Structural studies of microsporogenesis in fertile and male-sterile onions (Allium cepa L.) containing the cms-S cytoplasm

Summary The structure of anther tissues has been studied during microsporogenesis in male-sterile and -fertile onions. Three types of abnormal tapetal behaviour have been observed within the single line II/3ms containing the cms-S cytoplasm: type 1, the premature breakdown of the tapetum at the tetrad stage, type 2, the hypertrophy of the tapetum after the diad stage followed by its premature autolysis and, type 3, in which the tapetum remains in good condition but for an abnormally long period of time. Tapetal autolysis proceeds in the same manner in both male-steriles and -fertiles with only the stage at which it occurs differing between the types of plants. Mitochondria were prominent in the tapetal tissue of all onion types throughout all stages of microsporogenesis and were still visible during the last stages of tapetal autolysis. In a detailed study of type 2 behaviour, no differences in mitochondrial volumes were found until the tapetum hypertrophied.     

DNA polymorphism in Allium cepa cytoplasms and its implications concerning the origin of onions

Summary Mitochondrial and chloroplast DNA was isolated from fertile and cytoplasmic male sterile cultivars of cultivated onions. Restriction fragment length polymorphism led to the distinction between cytoplasms S and M. Mitochondrial DNA patterns from S cytoplasms appeared dentical and characterized mostly male sterile lines. An open-pollinated variety was found to bear this cytoplasm and thought to be the origin of S types. Mitochondrial DNA patterns from M cytoplasms were subdivided into four types, M₁ and M₂ corresponding to normal N cytoplasm, M₃ and M₄ probably corresponding to T cytoplasms. S and M cytoplasms were also distinguished by chloroplast DNA restriction patterns. Our results confirm previous genetic distinction between S, N and T cytoplasms.     

Asymmetric hybridization between cytoplasmic male-sterile (CMS) and fertile rice (Oryza safiva L.) protoplasts

Summary ⁶⁰Co-irradiated protoplasts of the cytoplasmic male-sterile line A-58 CMS (Oryza saliva L.) were electrofused with iodoacetamide (IOA)-treated protoplasts of the fertile (normal) rice cultivar Fujiminori. Seven of the colonies that formed were identified as cytoplasmic hybrids (cybrids): they all had the peroxidase isozymes of the fertile Fujiminori parent, but contained four plasmid-like DNAs (Bl, B2, B3 and B4) from the sterile A-58 CMS parent in their mitochondrial genomes. In addition, digestion of cybrid mtDNA gave a set of restriction fragments that differed from those of the parents.     

Molecular analysis of cytoplasmic male sterility in chives (Allium schoenoprasum L.)

The mitochondria of chive plants with normal N or male-sterile S cytoplasms have been examined by restriction fragment analysis and Southern hybridizations of mitochondrial DNA (mtDNA) and in organello protein biosynthesis. Restriction fragment patterns of the mtDNA differed extensively between N-and S-cytoplasms. The percentage of fragments with different mobility varied between 44–48% depending on the restriction enzyme used. In contrast to mtDNA, the restriction fragment patterns of the chloropolast DNA from N- and S-cytoplasms were identical. The organization of the analyzed mitochondrial genes coxII, coxIII, nad1 and nad3 was different in N- and S-cytoplasms. Comparison of mitochondrial proteins analyzed by in organello translation revealed an 18-kDa protein present only in S-cytoplasm. The restorer gene X suppressed the synthesis of that protein in S-cytoplasm. Thus, the 18-kDa protein seems to be associated with the cytoplasmic male-sterile phenotype.     

Molecular characterization of mitochondrial DNA of different subtypes of male-sterile cytoplasms of the sugar beet Beta vulgaris L.

Summary Mitochondrial (mt) DNA from eight cytoplasmic male-sterile (cms) lines of sugar beet from different breeding stations was investigated by restriction fragment analysis and Southern hybridization. All cms lines showed similar but not identical restriction and hybridization signal patterns, readily distinguishable from those of fertile (N) cytoplasm. Digestion of the mtDNA with BamHI, EcoRI, SalI, and XhoI revealed distinct differences between the sterile lines, and six subtypes of the S cytoplasm could be distinguished. Differences between the sterile lines were confirmed by hybridization with a gene probe revealing minor, line-specific hybridization signals. The data presented provide evidence for the existence of considerable variation within the only commercially used source of cms in the sugar beet, the Owen's type of cytoplasm.     

Cytoplasmic male sterility in hybrids of sterile wild beet (Beta vulgaris ssp. maritima) and O-type fertile sugar beet (Beta vulgaris L.): molecular analysis of mitochondrial and nuclear genomes

The organization of the mitochondrial genome of B3, B4 and B5generations of hybrids created by backcrossing sterile wild beet Betamaritima with a fertile O-type sugar beet line was studied usingrestriction fragment length polymorphism (RFLP) analysis. Random amplifiedpolymorphic DNA (RAPD) analysis was used to study restoration of the fertile(O-type) sugar beet genotype in hybrids after multiple backcrossings.Restriction of mtDNAs from the cytoplasm of B. maritimaandhybrids revealed BamHI, EcoRI andXhoI restriction patterns different from those for sterileand fertile sugar beet lines. The most conspicuous feature of our accession ofsterile wild beet mtDNA was the absence of the 10.7-kbEcoRI fragment detected in the cytoplasm of S-type sterileB. maritima and sugar beet. The hybridization of digestedmtDNAs with coxII, atpA andatp6 homologous probes revealed alterations within thesegene loci that distinguished wild beet and hybrids from sugar beets.Characteristic hybridization profiles for the wild beet and B3, B4 and B5hybrids were observed for all probes regardless of the restrictase used todigest mtDNA. Notable changes in atpA andatp6 genes resulted when probes that comprised the5flanking sequences of these genes and a small part of the coding sequences wereused. RFLP analysis of the sterile B. maritimamitochondrial genome further supported the unique character of this source ofwild beet sterility. The genotypic differences between hybrids and parentalaccessions were determined by scoring PCR-RAPD reaction products for nineselected primers. The diversity of the B. maritimagenotyperesulted in a lower genetic similarity index in comparison with hybrids,sterileand fertile lines of sugar beet. The dendrogram obtained after cluster analysisdistinguished hybrids as a group that differed from wild beet and themaintainersugar beet line used for backcrossing. These results may indicate incompleterestoration of the fertile sugar beet genotype in hybrids.     

Microsporogenesis and tapetal development in fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.)

Summary The development of sporogenous and tapetal cells in the anthers of male-fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.) plants was studied using light and transmission electron microscopy. In general, male-sterile anthers showed a much greater variability in developmental pattern than male-fertile anthers. The earliest deviation from normal anther development was observed to occur in sterile anthers at meiotic early prophase: there was a degeneration or irregular proliferation of the tapetal cells. Other early aberrant events were the occurrence of numerous small vesicles in the microspore mother cells (MMC) and a disorganized chromatin condensation. Deviations that occurred in sterile anthers at later developmental stages included: (1) less distinct inner structures in the mitochondria of both MMC and tapetal cells from middle prophase onwards. (2) dilated ER and nuclear membranes at MMC prophase, in some cases associated with the formation of protein bodies. (3) breakdown of cell walls in MMCs and tapetal cells at late meiotic prophase. (4) no massive increase in tapetal ER at the tetrad stage. (5) a general dissolution of membranes, first in the MMC, then in the tapetum. (6) abortion of microspores and the occurrence of a plasmodial tapetum in anthers reaching the microspore stage. (7) no distinct degeneration of tapetal cells after microspore formation. Thus, it seems that the factors that lead to abortive microsporogenesis are structurally expressed at widely different times during anther development. Aberrant patterns are not restricted to the tetrad stage but occur at early prophase.     

Cytoplasmic male sterility in rapeseed (Brassica napus L.)

Summary Restriction patterns of chloroplast (cp) and mitochondrial (mt) DNA in Brassica napus rapeseed reveal the alloplasmic nature of cytoplasmic male sterility in this crop. Both the Shiga and Bronowski systems probably exploit cytoplasmic diversity in B. napus cultivars arising from introgression of cytoplasm from the other rapeseed species, B. campestris. Nuclear genes specific to these systems do not cause sterility in maintainers (Bronowski and Isuzu-natane) because they have a campestris cytoplasm, but give rise to sterility in napus cytoplasms. In the course of hybridization to napus cultivars a line with the triazine resistant cytoplasm (a campestris cytoplasm) has undergone an alteration in the mt genome rendering its restriction pattern more similar than previously to that of napus. The alteration may be an inversion between 7.2 and 3.4 kb in length.     

The use of mitochondrial DNA polymorphism in the classification of individual onion plants by cytoplasmic genotypes

Summary Individual plants of a Japanese onion variety Sapporo-ki, which is characterized by the occasional occurrence of male-sterile plants, have been investigated for mitochondrial (mt) DNA polymorphism. Male-fertile and the Jones' cytoplasmic male-sterile (CMS) onions were also included for comparison. Southern blot hybridization with rrn26, cox-I, cox-II, cob, atpA and atp9 genes as probes revealed the two classes of mtDNA variation within a population of Sapporo-ki: Out of the 41 plants examined 19 contained mtDNA typical of malefertile plants, and 22 individuals contained mtDNA typical of the Jones' CMS genotype. Our results thus indcate that the use of the mitochondrial gene probes may greatly facilitate the classification of individual plants by cytoplasmic genotypes.     

Identification of cytoplasms using the polymerase chain reaction to aid in the extraction of maintainer lines from open-pollinated populations of onion

S-cytoplasm is the most common source of cytoplasmic-genic male sterility (CMS) used to produce hybrid-onion seed. Identification of the cytoplasm of a single plant takes from 4 to 8 years and is complicated by the segregation of a nuclear gene that restores fertility. Although CMS in onion may be due to an incompatibility between the mitochondrial and nuclear genomes, Southern analyses of DNA from individual plants from crosses of S- and N-cytoplasmic plants supported maternal inheritance of the chloroplast and mitochondrial DNA and, therefore, polymorphisms in the chloroplast DNA may be used to classify cytoplasms. Amplification by the polymerase chain reaction of a fragment that carries an autapomorphic 100-bp insertion in the chloroplast DNA of N-cytoplasm offers a significantly quicker and cheaper alternative to crossing or Southern analysis. Molecular characterization of N- and S-cytoplasms and frequencies of the nuclear non-restoring allele allow onion breeders to determine the proportion of plants in open-pollinated populations that maintain CMS and can significantly reduce the investment required to identify individual maintainer plants.     

Agrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots

This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the uidA gene coding for -glucuronidase was used as an indicator in the optimization of the protocol. Subspecies (onion and shallot) and cultivar were important factors for a successful transformation: shallot was better than onion and for shallot cv. Kuning the best results were obtained. Also, it was found that constantly reducing the size of the calli during subculturing and selection by chopping, thus enhancing exposure to the selective agent hygromycin, improved the selection efficiency significantly. Furthermore, callus induction medium and co-cultivation period showed a significant effect on successful stable transformation. The usage of different Agrobacterium strains, callus ages, callus sources and osmotic treatments during co-cultivation did not influence transformation efficiency. The highest transformation frequency (1.95%), was obtained with shallot cv. Kuning. A total of 11 independent transformed callus lines derived from zygotic embryos were produced: seven lines from shallot and four lines from onion. Large differences in plantlet production were observed among these lines. The best line produced over 90 plantlets. Via PCR the presence of the uidA and hpt (hygromycin phosphotransferase) genes could be demonstrated in these putative transformed plants. Southern hybridization showed that most lines originated from one transformation event. However, in one line plants were obtained indicating the occurrence and rescue of at least three independent transformation events. This suggested that T-DNA integration occurred in different cells within the callus. Most transgenic plants only had one copy of T-DNA integrated into their genomes. FISH performed on 12 plants from two different lines representing two integration events showed that original T-DNA integration had taken place on the distal end of chromosomes 1 or 5. A total of 83 transgenic plants were transferred to the greenhouse and these plants appeared to be diploid and normal in morphology.     

Chloroplast DNA polymorphism in fertile and male-sterile cytoplasms of sorghum (Sorghum bicolor (L.) Moench)

Summary Restriction endonuclease patterns of chloroplast DNA (cpDNA) were consistently distinguishable between fertile and male-sterile cytoplasms of sorghum [Sorghum bicolor (L.) Moench], whereas no differences in restriction patterns of cpDNA among male-sterile (A₁) lines, including six isocytoplasmic strains, were revealed in this study. It is suggested that chloroplast DNA may contribute to the male sterility of A₁ lines used currently in hybrid sorghum production.This research was supported by a research grant from Kansas Grain Sorghum Commission, Kansas Board of Agriculture. Contribution 90-293-J from the Kansas Agricultural Experiment Station     

A new type of cytoplasmic male sterility in rye (Secale cereale L.): analysis of mitochondrial DNA

Summary Mitochondrial (mt) DNA of a new type of rye cytoplasm (Gülzow, G) that induces cytoplasmic male sterility (CMS) was analyzed and compared with rye mtDNAs of different origins MtDNA of the G type was easily distinguishable from mtDNA of another CMS source, Pampa (P) type, and from mtDNA of fertile lines with respect to restriction fragment patterns and hybridization with mitochondrial genes. The results of the molecular analyses indicate a close, but not identical relationship between the mtDNA of the G type cytoplasm and that of cv Pluto.     

Mitochondrial DNA polymorphism in male-sterile cytoplasm of rice

Summary Mitochondrial DNAs (mtDNAs) were isolated and purified from ten strains of rice plants with male-sterile cytoplasm. The mtDNAs were digested with the restriction endonuclease PstI and the fragment patterns produced were analysed by 0.7% agarose gel electrophoresis. Restriction fragment length polymorphism was observed among the mtDNAs analysed; there were seven different patterns for the ten examined. Our results indicate that there are a variety of mtDNAs in cytoplasmically male-sterile rice.     

Rapid hybridization-based assays for identification by DNA probes of male-sterile and male-fertile cytoplasms of the sugar beet Beta vulgaris L.

Summary Methods are described whereby hybridization of mitochondrial (mt) DNA with different DNA probes can definitely distinguish male-fertile and and male-sterile (cms) cytoplasms of sugar beet Beta vulgaris L. We have developed two types of miniassays. (1) Comparative methods requiring the isolation and restriction of total cellular DNA, hybridization with cloned mtDNA fragments from either fertile or male-sterile cytoplasms, and comparison of the hybridization patterns to the fertile-and sterile-specific patterns of mtDNA of sugar beet for the given mtDNA probe. For these analyses, we routinely used 1 g of plant material to determine the type of cytoplasm. (2) Noncomparative (plus-minus) methods requiring neither the isolation of pure DNA nor restriction, electrophoresis, or Southern blotting. Instead, alkaline-SDS plant extracts from as little as 50 mg of plant material were dot-blotted and hybridized with fertile-specific (mitochondrial minicircular DNA) and/or cms-specific probes (consisting of a 2.3-kb mtDNA sequence exclusively occurring in the cms cytoplasm). The assays are simple to perform, give definitive results, are nonde-structive to the plants, and may be used in mass screening of sugar beet populations for hybrid production or in in vitro culture processes.     

Comparative restriction endonuclease analysis and molecular cloning of plastid DNAs from wild species and cultivated varieties of the genus Beta (L.)

Summary A phyletic tree of the genus Beta has been constructed based on EcoRI and PstI plastid DNA restriction patterns of eight species from three sections of the genus. In contrast to the remarkable morphological variability of the varieties of B. vulgaris the restriction patterns of the plastid DNA of this species were found to be almost identical. The comparison of plastic DNAs of B. vulgaris crassa fertile and sterile lines with 13 different restriction enzymes revealed only a single fragment polymorphism in the HindIII patterns. Hybridization analyses in the plastidal rDNA region revealed an interesting loss of an EcoRI restriction site in all cultivated B. vulgaris varieties in contrast to wild species. The results of the construction of clone banks for SalI and BamHI fragments of plastid DNA from fertile B. vulgaris crassa are reported and difficulties in the cloning of specific fragments are discussed.