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.     

Production of herbicide-resistant transgenic sweet potato plants through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> method

Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding β-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l⁻¹ PPT, respectively. Soil-grown plants were obtained 28–36 weeks after Agrobacterium-mediated transformation. Genetic transformation of the regenerated plants growing under selection was demonstrated by PCR, and Southern blot analysis revealed that one to three copies of the transgene were integrated into the plant genome of each transgenic plant. Expression of the bar gene in transgenic plants was confirmed by RT-PCR and application of herbicide. Transgenic plants sprayed with Basta containing 900 mg l⁻¹ of glufosinate ammonium remained green and healthy. The transformation frequency was 2.8% determined by herbicide application which was high when compared to our previous biolistic method. In addition, possible problems with multiple copies of transgene were also discussed. We therefore report here a successful and reliable Agrobacterium-mediated transformation of the bar gene conferring herbicide-resistance and this method may be useful for routine transformation and has the potential to develop new varieties of sweet potato with several important genes for value-added traits such as enhanced tolerance to the herbicide Basta.     

<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.     

Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling

To improve the transformation efficiency of wheat (Triticum aestivum L.) mediated by Agrobacterium tumefaciens, we explored the possibility of employing the basal portion of wheat seedling (shoot apical meristem) as the explants. Three genotypes of wheat were transformed by A. tumefaciens carrying β-1, 3-glucanase gene. After vernalization, the seeds to be transformed were germinated. When these seedlings grew up to 2∼5 cm, their coleoptile and half of the cotyledon were cut out, and the basal portions were infected by A. tumefaciens. A total 27 T₀ transgenic plants were obtained, and the average transformation efficiency was as high as 9.82%. Evident segregation occurred in some of the T₁ plants, as was indicated by PCR and Southern blotting analysis. Investigation of the T₂ plants revealed that some transformed plants had higher resistance to powdery mildew than the controls. Northern blotting revealed that β-1, 3-glucanase gene was normally expressed in the T₂ plants, which showed an increased resistance to powdery mildew. The results above indicate that the exogenous gene has been successfully integrated into the genome of wheat, transmitted and expressed in the transgenic progeny. From all the results above, it can be concluded that Agrobacterium inoculum to the basal portion of wheat seedling is a highly efficient and dependable transformation method. It can be developed into a practicable method for transfer of target gene into wheat.Tong-Jin Zhao and Shuang-Yi Zhao contributed equally to this paper.     

Expression of a chimeric GUS gene construct as a tool to study nodule morphogenesis in chicory leaves

Leaves from micropropagated chicory plantlets were cultivated in vitro to regenerate organogenic nodules. The nodules were transformed with a 2.3 kb fragment of an apple calmodulin promoter region fused to the coding sequence of uidA reporter gene. Histochemical detection of β-glucuronidase expression in transgenic regenerants showed that vessel-associated cells were strongly stained. Previous histological investigations have shown that the differentiation of vascular elements is essential to nodule and bud-derived-nodule development. Therefore, β-glucuronidase activity was tested in a single transgenic chicory line during nodule morphogenesis and bud regeneration. The vascular connections between leaves and nodules, then between nodules and buds were stained blue, indicating an alternative system for determining the histological origin of nodules and adventitious buds from chicory leaf explants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Promoter of the rolC gene of Agrobacterium rhizogenes can be strongly regulated in glandular cell of transgenic tobacco

A 577-bp promoter segment of Agrobacterium rhizogenes rolC, previously known as the phloem-specific gene expression promoter, was fused to the 5′ end of a reporter gene, β-glucuronidase (GUS), uidA. This rolC-promoter-driven expression of the GUS gene was found to be significantly strong in glandular cells in transgenic tobacco plants. Analysis of this segment of the promoter sequence revealed a myb response element.     

Generation of large numbers of transgenic Kentucky bluegrass (Poa pratensis L.) plants following biolistic gene transfer

A very efficient transformation system, using biolistic bombardment, has been developed for the production of transgenic plants of Kentucky bluegrass (Poa pratensis L.). Embryogenic calli, initiated from immature embryos, were transformed either with pAct1IHPT-4 containing the hygromycin phosphotransferase (hpt) gene or with pDM803 containing the phosphinothricin acetyltransferase (bar) gene and the β-glucuronidase (uidA) gene. In total 119 independent transgenic plants were recovered from 153 hygromycin-resistant lines. Bialaphos selection yielded a total of 99 bialaphos-resistant lines and from these 34 independent transgenic plants were recovered. Southern blot analysis demonstrated the independent nature of the transgenic plants and also revealed a complex transgene integration pattern with multiple insertions. The first two author contributed equally to this work     

Plant endogenous β-glucuronidase activity: how to avoid interference with the use of theE. coli β-glucuronidase as a reporter gene in transgenic plants

We have detected a plant β-glucuronidase activity, present in several tissues and organs of plant species belonging to different families. The fluorimetric β-glucuronidase assay was used to partially characterize this activity in post-ribosomal supernatants of tobacco leaves. The tobacco activity is very stable at low temperatures, but quickly inactivated above 45°C. It is relatively resistant to proteases and insensitive to-SH group reagents and to ionic conditions. It does not require, nor is it inhibited by, divalent cations. Although these properties are shared by theEscherichia coli β-glucuronidase, the two activities can be distinguished by: (i) their different sensitivity to the specific inhibitor saccharic acid-1,4-lactone; (ii) their different thermal stability (iii) their different pH optima (5.0 for the plant activity and close to neutral for the bacterial enzyme). Therefore, under appropriate experimental conditions, it should be possible to assay theE. coli β-glucuronidase in transgenic plants without interference from the endogenous plant activity.     

Genetic transformation of a hepatoprotective plant, <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Phyllanthus amarus Schum & Thonn. is a source of various pharmacologically active compounds such as phyllanthin, hypophyllanthin, gallic acid, catechin, and nirurin, a flavone glycoside. A genetic transformation method using Agrobacterium tumefaciens was developed for this plant species for the first time. Shoot tips of full grown plants were used as explants for Agrobacterium-mediated transformation. Transgenic plants were obtained by co-cultivation of shoot tips explants and A. tumefaciens strain LBA4404 containing the pCAMBIA 2301 plasmid harboring neomycin phosphotransferase II (NPT II) and β-glucuronidase encoding (GUS) genes in the T-DNA region in the presence of 200 μM acetosyringone. Integration of the NPT II gene into the genome of transgenic plants was verified by PCR and Southern blot analyses. Expression of the NPT II gene was confirmed by RT-PCR analysis. An average of 25 explants was used, out of which an average of 19 explants produced kanamycin-resistant shoots, which rooted to produce 13 complete transgenic plants.     

<Emphasis Type="Italic">WUS</Emphasis> and <Emphasis Type="Italic">STM</Emphasis>-based reporter genes for studying meristem development in poplar

We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5′ flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50–60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common in other organs, including in leaf veins (40 and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems as explants showed that expression was detectable prior to visible shoot development, starting 3–15 days after explants were placed onto callus inducing medium. A minority of WUS and STM events also showed expression in the cambium, phloem, or xylem of regenerated, greenhouse grown plants undergoing secondary growth. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Both paralogs of poplar STM were down-regulated threefold to sixfold during early callus initiation. We identified 15–35 copies of cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the events recovered may be useful for studying the process of primary and secondary meristem development, including treatments intended to stimulate meristem development to promote clonal propagation and genetic transformation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transgenic Plants of Colonial Bentgrass from Embryogenic Callus via Agrobacterium-mediated Transformation

Colonial bentgrass (Agrostis tenuis Sibth. Fl. Oxen.) is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable and repeatable approach in transforming the grass using Agrobacterium (strain LBA4404), in which -glucuronidase (gus) gene was used as a reporter and hygromycin phosphotransferase (hpt) gene as a selectable marker. This vector was effective in transforming 7-week-old calluses derived from mature seeds cultured on MS medium supplemented with 2,4-D. A two-step solid medium selection with increasing hygromycin concentration (from 50 to 70 mg l^–1) was used to obtain resistant calluses. Hundreds of transgenic plants have been produced from several independent transformed calluses. The presence of functional -glucuronidase (GUS) was detected in hygromycin-resistant calluses, young leaves and roots of transgenic plants. The transgenic plants collected from greenhouse showed strong resistance to 50 mg l^–1 hygromycin solution. Four putative transgenic plants and one control plant were randomly chosen and analyzed by Southern blot analysis. Bands corresponding to the hpt gene were clearly shown in transgenic plants.     

<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.     

Comparison of different techniques for gene transfer into mature and immature tobacco pollen

The particle gun, cocultivation withAgrobacterium tumefaciens, and imbibition in DNA solutions were compared as methods to transfer DNA into mature and immature pollen ofNicotiana tabacum. Bombardment of mature pollen with the β-glucuronidase gene cloned behind the pollen-specific PA2 promoter of the chalcone isomerase gene ofPetunia hybrida resulted in the expression of the β-glucuronidase gene in 0.025% of the pollen grains. Bombardment of younger stages followed byin vitro maturation also resulted in the formation of mature pollen that expressed β-glucuronidase, although at a lower frequency. Cocultivation of pollen duringin vitro maturation orin vitro germination withAgrobacterium tumefaciens did not yeild β-glucuronidase-expressing pollen. In these cases, an intron-containing β-glucuronidase gene was used which effectively prevented β-glucuronidase expression in the bacteria. Imbibition of mature, dry pollen in various DNA solutions of the same constructs also did not lead to the formation of β-glucuronidase expressing pollen.     

Genetic transformation of golden pothos (<Emphasis Type="Italic">Epipremnum aureum</Emphasis>) mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

To establish a procedure for Agrobacterium tumefaciens-mediated transformation of golden pothos (Epipremnum aureum) plants, the effects of selection antibiotics and the preculture period of stem explants before A. tumefaciens infection were examined. Explants were co-cultivated with A. tumefaciens EHA105, harboring the plasmid pGWB2/cGUS, on a somatic embryo-inducing medium supplemented with acetosyringone. Resulting transgenic somatic embryos were screened on an antibiotic selection medium, and the transgenic pothos plants were regenerated on a germination medium. Hygromycin was the optimum selection antibiotic tested. The preculture period significantly affected the transformation efficiency, with explants precultured for one-day showing the best efficiency (5–30%). Both transformed hygromycin-resistant embryos and regenerated plants showed β-glucuronidase activity. Southern blot analysis confirmed transgene integration into the pothos genome. This reproducible transformation system for golden pothos may enable the molecular breeding of this very common indoor plant.     

Efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation using embryogenic suspension cultures in sweetpotato, <Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.

Efficient Agrobacterium tumefaciens-mediated transformation was achieved using embryogenic suspension cultures of sweetpotato (Ipomoea batatas (L.) Lam.) cv. Lizixiang. Cell aggregates from embryogenic suspension cultures were cocultivated with the A. tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 with gusA and hygromycin phosphotransferase II gene (hpt II) genes. Selection culture was conducted using 25 mg l⁻¹ hygromycin. A total of 2,218 plants were regenerated from the inoculated 1,776 cell aggregates via somatic embryogenesis. β-glucuronidase (GUS) assay and PCR, dot blot and Southern blot analyses of the regenerated plants randomly sampled showed that 90.37% of the regenerated plants were transgenic plants. The number of integrated T-DNA copies varied from 1 to 4. Transgenic plants, when transferred to soil in a greenhouse and a field, showed 100% survival. No morphological variations were observed in the ex vitro transgenic plants. These results exceed all transformation experiments reported so far in the literature in quantity of independent events per transformation experiment in sweetpotato.     

An improved procedure for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of trifoliate orange (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.) via indirect organogenesis

Highly efficient Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata (L.) Raf.) was achieved via indirect shoot organogenesis. Stable transformants were obtained from epicotyl segments infected with Agrobacterium strain EHA 105 harboring the binary vector pBI121, which contained the neomycin phosphotransferase gene (NPTII) as a selectable marker and the β-glucuronidase (GUS) gene as a reporter. The effects of regeneration and selection conditions on the transformation efficiency of P. trifoliata (L.) Raf. have been investigated. A 7-d cocultivation on a medium with 8.86 μM 6-benzylaminopurine (BA)+1.43 μM indole-3-acetic acid (IAA) was used to improve callus formation from epicotyl segments after transformation. A two-step selection strategy was developed to select kanamycin-resistant calluses and to improve rooting of transgenic shoots. Transgenic shoots were multiplied on shoot induction medium with 1.11 μM BA + 5.71 μM IAA. Using the optimized transformation procedure, transformation efficiency and rooting frequency reached 417% and 96%, respectively. Furthermore, the number of regenerated escape shoots was dramatically reduced. Stable integration of the transgenes into the genome of transgenic citrus plants was confirmed by GUS histochemical assay, PCR, and Southern blot analysis.     

Transformation of maize by 2,4-dihydroxy-7-methoxy-2H-1,4-benzo- xazin-3(4H)-one resistant Agrobacterium strains

One of the important factors responsible for recalcitrance of maize tissue towards Agrobacterium-mediated transformation is the presence of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), an inhibitory metabolite found in maize cells. DIMBOA-resistant strains of Agrobacterium tumefaciens were used to transfer genes coding for GUS (-glucuronidase) and NPTII (neomycine phosphotransferase II) in maize shoot apical meristems derived from 20 day-old seedlings and immature embryos. GUS expression was higher (21–34%) in the apical meristem and was dependent on the type of infecting strain and explant-age. The PCR analysis of selected tissues confirmed the presence of GUS gene in the transformed cells.     

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.