Susceptibility of embryogenic and organogenic tissues of maritime pine (Pinus pinaster) to antibiotics used in Agrobacterium-mediated genetic transformation

The effects of antibiotics commonly used in Agrobacterium-mediated transformation were studied on Pinus pinaster tissues. Embryogenic tissue growth from three embryogenic lines and adventitious bud induction from cotyledons from three open-pollinated seed families were analysed. Cefotaxizme, carbenicillin and timentin commonly used for Agrobacterium elimination, at concentrations of 200–400 mg l ⁻¹ did not inhibit the embryogenic tissue growth on filter paper nor as clumps. Adventitious bud induction and bud number were significantly reduced for one of the tested families when using 400 mg l⁻¹ cefotaxime or timentin. The selection agent kanamycin significantly inhibited growth of embryogenic tissue on filter paper in all the embryogenic lines␣and concentrations tested (20–50 mg l⁻¹). Kanamycin also inhibited growth of embryogenic clumps after two subcultures at 5–50 mg l⁻¹. In␣cotyledons, kanamycin inhibited adventitious bud␣formation in the three seed families used, regardless of the concentrations tested (5–25 mg l⁻¹). There was a significant effect of the seed family on the bud induction and the number of adventitious buds produced. From the results obtained, we propose the use of timentin to eliminate Agrobacterium in transformation experiments, at concentrations of 400 mg l⁻¹ for embryogenic tissues and of 300 mg l⁻¹ for cotyledons. For selection of transformed tissues carrying the kanamycin resistance gene, kanamycin should be used at 20 mg l⁻¹ for embryogenic tissues on filter paper, at 5 mg l⁻¹ when clumps are in direct contact with the selection medium, and bellow 5 mg l⁻¹ for adventitious bud induction.     

Douglas fir embryogenic tissue initiation

Somatic embryogenesis (SE) is expected to play an important role in the future of US forests by providing increased productivity, sustainability, and uniformity. For broad scale implementation to occur, SE technology must work with a variety of genetically diverse trees. Douglas fir (Pseudotsuga menziesii (Mirb) Franco) is the dominant tree in the Pacific Northwest and has great economic and recreational value. We have developed a highly effective medium for initiation of embryogenic tissue of Douglas fir that contains ABA, biotin, brassinolide, folic acid, MES, pyruvic acid and can be used as a gelled medium or in a gelled-liquid medium overlay system. When tested with many high-value crosses over 2 years, initiation tests averaged initiation in the range of 40–57%. Additionally, a time- and labor-saving tetrazolium chloride embryo staining technique was developed to evaluate seed health and screen out seed sources likely to perform poorly in the initiation process.     

Agrobacterium-mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plants

Summary Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them confirmed the integration of the T-DNA.Abbreviations GUS ß-glucuronidase - X-Gluc 5-bromo-4-chloro-3indolyl ß-D-glucuronic acid - NPT II neomycin phosphotransferase II     

Effect of basal medium, growth regulators and Phytagel concentration on initiation of embryogenic cultures from immature zygotic embryos of loblolly pine (Pinus taeda L.)

Low initiation frequency is one of the main barriers in applying somatic embryogenesis to the clonal production of Pinus species. Factors affecting initiation, including basal medium, plant growth regulators, and Phytagel concentration, have been investigated in loblolly pine (Pinus taeda L.). BM₁ basal medium proved superior to DCR₁ and LP (LP basal salts plus BM₁ organic nutrients). No extrusion from megagametophytes was exhibited on LP medium. The combination of 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l 6-benzylaminopurine (BA) resulted in a higher extrusion frequency than that of 11 mg/l 2,4-D, 4.5 mg/l BA and 4.3 mg/l kinetin. Phytagel at 1 g/l resulted in the highest explant browning, but the lowest extrusion frequency, while 4 g/l Phytagel induced some dry embryogenic extrusions. Phytagel at 2 g/l was regarded as the best level for initiation of embryogenic cultures. Received: 23 December 1996 / Revision received: 22 July 1997 / Accepted: 2 September 1997     

Recovery of plants from cryopreserved embryogenic cell suspensions of Pinus caribaea

Summary Embryogenic cell suspension cultures of Pinus caribaea var. hondurensis have been cryopreserved in liquid nitrogen for up to four months, using sucrose and dimethylsulfoxide as cryoprotectants. Post-thaw growth was obtained after a short lag phase. Removal of the remaining liquid around the cells using a filter disc favoured subsequent regrowth of the cells. These reestablished cultures maintained an embryogenic potential similar to non-frozen cultures. The embryos produced were able to regenerate into plants, which are now growing in a greenhouse.Abbreviations BA 6-benzyladenine - DMSO dimethylsulfoxide - FDA fluorescein diacetate - MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - PAR photosynthetically active radiation     

Establishment and characterization of Pinus pinaster suspension cell cultures

We report the establishment of a Pinus pinaster (Ait.) cell suspension culture in a modified MS medium supplemented with 2 mg ml⁻¹ 2,4-D and 1 mg ml⁻¹ BA. Calli were obtained from seedling root segments and established a friable isodiametric cell suspension, suitable for in vitro studies of maritime pine at the cellular level. Growth (dry weight), cell viability, pH, and nutrient consumption: carbon source (sucrose, fructose and glucose), nitrogen source (ammonia and nitrate) and phosphate were monitored over 24 h. Suspension cells exhibited a 15-day exponential growth stage, during which a biphasic consumption profile was observed for all nutrients. Phosphate was the first limiting nutrient and preferable consumption was observed for glucose over fructose and nitrate over ammonium.     

Induction of embryogenic tissue and maturation of somatic embryos in Pinus brutia TEN

Embryogenic tissues (ET) of Turkish red pine (Pinus brutia TEN) were initiated from immature precotyledonary zygotic embryos sampled from 15 different plus trees. Seven collections were made weekly from June 10 to July 22, 2003. DCR basal medium supplemented with 13.6 μM 2,4–dichlorophenoxyacetic acid (2,4-D) and 2.2 μM benzylaminopurine (BAP) was used for initiation and maintenance of ET. Overall initiation frequency of ET in the study was 11.6%, initiation rates ranging between 4.7% and 24.1% per tree. Out of 12,940 explants tested, 3.4% were converted into established cell lines (ECL) following five subcultures. Of the maturation treatments tested, 80 μM ABA, sucrose (3%) and maltose (3% and 6%), and 3.75% PEG combined with 1% gellan gum were the most suitable combination for somatic embryo maturation.     

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 embryogenic competency and morphological changes during somatic embryogenesis in Iris pseudacorus

Embryogenic callus was obtained from bulb segments of Iris pseudacorus on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with kinetin. When early globular somatic embryos were subcultured onto MS medium with 4.52 μM 2,4-D, high frequency of somatic embryogenesis was obtained. Deprivation of 2,4-D was required for maturation. Mature somatic embryos had an elongated scutellum with a notch on the base of scutellum. Separation of embryos from embryo clusters was necessary to enhance the frequency of germination. Germination was stimulated by separation of embryos from embryo clusters and transfer onto fresh half-strength MS medium with 3% sucrose. After acclimation in artificial soil in greenhouse for 2 months, 96.4% of plantlets survived.     

Synthetic seed production from somatic embryos of Pinus radiata

Pinus radiata is one of the most important forestry species in the southern hemisphere. This work describes the regeneration of this plant via somatic embryogenesis from immature zygotic embryos. To improve this process, somatic embryogenic cell suspensions were established in liquid media for the generation of material for embryo maturation. Each developmental stage of these suspensions was characterized by microscopy and their growth phases quantified. An alginate-containing medium was used as an encapsulation method for the somatic embryos that were then germinated as artificial seeds in vitro. The protocols described in this work are both useful and reliable for industrial purposes.     

Plant regeneration from embryogenic cell suspension-derived protoplasts of rubber

Embryogenic cell suspensions of rubber derived from immature inflorescences and inner integuments of immature fruits released 3.1 ± 0.2 × 10⁷ protoplasts g^-1 f. wt. (mean ± s.e.m, n = 10) and 3.2 ± 0.2 × 10⁷ protoplasts g^-1 f. wt., with mean viabilities of 83 ± 2% and 77 ± 8%, respectively. Sustained mitotic division was observed only when protoplasts were cultured in KPR liquid medium on nitrocellulose membranes overlying the same semi-solid medium containing Lolium multiflorum nurse cells. Protoplast-derived cell colonies were produced within 2 months of culture. Protoplast-derived cell colonies proliferated, upon subculture to MS-based regeneration medium, with 40% of the protoplast-derived calli developing somatic embryos. The latter germinated into plants on the same medium after 3 months of culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Simplified and improved somatic embryogenesis for clonal propagation of Pinus pinaster (Ait.)

In this study, several improvements and simplifications of SE protocols in Pinus pinaster (Ait.), a species of economic importance in the regions of Western Europe, are described. These improvements pertained to all stages of SE including high initiation frequencies in eight control pollinated seed families, relatively high somatic embryo maturation yield when cells were coated with particles of activated charcoal and a rapid production of plants directly in a shade house. The SE initiation frequency from isolated zygotic embryos was high (up to 100%) and plants were produced from 11 embryogenic lines representing all crosses. Based on these results, the estimated number of somatic embryos required to produce 1,000 plants varied from slightly more than the required number of plants to more than double this number depending on the line. Such an estimate is critical in developing plant production strategy when a number of embryogenic lines are considered for production of clonal plants.     

Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.)

Efficient Agrobacterium tumefaciens-mediated transformation was developed using embryogenic suspension cell cultures of elite sweet potato (Ipomoea batatas [L.] Lam.) cultivars, including Ayamurasaki, Sushu2, Sushu9, Sushu11, Wanshu1, Xushu18 and Xushu22. Embryogenic suspension cultures were established in LCP medium using embryogenic calli induced from apical or axillary buds on an induction medium containing 2 mg l⁻¹ 2,4-D. Suspension cultures were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with the hpt gene as a selectable marker and an intron-interrupted uidA gene as a visible marker. Several key steps of the sweet potato transformation system have been investigated and optimized, including the appropriate antibiotics and their concentrations for suppressing Agrobacterium growth and the optimal doses of hygromycin for transformant selection. A total of 485 putative transgenic plant lines were produced from the transformed calli via somatic embryogenesis and germination to plants under 10 mg l⁻¹ hygromycin and 200 mg l⁻¹ cefotaxime. PCR, GUS and Southern blot analyses of the regenerated plants showed that 92.35% of them were transgenic. The number of T-DNA insertions varied from one to three in most transgenic plant lines. Plants showed 100% survival when 308 transgenics were transferred to soil in the greenhouse and then to the field. Most of them were morphologically normal, with the production of storage roots after 3 months of cultivation in the greenhouse or fields. The development of such a robust transformation method suitable to a range of sweet potato genotypes not only provides a routine tool for genetic improvement via transgenesis but also allows us to conduct a functional verification of endogenous genes in sweet potato.     

The origin and development of somatic embryos following cryopreservation of an embryogenic suspension culture ofPicea sitchensis

Summary The development of somatic embryos in an embryogenic suspension culture ofPicea sitchensis was followed every day for two weeks after thawing from liquid nitrogen (LN₂). Only a few cells, primarily located at the periphery of the embryonic region of the embryos, survived cryopreservation in LN₂. Surviving cells were classified into two groups: embryogenic cells (EC) and non-embryogenic cells (NEC), based on their morphology and embryogenic competence. The dense cytoplasmic EC underwent organized growth and differentiation with first divisions occurring after 24 h, and embryo formation 6–8 days after thawing from LN₂. No evidence of asymmetrical divisions or free-nuclear stages was found during somatic embryo formation. NEC had less dense cytoplasm with numerous small vacuoles. One to five days after thawing the NEC became progressively more vacuolated and elongated. Histological examination revealed no mitotic activity in NEC, and six days after thawing NECs were seen as single cells or unorganized cell aggregates. Two weeks after thawing the appearance of the cryopreserved cultures was comparable to that of the untreated cultures.Abbreviations EC embryogenic cells - ECC embryogenic cell clusters - FDA fluorescein diacetate - GMA glycol methacrylate - LN₂ liquid nitrogen (–196°C) - NEC non-embryogenic cells     

Brassinolide improves embryogenic tissue initiation in conifers and rice

Somatic embryogenesis (SE), the most promising technology for the large-scale production of high-value coniferous trees from advanced breeding and genetic engineering programs, is expected to play an important role in increasing productivity, sustainability, and the uniformity of future U.S. forests. To be successful for commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine (Pinus taeda L.), our main focus species, is often recalcitrant for desirable genotypes. Initiation percentages of loblolly pine, Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco], and Norway spruce (Picea abies L., Karst.) were improved through the use of brassinolide. Brassinosteroids, which include brassinolide, are a relatively new group of natural plant growth regulators that are found in many plant species. They have been shown to have diverse, tissue-specific, and species-specific effects, including the stimulation of cell elongation and ethylene production and increasing resistance to abiotic stress. In our media, brassinolide was effective at concentrations ranging from 0.005–0.25 M. Using control medium (no brassinolide) and brassinolide-supplemented (0.1 M) medium, we achieved improved initiation percentages in loblolly pine, Douglas-fir, Norway spruce, and rice—15.0% to 30.1%, 16.1% to 36.3%, 34.6% to 47.4%, and 10%, respectively. Brassinolide increased the weight of loblolly pine embryogenic tissue by 66% and stimulated initiation in the more recalcitrant families of loblolly pine and Douglas-fir, thus compensating somewhat for genotypic differences in initiation. Initiation percentages in loblolly pine were improved through the combination of modified 1/2-P6 salts, 50 mg/l activated carbon (AC), adjusted levels of Cu and Zn (to compensate for adsorption by AC), 1.5% maltose, 2% myo-inositol (to raise the osmotic level, partially simulating the megagametophyte environment), 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l -naphthaleneacetic acid, 0.63 mg/l 6-benzylaminopurine, 0.61 mg/l kinetin, 3.4 mg/l silver nitrate, 10 M cGMP, 0.1 M brassinolide, and 2 g/l Gelrite. Across 12 open-pollinated families of loblolly pine, initiation percentages ranged from 2.5% to 50.7%, averaging 22.5%.Abbreviations AC Activated carbon - BA 6-Benzylaminopurine - 8-Br-cGMP Guanosine 3,5-cyclic monophosphate, 8-bromo-, sodium salt - 2,4-D 2,4-Dichlorophenyloxyacetic acid - NAA -Naphthaleneacetic acid Communicated by G.C. Phillips     

A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): Embryogenic calli as a source to generate large numbers of transgenic plants

A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3–6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.V.G. Sunnichan and R. Kumria contributed equally to this investigation     

Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation

The availability of a system for direct transfer of anti-fungal candidate genes into American chestnut (Castanea dentata), devastated by a fungal blight in the last century, would offer an alternative or supplemental approach to conventional breeding for production of chestnut trees resistant to the blight fungus and other pathogens. By taking advantage of the strong ability of embryogenic American chestnut cultures to proliferate in suspension, a high-throughput Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into the tree was established. Proembryogenic masses (PEMs) were co-cultivated with A. tumefaciens strain AGL1 harboring the plasmid pCAMBIA 2301, followed by stringent selection with 50 or 100 mg/l Geneticin. A protocol employing size-fractionation to enrich for small PEMs to use as target material and selection in suspension culture was applied to rapidly produce transgenic events with an average efficiency of four independent transformation events per 50 mg of target tissue and minimal escapes. Mature somatic embryos, representing 18 transgenic events and derived from multiple American chestnut target genotypes, were germinated and over 100 transgenic somatic seedlings were produced and acclimatized to greenhouse conditions. Multiple vigorous transgenic somatic seedlings produced functional staminate flowers within 3 years following regeneration.     

Improved somatic embryogenesis of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) with focus on induction parameters and efficient plant regeneration

A study of four parameters (induction medium, floral explant, developmental stage and year) was carried out to determine the best combination for the embryogenesis induction of eight grapevine (Vitis vinifera L.) cultivars. Anthers and ovaries were extracted from flower buds at three developmental phases and incubated in two induction media over two consecutive years. As average, the percentage of embryogenesis on Nitsch and Nitsch-derived medium (9.1%) was higher than in Murashige and Skoog-derived medium (5.9%) and embryogenesis from ovaries (10.1%) was 2-fold higher than from anthers (4.9%). Earlier flower developmental stages (II–III) favored embryogenic induction from anthers, while later stages (III–V) did it from ovaries. Induction of embryogenic cultures was genotype dependent. Two years after the establishment of the embryogenic lines, an average of 48.0% of the pro-embryogenic masses were viable and suitable to initiate cell suspensions. Embryogenic cultures of four genotypes showed a high percentage of conversion from embryos to plants: Albariño (61.8%), Garnacha (48.8%), Tempanillo (71.0%) and Sultanina (69.0%). Moreover, cell suspensions were competent for transient transformation based on β-glucuronidase assay, as up to 6,387 blue spots per Petri plate after Biolistic bombardment were obtained. Here, we present the advantage of ovaries over anthers for the embryogenesis induction of several grapevine cultivars. This is the first report of embryogenesis from the cultivars Albariño, Verdejo and Muscat Hamburg as well as transient transformation of Albariño and Tempranillo.     

Cryopreservation of embryogenic tissue of a range of genotypes of sweet potato (Ipomoea batatas [L] Lam.) using an encapsulation protocol

Embryogenic tissue of nine sweet potato [Ipomoea batatas (L.) Lam] genotypes from Asia, Africa and the Americas was established from in vitro axillary buds on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid. Embryogenic aggregates, 1.0–2.0 mm in diameter, were encapsulated in alginate gel, precultured on medium containing elevated levels of sucrose and dehydrated prior to rapid freezing in liquid nitrogen. The maximum survival of embryogenic tissue ranged from 4% to 38%, depending on the genotype. With the incorporation of a slow-cooling step, survival was generally much higher than that obtained after rapid freezing alone. Five of eight genotypes tested with this protocol gave survival percentages in excess of 55%, and a further two in excess of 33%, all after evaporative dehydration. The most effective sucrose treatment(s), however, varied with the genotype. Received: 7 October 1996 / Revision received: 16 December 1996 / Accepted 27 January 1997