The Journal of Heredity 2002:93(6)
© 2002 The American Genetic Association 93:444-447
Brief Communication |
A New Search for Restorer Cytoplasm: The Restorer Cytoplasm for the Gene ms10 Most Probably Does Not Exist in Maize
From the Maize Research Institute, Zemun Polje, Slobodana Bajica 1, 11080 Zemun, Yugoslavia.
Address correspondence to M.B. Vidakovic at the address above, or telephone: 00-381-11-3756-704.
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Abstract |
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Since the suggestion concerning the hypothesis of the existence of restorer cytoplasms for some of the known and currently available male sterile (ms) genes in maize, a relatively limited amount of research effort has been made in order to test the hypothesis. Considering the importance of such a phenomenon, we designed a large, two-part experiment to test the idea more seriously. In the first part, 50 randomly chosen, medium-late open-pollinated (OP) varieties of maize from the Zemun Polje (ZP) collection were tested for the presence of the restorer cytoplasm in some currently known ms-genes in maize (ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, ms11, ms12, ms13, ms17, ms22, ms23, and ms24). In the second part, the whole ZP collection of maize germplasm (more than 4,000 entries) was tested for the presence of the restorer cytoplasm for the gene ms10. After the first basic screening of OP varieties, more than 70 nonsegregating "candidates" were identified; however, after additional screening of the collection and the direct testing with respective homozygous ms-testers, all of them showed segregation, indicating that the restorer cytoplasm does not exist, especially the gene ms10. While performing this experiment, we discovered almost a hundred sources of male sterile cytoplasm, which were distinguished by their overwhelming frequency of male sterile plants in segregating test progenies.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The problems encountered with the use of cytoplasmic male sterility in maize (Zea mays L.) hybrid seed production have forced researchers to look for alternative solutions to the problem. The use of different ms-genes existing in maize is always seriously considered, under the precondition that production of male sterile seed could be solved. Different authors have suggested different approaches, but none of them found a place in practice.
Patterson (1971) proposed the use of ms-genes closely linked to chromosomal aberrations (duplications and deficiencies). According to this approach, the male fertile maintainer should be the genotype having the ms-gene in the triplicated condition (ms/ms; Ms), where the Ms gene is present as a dominant allele on a duplicated part of the chromosome closely linked to the point of deficiency. This genotype should be the maintainer, multiplying itself by selfing. When used as a pollinator for all sterile female rows (ms/ms homozygotes), the pollen grains bearing Ms alleles would be abortive, due to the presence of chromosomal deficiency (differential viability of egg and pollen cells). Patterson found suitable translocations and implemented this system for the genes ms1, ms2, ms6, and ms10, but proved that the theoretically expected ratio of male sterile versus fertile plants in the progeny of selfed maintainers was not attained. In addition, the maintainer plants were weak and, as such, were not suitable for practical use.
Even before Patterson, Ramage (1965) proposed an essentially similar system, in which an extra chromosome would carry the dominant allele of the ms-gene. Marshall and Ellison (1988) suggested the use of gametophytic factor (Ga), in which the recessive allele of the Ga-gene, linked to the dominant allele of the ms-gene, should have the role of chromosomal aberration in Patterson's system. This idea suffers the same disadvantage as Patterson's by having 50% of male sterile plants in the progeny of the selfed maintainer. This system was never implemented, due to the lack of genic linkage of any of Ga and Ms-genes.
Van Mellaert (1993) proposed the linking of the dominant ms-gene allele to a gene susceptible to a herbicide. This idea was realized through the use of the barnase gene, which behaves as the dominant gene for male sterility, and the barstar gene, whose product inactivates the enzyme barnase and is linked to the gene for susceptibility to the herbicide Basta. The treatment of segregating female rows in hybrid seed production would eliminate the male fertile plants. This idea is not new, because Wiebe (1964) discovered the close linkage between the loci of the gene ms16 and a gene for susceptibility to DDT in barley (Hordeum vulgare L.). Through the DDT treatment, he was able to eliminate up to 90% of male fertile plants in barley.
Albertsen et al. (1993) suggested the use of genetic engineering to link a certain structural gene, responsible for pollen production, to a promoter that is not functional in the absence of a certain inducing substance. The plants with such genic constitution would be male sterile and would serve as female parents in hybrid seed production. These constitutively male sterile plants would be maintained by selfing after spraying with the inducing substance. Recently they reported that dicamba (3,6-dichlor-o-anizil acid), a selective systemic herbicide for maize, proved to fulfill these requirements. This is not a new idea, because a similar approach was proposed by Kasembe (1967) and Hockett et al. (1978).
Hermsen (1965) reasoned that the existence of different types of cytoplasmic male sterility in maize, the respective restorer genes for each of them, and the whole series of nuclear genes causing male sterility might mean that a restorer cytoplasm could exist for some or all ms-genes. This type of cytoplasm, introduced in ms/ms genotype, could suppress its male sterility and restore fertility. Hermsen (1965, 1968) argued that this type of cytoplasm had a low probability to be discovered by chance, mainly due to limited experimentation with the ms-genes and the fact that, in such crosses, male sterile segregants would be used as female parents, eliminating the possibility of unintentional testing of different cytoplasms. A similar idea was proposed and tested earlier by Kohel and Richmond (1963) in cotton and by Rutger and Jensen (1967) in barley, but both experiments were negative. Hermsen (1968) stated that these negative results should not be discouraging, because the experiments were carried out with only one ms-locus on a limited number of varieties.
Washnok (1972) tested 25 maize open-pollinated varieties of diverse origin with 12 different ms-genes. The results of these tests were also negative, and any interest in the further research for the restorer cytoplasm dropped sharply.
Burnham et al. (1981), working with flax (Linum usitatissimum L.), found differences in segregating patterns of F2 generations originating from reciprocal crosses of some flax varieties. For example, when large-seeded flax from Crete was used as female parent with many other stocks as male parents, the produced F2 generations segregated to male sterile and male fertile plants, while the F2 generations of the same reciprocal crosses did not segregate, giving only male fertile plants. They concluded that their results could be equally explained by two possible hypotheses:
- By the interaction of the male sterile cytoplasm and nuclear restorer genes
- By the presence of the restorer cytoplasm that suppressed the expression of the ms-gene and restored fertility. Due to complex nuclei-cytoplasmic interactions in flax, they were not able to discern between the two possibilities and suggested that the answer should be studied in a species like barley or maize.
- Our decision to study the problem wasprovoked by several reasons:
- The amount of research work was insufficient to give a conclusive negative answer to Hermsen's hypothesis.
- The evidence of the substantial importance of cytoplasm in overall inheritance was continually increasing.
- The overall cytoplasmic variability was well defined.
- The analogy, already considered by Hermsen, strongly suggested that the restorer cytoplasm could exist as a fourth missing point of the quadruple: male sterile cytoplasms/restorer genes//nuclear ms-genes/RESTORER CYTOPLASM?
- By the presence of the restorer cytoplasm that suppressed the expression of the ms-gene and restored fertility. Due to complex nuclei-cytoplasmic interactions in flax, they were not able to discern between the two possibilities and suggested that the answer should be studied in a species like barley or maize.
In short, we considered that, if certain types of cytoplasms can cause male sterility, strong susceptibility to pathogens, tolerance of environmental stress conditions, and modification of phenotypic expression of a series of quantitative traits, there should be no reason why male sterility caused by nuclear genes could not be suppressed by certain types of cytoplasm.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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According to Hermsen (1965, 1968) the search for "fertile" (F) cytoplasm should be based on the assumptions listed in Table 1.
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For an extensive testing of the hypothesis, the whole Zemun Polje (ZP) collection of maize germplasm—more than 4,000 different genotypes—was tested for the presence of ms10 restorer cytoplasm. All ms stocks used in this experiment were from E. B. Patterson, Agronomy Department of the University of Illinois, USA.
The ZP experiment started in 1993 by crossing 4,002 different entries, as female parents, to heterozygous testers ofMs10/ms10 genic constitution in space isolation. Because the tested material was extremely variable in respect to its earliness, the male tester was prepared as a mixture of genotypes with continuous variation in its length of vegetation. This was achieved by crossing male sterile (ms10/ms10) segregants of inbreds A632 and B73 to a series of genotypes, ranking from very early to extremely late. The mixture of the male tester provided pollen shedding during a period longer than one month.
Simultaneously, as a pilot experiment, crossing of 50 randomly chosen OP varieties to a tester heterozygous for the genes ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, ms11, ms12, ms13, ms17, ms22, ms23, and ms24 was done by hand pollination.
Instead of selfing the produced F1 generations, the two ears of each F1 generation were planted ear-to-row and crossed again as female parents with the same (above specified) male parents. Two ears of each F1 generation were taken, on the assumption that cytoplasmic variability within tested genotypes is small. The average sample of a minimum of 20 backcrossed ears, originating from each single F1 plot, was taken for screening. Under these conditions, the probability of random loss of the ms gene was 1/220, or <10-6. Each screening plot contained 60 plants with the frequency of male sterile segregants being 1/8 and the expected frequency of random escapes (all fertile plants) being 7/860. Therefore, the total probability of disappearance of male sterile segregants in the BC1 progeny was equal to the sum:
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The experiment was planted at the time of the first screening (1995), with up to 60 plants in each of 8,004 plots for observation. During pollination each plot was monitored once every two days. The plots exhibiting male sterile phenotypes were observed, and each male sterile plant was marked with a red tag in order to be easily recognized. We considered this necessary because of the possibility that partially restoring cytoplasm may also exist and, as such, can also be used as a possible maintainer of ms/ms fertile stock.
The test progenies not segregating for male sterile (ms10/ms10) plants were observed, while those with an excessive number of male sterile plants were suspected for the presence of male sterile cytoplasm.
In 1996 the reserve seeds of tested progenies that did not segregate in 1995 were planted again in a population of 200 plants for additional observation and direct testing with ms10/ms10 female testers. A substantial portion of the previously noted "candidates" showed segregation, but a few of them did not. Ten to 15 plants of each of the nonsegregating progenies were individually selfed and tested with ms10/ms10 male sterile female testers. The same procedure was followed with testing of 50 randomly chosen OP populations for the presence of the restorer (fertile) cytoplasm for any of the previously cited ms genes.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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After the first screening, 68 "candidates" for the gene ms10, one for ms12, two for ms13, three for ms17, and one for ms24 were observed. In most cases these "candidates" were representatives of only one F1 ear from the respective population (sample), but in a few instances progenies of both F1 ears did not segregate. However, after planting these candidates in a population of 200 plants, only four candidates for the gene ms10 did not segregate. None of these progenies, after selfing and testing of individual plants to the ms10/ms10 tester, gave 100% male sterile test progeny and 100% fertile selfed progeny, indicating the absence of the restorer (fertile) cytoplasm.
Considering the amount and the genetic variability of the tested material (including inbred lines, domestic Yugoslav OP varieties, varieties from all major maize-growing countries in the world, synthetic varieties, and tropical materials), we reached a conclusion that a restorer cytoplasm for the gene ms10 does not exist in maize. We cannot draw the same conclusion for other ms-genes, due to a very limited number of tested samples (only 50 Yugoslav OP varieties).
While performing this experiment we discovered almost a hundred sources of male sterile cytoplasms distinguishable by their overwhelming frequency of male sterile plants in segregating test progenies.
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Footnotes |
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J. Perry Gustafson
Received May 2, 2002
Accepted October 29, 2002
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Albertsen MC, Fax TW, and Trimnell MR, 1993. Cloning and utilising a maize nuclear male sterility gene. Proc Annu Corn Sorghum Ind Res Conf. 48:224-233.
Burnham CR, Phillips RL, and Albertsen MC, 1981. Inheritance of male sterility in flax involving nuclear-cytoplasmic interaction, including methods of testing for cytoplasmic restoration. Crop Sci. 21:659-663.
Hermsen JG, 1965. Towards a more efficient utilization of genic male sterility in breeding hybrid barley and wheat. Euphytica. 14:221-224.[CrossRef]
Hermsen JG, 1968. A discussion on cytoplasmic restoration of ms-sterility. Euphytica. :(Suppl. 1): 63-67.
Hockett EA, Baenziger PS, and Steffens GL, 1978. A proposal for increased research on chemical induction of fertility in genetic male sterile barley. Euphytica. 27:109-111.[CrossRef]
Kasembe JNR, 1967. Phenotypic restoration of fertility in a male sterile mutant by treatment with gibberellic acid. Nature. 215:668.[Medline]
Kohel RJ, and Richmond TR, 1963. Tests for cytoplasmic-genetic interaction involving a genetic male sterile stock of the genotype ms2 ms2. Crop Sci. 3:361-362.
Marshall DR, and Ellison FW, 1988. The potential use of gametophytic factors in the production of F1 hybrid varieties. Euphytica. 39:75-81.
Patterson EB, 1971. Proposed procedures for the use of genic male sterility in hybrid maize production. Illinois Corn Breeders School, March 11, 1971.
Ramage RT, 1965. Balanced tertiary trisomics use in hybrid seed production. Crop Sci. 5:177-178.
Rutger JN, and Jensen NF, 1967. On the use of genetic male sterility in screening for cytoplasmic-genetic sterility in barley. Euphytica. 16:350-353.[CrossRef]
Van Mellaert H, 1993. Engineered pollen control in corn. Proc Annu Corn Sorghum Ind Res Conf. 48:234-240.
Washnok RF, 1972. Cytoplasmic restoration of ms-sterility. Maize Genetic Coop Newsletter. 46:25-27.
Wiebe GA, 1964. A proposal for hybrid barley. Barley Newsletter. 8:16.
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