A high-efficiency regeneration/transformation system was developed for three elite aspen hybrids (Populus tremuloides × P.tremula, P.tremuloides × P.davidiana, and P. × canescens × P. grandidentata). On both modified MS medium for aspen (MSA) and Woody Plant Medium (WPM) supplemented with zeatin (2.0 mg/L) and NAA (1.0 mg/L), nearly 100% leaf explants formed calli, of which 80% to 100% regenerated into shoots on both media with 2.0mg/L zeatin and 0.01 mg/L TDZ. Bacterial concentration, pH value of the co-cultivation medium, and acetosyringone were evaluated for enhancing transformation efficiency. Agrobacterial concentration at 1.0 Absorbance at OD600 was better than at 0.1, 0.5 Abs, yielding 80% and 75% of callus induction rates from agrobacterium harboring CaMV35s and Heat shock promoter constructs, respectively. The pH of co-cultivation medium, ranging 5.0 to 5.9, did not have any effect on transformation frequency. Acetosyringone was added to the co-cultivation medium and/or to the callus induction medium, the induction of kanamycin-resistant callus increased from 70% to 80% to 90% to 100%, and the size of callus also increased. Acetosyringone had no effect on shoot regeneration from kanamycin-resistant calli. Regenerated aspen shoots were screened on the kanamycin-containing medium, and confirmed by GUS histochemical assay. The GUS-positive plants were further confirmed by polymerase chain reaction, showing that the nptII, uidA, and rolB genes were integrated into the aspen genomes.
Wenhao Dai, Zong-Ming Cheng, and Wayne Sargent
Chen-Yu Lin, Kan-Shu Chen, Hsuan-Ping Chen, Hsiang-I Lee, and Ching-Hsiang Hsieh
that no curds would initiate in tropical cauliflower cultivars at temperatures greater than 25 °C and 29 °C. Zenkteler et al. (2012) indicated that devernalized temperatures affected the structure of the shoot meristem during transformation from the
Alexander Vainstein, Morly Fisher, and Meira Ziv
The applicability of β- glucuronidase and chloramphenicol acetyltransferase reporter genes to a carnation (Dianthus caryophyllus L.) transformation procedure, was analyzed. Transgenic tobacco (Nicotiana tabacum L.) plants expressing the respective reporter genes were prepared and used as the enzyme source. Carnation leaf extract strongly inhibited enzymatic activity of β- glucuronidase, but not that of chloramphenicol acetyltransferase. One or more carnation phenolic compounds, acting in a noncompetitive manner, is suggested as the cause of the observed inhibition of fluorometrically assayed β- glucuronidase activity. This inhibition was eliminated by treating the carnation leaf extract with polyvinylpolypyrrolidone.
Joyce Van Eck, Franzine Smith, Ala D. Blowers, and John Sanford
Particle bombardment was investigated as a potential transformation method for Easter lily. Bulb scale explants from Lilium longiflorum Thunb. `Nellie White' were used as target material. The uidA (or gusA) reporter gene for ß- glucuronidase (GUS) expression was used in all particle bombardments to assess efficiency of gene delivery. Parameters examined to achieve optimal levels of transient GUS expression included gene promoter, helium pressure (particle velocity), and target distance. The highest level of transient GUS expression (as measured by number of indigo-stained cells/scale explant) was observed with the rice actin 1 (Act1) promoter, a helium pressure of 1500 psi, and target distances of 9 to 12 cm. Parameters considered for recovery of stable transform ants included the choice of selective agent (phosphinothricin or hygromycin) and their respective selectable resistance genes (phosphinothricin acetyltransferase and hygromycin phosphotransferase), and preculture time of scale explants prior to bombardment. Polymerase chain reaction analysis will be used to screen putative stable transformants, and from this data it will be determined which parameters yielded the highest transformation rates.
Ellen B. Peffley and Melanie A. Hart
Particle bombardment was investigated as a potential transformation method for onion. Seeds of Allium cepa `TG 1015' were planted onto BDS medium and placed in a dark incubator at 25C for germination. Two to 3 weeks after the seeds were germinated, meristems (1 to 2 mm) were excised and placed onto BDS medium containing 2 mg 2,4-D/liter for callus initiation. Callus was transferred monthly onto fresh BDS medium containing 2,4-D until bombardment. The reporter gene for B-glucuronidase (GUS) expression was used to assess efficiency of gene delivery in all particle bombardments. Characteristics examined were target distance and helium pressure (particle velocity). Tissues were subjected to the 5-bromo-4-chloro-3-indoyl-B-D-glucuronide (xgluc) test for detection of GUS activity. Measurements were taken on particle dispersion as affected by target distance and helium pressure. GUS expression was detected in putatively transformed tissues.
Abraham Cruz-Mendívil, Javier Rivera-López, Lourdes J. Germán-Báez, Melina López-Meyer, Sergio Hernández-Verdugo, José A. López-Valenzuela, Cuauhtémoc Reyes-Moreno, and Angel Valdez-Ortiz
first transformation protocol was reported in 1986 ( McCormick et al., 1986 ), this procedure is still far from being a routine technique. Tomato gene transfer is affected by several factors such as Agrobacterium strain and concentration, infection
Sumontip Bunnag, Ruth C. Martin, David W.S. Mok, and Machteld C. Mok
Quince (Cydonia oblonga) is widely used as a dwarfing rootstock for pear (Pyrus communis). We have devised procedures for Agrobacterium-mediated gene transfer using leaf discs of quince. The following factors were particularly important for recovery of shoots after incubation of leaf discs with Agrobacterium: an efficient regeneration system, the use of an effective antibiotic to eliminate Agrobacterium while maintaining a high regeneration frequency, and the choice of selectable markers. The regeneration frequency of control leaf discs on a medium containing 30 μM thidiazuron and 0.3 μM naphthaleneacetic acid was 100%. The frequency decreased linearly with increasing concentrations of antibiotics. Timentin. which consists of ticarcillin and a 6-lactamase inhibitor, was more effective in eliminating Agrobacterium than cefotaxime and carbenicillin. Vectors with the bar gene (bialaphos resistance) were better than those with the npt 11 gene (kanamycin resistance), since kanamycin bleached the leaf discs, resulting in poor regeneration. Bialaphos-resistant quince shoots which were positive in Southern hybridization have been obtained. The same procedures are being applied for transformation of pear.
J.M. Van Eck and E.D. Earle
Regeneration from VFNT Cherry tomato was optimized prior to transformation. Cotyledon and hypocotyl sections from 7-day-old in vitro grown VFNT Cherry tomato were cultured on medium containing MS salts, B5 vitamins and the following per liter: sucrose, 30g; zeatin, 1 mg; IAA, 0.1 mg; agar, 8.0g or GelriteR, 2.2g. Culture conditions investigated included cotyledon and upper hypocotyl explant lengths, light vs. dark germination, and agar vs. GelriteR. The conditions which resulted in the highest average number of shoots per explant were cotyledon basal explants 2 mm in length, 3.95; cotyledons from dark germination, 6.2; and hypocotyls from light germination, 8.6. An equal number of shoots regenerated on medium containing agar or GelriteR, however, shoots regenerated on medium containing agar were more vigorous. Cotyledon and hypocotyl sections were cocultivated with the Agrobacterium tumefaciens binary vector pBI121 containing the neomycin phosphotransferase II (NPTII) and B-glucuronidasc (GUS) genes. Transformants were selected by regeneration and rooting on medium containing kanamycin. Southern blot and PCR analysis indicated regeneranrs contain the NPTII and GUS genes. Mapping the chromosomal location of the NPTII gene is in progress.
John Norelli, JoAnn Mills, and Herb Aldwinckle
In vitro–grown leaves of Malus ×domestica Borkh. cv. Royal Gala were either crush-wounded with forceps, cut, or left whole, and then inoculated with A. tumefaciens strain EHA105 (p35SGUS_INT), with and without vacuum infiltration. Transformation was quantified 13 days after inoculation by determining the rate of β-glucuronidase (GUS) activity. Leaf wounding by crushing with nontraumatic forceps significantly increased transformation when compared with cutting leaves. Vacuum infiltration of inoculum had no effect on transformation.
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.