利用Inverse PCR快速筛选单个T—DNA拷贝转基因水稻植株的方法

In order to obtain single T-DNA copy transgenic rice, we have established a quick method to estimate the T-DNA copy number in transgenic rice using inverse PCR (IPCR). IPCR was used to amplify junction fragments, i.e. plant genomic DNA sequences flanking the known T-DNA sequences, which will help to estimate the T-DNA copy number in transgenic rice. We have analyzed 20 transgenic plants of 15 transgenic lines. Most plants (12) contain one integrated T-DNA copy per genome, 3 plants contain two and 1 plant contains 3 copies. In 4 transgenic plants no T-DNA copies could be detected using this method. The IPCR results were further tested by Southern analysis and sequence analysis.     

利用Inverse PCR快速筛选单个T-DNA拷贝转基因水稻植株的方法

为了获得单个T-DNA插入拷贝的植株, 我们建立了一套利用Inverse PCR(IPCR)快速检测转基因水稻中T-DNA拷贝数的方法。用IPCR的方法可以扩增出与已知T-DNA序列相邻的水稻基因组DNA未知序列,由此推测转基因水稻植株中T-DNA的拷贝数。我们共对15个转化株系20棵不同植株的DNA进行了IPCR检测。其中12株表现为T-DNA单拷贝插入,3株为双拷贝插入,1株为三拷贝插入。另外4株未检测到T-DNA插入拷贝。IPCR分析结果经过Southern杂交和测序的验证。     

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T₁ populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.     

Single-copy T-DNA insertions in Arabidopsis are the predominant form of integration in root-derived transgenics,whereas multiple insertions are found in leaf discs

Different patterns of T-DNA integration in Arabidopsis were obtained that depended on whether a root or a leaf-disc transformation method was used. An examination of 82 individual transgenic Arabidopsis plants, derived from 15 independent Agrobacterium-mediated transformations in which different cointegrate and binary constructs were used, indicated that the transformation method had a significant influence on the type and copy number of T-DNA integration events. Southern hybridizations showed that most of the transgenic plants produced by a leaf-disc method contained multiple T-DNA insertions (89%), the majority of which were organized as right-border inverted repeat structures (58%). In contrast, a root transformation method mostly resulted in single T-DNA insertions (64%), with fewer right-border inverted repeats (38%). The transformation vectors, including cointegrate and binary types, and the plant selectable markers, hygromycin phosphotransferase and dihydrofolate reductase, did not appear to influence the T-DNA integration patterns.     

Number and Accuracy of T-DNA Insertions in Transgenic Banana (Musa spp.) Plants Characterized by an Improved Anchored PCR Technique

Nineteen transgenic banana plants, produced via Agrobacterium-mediated transformation, were analyzed for the integration of T-DNA border regions using an improved anchored PCR technique. The method described is a relatively fast, three-step procedure (restriction digestion of genomic DNA, ligation of ‘vectorette’-type adaptors, and a single round of suppression PCR) for the amplification of specific T-DNA border-containing genomic fragments. Most transgenic plants carried a low number of inserts and the method was suitable for a detailed characterization of the integration events, including T-DNA border integrity as well as the insertion of non-T-DNA vector sequences, which occurred in 26% of the plants. Furthermore, the particular band pattern generated by four enzyme/primer combinations for each individual plant served as a fingerprint, allowing the identification of plants representing identical transformation events. Genomic Southern hybridization and nucleotide sequence analysis of amplification products confirmed the data obtained by anchored PCR. Sequencing of seven right or left border junction regions revealed different T-DNA processing events for each plant, indicating a relatively low frequency of precisely nicked T-DNA integration among the plants studied.     

Study of the factors influencing Agrobacterium-mediated transformation of pea (Pisum sativum L.)

Factors influencing the efficiency of Agrobacterium-mediated transformation of pea were tested using highly efficient, direct regeneration system. The virulence of three Agrobacterium strains (octopine LBA 4404, nopaline C58C1 and succinamopine, hypervirulent EHA 105) clearly varied giving 1 transgenic plant per 100 explants for LBA 4404, 2.2 for C58C1 and 8.2 for EHA 105. To test the efficacy of selection agents we used the hypervirulent EHA 105 strain carrying pGPTV binary vector with one of four different selection genes: nptII, hpt, dhfr or bar. The mean number of transgenic, kanamycin-resistant plants for two cultivars tested was 4.2 per 100 explants and was slightly higher than the number of phosphinothricin-resistant plants (3.6 plants per 100 explants). The proportion of transgenics among kanamycin-selected plants was also higher than among phosphinothricin-resistant plants (35% and 28% respectively). There was no regeneration on hygromycin or methotrexate media (transformation with hpt and dhfr genes). Acetosyringone had no apparent influence on efficiency of transformation with hypervirulent EHA 105 strain, however it did affect the rate of transformation when moderately virulent C58C1 was used. Recovery of transgenic plants was enhanced after application of 5-azacytidine. The presence of integrated T-DNA was checked by PCR and confirmed by Southern hybridization. T-DNA was stably transmitted to the next generation.     

Bacterial transposons are co-transferred with T-DNA to rice chromosomes during Agrobacterium-mediated transformation

Agrobacterium tumefaciens is widely utilized for delivering a foreign gene into a plant's genome. We found the bacterial transposon Tn5393 in transgenic rice plants. Analysis of the flanking sequences of the transferred-DNA (T-DNA) identified that a portion of the Tn5393 sequence was present immediately next to the end of the T-DNA. Because this transposon was present in A. tumefaciens strain LBA4404, but not in EHA105 and GV3101, our findings indicated that Tn5393 was transferred from LBA4404 into the rice genome during the transformation process. We also noted that another bacterial transposon, Tn5563, is present in transgenic plants. Analyses of 331 transgenic lines revealed that 26.0% carried Tn5393 and 2.1% contained Tn5563. In most of the lines, an intact transposon was integrated into the T-DNA and transferred to the rice chromosome. More than one copy of T-DNA was introduced into the plants, often at a single locus. This resulted in T-DNA repeats of normal and transposon-carrying TDNA that generated deletions of a portion of the T-DNA, joining the T-DNA end to the bacterial transposon. Based on these data, we suggest that one should carefully select the appropriate Agrobacterium strain to avoid undesirable transformation of such sequences.     

T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation

Transgenic Arabidopsis and tobacco plants (125) derived from seven Agrobacterium-mediated transformation experiments were screened by polymerase chain reaction and DNA gel blot analysis for the presence of vector `backbone' sequences. The percentage of plants with vector DNA not belonging to the T-DNA varied between 20% and 50%. Neither the plant species, the explant type used for transformation, the replicon type nor the selection seem to have a major influence on the frequency of vector transfer. Only the border repeat sequence context could have an effect because T-DNA vector junctions were found in more than 50% of the plants of three different transformation series in which T-DNAs with octopine borders without inner border regions were used. Strikingly, many transgenic plants contain vector backbone sequences linked to the left T-DNA border as well as vector junctions with the right T-DNA border. DNA gel blots indicate that in most of these plants the complete vector sequence is integrated. We assume that integration into the plant genome of complete vector backbone sequences could be the result of a conjugative transfer initiated at the right border and subsequent continued copying at the left and right borders, called read-through. This model would imply that the left border is not frequently recognized as an initiation site for DNA transfer and that the right border is not efficiently recognized as a termination site for DNA transfer.     

Agrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots

This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the uidA gene coding for -glucuronidase was used as an indicator in the optimization of the protocol. Subspecies (onion and shallot) and cultivar were important factors for a successful transformation: shallot was better than onion and for shallot cv. Kuning the best results were obtained. Also, it was found that constantly reducing the size of the calli during subculturing and selection by chopping, thus enhancing exposure to the selective agent hygromycin, improved the selection efficiency significantly. Furthermore, callus induction medium and co-cultivation period showed a significant effect on successful stable transformation. The usage of different Agrobacterium strains, callus ages, callus sources and osmotic treatments during co-cultivation did not influence transformation efficiency. The highest transformation frequency (1.95%), was obtained with shallot cv. Kuning. A total of 11 independent transformed callus lines derived from zygotic embryos were produced: seven lines from shallot and four lines from onion. Large differences in plantlet production were observed among these lines. The best line produced over 90 plantlets. Via PCR the presence of the uidA and hpt (hygromycin phosphotransferase) genes could be demonstrated in these putative transformed plants. Southern hybridization showed that most lines originated from one transformation event. However, in one line plants were obtained indicating the occurrence and rescue of at least three independent transformation events. This suggested that T-DNA integration occurred in different cells within the callus. Most transgenic plants only had one copy of T-DNA integrated into their genomes. FISH performed on 12 plants from two different lines representing two integration events showed that original T-DNA integration had taken place on the distal end of chromosomes 1 or 5. A total of 83 transgenic plants were transferred to the greenhouse and these plants appeared to be diploid and normal in morphology.     

Agrobacterium-mediated transformation of élite indica and japonica rice cultivars

A rapid, efficient, routine system has been established forAgrobacterium tumefaciens-mediated production of hundreds of fertile transgenic plants from commercially important rice cultivars, including an indica cultivar, Pusa Basmati 1. Calli induced from embryos of mature rice seeds were cocultivated withA. tumefaciens strain LBA4404 carrying the plasmid pTOK233, then exposed to hygromycin selection followed by an efficient regeneration system. Based on the total number of calli co-cultivated, the transformation frequencies of independent transgenic rice plants including cultivars Pusa Basmati 1, E-yi 105, E-wan 5 and Zhong-shu-wan-geng, were 13.5, 13.0, 9.1, and 9.3%, respectively. T1 seeds were harvested within 7–8 mo of initiation of mature embryo cultures. Data from Southern hybridization analysis proved that foreign genes on T-DNA were stably integrated into the rice genome at low copy/site numbers. Mendelian inheritance of the transgenes was confirmed in T1 progeny.     

Removal of the Selectable Marker Gene from Transgenic Tobacco Plants by Expression of Cre Recombinase from a Tobacco Mosaic Virus Vector through Agroinfection

Selection markers are often indispensable during the process of plant transformation, but dispensable once transgenic plants have been established. The Cre/lox site-specific recombination system has been employed to eliminate selectable marker genes from transgenic plants. Here we describe the use of a movement function-improved Tobacco Mosaic Virus (TMV) vector, m30B, to express Cre recombinase for elimination of the selectable marker gene nptII from transgenic tobacco plants. The transgenic tobacco plants were produced by Agrobacterium-mediated transformation with a specially designed binary vector pGNG which contained in its T-DNA region a sequence complex of 35S promoter-lox-the gfp coding sequence-rbcS terminator-Nos promoter-nptII-Nos terminator-lox-the gus coding region-Nos terminator. The expression of the recombinant viral vector m30B:Cre in plant cells was achieved by placing the viral vector under the control of the 35S promoter and through agroinoculation. After co-cultivating the pGNG-leaf discs with agro35S-m30B:Cre followed by shoot regeneration without any selection, plants devoid of the lox-flanked sequences including nptII were obtained with an efficiency of about 34% as revealed by histochemical GUS assay of the regenerants. Three of 11 GUS expressing regenerants, derived from two independent transgenic lines containing single copy of the pGNG T-DNA, proved to be free of the lox-flanked sequences by Southern blot analysis. Excision of the lox-flanked sequences in the three plants could be attributed to transient expression of Cre from the viral vector at the early stage of co-cultivation, since the cre sequence could not be detected in the viral RNA molecules accumulated in the plants, nor in their genomic DNA. The parental marker-free genotype was inherited in their selfed progeny, and all of the progeny were virus-free, apparently because TMV is not seed-transmissible. Therefore, expression of Cre from a TMV-based vector could be used to eliminate selectable marker genes from transgenic tobacco plants without sexual crossing and segregation, and this strategy could be extended to other TMV-infected plant species and applicable to other compatible virus–host plant systems.     

T-DNA structure and gene expression in petunia plants transformed by Agrobacterium tumefaciens C58 derivatives

Summary We have previously described substantial variation in the level of expression of two linked genes which were introduced into transgenic petunia plants using Agrobacterium tumefaciens. These genes were (i) nopaline synthase (nos) and (ii) a chimeric chlorophyll a/b binding protein/octopine synthase (cab/ocs) gene. In this report we analyze the relationship between the level of expression of the introduced genes and T-DNA structure and copy number in 40 transgenic petunia plants derived from 26 transformed calli. Multiple shoots were regenerated from 8 of these calli and in only 6 cases were multiple regenerated shoots from each callus genotypically identical to each other. Many genotypes showed no nos gene expression (22/28). Most of the plants (16/22) which lacked nos gene expression did contain nos-encoding DNA with the expected restriction enzyme map. Similarly, amongst the genotypes showing no cab/ocs gene expression, the majority (11/28) did not show any alterations in restriction fragments corresponding to the expected cab/ocs coding sequences (10/11). Approximately half of the plants carried multiple copies of T-DNA in inverted repeats about the left or right T-DNA boundaries. No positive correlation was observed between the copy number of the introduced DNA and the level of expression of the introduced genes. However, plants with high copy number complex insertions composed of multiple inverted repeats in linear arrays usually showed low levels of expression of the introduced genes.     

Characterization of rice transformed via an Agrobacterium-mediated inflorescence approach

Rice inflorescences were inoculated with Agrobacterium tumefaciens strain LBA4404 carrying plasmid pJD4 with application of vacuum infiltration. After co-cultivation, callus was initiated and subjected to hygromycin selection, and plants were regenerated from resistant callus lines. Based on the total number of co-cultivated inflorescences bearing flowers 1 to 3 mm in length, the average frequency for recovering independent transgenic rice plants was at least 30%. Seeds from selfed R0 plants were harvested within 6 months after initiation of the experiments. Genomic DNA blot analysis showed that genes in the T-DNA of the binary plasmid were stably integrated into the rice genome, typically at low copy number. In most, but not all, cases the transgene was transmitted to R1 progeny at a frequency characteristic for Mendelian inheritance of a single dominant trait. For selfed progeny of one two-locus insertion line, reactivation of GUS expression was observed for a single copy locus that segregated from a silenced multicopy locus. For this line and some additional plants, fluorescence in situ hybridization was used to visualize the chromosomal location of the transgene insert.     

Insights into recognition of the T-DNA border repeats as termination sites for T-strand synthesis by Agrobacterium tumefaciens

The recognition of the T-DNA left border (LB) repeat is affected by its surrounding sequences. Here, the LB regions were further characterized by molecular analysis of transgenic plants, obtained after Agrobacterium tumefaciens-mediated transformation with T-DNA vectors that had been modified in this LB region. At least the 24-bp LB repeat by itself was insufficient to terminate the T-strand synthesis. Addition of the natural inner and/or outer border regions to at least the LB repeat, even when present at a distance, enhanced the correct recognition of the LB repeat, reducing the number of plants containing vector backbone sequences. In tandem occurrence of both the octopine and nopaline LB regions with their repeats terminated the T-strand synthesis most efficiently at the LB, yielding a reproducibly high number of plants containing only the T-DNA. Furthermore, T-strand synthesis did not terminate efficiently at the right border (RB) repeat, which might indicate that signals in the outer RB region inhibit the termination of T-strand synthesis at the RB repeat.     

Extracellular enzymes ofErwinia carotovora eliminate the need for azacytidine treatment for high frequency transformation ofArabidopsis thaliana

Summary This paper reports a part of our studies on large-scale T-DNA-mediated gene tagging inArabidopsis thaliana. To enhance the chance of tagging specific stress-responsive genes of this species by monitoring the preferential insertion of the T-DNA into the actively transcribed loci, we exposed the root explants to low temperature (LT), abscisic acid (ABA), and extracellular enzymes (EXE) of the plant pathogenErwinia carotovora prior to transformation byAgrobacterium tumefaciens. Both LT and ABA reduced the frequency of transformation; with these treatments, the average transformation frequencies were 8.1% and 2.6%, respectively. However, in explants pretreated with EXE the transformation frequency was 89.0%, similar to that obtained in control materials (92.6%). Transgenic calli developed from these explants did not require any treatment with azacytidine (azaC) for efficient shoot regeneration. Furthermore, this treatment enhanced multiple insertion of the T-DNA into the plant genome; within a population of EXE-treated transgenic plants, the number of lines harboring at least three copies of the integrated T-DNA was much higher (61%) than that observed in an untreated population (34%).     

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.     

Chromosomal mapping of T-DNA inserts in transgenicPetunia byin situ hybridization

The chromosomal location of T-DNa inserts in ten independently derived and confirmed transgenic plants ofP. hybrida was detected byin situ hybridization. Nine transgenic plants had the T-DNA inerts at single sites distributed among each of the seven chromosomes; in one plant the T-DNA inserts were detected on two different chromosomes. Although the T-DNA inserts were integrated randomly among the chromosomes, seven of the 11 total inserts were located at or near the telomere. Thus, T-DNA inserts appear to have potential for tagging chromosomes and chromosome fragments.     

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 EHA105 (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 66bp 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.     

Chromosomal localization of foreign genes in Petunia hybrida

Summary A F1 hybrid of Petunia hybrida, heterozygous for at least one marker on each of the seven chromosomes, was transformed with a modified strain of Agrobacterium tumefaciens in which the phytohormone biosynthetic genes in the transferred DNA (T-DNA) were replaced with a NOS/NPTII/NOS chimeric gene and a wildtype nopaline synthase (NOS) gene. The chimeric gene, which confers kanamycin resistance, was used as selectable marker during the transformation process and the NOS gene was used as a scorable marker in the genetic studies. After plants had been regenerated from the transformed tissues, the transgenic plants that expressed both of these markers were backcrossed to the parental lines. The offspring were examined for the segregation of the NOS gene and the Petunia markers. Genetic mapping was thus accomplished in a single generation.By Southern hybridization analysis we confirmed the presence of the expected T-DNA fragments in the transformed plants. Four out of the six plants presented here, had just one monomeric T-DNA insertion. The sizes of the plant/T-DNA junction fragments suggest that the integration occurred in different sites of the Petunia genome. One transformant gave a more complicated hybridization pattern and possibly has two T-DNA inserts. Another transgenic plant was earlier reported (Fraley et al. 1985) to have two, possibly tandemly repeated T-DNAs.Data is presented on the genetic localization of the T-DNA inserts in six independently obtained transgenic plants. The T-DNA inserts in three plants were mapped to chromosome I. However, the distances between the NOS gene and the marker gene on this chromosome were significantly different. In another transgenic plant the NOS gene was coinherited with the marker on chromosome IV. Two other transgenic plants have the T-DNA insert on chromosome III. A three point cross enabled us to determine that both plants have the NOS gene distally located from the peroxidaseA (prxA) marker and both plants showed about 18% recombination. However, Southern hybridization analysis shows that the sizes of the plant/T-DNA junction fragments in these transgenic plants are different, thus suggesting that the integrations occurred in different sites.     

Desiccation of plant tissues post-<Emphasis Type="Italic">Agrobacterium</Emphasis> infection enhances T-DNA delivery and increases stable transformation efficiency in wheat

Summary Factors influencing the Agrobacterium-mediated transformation of both monocotyledonous and dicotyledonous plant species have been widely investigated. These factors include manipulating Agrobacterium strains and plasmids, growth conditions for vir gene induction, plant genotype, inoculation and co-culture conditions, and the selection agents and their application regime. We report here a novel physical parameter during co-culture, desiccation of plant cells or tissues post-Agrobacterium infection, which greatly enhances transfer DNA (T-DNA) delivery and increases stable transformation efficiency in wheat. Desiccation during co-culture dramatically suppressed Agrobacterium growth, which is one of the factors known to favor plant cell recovery. Osmotic and abscisic acid treatments and desiccation prior to inoculation did not have the same enhancement effect as desiccation during co-culture on T-DNA delivery in wheat. An efficient transformation protocol has been developed based on desiccation and is suitable for both paromomycin and glyphosate selection. Southern analysis showed approximately 67% of transgenic wheat plants received a single copy of the transgene.