The transfer of 'Polima' cytoplasmic male sterility from oilseed rape (Brassica napus) to broccoli (B. oleracea) by protoplast fusion.

Protoplast fusion was utilised to transfer Polima type cytoplasmic male sterility (CMS) from Brassica napus, canola cv. Polima Karat (Pol-Karat) to B. oleracea, broccoli, var. "Green Comet". Southern and RFLP analysis confirmed that four cybrids possessed nuclear genomes of broccoli with Polima mitochondria and chloroplasts. A fifth cybrid was a nuclear hybrid between broccoli and Pol-Karat, with Polima mitochondria and chloroplasts of broccoli. The broccoli type cybrids were morphologically similar to "Green Comet", while the hybrid type was an intermediate of the two fusion parents. Flowers on the cybrids were distinctive in that although they possessed a morphology typical of Polima, they had very reduced petals. The broccoli type cybrids exhibited some female fertility, albeit low, establishing potential for F1 hybrid production.

A rapid, single leaf, nucleic acid assay for determining the cytoplasmic organelle complement of rapeseed and related Brassica species.

An assay is described whereby Eco RI restriction fragment length polymorphisms of mitochondrial and chloroplast DNAs can definitively identify cytoplasms of interest in Brassica crop development. Restrictable mitochondrial and chloroplast DNA is extracted from as little as 2-3 g and 0.5 g leaf tissue, respectively, and the donor plants are able to continue to develop in a normal manner. An unknown cytoplasm can be identified in three days, which is a considerable saving in time and labor compared to the several years required by traditional methods. The assay is very inexpensive and should be established as a routine procedure in laboratories involved in sexual or somatic Brassica hybrid production.

The combination of polima cms and cytoplasmic triazine resistance in Brassica napus.

Protoplast fusion was used to combine cytoplasmic triazine resistance (ctr) and Polima type cytoplasmic male sterility (cms) in Brassica napus. The cybrids produced constitute the major biological input required for the production of commercial single-cross hybrid rapeseed bearing cytoplasmic triazine resistance. The results also indicate that Polima cms is associated with the mitochondrial genome.

Terminal protein association and sequence homology in linear mitochondrial plasmid-like DNAs of sorghum and maize.

The linear extrachromosomal mitochondrial plasmid-like DNAs from the Ru cytoplasm of maize, and M35-1 and IS1112C cytoplasms of sorghum, possess 5' terminally-attached proteins. These molecules required proteinase K treatment for mobility in agarose gels and were susceptible to exonuclease III but not lambda exonuclease cleavage. Hybridizations, under stringent conditions, indicated that the sorghum plasmid-like DNAs, N1 and N2, did not possess DNA sequence homology to cloned central regions of S1 and S2, the linear mitochondrial plasmid-like DNAs present in S cytoplasm of maize. In addition, a novel 4.2kb, DNAase sensitive, RNAase insensitive band, exhibiting homology to internal sequences from maize S2, was observed in the sorghum IS1112C cytoplasm only.

Mitochondrial DNA rearrangements in somatic hybrids of Solanum tuberosum and Solanum brevidens.

Thirty somatic hybrids between Solanum tuberosum and Solanum brevidens were analysed for mitochondrial and chloroplast genome rearrangements. In all cases, the chloroplast genomes were inherited from one of the parental protoplast populations. No chloroplast DNA alterations were evident but a range of mitochondrial DNA alterations, from zero to extensive intra- and inter-molecular recombinations, were found. Such recombinations involved specific 'recombination hot spots' in the mitochondrial genome. Not all hybrids regenerated from a common callus possessed identical mitochondrial genomes, suggesting that sorting out of mitochondrial populations in the callus may have been incomplete at the plant regeneration stage. Sorting out of organelles in planta was not observed.

The introduction of CMS mitochondria to triazine tolerant Brassica napus L., var. "Regent", by micromanipulation of individual heterokaryons.

Cytoplasmic triazine tolerance and cytoplasmic male sterility traits were combined in the nuclear genomic background of the Brassica napus variety 'Regent', following protoplast fusion, selection of fusion products by manual micro-manipulation, and culture in a Nicotiana tabacum nurse system. Whole plant cybrid regenerants were morphologically normal and produced seed on pollination, demonstrating their potential for incorporation into a breeding program.

Restriction endonuclease studies on the chloroplast and mitochondrial DNAs of alfalfa (Medicago sativa L.) protoclones.

Alfalfa protoclones were regenerated from the mesophyll protoplasts of two cloned source plants (parents), RS-K1 and RS-K2, initiated from Regen S seed. Because of the high frequency of karyotypic upset previously observed in these plants, chloroplast DNAs (cpDNA) from 23 protoclones and mitochondrial DNAs (mtDNA) from 20 protoclones were examined by restriction endonuclease analysis in order to assess recombination in their cytoplasmic genomes. Seven and four endonucleases were separately used for cpDNA and mtDNA analysis, respectively. Data were consistent with no, or a low frequency of, major sequence rearrangements in either the chloroplast or the mitochondrial genomes as a result of protocloning. However, two types of cpDNA were detected in the 23 protoclones, with only one protoclone possessing the cpDNA type of the cloned parental populations sampled. Possible explanations include a preferential selection during protocloning for one of two parental cpDNA types, an in planta sorting out of cpDNA types in the parental material or both.

Variable presence of the 1.94kb mitochondrial plasmid in maize S cytoplasm and its relationship to cytoplasmic male sterility.

Mitochondrial DNA (mtDNA) was isolated from over 100 different maize nucleo-cytoplasmic combinations. DNA preparations were assayed for the presence of the 1.94kb mitochondrial plasmid by agarose gel electrophoresis and hybridization to a recombinant clone of the plasmid. The plasmid was present in all tested inbreds which carried N, male fertile, cytoplasm or the cytoplasmically male sterile (cms) groups,cms-T andcms-C. However, members of thecms-S group differed with respect to the presence of the plasmid. Cytoplasms I, J and S possessed the plasmid, whereas cytoplasms B, CA, D, G, H, IA, ME, ML, PS, RD and VG did not.Cms-S group lines which had spontaneously reverted to fertility (nuclear and cytoplasmic revertants) did not exhibit a concomitant change in 1.94kb plasmid levels, although all such lines showed the previously reported alteration in levels of the linear mtDNAs, S1 and S2. The presence or absence of the plasmid was not correlated with (i) frequency of reversion to fertility, (ii) the degree of male sterility expressed, (iii) the presence or absence of standard nuclear restorer to fertility genes and (iv) nuclear genotype. Latin American races carrying RU cytoplasm possessed the plasmid, as did sweet corn varieties. The relevance of the data tocms and evolution of thecms-S group is discussed.

Transposon Tn5 mutagenesis in Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica.

In matings between Escherichia coli 2492(pJB4JI) and Erwinia carotovora subsp. carotovora Ecc71 and E. carotovora subsp. atroseptica Eca12, Kmr Gms transconjugants were obtained at high frequencies, indicating instability of the Mu-containing plasmid pJB4JI and transposition of Tn5 into the recipient genome. This was verified by Southern blot hybridization with pRZ102 DNA containing Tn5 as the 32P-labeled probe. Examination of Kmr Gms transconjugants of Ecc71 and Eca12 disclosed that a proportion (2 to 3%) were either auxotrophic or defective in catabolism of specific carbohydrates. Spontaneous prototrophic revertants were obtained for all markers with the exception of ilv, tyr, and suc. Genetic and physical data indicate that scattered insertions of Tn5 from pJb4JI into the chromosome of Ecc71 and Eca12 produced a variety of altered phenotypes due mostly to single insertions of Tn5 not accompanied by Mu DNA.

Somatic hybridization in the genus Solanum: S. tuberosum and S. brevidens.

Somatic hybrid plants were regenerated from fused mesophyll protoplasts of an albino potato (Solanum tuberosum spp. tuberosum) variant and Solanum brevidens, a non-tuber bearing species which is sexually incompatible with S. tuberosum. These somatic hybrid plants represent the first example of direct hybridization between potato and members of the taxonomic group Etuberosa, and offer the potential for introgressing valuable germplasm from Solanum species outside the sexually compatible range into a worldwide crop species.

Cytoplasmic DNA variation in a potato protoclonal population.

Mitochondrial DNA variation was detected in potato plants (protoclones) regenerated from leaf mesophyll protoplasts. Two forms of variation were evident; (1) DNA sequence alterations within the high molecular weight mitochondrial chromosome and (2) the appearance of an additional low molecular weight mitochondrial DNA species. Variation in chloroplast DNA was not detected. The data suggests that protocloning can introduce molecular diversity into mitochondrial genomes and thereby assist in overcoming the cytoplasmic genetic uniformity prevalent in most major crops.

Examination of the mitochondrial genome of revertant progeny from S cms maize with cloned S-1 and S-2 hybridization probes.

We have examined the molecular rearrangement of mitochondrial DNAs in each of several fertile revertants that arose spontaneously from S-type cytoplasmically male-sterile maize. Cloned segments of S-1 and S-2 DNAs (plasmid-like DNAs characteristic of the mitochondrial DNAs of S-type lines) were hybridized to untreated and restriction endonuclease-treated mitochondrial DNAs from fertile plants carrying normal cytoplasm, from cytoplasmically male-sterile plants, and from plants that had cytoplasmically reverted to fertility. The relative intensity of hybridization with S-1 and S-2 probes was different among the fertile, sterile, and revertant lines. The sizes of some restriction endonuclease fragments from the fertile revertant lines that hybridize with the S-2 probe differ from those of the sterile parental lines. Preferential synthesis of high molecular weight components of the mitochondrial genome carrying S-1 and S-2 sequences, concomitant with cessation in apparent autonomous replication of discrete S-1 and S-2 DNAs and their replicative intermediates (described here), could accommodate the hybridization data. The results suggest but do not prove that S-2 sequences are transposed coincident with the sterile plant's reversion to fertility. The inserted segments could arise from sequences already present in the high molecular weight DNA or from the lower molecular weight linear components of the mitochondrial genome. Putative target sites of insertion of S-1 and S-2 sequences would be multiple and separate for each. Reversion of S-type cytoplasmically male-sterile plants to fertility does not restore the organization of the mitochondrial genome to that of a normal fertile plant.

Mitochondrial DNA variation in races of maize indigenous to Mexico.

Mitochondrial DNAs have been examined in accessions of 25 Mexican races of maize and compared with the mitochondrial DNAs previously found in inbred lines from the USA. Many variants were found. Low molecular weight DNA components, not previously found in US lines, were found in many of the accessions. Accessions classified as belonging to the same race, and plants from a single accession, sometimes had different mitochondrial genomes. Mitochondrial genomes similar to those in T and S cytoplasms were found in Mexican accessions.A low molecular weight linear DNA species has partial homology with a sequence in the high molecular weight mitochondrial genome. All plants with a shorter version of the linear molecule had a correspondingly altered region of homology in the high molecular weight genome.There is evidence that the geographical distribution of mitochondrial DNA types within Mexico is not random. One type, found in the oldest races, appears to be widely dispersed but another less common type appears to be confined largely to coastal regions. The potential value of these findings in maize breeding and for evolutionary studies is discussed.

S1 and S2, the linear mitochondrial DNAs present in a male sterile line of maize, possess terminally attached proteins.

S1 and S2 are short linear mitochondrial DNA molecules found in a particular male sterile cytoplasm of maize. We show here that these DNA molecules and two other related linear DNA species found in maize mitochondria, have proteins attached, probably covalently, to their 5' ends. This is the first demonstration of such a linear DNA-terminal protein association in higher eukaryotes. Such proteins may be involved in priming replication of these DNAs.

Mitochondrial DNA analyses of fertile and sterile maize plants derived from tissue culture with the texas male sterile cytoplasm.

Maize plants carrying Texas (T) cytoplasm are male-sterile and sensitive to Drechslera maydis race T toxin, whereas plants carrying Normal (N) cytoplasm are male-fertile and resistant to the toxin. Some plants regenerated from T cytoplasm tissue cultures exhibit a N cytoplasm-like phenotype with respect to malefertility and toxin-resistance. Analysis of the high molecular weight mitochondrial DNA (mtDNA) of such regenerants has shown that the plants do not contain N cytoplasm mtDNA. However, their mtDNAs do show sequence differences from each other and from the mtDNA of plants possessing T cytoplasm. No single alteration detected correlates with the change to malefertility or toxin resistance. Sequence alterations were also evident in high molecular weight mtDNA isolated from a plant regenerated from N cytoplasm callus. No changes in low molecular weight mtDNA molecules were observed in regenerants from N or T cytoplasm callus.

Activation of Glyceraldehyde-Phosphate Dehydrogenase (NADP) and Phosphoribulokinase in Phaseolus vulgaris Leaf Extracts Involves the Dissociation of Oligomers.

Phosphoribulokinase (EC 2.7.1.19, ATP: d-ribulose-5-phosphate-1-phosphotransferase) resembles the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13, d-glyceraldehyde-3-phosphate: NADPH(+) oxidoreductase [phosphorylating]) of chloroplasts in that the activation of both of these enzymes involves the dissociation of oligomers (apparently tetrameric forms) with low catalytic activity to give protomers which possess higher catalytic activity. Gel filtration on Sepharose 6B has shown that the molecular weights of the oligomer and active protomer of phosphoribulokinase are, respectively, about 6.8 x 10(5) and 1.7 x 10(5), whereas the corresponding values for glyceraldehyde-3-phosphate dehydrogenase are 8.2 x 10(5) and 2.2 x 10(5). Activation of both enzymes occurs in response to either ATP, dithiothreitol, or cholate while the glyceraldehyde-3-phosphate dehydrogenase is also activated by NADPH. Activation/dissociation of these enzymes may involve conformational changes resulting from nucleotide binding, the reduction of sulfur bridges, and the cholate induced loosening of hydrophobic interactions.

Classification of normal and male-sterile cytoplasms in maize. I. Electrophoretic analysis of variation in mitochondrially synthesized proteins.

Male-sterile cytoplasms of maize have previously been classified into three groups (T, S and C) according to their fertility ratings in various inbred backgrounds. In earlier studies, mitochondria from three male-sterile cytoplasms, representing each of these three groups, have been found to synthesize characteristic variant polypeptides that distinguish them from each other and from those of normal (N) cytoplasm. In order to determine the extent of cytoplasmic variation, we have now analyzed the translation products of mitochondria from 28 additional cytoplasmic sources. The results show that on this basis 18 of the cytoplasms are identical to the USDA (S) cytoplasm, three are identical to the Texas (T) cytoplasm and two are identical to the C cytoplasm. The five remaining cytoplasms are indistinguishable from normal, male-fertile (N) cytoplasm. Our classification of the cytoplasms is in general agreement with those based on fertility restoration. However, of three cytoplasms that have previously remained unclassified, two (B and D) have now been assigned to the S group and one (LF) to the N group. No heterogeneity in mitochondrial translation products was detected within the normal or any of the three male-sterile groups. The usefulness of the analysis of mitochondrial translation products as a method for classifying normal and male-sterile cytoplasms is discussed.