Relationship between allelic state of T-DNA and DNA methylation of chromosomal integration region in transformed <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

T-DNA insertions are currently used as a tool to introduce, or knock out, specific genes. The expression of the inserted gene is frequently haphazard and up to now, it was proposed that transgene expression depends on the site of insertion within the genome, as well as the number of copies of the transgene. In this paper, we show that the allelic state of a T-DNA insertion can be at the origin of epigenetic silencing. A T-DNA insertional mutant was characterized to explore the function of AtBP80a′, a vacuolar sorting receptor previously associated with germination. Seeds homozygous for the T-DNA do not germinate, but this can be overcome by a cold treatment and maintained by the following generations. The non-germinating phenotype is only observed in homozygous seed produced by heterozygous plants indicating that it is correlated with the allelic state of the T-DNA in parental lines. Analysis of the region between the T-DNA insertion and the ATG codon of atbp80a′ showed that cytosine methylation is highly enhanced in chromatin containing the T-DNA. Data presented here show that an unpaired DNA region during meiosis could be at the origin of a de novo cytosine methylation mechanism.     

Vector integration in triple R gene transformants and the clustered inheritance of resistance against potato late blight

Genetic transformation with resistance (R) genes is expected to enhance resistance durability against pathogens, especially for potato, a vegetatively propagated crop with tetrasomic inheritance and a long-term breeding program. In this study, 128 potato transformants were analysed for the presence of vector T-DNA genes, borders and backbone sequences. They were harvested after transformation using a construct containing neomycin phosphotransferase II (nptII) and three R genes against potato late blight (Phytophthora infestans). Our analysis revealed that 45 % of the R gene-containing transformants possessed a low T-DNA copy number, without the integration of vector backbone and borders. The integration of vector backbone sequences was characterized using eight genes, and backbone gene tetA was selected for the early prediction of plants with backbone sequence integration. Three transformants, two plants harbouring one T-DNA copy and one plant harbouring three T-DNA copies, were crossed with susceptible cv. Katahdin. Based on our results, we conclude that all four T-DNA genes were inherited as one cluster and segregated in a Mendelian fashion. The three T-DNA inserts from the transformant harbouring three T-DNA copies were statistically proven to be un-linked and inherited into the offspring plants independently. All of the R genes were functionally expressed in the offspring plants as in their parental transformants. This functional gene stacking has important implications towards achieving more durable resistance against potato late blight.     

Hairy Root-activation Tagging: a High-throughput System for Activation Tagging in Transformed Hairy Roots

Activation tagging is a powerful technique for generating gain-of-function mutants in plants. We developed a new vector system for activation tagging of genes in “transformed hairy roots”. The binary vector pHR-AT (Hairy Root-Activation Tagging) and its derivative pHR-AT-GFP contain a cluster of rol (rooting locus) genes together with the right border facing four tandem repeats of the cauliflower mosaic virus (CaMV) 35S enhancer element on the same T-DNA. Transformation experiments using Arabidopsis, potato, and tobacco as model plants revealed that upon inoculating plants with Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants within a short period of time, and root culture lines were subsequently established. Molecular analyses of the pHR-AT-GFP-transformed Arabidopsis lines showed that expression of the genes adjacent to the T-DNA insertion site was significantly increased. This system may facilitate application of the activation-tagging approach to plant species that are recalcitrant to the regeneration of transgenic plants. High-throughput metabolic profiling of activation-tagged root culture lines will offer opportunities for identifying regulatory or biosynthetic genes for the production of valuable secondary metabolites of interest.     

Alternative selectable markers for potato transformation using minimal T-DNA vectors

Alternative selection systems for plant transformation are especially valuable in clonal crops, such as potato (Solanum tuberosum L.), to pyramid transgenes into the same cultivar by successive transformation events. We have modified the pGPTV series of binary vectors to construct pMOA1 to pMOA5, resulting in a series of essentially identical binary vectors except for the presence of different selectable marker genes. These selectable marker genes are tightly inserted between the left and right T-DNA borders and confer resistance to kanamycin (nptII), hygromycin (hpt), methotrexate (dhfr), phosphinothricin (bar), or phleomycin (ble). The T-DNA of all the vectors is based on the minimal features necessary for plant transformation, with no extraneous DNA segments that may be unacceptable to regulatory authorities for general release of transgenic plants. A series of unique restriction sites exists between the right border and each selectable marker gene for subsequent insertion of useful genes. We have also developed improved culture procedures for potato transformation and used the pMOA1 to pMOA5 binary vectors to define stringent selection conditions for each marker gene. Combining these advances improved the frequency of recovering transformed potato plants while maintaining a low frequency of escapes. The relative efficiency of recovering transgenic potato lines with each selectable marker gene can be summarised as: kanamycin resistance>hygromycin resistance>phosphinothricin resistance>phleomycin resistance>methotrexate resistance.     

Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics

We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice ( Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance ( hpt), green fluorescent protein ( gfp) and beta-glucuronidase ( gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75-95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30-40% of the T(0) plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T(1) progenies showed that 30-50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.     

Agrobacterium-Mediated Transient Gene Expression and Silencing: A Rapid Tool for Functional Gene Assay in Potato

Potato is the third most important food crop worldwide. However, genetic and genomic research of potato has lagged behind other major crops due to the autopolyploidy and highly heterozygous nature associated with the potato genome. Reliable and technically undemanding techniques are not available for functional gene assays in potato. Here we report the development of a transient gene expression and silencing system in potato. Gene expression or RNAi-based gene silencing constructs were delivered into potato leaf cells using Agrobacterium-mediated infiltration. Agroinfiltration of various gene constructs consistently resulted in potato cell transformation and spread of the transgenic cells around infiltration zones. The efficiency of agroinfiltration was affected by potato genotypes, concentration of Agrobacterium, and plant growth conditions. We demonstrated that the agroinfiltration-based transient gene expression can be used to detect potato proteins in sub-cellular compartments in living cells. We established a double agroinfiltration procedure that allows to test whether a specific gene is associated with potato late blight resistance pathway mediated by the resistance gene RB. This procedure provides a powerful approach for high throughput functional assay for a large number of candidate genes in potato late blight resistance.     

Generation of Marker- and Backbone-Free Transgenic Potatoes by Site-Specific Recombination and a Bi-Functional Marker Gene in a Non-Regular One-Border Agrobacterium Transformation Vector

A binary vector, designated PROGMO, was constructed to assess the potential of the Zygosaccharomyces rouxii R/Rs recombination system for generating marker- and backbone-free transgenic potato (Solanum tuberosum) plants with high transgene expression and low copy number insertion. The PROGMO vector utilises a constitutively expressed plant-adapted R recombinase and a codA-nptII bi-functional, positive/negative selectable marker gene. It carries only the right border (RB) of T-DNA and consequently the whole plasmid will be inserted as one long T-DNA into the plant genome. The recognition sites (Rs) are located at such positions that recombinase enzyme activity will recombine and delete both the bi-functional marker genes as well as the backbone of the binary vector, leaving only the gene of interest flanked by a copy of Rs␣and RB. Efficiency of PROGMO transformation was tested by introduction of the GUS reporter gene into potato. It was shown that after 21 days of positive selection and using 300 mgl⁻¹ 5-fluorocytosine for negative selection, 29% of regenerated shoots carried only the GUS gene flanked by a copy of Rs and RB. The PROGMO vector approach is simple and might be widely applicable for the production of marker- and backbone-free transgenic plants of many crop species.     

Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato

Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophila melanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant.     

Comparative transcriptome analysis reveals whole-genome duplications and gene selection patterns in cultivated and wild <Emphasis Type="Italic">Chrysanthemum</Emphasis> species

Key message

This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. T-DNA inserts are stable; no transgene rearrangements were observed. AmCYAN1 and PMI protein accumulation levels were maintained. There was no evidence that production of either protein declined across generations and no transgene silencing was observed in three commercial sugarcane varieties through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes over 4 years of field testing. Long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized.

Abstract

This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. These data are critical supporting information needed for successful commercialization of GM sugarcane. Here seventeen transgenic events, containing the AmCYAN1 gene driven by a CMP promoter and the E. coli PMI gene driven by either a CMP or Ubi promoter, were used to monitor T-DNA insert stability and consistency of transgene encoded protein accumulation through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes. The experiments were conducted in three commercial sugarcane varieties over 4 years of field testing. DNA gel blot analysis showed that the T-DNA inserts are stable; no transgene rearrangements were observed. Quantitative ELISA showed no evidence of decreasing AmCYAN1 and PMI protein levels across generations and no transgene silencing was observed. These results indicate that long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized.

     

The Frequency of Silencing in Arabidopsis Thaliana Varies Highly Between Progeny of Siblings and can be Influenced by Environmental Factors

In a collection of 111 transgenic Arabidopsis thaliana lines, silencing of the nptII gene was observed in 62 (56%) of the lines and three distinct nptII-silencing phenotypes were identified. Two T-DNA constructs were used, which differed in distance and orientation of the marker gene relative to the border sequences. Comparison of the sets of lines generated with each vector, indicate that the T-DNA construct configuration influence the incidence of lines displaying silencing, as well as the distribution of silencing phenotypes. Twenty lines were investigated more thoroughly. The frequency of silencing varied between siblings in 19 lines, including three lines containing a single T-DNA copy. The last line showed 100% silencing. The gus gene present in both constructs could be expressed in the presence of a silenced nptII gene. Investigation of methylation at a single site in the pnos promoter revealed partial methylation in multi-copy lines, but no methylation in single-copy lines. For 16 lines, the overall frequencies of silencing differed significantly between control plants and plants exposed to temperature stress; in 11 of these lines at the 0.1% level. In several cases, the frequency of silencing in progeny of stress-treated plants was higher than for the control group, while other lines showed higher frequencies of kanamycin-resistant progeny for the stress-treated sibling plants.     

而今迈步从头越: 马铃薯育种跨入“有种”时代

现代栽培马铃薯(Solanum tuberosum)是同源四倍体, 基因组高度杂合, 遗传组成复杂, 存在严重的自交衰退, 优异性状难以聚合, 使得育种周期漫长, 造成基于种子传代的马铃薯杂交育种难以突破。与此同时, 块茎无性繁殖导致马铃薯繁殖系数低、储运成本高和易携带病虫害等痼疾, 且种薯脱毒成本高, 限制其产业化发展。近期, 中国农业科学院深圳农业基因组研究所黄三文团队运用基因组设计理论和方法体系培育杂交马铃薯, 用二倍体育种替代四倍体育种, 实现了用杂交种子繁殖替代薯块繁殖, 创制了第1代高纯度的二倍体自交系和杂种优势明显的杂交马铃薯品系。该研究是马铃薯育种领域里程碑式的重大成果, 开启了基于基因组设计和种子迭代的马铃薯生物育种新纪元。     

DNA methylome analysis provides evidence that the expansion of the tea genome is linked to TE bursts

DNA methylation is essential for gene regulation, imprinting and silencing of transposable elements (TEs). Although bursts of transposable elements are common in many plant lineages, how plant DNA methylation is related to transposon bursts remains unclear. Here we explore the landscape of DNA methylation of tea, a species thought to have experienced a recent transposon burst event. This species possesses more transposable elements than any other sequenced asterids (potato, tomato, coffee, pepper and tobacco). The overall average DNA methylation levels were found to differ among the tea, potato and tomato genomes, and methylation at CHG sequence sites was found to be significantly higher in tea than that in potato or tomato. Moreover, the abundant TEs resulting from burst events not only resulted in tea developing a very large genome size, but also affected many genes involved in importantly biological processes, including caffeine, theanine and flavonoid metabolic pathway genes. In addition, recently transposed TEs were more heavily methylated than ancient ones, implying that DNA methylation is proportionate to the degree of TE silencing, especially on recent active ones. Taken together, our results show that DNA methylation regulates transposon silencing and may play a role in genome size expansion.     

Factors affecting the rate of T-DNA transfer from Agrobacterium tumefaciens to Nicotiana glauca plant cells

Different factors involved in the early steps of the T-DNA transfer process were studied by using a -glucuronidase gene (gusA) as a reporter in Nicotiana glauca leaf disc transformation experiments. The levels of transient expression of the gusA gene in leaf discs infected with several strains or vir mutants correlated well with their virulence phenotype, except for virC mutants. The rate of T-DNA transfer was shown to be stimulated in the case of non-oncogenic strains by the co-transfer of small amounts of oncogenic genes. It was found that the location of the T-DNA in the Agrobacterium genome affected the T-DNA transfer rate especially in virC mutants. The virC mutants transferred the gusA-containing T-DNA located on a binary vector more efficiently than the oncogenic T-DNA of the Ti plasmid. Although wild-type strains induced high levels of gusA expression early after infection, the gusA expression appeared to be lost late after infection in the infected leaf discs. In contrast, in leaf discs infected by virC mutants the level of gusA expression increased steadily in time. A model explaining these results is presented.     

Identification of further <Emphasis Type="Italic">Craterostigma plantagineum cdt</Emphasis> mutants affected in abscisic acid mediated desiccation tolerance

The resurrection plant (Craterostigma plantagineum) is desiccation tolerant. However, callus derived from this plant, when propagated in vitro, requires exogenously applied abscisic acid (ABA) in order to survive desiccation. Treatment of callus tissue with ABA induces most of the genes that are induced by dehydration in the whole plant. This property has been exploited for the isolation of mutants that show dominant phenotypes resulting from the ectopic expression of endogenous genes induced by the insertion of a foreign promoter. Here we describe new T-DNA tagged Craterostigma desiccation-tolerant (cdt) mutants with different molecular and physiological characteristics, suggesting that different pathways of desiccation tolerance are affected. One of the mutants, cdt-2, constitutively expresses known osmoprotective Lea genes in callus and leaf tissue. Further analysis of this mutant revealed that the tagged locus is similar to a previously characterised gene, CDT-1, which codes for a signalling molecule that confers desiccation tolerance. The nature of the T-DNA insertion provides insight into the mechanism by which the CDT-1/2 gene family functions in ABA signal transduction.