of yeast m i t o c h o n -
360
36 37
38 39 40
dria: c o m p l e t e t r a n s f o r m a t i o n of cells into respiratory deficient n o n - c h r o m o s o m a l " p e t i t e s " , Biochem. Biophys. Res. Commun., 30 (1968) 232--239. Steinert, M., Specific loss of kinetoplastic DNA in Trypa nos oma t i da e t re a t e d w i t h e t hi di um bromide. Exp. Cell Res., 55 (1969) 248--252. Travis, M., D.K. Stewart and G.W. Wilson, Nuclear and c y t o p l a s m i c chloroplast m u t a n t s i nduc e d by chemical mu tagens in Mimulus cardinalis: genetics and ultrastruct~re. Theor. Appl. Genet., 46 (1975) 67--77. Waring, M., Binding of drugs to supercoiled circular DNA: evidence for and against intercalation, Prog. Mol. Subcell. Biol., 2 (1971) 215--231. Washington, W.J., and S.S. Maan, Disease reaction of w h e a t with alien cytoplasms, Crop Sci., 14 (1974) 9 03--905. Ziv, M., A.H. Haievy and A. Ashri, P h y t o h o r m o n e s and light regulation of growth h a b i t in pe a nut s (Arachis hypogaea L.), Plant and Cell Phys., 14 (1973) 727--735.
Mutation Research, 51 ( 1 9 7 8 ) 3 4 7 - - 3 6 0 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
INDUCED PLASMON MUTATIONS A F F E C T I N G THE GROWTH HABIT OF PEANUTS, A. bypogaea L.
A. L E V Y * a n d A. A S H R I
The Hebrew University, Faculty of Agriculture, Rehovot (Israel) 21 N o v e m b e r 1977) ( A c c e p t e d 23 March 1 9 7 8 )
(Received
Summary The effectiveness of the acridines ethidium bromide (EB) and acriflavine in inducing plasmon mutations was compared with the alkylating agents ethyl methanesulphonate (EMS) and diethyl sulphate and to 7-rays. The growth habit (trailing versus bunch) of peanuts (A. hypogaea), controlled by geniccytoplasmic interactions, was utilized. Breeding tests distinguishing nuclear from plasmon mutations were developed and are described in detail. Plasmon mutations were induced, b u t there were differences in mutation yields between the cultivars and the mutagens. In the trailing line, T B R [ V 4 ] , 135 independent bunch mutations (in 1804 M2 families) were recovered: 28 bred true while 97 continued to segregate into M3 and M4. Of the 28, 14 were nuclear from an Hb to an hb allele while 14 were in the plasmon. Of the latter, 6 were induced by EMS, 7 by 7-rays and 1 by acriflavine. Somatic segregation of heteroplasmons, i.e. more plasmon mutations, could be responsible for many of the mutations that continued to segregate, b u t in some cases chromosomal aberrations might be involved. In the bunch cultivars there were 32 independent trailing mutations (in 3895 M2 families), one bred true for trailing, while the others continued to segregate into M3 and M4. Plasmon mutations could n o t be ascertained because of the continuing segregations, b u t these mutations manifested sorting o u t of heteroplasmons.
* Present address: Medicinal and Spice Crops Division, Agricultural Research Organization, Volcani Center, Bet-Dagan (Israel). Cultivars: DA, Dixie Anak; V4, Virginia Bet-Dagan No. 4; $9, Sepharadi No. 9; TBR[V4], True Breeding Runner [V4]. Mutagens: Act, acriflavine; DES, diethyl sulphate; EB, ethidium bromide; EMS, e t h y l m e t h a n e sulphonate. Plasmons: [O], "others"; [V4], "V4".
Abbreviations.
6
3 4
1804
7-26
Total
--
--
Act 2
Total
--
--
--
0.8
0.8 1.5
0.2
--
.
2.8
--
.
14
1 9
--
2
1
1
.
.
w
0.8
0.3 3.4
--
1.2
0.5
0.3
.
7
97
51 28
6
I
4
5.4
13.6 10.5
1.3
0.6
2.1 1.9
179
38 141
3716
532 216
755
77
197
511 1428
--
---
--1
--
--
--
1
--
Number
--
---
0.03
---
--
--
--
0.07
--
%
True breeding in M 3
2
1 1
29
11 3
1
1
7 3
3
Number
1.1
2.6 0.7
0.8
2.0 1.4
0.1
1.3
1.5
0.9 0.5
%
Segregating in M 3
MUTAGENICTREAT-
a DES, 1 1 4 . 8 m M ( 1 5 m l / l ) , 30 rain; EMS, 38.7 m M (4 m U D , 24 h; E B 1 = 1.26 m M ( 5 0 0 m g / l ) , 2 4 h; E B 2 = 1.26 m M , 72 h; A c r 1 = 0 . 3 8 m M ( i 0 0 m g / l ) , 48 h; A c t 2 = 0.11 m M ( 3 0 mg/1), 13 d a y s ; 7 - 1 4 = g a m m a i r r a d i a t i o n , 1 4 k~ad; 7 - 2 6 = g a m m a i r r a d i a t i o n , 26 krad.
--
EBI
Developing embryos
14
1
--
--
--
375 266
1
171
--
333 211
%
Number
%
Number
%
Number
Nuclear
Cytoplasmic
No.
Segregating in M 3
BY DIFFERENT
Trailing mutations
True breeding in M 3
INDUCED
No.
MUTATIONS
M2 family
AND TRAILING
Bunch mutations
OF BUNCH
M2 family
M2 FAMILIES)
Bunch cultivars
(% O F T O T A L
Trailing TBR V4
448
7-14
Act
EB2
EB1
EMS
DES
Mature seeds
Treatmerit
NATURE AND FREQUENCY MENTS a
TABLE 1
00
co
349 Introduction
Natural plasmon mutations and divergence have taken part in the evolutionary process [17,32,34]. Studies on the induction of plasmon mutations were undertaken fairly recently, mostly in microorganisms [17,24,30,33] and a few in higher plants dealing with plastid mutations [15,37] or cytoplasmic male sterility [ 16,25]. Such mutations affect the fertility and/or the viability of the plants, thus making genetic analysis of the mutants difficult. EB and acriflavine, reported to be highly effective in inducing plasmon mutations in yeast [35], Chlamydomonas [1] and Trypanosoma [36], have n o t been studied in higher plants. Hence, a study was initiated in 1971 with the bunch versus trailing growth habit of peanuts which is controlled by geniccytoplasmic interactions [2,3], to evaluate the potency of 7-rays, the acridines EB and acriflavine and the alkylating agents EMS and DES in inducing plasmon mutations. This system has the advantages of clear phenotypic differences, cleistogamy and full fertility of the plants. The initiation of the research was stimulated by the natural plasmon variation found in this system [4]. Recently, EB treatments using the procedures developed in the present study [27] were reported to induce cytoplasmic male sterility mutations in P e n n i s e t u m american u m [10]. Apart from the economically important cytoplasmic male sterility used in several crop plants for the production of hybrid cultivars, other agronomic characters affecting yield and resistance to pathogens are cytoplasmically inherited [22,39]. Studies on plasmon-induced mutations in higher plants could, therefore, have important consequences in breeding and genetic vulnerability, as well as theoretical implications for the role of the plasmon in evolution and divergence. Materials and methods Fully mature seeds were irradiated with 7-rays at doses of 14 and 26 krad from a 6°Co source at the Soreq Nuclear Research Centre at a dose rate of 27 krad/h. The acridines EB and acriflavine, and the alkylating agents EMS and DES, were applied to mature seeds or developing embryos attached to the plants. The concentrations were determined after preliminary physiological sensitivity trials [5,26]. The seeds were pre-soaked in deionized water for 1 h at room temperature. During the treatments (Table 1) air was bubbled into the solutions. After the treatments, the seeds were rinsed for 30 min in running tap water, sown immediately and watered well. The developing embryos, at different stages of development, were treated as described earlier [5,27]. 5 pure-line, homozygous cultivars, which represented different botanical types of peanuts [19] according to their branching and reproductive patterns, were treated with the mutagens. The bunch cultivars were V4, DA, $9 and Congo. V4 has the [V4] plasmon and the nuclear genotype HblHblhb2hb:hbshbs [3]; DA, $9 and Congo have the [O] plasmon and the nuclear genotype hblhblHb2Hb2hbshbs [3]. $9 and V4 also served as testers for the growth habit mutations (see Figs. 1--4 below). The trailing line used was TBR[V4] which has the [V4] plasmon and is HblHblHb2Hb2hbshbs [3]. The M~, M2 and where necessary, Ms and M4 plants of the different muta-
350 genic treatments, were studied several times during the growing season for their growth-habit mutations, namely bunch mutants in the trailing cultivar and trailing ones in the bunch cultivars. (For details see Levy and Ashri [27].) The mutants were screened by their pod and seed characteristics to be true to type and n o t chance outcrosses. Border rows and seed-increase plots of the different cultivars grown for several years served as controls for spontaneous mutations.
Results and discussion
Genetic tests distinguishing cytoplasmic from nuclear mutations Breeding tests based on the genetic models proposed by Ashri [2,3], were (I) C y t o p l a s m i c m u t a t i o n s : [ V 4 ] -~ [ m l]
? V4, bunch [ V 4 ] H b i H b 1h b 2 h b 2 h b s h b 5
TBR[V4] (trailing) [V4] Hb 1HblHb2Hb2hb5hb 5
X
Bunch mutant [ml] Hb 1Hbl Hb2Hb2hbshb 5
F 1 trailing [ V 4 ] H b l I - I b l I - I b 2 h b 2 h b s h b 5
®,
F 2 43-I V 4 ] H b l H b l H b 2 - h b s h b 5 I[V4]
HblHblhb2hb2hbshb
trailing 5 bunch
R e c i p r o c a l cross:
F 1 b u n c h [ m 1] H b l H b l H b 2 h b 2 h b s h b ×~
5
F 2 [ml] trailing: bunch ? C o n c l u s i o n . R e c i p r o c a l d i f f e r e n c e s a r e e x p e c t e d in t h e F 1 . F 2 s e g r e g a t i o n will d e p e n d u p o n t h e i n t e r a c t i o n o f t h e m u t a n t c y t o p l a s m a n d t h e d i f f e r e n t loci.
(II) N u c l e a r m u t a t i o n (a) H b 2 ~ h b 2
TBR[V4] trailing [ V 4 ] H b i H b I H b 2 H b 2 h b 5hb 5 Bunch mutant IV4] Hb iHblhb2hb2hbshb 5
Q V4 bunch [V4] Hb iHb lhb2hb2hb 5hb 5
F 1 bunch IV4] HblHblhb2hb2hbshb5
®~
F 2 all b u n c h I V 4 ] I-Ibl H b l h b 2 h b 2 h b s h b 5 R e c i p r o c a l croas. T h e s a m e F I a n d F 2 r e s u l t s will b e o b t a i n e d . C o n c l u s i o n . N o r e c i p r o c a l d i f f e r e n c e s a r e e x p e c t e d in F 1 a n d F 2 ; b u n c h r e c i p r o c a l F I ' s i n d i c a t e recessive m u t a t i o n f r o m H b 2 t o h b 2.
(b) H b I -+ h b I
TBR[V4] trailing
9 V4 bunch [V4] HblHblhb2hb2hbshb5
X
Bunch mutant [V4] hblhblHb2Hb2hbshb5
F I trailing I V 4 ] H b l h b l H b 2 h b 2 h b s h b
®¢
S
F 2 I-~[V4] I-Ibl-Hb2-hbshb $ trailing i-~ all r e m a i n i n g g e n o t y p e s ; b u n c h R e c i p r o c a l cross. T h e s a m e F I a n d F 2 r e s u l t s will b e o b t a i n e d C o n c l ~ l o n . N o r e c i p r o c a l d i f f e r e n c e s a r e e x p e c t e d in F 1 a n d F 2 ; t r a i l i n g r e c i p r o c a l F l ' s i n d i c a t e recessive m u t a t i o n f r o m H b I t o h b I .
Fig. 1. B r e e d i n g t e s t s d i s t i n g u i s h i n g c y t o p l a s m i c f r o m n u c l e a r m u t a t i o n s t h a t c h a n g e g r o w t h h a b i t f r o m t r a i l i n g t o b u n c h . V 4 w a s u s e d as a t e s t e r ( V 4 p l a s m o n ) .
351 (I) C y t o p l a s m i c m u t a t i o n s : [V4] -+ [ m l ] TBR[V 4] Wailing [V4] Hb i H b iHb21-Ib2hb 5hb 5 $9, bunch [O] h b l h b l H b 2 H b 2 h b s h b 5
X
Bunch mutant [ml] HblHblHb2Hb2hbshb S
F 1 trailing [O] H b l h b l H b 2 H b 2 h b s h b 5 (II) Nuclear m u t a t i o n (a) Hb I --~hb I
TBR [V4] trailing 9 S9,bunch [O] h b l h b l H b 2 H b 2 h b s h b 5
X
Bunch mutant [V4] hb l h b l H b 2 H b 2 h b s h b 5
F 1 b u n c h [O] h b l h b l H b 2 H b 2 h b s h b 5
®~
F 2 all b u n c h [O] h b l h b l H b 2 H b 2 h b s h b 5 (b) Hb 2 --~ hb 2 TBR [V4] trailing 9 $9, b u n c h [O] b b l b b l H b 2 H b 2 h b s h b 5
X
Bunch mutant IV4] H b l H b l h b 2 h b 2 h b s h b $
F 1 b u n c h [O] H b l h b l H b 2 h b 2 h b 5 h b 5
®~
F 2 5 Wailing: 11 b u n c h
Conclusion. Trailing F 1 h y b r i d s indicate a p l a s m o n m u t a t i o n w h e r e a s b u n c h F 1 's indicate recessive m u t a t i o n in Hb I or Hb 2 loci. T h e l o c u s i n v o l v e d can be identified b y the presence or absence o f segregation in t h e F 2 .
Fig. 2. Breeding tests distinguishing c y t o p l a s m i c f r o m nuclear m u t a t i o n s that change g r o w t h habit f r o m trailing to b u n c h w h e n $9 is the female parent tester (O p l a s m o n ) .
developed to distinguish nuclear mutations from cytoplasmic ones according to the growth habit of the F1 hybrids and sometimes F2 progenies (Figs. 1--4). When bunch mutations from the trailing cultivar are crossed reciprocally to V4, the nature of the mutations can be determined by the F~ hybrid phenotypes (Fig. 1). This is also true when $9 is the female parent tester (Fig. 2). It should be noted that when V4 is used as a tester reciprocally the plasmon is studied, whereas with $9 as a female tester, only the nuclear genotype is checked. For trailing mutations, $9 as a female parent tester facilitates a rapid and sharp determination of the nature of the mutations (Fig. 4); cytoplasmic mutations produce bunch F~ hybrids whereas nuclear ones yield trailing F~'s. Therefore, reciprocal crosses are not necessary. If V4 is the tester, no phenotypic F~ differences are expected, and F2 segregations are required to distinguish between nuclear and cytoplasmic mutations (Fig. 3). Since the genetic system controlling growth habit in peanuts may be more complex and additional loci with different interactions might be involved [3,21], the identification o f the mutations should be based essentially on the FI results, especially on reciprocal crosses between the mutants and V4.
Nature of bunch mutations from the trailing cultivar No growth habit mutations were detected in the M1. From the different
352
(I) C y t o p l a s m i c m u t a t i o n : [ O ] -+ [ m 2 ]
9 V4, bunch [O] HblHblhb2hb2hbshb
5
X
Bunch cultivar [O] hbl hbl Hb2 Hb2hbshb5 t Trailing mutant [ m 2] h b l h b l H b 2 H b 2 h b s h b 5
F 1 trailing [ V 4 ] H b l h b l H b 2 h b 2 h b s h b 5
®~
F2 1~ I V 4 ] H b l - H b 2 - h b s h b 5 t r a i l i n g ~6all remaining genotypes; bunch R e c i p r o c a l cross
F 1 trailing [ m 2 ] H b l h b l H b 2 h b 2 h b s h b
®~
5
F2 1 [ m 2 ] h b l h b l h b 2 h b 2 h b s h b 5 ' b u n c h i5 I--6 all r e m a i n i n g g e n o t y p e s ; t r a i l i n g T h e F 2 s e g r a t i o n o f 1 : 1 5 is e x p e c t e d if H b 1 a n d H b 2 b e h a v e as d u p l i c a t e l o c i in t h e [ m 2 ] p l a s m o n . However, other interactions and therefore different segregations can be obtained. C o n c l u s i o n . N o r e c i p r o c a l d i f f e r e n c e s a r e e x p e c t e d in F 1. S u c h d i f f e r e n c e s m i g h t a p p e a r in F 2.
(II) N u c l e a r m u t a t i o n (a) h b 1 "-~ H b I Bunch cultivar [O] hblhbl Hb2Hb2hbshbs V4, bunch [V4] HblHblhb2hb2hbshb
5
X
Trailing mutant [O] HblHblHb2Hb2hbshb5
F 1 trailing [ V 4 ] H b l I - I b l H b 2 h b 2 h b s h b 5
@;
F 2 ~ [V4] HblHblHb2-hbshbs, I IV4] Itblltblhb2hb2hbshbs,
trailing bunch
R e c i p r o c a l cross. T h e s a m e F 1 a n d F 2 r e s u l t s will b e o b t a i n e d . (b) h b S -~ H b $ Bunch cultivar
V4, bunch [V4] HblHblhb2hb2hbshbs
×
Trailing mutant [O] hblhblHb2Hb2Hb$Hb5
F 1 trailing [ V 4 ] H b l h b l I - l " b 2 h b 2 H b s b b 5
®~ F2
b u n c h , all g e n o t y p e s h o m o z y g o u s recessive f o r t w o l o c i o r m o r e . 27 3-'2 t r a i l i n g , all r e m a i n i n g g e n o t y p e s .
R e c i p r o c a l cross F 1 trailing [ O ] H b l h b l H b 2 h b 2 H b $ h b 5
®~ F2
b u n c h , g e n o t y p c s h a v i n g t w o d o m i n a n t alleles o r less in a n y c o m b i n a t i o n . 21 t r a i l i n g , all r e m a i n i n g g e n o t y p e s . ~-~
C o n c l u s i o n . N o r e c i p r o c a l d i f f e r e n c e s are e x p e c t e d in F 1. D i f f e r e n c e s in F 2 s e g r e g a t i o n s c a n b e u s e d f o r t h e i d e n t i f i c a t i o n o f t h e locUS i n v o l v e d .
Fig. 3 . B~eedlng t e s t s d i s t i n g u i s h i n g c y t o p l a s m i c f r o m n u c l e a r m u t a t i o n s t h a t c h a n g e g r o w t h h a b i t in c u l t i v a r s $ 9 , C o n g o a n d D A f r o m b t m e h t o t r a i l i n g . V 4 w a s u s e d as a t e s t e r .
353
(I) C y t o p l a s m i c m u t a t i o n : [O] --~[m2] B u n c h cultivar
[O] h b l h b l H b 2 H b 2 h b s h b 5 9 $9, b u n c h [O] b b l h b l H b 2 H b 2 h b s h b 5
Trailing m u t a n t
X
[m2] h b l h b l H b 2 H b 2 h b s h b s
F 1 b u n c h [O] h b l h b l H b 2 H b 2 h b s h b 5 (II) N u c l e s x m u t a t i o n (a) hb 1 --~ Hb 1 B u n c h cultivar
9 $9, b u n c h [O] hb l h b l Hb2 H b 2 h b s h b 5
Trailing m u t a n t
× [O] Hbl Hbl Hb2Hb2hb5hb5 ¢ F 1 trailing [O] H b l h b l H b 2 H b 2 h b s h b 5
(b) hb 5 ~ I-Ib5 B u n c h cultivar
¢ 9 $9, b u n c h [O] h b l h b l H b 2 H b 2 h b s h b 5
Trailing m u t a n t
X
[O] h b l h b l H b 2 H b 2 H b s H b s
F 1 trailing [O] h b l h b l H b 2 H b 2 H b s h b 5
C o n c l u s i o n . B u n c h F 1 h y b r i d s i n d i c a t e a p l a s m o n m u t a t i o n w h e r e a s trailing F 1 's indicate a d o m i n a n t m u t a t i o n in h b I or h b 5. T h e l o c i i n v o l v e d can b e distinguished b y a n o t h e r t e s t e r having t h e G p l a s m o n
[3]. Fig. 4. B r e e d i n g t e s t s d i s t i n g u i s h i n g c y t o p l a s m i c f r o m n u c l e a r m u t a t i o n s w h i c h c h a n g e t h e g r o w t h h a b i t f r o m t h e b u n c h cultivars $ 9 , C o n g o and D A t o trailing w h e n $ 9 is t h e f e m a l e p a r e n t tester.
mutagenic treatments of T B R [ V 4 ] , 137 independent bunch mutations were obtained in the M2 progeny rows. All the bunch mutations, except two that were sterile and 10 that were nearly sterile, were progeny tested in the M3:28 gave uniform bunch progenies while 97 segregated for bunch versus trailing in different ratios (see below}. M3 plants from the 28 true-breeding bunch mutations were crossed reciprocally to V4 and/or to $9 as the female parent. According to the phenotypes of their F1 hybrids with the testers, 14 of them are plasmon mutations (Table 2): 10 mutations crossed to V4 gave reciprocal differences in the F1; since the original cultivar TBR[V4] contained the [V4] plasmon, no reciprocal differences were expected if the mutations did not affect the plasmon (Fig. 1). 4 additional uniform bunch mutations were crossed only to $9 as the female parent, and all the F~'s from these crosses were trailing as expected for plasmon mutations (Fig. 2, i). 3 mutations crossed to V4 were also crossed with S9 as the female parent (Table 2): in two of them, the F~ 's were trailing as expected for a plasmon mutation (Fig. 2, I); the Fl's of the third mutation were bunch, perhaps owing to a change in the genetic background of this mutation [18,21]. F2 populations of the crosses between the above 10 bunch mutations and V4 were studied for growth habit. Only two F2 populations segregated at the ratio of 3 trailing: 1 bunch expected with a plasmon mutation only (Fig. 1, I}. In the F2 populations of the other mutations, different ratios of trailing versus bunch plants were obtained (Table 2}. Such deviations from the expected ratio could
354
TABLE 2 THE ORIGIN OF BUNCH CYTOPLASMIC MUTATIONS FROM TBR[V4] INDUCED BY MATURESEED TREATMENTS AND THE BEHAVIOUR OF THEIR F 1 AND F 2 PROGENIES FROM CROSSES W I T H V 4 A N D / O R $9 Mutation No. 1973
Mutagen a
Tester
F! hybrids, number b and phenotypes with m u t a n t as
F 2 s e g r e g a t i o n b w i t h t h e m u t a n t as male
female
B male
female
T
T
26 200
94 137
5219 5318 5388
7-14 7-26 EMS
$9 $9 $9
3 T 6 T 1 T
. . .
. . .
. . .
5390 5415 5260
EMS Act 1 7-14
$9 V4 V4
1 T 4 T 4 T
. 1 B 3 B
.
.
5260 5261 5261
7-14 7-14 7-14
S9 V4 $9
6B 6 T 1 T
. 4 B .
5279 5308 5332
7-26 7-26 ")'-26
V4 V4 V4
2 T 4 T 5 T
1 B 3 B 4 B
142 246 282
111 232 308
-162 202
-103 162
5381 5394 c
EMS EMS
V4 V4
6 T 2 T
3 B 5 B
345 115
565 370
134 128
141 78
202 68
181 55
5394
EMS
$9
3 T
.
5404 5405 c
EMS EMS
V4 V4
4 T 1 T
5 B 1 B
. .
.
. . .
B
. . .
.
.
22 121
216 501
. 73
. 145
.
.
.
98
.
. 98 28
. 95
. 134 55
a See Table I for details of treatments. b B, B u n c h ; T, T r a i l i n g . c F i t n e s s o f F 2 s e g r e g a t i o n f o r 3 t r a i l i n g : l b u n c h w i t h t h e m u t a n t as m a l e p a r e n t : 5 3 9 4 , P ( 1 d f ) = 0 . 4 0 - 0 . 5 0 ; 5 4 0 5 , P ( 1 d r ) = 0.05---0.10.
result from varying intensities of haplontic selection or from additional changes in the genetic background of the mutants as found for other mutations in peanuts [ 1 8 , 2 1 ] . In this regard, it should be noted that many loci might be involved in the hormonal control of growth habit in peanuts [ 3 , 4 0 ] . 14 other true-breeding independent mutations gave, in reciprocal crosses with V4 and/or $9 as female parent, only bunch FI plants and are therefore nuclear mutations (Fig. 1, II; Fig. 2, II). 8 of them gave no reciprocal F, differences when crossed to V4; hence the mutation was from Hb~ to hb2 and their genotype is the same as V4. This assessment was reinforced by the lack of segregation for growth habit in two F2 populations obtained from these crosses: all the progenies were bunch. Moreover, 5 of the 8 mutations above were also crossed to $9 as the female parent, and all their F, 's were bunch as expected for a nuclear mutation (Fig. 2, II). The other 6 mutations were crossed only to $9 as female parent. They behaved as nuclear mutations because their F,'s were all bunch (Fig. 2, II), but the Hb locus or loci involved could n o t be determined w i t h o u t further test crosses. The other 97 independent bunch mutations in Ms progeny tests gave a wide range of segregations: from 1 to 90% of the plants were bunch in the different
355
u~
20
z
o F--
4c o o z
6 4
0
4~-~
,I - '10
%. BUNCH
2~-~0 ~-40 4t-50 54-60 6~-70 T~-80 8t-90
PLANTS
IN M3
POPULATION
Fig. 5. F r e q u e n c y d i s t r i b u t i o n o f t h e i n d e p e n d e n t s e g r e g a t i n g b u n c h m u t a t i o n s a c c o r d i n g t o t h e p r o p o r t i o n s o f b u n c h p l a n t s in t h e M 3 p r o g e n y r o w s . ( O n e m u t a n t M 2 I~lant per m u t a t i o n w a s t e s t e d in M 3.)
progeny rows (Fig. 5). 85 bunch plants from different segregating M3 families were progeny tested in M4:79 continued to segregate, usually with a preponderance of trailing progenies, while 6 gave bunch progenies only. 26 M3 trailing offspring of different bunch M2 plants were progeny tested in M4:23 gave uniform trailing M4 rows, while 3 segregated with a preponderance of trailing plants. The segregations of the bunch mutants and the M4 results of the above trailing-progeny tests cannot be interpreted as nuclear mutations or outcrosses in view of earlier findings showing that the bunch growth habit is recessive to
'10 z 0
23 LL 0 0 Z
t-t0 %
tt-20 TRAILING
24-30
31-40
PLANTS
41-50 IN
M3
51-60
I
~-70
71-80
81- 9 0
POPULATION
Fig. 6. F r e q u e n c y d i s t r i b u t i o n o f t h e i n d e p e n d e n t segregating trailing m u t a t i o n s a c c o r d i n g t o t h e p r o p o r t i o n s o f trailing plants in t h e M 3 p r o g e n y r o w s . ( O n e m u t a n t M 2 p l a n t per m u t a t i o n w a s t e s t e d in M3. )
356 the trailing one [ 2,21]. Neither can these results be interpreted by nuclear gene models in which dominance of the bunch growth habit [23] or other genie interactions [12] is assumed. The segregations of the offspring of the bunch mutants in the M3 (Fig. 5) and M4 can be interpreted in terms of somatic segregations [15,24]. Accordingly, in the segregating bunch mutations, the initial m u t a n t cells were heteroplasmic and, through sorting o u t of the m u t a t e d particles, bunch plants were recovered in M2. Bunch or trailing plants could be homoplasmic or heteroplasmic, depending on the proportions of the particles in the "mixed cells", as in plastid mutations [9,15,20]. Thus, different segregation ratios of bunch versus trailing offspring could then be obtained as in M3 and M4, depending u p o n the ratios of the particle types and their relative multiplication rates. Somatic segregation can also explain the behaviour of the FI hybrids between V4 and bunch or trailing M3 progenies from the 14 segregating bunch mutations, especially the reciprocal F1 differences and sibs' (from the same pods) segregations (Table 3). Some of the progenies of the segregating bunch mutants were semisterile. Therefore, the possible presence of chromosomal aberrations was checked in the PMC's of such mutants. Indeed, observations of c h r o m o s o m e stickiness at Mx and AI, asynchronized m o v e m e n t of the chromosomes at AI, and hexades with t w o micronuclei at TII suggest that induced chromosomal aberrations led to some of the growth habit changes. The aberrations were difficult to analyze because of the small size and large number of chromosomes (2n = 40) in cultivated peanuts.
TABLE 3 GROWTH HABIT LINES WITH V4
OF
FI'S
FROM
Mutation No. 1972
Growth habit of the mutant a
3577-18 b 3583-9 3618-1
CROSSES
OF
MUTANTS
SEGREGATING
F 1 hybrids, number and phenotypes male
female
B
1B, 2T
1B
T B
1T 5T
2B 2B
3608-1 3657-11 3789-11
B T T
1B IT IT
-2B --
3899-24 b 3929-15 3949-5 b
T T T
----
1T, 1B 2B 1T, 1B
3950-8 3966-5 3946-18
T T T
1T -1T
3B 1B 2B
4693-19 5042-18
B B
-4T
2B 1B
a B , B u n c h ; T, T r a i l i n g . b N o t e s e g r e g a t i o n o f sibs.
FROM
BUNCH
a w i t h m u t a n t as
M3
357
Nature of trailing mutations from the bunch cultivars 32 independent trailing mutations were obtained in the M2 from the different bunch cultivars. All but one of them segregated in the M3 for buhch versus trailing, usually with more bunch progenies (Fig. 6). In 6 trailing mutations, segregation for pod or shoot traits characteristic of other cultivars was observed in the M3; therefore, cross pollination was suspected, and studies with them were discontinued. The continued segregation of the trailing mutants made it difficult to determine their genotype by appropriate test crosses (Figs. 3, 4). However, 13 M3 trailing plants from different mutations were crossed reciprocally to the testers, and their FI and F2 behaviour is summarized in Table 4. The M3 segregations and F1 results of most of the trailing mutations can be interpreted by a nuclear mutation at one of the Hb loci or by appropriate cross-pollination of the M~ plants. However, some of the trailing-mutant results are compatible with somatic segregation, as discussed for the bunch mutants above. Since most of the trailing M2 mutants segregated in the M3, they could have been heteroplasmic or heterozygous for Hb~ or Hbs. In the latter, the segregation expected in M3 was 3 trailing: 1 bunch, but most M3 progeny rows gave different ratios (Fig. 6). Such results could be obtained from sorting out of plasmon mutations, or from changes in the nuclear genetic background [18,21]. It was not possible to eliminate one of the above alternatives with the
TABLE 4 T H E O R I G I N OF T R A I L I N G M U T A T I O N S AND T H E B E H A V I O U R OF T H E I R F 1 A N D F 2 PROGEN I E S F R O M C R O S S E S W I T H V 4 A N D / O R $9 Mutation No. 1973
5146 5590 c 5644
Origin cultivar
V4 V4 $9
Mutagenic treatment a
Act 2 EMS EB1
Tester
V4 $9 $9
F I hybrids, number b and p h e n o t y p e s w i t h m u t a n t as
F 2 segregation b with m u t a n t as
male
female
B
1B 2B 3T
2B 3T .
. 109 .
male
.
female T .
B . 77
. 107
106
89 118
156 148
95 100
249
344
0
70
25
311
0
.
.
5532
Congo
EMS
$9
2B
IT, I B
.
.
.
.
5518 5548
Congo Congo
7-26 7-26
$9 $9
2T, 1 B --
2T, 2 B 2T
. .
. .
. .
. .
5556 5559
Congo Congo
Act 1 Acr 1
V4 V4
5T 2T
5B 4B
176 128
5559
Congo
Act 1
$9
7T
2B
5427 5428
DA DA'
7-14 7-14
$9 $9
6B 2B
. 6B
147
5429
DA
7"14
V4
IT
1B
104
5442
DA
7-14
$9
--
2B
.
5443
DA
7-14
$9
1 T , 2B
2B
225 d
.
. .
. .
. .
.
106 .
T
.
0
.
a S e e Table 1 for d e t a i l s o f t r e a t m e n t s . A c t 2, t r e a t m e n t o f d e v e l o p i n g e m b r y o s w i t h acriflavine 0 . 1 1 m M for 1 3 d a y s . All o t h e r t r e a t m e n t s w e r e o n m a t u r e seeds. b B, B u n c h ; T, Trailing. c M u t a t i o n t h a t b r e d true in t h e M 3. All o t h e r trailing m u t a t i o n s s e g r e g a t e d in t h e M 3. d S e g r e g a t i o n o f a b u n c h F 1 plant.
358 data furnished by the F1 hybrids of the trailing mutants and the known testers. The reciprocal differences between the F1 hybrids of the true breeding mutation No. 5590 (Table 4) cannot be attributed to a nuclear mutation and may again indicate a plasmon mutation.
Concluding remarks Most of the true-breeding plasmon mutations were recovered in the trailing cultivar TBR[V4] after v-ray and EMS treatments of mature seeds (Table 1). The EB treatments proved highly effective in inducing various shoot mutations [27]; however, all the true-breeding growth-habit mutations obtained from the EB treatments were nuclear mutations (Table 1). In pearl millet (P. americanum), EB induces a high frequency of cytoplasmic male sterile mutations [10]; and in microorganisms, EB and acriflavine are highly efficient in the induction of cytoplasmic mutations [1,29,35]. Therefore it was surprising that in the growth habit of peanuts, even severe treatments with these two mutagens were less efficient than EMS and v-rays in inducing such mutations. The effectiveness of EMS and v-rays is in agreement with other studies reporting the induction of cytoplasmic male sterility in barley by EMS and X-rays [16], in sorghum by alkylating agents [28], in sugar beet with v-rays [25] and plastid mutations by EMS in tobacco [15]. The differences in the frequency of plasmon mutations between the mutagens may result from their different modes of action [6,14] or from different intensities of diplontic selection [13,24]. EB has been reported to have a great affinity for circular DNA [31,38]. In view of its low efficiency in inducing plasmon mutations in the growth habit of peanuts, it is tempting to suggest that the cytoplasmic DNA component affecting this system might not be circular. From the treatments of developing embryos, small M1 and M2 populations were obtained. Therefore, it is difficult to compare treatment of mature seeds with treatment of embryos for the induction of plasmon mutations. On the other hand, the differences between the cultivars in the frequency of such mutations underline the genotypic influence on plasmon mutations which is known for nuclear mutations [7,8].
Acknowledgements This research was financed in part by a grant to A.A. made by the U.S. Department of Agriculture Research Service, authorized by P.L. 480, Project No. A10-CR-77, Grant No. FG-Is-281. The report is based in part on the dissertation of A.L. submitted to the Hebrew University in partial fulfilment of the requirements for the Ph.D. degree. We acknowledge with thanks the technical assistance of Y. Berg, A. Cohen, M. Kochavi and R. Offenbach in various phases of this research. References 1 Alexander, N.J., N.W. Gilham and J.E. B o y n t o n , The m i t o c h o n d r i a l g e n o m e o f C h l a m y d o m o n a s : i n d u c t i o n o f m i n u t e c o l o n y m u t a t i o n s b y acriflavine and their inheritance, Mol. Gen. Genet., 130 (1974) 275--290.
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