Alteration of mitochondrial genomes containing atpA genes in the sexual progeny of cybrids between Raphanus sativus cms line and Brassica napus cv. Westar

Summary We have investigated the fate of the mitochondrial genomes of cybrids derived from donor-recipient protoplast fusion between X-irradiated Raphanus sativus (cms line) and iodoacetamide-treated Brassica napus cv. Westar. Two out of ten fusion products were male-sterile with the diploid chromosome number of B. napus. The mitochondrial (mt) genomes of the cybrids and their progeny were further analyzed by DNA-DNA hybridizaion using the pea mitochondrial ATPase subunit gene (atpA) as a probe. One cybrid, 18-3, had a 3.0 kb fragment characteristic of B. napus and a 2.0 kb non-parental fragment when the BamHI-digested DNA was hybridized with the probe. In the first-backcrossed progeny of this cybrid, the hybridization pattern was not stably inherited. A 4.0 kb radish fragment, not detectable in the cybrid, appeared in one of the BC₁ generation siblings, and the 2.0 kb non-parental fragment was lost in another. The hybridization patterns in BC₁ progeny siblings of cybrid 12-9 were also varied. The alteration of mtDNA in the cybrid progeny continued to the BC₂ generation. There was no clear evidence of a heteroplasmic state or of sub-stoichiometric molecules in the mt genome of cybrid 18-3. A possible cause of the observed alteration in the mt genome is discussed.     

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

Summary 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.     

Linkage between an isozyme marker and a restorer gene in radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Summary Co-segregation studies of isozyme markers and male fertility restoration showed that a restorer gene from radish was introduced into rapeseed along with an isozyme marker (Pgi-2). The radish chromosome segment carrying these genes was introgressed into rapeseed through homoeologous recombination, substituting for some of the rapeseed alleles. By crossing heterozygous restored plants to male-sterile lines and to maintainers, tight linkage was found between the restorer gene and the marker. The recombination fraction was estimated at 0.25 ± 0.02%. Although few restored plants lacked the radish isozyme marker, it was still possible to distinguish male-fertile from male-sterile plants by their PGI-2 patterns. Furthermore, homozygous and heterozygous restored plants could be separated by specific PGI-2 phenotypes. Thus, the Pgi-2 marker is now currently used in restorer breeding programs.     

Transfer of Ogu cytoplasmic male sterility to Brassica juncea and improvement of the male sterile line through somatic cell fusion

Male sterility conferred by ogu cytoplasm of Raphanus sativus has been transferred to Brassica juncea cv RLM 198 from male-sterile B. napus through repeated backcrossing and selection. The male-sterile B. juncea is, however, highly chlorotic and late. It has low female (seed) fertility and small contorted pods. To rectify these defects, protoplasts of the male sterile were fused with normal RLM 198 (green, self fertile). Four dark green, completely male-sterile plants were obtained and identified as putative cybrids. All the plants were backcrossed three times with RLM 198. Mitochondrial and chloroplast DNA analysis of backcross progeny confirmed hybridity of the cytoplasm. The restriction pattern of the chloroplast DNA of progeny plants of three cybrids (Og 1, Og 2, Og 3) was similar to that of the green self-fertile RLM 198 and indicated that the correction of chlorosis resulted from chloroplast substitution. The chloroplast DNA of the lone progeny plant of the fourth cybrid (Og 10) could not be analyzed because the plant was stunted and had only a few leaves. When total cellular DNA was probed with mitochondrial probes coxI and atpA it was found that the cybrids had recombinant mitochondria. The chlorosis-corrected plants were early flowering and had vastly improved seed fertility.     

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.     

Introduction of a gene from fertility restored radish (Raphanus sativus) into Brassica napus by fusion of X-irradiated protoplasts from a radish restorer line and iodacetoamide-treated protoplasts from a cytoplasmic male-sterile cybrid of B. napus

To establish a cytoplasmic male-sterile/restored fertility (cms-Rf) system for F₁ seed production in Brassica napus, we transferred a gene from fertillity restored radish to B. napus by protoplast fusion. X-irradiated protoplasts, isolated from shoots of Raphanus sativus cv Kosena (Rf line), were fused with iodoacetamide-treated protoplasts of a B. napus cms cybrid. Among 300 regenerated plants, six were male-fertile. The fertile plants were characterized for petal color, chromosome number and the percentage of viable pollen grains. Three fertile plants had aneuploid chromosome numbers and white or cream petals, which is a dominant marker in radish. Of these three plants, one which had 2n = 47 chromosomes and white petals was used for further backcrosses. After two backcrosses, chromosome number and petal color became identical to that of B. napus. No female sterility was observed in the BC₃ generations.     

Identification of RAPD markers linked to a fertility restorer gene for theOgura radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in theOgura radish cytoplasmic male sterility of rapeseed. A total of 138 arbitrary 10-mer oligonucleotide primers were screened on the DNA of three pairs of bulks, each bulk corresponding to homozygous restored and male sterile plants of three segregating populations. Six primers produced repeatable polymorphisms between paired bulks. DNA from individual plants of each bulk was then used as a template for amplification with these six primers. DNA polymorphisms generated by four of these primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertility restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragments did not share any homology. Southern hybridization of labelled RAPD fragments on digested genomic DNA from the same three pairs of bulks revealed fragments specific to either the male sterile bulks or to the restored bulks and a few fragments common to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments that were completely linked to the restorer locus have been cloned and sequenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of restorer lines used in breeding programs and is the first step towards map-based cloning of the fertility restorer allele.     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Intergeneric hybridization between Brassica napus and Sinapis pubescens,and the cytology and crossability of their progenies

The cytological possibility of gene transfer from Sinapis pubescens to Brassica napus was investigated. Intergeneric hybrids between Brassica napus (2n = 38) and Sinapis pubescens (2n = 18) were produced through ovary culture. The F₁ hybrids were dihaploid and the chromosome configurations were (0–1) _III + (2–11) _II + (5–24) _I . One F₂ plant with 38 chromosomes was obtained from open pollination of the F₁ hybrid. Thirty-one seeds were obtained from the backcross of the F₂ plant with B. napus. Five out of seven plants had 38 chromosomes, and the pollen stainability ranged from 0% to 81.4%. In the B₂ plants obtained from the backcross of B₁ plants with B. napus, 66.7% of the plants examined had 38 chromosomes. S. pubescens may become a gene source for the improvement of B. napus.     

Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus

Summary Under an intermediate temperature regime (23° C/18° C; day/night), microsporogenesis in stamens of the ogu cytoplasmic male-sterile (CMS) line of Brassica napus terminated by the tetrad stage, although in some cases degeneration of the sporogenous tissue occurred prior to meiosis. In most cases the tetrads were collapsed and bounded by a sparse exine, but contained many organelles. Also, the tapetum in CMS anthers was abnormal and often highly vacuolated by the tetrad stage. Under low temperature conditions (18° C/15° C; day/night), neither microsporogenous nor tapetal tissues were observed. In the normal stamens, no differences were observed under different temperature regimes. In conjunction with the adjoining paper, this study demonstrates that temperature conditions strongly affect the cytological processes associated with microsporogenesis in the CMS anthers.     

Intergeneric somatic hybrid production through protoplast fusion between Brassica juncea and Diplotaxis muralis

Summary The need to transfer genetic traits from Diplotaxis muralis (2n=42) to Brassica juncea (2n=36), a major oil seed crop of the tropical world, was realised. Since the two plant types are sexually incompatible, attempts were made to evolve parasexual hybrids as the result of protoplast fusion. Protoplasts of hypocotyl-derived calli of two cultivars of B. juncea were fused with normal and -irradiated mesophyll protoplasts of Diplotaxis muralis. Regeneration of 110 plants from the fused products was successfully achieved. Upon analysis of some of them, we realised that true somatic hybrids and partial somatic hybrids had been generated. Thus the primary goal of evolving intergeneric hybridisation products between these two plant types was fulfilled.     

Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus

Summary In the ogu cytoplasmic male-sterile (CMS) line of Brassica napus, stamen morphology was influenced by temperature conditions. Under a high temperature regime (27° C/23° C; day/ night) CMS stamens had a near-normal morphology, but microsporogenesis proceeded to a maximum of the microspore stage. However, compared to the normal stamens, the occurrence of sporopollenin-like deposits in the tapetum and deposition of exine on the microspores was sparse. Also, the tapetal cells of the CMS line were often highly vacuolate and failed to degenerate at the same stage as the normal. Ultrastructural changes in the mitochondrial matrix and cristae plus dilation of the endoplasmic reticulum, which occurred during development in sporogenous tissues of the normal line, were often lacking or mistimed in the mutant. Due to extensive variation, even between adjacent locules, the cytological differences between the normal and CMS anthers cannot be ascribed as the cause of male sterility in the ogu CMS line of B. napus, rather they may be the consequence of it.     

Somatic transfer of cytoplasmic traits in Brassica napus L. by haploid protoplast fusion

Summary Fusions between protoplasts from haploid cytoplasmic atrazine resistant (CATR) and haploid cytoplasmic male sterile (CMS) Brassica napus plants were used to produce a diploid CMS/CATR cybrid. The hybrid nature of the cytoplasm was confirmed by comparing the EcoRI restriction fragment patterns of chloroplast and mitochondrial DNA from the cybrid with the parental patterns. The advantages of using haploid protoplasts for fusion experiments as well as the utilization of the CMS/CATR cybrid for hybrid seed production are discussed.     

Transfer of hygromycin resistance into Brassica napus using total DNA of a transgenic B. nigra line

The successful transfer of a marker gene (hpt gene) from Brassica nigra into B. napus via direct gene transfer was demonstrated. Total DNA was isolated from a hygromycin-resistant callus line, which contained three to five copies of the hpt gene. This line had been produced via direct gene transfer with the hygromycin resistance-conferring plasmid pGL2. The treatment of B. napus protoplasts with genomic DNA of B. nigra (Hyg^R) resulted in relative transformation frequencies of 0.1–0.4%. Similar transformation rates were obtained in direct gene transfer experiments using B. napus protoplasts and plasmid pGL2.     

Synthesis of male sterile,triazine-resistant Brassica napus by somatic hybridization between cytoplasmic male sterile B. oleracea and atrazine-resistant B. campestris

Summary Fusion of leaf protoplasts from an inbred line of Brassica oleracea ssp. botrytis (cauliflower, n=9) carrying the Ogura (R1) male sterile cytoplasm with hypocotyl protoplasts of B. campestris ssp. oleifera (cv Candle, n=10) carrying an atrazine-resistant (ATR) cytoplasm resulted in the production of synthetic B. napus (n=19). Thirty-four somatic hybrids were produced; they were characterized for morphology, phosphoglucose isomerase isoenzymes, ribosomal DNA hybridization patterns, chromosome numbers, and organelle composition. All somatic hybrids carried atrazine-resistant chloroplasts derived from B. campestris. The mitochondrial genomes in 19 hybrids were examined by restriction endonuclease and Southern blot analyses. Twelve of the 19 hybrids contained mitochondria showing novel DNA restriction patterns; of these 12 hybrids, 5 were male sterile and 7 were male fertile. The remaining hybrids contained mitochondrial DNA that was identical to that of the ATR parent and all were male fertile.     

Proteomic approach for investigation of cytoplasmic male sterility (CMS) in Brassica

The alloplasmic ‘Tournefortii’ CMS system fromB. napus was chosen for a comprehensive investigation of the protein content of the mitochondrial compartment in male sterile and fertile near isogenic lines. As proteins involved in the male sterile phenotype may be expressed in a tissue-specific manner, mitochondrial preparations were made from different tissues and checked for the purity of the organelles with a new and highly sensitive method (BN-PAGE). InBrassica, only mitochondrial preparations from 4-5-day-old seedlings and from flower buds were found to be sufficiently pure. High resolution, two-dimensional gel electrophoresis of total protein from anthers and from mitochondrial protein of male sterile and fertile seedlings of near isogenic lines revealed distinct differences in the protein patterns. Several explanations for the occurrence of these differences, which may be the starting point for a detailed molecular analysis and a better understanding of the CMS syndrome, are discussed.     

Opportunities for gene transfer from transgenic oilseed rape (Brassica napus) to related species

Before novel transgenic plant genotypes are grown outside containment facilities and evaluated under field conditions, it is necessary to complete a risk assessment to consider the possible consequences of that release. An important aspect of risk assessment is to consider the likelihood and consequences of the transgene being transferred by cross-pollination to related species, including other crops, weeds and ruderal populations. The purpose of this report is to review the literature to assess the ease with whichBrassica napus can hybridize with related species. The evidence for hybridization is considered at three levels: a) by open pollination, b) by hand pollination and c) by the use ofin vitro ovule and embryo rescue techniques; and also examines the fertility and vigour of the F₁, F₂ and backcross generations. Four species are reported to hybridize withB. napus by open pollination:B. rapa andB. juncea using fully fertile parents; andB. adpressa andR. raphanistrum using a male-sterileB. napus parent. Seventeen species are reported to form hybrids (including the four species above) withB. napus when pollination is carried out manually. At least 12 of these species were unable to form F₂ progeny, and eight were unable to produce progeny when the F₁ was backcrossed to one of the parental species. Many factors will influence the success of hybridization under field conditions, including: distance between the parents, synchrony of flowering, method of pollen spread, specific parental genotypes used, direction of the cross and the environmental conditions. Even where there is a possibility of hybridization betweenB. napus and a related species growing in the vicinity of a release, poor vigour and high sterility in the hybrids will generally mean that hybrids and their progeny will not survive in either an agricultural or natural habitat.     

The transfer of cytoplasmic male sterility to winter-type oilseed rape (Brassica napus L.) by protoplast fusion

‘Polima’ cytoplasmic male sterility (cms) was transferred in one step from spring to winter lines of oilseed rape (Brassica. napus L.) by protoplast fusion. The transfer was confirmed by restriction fragment length polymorphism (RFLP) analysis of cytoplasmic DNA. Amphidiploid individuals with normal female fertility were recovered. The regenerated plants retained the winter habit. All traits studied were stable through subsequent sexual generations. The introduction of cms to winter lines by a conventional backcross normally takes 3 years; a similar alloplasmic substitution was achieved in approximately 9 months by protoplast fusion.     

Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium

Summary The response of oilseed rape cultivars to infection with Agrobacterium tumefaciens and A. rhizogenes and the possibility of regenerating genetically transformed oilseed rape plants were examined. The frequency at which Agrobacterium induced galls or hairy-roots on in vitro cultured plants ranged from 10% to 70%, depending on the cultivar. From galls induced by the tumorigenic strain T37, known to be strongly shoot inducing on tobacco, roots developed frequently. Occasionally, shoots formed and some of these produced tumour cell specific nopaline. Attempts to grow the transformed shoots into plants have so far been unsuccessful. Whole plants transformed with Ri-T-DNA, however, were regenerated. These had crinkled leaves and abundant, frequently branching roots that showed reduced geotropism, similar to previously isolated Ri T-DNA transformed tobacco and potato plants. The transformed oilseed rape plants flowered, but failed to form seeds.     

Interspecific somatic hybridization between Brassica napus and Brassica hirta (Sinapis alba L.)

Summary Somatic hybridization between Brassica napus and B. hirta (or Sinapis alba) is described. No cybrid plant with B. napus nucleus exhibiting cytoplasmic male sterility was recovered. Somatic hybrids were identified morphologically and, for some of them, by cytological observations. They were also characterised by Southern hybridization of nuclear rDNA. Chloroplast and mitochondrial DNA restriction analysis showed that 2 plants out of 14 have B. hirta ctDNA, one the B. napus mtDNA and the other a hybrid. Nine possess B. napus ctDNA with a hybrid mtDNA. For six of them, mtDNA patterns present novel bands, suggesting intergenomic recombination during fusion. These hybrids will be included in the breeding program.