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Ann C. Smigocki and Freddi A. Hammerschlag

Immature `Redhaven' peach [Prunus persica (L.) Batsch] embryos were infected with a shooty mutant strain of Agrobacterium tumefaciens, tms328::Tn5, which carries an octopine-type Ti plasmid with a functional cytokinin gene and a mutated auxin gene. Shoots were regenerated from embryo-derived callus that was initiated on MS medium lacking phytohormones. Shoots exhibited increased frequency of branching and were more difficult to root than the noninfected. Transcripts of the tms328::Tn5-cytokinin gene were detected using northern analyses of total plant RNA. Polymerase chain reaction of genomic DNA and cDNA resulted in amplification of DNA fragments specific for the cytokinin gene, as determined by restriction enzyme and Southern analyses. The concentrations of the cytokinins zeatin and zeatin riboside in the leaves of regenerated plants were on the average 51-fold higher than in leaves taken from nontransformed plants. None of the shoots or callus tissues were postive for octopine. The expression of the T-DNA encoded cytokinin gene promotes growth of peach cells in the absence of phytohormones, thus serving as a marker for transformation. In addition, this gene appears to promote morphogenesis without an auxin inductive step.

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Ahmad A. Omar, Wen-Yuan Song, James H. Graham, and Jude W. Grosser

Citrus canker disease caused by the bacterial pathogen Xanthomonas axonopodis pv. citri is becoming a worldwide problem. Xa21 gene is a member of the Xa21 gene family of rice, which provides broad spectrum Xanthomonas resistance in rice. `Hamlin' sweet orange [Citrus sinensis (L.) Osbeck) is one of the leading commercial cultivars in Florida because of its high yield potential and early maturity. `Hamlin' also has a high regeneration capacity from protoplasts and is often used in transformation experiments. Since the citrus canker pathogen is in the same genus, this gene may have potential to function against canker in citrus. The wild-type Xa21 gene contains an intron, and there are some questions whether dicot plants can process genes containing monocot introns (the cDNA is intron-free). Plasmids DNA, encoding the non-destructive selectable marker EGFP (Enhanced Green Fluorescent Protein) gene and the cDNA of the Xa21 gene were transformed or co-transformed into `Hamlin' orange protoplasts using polyethylene glycol. More than 200 transgenic embryoids were recovered. More than 400 transgenic plants were developed from 75 independent transgenic events. PCR analysis revealed the presence of the cDNA of the Xa21 and the GFP genes in the transgenic plants. Some of the plants have the GFP only. Southern analysis is showing integration of the cDNA into different sites ranges from one to five sites. Western analysis is showing the expression of the cDNA of the Xa21 gene in the transgenic citrus plants. This is the first time that a gene from rice has been stably integrated and expressed in citrus plants. Canker challenge assay is in progress.

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M. S. Strefeler

Genetic transformation of cut roses may greatly facilitate cultivar improvement programs by shortening the time required to introduce new genes into elite germplasm. The biolistic process offers a very promising method for the genetic transformation of roses.

The biolistic process uses high velocity mircoprojectiles (gold or tungsten) to carry foreign DNA into cells. This process has been shown to be useful for genetic transformation of many organisms. The first step in taking advantage of this process is to optimize the factors which affect transformation efficiency.

Several factors that have a significant affect on transformation efficiency were examined in an effort to optimize the biolistic process for gene transfer in roses. The factors examined were type of tissue (leaf segments, petioles, callus, etc), bombardment distance, the number of bombardments, DNA construct and microcarrier velocity.

The reporter gene, GUS, was used for determining transformation efficiency in this study. GUS was carried on several plasmid constructs which also contained antibiotic resistance (kanamycin or streptomycin. Efficiency of gene transfer was determined by calculating the number of transiently expressing GUS cells for each combination of factors.

Results of this study will be discussed and summarized.

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Kathryn Kamo, Alan Blowers, Franzine Smith, Joyce Van Eck, and Roger Lawson

More than 100 transgenic Gladiolus plants were recovered after particle bombardment of regenerable suspension cells and callus. For transformation, Gladiolus callus and suspension cells were co-bombarded with phosphinothricin acetyltransferase-(PAT) and ß- glucuronidase (GUS) -expressing plasmids. Stably transformed calli were selected on medium containing either phosphinothricin (PPT) or bialaphos followed by transfer to a regeneration medium to recover transgenic plants. Stable transformation was confirmed by detection of the PAT gene by DNA gel blot analysis and by enzymatic assays to measure GUS activity. In general, particle bombardment of regenerable suspension cells rather than callus resulted in the largest number of transformants. The rate of co-expression for GUS in PPT-resistant plants was high (≈ 70%). Promoters that are typically more efficient in dicotyledonous plants were very active in Gladiolus, a monocotyledonous bulb plant. Establishment of an efficient transformation protocol for Gladiolus will now allow the introduction of transgenes to confer resistance to the viral and fungal pathogens that decrease Gladiolus production.

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Kalyani Dias, Suzanne M.D. Rogers, and Ronald J. Newton

Citrus is one of the major horticultural cash crops in the Southern United States. Several attempts have been made to genetically improve present citrus cultivars using biotechnology. We report transformation of citrus (Citrus sinensis (L.) Osbeck `Hamlin') suspension cultures using microprojectile bombardment.

A thin layer of seven day-old suspension cultures, grown in HH medium, were transferred to Whatman #1 filter paper at a cell density of 0.15 mg/ml fresh weight. The cells were bombarded with 1.112μM diameter, DNA-coated, tungsten particles using the Dupont PDS 1000 biolistic system. Plasmid PRT 99 GUS containing the marker genes β-Glucuronidase (GUS) and NPT II, with a 35S promoter, and NPT II terminators was used in transformation.

GUS activity was monitored on a time scale. Expression of GUS was observed after 48 hours of bombardment. Further studies are being done to enhance the transformation efficiency.

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Ravindra K. Hajela, Neerja Hajela, Mark G. Bolyard, Wayne M. Barnes, and Mariam B. Sticklen

A simple gene transfer method based on Agrobacterium -mediated transformation of adventitious multiplication of Juneberry (Amelanchier laevis L.) basal shoots is described. Evidence is presented for successful integration and expression of a transformed gene in greenhouse-grown transgenic plants. This method can transform woody perennials that are difficult to regenerate from leaf disks, protoplasts, or other tissue culture regimens.

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M.J. Bosela, J.P. Schnurr, Z.-M. Cheng, and W.A. Sargent

Three elite hybrid aspen, Populus grandidentata × P. canescens, P. tremuloides × P. tremula, and P. tremuloides × P. davidiana, have been transformed with Agrobacterium tumefaciens strains LBA4404 and EHA105 carrying kanamycin resistance and GUS genes. The leaves of micropropagated shoots were co-cultivated with Agrobacterium for 65 to 72 hr and then transferred to callus-induction medium with 80–120 mg/L kanamycin in the dark. After 2 weeks, the leaves were transferred to shoot-induction medium under 18-hr photoperiod. Regenerated shoots were verified for transformation by histochemical staining and PCR. Transformed shoots rooted and were transplanted to soil. The three hybrid clones differed widely in their medium requirements for regeneration and in their competence for transformation. The leaves of P. grandidentata × P. canescens callused vigorously on a wide variety of media. In a typical transformation experiment, 30% to 60% of infected leaves produced putatively transformed calli (up to 10 calli per leaf). The origin of these calli and the frequency of shoot formation depended on the Agrobacterium strains. The calli from EHA105-infected leaves produced shoots within six weeks of co-cultivation and at high frequencies (70% to 90%). However, the calli from LBA4404-infected leaves produced shoots more slowly and at much lower frequencies (5% to 10%). Delaying selection for 2 weeks was found to lower the transformation frequency. Putatively transformed calli were obtained from P. tremuloides × P. tremula, and P. tremuloides × P. davidiana hybrids at frequencies of only 2% to 3%. The calli regenerated from P. tremuloides × P. davidiana leaves were very small, but they continued to grow upon being transferred to shoot-induction media and have started to produce shoots. The calli from leaves of P. tremuloides × P. tremula were much larger and they produced shoots more quickly. This transformation protocol is currently being used to introduce rooting genes into these hybrids to improve their rooting from hardwood cuttings.

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Leighan Howard, Philip Stewart, Amit Dhingra, Craig Chandler, and Kevin Folta

Cultivated strawberry (Fragari×ananassa) is a valuable crop, yet has benefitted little from recent advances in biotechnology and genomics. A high-throughput system for transformation and regeneration would hasten elucidation of gene function for strawberry and possibly the Rosaceae in general. In this report, a protocol for high-frequency octoploid strawberry transformation and regeneration is presented. The protocol uses leaf, petiole, and stolon as explants from a newly selected genotype, `Laboratory Festival #9'. This genotype was selected from progeny of a `Strawberry Festival' self-cross exclusively for its rapid regeneration and robust growth in culture. Direct organogenesis has been achieved from the leaf or from prolific callus with multiple shoots being visible in as few as 14 days. Over 100 viable regenerants may be obtained from a single leaf explant of about 3-cm2 size. This laboratory-friendly genotype allows high-throughput, statistically relevant, studies of gene function in the octoploid strawberry genetic background as well as generation of large transgenic populations.

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Vellicce Gabriel Ricardo, Yamilet Coll, Atilio Castagnaro, and Juan Carlos Diaz Ricci

A protocol for shoot regeneration of strawberry (Fragaria ×ananassa Duch. `Pajaro') leaf disks was developed. In Murashige and Skoog basal medium with 3% of sucrose (w/v), BA (1 mg·L-1), and 2,4-D (0.1 mg·L-1), 70% of the cultivated leaf explants regenerated plants. This regeneration system was used for genetic transformation of strawberry with Agrobacterium tumefaciens strain LBA 4404 carrying the binary vector plasmid pBI121 that contains the npt II (neomycin phosphotransferase) and uidA (ß-D glucuronidase) genes. A transformation rate of 6.6%, calculated as the number of leaf disks able to regenerate kanamycin-resistant plants/total leaf disks infected, was obtained. The integration of both marker genes was evaluated in each transformed line by PCR (polymerase chain reaction) amplification of the npt II and uidA genes. High expression levels of the uidA gene were found in leaves, flower, and fruits of the transgenic lines. The protocol here reported may represent a way to conduct transformation research in strawberries. Chemical names used: benzyladenine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D).

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Xiaojian Ye, Susan K. Brown, Ralph Scorza, John Cordts, and John C. Sanford

Physical and biological parameters affecting the efficiency of biolistic transformation of peach were optimized using ß-glucuronidase (GUS) as a reporter gene, such that efficiency of transient GUS expression in peach embryo-derived callus was increased markedly. Transient expression was also obtained in embryonic axes, immature embryos, cotyledons, shoot tips, and leaves of peach. Stable expression of a fusion gene combining neomycin phosphotransferase (NPTII) and ß-glucuronidase activities has been achieved in peach embryo calli. Sixty-five kanamycin-resistant callus lines were obtained from 114 pieces of bombarded calli after 4 months of selection. Nineteen of the 65 putative transformant lines produced shoot-like structures. Seven lines were examined to confirm stable transformation using the colorimetric GUS assay and PCR analysis. All seven lines showed GUS activity. PCR analysis confirmed that, in most of the putative transformants, the chimeric GUS/NPTII gene had been incorporated into the peach genome. The transgenic callus lines were very weakly morphogenic, presumably because the callus was 5 years old and no transgenic shoots developed from this callus. Results of this research demonstrate the feasibility of obtaining stable transgenic peach tissue by biolistic transformation.