Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.)

Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hübner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot.     

The development of a reproducible Agrobacterium tumefaciens transformation system for garlic (Allium sativum L.) and the production of transgenic garlic resistant to beet armyworm (Spodoptera exigua Hübner)

This paper describes the development of a reliable transformation system for garlic (Allium sativum L.) and its application in producing insect resistant GM garlic lines. The transformation system is based on Agrobacterium tumefaciens as a vector, using young callus derived from different callus sources: callus induced from both apical and non-apical root segments of in vitro plantlets, true garlic seeds and bulbils. Two different reporter genes were used in our garlic transformation experiments, namely the gusA gene coding for -glucuronidase and the gfp gene coding for green fluorescent protein. A total of seven independent transformed callus lines derived from different callus sources were obtained. The advantage of the system developed is the short time period needed for completion of the protocol (about 6 months) and the year-round availability of high quality callus from in vitro roots. The highest transformation frequency in a single experiment (1.47%), was obtained using garlic cv. 'Printanor'. Differences existed between cultivars in transformation frequency but were not significant. The same was found for the plasmids used in transforming garlic. Via PCR the presence of the gusA, hpt (hygromycin phosphotransferase) and gfp genes could be demonstrated in putative transformed in vitro plants. Southern hybridization showed that the reporter gene gusA and the selective gene hpt were stably integrated into the garlic genome. After transfer to the greenhouse of in vitro regenerants, transgenic garlic harbouring the gusA gene survived and grew well, whereas the gfp transgenic garlic gradually died under these conditions.Using this protocol transgenic garlic resistant to beet armyworm using the cry1Ca and H04 resistance genes from Bacillus thuringiensis were developed. Via Southern hybridization it was shown that the cry1Ca sequence was stably integrated into the garlic genome. After transfer of the transgenic in vitro garlic plants to the greenhouse, the cry1Ca plants developed normally and grew well to maturity with normal bulbs. However, all transgenic in vitro H04 garlic plants did not survive after transfer to the greenhouse. Transgenic cry1Ca garlic plants proved completely resistant to beet armyworm in a number of in vitro bio-assays. This finding will facilitate the development of new garlic cultivars resistant to beet armyworm.     

Ectopic Expression of an <Emphasis Type="Italic">FT</Emphasis> Homolog from <Emphasis Type="Italic">Citrus</Emphasis> Confers an Early Flowering Phenotype on Trifoliate Orange (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.)

Citrus FT (CiFT) cDNA, which promoted the transition from the vegetative to the reproductive phase in Arabidopsis thaliana, when constitutively expressed was introduced into trifoliate orange (Poncirus trifoliata L. Raf.). The transgenic plants in which CiFT was expressed constitutively showed early flowering, fruiting, and characteristic morphological changes. They started to flower as early as 12 weeks after transfer to a greenhouse, whereas wild-type plants usually have a long juvenile period of several years. Most of the transgenic flowers developed on leafy inflorescences, apparently in place of thorns; however, wild-type adult trifoliate orange usually develops solitary flowers in the axils of leaves. All of the transgenic lines accumulated CiFT mRNA in their shoots, but there were variations in the accumulation level. The transgenic lines showed variation in phenotypes, such as time to first flowering and tree shape. In F₁ progeny obtained by crossing ‘Kiyomi’ tangor (C. unshiu × sinensis) with the pollen of one transgenic line, extremely early flowering immediately after germination was observed. The transgene segregated in F₁ progeny in a Mendelian fashion, with complete co-segregation of the transgene and the early flowering phenotype. These results showed that constitutive expression of CiFT can reduce the generation time in trifoliate orange.     

Genetic transformation of prickly-pear cactus (Opuntia ficus-indica) by Agrobacterium tumefaciens

A system for genetic transformation of an elite prickly pear cactus (Opuntia ficus-indica L., cultivar Villa Nueva) by Agrobacterium tumefaciens was developed. Beginning with direct bacterial infection by using a hypodermic syringe to the meristematic tissue termed areoles, transgenic plants were obtained by selection with 100 mg l⁻¹ kanamycin. Transient and stable GUS activities were monitored on kanamycin-resistant shoots and regenerated plants, respectively. Genetic transformation of regenerated plants growing under selection was demonstrated by PCR and Southern blot analysis; transgene copy number in the genome of transgenic plants ranged from two to six, while the transformation frequency obtained by the system reported here was of 3.2%. This method may be useful for routine transformation and introduction of several important genes in prickly pear cactus.     

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.     

Overexpression of the Arabidopsis gai gene in apple significantly reduces plant size

Genetic engineering is an attractive method to obtain dwarf plants in order to eliminate the extensive use of growth retardants in horticultural crop production. In this study, we evaluated the potential of using the Arabidopsis gai (gibberellic acid insensitive) gene to dwarf apple trees. The gai gene under 35S promoter was introduced in the apple rootstock A2 and the cultivars Gravenstein and McIntosh through Agrobacterium-mediated transformation. One transgenic clone was recovered for Gravenstein and McIntosh, and several transgenic clones for A2, confirmed by Southern blot analysis. Two weak bands were detected by Southern blot analysis in all the untransformed controls, possibly indicating the existence of the internal GAI gene in apple. Most of the transgenic plants showed reduced growth in vitro. Growth analyses in the greenhouse showed a clear reduction in stem length, internode length and node number for the dwarf clones. The normal phenotype of some transgenic clones appears to be associated with silencing of the introduced gai gene, confirmed by RT–PCR analysis. In general, transgenic clones showed reduced rooting ability, especially for the extremely compact ones.     

转OvDREB2B基因陆地棉鉴定及其对水分渗透胁迫的分析

通过花粉管通道技术,以该实验室自育陆地棉品系TH₁和TH₂为材料,将诸葛菜(Orychophragmus vidaceus)抗逆转录因子OvDREB2B基因构建到植物表达载体后,导入棉花基因组,经卡那霉素筛选和分子鉴定表明目的基因已整合到棉花基因组中并表达。将T₁代转基因植株和受体对照在温室中栽培,待植株生长至四叶一心时,用不同渗透势的PEG-6000水溶液进行渗透胁迫处理,分析探讨转基因植株的抗旱效果及其抗旱机理。结果显示:当渗透势为0和0.5 MPa处理时,转基因植株和对照无明显差异;当渗透势为0.8 MPa和1.1 MPa处理时转基因植株较对照抗旱性明显提高。当渗透势为1.1 MPa处理96 h时,对照植株F_v/F_m降至0.2左右,而转基因植株仍正常生长,F_v/F_m值约为0.51,而且初始荧光（F₀）值、净光合速率（P_n）、胞间CO₂浓度（C_i）、蒸腾速率（T_r）等一系列参数转基因植株都明显优于对照,表明DREB2B基因能够提高棉花对水分胁迫的耐受性。     

Highly efficient transformation of the GFP and MAC12.2 genes into precocious trifoliate orange (Poncirus trifoliata [L.] Raf), a potential model genotype for functional genomics studies in Citrus

Precocious trifoliate orange (Poncirus trifoliata [L.] Raf), an extremely early flowering mutant of P. trifoliata, is an attractive model for functional genomics research in Citrus. A procedure for efficient regeneration and transformation of this genotype was developed by using green fluorescent protein (GFP) gene as visual marker and etiolated stem segments as explants. In vivo monitoring of GFP expression permitted a rapid and easy discrimination of transgenic shoots and escapes. Transformation efficiency was 20.7% and the transformants were identified by polymerase chain reaction (PCR) and Southern blot analysis. Moreover, the transgenic lines expressed variable amounts of the GFP gene as revealed by real-time PCR analysis. Fifteen transgenic plants flowered 18 months after transfer to the greenhouse and six of them set fruits. GFP expression was also observed in the transgenic flowers and fruits. To test the utility of this system for functional genomics studies, an Arabidopsis thaliana MAC12.2 gene with the potential to produce seedless fruits was introduced into this genotype, and the traits of the transgenic fruits were characterized. The successful transformation of this perennial woody genotype with extremely short juvenility will allow us to test the function of cloned genes in citrus, the improvement of which is hindered by a long juvenility period.     

Agrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots

This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the uidA gene coding for -glucuronidase was used as an indicator in the optimization of the protocol. Subspecies (onion and shallot) and cultivar were important factors for a successful transformation: shallot was better than onion and for shallot cv. Kuning the best results were obtained. Also, it was found that constantly reducing the size of the calli during subculturing and selection by chopping, thus enhancing exposure to the selective agent hygromycin, improved the selection efficiency significantly. Furthermore, callus induction medium and co-cultivation period showed a significant effect on successful stable transformation. The usage of different Agrobacterium strains, callus ages, callus sources and osmotic treatments during co-cultivation did not influence transformation efficiency. The highest transformation frequency (1.95%), was obtained with shallot cv. Kuning. A total of 11 independent transformed callus lines derived from zygotic embryos were produced: seven lines from shallot and four lines from onion. Large differences in plantlet production were observed among these lines. The best line produced over 90 plantlets. Via PCR the presence of the uidA and hpt (hygromycin phosphotransferase) genes could be demonstrated in these putative transformed plants. Southern hybridization showed that most lines originated from one transformation event. However, in one line plants were obtained indicating the occurrence and rescue of at least three independent transformation events. This suggested that T-DNA integration occurred in different cells within the callus. Most transgenic plants only had one copy of T-DNA integrated into their genomes. FISH performed on 12 plants from two different lines representing two integration events showed that original T-DNA integration had taken place on the distal end of chromosomes 1 or 5. A total of 83 transgenic plants were transferred to the greenhouse and these plants appeared to be diploid and normal in morphology.     

Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens-mediated transformation

Lolium temulentum L. (Darnel ryegrass) has been proposed to be used as a model species for functional genomics studies in forage and turf grasses, because it is a self-fertile, diploid species with a short life cycle and is closely related to other grasses. Embryogenic calluses were induced from mature embryos of a double haploid line developed through anther culture. The calluses were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harboring pCAMBIA1301 and pCAMBIA1305.2 vectors were used to infect embryogenic callus pieces. Hygromycin was used as a selection agent in stable transformation experiments. Hygromycin resistant calluses were obtained after 4–6 weeks of selection and transgenic plants were produced in 10–13 weeks after Agrobacterium-mediated transformation. Fertile plants were readily obtained after transferring the transgenics to the greenhouse. Transgenic nature of the regenerated plants was demonstrated by Polymerase chain reaction (PCR), Southern hybridization analysis, and GUS staining. Progeny analysis showed Mendelian inheritance of the transgenes. The transformation system provides a valuable tool for functionality tests of candidate genes in forage and turf grasses.     

<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.     

Expression of the B subunit of <Emphasis Type="Italic">E. coli</Emphasis> heat-labile enterotoxin in tobacco using a herbicide resistance gene as a selection marker

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l^-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants.     

Extended flower longevity of Petunia hybrida plants transformed with boers,a mutated ERS gene of Brassica oleracea

Petunia x hybridaHort.Vilm.-Andr. was transformed with boers, a mutatedallele of BOERS, an ethylene receptor sensor gene ofBrassica oleracea.boers was obtained by removing anEcoRI cutting site with a silent mutation at Gly-521 andintroducing a point mutation at Ile-62, replacing isoleucine withphenylalanine. Transformation was Agrobacterium tumefaciens mediated.Hygromycin resistant regenerants were tentatively confirmed as transformants byPCR's for HPH and boers and moredefinitively by Southern hybridization of genomic DNA with pBOERS4421. Flowersof transgenic plants retained turgidity and pigmentation considerably longerthan those of untransformed controls, whether left undisturbed on plants orexcised and placed in water. Furthermore, flowers were unaffected by exposureto exogenous ethylene. Excised shoots of transgenic plants released considerablymore ethylene than those of untransformed plants. Transformed plants alsoproduced apparently larger flowers. Unexpectedly higher mortality was observed,suggesting that the ethylene insensitive petunia plants were also lower indisease resistance.     

Rapid and reproducible Agrobacterium-mediated transformation of sorghum

A rapid and reproducible Agrobacterium-mediated transformation protocol for sorghum has been developed. The protocol uses the nptII selectable marker gene with either of the aminoglycosides geneticin or paromomycin. A screen of various A. tumefaciens strains revealed that a novel C58 nopaline chromosomal background carrying the chrysanthopine disarmed Ti plasmid pTiKPSF₂, designated NTL₄/Chry5, was most efficient for gene transfer to sorghum immature embryos. A NTL₄/Chry5 transconjugant harboring the pPTN290 binary plasmid, which carries nptII and GUSPlus ^TM expression cassettes, was used in a series of stable transformation experiments with Tx430 and C2-97 sorghum genotypes and approximately 80% of these transformation experiments resulted in the recovery of at least one transgenic event. The transformation frequencies among the successful experiments ranged from 0.3 to 4.5%, with the average transformation frequency being approximately 1% for both genotypes. Over 97% of the transgenic events were successfully established in the greenhouse and were fully fertile. Co-expression of GUSPlus ^TM occurred in 89% of the transgenic T₀ events. Seed set for the primary transgenic plants ranged from 145 to 1400 seed/plant. Analysis of T₁ progeny demonstrated transmission of the transgenes in a simple Mendelian fashion in the majority of events.     

Promotion of flowering and morphological alterations in Atropa belladonna transformed with a CaMV 35S-rolC chimeric gene of the Ri plasmid

Summary Kanamycin-resistant plants of belladonna (Atropa belladonna) were obtained after Agrobacterium mediated transformation. When a rolC gene, which is one of the loci located on Ri plasmid of Agrobacterium rhizogenes, was co-introduced with a kanamycin resistant (NPT II) gene under control of a cauliflower mosaic virus 35S promoter, the rolC gene was expressed strongly in leaves, flowers, stems and roots. The transformed plants exhibited dramatic promotion of flowering, reduced apical dominance, pale and lanceolated leaves and smaller flowers. On the other hand, when native rolC gene was co-introduced with NPT II, the transgenic plants obtained did not exhibit the altered phenotypes observed in 35S-rolC transformants, and the expression level of the rolC gene was much lower than in 35S-rolC transformants. These results suggest that the morphological changes in transgenic Atropa belladonna were related to the degree of expression of the rolC gene.Abbreviations native rolC rolC gene under control of its own promoter - 35S-rolC rolC gene under control of a cauliflower mosaic viras 35S promoter     

Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens

A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in the T-DNA. The efficiency of gene transfer varied with the kinds of gelling agents and the basic compositions of co-cultivation media. The highest activity of GUS after co-cultivation was observed when NB medium solidified with agarose was used. For the subsequent cultures, two types of media (modified NB and CC) were chosen to recover hygromycin-resistant cells efficiently. The transformation protocol thus developed worked very well in all of the varieties tested in this study, and the transformation frequency (number of independent hygromycin-resistant and GUS-positive plants per embryo) reached more than 30% in IR8, IR24, IR26, IR36, IR54, IR64, IR72, Xin Qing Ai 1, Nan Jin 11, and Suewon 258. Most of the transformants (T₀) were normal in morphology and fertile. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the T₀ and T₁ generations. For the recovery of multiple independent transgenic events from a single immature embryo, procedures were developed to section the embryo into as many as 30 pieces after non-selective cultures following co-cultivation. Transformants were then obtained from the pieces cultured on the selective media, and, in the highest case, more than seven independent transgenic plants per original embryo (transformation frequency of 738%) were produced. Thus, the efficiency of transformation was remarkably improved.     

A family 19 chitinase (Chit30) from Streptomyces olivaceoviridis ATCC 11238 expressed in transgenic pea affects the development of T. harzianum in vitro

Embryo axes excised from mature seeds of pea (Pisum sativum L.) cv. ‘Sponsor’ were used as explants for Agrobacterium-mediated transformation using pGreenII 0229 binary vectors. The vectors harbored a chimeric chitinase gene (chit30), driven by the constitutive 35S promoter or the elicitor inducible stilbene synthase (vst) promoter from grape (Vitis vinifera L.). The secretion signal of the bacterial chitinase gene from Streptomyces olivaceoviridis ATCC 11238 (DSM 41433) was replaced by the A. thaliana basic chitinase leader sequence. Functional properties of the recombinant gene were tested in tobacco as a model system before the long process of pea transformation was undertaken. Several transgenic pea clones were obtained and the transgenic nature confirmed by different molecular methods. The accumulation and activity of chitinase in stably transformed plants were examined by Western blot analysis and in-gel assays, which showed the presence of an additional 3 isoform bands. Using in vitro bioassays with Trichoderma harzanium as a model, we found an inhibition or delay of hyphal extension, which might indicate enhanced antifungal activity compared with non-transformed pea plants. Up to the 4th generation, the transgenic plants did not show any phenotypic alterations compared with non-transgenic control plants.     

Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection with Agrobacterium tumefaciens

The efficiency of transformation was improved by treating immature embryos with heat and centrifugation before infection with Agrobacterium tumefaciens in rice and maize. Because the effects were detected both in the levels of transgene expression after co-cultivation and in the number of independent transgenic plants obtained per embryo, conditions were first optimized based on the transgene expression, and then transformants were produced. The optimal conditions varied considerably depending on species and genotypes, but reasonably good parameters were identified for Japonica rice, Indica rice or maize. As a general tendency, the effect of centrifugation was greater than that of heat in Japonica rice, whereas that of heat was greater than that of centrifugation in Indica rice and maize A188, and the combination of the treatments was the most effective in all of the genotypes tested. The frequency of transformation was improved several fold in rice and maize. In addition, transformation of certain genotypes of maize, which were not transformable before, and transformation of maize with a less efficient vector, which could not transform maize before, became possible by these pre-treatments. In the highest case, 18 independent transgenic plants were obtained from a single immature embryo of Japonica rice. Although nothing is known about the mechanism, these pre-treatments seemed to render cells of rice and maize more competent for transformation mediated by A. tumefaciens.     

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.