Search Results
Embryogenic callus cultures of three genetically diverse cultivars of rose (Rosa hybrida L.), the floribunda `Trumpeter', the multiflora `Dr. Huey', and the hybrid tea `Tineké', were used to study the effect of various carbohydrates and osmotically active compounds on somatic embryo maturation and conversion. Cotyledonary-stage embryos were produced by dispersing callus in liquid medium followed by filtration to isolate globular-stage embryos. Quantitative experiments were conducted to determine maturation and conversion of the three rose cultivars in response to medium with sucrose, glucose, fructose, or maltose as the primary carbon source and also in response to various concentrations of either myo-inositol, polyethylene glycol, or mannitol in combination with 3% sucrose. Conversion of 27% was achieved for `Trumpeter' embryos following their maturation on 3% fructose. `Dr. Huey' embryos required maturation on medium containing 3% sucrose supplemented with either 2.5% or 5% mannitol for 36% and 61% conversion, respectively. Maturation of `Tineké' embryos on either 3% sucrose, 3% glucose, or 3% fructose resulted in a maximum 12% conversion.
Slow-growing compact calluses were initiated from bulb scales of Lilium longiflorum cv. Nellie White that had been cultured for at least 6 months on Murashige and Skoog (MS) medium with 9 μm dicamba. To develop a reliable selection system, the sensitivity of nontransformed calluses and in vitro plants to different selective agents such as phosphinothricin, kanamycin, geneticin, paromomycin, and hygromycin was tested when grown on MS medium. Nontransformed calluses showed high sensitivity to 0.5 mg·L−1 phosphinothricin, 25 mg·L−1 geneticin, and 5 mg·L−1 hygromycin. Nontransformed plants grown in vitro died on either 2 mg·L−1 phosphinothricin or 75 mg·L−1 hygromycin. Plants did not die when grown on either 200 mg·L−1 kanamycin or 100 mg·L−1 geneticin, and 100 mg·L−1 paromomycin stimulated plant growth. Transformation was achieved using biolistics on callus bombarded with either the bar-uidA fusion gene under control of the CaMV 35S promoter or npt II and uidA under control of the CaMV 35S promoter. One week after biolistic bombardment, callus bombarded with the bar-uidA fusion gene was cultured for 1 month on MS medium supplemented with 9 μm dicamba and 0.1 mg·L−1 phosphinothricin and then transferred to 0.2 mg·L−1 phosphinothricin for 1 month followed by 1.0 mg·L−1 for the next 4 months. Regenerating shoots and well-established plants were cultured on MS medium lacking hormones and with either 0.2 mg·L−1 or 2.0 mg·L−1 phosphinothricin, respectively. Callus bombarded with the npt II gene was cultured on MS medium with 50 mg·L−1 geneticin until shoots regenerated. Regenerated shoots were cultured on MS medium lacking hormones. Under optimal conditions, 10 transgenic plants were selected from seven plates of callus bombarded with the bar-uidA fusion gene using phosphinothricin for selection. Both Southern hybridization of genomic DNA and polymerase chain reaction analysis verified the presence of the transgene in transformed ‘Nellie White’ plants. Transgenic plants were phenotypically normal, and they were crossed with nontransformed plants of L. longiflorum cvs. Sakai, Yin tung, Sakai, and Flavo. The presence of the bar gene in 41% of the T1 progeny plants confirmed stable integration of the transgene into the genomic DNA of transgenic lily plants. β-glucuronidase expression resulting from the uidA gene was demonstrated in leaves and roots of some of the transgenic lily plants by histochemical staining, determination of the specific activity of the β-glucuronidase enzyme, and Northern hybridization.
A genetic transformation system has been developed utilizing Gladiolus suspension cells bombarded with high-velocity DNA coated particles. Transient β-glucuronidase (GUS) expression levels from the uidA gene were measured to assess efficiency of DNA delivery following particle bombardment. We have determined that 1) non-regenerable cells express higher levels of GUS activity than regenerable cells; 2) gold particles are superior to tungsten for DNA delivery; 3) maximum GUS expression occurs in suspension cells bombarded 10-13 days after subculture; and 4) incubation of bombarded cells in osmoticum-containing medium after particle bombardment enhances GUS expression levels. Several gene promoters have been evaluated for their ability to direct GUS expression in Gladiolus and a putative stably-transformed Gladiolus callus has been isolated.
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.
Over one hundred Gladiolus plants of the commercially important cultivars `Jenny Lee' and `Peter Pears' have been stably transformed following particle bombardment on regenerable callus, suspension cells, or cormel slices. The phosphinothricin acetyltransferase gene which confers phosphinothricin resistance was cotransformed with either antiviral genes or the uidA reporter gene coding for β- glucuronidase expression. Transformed plants were regenerated following selection on concentrations of phosphinothricin which varied with the type of tissue used for bombardment. Integration of foreign DNA was confirmed by polymerase chain reaction and gene expression.
The main goal of this research was to develop Campanula glaumerata `Acaulis' plants transformed with the isopentenyl transferase (ipt) gene for increased growth of the axillary buds and en-hanced insect resistance. Isopentenyl transferase is a first enzyme in the cytokinin biosynthetic pathway. For Campanula transformation, leaf discs were co-cultivated with Agrobacterium tumefacience LBA4404, which harbored the binary vector pBC34 (A. Smigocki, Beltsville, Md.) that codes for the nos-nptII gene and the ipt gene controlled by the CaMV35S promoter. The transformation frequency was about three times higher when leaf blade explants were infected with LBA4404 containing pBC34 as compared to infection with pGUSINT, which contains the gusint gene instead of the ipt gene. This difference in transformation frequency was attributed to expression of cytokinin from the ipt gene. Transgenic plant lines containing the ipt gene were verified by southern hybridization and divided into three groups by phenotype following culture in vitro on MS medium: 1) yellow/large leaves, no rooting; 2) green/large leaves, no rooting; 3) green/normal leaf size, rooting. These different phenotypes could be due to different levels of cytokinin expression in the transgenic plants.