ABSTRACT. Spontaneous mutations from sterile to fertile were demonstrated in
four different cytoplasmic male-sterile stocks of pearl millet. One stock, ASM-3,.
PLASMON MUTATIONS IN CYTOPLASMIC MALE-STERILE PEARL MILLET, PENNISETUM TYPHOIDES WILLIAM M. CLEMENT, JR.
Department of Biology, Vanderbilt Uniuersity, Nashuille, Tennessee 37235 Manuscript received July 1, 1974 ABSTRACT
Spontaneous mutations from sterile to fertile were demonstrated in four different cytoplasmic male-sterile stocks of pearl millet. One stock, ASM-3, was of African origin, and the other three, LMS-lA, ASM-5, and ASM-7, all had the same cyptoplasm of Indian origin, but differed in nuclear make-up. These reversions were shown to be plasmon mutations, rather than genic in nature. ASM-3 mutated at the lorwest rate, about 0.03/10 heads. LSM-IA, ASMB, and ASM-7 all had the same cytoplasm but mutated at the respective rates of 0.15, 0.26, and 1.02/100 heads, indicating that plasmon mutation rate is strongly influenced by the nuclear environment.
HE existence of cytoplasmic inheritance has been recognized almost as long Tas mendelian inheritance. CORRENSdescribed cytoplasmically inherited male sterility in the genus Cirsium in 1908. Since that time the inheritance of numerous cases of non-chromosomal genes has been reported in a wide variety of plants and animals, but the importance of these non-mendelian factors has not been appreciated by many biologists. Within the last few years increased attention has been focused on non-chromosomal genes. Cytoplasmically inherited male sterility has been developed and utilized extensively in crop plants, but our knowledge of the nature of these cytoplasmic genes is very limited. We know little of the number of copies, mutation rate, location, and relationship of cytoplasmic genes to chromosomal genes. The hereditary material is generally assumed to be the DNA of the chloroplast, mitochondria, or possibly other cytoplasmic DNA. Several different kinds of cytoplasmic male sterility are known in pearl millet (BURTONand ATHWAL 1967). Multiple sources of cytoplasmic male sterility are known in corn (BECKETT1971; SMITHet aZ.1971) as well as in other crop plants. Since there is cytoplasmic variability it is reasonable to assume that cytoplasmic mutations must occur. This paper deals with spontaneous mutations of cytoplasmic male sterility in pearl millet, Pennisetum typhoides (Burm.) . The term plasmon will be used to describe the extrachromosomal hereditary determinants, and plasmon mutation to describe the mutational change of these extrachromosomalfactcurs. BURTON (1972) reported several mutations from male-sterile to male-fertile in pearl millet and suggested that some of these were cytoplasmic and some were genic in nature. He did not report mutation rates, nor were his data critical in distinguishing between genic and plasmon mutations. Genetics 79:583-588 April, 1975
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WILLIAM M. CLEMENT, JR. MATERIALS A N D METHODS
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Four different male-sterile lines, representing two different sources of cytoplasm, were used in this experiment. One line, ASM-3, was derived from African material and the other three lines all carried the Tift 23A cytoplasm, derived from millet of Indian origin (BURTON1958). LMS-1A is the equivalent of Tift 23A, having been produced by direct seed increase from Tift 23A. ASM-5 and ASM-7 are both sterile single crosses from LMS-1A and two different malefertile non-restorer lines, and were produced by hand pollination the previous year. The three male-sterile lines carrying Tift 23A cytoplasm all produce small pink anthers which contain no pollen, and ASM-3 produces small yellow anthers with no pollen. The sterile anthers in either case are easily distinguishable from the fertile anthers which shed pollen. Anthesis generally occurs 2-3 days after the stigmas emerge. It starts about two-thirds of the way up the spike and proceeds in both directions until it is complete in 3-5 days. Each plant generally produces from 3-15 tillers which begin flowering within a few days of each other. For details of flowering and pollination procedures see BURTON (1968). In this experiment all four lines were in flower at the same time and thus were subjected to the same natural environmental conditions. Each morning every head was examined and if a sector of fertile anthers was found, that head w a s covered with a pollination bag. The next day, time and weather conditions permitting, pollen was collected in that bag and used to pollinate another head on a different male-sterile plant in the same line. Additional pollinations were made onto another male-sterile line whenever sufficient pollen was available. All pollinations were made onto heads which were bagged 2-3 days before the stigmas were out. A bag was replaced on the original head and seeds were harvested from these heads as well as from the crosses. Since there was no other millet flowering in the vicinity at this time most of the seeds produced on the mutant heads were selfs. RESULTS A N D DISCUSSION
In 1970 during the course of a “clean-up” program to eliminate male-fertile off types from LMS-lA, I noticed that several heads, on otherwise sterile plants, had sectors of fertile anthers (Figure 1 ) .It was not possible at that time to obtain an accurate count of the number of such heads, but it appeared to be in the order of a few per 1000 heads. The following year a systematic count of the chimeral frequency was made on all four different male-sterile lines, ASM-3, ASM-5, -4SM-7, and LMS-1A (Table 1). The size of the fertile sector varied from as small as only a few florets to as large as the entire head, and in a few cases several heads on the same plant. ’The size of the chimera is assumed to be directly related to the stage of development in which the mutation occurred. ASM-3 has the lowest mutation rate. Only 1 mutant sector was found in a total of 3,303 heads examined, or a mutation rate of about 0.03/100 heads. ASM-7 had the highest mutation rate, 1.17/100 heads, while ASM-5 and LMS-1A were intermediate, having respective rates of 0.26 and 0.15/100 heads. Since cytoplasmic male sterility in millet is a dual cytoplasmic-genic system it is necessary to determine if these mutations are cytoplasmic o r genic in nature. Information on this question is derived from the progeny of crosses and selfs obtained from the mutant chimeras. If the mutation is chromosomal in nature, the genotype of the chimeral sector should be Ms/ms in contrast to ms/ms for the rest of the head. Pollinations made onto other male-steriles would produce segregating progeny in approximately 1: 1 ratios. Selfed seeds on that head would be of two types: segregating 3: 1 if they were produced on the mutant sector, or I : 1 if produced on other parts of the inflorescence.
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CYTOPLASMIC MALE-STERILITY
FIGURE I.-Head of cytoplasmic male-sterile millet (ASM-5) showing fertile mutant sector.
If the mutation is plasmon in nature, then all cross progeny would be malesterile since they would have only cytoplasm of the maternal parent. In contrast, selfed seeds from the mutant sector would produce all fertile progeny and seeds from the other parts of the influorescence would produce only male-sterile progeny. The data are consistent with plasmon mutations (Table 2). In all cases, when pollen from a mutant sector of ASM-7 was crossed onto another head, either from the same plant or onto a different piant of ASM-7 or onto ASM-5, all progeny were male-sterile. Likcwise, only male-sterile progeny resulted from crosses TABLE 1 Frequencies of male-feriilesectors in four male-sierilelines of millet Number
Genetic stock
of plants
Total number of heads
ASM-3 LMS-1A ASM-5 ASM-7
660 652 975
3303 3,336 9,758
70
1,023
Number of
mutant sectors
I 5 26 12
Mutations/ 100 heads
0.03 0.15 0.26 I .a2
586
WILLIAM
M. CLEMENT,
JR.
TABLE 2 Progeny phenoteypes of various crosses and selfs of d e - f e r t i l e sectors Identification
ASM-5 x ASM-5 ASM-5 x ASM-7 ASM-7 X ASM-7 ASM-7 x ASM-5 ASMB @ ASM-7 @ ASMd @+ ASM-7 @+ ASM-3 x ASM-7 ASMB x ASM-5
Number of differentcrases
5 7
6 3 25 20 1
2 5 2
progeny phenotype
All sterile* All sterile All sterile All sterile Segregated Segregated All fertile All fertile Segregated @ 1:I Segregated @ 1:l
* One cross segregated, but same head crossed onto ASM-7 gave all fertile progeny.
+ Entire head was fertile.
of ASM-5 onto itself or ASM-7, with one exception. I n this case, an ASM-5 plant segregated. However, the same ASM-5 mutant crossed onto ASM-7 produced all male-sterile progeny. It is likely that segregation in this case was the result of pollination of a head that itself contained a mutant sector. Seeds produced in the mutant sector would be male-fertile, and those produced on the rest of the head would be male-sterile. This explains the results obtained when heads containing mutant sectors were selfed. Segregation in the resulting progeny ranged from about a 1 : 1 to about 30: 1 and was correlated with the size of the mutant sector. I n two cases the mutations included the entire head and their progeny were all fertile. Further verification of the plasmon mutation hypothesis was obtained by pollinating LMS-1A with pollen obtained from progeny of fertile mutations. I n all cases such crosses produced only male-sterile progeny. It is significant also that the plasmon mutations appear to be at least as stable as. or even more stable than, the male-sterile stocks. No reverse mutations have been noted at this time. I n all cases ASM-5 and ASM-7 mutants produced segregating progeny when crossed onto ASM-3, which carries a different source of cytoplasm. I n one case 28 sterile and 31 fertile plants were produced and in another, 20 sterile and 24 fertile. Either LMS-1A or the male lines used to make up the sterile single-crosses (ASM-5 and ASM-7) must carry restorer genes for the ASM-3 cytoplasm. The observed ratios are consistent with a single gene in the heterozygous condition, but subsequeEt testing is necessary t o be sure that this is the case. In this study only one mutant sector was found in ASM-3, and pollinations from it failed to produce seeds. However, during the course of other breeding work, several other fertile sectors have been found in ASM-3. When other ASM-3 plants were fertilized with pollen from these mutant sectors only male-sterile progeny are produced, providing verification of plasmon mutations in this cytoplasm. The evidence is strong that all mutants observed in this study were cytoplasmic and not genic in nature. Whether or not these changes are the result of mutations
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CYTOPLASMIC MALE-STERILITY
in cytoplasmic DNA or the result of somatic segregation of cytoplasmic components is not clear at this time. In either case, they act as B or maintainer lines and not as R or restorer lines, a result which simplifies the maintenance of malesterile seed stocks. Fertile mutations of nuclear genes would demand a very stringent program in order to propagate male-sterile stocks. Plasmon mutations have been demonstrated to occur in two different cytoplasms of millet. SINGHand LAUGHNAN (1972) reported similar mutations in maize. These observations suggest that these plasmon mutations are general in nature and are not peculiar to unique cytoplasms. The striking difference in mutation rate in three different genetic stocks, all of which carry the same cytoplasm, is especially interesting. If we exclude from consideration the possibility of cytoplasm contributions by the male parent, LMS-IA, ASM-5, and ASM-7 all carry identical cytoplasms and differ only in their nuclear gene content. Yet the plasmon mutation rate of ASM-7 is several times higher than that of LMS-1A and ASM-5 (Table 3). The nuclear genes thus have a direct effect on the mutation of cytoplasmic genes. I n light of the results of this study, the common assumption that the cytoplasm is the same for all stocks possessing the same maternal parentage is open to question. There is likely to be considerable cytoplasmic variability in divergent lines originating from the same cytoplasmic source, and plasmon-regulated characters should be amendable to selection. Inbred lines of hybrids of maize which carry the csm-T (Texas) source of cytoplasmic male sterility are susceptible to southern leaf blight, Helminthosproium maydis, race T. (SCHEIFELE, WHITEHEAD and ROWE 1970). The widespread use of this single cytoplasm in the United States ied to a serious outbreak of southern leaf blight in 1970. It is reasonable to assume that variation exists within cms-T cytoplasm, which could provide resistance to this disease. The cytoplasmic male-sterile system in pearl millet is an ideal one for study of plasmon mutations. Controlled pollination is easy, large numbers of seeds may be obtained from a single pollination, multiple crosses may be carried out on the same plant, mutation from sterile or fertile is easy to recognize, and seeds from such events may be saved for testing. It is possible in the LMS-1A stock to obtain lines which have the same nuclear genetic make-up and differ in cytoplasm only by a plasmon mutation. Such “isoplasmic” lines should be very useful in studies as to the nature of cytoplasmic mutations. The major contributions of this work are as follows. ( 1 ) Demonstration that mutations from male-sterile to male-fertile occur. (2) These are plasmon mutaTABLE 3 Genetic stocks compared
LMS-1A US. ASM-5 LMS-1A US.ASM-7 AMs-5 us. ASM-7
*** Significant at the 0.005 level.
x2
1 df
0.979 18.515*** 19.162** *
588
WILLIAM M. CLEMENT, JR.
lions. ( 3 ) They are not restricted to one unique cytoplasm, but occur in at least two different cytoplasms. (4)The frequency of plasmon mutation is strongly
influenced by the nuclear genotypz I wish to acknowledge the assistance and cooperation of Northrup, King & Co. Southern Research Center, Atmore, Alabama. LITERATURE CITED
BECKETT,J. B., 1971 Classification of male-sterile cytoplasms in maize (Zen mays L.). Crop Sci. 11: 724-727. BURTON,G. W., 1958 Cytoplasmic male-sterility in pearl millet (Pennisetum glaucurn) (L.) . R. Br. Agron. J. 50: 230-231. -, 1972 Natural sterility maintainer and fertility restorer mutants in Tift 23A cytoplasmic male sterile pearl millet, Pennisetum typhoides (Burm.) Stapf and Hubb. Crop Sci. 12: 280-282. BURTON,G. W. and D. S. ATHWAL,1967 Two additional sources of cytoplasmic male-sterility in pearl millet and their relationship to Tift 23A. Crop Sci. 7: 209-21 1. BURTON,G. W. and J. B. POWELL, 1968 Pearl millet breeding and cytogenetics. Advan. in Agron. 20: 49-89.
HOOKER, A. L., 1972 Southern leaf blight of corn-present status and future prospects. J. Environ. Quality 1 : 244-249. SCHEIFELE, G. L., W. WHITEHEAD and C. ROWE,1970 Increased susceptibility to southern leaf spot (Helminthosporium maydis) in inbred lines and hybrids of maize with Texas malesterile cytoplasm. Plant Dis. Rep. 54: 501-503. SINGH,A. and J. R. LAUGHNAN, 1972 Instability of S male-sterile cytoplasm i n maize. Genetics 71 : 607-620. SMITH,D. R., A. L. HOOKER, S. M. LIM and J. B. BEZKETT, 1971 Disease reaction of thirty sources of cytoplasmic male-sterile corn to Helminthosporium maydis race T’. Crop Sci. 11: 772-773. Corresponding editor: R. W. ALLARD
