Atrazine-resistant cauliflower obtained by somatic hybridization between Brassica oleracea and ATR-B. napus

Summary Somatic hybridization between Brassica oleracea ssp. botrytis (cauliflower, 2n=18), carrying the Ogura (R1) male-sterile cytoplasm and B. napus (2n= 38), carrying a male-fertile, atrazine-resistant (ATR) cytoplasm, yielded three hybrids (2n=56) and six cauliflower cybrids (2n=18), which were selected for resistance to the herbicide in vitro. The hybrids and cybrids were male fertile and self-compatible. They contained both chloroplasts and mitochondria from the ATR cytoplasm. We found no evidence for mtDNA recombination in any of the regenerated plants. Selfed progeny of the B. oleracea atrazine-resistant cybrids were evaluated for tolerance to the herbicide in the field. Resistant plants exposed to 0.56–4.48 kg/ha (0.5–4.0 pounds/acre) atrazine in the soil showed no damage at any herbicide level, whereas plants of a susceptible alloplasmic line were severely damaged at the lowest level of herbicide application and killed at all higher levels. These atrazine-resistant cauliflower may have potential horticultural use, especially in fields where atrazine carry over is a serious problem.     

Production and characterization of somatic hybrids between the Japanese radish and cauliflower

Summary Somatic hybrids between the Japanese radish and cauliflower (Brassica oleracea) were produced by protoplast electrofusion in order to introduce clubroot disease resistance in the Japanese radish (Raphanus sativus) into Brassica crops. After electrofusion of iodoacetamide-treated cauliflower protoplasts with untreated radish ones, culture was performed under conditions, that allowed only cauliflower protoplasts to regenerate. Out of 40 regenerated plants, 37 were morphologically of a hybrid type and 3 of a cauliflower type. On the basis of isozyme and RFLP analysis, all of the hybrid-type plants tested proved to be true hybrids. Of the 10 true hybrids tested, 9 were found to contain chloroplasts similar to those found in the Japanese radish, while only 1 contained those of the cauliflower. Using two mitochondrial genes as probes, we were able to show that 3 hybrids contained mitochondria of the Japanese radish, with some modification, while 7 hybrids had either parental or new patterns. All of the hybrid-type plants showed resistance to clubroot disease as high as that found in the Japanese radish. Some hybrids were self-fertile. All of the self-fertile hybrids were found to contain 36 chromosomes, indicating that they were amphidiploids. In addition, a few seeds were obtained from a backcross of the self-fertile hybrids to both parents.     

Increased totipotency of protoplasts from Brassica oleracea plants previously regenerated in tissue culture

Factors affecting the division of cells derived from leaf and cotyledon protoplasts from Brassica oleracea L. var. italica (Green Comet hybrid broccoli) were examined to optimize conditions for plant regeneration and to determine whether there was a genetic basis for improved regeneration from protoplasts derived from plants previously regenerated from tissue cultures [15]. When leaf protoplasts from different plants grown from hybrid seed were isolated and cultured simultaneously, division efficiencies of 1–95% were obtained. Cells from some plants showed high division efficiencies in consecutive experiments while cells from other plants had consistently low division rates. More plants from hybrid seed gave high division efficiencies when cotyledon protoplasts were used. However, cotyledon or leaf protoplasts from selfed progeny of regenerated plants produced more vigorous calli and more shoots than protoplasts from hybrid seed. These results suggest that there may be a genetic component to the increased totipotency of Brassica oleracea protoplasts.     

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.     

Synthesis of Brassica oleracea/Brassica napus somatic hybrid plants with novel organeile DNA compositions

Summary Broccoli (Brassica oleracea L. italica) hypocotyl protoplasts were fused with mesophyll protoplasts of two B. napus lines, one carrying the Ogura (ogu) cms cytoplasm, and the other carrying a hybrid cytoplasm consisting of ogu mitochondria combined with triazine-tolerance-conferring chloroplasts from ctr cytoplasm. Two male-sterile somatic hybrids were recovered from the fusion of broccoli protoplasts with those of ogu/ctr cybrid B. napus. The ogu mtDNAs and ctr cpDNAs were not altered in these hybrids. Four male-sterile plants were recovered from the somatic hybridization of broccoli with ogu cms B. napus. Three of these possessed mitochondrial genomes that appeared to have resulted from recombination between the ogu and normal B. oleracea (ole) mtDNAs, while the fourth possessed an unrearranged ogu mtDNA. All four of these plants had B. oleracea cpDNA, and none displayed the seedling chlorosis associated with ogu chloroplasts. Most of the plants recovered from these fusions had the chromosome number expected of B. oleracea + B. napus hybrids (2n = 56). The novel cytoplasms may prove to be useful for the molecular analysis of Brassica cms and for the production of hybrid Brassica.     

Synthesis of Ogura male sterile rapeseed (Brassica napus L.) with cold tolerance by protoplast fusion and effects of atrazine resistance on seed yield

Twenty-one cold-tolerant, male sterile Brassica napus somatic hybrids were produced by protoplast fusion. The fusion partners were a coldsensitive, Ogura cytoplasmic male sterile cauliflower inbred (B. oleracea var. botrytis inbred NY7642A) and a cold-tolerant, fertile canola-type B. rapa cv. Candle. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Organellar analyses revealed a very strong bias for Brassica over Raphanus chloroplasts. Cold tolerance was confirmed by cold chamber studies and chloroplast DNA analyses. Good female fertility with 21.4 ± 3.1 seeds/pod was observed in the field using natural pollination vectors. Total seed yield was significantly greater for the atrazine-sensitive somatic hybrids produced in this study than for atrazine-resistant isolines.Abbreviations CMS cytoplasmic male sterility - IA iodoacetate - cpDNA chloroplast DNA     

The morphology, cytology, and C-banded karyotypes of Brassica campestris, B. oleracea, and B. napus plants regenerated from protoplasts

The behaviour of Brassica campestris (2n=20, AA), B. oleracea (2n=18, CC), and B. napus (2n=38, AACC) were studied during a tissue-culturing process. Hypocotyl-protoplasts were cultivated into calli from which new plants were regenerated. The regenerated plants were compared, and mitotic root-tip cells were C-banded and karyotyped. A majority of the plants were tetraploid. The meioses were studied in the PMCs. A number of abberations were observed, mainly due to faulty spindle function. There was a difference between the three species in that B. campestris performed the most poorly with many fewer regenerated plants. These plants were more morphologically disturbed and had more problems during pollen production than B. oleracea and B. napus plants.     

A new source of cytoplasmic male sterility in maize induced by the nuclear gene,iojap

Summary Cytoplasmic male sterility (cms) was found in plants derived from the F₂ progeny of fertile, normal cytoplasm plants of the inbred R181 pollinated with a genetic stock carrying the recessive nuclear gene, iojap. The male sterile plants were maintained by back-crossing with the inbred W182BN which maintains all known sources of cytoplasmic male sterility. The new male sterile progeny were found to exhibit stable male sterility under field conditions in two environments. However, they were partially fertile in the hot, dry summer of 1983 at Aurora, NY. It was found that these lines were restored by lines that characteristically restore cms S group cytoplasms. Pollen phenotype studies indicated that the restoration was gametophytic in nature, also characteristic of the cms S group. Agarose gel electrophoresis of undigested mitochondrial DNA (mtDNA) from these steriles indicated that these lines have the S-1 and S-2 episomes characteristic of the cms S group. Restriction endonuclease digest patterns of mtDNA from these sterile lines digested with BamH I indicated that these steriles fit into the CA subgroup of the cms S group. The new source of cms has been designated cms Ij-1.     

Regeneration of transgenic vegetable brassicas (Brassica oleracea andB. campestris) via Ri-mediated transformation

A procedure for the production of fertile transgenic brassicas via Ri-mediated transformation is reported in this paper. Transgenic hairy root lines were selected for 12 vegetable brassica cultivars and lines representing six varieties: broccoli, Brussels sprouts, cabbage, cauliflower, rapid-cycling (allBrassica oleracea) and Chinese cabbage (B. campestris). Leaf explants or petioles of intact cotyledons were co-cultivated withAgrobacterium strain A4T harbouring various binary vectors. The T-DNA region of all binary vectors contained a neomycin phosphotransferase II gene for kanamycin resistance, in addition to other genes. Hairy root lines grew prolifically on hormone-free medium containing kanamycin. Transgenic shoots were regenerated from all cultivars either spontaneously or after transfer of hairy roots to a hormone-containing medium. Southern analysis confirmed that the plants were transgenic. Plants from all brassica types were successfully transferred to greenhouse conditions. Plants were fertile and segregation analysis confirmed transmission of traits to progeny.Abbreviations BA 6-Benzylaminopurine - GUS -Glucuronidase - LS Linsmaier and Skoog medium - NAA I-Naphthaleneacetic acid - NPTII Neomycin phosphotransferase II - TDZ thidiazuron     

The distribution of BoCAL-a alleles in Brassica oleracea is consistent with a genetic model for curd development and domestication of the cauliflower

The characteristic curd of cauliflower (Brassica oleracea var. botrytis L.) consists of proliferating, arrested inflorescence and floral meristems. However, the origins and events leading to the domestication of this important crop trait remain unclear. A similar phenotype observed in the ap1-1/cal-1 mutant of Arabidopsis thaliana led to speculation that the orthologous genes from B. oleracea may be responsible for this characteristic trait. We have carried out a detailed molecular and genetic study, which allows us to present a genetic model based on segregation of recessive alleles at specific, mapped loci of the candidate genes BoCAL and BoAP1. This accounts for differences in stage of arrest between cauliflower and Calabrese broccoli (B. oleracea var. italica Plenck), and predicts the intermediate stages of arrest similar to those observed in Sicilian Purple types. Association of alleles of BoCAL-a with curding phenotypes of B. oleracea is also demonstrated through a survey of crop accessions. Strong correlations exist between specific alleles of BoCAL-a and discrete inflorescence morphologies. These complementary lines of evidence suggest that the cauliflower curd arose in southern Italy from a heading Calabrese broccoli via an intermediate Sicilian crop type. PCR-based assays for the two key loci contributing to curd development are suitable for adoption in marker-assisted selection.     

Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay

In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers T_L sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T₀ plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T₁ progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri T_L-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of T_L-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli.     

Detection and analysis of QTLs based on RAPD markers for polygenic resistance to Plasmodiophora brassicae Woron in Brassica oleracea L.

Resistance to Plasmodiophora brassicae Woron, the causal fungus of clubroot, was examined in an F₂ population of a cross between a clubroot-resistant kale (Brassica oleracea L. var. acephala) and a susceptible cauliflower (Brassica oleracea L. var. botrytis). QTL detection was performed with RAPD markers. Two resistance notations, carried out at different times after inoculation, were used. Three markers were associated with these two notations and three were specifically linked to only one notation. QTL analysis suggests the existence of at least two genetic mechanisms implicated in the resistance phenomenon.     

Reciprocal transfer of male sterile and normal plasmons in Petunia

Summary The goal in this experiment was to achieve direct plasmon transfer via cell fusion. Two lines were used — a normal fertile line of P. hybrida, and a cytoplasmic male sterile (cms) line with the nuclear background of P. parodii. Two plants phenotypically similar to the original male sterile line were developed from protoplasts, but instead of being cms they were male fertile. On the other hand, two plants typical of the original normal line developed from protoplasts, but they were cms instead of fertile. Chromosome counts were done and in all cases the expected diploid number (=14) was found. Genetic analysis showed that sorting out of cms and fertile segregants was evident in the first and second backcross of the cms cybrids. The fertile type cybrids were stable fertile for several generations of selfing and proper backcrossing. These results are discussed in the light of an earlier fusion experiment in which these two parental lines were involved.Contribution from the Department of Plant Genetics and Breeding, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 991-E, 1984 series     

Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (cms line) and Brassica napus through cytoplast-protoplast fusion

Summary Cytoplasts isolated from hypocotyl protoplasts of Raphanus sativus cv Kosena (cms line) by ultracentrifugation through Percoll/mannitol discontinuous gradient were fused with iodoacetamide(IOA)-treated protoplasts of Brassica napus cv Westar. Seventeen randomly selected regenerated plants were characterized for morphology and chromosome numbers. All of the regenerated plants had morphology identical to B. napus and 10 of them possessed the diploid chromosome number of B. napus. The remaining plants had chimeric or aneuploid chromosome numbers. The mitochondrial genomes in the 10 fusion products possessing the diploid chromosome numbers of B. napus were examined by Southern hybridization analysis. Four of the 10 plants contained mitochondrial DNA showing novel hybridization patterns. Of these 4 plants, 1 was male sterile, and 3 were male fertile. The remaining plants showed mitochondrial DNA patterns identical to B. napus and were male fertile.     

The use of phenotypic markers to identify Brassica oleracea genotypes for routine high-throughput Agrobacterium-mediated transformation

Doubled haploid (DH) genotypes from a genetic mapping population of Brassica oleracea were screened for ease of transformation. Candidate genotypes were selected based on prior knowledge of three phenotypic markers: susceptibility to Agrobacterium tumefaciens, shoot regeneration potential and mode of shoot regeneration. Mode of regeneration was found to be the most significant of the three factors. Transgenic plants were successfully obtained from genotypes that regenerated multiple shoots via a distinct swelling or callus phase. The absence of tissue culture blackening (associated with genotypes that formed callus) was found to be critical for transformation success. Transgenic shoots were obtained from genotypes that regenerated via an indirect callus mode, even when susceptibility to Agrobacterium was low. The most efficient genotype (DH AG1012) produced transgenic shoots at an average rate of 15% (percentage of inoculated explants giving rise to transgenic plants). The speed and efficiency of regeneration enabled the isolation of transgenic shoots 5–6 weeks after inoculation with A. tumefaciens. This line was also self-compatible, enabling the production of seed without the need for hand-pollination. A genetically uniform DH genotype, with an associated genetic map, make DH AG1012 highly desirable as a potential model B. oleracea genotype for studying gene function. The possibility of applying the same phenotypic tissue culture markers to other Brassica species is discussed.     

Selection of ethionine-resistant variants with increased accumulation of methionine from embryogenic protoplasts of the forage legume <Emphasis Type="Italic">Astragalus adsurgens</Emphasis>

In vitro selection was carried out to obtain ethionine-resistant plants with increased contents of free methionine in the vegetative tissues of the forage legume Astragalus adsurgens Pall. Three-week-old cell colonies were derived from protoplasts mutagenized with N-methyl-N-nitrosoguanidine from embryogenic callus and were selected with 0.6mM ethionine. Four colony lines were isolated and their resistance to ethionine was 7–8 times that of the wild-type callus. No plant regeneration occurred on these colony lines in the differentiation medium containing ethionine. Only one colony line (R-1) regenerated plants through somatic embryogenesis in the absence of ethionine. Stem and leaf segments from the regenerated plants showed the same potential to produce callus in the presence of ethionine as in the absence of ethionine. The formed callus kept continuously growing in ethionine-containing medium. Free amino acid analysis revealed that colony line R-1, its regenerated plants and callus from the regenerated plants accumulated methionine at levels at 5–9 times higher than in wild-type. These results suggested that ethionine resistance and methionine over-accumulation were also expressed at plant level. Thus, the obtained resistant colony line that could regenerate plants with over-accumulation of methionine might provide an alternative approach to improve the nutritional quality of this forage.     

An extensive hairy root production precedes shoot regeneration in protoplast-derived calli of cauliflower (Brassica oleracea var. botrytis)

Summary Protoplasts isolated from mesophyll tissues of cauliflower (Brassica oleraceavar. botrytis) were induced to divide in culture, with 2% of them producing calli. Upon transfer to a regeneration medium containing a low auxin/cytokinin balance (0.02mg/l 1-naphthaleneacetic and 2mg/l 6-benzylaminopurine), they displayed an extensive production of hairy roots before the regeneration of shoots. Negative effects of root differentiation on the subsequent caulogenesis by such calli were not observed, since 97% of the calli regenerated hairy roots and 93% gave shoots.Abbreviations NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4 D 2,4-dichlorophenoxyacetic acid - GA3 gibberellic acid - MES 2-N-morpholinoethane sulfonate     

A new recombined double low restorer line for the Ogu-INRA cms in rapeseed (Brassica napus L.)

A major objective of breeders using the Ogu-INRA cytoplasmic male sterility (cms) system in rapeseed (Brassica napus L.) is to obtain double low restorer lines with a shorter introgression and a good agronomic value. The development of low glucosinolate content (low GC) restorer lines often occurs through the deletion of a part of the introgression. One of these lines has lost the radish Pgi-2 allele expression, without recovering that of the rapeseed Pgi-2 allele. This line shows a defect in the meiotic transmission of the restorer gene Rfo and a very poor agronomic value. We initiated a programme to force non-spontaneous recombination between this Rfo-carrying introgression and the rapeseed homologous chromosome from a low GC B. napus line. Gamma ray irradiation was used to induce chromosome breakage just prior meiosis aiming at just such a recombination. Low GC cms plants were crossed with the pollen of irradiated plants that were heterozygous for this introgression. The F₂ families were scored for their vigour, transmission rate of Rfo and female fertility. One family of plants, R2000, showed an improved behaviour for these three traits. This family presented a unique combination of molecular markers when compared to other rapeseed restorers analysed, which suggests that the recombination event allowed the recovery of B. oleracea genetic information that was originally replaced by the radish introgression in the original restorers. This resulted in a duplicated region (originating from radish and B. oleracea) on the chromosome carrying the introgression in the R2000 family.Electronic Supplementary Material Supplementary material is available for this article at     

Tolerance and antibiosis resistance to cabbage root fly in vegetable Brassica species

Four accessions of the wild species Brassica fruticulosa Cyrillo (Brassicaceae) were studied in order to identify its tolerance and antibiosis resistance to the cabbage root fly, Delia radicum L. (Diptera: Anthomyiidae), in comparison to a widely cultivated cauliflower cultivar and a rapid cycling Brassica oleracea L. line. Antibiosis was prominent, as the insects reared on resistant accessions showed reduced individual pupal weight, total pupal weight, adult dry weight, and the longest average fly eclosion time. Host plant resistance, however, did not affect the sex ratio of adult flies. A study of the root architecture of plants with and without root fly inoculation revealed differences in the structure within B. oleracea accessions. A long main root and a high number of lateral roots appeared to be important characteristics for a Brassica type, with a higher tolerance level to cabbage root fly attack.     

Production and analysis of intergeneric somatic hybrids between <Emphasis Type="Italic">Brassica oleracea</Emphasis> and<Emphasis Type="Italic"> Matthiola incana</Emphasis>

The gene pool of Brassica oleracea was enriched via intergeneric somatic hybridization between B. oleracea (2n = 18) and Matthiola incana (2n = 14). One hundred and eighteen plants were obtained from 96 calli. Only four plants (H1, H2, H3 and H4) showed an intermediate phenotype from the parents; among these, H1 and H3 arose from the same callus. Random amplified polymorphic DNA (RAPD), sequence-related amplified polymorphism (SRAP), and cytological analyses confirmed that H1 and H3 were hybrids. The nuclear DNA content of the regenerated plants was determined by flow cytometry. More than half of the plants contained a nuclear DNA content of 1.3 to 3.9 pg/cell, which was higher than the content of B. oleracea but lower than that of M. incana. H1 contained 4.89 ± 0.02 pg of DNA per cell, while H3 nuclear DNA content was estimated at 4.87 ± 0.06 pg/cell. Cytological study of the root-tip cells revealed that the majority of the H1 and H3 hybrid cells contained 28 chromosomes.