Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants

 The green fluorescent protein (GFP) from Aequorea victoria has been introduced into three different citrus genotypes [Citrus aurantium L., C. aurantifolia (Christm.) Swing. and C. sinensis L. Osbeck×Poncirus trifoliata (L.) Raf.] which are considered recalcitrant to transformation, mainly due to low transformation frequencies and to the regeneration of escape shoots at high frequencies from the Agrobacterium-inoculated explants. High-level GFP expression was detected in transgenic cells, tissues and plants. Using GFP as a vital marker has allowed us to localize the sites of transgene expression in specific cells, always occurring in callus tissue formed from the cambium of the cut ends of explants. Whereas green fluorescent shoots regenerated in all cases from this callus, most escapes regenerated directly from explants with almost no callus formation. Thus, development of callus from cambium is a prerequisite for citrus transformation. Furthermore, in vivo monitoring of GFP expression permitted a rapid and easy discrimination of transgenic and escape shoots. The selection of transgenic shoots could be easily favored by eliminating the escapes and/or by performing shoot-tip grafting of the transgenic buds soon after their origin. GFP-expressing shoots have also been observed in citrus explants co-cultivated with Agrobacterium but cultured in a medium without the selective agent kanamycin. This opens the possibility to rescue the transgenic sectors and to regenerate transgenic plants without using selectable marker genes conferring antibiotic or herbicide resistance, which is currently a topic of much discussion for the commercialization of transgenic plants. Received: 28 October 1998 / Accepted: 28 November 1998     

Selection of transgenic <Emphasis Type="Italic">Petunia</Emphasis> plants using the green fluorescent protein (GFP)

Selectable marker genes are needed for efficient transformation of plants. The present study focused on testing the applicability of green fluorescent protein (GFP) for selecting transgenic Petunia hybrida plants without applying antibiotics or herbicides. Based on a transient gene expression assay, the efficiency of two gfp genes, mGFP-4 and smRS-GFP, was compared. Two days after infiltration of Agrobacterium tumefaciens, GFP expression was recorded in leaf epidermal cells. The intensity of smRS-GFP fluorescence was higher than that of mGFP-4 and easier to distinguish from other unspecific fluorescent signals in Petunia. Transformations using the pMen65smRS-GFP vector, which contained the neomycin phosphotransferase II (nptII) gene, resulted in callus and shoots that visually and clearly expressed detectable GFP levels; in addition, this vector made it possible to exclusively select transformed plants using GFP. The transformation efficiencies achieved by using GFP selection versus combined kanamycin and GFP selection (nptII+GFP) were compared in four Petunia genotypes with a transformation experiment with four replications. In three out of four Petunia cultivars a higher transformation frequency was achieved by using nptII+GFP selection. Southern blot hybridisation revealed single and multiple integrations of smRS-GFP in Petunia. Single copy plants showed intensive expression in all parts of the plants, whereas a higher copy number led to only weak or partial expression of smRS-GFP allowing the visual selection of single copy events. Thus, it is possible to select transgenic Petunia plants based on their GFP expressions without applying antibiotics or herbicides.     

Improved <Emphasis Type="Italic"> Agrobacterium </Emphasis>-mediated transformation of sunflower (<Emphasis Type="Italic"> Helianthus annuus </Emphasis>L.): assessment of macerating enzymes and sonication

Agrobacterium -mediated transformation of shoot apices of sunflower (Helianthus annuus L.) was evaluated following wounding by cell-wall-digesting enzymes and sonication. The frequency of explants with regenerated shoots expressing GUS (beta-glucuronidase) or GFP (green fluorescent protein) increased following treatment with the macerating enzymes cellulase Onozuka R-10 and pectinase Boerozym M5, whereas treatment with macerozyme R-10 had a negative effect. When a combination of cellulase (0.1%) and pectinase (0.05%) was used, the rate of explants with uniformly GUS-positive shoots increased at least twofold. The transient expression of reporter genes was also enhanced using sonication (50 MHz; 2, 4 and 6 s), but stable expression in regenerated shoots following 4 weeks of selection did not increase with this treatment. Enzyme treatment alone (0.1% cellulase and 0.05% pectinase) was superior to a combined treatment of sonication and enzymes with respect to stable transformation. Polymerase chain reaction analyses of shoots recovered by grafting from transformation experiments using GFP as the reporter gene demonstrated the stable integration of the transgene. Regenerated plants were fertile and seeds could be harvested.     

Expression of Brassica oleracea FtsZ1-1 and MinD alters chloroplast division in Nicotiana tabacum generating macro- and mini-chloroplasts

FtsZ1-1 and MinD plastid division-related genes were identified and cloned from Brassica oleracea var. botrytis. Transgenic tobacco plants expressing BoFtsZ1-1 or BoMinD exhibited cells with either fewer but abnormally large chloroplasts or more but smaller chloroplasts relative to wild-type tobacco plants. An abnormal chloroplast phenotype in guard cells was found in BoMinD transgenic tobacco plants but not in BoFtsZ1-1 transgenic tobacco plants. Transgenic tobacco plants bearing the macro-chloroplast phenotype had 10 to 20-fold increased levels of total FtsZ1-1 or MinD, whilst the transgenic tobacco plants bearing the mini-chloroplast phenotype had lower increased FtsZ1-1 or absence of detectable MinD. We also described for the first time, plastid transformation of macro-chloroplast bearing tobacco shoots with a gene cassette allowing for expression of green fluorescent protein (GFP). Homoplasmic plastid transformants from normal chloroplast and macro-chloroplast tobacco plants expressing GFP were obtained. Both types of transformants accumulated GFP at ~6% of total soluble protein, thus indicating that cells containing macro-chloroplasts can regenerate shoots in tissue culture and can stably integrate and express a foreign gene to similar levels as plant cells containing a normal chloroplast size and number.     

Transformation of homozygous diploid potato with an Agrobacterium tumefaciens binary vector system by adventitious shoot regeneration on leaf and stem segments

Transformed potato (Solanum tuberosum) plants were obtained from homozygous diploid potato by using a transformation procedure in combination with an adventitious shoot regeneration method. Leaf and stem explants were inoculated with an Agrobacterium tumefaciens strain which contained a binary vector (pVU 1011) carrying the neomycin phosphotransferase gene. Shoot regeneration most effectively on stem explants, occurred within six weeks directly from the explants without introducing a callus phase. A strong seasonal influence on transformation efficiencies was observed. Analysis of a number of randomly selected regenerated shoots for their ability to root and form shoots on kanamycin-containing medium shows that over 90% of the regenerated shoots obtained are transformed. In a number of shoots transformation was confirmed by a test for the presence and expression of the NPT-II gene.     

Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.)

Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate “escapes” and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained at frequencies of 3–11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic calli were regularly monitored under the microscope with blue light when they were transferred to media without selective agents. Green fluorescent shoots were obtained at frequencies of 2–5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has potential for the development of selectable marker-free transgenic sugar beet.     

Highly efficient transformation of the GFP and MAC12.2 genes into precocious trifoliate orange (Poncirus trifoliata [L.] Raf), a potential model genotype for functional genomics studies in Citrus

Precocious trifoliate orange (Poncirus trifoliata [L.] Raf), an extremely early flowering mutant of P. trifoliata, is an attractive model for functional genomics research in Citrus. A procedure for efficient regeneration and transformation of this genotype was developed by using green fluorescent protein (GFP) gene as visual marker and etiolated stem segments as explants. In vivo monitoring of GFP expression permitted a rapid and easy discrimination of transgenic shoots and escapes. Transformation efficiency was 20.7% and the transformants were identified by polymerase chain reaction (PCR) and Southern blot analysis. Moreover, the transgenic lines expressed variable amounts of the GFP gene as revealed by real-time PCR analysis. Fifteen transgenic plants flowered 18 months after transfer to the greenhouse and six of them set fruits. GFP expression was also observed in the transgenic flowers and fruits. To test the utility of this system for functional genomics studies, an Arabidopsis thaliana MAC12.2 gene with the potential to produce seedless fruits was introduced into this genotype, and the traits of the transgenic fruits were characterized. The successful transformation of this perennial woody genotype with extremely short juvenility will allow us to test the function of cloned genes in citrus, the improvement of which is hindered by a long juvenility period.     

Agrobacterium tumefaciens-mediated transformation of the legume Astragalus sinicus using kanamycin resistance selection and green fluorescent protein expression

To develop an efficient protocol for the transformation of the legume Astragalus sinicus (Chinese milk vetch), cotyledon segments were infected with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBINm-gfp5-ER which carries the gfp5 gene encoding green fluorescent protein and the kanamycin (Km) resistance gene nptII. The infected explants were cultured on shoot regeneration (SR) medium containing 1.0 mg l^–1 -naphthaleneacetic acid (NAA) and 1.0 mg l^–1 thidiazuron (TDZ). Putative transformed shoots were selected on SR medium containing 75 g ml^–1 Km, 200 g ml^–1 Timentin, and transformation was monitored by observation of GFP expression under a dissecting fluorescence microscope with appropriate filters. The identification of GFP-expressing shoots or callus in combination with Km selection allowed the visual selection of growing transgenic cells and shoots with no escapes. Plants were regenerated from seven independent transgenic events and five plants have set seed. GFP expression segregated in the T₁ seedlings of the two lines tested in a 3 – 1 ratio. In addition to the GFP expression of the transgenic plants, the transgenic nature of individual plants was confirmed by Southern and Western blot analyses.     

Transgenic peach plants (<Emphasis Type="Italic">Prunus persica</Emphasis> L.) produced by genetic transformation of embryo sections using the green fluorescent protein (GFP) as an <Emphasis Type="Italic">in vivo</Emphasis> marker

The main obstacle to genetic engineering of fruit tree species is the regeneration of transformed plantlets. Transformation events in peach (Prunus persica L.) have been reported using particle bombardment or Agrobacteriummediated transformation of immature embryos. However, the regeneration of plants from transgenic tissues is still difficult and the recovery of non-chimeric plants has not been reported to date. In this paper we describe an efficient, reliable transformation and regeneration system to produce transgenic peach plants using embryo sections of mature seeds as starting material. This represents an important advantage due to the availability of such material throughout the year. A. tumefaciens strain C58 (pMP90) containing the binary plasmid pBin19 was used as vector system for transformation. We used the Nospro-nptII-Noster cassette as a selectable marker and the CaMV35Spro-sgfp-CaMV35Ster cassette as a vital reporter gene coding for an improved version of the green fluorescent protein (sGFP). In vitro cultured embryo sections were Agrobacterium-cocultivated and, after selection, transgenic shoots were regenerated. Shoots that survived exhibited high-level of sGFP expression mainly visible in the young leaves of the apex. In vivo monitoring of GFP expression permitted an early, rapid and easy discrimination of both transgenic and escape buds. After elimination of escapes, transgenic shoots were rooted in vitro and the recovered plantlets were screened using PCR amplification. Southern analysis confirmed stable genomic integration of the sgfp transgene. The high levels of GFP expression were also maintained in the second generation of transgenic peach plants.     

In Planta visual monitoring of green fluorescent protein in transgenic rice plants

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a widely used reporter that can be directly visualized in the living cells in both animals and plants. We inserted a synthetic gene (sgfp) encoding a modified form of the GFP into expression vector, Act1-sgfp for the direct expression of GFP which is easily detectable in rice plants. Green fluorescence emitted from GFP could be visualized in calli, dry seeds, roots and seedlings with green shoots of transgenic rice plants. In our visualization system with a charge-coupled device camera, band-pass filters and a light source, the presence of red chlorophyll autofluorescence from chloroplasts did not alter the green fluorescence of GFP. These results demonstrate that GFP could be used as a non-destructive visual selection marker for examining gene expression in transformed calli, dry seeds and young plants.     

Protoplast transformation and regeneration of transgenic Valencia sweet orange plants containing a juice quality-related pectin methylesterase gene

Valencia orange [Citrus sinensis (L.) Osbeck] is the leading commercial citrus species in the world for processed juice products; however, the presence of thermostable pectin methylesterase (TSPME) reduces its juice quality. A long-term strategy of this work is to eliminate or greatly reduce TSPME activity in Valencia orange. Previous work resulted in the isolation of a putative TSPME gene, CsPME4, associated with a thermostable protein fraction of Valencia orange juice. To begin research designed to overexpress CsPME4 to verify the thermostability of the protein product and/or to downregulate the gene, a sense gene cassette containing a gene-specific sequence from a putative TSPME cDNA and the enhanced green fluorescent protein (GFP) as a selectable marker was constructed (M2.1). In the work reported here, M2.1 plasmid DNA was transformed (polyethylene glycol-mediated) into protoplasts isolated from an embryogenic suspension culture of Valencia somaclone line B6-68, in an effort to obtain transgenic Valencia lines. A vigorous transformed line was identified via GFP expression, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. One transgenic proembryo expressing GFP was recovered and multiple shoots were regenerated. The recovery of multiple transgenic plants was expedited by in vitro grafting. Polymerase chain reaction analysis revealed the presence of the PME gene in transgenic plants, and subsequent Southern blot analysis confirmed the presence of the eGFP gene. These transgenic plants show normal growth and minor morphological variation. The thermostability of PME in these plants will be assessed after flowering and fruit set. This is the first successful transfer of a target fruit-quality gene by protoplast transformation with recovery of transgenic plants in citrus. This method of transformation has the advantage over Agrobacterium-mediated transformation in that it requires no antibiotic-resistance genes.     

Green fluorescent protein as a visual marker for wheat transformation

 Wheat (Triticum aestivum L.) transformation via particle bombardment is now established in many laboratories, but transformation efficiencies are still largely low and the highest efficiencies can only be obtained with certain genotypes. For rapid optimization and improvement of wheat transformation protocols, a non-destructive marker which permits early detection of transformed cells is needed. We have assessed the ability of a modified version of the Aequorea victoria green fluorescent protein (GFP) to act as a marker for detecting transformed cells and tissues of wheat. Multicellular clusters emitting green fluorescence were observed 14 days after particle bombardment with a sGFPS65T gene construct, and gfp-expressing shoots (often with expressing roots) could be observed as early as 21 days after bombardment. These shoots can be removed from the callus and grown further until they are ready to transfer to soil. Transgenic wheat plants could be selected on the basis of gfp expression alone although the inclusion of antibiotic resistance as a selectable marker could improve the efficiency. Using sgfpS65T as a marker gene in an experiment comparing bombardment parameters allowed the rapid identification of variables that could be targeted for optimization. Received: 29 March 2000 / Accepted: 29 March 2000     

Use of green fluorescent protein as A non-destructive marker for peanut genetic transformation

Summary The ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The brightest and most dense fluorescent signals observed during transient expression were from p524EGFP. 1 and EYFP. Optimized bombardment conditions consisted of 0.6 μm diameter gold particles, 12410 kPa bombardment pressure, 95 kPa vacuum pressure, and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny.     

The use of green fluorescent protein (GFP) improves Agrobacterium-mediated transformation of ‘Spadona’ pear (Pyrus communis L.)

An efficient and reproducible system for Agrobacterium-mediated transformation of the pear (Pyrus communis L.) cultivar Spadona was developed. Leaf explants of in vitro propagated plants were cocultivated with the disarmed Agrobacterium strain EHA105 harboring the plasmid pME504, carrying the uidA-intron and nptII genes. Under selective conditions, 5% of the plantlets regenerated and were positively stained for GUS. However, most of the GUS-positive plants re-callused and subsequently died, leaving only 0.3–0.8% of these plantlets to reach maturity. In order to identify transformed shoots at early stages of regeneration, we introduced the green fluorescent protein (GFP) into the pear cultivar Spadona using the plasmid PZP carrying the nuclear-targeted GFP and nptII genes. High expression levels of GFP were detected in transgenic cells as early as 7 days after transformation. GFP marked-callii and transformed plants were observed after 14 and 24 days, respectively. Fluorescence microscopy screening of transformed plant material, under the selection of kanamycin, increased the transformation frequency to 3.0–4.0%. We conclude that the introduction of GFP improves the selection of transformed plants of Spadona pear.     

Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system

A dual-marker plasmid containing the selectable marker gene, manA, and the reporter gene, sgfp, was used to transform immature sorghum embryos by employing an Agrobacterium-mediated system. Both genes were under the control of the ubi1 promoter in a binary vector pPZP201. The Escherichia coli phosphomannose isomerase (PMI) gene, pmi, was used as the selectable marker gene and mannose was used as the selective agent. The sgfp gene encoding green fluorescence protein (GFP) was the reporter gene and served as a visual screening marker. A total of 167 transgenic plants were obtained from nine different embryogenic callus lines grown on a selection medium containing 1%-2% mannose. Embryoids and shoots regenerated via embryogenesis, that showed strong GFP fluorescence, were selected from two sorghum genotypes: C401, an inbred line, and Pioneer 8505, a commercial hybrid. The GFP accumulation in transgenic plants was observed with a dissecting stereomicroscope. The integration and expression of the manA gene was confirmed by Southern blot and Western blot analyses, and the feasibility of manA selection was demonstrated by the chlorophenol red (CPR) assay. Our results indicated that transgenes segregated in the Mendelian fashion in the T1 generation. The conversion of mannose to a metabolizable fructose carbon source is beneficial to plants. In addition, except in soybean and a few legumes, no endogenous PMI activity has been detected in plant species, indicating that PMI is useful in the transformation of sorghum. In addition, PMI has no sequence homology to known allergens. Optimization of this selection system for sorghum transformation provides an efficient way to produce transgenic plants without using antibiotic or herbicidal agents as selectable markers, and our results showed that the transformation efficiency reached 2.88% for Pioneer 8505 and 3.30% for C401, both values higher than in previously published reports.     

Evaluation of green fluorescent protein as a reporter gene and phosphinothricin as the selective agent for achieving a higher recovery of transformants in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L. cv. Poinsett76) via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Efficient Agrobacterium tumefaciens-mediated transformation and a higher recovery of transformed plants of cucumber cv. Poinsett76 were achieved via direct organogenesis from cotyledon explants. Stable transformants were obtained by inoculating explants with A. tumefaciens strains EHA105 or LBA4404, both harboring the binary vector pME508, which contains the neomycin phosphotransferase II (nptII) and phosphinothricin resistance genes (bar) conferring resistance to kanamycin and PPT, respectively, as selectable markers and the sgfp-tyg gene for the green fluorescent protein (GFP) as a visual marker driven by the constitutive CaMV35S promoter in the presence of acetosyringone (50 μM). Transformed shoots were obtained on MS Murashige and Skoog (Plant Physiol. 15: 473–497, 1962) medium supplemented with 1 mg L⁻¹ benzyladenine (BA), 20 mg L⁻¹ l-glutamine and 2 mg L⁻¹ phosphinothricin (PPT) or 100 mg L⁻¹ kanamycin. The regenerated shoots were examined in vivo using a hand-held long wave UV lamp for GFP expression. The GFP screening helped identify escapes and chimeric shoots at regular intervals to increase the growth of transformed shoots on cotyledon explants. Elongation and rooting of putative transformants were achieved on PPT (2 mg L⁻¹) containing MS media with 0.5 mg L⁻¹ gibberellic acid (GA₃) and 0.6 mg L⁻¹ indole butyric acid (IBA), respectively. PCR and Southern analyses confirmed the integration of the sgfp gene into the genome of T₀ and the progenies. T₁ segregation of transgenic progeny exhibited Mendelian inheritance of the transgene. The use of EHA105 resulted in 21% transformation efficiency compared to 8.5% when LBA4404 was used. This higher rate was greatly facilitated by PPT selection coupled with effective screening of transformants for GFP expression, thus making the protocol highly useful for the recovery of a higher number of transgenic cucumber plants.     

Optimizing Agrobacterium-mediated transformation of grapevine

Summary A translational fusion between the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTH) genes was used to optimize parameters influencing Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless. The corresponding bifunctional protein produced from this EGFP/NPTH fusion gene allowed for a single promoter to drive expression of both green fluorescence and kanamycin resistance, thus conserving promoter resources and climinating potential promoter-promoter interactions. The fusion gene, driven by either a double cauliflower mosaic virus 35S (CaMV 35S) promoter or a double cassava vein mosaic virus (CsVMV) promoter, was immobilized into Agrobacterium strain EHA 105. Somatic embryos capable of direct secondary embryogenesis were used as target tissues to recover transgenic plants. Simultaneous visualization of GFP fluorescence and kanamycin selection of transgenic cells, tissues, somatic embryos, and plants were achieved. GFP expression and recovery of embryogenic culture lines were used as indicators to optimize transformation parameters. Preculturing of somatic embryos for 7 d on fresh medium prior to transformation minimized Agrobacterium-induced tissue browning/necrosis. Alternatively, browning/necrosis was reduced by adding 1 gl⁻¹ of the antioxidant dithiothreitol (DTT) to post co-cultivation wash media. While combining preculture with antioxidant treatments did not result in a synergistic improvement in response, either treatment resulted in recovery of more stable embryogenic lines than did the control. A 48h co-cultivation period combined with 75 mgl⁻¹ kanamycin in selection medium was optimal. DNA analysis confirmed stable integration of transgenes into the grape genome: 63% had single gene insertions, 27% had two inserts, and 7 and 3% had three and four inserts, respectively. Utilizing optimized procedures, over 1400 stable independent transgenic embryogenic culture lines were obtained, of which 795 developed into whole plants. Transgenic grapevines have exhibited normal vegetative morphology and stable transgene expression for over 5 yr.     

Agrobacterium-mediated barley (Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA∝barley genomic DNA junctions

Agrobacterium-mediated barley transformation promises many advantages compared to alternative gene transfer methods, but has so far been established in only a few laboratories. We describe a protocol that facilitates rapid establishment and optimisation of Agrobacterium-mediated transformation for barley by instant monitoring of the transformation success. The synthetic green fluorescent protein (sgfpS65T) reporter gene was introduced in combination with thehpt selectable marker gene into immature embryos of barley (Hordeum vulgare L.) by cocultivation with Agrobacterium tumefaciens strain AGLO harboring binary vector pYF133. Using green fluorescent protein (GFP) as a non-destructive visual marker allowed us to identify single-cell recipients of T-DNA at an early stage, track their fate and evaluate factors that affect T-DNA delivery. GFP screening was combined with a low level hygromycin selection. Consequently, transgenic plantlets ready to transfer to soil were obtained within 50 days of explant culture. Southern blot- and progeny segregation analyses revealed a single copy T-DNA insert in more than half of the transgenic barley plants. T-DNA/barley genomic DNA junctions were amplified and sequenced. The right T-DNA ends were highly conserved and clustered around the first 4 nucleotides of the right 25 bp border repeat, while the left T-DNA ends were more variable, located either in the left 25 bp border repeat or within 13 bp from the left repeat. T-DNAs were transferred from Agrobacterium to barley with exclusion of vector sequence suggesting a similar molecular T-DNA transfer mechanism as in dicotyledonous plants.     

Stable transformation of sunflower (Helianthus annuus L.) using a non-meristematic regeneration protocol and green fluorescent protein as a vital marker

Stable transformation of sunflower was achieved using a non-meristematic hypocotyl explant regeneration protocol of public inbred HA300B. Uniformly transformed shoots were obtained after co-cultivation with Agrobacterium tumefaciens carrying a gfp (green fluorescent protein) gene containing an intron that blocks expression of gfp in Agrobacterium. Easily detectable, bright green fluorescence of transformed tissues was used to establish an optimal regeneration and transformation procedure. By Southern blot analysis, integration of the gfp and nptII genes was confirmed. Stable transformation efficiency was 0.1%. From 68 T₁ plants analyzed, 17 showed transmission of transgene DNA and 15 of them contained the intact gfp gene. Expression of gfp was detected in 10 T₁ plants carrying the intact gfp gene using a fluorimetric assay or western blot analysis. Expression of the nptII gene was confirmed in 13 T₁ plants. The transformation system enables the rapid transfer of agronomically important genes.     

Green fluorescent protein as a visual selection marker for coffee transformation

The green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined with low level of antibiotics selection improved the transformation efficiency and increased the number of transformed coffee plants compared to selection in the presence of antibiotics. Molecular analysis confirmed the presence of the sgfp-S65T coding region in the regenerated plants. Visual screening of transformed cells using GFP by Agrobacterium-mediated transformation techniques was found to be efficient and therefore has the potential for development of selectable marker-free transgenic coffee plants.