Intergeneric hybridization between Diplotaxis siifolia,a wild species and crop brassicas

Summary Attempts were made to obtain intergeneric hybrids between Diplotaxis siifolia, a wild species, and cultivars of Brassica (B. campestris, B. juncea, and B. napus). The crosses showed unilateral incompatibility. When the wild species was used as female parent, pollen germination and pollen tube growth were normal, but hybrid seeds aborted due to post-fertilization barriers. Reciprocal crosses (cultivars as female parent) showed strong pre-fertilization barriers; although pollen grains showed germination, pollen tubes failed to enter the stigma. Hybrids were realized in two of the crosses, D. siifolia x B. juncea and D. siifolia x B. napus, through ovary culture. The hybrids were multiplied in vitro by multiplication of axillary shoots, or somatic embryogenesis. Detailed studies were carried out on the hybrid D. siifolia x B. juncea. F₁ hybrids had shrivelled anthers and were pollen sterile. Amphiploids of this hybrid showed 60% pollen fertility and produced seeds upon self-pollination as well as backcross pollination with the pollen of B. juncea.     

Transfer of resistance to the beet cyst nematode (Heterodera Schachtii Schm.) from Sinapis alba L. (white mustard) to the Brassica napus L. gene pool by means of sexual and somatic hybridization

Summary Sexual and somatic hybrid plants have been produced between Sinapis alba L. (white mustard) and Brassica napus L. (oil-seed rape), with the aim to transfer resistance to the beet cyst nematode Heterodera schachtii Schm. (BCN) from white mustard into the oil-seed rape gene pool. Only crosses between diploid accessions of S. alba (2n = 24, S_a1S_a1) as the pistillate parent and several B. napus accessions (2n = 38, AACC) yielded hybrid plants with 31 chromosomes. Crosses between tetraploid accessions of S. alba (2n = 48, S_a1S_a1S_a1S_a1) and B. napus were unsuccessful. Somatic hybrid plants were also obtained between a diploid accession of S. alba and B. napus. These hybrids were mitotically unstable, the number of chromosomes ranging from 56 to more than 90. Analysis of total DNA using a pea rDNA probe confirmed the hybrid nature of the sexual hybrids, whereas for the somatic hybrids a pattern identical to that of B. napus was obtained. Using chloroplast (cp) and mitochondrial (mt) DNA sequences, we found that all of the sexual F₁ hybrids and somatic hybrids contained cpDNA and mtDNA of the S. alba parent. No recombinant mtDNA or cpDNA pattern was observed. Three BC₁ plants were obtained when sexual hybrids were back-crossed with B. napus. Backcrossing of somatic hybrids with B. napus was not successful. Three sexual hybrids and one BC₁ plant, the latter obtained from a cross between a sexual hybrid and B. napus, were found to show a high level of BCN resistance. The level of BCN resistance of the somatic hybrids was in general high, but varied between cuttings from the same plant. Results from cytological studies of chromosome association at meiotic metaphase I in the sexual hybrids suggest partial homology between chromosomes of the AC and S_a1 genomes and thus their potential for gene exchange.     

Hybridisation between Brassica napus L. and Raphanus raphanistrum L. under agronomic field conditions

The frequency of hybridisation between Brassica napus L. and Raphanus raphanistrum L. under agronomic conditions was assessed in field experiments, where R. raphanistrum were randomly planted at two different densities into large plots of B. napus. An acetolacate synthase (ALS)-inhibiting herbicide-resistant trait was used to detect potential hybrid individuals. No hybrids were detected amongst 25,000 seedlings grown from seed collected from R. raphanistrum plants. Two hybrids were obtained from more than 52-million B. napus seedlings. Both hybrids were characterised as amphidiploids (AACCRrRr, 2n = 56) and were fertile. The frequency of hybridisation into B. napus in this experiment using male-fertile B. napus was 4 × 10^–8. Received: 31 August 2000 / Accepted: 23 January 2001     

Gene dispersal from transgenic crops

The risk of release of genetically modified oilseed rape (Brassica napus) was investigated in relation to interspecific gene flow with hoary mustard (Hirschfeldia incana). Microscopic studies showed polymorphism within the population of hoary mustard for pollen germination on oilseed rape flowers. The transgenic herbicide-resistant and a commercial cultivar of oilseed rape were not different for pollen behaviour and ovule fertilization. Pollen tube growth was slow and erratic in interspecific crosses. Fertilization efficiency of oilseed rape and hoary mustard pollen in interspecific crosses was 15% and 1.3%, respectively, of that in intraspecific crosses. This unequal efficiency in reciprocal crosses was confirmed by hybrid seed set in pods. There was no post-zygotic barrier to the development of hybrid embryos in hoary mustard pods. Up to 26 spontaneous hybrids per male sterile oilseed rape plant, and one per hoary mustard plant, were obtained in field experiments. Hybrids were identified by isozyme electrophoresis, morphology and cytology. All hybrids were triploid with 26 chromosomes, and had low fertility. They produced 0.5 seeds per plant after spontaneous backcrossing with hoary mustard. Some of these descendants were produced from unreduced gametes. Our results suggest that gene flow is likely to occur, but its actual frequency under crop growing conditions remains to be estimated.     

Wide hybridization in Brassica

Summary Crosses were made to obtain interspecific hybrids between B. fruticulosa (wild species , 2n = 16) × B. campestris (cultivar , 2n = 20). Although many pollen grains germinated and their tubes entered the style, only about 30% of the ovules received pollen tubes. Fertilized ovules aborted at various stages of development. A few hybrid seeds resulted from hand pollinations in the field, and they showed poor germination and seedling establishment. The in vitro culture of ovaries, ovules, and seeds increased the frequency of obtaining hybrid seeds and plants: the most effective method was ovary culture followed by ovule culture. The hybrid nature of the plants was confirmed through morphological, cytological, and electrophoretic studies. A meiotic analysis of F₁ hybrids (2n = 18) showed that they had 0–5 bivalents and were completely pollen sterile. Electrophoretic analysis of leaf esterases and acid phosphatases of F₁ hybrids revealed bands derived from each parent. Induced amphidiploids of F₁ hybrids contained mostly bivalents, and had about 50% fertile pollen.     

Effects of host plant species on the interaction between the parasitic wasp Diospilus capito and pollen beetles (Meligethes spp.)

1 Field studies were conducted in central Sweden to establish whether two host plants with high and low suitability for pollen beetles (Meligethes aeneus Fabr. [Coleoptera: Nitidulidae] and Meligethes viridescens Fabr.) affected the parasitoid Diospilus capito Nees [Hymenoptera: Braconidae]. 2 Samples of larvae were taken from fields with plots of white mustard, Sinapis alba L. and spring rape, Brassica napus L. in 1997 and 1998. Levels of parasitism and the survival and size of D. capito were measured to determine any influence of the host plant species. Survival and weights of pollen beetles from S. alba and B. napus were also measured. 3 Levels of parasitism between 8% and 29% were recorded. There was a significantly higher likelihood of being parasitized by D. capito for beetle larvae developing on S. alba than on B. napus. We argue that semiochemical or morphological properties of plant species could be responsible for differential parasitism by D. capito. 4 Neither survival of D. capito nor parasitoid size differed from hosts developing on S. alba or B. napus. Pollen beetle emergence was the same for both plant species, but beetles that developed on S. alba weighed less than those from B. napus. Results suggest that the partial resistance of S. alba to the pollen beetle will have no negative effects on the parasitoid D. capito.     

Transfer of auxinic herbicide resistance from Brassica kaber to Brassica juncea and Brassica rapa through embryo rescue

Auxinic herbicides are widely used in agriculture to selectively control broadleaf weeds. Prolonged use of auxinic herbicides has resulted in the evolution of resistance to these herbicides in some biotypes of Brassica kaber (wild mustard), a common weed in agricultural crops. In this study, auxinic herbicide resistance from B. kaber was transferred to Brassica juncea and Brassica rapa, two commercially important Brassica crops, by traditional breeding coupled with in vitro embryo rescue. A high frequency of embryo regeneration and hybrid plant establishment was achieved. Transfer of auxinic herbicide resistance from B. kaber to the hybrids was assessed by whole-plant screening of hybrids with dicamba, a widely used auxinic herbicide. Furthermore, the hybrids were tested for fertility (both pollen and pistil) and their ability to produce backcross progeny. The auxinic herbicide-resistant trait was introgressed into B. juncea by backcross breeding. DNA ploidy of the hybrids as well as of the backcross progeny was estimated by flow cytometry. Creation of auxinic herbicide-resistant Brassica crops by non-transgenic approaches should facilitate effective weed control, encourage less tillage, provide herbicide rotation options, minimize occurrence of herbicide resistance, and increase acceptance of these crops.     

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.     

Production and cytogenetics of intergeneric hybrids between Brassica napus and Orychophragmus violaceus

The intergeneric hybrid between Brassica napus and Orychophragmus violaceus was obtained by means of embryo culture technique with the latter as the pollen parent. The hybrid was morphologically intermediate between its parents, but could produce a lot of seeds when selfed. Somatic separation of the genomes from the two parental species was observed during the mitotic divisions of some of the hybrid cells. Thus, the hybrid became the mixoploid in nature, consisting of haploid and diploid cells of B. napus, and a nuclear — cytoplasmic hybrid, with the cytoplasm of B. napus and the nuclei of O. violaceus, and the hybrid cells. Pollen mother cells with 19, 12 and 6 bivalents, respectively, were produced by the hybrid. From the selfed progeny of the hybrid, mainly two kinds of plants, B. napus and the hybrid, were found. The hybrid plants of the selfed progeny again produced two kinds of plants, B. napus and the hybrid.     

Spontaneous hybridization between a male-sterile oilseed rape and two weeds

Spontaneous interspecific hybrids were produced under natural conditions (pollination by wind and bees) between a male-sterile cybrid Brassica napus (AACC, 2n = 38) and two weeds Brassica adpressa (AdAd, 2n = 14) and Raphanus raphanistrum (RrRr, 2n = 18). After characterization by chromosome counts and isozyme analyses, we observed 512 and 3 734 inter-specific seeds per m² for the B. napus-B. adpressa and B. napus-R. raphanistrum trials respectively. Most of the hybrids studied had the expected triploid structure (ACX). In order to quantify the frequency of allosyndesis between the genomes involved in the hybrids, their meiotic behavior was compared to a haploid of B. napus (AC). For the B. napus-B. adpressa hybrids, we concluded that probably no allosyndesis occurred between the two parental genomes, and that genetic factors regulating homoeologous chromosome pairing were carried by the B. adpressa genome. For the B. napus-R. raphanistrum hybrids, high chromosome pairing and the presence of multivalents (in 9.16% of the pollen mother cells) indicate that recombination is possible between chromosomes of different genomes. Pollen fertility of the hybrids ranged from 0 to 30%. Blackleg inoculation tests were performed on the three parental species and on the interspecific hybrids. BC₁ production with the weeds and with rapeseed was attempted. Results are discussed in regard to the risk assessment of transgenic rapeseed cultivation, F₁ hybrid rapeseed variety production, and rapeseed improvement.     

Production of fertile intergeneric somatic hybrids between Brassica napus and Sinapis arvensis for the enrichment of the rapeseed gene pool

Somatic hybrids between Brassica napus (oilseed rape) and its wild relative Sinapis arvensis (Xinjiang wild mustard) from northwestern China were produced by fusing mesophyll protoplasts. Fifty-four plants were identified as symmetric hybrids and four as asymmetric hybrids by random amplified polymorphic DNA analysis and nuclear DNA content. The morphology of investigated 58 hybrid plants resembled characters from both parental species. Highly fertile hybrids were recovered where the fertility was associated with the choice of B. napus genotype. Enhanced disease resistance to Leptosphaeria maculans was found in S. arvensis and in the hybrid offspring. This plant material has great potentials not only for use as a bridge for the introduction of a number of valuable traits from the wild species to Brassica crops but also for breeding new varieties with improved blackleg resistance.     

Production of wide hybrids and backcross progenies between Diplotaxis erucoides and crop brassicas

Intergeneric hybrids were produced between D. erucoides (), a wild species, and four cultivated species of Brassica, B. campestris, B. juncea, B. napus and B. oleracea, through embryo rescue. The hybrid nature of these plants was confirmed through morphological and cytological studies. Backcross pollinations with the pollen of the respective cultivars yielded BC progenies in the hybrids D. erucoides x B. juncea and D. erucoides x B. napus but not in D. erucoides x B. campestris and D. erucoides x B. oleracea. The hybrid D. erucoides x B. campestris was also used as a bridge species and crossed with B. juncea to raise the hybrid and backcross progenies. F₂ progenies were more amenable than f₁ hybrids for raising backcross progenies. Although D. erucoides is considered to be a close relative of B. campestris and B. oleracea, incompatibility barriers of this species with different cultivars do not reflect this relationship.     

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.     

Brassica naponigra,a somatic hybrid resistant to Phoma lingam

Summary Brassica napus and B. nigra were combined via protoplast fusion into the novel hybrid Brassica naponigra. The heterokaryons were identified by fluorescent markers and selected by flow sorting. Thirty hybrid plants were confirmed by isozyme analysis to contain both B. nigra and B. napus chromosomes; of these, 20 plants had the sum of the parental chromosome numbers. A non-random segregation of the chloroplasts was found in the hybrids. Of 14 hybrid plants investigated, all had the B. napus type of chloroplast. The resistance to Phoma lingam found in the B. nigra cultivar used in the fusion experiments was expressed in 26 of the hybrid plants. The hybrids obtained in this study contain all of the three Brassica genomes (A, B and C) and have thus created unique possibilities for genetic exchanges between the genomes. Since most of the plants were fertile as well as resistant to P. lingam, they have been incorporated into conventional rapeseed breeding programs.     

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.     

Herbicide resistant rice (Oryza sativa L.): Global implications for weedy rice and weed management*

Rice cultivars resistant to broad‐spectrum herbicides have been developed and their commercial release is imminent, especially for imidazolinone and glufosinate resistant varieties in the USA and Latin America. Glyphosate‐resistant rice should follow within a few years. Rice growers throughout the world could benefit from the introduction of herbicide‐resistant rice cultivars that would allow in‐crop, selective control of weedy Oryza species. Other perceived benefits are the possibility to control ‘hard‐to‐kill’ weed species and weed populations that have already evolved resistance to herbicides currently used in rice production, especially those of the Echinochloa species complex. Weed management could also be improved by more efficient post‐emergence control. Introduction of herbicide resistant rice could also bring areas heavily infested with weedy rice that have been abandoned back to rice production, allow longer term crop rotations, reduce consumption of fossil fuels, promote the replacement of traditional chemicals by more environmentally benign products, and provide more rice grain without adding new land to production. There are also concerns, however, about the impact of releasing herbicide‐resistant rice on weed problems. Of most concern is the possibility of rapid transfer of the resistance trait to compatible weedy Oryza species. Development of such herbicide resistant weedy rice populations would substantially limit the chemical weed management options for farmers. Herbicide‐resistant rice volunteers also could become problematic, and added selection pressure to weed populations could aggravate already serious weed resistance problems. Because of the risk of weedy Oryza species becoming resistant to broad‐spectrum herbicides, mitigating measures to prevent gene flow, eventually attainable by both conventional breeding and molecular genetics, have been proposed. With commercialisation of the first herbicide resistant varieties planned for 2001, these mitigating measures will not be available for use with this first generation of herbicide resistant rice products. Release of herbicide resistant rice should depend on a thorough risk assessment especially in areas infested with con‐specific weedy rice or intercrossing weedy Oryza species. Regulators will have to balance risks and benefits based on local needs and conditions before allowing commercialisation of herbicide‐resistant rice varieties. If accepted, these varieties should be considered as components of integrated weed management, and a rational herbicide use and weedy rice control should be promoted to prevent losing this novel tool.     

Direct and indirect responses to selection on pollen size in Brassica rapa L.

Pollen size varies little within angiosperm species, but differs extensively between species, suggesting the action of strong selection. Nevertheless, the potential for genetic responses of pollen size to selection, as determined by additive genetic variance and genetic correlations with other floral traits, has received little attention. To assess this potential, we subjected Brassica rapa to artificial selection for large and small pollen during three generations. This selection caused significant divergence in pollen diameter, with additive genetic effects accounting for over 30% of the observed phenotypic variation in pollen size. Such heritable genetic variation suggests that natural selection could effect evolutionary change in this trait. Selection on pollen size also elicited correlated responses in pollen number (–), flower size (+), style length (+), and ovule number (+), suggesting that pollen size cannot evolve independently. The correlated responses of pollen number, flower size and ovule number probably reflect the genetically determined and physically constrained pattern of resource allocation in B. rapa. In contrast, the positive correlation between pollen size and style length may represent a widespread gametic‐phase disequilibrium in angiosperms that arises from nonrandom fertilization success of large pollen in pistils with long styles.     

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.     

Somatic hybrids between Brassica juncea (L). Czern. and Diplotaxis harra (Forsk.) Boiss and the generation of backcross progenies

An attempt to transfer genes from droughttolerant Diplotaxis harra, a wild relative of Brassica species, to an elite oil-yielding cultivar, B-85, of mustard (Brassica juncea) was made through protoplast fusion, as the two plant systems are sexually incompatible. By following the standard protocol for PEG-mediated protoplast fusion followed by high pH, high Ca⁺⁺, DMSO treatment and appropriate cell-culture technique, 16 presumptive somatic hybrid plants could be regenerated. Chromosomal analysis of four such somatic hybrids revealed that three of them were asymmetric. Analysis of morphological characters, meiotic chromosomes, and esterase isoenzyme pattern revealed that all the somatic hybrids were different from each other. Furthermore four chromosomes of each genome could undergo homoeologous pairing at meiosis indicating the possibilities for genetic recombination and chromosomal rearrangements. Irregular distribution of chromosomes at anaphase-II at meiosis has been a consistent feature of these plants. Eventually, pollen of all the somatic hybrids showed complete infertility preventing the recovery of any selfed seed. Nevertheless, ovule fertility of one somatic hybrid was not totally impaired as it had set some seeds upon backcrossing with the B. juncea parent. The esterase isoenzyme banding pattern of 24 individual progeny plants of this backcross provided evidence for their recombinant nature. It was thus confirmed that a transfer of genetic traits from Diplotaxis harra to B. juncea had indeed taken place. Furthermore, it was conceptualised that a transfer of alien genes through the protoplast-fusion technique is primarily possible in situations where meiotic pairing of the chromosomes of the two participating genomes generates recombinant gametocytes which can pass through subsequent filial generations.     

大白菜与紫甘蓝种间杂种的获得与鉴定

以不同类型的大白菜与紫甘蓝为材料,运用蕾期授粉结合胚挽救技术获得大白菜与紫甘蓝的种间杂种,并对其进行细胞学鉴定.结果表明,大白菜与紫甘蓝杂交,获得了57株F1代幼苗;对根尖染色体数量进行观察和杂种花粉特性调查发现,其中47株具有预期的染色体数目,2n =19,鉴定为真杂种,其花粉败育;另有6株具有38条染色体,鉴定为种间异源双二倍体,应该是发生了染色体自然加倍,其花粉可育.可育的杂种F1代与大白菜回交,获得了大白菜-紫甘蓝BC1代材料.田间观测结果显示,杂种F1代植株综合性状均介于双亲之间,BC1代植株包球明显,综合性状偏向母本大白菜.