A comparison of cytoplasmic revertants to fertility from different CMS-S maize sources

Summary The mitochondrial genome organizations of a number of independent culture-derived fertile CMS-S revertants with the nuclear genotype W182BN were compared to spontaneous field revertants with the genotypes WF9, M825/Oh07 and 38-11. Regions of the genome around sequences homologous to the terminal repeats of the linear S1 and S2 episomes characteristic of CMS-S mitochondria were used as hybridization probes on Southern blots of BamHI and SalI digested mitochondrial DNA. The results obtained suggest that the nuclear, not the cytoplasmic, genotype of the parent plant affects the type of novel mitochondrial DNA organization found in the revertant. The DNA reorganization during reversion from CMS-S in tissue culture appears to be similar to that observed in spontaneous revertants obtained during the normal plant life-cycle. Unlike the situation for reversion from CMS-T, no common DNA sequence or reading frame appeared to be lost or disrupted in revertants.     

Somaclonal-variation-induced aluminum-sensitive mutant from an aluminum-inbred maize tolerant line

Somaclonal-variation-induced multiple mutations were observed in a progeny of the S1587 plant, regenerated from type I calli of the aluminum-tolerant inbred maize line Cat-100-6. After five generations of self-pollination, 14 progeny families of the S1587 somaclone were found to show aluminum toxicity symptoms with altered root tip morphology and reduced primary root growth. The most sensitive progeny, S1587-17, was crossed to the Cat-100-6 inbred line. The parental lines and the F1 were tested in nutrient solutions containing an aluminum activity gradient of 0–93 ⋅ 10^–6. The heterozygote behaves like the tolerant parent at aluminum activities up to 40 ⋅ 10^–6 and showed an intermediate phenotype at higher aluminum concentrations. Histological sections of aluminum-treated roots from tolerant and sensitive plants stained with hematoxylin, an aluminum marker, showed a progressive destruction of the root tip of the aluminum-sensitive genotype over time and indicated that tolerance in Cat-100-6 could be due to an aluminum exclusion mechanism. Segregation analysis of the F2 and backcross to the sensitive parent based on root morphology of plants subjected to an aluminum activity of 30 ⋅ 10^–6 showed the typical 3:1 and 1:1 tolerant:sensitive segregation ratios, respectively, indicating that tolerance in the Cat-100-6 inbred maize line is controlled by a single nuclear, semidominant gene, named Alm1. Received: 9 May 1996 / Revision received: 24 February 1997 / Accepted: 8 March 1997     

RFLP analysis of mitochondrial DNA from cytoplasmic male-sterile lines of pearl millet

Mitochondrial DNA (mtDNA) from 13 cytoplasmic male-sterile (cms) lines from diverse sources were characterized by Southern blot hybridization to pearl millet and maize mtDNA probes. Hybridization patterns of mtDNA digested with PstI, BamHI, SmaI or XhoI and probed with 13.6-, 10.9-, 9.7- or 4.7-kb pearl millet mtDNA clones revealed similarities among the cms lines 5141 A and ICMA 1 (classified as the S-A1 type of cytoplasm based on fertility restoration patterns), PMC 30A and ICMA 2. The remaining cms lines formed a distinct group, within which three subgroups were evident. Among the maize mitochondiral gene clones used, the coxI probe revealed two distinct groups of cytoplasms similar to the pearl millet mtDNA clones. The atp9 probe differentiated the cms line 81 A4, derived from P. glaucum subsp. monodii, while the coxII gene probe did not detect any polymorphism among the cms lines studied. MtDNA digested with BamHI, PstI or XhoI and hybridized to the atp6 probe revealed distinct differences among the cms lines. The maize atp6 gene clone identified four distinct cytoplasmic groups and four subgroups within a main group. The mtDNA fragments hybridized to the atp6 gene probe with differing intensities, suggesting the presence of more than one copy of the gene in different stoichiometries. Rearrangements involving the coxI and/or rrn18-rrn5 genes (mapped within the pearl millet clones) probably resulted in the S-A1 type of sterility. Rearrangements involving the atp6 gene (probably resulting in chimeric form) may be responsible for male sterility in other cms lines of pearl millet.     

Mitochondrial sensitivity to Drechslera maydis T-toxin and the synthesis of a variant mitochondrial polypeptide in plants derived from maize tissue cultures with texas male-sterile cytoplasm

Summary Tissue cultures of maize carrying cms-T cytoplasm have been found to regenerate fertile, T-toxin resistant plants, with and without a selective treatment with T-toxin. Progeny of these plants were tested for mitochondrial sensitivity to T-toxin and the translation products synthesised by isolated mitochondria were analysed. The results confirm previous indications of a close correlation between susceptibility to T-toxin and the synthesis of a variant 13,000 M_r mitochondrial polypeptide. Interestingly, there appeared to be a critical level at about 33% maximum synthesis of the 13,000 M_r polypeptide above which male sterility and sensitivity to T-toxin are jointly expressed. The possibility that there is a causal link between synthesis of this additional mitochondrial polypeptide, pollen abortion and sensitivity to T-toxin is discussed.     

Molecular analysis of mitochondria from a fertility restorer line of maize

Summary Inbred line Ky21 carries nuclear genes which restore fertility to all three cytoplasmic male sterile (cms) types of maize: T, C and S. By substituting the Ky21 cytoplasm into a nuclear background lacking all of the nuclear restorer genes, we have demonstrated that Ky21 contains a fertile (normal) cytoplasm. Gel electrophoresis of mitochondrial DNA from Ky21 demonstrated an approximately 2.1 kb plasmid and no evidence for a 2.35 kb plasmid found in many normal cytoplasms of North American lines of maize. A 2.1 kb plasmid had been reported to be diagnostic for the T-type cms. However, the restriction endonuclease digestion pattern of Ky21 mtDNA more closely resembled that of normal lines than T-cms. Furthermore, mitochondria of Ky21 plants did not synthesize a 13 kilodalton polypeptide, which has only been found to be synthesized by T-type mitochondria. From these molecular criteria, as well as from the genetic analysis, we conclude that the mitochondria of the Ky21 universal restorer line are normal. In having a shorter form of a linear mtDNA plasmid, Ky21 resembles cytoplasms found in Mexican races of maize.     

离体条件下核不育水稻突变为细胞质雄性不育

地水稻无花粉型核不育系南广占的体细胞无性系典败变异株ＮＴ１和ＮＴ２经过多代回交已经转成类似野败型的核质互作雄性不育系。ＮＴ１和ＮＴ２的恢保关系与野败型的一致,即野败型的恢复系和保持系同样可作ＮＴ１和ＮＴ２的恢复系和保持系,ＮＴ１现已回交１０代,不育性能稳定。     

In vitro selection for methomyl resistance in CMS-T maize

Summary Many plants resistant to methomyl (Lannate), an insecticide which selectively damages maize with the Texas (T) type of cytoplasmic male sterility (CMS-T), were obtained by in vitro selection and also without selection. The selection procedure used 0.6–0.7mM methomyl and callus from CMS-T versions of several field and sweet corn genotypes (W182BN, Wf9, P39, MDM1, SW1 and hybrids of SW1, IL766A1, IL766A2, and 442 with W182BN-N). Addition of 1 mM methomyl to the regeneration medium greatly reduced recovery of methomyl-sensitive escapes. Resistance was linked with reversion to male fertility and maternally inherited. Most progeny of resistant plants exhibited stable maternally inherited resistance for two generations in field tests. First-generation progeny of seven culture-derived plants segregated for resistance and sensitivity; this suggests that ears of these seven regenerants were cytoplasmically chimeral. Resistance to methomyl was associated with resistance to T toxin from Helminthosporium maydis race T and with changes in mitochondrial physiology. Prolonged culture (14–16 months versus 6–8 months) increased the frequency of resistance among both selected and non-selected regenerants. Little or no resistance was found among regenerants from certain genotypes. Selection with methomyl may be useful for production of improved sweet corn lines and as a source of mitochondrial mutants. This system is also convenient for studies of the effects of nuclear background and of culture and selection systems on the generation of cytoplasmic mutants.     

Reorganization of mitochondrial genomes of cytoplasmic revertants in cms-S inbred line WF9 in maize

Summary Cytoplasmic reversion to fertility in cms-S maize has been previously correlated with changes in mitochondrial genome organization, specifically with loss of the autonomously replicating linear plasmid-like DNAs, S1 and S2, and with accompanying alterations in the high molecular weight mtDNA (main genome) that specifically involved S1 and S2 sequences. These studies, however, dealt with cytoplasmic revertants occurring in the cms-VG M825 inbred line and in the cms-VG M825/Oh07 F₁ hybrid. This paper deals principally with patterns of mitochondrial DNA reorganization accompanying cytoplasmic reversion to fertility in the WF9 inbred line nuclear background. Here the free S1 and S2 plasmid-like DNAs are retained in the revertants. Mitochondrial DNA analysis by Southern hybridization using cloned fragments of S1 and S2 shows altered organization around S-homologous regions in the main mitochondrial genome of revertants as compared with that of the male-sterile parental controls, but the pattern of main genome changes involving these regions differs from that of the cytoplasmic revertants that occurred in M825 and M825/Oh07 backgrounds. Similar experiments using a clone of the cytochrome oxidase I (COX I) gene of maize as a probe indicate that reorganization in this region is also involved in the changes in mtDNA that accompany cytoplasmic reversion to male fertility in cms-S WF9. The heterogeneity in patterns of reorganization of the main mtDNA genome that accompany cytoplasmic reversion in the same and different nuclear backgrounds are discussed in relation to cytoplasmic male sterility (CMS).     

Mitochondrial DMA variation in plants regenerated from embryogenic callus cultures of CMS triticale

The mitochondrial DNA (mtDNA) organization of primary hexaploid cytoplasmic male-sterile (CMS) triticale regenerants containing Triticum timopheevi cytoplasm was analysed by hybridization experiments and compared with the mitochondrial genome organization of the corresponding regenerants with maintainer cytoplasm. Callus cultures had been derived from immature embryos, and 623 triticale plants were regenerated via somatic embryogenesis after three to four subcultures. The chondriome of 159 regenerants was investigated with regard to somaclonal variation. Six different mitochondrial gene probes and four different restriction enzymes were used for Southern blot analyses by the non-radioactive digoxigenin labeling technique. Alloplasmic regenerants showed a gain or loss of hybridization signals up to a high percentage, while euplasmic ones revealed only minor variability with respect to band stoichiometries. In 24 cases rearrangements in the mtDNA were proved. We suppose that recombination processes and selective amplification events are responsible for these findings.     

小麦胞质突变型雄性不育系85EA与其保持系线粒体DNA的比较研究

８５ＥＡ是通过电子束辐照获得的胞质突变型小麦不育要用ＲＦＬＰ和ＲＡＰＤ技术对８５ＥＡ及春保持系的线粒体ＤＮＡ进行了比较研究。ＲＦＬＰ分析表明８５ＥＡ线粒体基因组中ｃｏｘⅡ基因的位置结构与保持系发生了变化；ＲＡＰＤ分析中引物ＯＰＤ－１５扩增产物在不育系和保持系间有明显差异，不育系的扩增产物比保持系多１条分子量为０．６ｋｂ的特展览 要带，用Ｔ－ｅａｓｙ ｖｅｃｔｏｒ克隆该不育系特异条带并命名为ＯＰＤ－     

Cytoplasmic male sterility (CMS) in Lolium perenne L.: 1. Development of a diagnostic probe for the male-sterile cytoplasm

Analysis of reciprocal crosses between nonrestoring fertile genotypes and restored male-sterile genotypes of Lolium perenne confirmed the cytoplasmic nature of the sterility trait. This prompted a search for a molecular probe that could be used to distinguish between fertile and cytoplasmic male-sterile (CMS) cytoplasms. We describe the identification and cloning of a 4.5-kb BamHI-HindIII restriction fragment from the mtDNA of the CMS line. The cloned fragment (pCMS45) failed to hybridise to sequences in the mtDNA of fertile lines and was thus capable of unambiguously distinguishing between fertile and CMS cytoplasms. The use of pCMS45 as a diagnostic probe provided a simple test for positive identification of young non-flowering plants carrying the CMS cytoplasm and also permitted confirmation at the molecular level of the maternal transmission of the CMS trait suggested by the genetic data.     

Molecular analysis of genomic stability of mitochondrial DNA in tissue cultured cells of maize

Summary Mitochondrial DNA (mtDNA) of the Black Mexican sweet line of maize isolated from tissue cultured cell suspension cultures and young seedlings was examined. Restriction fragments generated by two endonucleases were comparatively analyzed by visualization of ethidium bromide stained gels as well as by membrane hybridization with nick-translated DNA probes of plasmid-like S1 and S2 DNA. Although no major molecular alterations were seen in tissue cultured cells, the samples were clearly not identical. The variation was mainly in the stoichiometry of several restriction fragments. Hybridization analyses with S1 and S2 probes show no evidence of molecular rearrangement in this part of the genome in tissue cultured cells. Minor variations in restriction patterns could reflect alterations in frequency of circular mtDNA molecules, perhaps related to nuclear alterations during the extended period of culture.     

A tissue culture induced Adh1 null mutant of maize results from a single base change

Summary We have found a null Adh1 allele which arose as a somaclonal variant following tissue culture of maize embryos carrying Adh1-1S and Adh1-1F alleles. Cloning and sequencing shows that the mutant allele derives from Adh1-1S and that there has been a single base change in the coding region of the gene which converts and AAG lysine codon to a TAG stop codon. The rate of nucleotide substitution (two per 218 embryos cultured) is much greater than normal mutation rates.     

Fertility restoration and mitochondrial nucleic acids distinguish at least five subgroups among cms-S cytoplasms of maize (Zea mays L.)

Summary Differences in fertility restoration and mitochondrial nucleic acids permitted division of 25 accessions of S-type male sterile cytoplasm (cms-S) of maize into five subgroups: B/D, CA, LBN, ME, and S(USDA). S cytoplasm itself (USDA cytoplasm) was surprisingly not representative of cms-S, since only two other accessions, TC and I, matched its mitochondrial DNA pattern. CA was the predominant subgroup, containing 18 of the 25 accessions. The B/D and ME subgroups were the most fertile and LBN the most sterile. The exceptional sterility of LBN cytoplasm makes it the most promising of the 25 cms-S accessions for the production of hybrid seed. The most efficient means of quantifying the fertility of the subgroups was analysis of pollen morphology in plants having cms-S cytoplasm and simultaneously being heterozygous for nuclear restorer-of-fertility (Rf) genes. This method took advantage of the gametophytic nature of cms-S restoration. The inbred NY821LERf was found to contain at least two restorer genes for cms-S. Fertility differences were correlated with mitochondrial nucleic acid variation in the LBN, ME, and S (USDA) subgroups.Paper No. 9498 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC     

Somaclonal variation in a maize inbred line is not associated with changes in the number or location of Ac-homologous sequences

Summary Somaclonal variation (tissue culture-induced mutations) may result, in some instances, from the activation of transposable elements. This study was conducted to determine whether somaclonal variants in the Zea maize L. inbred line FR27rhm were associated with movement of the transposable element Activator (Ac). Ten variants, seven of which from genetic analyses fit a single recessive gene model and three which did not due to a low number of mutant plants, were selected for analysis. Total DNA from these and from uncultured FR27rhm seedlings were examined by Southern blot analysis using the internal 1.6-kb Hind III fragment derived from the cloned Ac7 element as a probe. By using a restriction endonuclease which does not cut within the element, the number and distribution of the copies of Acrelated sequences in the FR27rhm genome could be determined. From the number of bands seen in the blots, we conclude that the FR27rhm inbred contains large numbers of Ac -related sequences. However, the pattern of bands seen in the ten variants and in the uncultured seedlings were identical, indicating that there had been no movement of any of the Acrelated sequences to cause the tissue culture-induced mutations.Contribution from Department of Agronomy, University of Illinois, Urbana, IL 61801, supported by funds from the Illinois Agricultural Experiment Station and Illinois Foundation Seeds     

普通小麦三种细胞质雄性不育系线粒体DNA的比较研究

对细胞质分别来源于粘果山羊草（Ａｅ．ｋｏｔｓｃｈｙｉ）、偏凸山羊草（Ａｅ．ｖｅｎｔｒｉｃｏｓａ）、提莫菲维（Ｔ．ｔｉｍｏｐｈｅｅｖｉ）的３种普通小麦雄性不育系,其相应保持系和共有的一种恢复系的ｍｔＤＮＡ进行了ＲＦＬＰ比较分析。发现Ｋ型和Ｖ型不育系的ｍｔＤＮＡ在组织结构上不同于Ｔ型,说明Ｋ、Ｖ型不育系是有别于Ｔ型的两种新不育类型。Ｋ型、Ｖ型不育系的ｍｔＤＮＡ与保持系和恢复系显著不同,推测ｍｔＤＮＡ与小麦细胞质雄性不育性有关。实验同时发现Ｔ型不育系与其保持系的ｍｔＤＮＡ非常相似,对这种相似性的原因进行了讨论。