Analysis of a <Emphasis Type="Italic">LEA</Emphasis> gene promoter via <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the desiccation tolerant plant <Emphasis Type="Italic">Lindernia brevidens</Emphasis>

An Agrobacterium tumefaciens-based transformation procedure was developed for the desiccation tolerant species Lindernia brevidens. Leaf explants were infected with A. tumefaciens strain GV3101 harbouring a binary vector that carried the hygromycin resistance gene and an eGFP reporter gene under the control of a native dehydration responsive LEA promoter (Lb2745pro). PCR analysis of the selected hygromycin-resistant plants revealed that the transformation rates were high (14/14) and seeds were obtained from 13/14 of the transgenic lines. A combination of RNA gel blot and microscopic analyses demonstrated that eGFP expression was induced upon dehydration and ABA treatment. Comparison with existing procedures used to transform the well studied resurrection plant and close relative, Craterostigma plantagineum, revealed that the transformation process is both rapid and leads to the production of viable seed thus making L. brevidens a candidate species for functional genomics approaches to determine the genetic basis of desiccation tolerance.     

Use of fused <Emphasis Type="Italic">gfp</Emphasis> and <Emphasis Type="Italic">gus</Emphasis> reporters for the recovery of transformed <Emphasis Type="Italic">Medicago truncatula</Emphasis> somatic embryos without selective pressure

We developed an alternative methodology for in vitro selection of transgenic Medicago truncatula cv. Jemalong plants using a bifunctional construct in which the coding sequences for the green fluorescent protein (GFP) and the β-glucuronidase protein (GUS) are fused. An Agrobacterium-mediated transformation protocol was used followed by regeneration via somatic embryogenesis in the dark, to avoid the synthesis and the consequent autofluorescence of chlorophyll. This method is a clear advantage over antibiotic and herbicide selection in which survival of non-transformed tissue is commonly reported, with the reassurance that all the somatic embryos selected as GFP positive are transformed. This was subsequently corroborated by the detection of GUS activity in leaves, stems and roots of the regenerated plants. Without antibiotic selection, and performing the embryo induction in the dark, it was possible to attest the advantage of using GFP as an in vivo detectable reporter for early embryo selection. The fusion with the GUS coding sequence provided additional evidence for the transformation of the previously selected embryos.     

Efficient <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of oilseed mustard [<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern.] using leaf piece explants

Leaf piece explants of five Brassica juncea (L.) Czern. cultivars were transformed with an Agrobacterium tumefaciens strain EHA105 harboring the plasmid pCAMBIA1301, which contains the β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes under the control of cauliflower mosaic virus 35S (CaMV35S) promoter. Transgenic plants were regenerated on Murashige and Skoog (MS) medium fortified with 8.87 μM 6-benzylaminopurine, 0.22 μM 2,4-dichlorophenoxyacetic acid, and 20 μM silver nitrate in the presence of 30 mg/l hygromycin. When co-culture took place in the presence of 100 μM acetosyringone, the efficiency of stable transformation was found to be approximately 19% in the T ₀ generation, with the transgenic plants and their progeny showing constitutive GUS expression in different plant organs. Southern blot hybridization of uidA and hpt genes confirmed transgene integration within the genome of transformed plants of each cultivar. Inheritance of hpt gene for single copy T-DNA inserts showed a 3:1 pattern of Mendelian segregation in progeny plants through germination of T ₁ seeds on MS medium containing 30 mg/l hygromycin. The protocol described here reports superior transformation efficiency over previously published protocols and should contribute to enhanced biotechnology applications in B. juncea.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Melastoma malabathricum</Emphasis> and <Emphasis Type="Italic">Tibouchina semidecandra</Emphasis> with sense and antisense dihydroflavonol-4-reductase (DFR) genes

Genetic engineering of a wide variety of plant species has led to the improvement of plant traits. In this study, the genetic transformation of two potentially important flowering ornamentals, Melastoma malabathricum and Tibouchina semidecandra, with sense and antisense dihydroflavonol-4-reductase (DFR) genes using the Agrobacterium-mediated method was carried out. Plasmids pBETD10 and pBETD11, each harbouring the DFR gene at different orientations (sense and antisense) and selectable marker nptII for kanamycin resistance, were used to transform M. malabathricum and T. semidecandra under the optimized transformation protocol. Putative transformants were selected in the presence of kanamycin with their respective optimized concentration. The results indicated that approximately 4.0% of shoots and 6.7% of nodes for M. malabathricum regenerated after transforming with pBETD10, whereas only 3.7% (shoots) and 5.3% (nodes) regenerated with pBETD11 transformation. For the selection of T. semidecandra, 5.3% of shoots and 9.3% of nodes regenerated with pBETD10 transformation, while only 4.7% (shoots) and 8.3% (nodes) regenerated after being transformed with pBETD11. The presence and integration of the sense and antisense DFR genes into the genome of M. malabathricum and T. semidecandra were verified by polymerase chain reaction (PCR) and nucleotide sequence alignment and confirmed by southern analysis. The regenerated putative transformants were acclimatized to glasshouse conditions. Approximately 31.0% pBETD10-transformed and 23.1% pBETD11-transformed M. malabathricum survived in the glasshouse, whereas 69.4% pBETD10-transformed and 57.4% pBETD11-transformed T. semidecandra survived. The colour changes caused by transformation were observed at the budding stage of putative T. semidecandra transformants where greenish buds were produced by both T. semidecandra harbouring the sense and antisense DFR transgenes. Besides that, the production of four-petal flowers also indicated another morphological difference of putative T. semidecandra transformants from the wild type plants which produce five-petal flowers.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

The transformation of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.): applicable methods of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated gene transfer

Three methods of transformation of pea (Pisum sativum ssp. sativum L. var. medullare) were tested. The most efficient Agrobacterium tumefaciens-mediated T-DNA transfer was obtained using embryonic segments from mature pea seeds as initial explants. The transformation procedure was based on the transfer of the T-DNA region with the reporter gene uidA and selection gene bar. The expression of β-glucuronidase (GUS) in the regenerated shoots was tested using the histochemical method and the shoots were selected on a medium containing phosphinothricin (PPT). The shoots of putative transformants were rooted and transferred to non-sterile conditions. Transient expression of the uidA gene in the tissues after co-cultivation and in the course of short-term shoot cultivation (confirmed by histochemical analysis of GUS and by RT-PCR of mRNA) was achieved; however, we have not yet succeeded in proving stable incorporation of the transgene in the analysed plants.     

Development of genotype-independent regeneration system for transformation of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> ssp. <Emphasis Type="Italic">indica</Emphasis>)

Rice (Oryza sativa ssp. indica) is an important economic crop in many countries. Although a variety of conventional methods have been developed to improve this plant, manipulation by genetic engineering is still complicated. We have established a system of multiple shoot regeneration from rice shoot apical meristem. By use of MS medium containing 4 mg L⁻¹ thidiazuron (TDZ) multiple shoots were successfully developed directly from the meristem without an intervening callus stage. All rice cultivars tested responded well on the medium and regenerated to plantlets that were readily transferred to soil within 5–8 weeks. The tissue culture system was suitable for Agrobacterium-mediated transformation and different factors affecting transformation efficiency were investigated. Agrobacterium strain EHA105 containing the plasmid pCAMBIA1301 was used. The lowest concentration of hygromycin B in combined with either 250 mg L⁻¹ carbenicillin or 250 mg L⁻¹ cefotaxime to kill the rice shoot apical meristem was 50 mg L⁻¹ and carbenicillin was more effective than cefotaxime. Two-hundred micromolar acetosyringone had no effect on the efficiency of transient expression. Sonication of rice shoot apical meristem for 10 s during bacterial immersion increased transient GUS expression in three-day co-cultivated seedlings. The gus gene was found to be integrated into the genome of the T₀ transformant plantlets.     

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     

A large-scale<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A large-scale transformation procedure handling an adequate number of stable transformants with highly efficient positive-negative selection is a necessary prerequisite to successful gene targeting by homologous recombination, as the integration of a transgene by somatic homologous recombination in higher plants has been reported to be 10^-3 to 10^-5 compared with random integration by non-homologous end joining. We established an efficient and large-scale Agrobacterium-mediated rice transformation protocol that generated around 10³ stable transformants routinely from 150 seeds and a strong positive-negative selection procedure that resulted in survivors at 10^-2 using the gene for diphtheria toxin A fragment as a negative marker. The established transformation procedure provides a basis for efficient gene targeting in rice.Abbreviations AS: Acetosyringone - 5-FU: 5-Fluorouracil - FW: Fresh weight - GT: Gene targeting - HR: Homologous recombination - NHEJ: Non-homologous end joining Communicated by H. Ebinuma     

Optimization of conditions for transient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated gene expression assays in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The β-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85–90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.     

Genetic transformation of <Emphasis Type="Italic">Torenia fournieri</Emphasis> using the PMI/mannose selection system

Transgenic torenia plants were obtained using the selectable marker gene phosphomannose isomerase (manA), which encodes the enzyme phosphomannose isomerase (PMI) to enable selection of transformed cells on media containing mannose. We found that shoot organogenesis in torenia leaf explants was effectively suppressed on medium supplemented with mannose, which indicated that torenia cells had little or no PMI activity and could not utilize mannose as a carbon source. Leaf pieces from in vitro-germinated plants were inoculated with Agrobacterium tumefaciens EHA105 containing the binary vector pKPJ with both hpt and ManA genes, and subsequently selected on shoot induction (SI) medium (half strength MS basal + 4.4 μM BA + 0.5 μM NAA) supplemented with 20 g l⁻¹ mannose and 5 g l⁻¹ sucrose as carbon sources. Transformed plants were confirmed by PCR and Southern blot. The transgene expression was evaluated using Northern blot and the chlorophenol red assay. The transformation efficiency ranged from 7% to 10%, which is 1–3% higher than that obtained by selection with hygromycin. This system provides an efficient manner for selecting transgenic flower plants without using antibiotics or herbicides.     

Development of a shoot regeneration protocol for genetic transformation in <Emphasis Type="Italic">Pelargonium zonale</Emphasis> and <Emphasis Type="Italic">Pelargonium peltatum</Emphasis> hybrids

The attempts of this investigation were to develop a system for plant regeneration from explants of adult plants and its use for genetic transformation of important commercial Pelargonium zonale hybrid and P. peltatum hybrid cultivars. To this aim, leaf blade and petiole explants of eight cultivars were cultured on modified MS (Murashige and Skoog, 1962) medium with two concentrations of TDZ, BA, and zeatin (5 and 20 M). Petiole explants showed a higher regeneration response than leaf blade explants and TDZ was the most effective cytokinin. Petioles of 16 cultivars were incubated on medium containing 5, 10, 15, and 20 M TDZ, respectively, in order to identify the most effective TDZ concentration. For the majority of genotypes 10 M TDZ resulted in the best regeneration response. Cefotaxim at 500 mg l ^–1 had no effect on shoot regeneration and did not show interaction with glufosinate. For selection of transgenic cells, a concentration of 2.5 M glufosinate was shown to be appropriate. LBA4404 and EHA101 Agrobacterium strains carrying pIBGUS vector with pat gene as selectable marker gene and GUS gene as reporter gene were compared in transformation studies. With regard to GUS expression in petiole explants 16 days after coculture, better results were obtained with EHA 101 than with LBA 4404.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

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.     

Improved genetic transformation protocol for cork oak (<Emphasis Type="Italic">Quercus suber</Emphasis> L.)

An optimized protocol for Agrobacterium tumefaciens-mediated transformation of mature Quercus suber L. embryogenic masses is reported. In this work several variables were tested. Plant genotype, explant type and time elapsed between the last subculture and inoculation, i.e. the explant preculture period, were found to be very important. Interaction between inoculum density and cocultivation period influenced the transformation efficiency as well. A transformation efficiency (i.e. percentage of the inoculated explants that yielded independent transgenic embryogenic lines) of up to 43% was obtained, greatly improving the previously described method for plant transformation of adult-selected cork oak. It was also shown that this protocol could be applied to various genotypes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Echinacea</Emphasis> biotechnology: Challenges and opportunities

Echinacea, better known as purple coneflower, has received a global attention because of its increasing medicinal value. There is enormous potential for the discovery of new medicinal compounds in this species and an immediate need for techniques to facilitate the production of high quality, chemically consistent plant material for drug development and clinical trials. In vitro tissue culture of Echinacea can play a vital role in the development of novel germplasm, rapid multiplication, and genetic modifications for an enhanced phytochemical production. Recent establishment of liquid culture techniques, large-scale bioreactors, and Agrobacterium-mediated transformation are changing the production parameters of the Echinacea species. This review provides an overview of the recent developments in in vitro technologies and challenges that remain in the Echinacea biotechnology.     

<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.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies.     

Expression of the glutathione <Emphasis Type="Italic">S</Emphasis>-transferase gene (NT107) in transgenic <Emphasis Type="Italic">Dianthus superbus</Emphasis>

Using an Agrobacterium-mediated transformation method based on wounding cultured immature seeds with carborundum (600 mesh) in liquid, auxin-regulated tobacco glutathione S -transferase (GST) (NT107) constructs were used to transform Dianthus superbusL. A 663 bp DNA band was found in the transgenic plant genome by PCR analysis using NT107-1 and NT107-2 primers, and a Southern blot analysis showed that the DIG-labelled GST gene was hybridized to the expected amplified genomic DNA fragment from transgenic D. superbus. An overexpression of NT107 led to a twofold increase in GST-specific activity compared to the non-transgenic control plants, and the GST overexpression plants showed an enhanced acclimatization in the soil. To investigate whether an increased expression of GST could affect the resistance of photosynthesis to environmental stress, these plants were subjected to drought and various light intensities from 100 to 3000 mol m^–2s^–1. Copper accumulation and the translocation rate were also analysed in the transgenic lines, and the GST overexpression plants were found to synthesize phytochelatin (PC), which functions by sequestering and detoxifying excess copper ions.These two authors contributed equally to this work