<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated genetic transformation in tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> [L.] O. Kuntze)

We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7 d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L giberellic acid (GA₃). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern hybridization to confirm gene integration.     

Transgenic plants of coffee Coffea canephora from embryogenic callus via Agrobacterium tumefaciens-mediated transformation

 Embryogenic calli were induced from leaf explants of coffee (Coffea canephora) on McCown's woody plant medium (WPM) supplemented with 5 μM N⁶–(2-isopentenyl)-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransferase II genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentration (5, 50, 100 mg/l). The embryogenic calli surviving on medium containing 100 mg/l hygromycin showed a strong GUS-positive reaction with X-Gluc solution. Somatic embryos were formed from these putative transgenic calli and germinated on WPM medium with 5 μM 2-iP. Regenerated small plantlets with shoots and roots were transferred to medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin for final selection of transgenic plants. The selected plantlets exhibited strong GUS activity in leaves and roots as indicated by a deep blue color. GUS and HPT genes were confirmed to be stably integrated into the genome of the coffee plants by the polymerase chain reaction. Received: 14 December 1998 / Revision received: 12 March 1999 / Accepted: 24 March 1999     

Introduction of Foreign Gene into Small Cell Groups Digested Partial-Enzymically in Rice and Regeneration of Transgenic Plants

Suspension cultures of small cell groups (SCG; ca. 50–100 cells per group) were established from calli of Japonica rice Fang 7 and Hl24. The SCG were partially digested and transformed by plasmid pBll21 harboring the NPT-II (neomycin phosphotransferase) and GUS (betaglucuronidase) genes. Plasmid DNA was introduced into cells' by PEG, electroporation and PEG plus electroporation. NTP-II and GUS activity assay showed that the report genes were expressed in transformed cells. Transgenic plants were regeneiated possessing GUS activity due to the integration of intact foreign DNA into their genome as evidanced by hybridization. The results prove that the partially digested SCG is a potential, feasible system as receptor for gene transfer, especially for plants which are difficult for protoplast culture and plant regeneration from protoplasts.     

Analysis and optimisation of DNA delivery into wheat scutellum and Tritordeum inflorescence explants by tissue electroporation

Wheat scutella and tritordeum inflorescences were transformed by tissue electroporation with plasmid DNA containing a β-glucuronidase (GUS) gene (gus A) under the control of the rice actin1 promoter. Factors affecting electroporation efficiency were analysed. Important factors were electroporation voltage and pulse length, the volume of electroporation buffer, the osmoticum of electroporation buffer and medium, the osmoticum of pre-electroporation culture medium, and pre-electroporation incubation time and temperature. Maximum transient gene expression was obtained with a single pulse of 550 V/cm from a 960-μF capacitor, using 200 μl of electroporation buffer, after 2–3 h culture on media with 357 mOsm for wheat scutella or 1 day on media with 222 mOsm for tritordeum inflorescences, and 0.5–1 h pre-electroporation incubation with DNA at 24 °C. Under these conditions, up to 90% of the explants showed GUS expression, and up to 149 expression signals were recorded per replicate. Electroporated explants showed high rates of survival and retained the ability to regenerate plants via somatic embryogenesis. Received: 9 August 1996 / Revision received: 11 September 1997 / Accepted: 2 July 1998     

Transformation of protoplasts and intact cells from slowly growing embryogenic callus of wheat (Triticum aestivum L.)

A procedure for culturing protoplasts from slowly growing embryogenic calli of wheat was developed. The procedure was dependent on the ability to isolate large numbers of culturable protoplasts from slowly growing embryogenic callus. Approximately 68% of the isolated protoplasts divided, and 22% formed colonies; of the latter, 67% continued to proliferate. Plating efficiency was reduced when protoplasts were transformed by polythylene glycol, electroporation, and/or Agrobacterium. Intact cells were also directly transformed by electroporation. Direct electroporation of the Agrobacterium binary vector into intact cells resulted in a significant increase of GUS activity over the control.     

Regeneration of transgenic plants of grapevine (Vitis vinifera L.) via Agrobacterium rhizogenesmediated transformation of embryogenic calli

Genetically transformed roots and calli were induced from leafsegments of grapevine (Vitis vinifera L. cv. Koshusanjaku) afterco-cultivation with wild-type Agrobacterium rhizogenes strains,but plant regenera tion from them was not achieved. On the otherhand, transgenlc grapevine plants were obtained via somaticembryogenesis after co-cultivation of embryogenic calli withan engineered A. rhizogenes strain including both the neomycinphosphotransferase II (NPT II) and the ß-glucuronidase(GUS) genes, followed by selection of secondary embryos forkanamycin resistance. All these plants showed GUS gene expressionrevealed by histochemical assay. Southern blot analysis revealedthe stable integration of the GUS cording region in their genome.Transformants containing Ri T-DNA exhibited various phenotypes:most of them showed a typical Ri-transformed phenotype suchas wrinkled leaves, while the others looked normal. Key words: Agrobacterium rhizogenes, grapevine, transgenic plants, Vitis vinifera     

Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium × formolongi

An efficient system for Agrobacterium-mediated transformation of Lilium × formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 μM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.     

Electroporation-mediated transient gene expression in intact cells of sweetpotato

Summary Transient expression of the β-glucuronidase (GUS) gene has been studied in leaf-derived embryogenic callus of sweetpotatoIpomoea batatas L. (Lam.) by electroporation. The influence of several factors including electric field strength, buffer composition, time course of transientGUS gene expression, DNA concentration, enzyme, and polyethylene glycol (PEG) treatment was examined onGUS gene expression (number of blue spots). MaximumGUS gene expression (an average of 90 blue spots/fifty mg fresh weight callus tissue) was observed after 48 h when callus pieces were preincubated with electroporation (EPR) buffer for 1 h, followed by electroporation with a single electric pulse of 500 V/cm discharged from a 960-μF capacitor in the presence of 20 μg DNA/ml and 8.3 μl NaCl (3M). Changing the electroporation buffer conductivity (by varying the buffer composition with low-high salt concentrations), had only slight effect on the number of blue spots. Similarly, the time course study ofGUS gene expression revealed that GUS activity could be detected 12 h after electroporation with a maximum activity after 72 h (112 blue spots). Increasing the amount of DNA from 5 to 50 μg/ml in the EPR buffer had a slight effect on the expression frequency (from 20–110 blue spots, and 112 blue spots with 20 μg/ml). The number of blue spots was increased by enzymatic wounding of callus pieces for 10 min and by addition of 200 μl PEG 4000 (15%) before electroporation. These results suggest that intact cell electroporation can be used for producing transgenic sweetpotato tissue.     

Expression ofArabidopsis tryptophan biosynthetic pathway genes: effect of the 5′ coding region of phosphoribosylanthranilate isomerase gene

There are three non-allelic isogenes encoding phosphoribosylanthranilate isomerase (PAI) inArabidopsis thaliana. The expression plasmids were constructed by fusion of the GUS reporter gene to the three PAI promoters with or without the 5′ region encoding PAI N-terminal polypeptides and transferred into Arabidopsis plants byAgrobacterium tumefaciens. Analysis of GUS activity revealed that the PAI 5′ coding region was necessary for high expression of GUS activity. GUS activity in transgenic plants transformed with the expression plasmids containing the 5′ coding region of PAH or PAI3 was 60–100-fold higher than that without the corresponding 5′ region. However, the effect of 5’ coding region of PAI2 gene on the GUS activity was very small (only about 1 time difference). The GUS histochemical staining showed a similar result as revealed by GUS activity assay. It was expressed in the mesophyll cells and guard cells, but not in the epidermic cells, indicating that the N-terminal polypeptides encoded by the 5′ region of PAI genes have the function of PTP.     

Production of bialaphos-resistant Nierembergia repens by electroporation

Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient β-glucuronidase (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and 100 μF in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos, on which they formed adventitious shoots 1–2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos solution at 10.0 mg/l.     

Transgenic coffee fruits from <Emphasis Type="Italic">Coffea arabica</Emphasis> genetically modified by bombardment

The genetic modification of Coffea arabica fruits is an important tool for the investigation of physiological characteristics and functional validation of genes related to coffee bean quality traits. In this work, plants of C. arabica cultivar Catuaí Vermelho were successfully genetically modified by bombardment of embryogenic calli. Calli were obtained from 90% of the leaf explants cultivated in a callogenesis-inducing medium modified with 20 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting calli were bombarded with the pBI426 vector containing a uidA and nptII gene fusion that was driven by the double CaMV35s promoter. Kanamycin-selected embryos were positive for β-glucuronidase (GUS) activity in histochemical assays and for target gene amplification by polymerase chain reaction. Integration of the nptII gene was confirmed by Southern blot and showed a low copy number (one to three) of insertions. Transformed plants showed normal development and settled fruits. GUS expression was assessed in the flower and fruit organs demonstrating the capacity of the double CaMV35s promoter to drive long-term stable expression of uidA in C. arabica fruit tissues. Moreover, we obtained a T₁ progeny presenting 3:1 Mendelian segregation of the uidA gene. This investigation is the first to report exogenous gene expression in coffee fruits and transgenic inheritance in C. arabica plants.     

Introduction of <Emphasis Type="Italic">OsglyII</Emphasis> gene into <Emphasis Type="Italic">Oryza sativa</Emphasis> for increasing salinity tolerance

Mature seed-derived embryogenic calli of indica rice (Oryza sativa L. cv. PAU201) were induced on semisolid Murashige and Skoog medium supplemented with 2.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 0.5 mg dm⁻³ kinetin + 560 mg dm⁻³ proline + 30 g dm⁻³ sucrose + 8 g dm⁻³ agar. Using OsglyII gene, out of 3180 calli bombarded, 32 plants were regenerated on medium containing hygromycin (30 mg dm⁻³). Histochemical GUS assay of the hygromycin selected calli revealed GUS expression in 50 % calli. Among the regenerants, 46.87 % were GUS positive. PCR analysis confirmed the presence of the transgene of 1 kb in 60 % of independent plants. Further, these plants have been grown to maturity in glasshouse. In vitro screening for salt tolerance showed increase in fresh mass of OsglyII putative transgenic calli (185.4 mg) as compared to control calli (84.2 mg) on 90 mM NaCl after 15 d. When exposed to 150 mM NaCl, OsglyII putative transgenic plantlets showed normal growth while the non-transgenic control plantlets turned yellow and finally did not survive.     

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.     

Expression of green-fluorescent protein gene in sweet potato tissues

Green-fluorescent protein (GFP) gene expression, transient and stable after electroporation and particle bombardment, was analyzed in tissues of sweet potato cv.Beauregard. Leaf and petiole tissues were used for protoplast isolation and electroporation. After 48 h, approximately 25–30% of electroporated mesophyll cell protoplasts regenerated cell walls, and of these, 3% expressed GFP. Stable expression of GFP after four weeks of culture was observed in 1.0% of the initial GFP positive cells. In a separate experiment, we observed 600–700 loci expressing GFP 48 h after bombarding leaf tissue or embryogenic calli, and stable GFP-expressing sectors were seen in leaf-derived embryogenic calli after four weeks of protoplast culture without selection. These results demonstrate GFP gene expression in sweet potato tissues. Screening for GFP gene expression may prove useful to improve transformation efficiency and to facilitate detection of transformed sweet potato plants.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

Production of herbicide-resistant sweet potato plants transformed with the <Emphasis Type="Italic">bar</Emphasis> gene

Herbicide-resistant sweet potato plants were produced through biolistics of embryogenic calli derived from shoot apical meristems. Plant materials were bombarded with the vectors containing the β-glucuronidase gene (gusA) and the herbicide-resistant gene (bar). Selection was carried out using phosphinothricin (PPT). Transformants were screened by the histochemical GUS and Chlorophenol Red assays. PCR and Southern-blot analyses indicated the presence of introduced bar gene in the genomic DNA of the transgenic plants. When sprayed with Basta, the transgenic sweet potato plants was tolerant to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to sweet potato.     

Regeneration of transgenic Picea glauca, P. Mariana, and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens

Summary Transgenic plants of three Picea species were produced after coculture of embryogenic tissue with the disarmed strain of Agrobacterium tumefaciens C58/pMP90/pBIV10 and selection on medium containing kanamycin. In addition to the nptII selectable gene (conferring resistance to kanamycin), the vector carried the uidA (β-glucuronidase) marker gene. Transformation frequencies were dependent on the species, genotype, and post-cocultivation procedure. Of the three species tested, P. mariana was transformed at the highest frequency, followed by P. glauca and P. abies. The transgenic state of the embryogenic tissue was initially, confirmed by histochemical β-glucuronidase (GUS) assay followed by Southern hybridization. One to over five copies of T-DNA were detected in various transgenic lines analyzed. Transgenic plants were regenerated for all species using modified protocols for maturation and germination of somatic embryos.     

Introduction of a chimeric gene encoding an oryzacystatin-β-glucuronidase fusion protein into rice protoplasts and regeneration of transformed plants

In order to construct transgenic rice plant with an introduced oryzacystatin (OC)--glucuronidase (GUS) fusion gene, we first introduced it into rice protoplasts by electroporation, together with a marker gene conferring hygromycinresistance (pUC-HPH). In a transient assay using the transfected protoplasts, both OC and GUS activities were detected. The GUS activity was higher when the OC-GUS fusion protein was expressed than when only a single GUS protein was expressed. Next, to isolate stable transformants, hygromycin-resistant calli were selected. Forty one out of 116 hygromycin-resistant calli expressed a 2.2 kb mRNA transcribed from the chimeric gene and their extracts exhibited the activities of both OC and GUS. Finally, the transgenic calli were regenerated into rice plants whose tissues (leaves, roots and seeds) exhibited GUS activity probably derived from the fusion protein.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of embryogenic cell suspension cultures of <Emphasis Type="Italic">Santalum album</Emphasis> L.

An efficient method for Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L. is described. Embryogenic cell suspension cultures derived from stem internode callus were transformed with Agrobacterium tumefaciens harbouring pCAMBIA 1301 plant expression vector. Transformed colonies were selected on medium supplemented with hygromycin (5 mg/l). Continuously growing transformed cell suspension cultures were initiated from these colonies. Expression of β-glucuronidase in the suspension cultures was analysed by RT-PCR and GUS histochemical staining. GUS specific activity in the transformed suspension cultures was quantified using a MUG-based fluorometric assay. Expression levels of up to 105,870 pmol 4-MU/min/mg of total protein were noted in the transformed suspension cultures and 67,248 pmol 4-MU/min/mg of total protein in the spent media. Stability of GUS expression over a period of 7 months was studied. Plantlets were regenerated from the transformed embryogenic cells. Stable insertion of T-DNA into the host genome was confirmed by Southern blot analysis. This is the first report showing stable high-level expression of a foreign protein using embryogenic cell suspension cultures in S. album. U. K. S. Shekhawat and T. R. Ganapathi contributed equally to this work.     

Electroporation of embryogenic protoplasts of sweet orange (<Emphasis Type="Italic">Citrus sinensis</Emphasis> (L.) Osbeck) and regeneration of transformed plants

Summary Electroporation conditions were optimized for the transfection of protoplasts isolated from an embryogenic cell line of sweet organe [Citrus sinensis (L.) Osbeck ev. Hamlin]. Electric field strength (375–450 V cm⁻¹) vector DNA concentration (100 μgml⁻¹), carrier DNA concentration (100 μgml⁻¹), electroporation buffer (pH 8), and preelectroporation heat shock of protoplasts (5 min at 45°C) were optimized. The plasmid vector pBI221 containing the β-glucuronidase (GUS) coding sequence under the control of the CaMV 35S promoter was used and GUS activity was measured 24h after electroporation. All variables significantly affected transfection efficiency and when optimal conditions for each were combined. GUS activity was 7714 pmol 4-methylumbelliferone (MU) mg⁻¹ (protein) min⁻¹. Protoplasts were then electroporated in the presence of green fluorescent protein (GFP) expression vectors pARS101 or pARS108. Green fluorescent embryos were selected, plants regenerated, and integration of the transgene was confirmed by Southern blot analysis. Both plasmids were constructed using EGFP, a GFP variant 35 times brighter than wtGFP, having a single, red-shifted excitation peak, and optimized for human codon-usage. pARS101 was constructed by placing EGFP under the control of a 35S–35S promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus. pARS108 was constructed similarly except sequences were added for transport and retention of EGFP in the lumen of the endoplasmic reticulum. Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the US Department of Agriculture and does not imply its approval to the exclusion of other products or veudors that may also be suitable.