An improved protocol for<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Antirrhinum majus</Emphasis> L.

Efficient Agrobacterium -mediated transformation of Antirrhinum majus L. was achieved via indirect shoot organogenesis from hypocotyl explants of seedlings. Stable transformants were obtained by inoculating explants with A. tumefaciens strain GV2260 harboring the binary vector pBIGFP121, which contains the neomycin phosphotransferase gene (NPT II) as a selectable marker and the gene for the Green Fluorescent Protein (GFP) as a visual marker. Putative transformants were identified by selection for kanamycin resistance and by examining the shoots using fluorescence microscopy. PCR and Southern analyses confirmed integration of the GFP gene into the genomes of the transformants. The transformants had a morphologically normal phenotype. The transgene was shown to be inherited in a Mendelian manner. This improved method requires only a small number of seeds for explant preparation, and three changes of medium; the overall transformation efficiency achieved, based on the recovery of transformed plants after 4–5 months of culture, reached 8–9%. This success rate makes the protocol very useful for producing transgenic A. majus plants.Communicated by G. Jürgens     

Transfer of a minimal linear marker-free and vector-free <Emphasis Type="Italic">smGFP</Emphasis> cassette into soybean via ovary-drip transformation

A tissue culture-independent plant transformation method, called ovary-drip transformation, was established in which a minimal linear gene cassette [35S CaMV promoter, open reading frame of soluble modified green fluorescent protein (smGFP), and NOS terminator] was transformed into soybean. The method is characterized by directly dripping a DNA solution, which is supplemented with a surfactant, onto the ovary wound 6–8 h after self-pollination. The growth of the pollen tube was measured after self-pollination. The movement of smGFP across the passageway toward the embryo sac was monitored using fluorescein isothiocyanate-labeled DNA. The transformation frequency reached 3.2% by PCR analysis. Southern analysis of the primary transformants denoted the integration of a single site smGFP. The transgenic plants exhibited a high level of smGFP expression which was visible in the immature embryos of the transgenic soybean.     

Genetic transformation of <Emphasis Type="Italic">Agave salmiana</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and particle bombardment

Agave salmiana was transformed using two different protocols: co-cultivation with Agrobacterium tumefaciens and particle bombardment. The uidA (β-glucuronidase) gene was used as a reporter gene for both methods whereas the nptII and bar genes were used as selectable markers for A. tumefaciens and biolistic transformation respectively. Previous reports for in vitro regeneration of A. salmiana have not been published; therefore the conditions for both shoot regeneration and rooting were optimized using leaves and embryogenic calli of Agave salmiana. The transgenes were detected by Polymerase Chain Reaction (PCR) in 11 month old plants. The transgenic nature of the plants was also confirmed using GUS histochemical assays. Transformation via co-cultivation of explants with Agrobacterium harbouring the pBI121 binary vector was the most effective method of transformation, producing 32 transgenic plants and giving a transformation efficiency of 2.7%. On the other hand, the biolistic method produced transgenic calli that tested positive with the GUS assay after 14 months on selective medium while still undergoing regeneration.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Cry1C,Cry2A</Emphasis> and <Emphasis Type="Italic">Cry9C</Emphasis> genes into <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> and plant regeneration

Three constructs harbouring novel Bacillus thuringiensis genes (Cry1C, Cry2A, Cry9C) and bar gene were transformed into four upland cotton cultivars, Ekangmian10, Emian22, Coker201 and YZ1 via Agrobacterium-mediated transformation. With the bar gene as a selectable marker, about 84.8 % of resistant calli have been confirmed positive by polymerase chain reaction (PCR) tests, and totally 50 transgenic plants were regenerated. The insertions were verified by means of Southern blotting. Bioassay showed 80 % of the transgenic plantlets generated resistance to both herbicide and insect. We optimized conditions for improving the transformation efficiency. A modified in vitro shoot-tip grafting technique was introduced to help entire transplantation. This result showed that bar gene can replace antibiotic marker genes (ex. npt II gene) used in cotton transformation.     

Transformation of wheat with the HMW-GS <Emphasis Type="Italic">1Bx14</Emphasis> gene without markers

High molecular weight (HMW) glutenin polypeptides are critical contributors to the visco/elastic properties responsible for the processing characteristics and utilizations of wheat flour. In order to improve bread making quality of flour and produce transgenic plants free of selectable markers, a linear DNA construct consisting of a minimal expression cassette with the HMW-GS 1Bx14 gene was transformed into wheat cultivar Mianyang19 by microprojectile bombardment. The transform ants were selected by PCR instead of herbicidal markers. Seven transgenic plants were identified from a total of 1219 transformants, yielding a transformation frequency of 0.28%. An SDS-PAGE analysis confirmed that the 1Bx14 gene was expressed in three T1 seeds of the transgenic plants. Our results demonstrated that it is feasible to obtain marker-free trans-formants using the linear-expression-cassette-transformation approach coupled with PCR selection.     

Generation of marker-free transgenic maize by regular two-border <Emphasis Type="Italic">Agrobacterium</Emphasis> transformation vectors

By introducing additional T-DNA borders into a binary plasmid used in Agrobacterium-mediated plant transformation, previous studies have demonstrated that the marker gene and the gene of interest (GOI) can be carried by independent T-strands, which sometimes integrate in unlinked loci in the plant genome. This allows the recovery of marker-free transgenic plants through genetic segregation in the next generation. In this study, we have found that by repositioning the selectable marker gene in the backbone and leaving only the GOI in the T-DNA region, a regular two-border binary plasmid was able to generate marker-free transgenic maize plants more efficiently than a conventional single binary plasmid with multiple T-DNA borders. These results also provide evidence that both the right and left borders can initiate and terminate T-strands. Such non-canonical initiation and termination of T-strands may be the basis for the elevated frequencies of cotransformation and unlinked insertions.     

<Emphasis Type="Italic">Botrytis cinerea</Emphasis>-resistant marker-free <Emphasis Type="Italic">Petunia hybrida</Emphasis> produced using the MAT vector system

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a controversial issue and a serious problem for their public acceptance and commercialization. The MAT (multi-auto-transformation) vector system has been one of the strategies developed to excise the selection marker gene and produce marker-free transgenic plants. In an attempt to produce transgenic marker-free Petunia hybrida plants resistant to Botrytis cinerea (gray mold), we used the ipt gene as a selectable marker gene and the wasabi defensin (WD) gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), as a gene of interest. The WD gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacterium tumefaciens strain EHA105. Infected leaf explants of P. hybrida were cultured on hormone- and antibiotic-free MS medium. Extreme shooty phenotype (ESP)/ipt shoots were produced by the explants infected with the pMAT21-WD. The same antibiotic- and hormone-free MS medium was used in subcultures of the ipt shoots. Ipt shoots subsequently produced morphologically normal shoots. Molecular analyses of genomic DNA from the transgenic plants confirmed the integration of the gene of interest and excision of the selection marker. Expression of the WD gene was confirmed by northern blot and western blot analyses. A disease resistance assay of the marker-free transgenic plants exhibited enhanced resistance against B. cinerea strain 40 isolated from P. hybrida.     

Isopentenyl transferase gene expression offers the positive selection of marker-free transgenic plant of <Emphasis Type="Italic">Kalanchoe blossfeldiana</Emphasis>

The technologies allowing the production of transgenic plants without selectable marker genes, is of great interest in public and environmental safety. For generating such marker-free transgenic plants, possibility has been offered by Multi-Auto-Transformation [MAT] vector system, which combines positive selection, using the isopentenyl transferase (ipt) gene, with a site-specific recombination that generates marker-free plants. In this study Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pMAT21, containing lacZ, gus genes and the removable cassette in the T-DNA region was used to produce marker-free transgenic Kalanchoe blossfeldiana Poelln., employing ipt gene as the selectable marker gene. Co-cultivated explants were cultured on hormone- and selective agent-free MS medium, and 85% of the regenerated shoots showed ipt-shooty phenotype with GUS expression. Forty-one morphologically normal shoots were produced during the subculture. More than ninety percent of the normal shoots were ipt ⁻, gus ⁻ but lacZ ⁺ as determined by PCR analyses. These results indicate that the ipt phenotype was clearly distinguishable from non-transgenic as well as transgenic marker-free shoots. This study opens interesting perspective for the generation of marker-free transgenic K. blossfeldiana with objective useful transgene.     

利用子房滴注法获得无载体骨架序列和选择标记的转基因玉米

转基因植物中的载体骨架序列和选择标记基因是引起生物安全性争论的根本原因, 最直接、最有效的解决方法是在转化过程中不使用载体骨架序列和选择标记基因。本研究建立并优化了玉米子房滴注转化法, 其操作要点是将DNA转化溶液直接滴加在完全去除花柱的子房上。利用子房滴注法将无载体骨架序列和选择标记的线性GFP基因表达框转化玉米。PCR结果表明: 适合子房滴注法转化的玉米品种为9818, 最佳转化时间为授粉后18~20 h, 在此条件下得到最高的PCR阳性率, 为3.01%; Southern blotting结果表明外源基因的整合方式简单(1~2条杂交带); RT-PCR结果表明转基因植株中GFP基因能够在RNA水平上正常表达; 在转基因植株的根和幼胚中观察到GFP表达。     

Marker-Free Transgenic Chrysanthemum Obtained by <Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Transformation with Twin T-DNA Binary Vectors

In this study, a superbinary vector was constructed to evaluate the potential of a twin T-DNA system for generating selectable marker-free transgenic chrysanthemum plants. The first T-DNA of the pCAMBIA 1300 vector contained the hygromycin phosphotransferase (hpt) selectable marker gene, while the second T-DNA carried the β-glucuronidase gene (uidA) and featuring the gene of interest. The two T-DNA regions were placed adjacent to each other with no intervening region. This vector was then used to transform transversal thin cell layers (1–2 mm thick) of internodal stem segments of chrysanthemum via Agrobacterium-mediated transfer. Putative transgenic plants were obtained and analyzed for presence and integration of the transgene using polymerase chain reaction amplification and Southern blotting. The primary cotransformation frequency was calculated at 38.4%. A total of 17 hpt-resistant/gus-positive T0 plants were evaluated for segregation in the next generation (T1), and among those approximately 15.7% carried the transgene. Overall, the two T-DNA system appeared to be a useful approach to generate marker-free transgenic chrysanthemum plants, thereby eliminating public concerns regarding proliferation of antibiotic and herbicide resistance genes into the environment.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the genome-sequenced poplar clone,nisqually-1 (<Emphasis Type="Italic">Populus trichocarpa</Emphasis>)

The US Department of Energy recently released a 6.8X draft of the genome sequence for Nisqually-1, a genotype of black cottonwood (Populus trichocarpa). To improve its utility for functional genomics research, having an efficient means for transformation and regeneration is necessary. To examine several parameters known to affect the transformation rate, we cocultivated leaf disc and stem explants with a strain ofAgrobacterium tumefaciens harboring a binary plasmid vector containing genes for both neomycin phosphotransferase (NPTII) and β-glucuronidase (GUS). Shoot regeneration from stem explants was observed in the presence of kanamycin when thidiazuron was incorporated in the selection medium. Transformation efficiency was influenced by the level of thidiazuron to which explants were exposed during the early stages of shoot induction. Histochemical assays revealed expression of theGUS gene in leaf, stem, and root tissues of transgenic plants. Polymerase chain reaction confirmed the presence of both selectable marker and reporter genes in all lines that stained positive for β-glucuronidase activity. By use of our modified protocol, transgenic plants were recovered within 6 mo at an efficiency of 6%, adequate to produce a large number of transgenic events with modest effort.     

Evaluation of selection strategies alternative to <Emphasis Type="Italic">nptII</Emphasis> in genetic transformation of citrus

The neomycin phosphotransferase (nptII) selection system has proved successful in citrus transformation; however, it may be recommendable to replace it given the pressure exerted against antibiotic-resistance selectable marker genes in transgenic plants. The present work investigates three different selection alternatives, comparing them to nptII selection in two citrus genotypes, Carrizo citrange and Pineapple sweet orange. The first method used the beta-glucuronidase (uidA) reporter marker gene for selection; the second attempted to generate marker-free plants by transforming explants with a multi-auto-transformation (MAT) vector, combining an inducible R/RS-specific recombination system with transgenic-shoot selection through expression of isopentenyl transferase (ipt) and indoleacetamide hydrolase/tryptophan monooxygenase (iaaM/H) marker genes; while the third exploited the phosphomannose isomerase (PMI)/mannose conditional positive selection system. Firstly, GUS screening of all regenerated shoots in kanamycin-free medium gave 4.3% transformation efficiency for both genotypes. Secondly, workable transformation efficiencies were also achieved with the MAT system, 7.2% for citrange and 6.7% for sweet orange. This system affords an additional advantage as it enables selectable marker genes to be used during the in vitro culture phase and later removed from the transgenic plants by inducible recombination and site-specific excision. Thirdly, the highest transformation rates were obtained with the PMI/mannose system, 30% for citrange and 13% for sweet orange, which indicates that this marker is also an excellent candidate for citrus transformation.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of meadow fescue (<Emphasis Type="Italic">Festuca pratensis</Emphasis> Huds.)

Meadow fescue (Festuca pratensis Huds.) is an important cool-season forage grass in Europe and Asia. We developed a protocol for producing meadow fescue transgenic plants mediated by Agrobacterium tumefaciens transformation. Embryogenic calli derived from mature embryos were transformed with A. tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. In total, 40 independent transgenic plants were recovered from 45 bialaphos-resistant callus lines and an average transformation efficiency of 2% was achieved. The time frame from infection of embryogenic calli with Agrobacterium to transferring the transgenic plants to the greenhouse was 18 weeks. In a study of 11 BASTA-resistant transgenic lines, the uidA gene was expressed in 82% of the transgenic lines. Southern blot analysis revealed that 82% of the tested lines integrated one or two copies of the uidA gene. C. Gao and J. Liu contributed equally to the work.     

Genetic transformation of apple (<Emphasis Type="Italic">Malus</Emphasis> x <Emphasis Type="Italic">domestica</Emphasis>) without use of a selectable marker gene

Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, antibiotic or herbicide resistance marker genes are preferred because they tend to be most efficient. Due mainly to consumer and grower concerns, considerable effort is being put into developing strategies (site-specific recombination, homologous recombination, transposition, and cotransformation) to eliminate the marker gene from the nuclear or chloroplast genome after selection. For the commercialization of genetically transformed plants, use of a completely marker-free technology would be desirable, since there would be no involvement of antibiotic resistance genes or other marker genes with negative connotations for the public. With this goal in mind, a technique for apple transformation was developed without use of any selectable marker. Transformation of the apple genotype “M.26” with the constructs pPin2Att35SGUSintron and pPin2MpNPR1 was achieved. In different experiments, 22.0–25.4% of regenerants showed integration of the gene of interest. Southern analysis in some transformed lines confirmed the integration of one copy of the gene. Some of these transformed lines have been propagated and used to determine the uniformity of transformed tissues in the plantlets. The majority of the lines are uniformly transformed plants, although some lines are chimeric, as also occurs with the conventional transformation procedure using a selectable marker gene. A second genotype of apple, “Galaxy,” was also transformed with the same constructs, with a transformation efficiency of 13%.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) with a novel stilbene synthase gene from Chinese wild <Emphasis Type="Italic">Vitis pseudoreticulata</Emphasis>

A novel stilbene synthase gene (STS), cloned from Chinese wild Vitis pseudoreticulata (W. T. Wang) and responsible for synthesis of the phytoalexin resveratrol in grapevine, was successfully transferred into V. vinifera L. cv. Thompson Seedless via Agrobacterium tumefaciens-mediated transformation. Using transformation procedures developed in the present study, 72% GFP-positive germinated embryos were produced with about 38% of transformed embryos regenerated into normal plantlets. Integration of the STS gene into the transgenic plants was verified by PCR and Southern blot analysis. Expression of the STS gene was detected by high performance liquid chromatography (HPLC), which showed that the resveratrol concentration in the transgenic plants was 5.5 times higher than that in non-transformed control plants. Chaohong Fan and Ni Pu contributed equally to this work.     

Development of marker-free transgenic sorghum [<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench] using standard binary vectors with <Emphasis Type="Italic">bar</Emphasis> as a selectable marker

We report an Agrobacterium-mediated transformation system that can generate marker-free transgenic sorghum [Sorghum bicolor (L.) Moench] from a public line [P898012] using standard binary vectors with bar as a selectable marker. Eight co-cultivation conditions were examined for their effect on transformation. The average transformation frequencies were 0.4 and 0.7% for pZY101-TC2 and pZY101-SKRS, respectively, derived from binary vector pZY102 and containing bar and target gene(s) in separate T-DNA regions. A low selection pressure (2.5 mg l⁻¹ DL-phosphinothrithin, PPT) was deployed during callus induction in combination with rapid selection to generate plants from 80 independent events, all but three of which were fertile and set seed. PCR and Southern analyses showed that 36 out of 80 events contained both bar and the target gene(s) (an average co-transformation frequency of 45%). Seedlings of the T1 generation transmitted T-DNAs with target gene(s) and bar gene independently, generating a fraction of progeny with only the target gene(s).     

Factors influencing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of monocotyledonous species

Summary Since the success of Agrobacterium-mediated transformation of rice in the early 1990s, significant advances in Agrobacterium-mediated transformation of monocotyledonous plant species have been achieved. Transgenic plants obtained via Agrobacterium-mediated transformation have been regenerated in more than a dozen monocotyledonous species, ranging from the most important cereal crops to ornamental plant species. Efficient transformation protocols for agronomically important cereal crops such as rice, wheat, maize, barley, and sorghum have been developed and transformation for some of these species has become routine. Many factors influencing Agrobacterium-mediated transformation of monocotyledonous plants have been investigated and elucidated. These factors include plant genotype, explant type, Agrobacterium strain, and binary vector. In addition, a wide variety of inoculation and co-culture conditions have been shown to be important for the transformation of monocots. For example, antinecrotic treatments using antioxidants and bactericides, osmotic treatments, desiccation of explants before or after Agrobacterium infection, and inoculation and co-culture medium compositions have influenced the ability to recover transgenic monocols. The plant selectable markers used and the promoters driving these marker genes have also been recognized as important factors influencing stable transformation frequency. Extension of transformation protocols to elite genotypes and to more readily available explants in agronomically important crop species will be the challenge of the future. Further evaluation of genes stimulating plant cell division or T-DNA integration, and genes increasing competency of plant cells to Agrobacterium, may increase transformation efficiency in various systems. Understanding mechanisms by which treatments such as desiccation and antioxidants impact T-DNA delivery and stable transformation will facilitate development of efficient transformation systems.     

Inducible Excision of Selectable Marker Gene from Transgenic Plants by the Cre/<Emphasis Type="Italic">lox</Emphasis> Site-specific Recombination System

In a plant transformation process, it is necessary to use marker genes that allow the selection of regenerated transgenic plants. However, selectable marker genes are generally superfluous once an intact transgenic plant has been established. Furthermore, they may cause regulatory difficulties for approving transgenic crop release and commercialization. We constructed a binary expression vector with the Cre/lox system with a view to eliminating a marker gene from transgenic plants conveniently. In the vector, recombinase gene cre under the control of heat shock promoter and selectable marker gene nptII under the control of CaMV35S promoter were placed between two lox P sites in direct orientation, while the gene of interest was inserted outside of the lox P sites. By using this vector, both cre and nptII genes were eliminated from most of the regenerated plants of primary transformed tobacco through heat shock treatment, while the gene of interest was retained and stably inherited. This autoexcision strategy, mediated by the Cre/lox system and subjected to heat shock treatment to eliminate a selectable marker gene, is easy to adopt and provides a promising approach to generate marker-free transgenic plants.     

Detection of vector- and selectable marker-free transgenic maize with a linear GFP cassette transformation via the pollen-tube pathway

The pollen-tube pathway is feasible to transform vector- and selectable marker-free linear gene cassettes into plants to address the biosafety issues. However, its transformation frequency is low and the screening of selectable marker-free transformants by PCR analysis is time-consuming and expensive. In this study, a linear GFP cassette (Ubi-GFP-nos) flanked by 25bp T-DNA borders was transformed into maize via the pollen-tube pathway. The forepart of each maize ear was divided into five segments (segments I-V) at an interval of two rows of kernels. The segments that were most likely to contain transgenic kernels were identified by monitoring GFP expression in the immature embryos. A total of 21 ears were transformed with the linear GFP cassette. Seven out of 19 ears exhibited positive GFP expression in the immature embryos. Transgenic kernels were primarily identified in segments III and IV. A total of 121 plants derived from kernels located within segments III and IV of the remaining two ears were screened by PCR analysis. Six plants (4.96%) showed the presence of the GFP cassette. Southern blot analysis showed that the transgenic plants had simple integration patterns. The identification of transgenic kernels would facilitate PCR screening for marker-free transgenic plants.