<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic tissues of hybrid firs (<Emphasis Type="Italic">Abies</Emphasis> spp.) and regeneration of transgenic emblings

A genetic transformation system has been developed for selected embryogenic cell lines of hybrids Abies alba × A. cephalonica (cell lines AC2, AC78) and Abies alba × A. numidica (cell line AN72) using Agrobacterium tumefaciens. The cell lines were derived from immature or mature zygotic embryos on DCR medium containing BA (1 mg l⁻¹). The T-DNA of plant transformation vector contained the β-glucuronidase reporter gene under the control of double dCaMV 35S promoter and the neomycin phosphotransferase selection marker gene driven by the nos promoter. The regeneration of putative transformed tissues started approximately 1 week after transfer to the selection medium containing 10 mg geneticin l⁻¹. GUS activity was detected in most of the geneticin-resistant sub-lines AN72, AC2 and AC78, and the transgenic nature of embryogenic cell lines was confirmed by PCR approach. Plantlet regeneration from PCR-positive embryogenic tissues has been obtained as well. The presence of both gus and nptII genes was confirmed in 11 out of 36 analysed emblings.     

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

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

<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.     

The role of BAP in somatic embryogenesis induction from seed explants of <Emphasis Type="Italic">Arachis</Emphasis> species from Sections <Emphasis Type="Italic">Erectoides</Emphasis> and <Emphasis Type="Italic">Procumbentes</Emphasis>

Somatic embryogenesis was induced from seed explants of Arachis archeri, A. porphyrocalix (Section Erectoides) and A. appressipila (Section Procumbentes) in response to 6-benzylaminopurine (BAP). Embryo axes first developed into single shoots in response to 4.4 μM BAP. Friable embryogenic calluses were produced from the hypocotyl region of these explants in response to different BAP concentrations. Embryonic leaflets also gave rise to friable calluses, but somatic embryos were only observed in explants of A. archeri and A. appressipila. Histological analyses revealed the presence of heart-shaped, torpedo and cotyledonary stages embryos, both as isolated and fused structures. A low frequency of embryo-to-plant conversion was achieved by inducing shoot development on medium solidified with 0.5% phytagel and supplemented with 1.5% or 3% sucrose. Rooting was induced on MS supplemented with indole-3-acetic acid (IAA).     

Plant regeneration in Stone pine (<Emphasis Type="Italic">Pinus pinea</Emphasis> L.) by somatic embryogenesis

Regeneration of plants by somatic embryogenesis (SE) was achieved in Stone pine (Pinus pinea), one of the most characteristic tree species of the Mediterranean ecosystem. The initial explants were megagametophytes containing zygotic embryos from five selected half-sib families collected at different dates over 2 consecutive years. Rates of extrusion and initiation of SE differed in both years. However, qualitative patterns were very similar: for most families, the responsive developmental window was from late cleavage polyembryony to early cotyledonary stage. The highest overall mean frequencies of extrusion and SE initiation (7 and 0.9%, respectively, for the five families and the eight 2006 collections) were obtained on a modified Litvay’s medium with 9 μM 2,4-D and 4.5 μM BAP, supplemented with L-glutamine and casein hydrolysate. Families showed large differences in frequencies of SE initiation from year to year. Only seven embryogenic lines were induced in 2005, representing three of the five families tested, whereas 34 lines from all the families were obtained in 2006. Proliferation of embryonal masses (EM) was significantly improved when they were subcultured after dispersing in liquid medium and collected on filter paper disks, instead of being subcultured as small clumps. This effect showed a significant interaction with genotype. Several preconditioning treatments and culture media combinations were tested for embryo development and maturation. The high proliferation rate of EM hampered somatic embryo development. However, up to 42 mature embryos from different lines of three of the five families were obtained, 23 of them germinated and seven converted into somatic seedlings.     

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.     

Comparative analysis of transgenic tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb.) plants obtained by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and particle bombardment

Agrobacterium-mediated transformation and particle bombardment are the two most widely used methods for genetically modifying grasses. Here, these two systems are compared for transformation efficiency, transgene integration and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The bar gene was used as a selectable marker and selection during tissue culture was performed using 2 mg/l bialaphos in both callus induction and regeneration media. Average transformation efficiency across the four callus lines used in the experiments was 10.5% for Agrobacterium-mediated transformation and 11.5% for particle bombardment. Similar transgene integration patterns and co-integration frequencies of bar and uidA were observed in both gene transfer systems. However, while GUS activity was detected in leaves of 53% of the Agrobacterium transformed lines, only 20% of the bombarded lines showed GUS activity. Thus, Agrobacterium-mediated transformation appears to be the preferred method for producing transgenic tall fescue plants.     

Osmotic sucrose enhancement of single-cell embryogenesis and transformation efficiency in <Emphasis Type="Italic">Oncidium</Emphasis>

Traditional breeding processes to genetically modify the long reproductive cycle and slow seed maturation of orchids have limits. We developed a more efficient protocol using particle bombardment to produce transgenic plants of Oncidium Sharry Baby OM8 (Orchidaceae). Pretreating protocorm-like bodies (PLBs) with 0.5 M sucrose for 2 h increased single-cell embryogenesis 3- to 4-fold; however, shoot formation was suppressed. In addition, new PLBs were regenerated from the entire sucrose-pretreated PLBs, whereas in untreated PLBs, this occurred only from the bases. Pretreated PLBs were bombarded with pSPFLP containing genes encoding a sweet pepper ferredoxin-like protein (pflp), hygromycin phosphotransferase (hpt) and -glucuronidase (GUS) driven by the cauliflower mosaic virus 35S promoter. Pretreated PLBs showed a 14.8-fold increase in GUS expression over the untreated PLBs 40days after bombardment. The presence of pflp and hpt transgenes in the 40 putatively stably transformed lines that produced 113 clones was confirmed by PCR analysis. Six lines (eight clones) were positive for both pflp and hpt transgenes. In addition, clones derived from these lines were either all positive or all negative for the two transgenes, which suggests homogeneity in pretreated PLBs with more single-cell embryogenesis. Thus, sucrose pretreatment enhanced the regeneration of PLBs, single-cell embryogenesis and efficiency of transformation.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of poinsettia, <Emphasis Type="Italic">Euphorbia pulcherrima</Emphasis>, with virus-derived hairpin RNA constructs confers resistance to <Emphasis Type="Italic">Poinsettia mosaic virus</Emphasis>

Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2-3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of gypsophila (<Emphasis Type="Italic">Gypsophila paniculata</Emphasis> L.)

As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium inoculation of highly regenerative stem segments. The transformation procedure employs stem explants derived from GA₃-pretreated mother plants and a two-step selection scheme. The GA₃ treatment was crucial for obtaining high gene-transfer frequencies (75–90% GUS-expressing explants out of total inoculated explants), as shown using three different gypsophila varieties. An overall transformation efficiency of five GUS-expressing shoots per 100 stem explants was demonstrated for cv. Arbel. The applicability of the transformation system to gypsophila was further reinforced by the generation of transgenic plants expressing Agrobacterium rhizogenes rolC driven by a CaMV 35S promoter. Transgenic gypsophila plantlets exhibited extensive rooting and branching, traits that could be beneficial to the ornamental industry.     

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

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

<Emphasis Type="Italic">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.     

Somatic embryogenesis in loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis> L.): improving culture initiation with abscisic acid and silver nitrate

Loblolly pine (Pinus taeda L.) culture initiation was improved by the addition of abscisic acid (ABA) (3.7 µM), silver nitrate (20 µM), and guanosine 3,5-cyclic monophosphate, 8-bromo-, sodium salt (10 µM) to the medium and by raising cytokinin levels in the presence of 50 mg/l activated carbon (AC). Basal medium contained modified 1/2-P6 salts, 50 mg/l AC, Cu and Zn added to compensate for adsorption by AC, 1.5% maltose, 2% myo-inositol, 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l -naphthaleneacetic acid (NAA), 0.55 mg/l 6-benzylaminopurine (BA), 0.53 mg/l kinetin, and 2 g/l Gelrite. Across 32 open-pollinated families initiation ranged from 0 to 53.4%, with an average of 17.9%. Further optimization of cytokinins to 0.63 mg/l BA and 0.61 mg/l kinetin along with the removal of ABA maintained initiation at 18.2% across 19 families. Survival of 2001 new initiations was tracked for 4–6 months. Survival averaged 28.8%. A test of 68 new initiations tracked closely for 4 months demonstrated that at least 80% of the cultures lost did not grow after transfer to the multiplication media, suggesting that many new initiations abort during the initiation process.Abbreviations ABA Abscisic acid - AC Activated carbon - BA 6-Benzylaminopurine - 8-Br-cGMP Guanosine 3,5-cyclic monophosphate, 8-bromo-, sodium salt - NAA -Naphthaleneacetic acid Communicated by G.C. Phillips     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

Studies on genetic transformation of olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) somatic embryos: I. Evaluation of different aminoglycoside antibiotics for <Emphasis Type="Italic">nptII</Emphasis> selection; II. Transient transformation via particle bombardment

As a first step to the establishment of a genetic transformation protocol for olive somatic embryos obtained from the seeds of cv. ‘Picual’, the efficiencies of different aminoglycoside antibiotics as selective agents to be used with the nptII marker gene, and the particle bombardment technique for transient transformation have been evaluated. Among the three antibiotics tested, paromomycin and kanamycin showed a similar inhibitory effect and, at 200 mg l⁻¹, both of them impaired callus growth after 8 weeks of culture. However, when isolated embryos were cultured in the presence of these antibiotics, a 20% of the embryos still remained viable at 400 mg l⁻¹. Neomycin was discarded as a selective agent since it showed only a moderate toxic effect. Contrary to solid medium, when olive callus was cultured in liquid medium supplemented with different paromomycin concentrations for 3 weeks, the callus growth was impaired at the lowest antibiotic concentration, 3 mg l⁻¹. Best conditions for transient transformation of olive callus using PDS-1000/He system were a 6 cm target distance and a 900 psi bombardment pressure. pCGU∆1 plasmid, containing the gus gene under the control of sunflower ubiquitin promoter yielded a significantly higher number of gus expression areas per bombarded explant than pGUSINT or pJGUS5 plasmids, where the gus gene is driven by CaMV35S promoter or CaMV35S with enhancer, respectively. Almost 45% of bombarded explants showed gus expression 12 weeks after bombardment.