Genetic transformation of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a promoterlessgus::nptII fusion gene based vector

We have generated putative promoter tagged transgenic lines inArachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated byAgrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4 mg/l in combination with 0.1 mg/l α-napthaleneacetic acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters enhancing genetic transformation viz. seedling age,Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation with CN explants from 6-day-old seedlings co-cultivated withAgrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation frequency was achieved with p35S GUSINT in Β-glucuronidase (GUS) assays. Among thein vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS percentage. We have generated over 141 putative T₀ plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression or blue spots in at least one plant part. The progeny of 15 T₀ plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression patterns were more or less similar in both T₀ and corresponding T₁ progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic lines in the present communication.     

Genetic transformation and regeneration of rubber tree (Hevea brasiliensis Muell. Arg) transgenic plants with a constitutive version of an anti-oxidative stress superoxide dismutase gene

Agrobacterium tumefaciens-mediated genetic transformation and the regeneration of transgenic plants was achieved in Hevea brasiliensis. Immature anther-derived calli were used to develop transgenic plants. These calli were co-cultured with A. tumefaciens harboring a plasmid vector containing the H. brasiliensis superoxide dismutase gene (HbSOD) under the control of the CaMV 35S promoter. The -glucuronidase gene (uidA) was used for screening and the neomycin phosphotransferase gene (nptII) was used for selection of the transformed calli. Factors such as co-cultivation time, co-cultivation media and kanamycin concentration were assessed to establish optimal conditions for the selection of transformed callus lines. Transformed calli surviving on medium containing 300 mg l^-1 kanamycin showed a strong GUS-positive reaction. Somatic embryos were then regenerated from these transgenic calli on MS2 medium containing 2.0 mg l^-1 spermine and 0.1 mg l^-1 abscisic acid. Mature embryos were germinated and developed into plantlets on MS4 medium supplemented with 0.2 mg l^-1 gibberellic acid, 0.2 mg l^-1 kinetin (KIN) and 0.1 mg l^-1 indole-3-acetic acid. A transformation frequency of 4% was achieved. The morphology of the transgenic plants was similar to that of untransformed plants. Histochemical GUS assay revealed the expression of the uidA gene in embryos as well as leaves of transgenic plants. The presence of the uidA, nptII and HbSOD genes in the Hevea genome was confirmed by polymerase chain reaction amplification and genomic Southern blot hybridization analyses.Communicated by L. Peña     

Plate flooding as an alternative <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for American chestnut somatic embryos

In an attempt to improve Agrobacterium-mediated transformation frequency of American chestnut somatic embryos, a novel method of inoculation/co-cultivation was developed. Plate flooding is a simple method where the Agrobacterium inoculum is poured onto the embryos while they remain on multiplication medium. This method tested the hypothesis that wounding tissues prior to co-cultivation was unnecessary or counterproductive. Two clones, WB296 and P1-1, were tested for differences in transformation efficiency as measured by the number of transformed embryogenic cell lines per Petri dish, the total number of transformed cell lines (embryos plus callus) and percentage of transformants that remained embryogenic. Plate flooding using clone WB296 produced significantly more transformed embryo cell lines and had a higher percentage of transformants remain embryogenic. The number of total transformed cell lines (embryos plus callus) was the same as obtained by other methods (desiccation, blot dry, sand abrasion, sonication and vacuum infiltration). With clone P1-1 there were no significant differences among the inoculation/co-cultivation treatments tested. Polymerase chain reaction and Southern hybridizations confirmed that the transgene of interest had been stably integrated into both American chestnut clones. Whole plants were regenerated from clone P1-1.     

Factors affecting Agrobacterium tumefaciens-mediated genetic transformation of Lycium barbarum L.

Summary Using the system for genetic transformation and transgenic plant regeneration via somatic embryogenesis (SE) of Lycium barbarum established in this laboratory, this study reports the optimization of the factors affecting the efficiency of transformation, including pre-culture period, leaf explant source, use of acetosyringone, strains and density of Agrobacterium, and temperature of co-cultivation. The optimized transformation protocol for L. barbarum included preculture of leaf explants from 3-wk-old seedlings for 3 d on the medium for callus induction followed by inoculation with Agrobacterium strain EHA101 (pIG121 Hm), co-cultivation for 3d at 24&#x00B0;C, and transfer to the selection regeneration medium with 50 mg l^&#x2212;1 kanamycin (Kan). Using this protocol, 65% L. barbarum explants gave rise to Kan-resistant and GUS-positive calli. In addition, the expression of introduced transgene (npt II) in clonal progeny was verified by formation of calli and somatic embryos from leaf segments of nine transgenic plants grown on the Kan-containing medium. All explants formed calli at 50 mg l^&#x2212;1 Kan and seven out of nine transgenic plants were found to possess callus-forming capacity even at 100 mg l^&#x2212;1 Kan. These calli also possessed higher SE potential on SE medium supplemented with 25 mg l^&#x2212;1 Kan.     

A co-transformation system to produce transgenic grapevines free of marker genes

A co-transformation system was developed to produce grapevines free of selectable marker genes. This was achieved by transforming Vitis vinifera L. ‘Thompson Seedless’ somatic embryos with a mixture of two Agrobacterium strains. The first strain contained a binary plasmid with an egfp gene of interest between the T-DNA borders. The second strain harbored the neomycin phosphotransferase (nptII) gene for positive selection and the cytosine deaminase (codA) gene for negative selection, linked together by a bi-directional dual promoter complex. Our technique included a short positive selection phase on medium containing 100 mg l⁻¹ kanamycin before subjecting cultures to prolonged negative selection on medium containing 250 mg l⁻¹ 5-fluorocytosine. We regenerated 25 stable EGFP expressing transgenic lines. PCR analysis confirmed 18 lines contained only the egfp gene, whereas the remaining contained both egfp and codA/nptII genes. Presumably, the 18 monogenic lines arose through cross protection by being in close proximity to cells that expressed nptII and thus detoxified kanamycin in the immediate vicinity. This is the first report for grapevine using a combination of positive and negative selection to produce transgenic plants that do not contain marker genes.     

Agrobacterium tumefaciens-mediated transformation of embryogenic tissue and transgenic plant regeneration in Chamaecyparis obtusa Sieb. et Zucc.

A genetic transformation procedure for Chamaecyparis obtusa was developed after co-cultivation of embryogenic tissues with disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the sgfp (synthetic green fluorescent protein) visual reporter and nptII (neomycin phoshotransferase II) selectable marker genes. The highest transformation frequency was 22.5 independent transformed lines per dish (250 mg embryogenic tissue) following selection on kanamycin medium. Transgenic plantlets were regenerated through the maturation and germination of somatic embryos. The intensity of GFP fluorescence, observed under a fluorescence microscope, varied from very faint to relatively strong, depending on the transgenic line or part of the transgenic plant. The integration of the genes into the genome of regenerated plantlets was confirmed by Southern blot analysis.     

Efficient transformation of papaya by coat protein gene of papaya ringspot virus mediated byAgrobacterium following liquid-phase wounding of embryogenic tissues with caborundum

Summary Generation of transgenic papaya (Carica papaya L.) has been hampered by the low rates of transformation achieved by conventionalAgrobacterium infection or microprojectile bombardment. We describe an efficientAgrobacterium-mediated transformation method based on wounding of cultured embryogenic tissues with carborundum in liquid phase. Embryogenic tissues were obtained from cultured immature zygotic embryos collected 75–90 days after pollination. The expressible coat protein (CP) gene of a Taiwan strain of papaya ringspot virus (PRSV) was constructed in a Ti binary vector pBGCP, which contained the NPT-II gene as a selection marker. The embryogenic tissues were vortexed with 600 mesh carborundum in sterile distilled water for 1 min before treating with the disarmedA. tumefaciens containing the pBGCP. Transformed cells were cultured on kanamycin-free medium containing 2,4-D and carbenicillin for 2–3 weeks and then on the kanamycin medium for 3–4 months. The developed somatic embryos were transferred to the medium containing NAA, BA and kanamycin and subsequently regenerated into normal-appearing plants. Presence of the PRSV CP gene in the putative transgenic lines was detected by PCR and the expression of the CP was verified by Western blotting. The transgene was nuclearly inherited as revealed by segregation analysis in the backcrossed R₁ progeny. From five independent experiments, the average successful rate of transformation was 15.9% of the zygotic embryos treated (52 transgenic somatic embryo clusters out of 327 zygotic embryos treated), about 10–100 times higher than the available methods previously reported. Thus, wounding highly regenerable differentiating tissues by carborundum vortexing provides a simple and efficient way for papaya transformation mediated byAgrobacterium.     

Enhancing the frequency of somatic embryogenesis following <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of immature cotyledons of soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill.]

Summary A characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation-competent cells/tissues on immature cotyledons of soybean [Glycine max (L.) Merrill.] under hygromycin selection was identified. This highly embryogenic immature cotyledon was characterized with emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5–8 mm in length, a 1∶1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-wk cocultivation period significantly increased the frequency of transgenic somatic embryos. Whereas, increasing the infection period of explants or subjecting explants to either wounding or acetosyringone treatments did not increase the frequency of transformation. An optimal selection regime was identified when inoculated immature cotyledons were incubated on either 10 or 25 mgl⁻¹ hygromycin for a 2-wk period, and then maintained on selection media containing 25 mgl⁻¹ hygromycin in subsequent selection periods. However, somatic embryogenesis was completely inhibited when inoculated immature cotyledons were incubated on a kanamycin selection medium. These findings clearly demonstrated that the tissue culture protocols for transformation of soybean should be established under both Agrobacterium and selection conditions.     

Development of highly efficient genetic transformation protocols for table grape Sugraone and Crimson Seedless at low <Emphasis Type="Italic">Agrobacterium</Emphasis> density

Highly efficient genetic transformation protocols and the regeneration of transgenic plants of Sugraone and Crimson Seedless grapevines (Vitis vinifera L.) were achieved from embryogenic calli co-cultured with low Agrobacterium tumefaciens densities. The sensitivity of embryogenic cultures to kanamycin, as well as the effect of Agrobacterium strains, C58(pMP90) or EHA105, and the bacterial concentration (0.06 or 0.2 at Optical Density OD₆₀₀) on transformation efficiency were studied. Embryogenic cultures showed different kanamycin sensitivities and the total suppression of embryo differentiation at 20 and 50 mg/l kanamycin for Crimson Seedless and Sugraone, respectively. sgfp gene expression was evaluated in callus co-cultured with each bacterial strain. Although GFP transient expression was higher with A. tumefaciens EHA105 in both cultivars at the beginning of the culture, there were no significant differences 28 days post-inoculation. However, the concentration of Agrobacterium did affected transformation efficiency: 0.06 OD₆₀₀ being more effective for the transformation of Crimson Seedless and 0.2 OD₆₀₀ for Sugraone. By following the optimised procedure, 21 and 26 independent transgenic plants were generated from Sugraone and Crimson Seedless respectively, three to five months post-infection. PCR analyses were carried out to verify the integration of the sgfp and nptII genes into grapevine genome and the stable integration of the sgfp gene was confirmed by Southern blot.     

An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.)

An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’.     

RETRACTED ARTICLE: Transgenic ramie [<Emphasis Type="Italic">Boehmeria nivea</Emphasis> (L.) Gaud.]: factors affecting the efficiency of <Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation and regeneration

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for ramie [Boehmeria nivea (L.) Gaud.] based on the examinations of several factors affecting plant transformation efficiency. The effects of Agrobacterium cell density, acetosyringone, co-cultivation temperature, co-cultivation duration, co-cultivation photoperiod and pH on stable transformation were evaluated. Agrobacterium at a concentration of OD = 0.5–0.8 improved the efficiency of transformation. Concentration of acetosyringone at 50 mg/L during co-cultivation significantly increased transformation efficiency. Co-cultivation at 20°C, in comparison to 15, 25 and 28°C, consistently resulted in higher transformation frequencies. A relatively short co-cultivation duration (3 days) was optimal for ramie transformation. Co-cultivation medium at pH 5.9 and co-cultivation in darkness both improved the transformation efficiencies of ramie. An overall scheme for producing transgenic ramie is presented, through which an average transformation rate from 10.5 to 24.7% in five ramie varieties was obtained. Stable expression and integration of the transgenes were confirmed by histochemical GUS assay, kanamycin painting assay, PCR and Southern blotting. This optimized transformation system should be employed for efficient Agrobacterium-mediated transformation of ramie. An erratum to this article can be found at     

Improving genetic transformation of European chestnut and cryopreservation of transgenic lines

The aim of the present work was to study the effect of the developmental stage of the somatic embryos and of the genotype on the genetic transformation of embryogenic lines of European chestnut (Castanea sativa Mill.) and the cryopreservation of the embryogenic lines that are generated. As an initial source of explants in the transformation experiments, it was found that the use of somatic embryos isolated in the globular stage or clumps of 2–3 embryos in globular/heart-shaped stages was more effective (30%) than when embryos at the cotyledonary stage were used (6.7%). All of the seven genotypes tested were transformed, and transformation efficiency was clearly genotype dependent. Three transgenic lines were successfully cryopreserved using the vitrification procedure, and the stable integration of the uidA gene into the transgenic chestnut plants that were regenerated subsequent to cryopreservation was demonstrated.     

Highly efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic cell suspensions of <Emphasis Type="Italic">Musa acuminata</Emphasis> cv. Mas (AA) via a liquid co-cultivation system

A high efficient protocol of Agrobacterium-mediated transformation of Musa acuminata cv. Mas (AA), a major banana variety of the South East Asia region, was developed in this study. Male-flower-derived embryogenic cell suspensions (ECS) were co-cultivated in liquid medium with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 carrying nptII and gusA gene in the T-DNA. Depending upon conditions and duration of co-cultivation in liquid medium, 0–490 transgenic plants per 0.5 ml packed cell volume (PCV) of ECS were obtained. The optimum duration of inoculation was 2 h, and the highest transformation frequency was achieved when infected ECS were co-cultivated in liquid medium first for 12 h at 40 rpm and then for 156 h at 100 rpm on a rotary shaker. Co-cultivation for a shorter duration (72 h) or shaking constantly at 100 rpm at the same duration gave 1.6 and 1.8 folds lower transformation efficiency, respectively. No transgenic plants were obtained in parallel experiments carried on semi-solid media. Histochemical GUS assay and molecular analysis in several tissues of the transgenic plants demonstrated that foreign genes were stably integrated into the banana genome. Compared to semi-solid co-cultivation transformation in other banana species, it is remarkable that liquid co-cultivation was much more efficient for transformation of the Mas cultivar, and was at least 1 month faster for regenerating transgenic plants.     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic cultures and plant regeneration in <Emphasis Type="Italic">Vitis rotundifolia</Emphasis> Michx. (muscadine grape)

A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM) and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l⁻¹ in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of transgenic cells, compared to 50 or 75 mg l⁻¹, which permitted the proliferation of more non-transformed cells. Transgenic plants of “Alachua” and “Carlos” were recovered after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization. Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots, inflorescences and the embryo and endosperm of developing berries.     

Production of transgenic <Emphasis Type="Italic">Podophyllum peltatum via Agrobacterium tumefaciens</Emphasis>-mediated transformation

Transgenic Podophyllum peltatum plants were successfully produced by Agrobacterium tumefaciens-mediated transformation. Embryogenic callus was co-cultivated with Agrobacterium tumefaciens harboring a binary vector pBI 121 carrying β-glucuronidase (GUS) and neomycinphosphotransferase (NPT II) gene. GUS-histochemical analysis revealed that, 50 μM acetosyringone treatments during Agrobacterium infection and 3 d co-cultivation with Agrobacterium showed enhanced transformation efficiency. Percentage of GUS positive callus increased rapidly as the subculture time proceeded on selection medium containing 100 mg dm⁻³ kanamycin. Kanamycin resistant somatic embryos were formed from embryogenic callus after cultivation with 11.35 μM abscisic acid (ABA) for 3 weeks and then on hormone-free selection medium. Somatic embryos were germinated and converted into plantlets on medium containing 2.89 μM gibberellic acid (GA₃). The integration of GUS and NPT II gene into transgenic plants was confirmed by polymerase chain reaction and Southern analysis.     

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.     

High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize

A high throughput genetic transformation system in maize has been developed with Agrobacterium tumefaciens mediated T-DNA delivery. With optimized conditions, stable callus transformation frequencies for Hi-II immature embryos averaged approximately 40%, with results in some experiments as high as 50%. The optimized conditions include N6 medium system for Agrobacterium inoculation, co-cultivation, resting and selection steps; no AgNo₃ in the infection medium and adding AgNo₃ in co-cultivation, resting and selection medium; Agrobacterium concentration at 0.5×10⁹ c.f.u. ml^–1 for bacterium inoculation; 100 mg l^–1 carbenicillin used in the medium to eliminate Agrobacterium after inoculation; and 3 days for co-cultivation and 4 days for resting. A combination of all of these conditions resulted in establishing a high throughput transformation system. Over 500 T₀ plants were regenerated and these plants were assayed by transgene expression and some of them were also analyzed by Southern hybridization. T₁ plants were analyzed and transmission of transgenes to the T₁ generation was verified. This represents a highly reproducible and reliable system for genetic transformation of maize Hi-II.     

Agrobacterium-mediated transformation of American chestnut (Castanea dentata (Marsh.) Borkh.) somatic embryos

These studies were designed to test if a binary vector containing the gfp, bar and oxalate oxidase genes could transform American chestnut somatic embryos; to see if a desiccation treatment during co-cultivation would affect the transformation frequency of different American chestnut somatic embryo clones; to explore the effects of more rapid desiccation; and to see if the antibiotics used to kill the Agrobacterium were interfering with the regeneration of the somatic embryos. Two days of gradual desiccation was found to significantly enhance transient GFP expression frequency. When this treatment was tested on six American chestnut clones, five were transformed and four of these remained embryogenic. Transformation was confirmed by Southern hybridization. Phenotypically normal transgenic shoots were regenerated and rooted. Vascular tissue specific expression of the oxalate oxidase gene was detected in one transgenic line. Carbenicillin, cefotaxime, and tricarcillin were found to not interfere with the regeneration of transformed embryos.     

Genetic transformation of commercial breeding lines of alfalfa (Medicago sativa)

Bio-engineering technologies are now routinely used for the genetic improvement of many agricultural crops. However, breeding lines of Medicago sativa are not easily amenable to genetic transformation and therefore cannot benefit from the molecular tools that have been developed for genetic manipulations. This paper describes a strategy that has been developed to transfer DNA into commercially important breeding lines of winter-hardy alfalfa via Agrobacterium infection. Three highly regenerative genotypes have been selected from ca 1000 genotypes within 11 breeding lines. They have been used as basic material for an extensive genetic transformation trial. Combinations of genotypes (11.9, 8.8, 1.5) expression vectors (pGA482, pGA643, pBibKan) and bacterial strains (C58, A281, LBA4404) were tested for their ability to produce stable transgenic material. Putative transgenic plantlets were further screened by nptII-specific PCR amplification, Southern hybridization and recallusing assays. One genotype (1.5) gave only one transformant out of 432 individual trials. With the two other genotypes, efficiency of transformation (kanamycin-resistant calluses obtained/explant tested) ranged from 0 to 0.92 depending on the strain/vector combination used. Statistical interactions underline the possibility of obtaining good genotype-strain-vector combinations for alfalfa transformation. Predicted transformation probability indicates that with strain LBA4404 containing the vector pGA482 and genotype 11.9, transformation efficiency is above 60% and 10% or more of the calluses retain embryogenic potential. PCR amplification and Southern hybridization of randomly chosen regenerated plantlets demonstrated that all embryos developing on 50 g ml^-1 kanamycin had a stable genomic insertion of nptII. Sexual crosses with untransformed genotypes showed that segregation of the transgenic trait followed Mendelian heredity.