First successful reduction of clinical allergenicity of food by genetic modification: Mal d 1-silenced apples cause fewer allergy symptoms than the wild-type cultivar.

BACKGROUND: Genetic modification of allergenic foods such as apple has the potential to reduce their clinical allergenicity, but this has never been studied by oral challenges in allergic individuals. METHODS: We performed oral food challenges in 21 apple-allergic individuals with Elstar apples which had undergone gene silencing of the major allergen of apple, Mal d 1, by RNA interference. Downregulation of Mal d 1 gene expression in the apples was verified by qRT-PCR. Clinical responses to the genetically modified apples were compared to those seen with the wild-type Elstar using a visual analogue scale (VAS). RESULTS: Gene silencing produced two genetically modified apple lines expressing Mal d 1.02 and other Mal d 1 gene mRNA levels which were extensively downregulated, that is only 0.1-16.4% (e-DR1) and 0.2-9.9% (e-DR2) of those of the wild-type Elstar, respectively. Challenges with these downregulated apple lines produced significantly less intense maximal symptoms to the first dose (Vmax1) than with Elstar (Vmax1 Elstar 3.0 mm vs 0.0 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043), as well as significantly less intense mean symptoms per dose (meanV/d) than with Elstar (meanV/d Elstar 2.2 mm vs 0.2 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043). Only one subject (5%) remained symptom-free when challenged with the Elstar apple, whereas 43% did so with e-DR1 and 63% with e-DR2. CONCLUSION: These data show that mRNA silencing of Mal d 1 results in a marked reduction of Mal d 1 gene expression in the fruit and reduction of symptoms when these apples are ingested by allergic subjects. Approximately half of the subjects developed no symptoms whatsoever, and virtually all subjects wished to consume the apple again in the future.

The mode of inheritance in tetraploid cut roses.

Tetraploid hybrid tea roses (Rosa hybrida) represent most of the commercial cultivars of cut roses and form the basis for breeding programmes. Due to intensive interspecific hybridizations, modern cut roses are complex tetraploids for which the mode of inheritance is not exactly known. The segregation patterns of molecular markers in a tetraploid mapping population of 184 genotypes, an F(1) progeny from a cross of two heterozygous parents, were investigated for disomic and tetrasomic inheritance. The possible occurrence of double reduction was studied as well. We can exclude disomic inheritance, but while our observations are more in line with a tetrasomic inheritance, we cannot exclude that there is a mixture of both inheritance modes. Two novel parental tetraploid linkage maps were constructed using markers known from literature, combined with newly generated markers. Comparison with the integrated consensus diploid map (ICM) of Spiller et al. (Theor Appl Genet 122:489-500, 2010) allowed assigning numbers to each of the linkage groups of both maps and including small linkage groups. So far, the possibility of using marker-assisted selection in breeding of tetraploid cut roses and of other species with a tetrasomic or partly tetrasomic inheritance, is still limited due to the difficulties in establishing marker-trait associations. We used these tetraploid linkage maps to determine associations between markers, two morphological traits and powdery mildew resistance. The knowledge on inheritance and marker-trait associations in tetraploid cut roses will be of direct use to cut rose breeding.

The potential of virus-induced gene silencing for speeding up functional characterization of plant genes

Virus-induced gene silencing (VIGS) has been shown to be of great potential in plant reverse genetics. Advantages of VIGS over other approaches, such as T-DNA or transposon tagging, include the circumvention of plant transformation, methodological simplicity and robustness, and speedy results. These features make VIGS an attractive alternative instrument in functional genomics, even in a high throughput fashion. The system is already well established in Nicotiana benthamiana; however, efforts are being made to improve VIGS in other species, including monocots. Current research is focussed on unravelling the mechanisms of post-transcriptional gene silencing and VIGS, as well as on finding novel viral vectors in order to broaden the host species spectrum. We examined how VIGS has been used to assess gene functions in plants, including molecular mechanisms involved in the process, available methodological elements, such as vectors and inoculation procedures, and we looked for examples in which the system has been applied successfully to characterize gene function in plants.

Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders.

Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA 105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66 bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration.

Transgenic caraway, Carum carvi L.: a model species for metabolic engineering.

Regeneration in caraway was obtained via two different routes. Hypocotyls showed delayed shoot formation after a callus phase and at relatively low frequencies. In contrast, high-frequency, direct regeneration occurred when cotyledonary node explants were used. Transient expression of ß-glucuronidase was monitored after inoculation of both explant types with Agrobacterium tumefaciens AGL0(pMOG410). Gene transfer was more efficient when using cotyledonary node explants. This explant type also proved to be the best for stable transformation resulting in transgenic plants. Several parameters determining regeneration and transformation efficiency were tested. The percentage of explants giving one to numerous transgenic plants could be as high as 13%. This system for the rapid production of many transgenic caraway plants opens up possibilities for studying metabolic engineering with this crop.

Stomatal Guard Cells Are Totipotent.

It has been successfully demonstrated, using epidermis explants of sugar beet (Beta vulgaris L.), that stomatal guard cells retain full totipotent capacity. Despite having one of the highest degrees of morphological adaptation and a unique physiological specialization, it is possible to induce a re-expression of full (embryogenic) genetic potential in these cells in situ by reversing their highly differentiated nature to produce regenerated plants via a callus stage. The importance of these findings both to stomatal research and to our understanding of cytodifferentiation in plants is discussed.

Microprotoplast-mediated transfer of single specific chromosomes between sexually incompatible plants.

Microprotoplast-mediated chromosome transfer (MMCT) through fusion of small (subdiploid) microprotoplasts of a transgenic triploid potato (Solanum tuberosum) cell line with leaf protoplasts of tobacco (Nicotiana tabacum) and the wild tomato species Lycopersicon peruvianum is reported. The microprotoplasts contained one or a few chromosomes. Monosomic addition plants were produced from the fusion products. We employed mass-scale induction of micronuclei in donor suspension cells of potato using the microtubule inhibitor Cremart. Protoplasts were isolated from micronucleated cells after incubation in a cell wall digesting enzyme mixture. The microprotoplasts were isolated from the micronucleated protoplasts by high-speed centrifugation. By using sequential filtration, small microprotoplasts containing one or few chromosomes were separated from the bigger subdiploid microprotoplasts. These small microprotoplasts were fused with recipient protoplasts of tobacco or tomato using polyethylene glycol. The selectable marker kanamycin resistance (KanR) and the reporter gene β-glucuronidase (gus), carried by the donor potato chromosome, were used for the selection of fusion products and the isolation of hybrid calli. Several monosomic addition plants were obtained within the short period of 3-4 months after fusion. These contained one potato chromosome carrying a single copy of gus and one or two copies of the neomycin phosphotransferase (nptII) gene conferring KanR, and the complete set of chromosomes of tobacco or tomato, as revealed by genomic in situ hybridization and Southern blot hybridization. The alien genes, gus and nptII, were stably expressed in both the tobacco and tomato backgrounds. They were transmitted to the progeny after backcrossing to tomato. Monosomic and disomic additions, and some introgression plants showing integration of gus and nptII in the tomato genome, were recovered in the first backcross progeny. The potential value of MMCT for the transfer of economically important traits, genome analysis, and gene expression is discussed. Key words : chromosome transfer, microprotoplast fusion, monosomic-disomic additions, sexual transmission, DNA integration, alien gene expression.

A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cells.

An optimized protocol has been developed for the efficient and rapid genetic modification of sugar beet (Beta vulgaris L.). A polyethylene glycol-mediated DNA transformation technique could be applied to protoplast populations enriched specifically for a single totipotent cell type derived from stomatal guard cells, to achieve high transformation frequencies. Bialaphos resistance, conferred by the pat gene, produced a highly efficient selection system. The majority of plants were obtained within 8 to 9 weeks and were appropriate for plant breeding purposes. All were resistant to glufosinate-ammonium-based herbicides. Detailed genomic characterization has verified transgene integration, and progeny analysis showed Mendelian inheritance.

Intergeneric transfer of a partial genome and direct production of monosomic addition plants by microprotoplast fusion.

Results are reported on the transfer of single, specific chromosomes carrying kanamycin resistance (Kan(R)) and ß-glucuronidase (GUS) traits from a transformed donor line of potato (Solanum tuberosum) to a recipient line of the tomato species Lycopersicon peruvianum through microprotoplast fusion. Polyethylene glycol-induced mass fusion between donor potato microprotoplasts containing one or a few chromosomes and normal recipient diploid L. peruvianum protoplasts gave several Kan(R) calli. A high frequency of plants regenerated from Kan(R) calli expressed both Kan(R) and GUS, and contained one or two copies of npt-II and a single copy of gus. Genomic in situ hybridization showed that several microprotoplast hybrid plants had one single potato donor chromosome carrying npt-II and gus genes and the complete chromosome complement of the recipient L. peruvianum (monosomic additions). Several monosomic-addition hybrid plants could be regenerated within the short time of 3 months and they were phenotypically normal, resembling the recipient line. These results suggest that the transfer of single chromosomes is tolerated better than is the transfer of the whole donor genome. The unique advantages of microprotoplast fusion are discussed: these include the direct production of monosomic addition lines for the transfer and introgression of economically important traits in sexually-incongruent species, the construction of chromosome-specific DNA libaries, high-resolution physical mapping and the identification of alien chromosome domains related to gene expression.

Computer-Assisted Identification of Protoplasts Responsible for Rare Division Events Reveals Guard-Cell Totipotency.

With the use of a computer-controlled microscope system to assist in the positioning and rapid relocation of large numbers of cultured cells, we were able to identify those protoplasts with the capacity to divide within a highly recalcitrant culture in which only a tiny fraction of the total population proceeds to produce viable microcalli. In the cultures used, comprising Beta vulgaris L. (sugar beet) leaf protoplasts, it was confirmed that these cells can be recognized solely on the basis of morphological characters. Therefore, a direct link exists between competence for cell division in vitro and cell type. Divergent callus morphologies and totipotent potential could also be ascribed to distinct protoplast types and hence to cells with a specific origin. The progenitors of the totipotent protoplasts in these cultures have been confirmed as being stomatal guard cells. Consequently, in plants even the most highly adapted living cells clearly retain and can reactivate all of the functional genetic information necessary to recreate the whole organism; an extreme degree of cytodifferentiation is, therefore, no hindrance to expressing totipotent potential. In addition to the considerable practical value of these findings, their implications concerning our understanding of both the control of gene expression and plant cell differentiation and its reversibility are of fundamental significance.

Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.).

Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4 x 10(6) cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.

Different CMS sources found in Beta vulgaris ssp maritima: mitochondrial variability in wild populations revealed by a rapid screening procedure.

Mitochondrial DNA (mtDNA) variation in natural Beta maritima populations has been characterized by way of Southern blot hybridizations of total DNA using non-radioactive probes and chemiluminescent detection. It was found that the previously described N ("normal") mitochondrial type could be subdivided into three subtypes. A new mitochondrial genotype (type R) was distinguished in addition to the previously described type S. Both are male-sterile cytoplasms and can produce a. segregation of sexual phenotypes in their progenies depending on the nuclear background. The populations contained at least two to four different mitochondrial genotypes.

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.

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, Sa1Sa1) 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, Sa1Sa1Sa1Sa1) 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 Sa1 genomes and thus their potential for gene exchange.

Improvement of protoplast culture protocols for Beta vulgaris L. (sugar beet).

The effects of NaCl, feeder cells and the embedding of protoplasts in calcium alginate have been investigated in an attempt to improve culture conditions of recalcitrant sugar beet (Beta vulgaris L.) mesophyll protoplasts. While the use of NaCl in all instances proved detrimental to protoplast development, the other two treatments had clear beneficial effects. Minimum plating densities, necessary to sustain cell division, could be reduced to <5% (<4000 protoplasts / ml) of the control levels and plating efficiencies could be significantly enhanced by approx. 10 fold. Plants could still be regenerated from soft calli derived from mesophyll protoplasts cultured under the modified conditions at a frequency of 20-30 %. In particular, the use of alginate is considered of potentially great importance for the further application of beet protoplasts for other aims e.g. asymmetric hybridization.

Isolation of DNA markers linked to a beet cyst nematode resistance locus in Beta patellaris and Beta procumbens.

In cultivated beet no useful level of resistance of the beet cyst nematode (BCN) Heterodera schachtii Schm. has been found, unlike the situation in wild species of the section Procumbentes. Stable introgression of resistance genes from the wild species into Beta vulgaris has not been achieved, but resistant monosomic additions (2n = 18 + 1), diploids of B. vulgaris with an extra alien chromosome carrying the resistance locus, have been obtained. Here we describe a new series of resistant monosomic fragment addition material of B. patellaris chromosome 1 (pat-1). We further describe the cloning of a single-copy DNA marker that specifically hybridizes with a monosomic addition fragment of approximately 8 Mb (AN5-90) carrying the BCN resistance locus. This marker and another fragment-specific, single-copy DNA marker probably flank the BCN locus on the addition fragment present in the AN5-203 material, which is approximately 19 Mb in size. Furthermore, several specific repetitive DNA markers have been isolated, one of which hybridizes to AN5-90 and also to DNA from a smaller DNA segment of Beta procumbens, present in line B883, carrying a BCN resistance locus introgressed into the B. vulgaris genome. This suggests that the specific repetitive marker is closely linked to the BCN locus.

Asymmetric somatic cell hybridization in plants. II. Electrophoretic analysis of radiation-induced DNA damage and repair following the exposure of sugarbeet (Beta vulgaris L.) protoplasts to UV and gamma rays.

As part of an investigation into whether it would be possible to use UV radiation as a suitable pretreatment of the donor cells in asymmetric hybridization experiments, the effects of this treatment on sugarbeet (Beta vulgaris L.) protoplast DNA have been determined and compared with those of gamma radiation. Both nuclear and mitochondrial DNAs have been examined. The dose ranges chosen had previously been determined to be potentially applicable for fusion experiments. Pulsed field gel electrophoresis and standard agarose gel electrophoresis have been used in combination with laser scanning densitometry to gain an insight into the precise nature and degree of DNA damage resulting from irradiation. It was observed that UV radiation introduced substantial modifications to sugarbeet DNA. Double-strand breaks were detected, the number of which was found to be directly proportional to the dose applied. Such breaks indicate that UV radiation results in substantial chromosome/chromatid fragmentation in these cells. Chemical modifications to the DNA structure could be revealed by a significant reduction in DNA hybridization to specific mitochondrial and nuclear DNA probes. Following gamma irradiation at equivalent biological doses (i.e. those just sufficient to prevent colony formation) much less damage was detected. Fewer DNA fragments were produced indicating the presence of fewer double-strand breaks in the DNA structure. In comparison to UV treatments, DNA hybridization to specific probes following gamma radiation was inhibited less. For both treatments, mitochondrial DNA appeared more sensitive to damage than nuclear DNA. The possibility that DNA repair processes might account for these differences has also been investigated. Results indicate either that repair processes are not involved in the effects observed or that DNA repair occurs so fast that it was not possible to demonstrate such involvement with the experimental system used. The general relevance of such processes to asymmetric cell hybridization is discussed.

Asymmetric somatic cell hybridization in plants. I. The early effects of (sub)lethal doses of UV and gamma radiation on the cell physiology and DNA integrity of cultured sugarbeet (Beta vulgaris L.) protoplasts.

An investigation into the possible application of UV radiation as a pretreatment for the donor cells in asymmetric plant cell hybridization protocols has been carried out. A comparison was made between the effects of UV doses in the range 700-4200 J/m2 and those of 60Co gamma radiation over the range 0.15-1 kGy on Beta vulgaris suspension cell protoplasts. The investigation had two aspects. Firstly, alterations to cell physiology (cell wall resynthesis, viability, division and colony formation) in irradiated protoplasts were examined during a 4-week culture period. Results have indicated that a dose of 700 J/m2 UV is necessary to prevent further cell division and colony formation in these cells. A dose of 0.15 kGy gamma radiation generally prevented colony formation, although some early cell division did occur (as was also observed even after 0.45 kGy had been applied). Membrane integrity, as measured after 6 days, using fluorescein diacetate staining, was not affected by either treatment within the dose ranges applied. Secondly, denaturing (alkaline) gel electrophoresis, in association with a pulsed field gel DNA preparation technique, was used to determine the degree of in vivo DNA damage following the radiation treatments. After UV radiation, considerable fragmentation of the DNA was observed, the extent of which was dose-dependent. Gamma radiation, however, appeared to result in fewer DNA lesions, with only the 1 kGy treatment revealing a pattern significantly altered from that of the control. These results augur well for the potential use of UV radiation in asymmetric fusion experiments.

Molecular aspects of cytoplasmic male sterility in perennial ryegrass (Lolium perenne L.): mtDNA and RNA differences between plants with male-sterile and fertile cytoplasm and restriction mapping of their atp6 and coxI homologous regions.

Lolium perenne L. male-sterile and fertile cytoplasms contain different mitochondrial genomes, as revealed by Southern hybridization with a number of heterologous mitochondrial probes. In addition, transcriptional patterns of atp6 and coxI genes distinguish both cytoplasmic types. The majority of the L. perenne sequences from male-sterile and fertile cytoplasm showing homology with these two genes has been cloned and mapped by restriction digestion. A complex genomic organization, especially concerning coxI homologous sequences, was found in the male-sterile cytoplasm. Furthermore, during the course of these studies tissue-culture-induced mtDNA mutations in a number of coxI-containing sequences were detected in regenerated plants.

Transfer of resistance to the beet cyst nematode (Heterodera schachtii Schm.) into the Brassica napus L. gene pool through intergeneric somatic hybridization with Raphanus sativus L.

An intergeneric somatic hybrid was obtained through PEG-induced protoplast fusion between Brassica napus L. (oil-seed rape, AACC, 2n=38) and a beet cyst nematode resistant genotype of Raphanus sativus L. (fodder radish, RR, 2n=18). The hybrid nature of the regenerated plant was confirmed by flow cytometric analysis, RFLP-analysis, and chromosome counts. Southern blot analysis of total DNA using pPhcPS1 (rbc-L) as probe indicated that the somatic hybrid contains chloroplasts of B. napus. The mitochondrial genome of the somatic hybrid was studied more extensively using several probes and restriction enzymes. The results indicate inter- or intraspecific mitochondrial DNA recombination. Resistance to the beet cyst nematode (Heterodera schachtii Schm., BCN) was expressed in the hybrid at a high level.