Franzine Smith

Biolistic transformation of prokaryotes: factors that affect biolistic transformation of very small cells.

Five bacterial species were transformed using particle gun-technology. No pretreatment of cells was necessary. Physical conditions (helium pressure, target cell distance and gap distance) and biological conditions (cell growth phase, osmoticum concentration, and cell density) were optimized for biolistic transformation of Escherichia coli and these conditions were then used to successfully transform Agrobacterium tumefaciens, Erwinia amylovora, Erwinia stewartii and Pseudomonas syringae pv. syringae. Transformation rates for E. coli were 10(4) per plate per 0.8 micrograms DNA. Although transformation rates for the other species were low (less than 10(2) per plate per 0.8 micrograms DNA), successful transformation without optimization for each species tested suggests wide utility of biolistic transformation of prokaryotes. E. coli has proven to be a useful model system to determine the effects of relative humidity, particle size and particle coating on efficiency of biolistic transformation.

Optimization of biolistic transformation of embryogenic grape cell suspensions

Embryogenic suspensions of ‘Chancellor’ (Vitis L. complex interspecific hybrid) were bombarded with tungsten particles coated with plasmid pBI426 encoding ß-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) which results in kanamycin resistance. Two d after bombardment, cultures were placed on semi-solid medium containing either 8.6 or 17.2 μM kanamycin. Factors that affect biolistic transformation rates were studied. Tungsten microprojectiles with a mean diameter of 1.07 μm (M10) resulted in more transient gene expression than 0.771 μm diameter particles. Using M10 particles, helium pressures of 1000 and 1200 psi yielded more GUS-expressing colonies per plate than did 800 psi 2 d following bombardment. The number of transformants present after 34 d was not affected by the helium pressure. The distance between the particle launch site and the target cells, and the number of days between the last cell subculture and bombardment, did not affect the numbers of transient and long term GUS expressing colonies. The addition of 3 g/l of activated charcoal to the post-bombardment medium increased long term GUS expression four fold. Wrapping the plates after bombardment with Parafilm increased long term GUS expression three fold compared with plates wrapped with a porous venting tape. With up to 850 transformed callus colonies per plate 23 d after bombardment, the biolistic device holds much promise as a method to achieve stable transformation of grapevines.

Stable transformation of Gladiolus by particle gun bombardment of cormels

Transgenic plants of Gladiolus were produced following particle bombardment of cormel slices. Plant cells were cotransformed with the gene for phosphinothricin acetyltransferase under control of the cauliflower mosaic virus 35S promoter and the uidA gene coding for β-glucuronidase (GUS) under control of the actin promoter isolated from rice. The optimum concentration for the first selection of transformed plants was 8 mg/l phosphinothricin which resulted in 14% of the bombarded cormel slices regenerating plants that were transformed as confirmed by polymerase chain reaction amplification. Polymerase chain reaction amplification, Southern hybridization and histochemical staining for GUS gene expression on plants after two selective screenings with phosphinothricin indicated that regenerated plants were transformed. Histochemical staining for GUS gene expression showed that the actin promoter resulted in GUS gene expression primarily in callus cells and root meristems. Leaves were typically chimeric for GUS gene expression.

Importance of Biotechnology to the Horticultural Plant Industry

Abstract Horticulture continues to provide variety, color and flavor to the foods we eat, enriches our lives with many aesthetically appealing products, and adds to sustaining a healthy environment. Biotechnology is introducing new tools to advance the many benefits of horticulture. This review explores some of the benefits of horticulture and provides examples of successes being realized in agricultural biotechnology. The review also introduces some of the exciting work that may some day be available for consumers. A future that includes more nutritious fruits and vegetables; fragrant and more colorful flowers; a broader selection of indoor plants; and thicker, greener lawns that require less mowing and water are quickly becoming more than just a vision. We hope after reading this text, you feel the same way.

Regeneration from long-term embryogenic callus of the Rosa hybrida cultivar kardinal

SummaryMedia components used for three stages of development: (1) callus maintenance, (2) maturation of embryos, and (3) conversion of embryos to plants were shown to affect regeneration of plants for the commercially important red rose cultivar Kardinal. Embryogenic callus was maintained for 5yr on either Schenk and Hildebrandt’s basal salts medium (SH) supplemented with 13.6 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or Murashige and Skoog’s basal salts medium (MS) supplemented with 18.1 μM dicamba and 0.46 μM kinetin. Maturation of embryos was three times higher using callus maintained on the SH medium supplemented with 2,4-D while conversion of cotyledonary-stage embryos to plants was significantly higher (10 times) using callus that had been maintained on MS medium with dicamba and kinetin. Maximum maturation (13.5%), and conversion (15.2%), occurred when callus was cultured on MS maturation medium without hormones. Cotyledonary-stage embryos cultured on MS conversion medium supplemented with abscisic acid (5–20 μM) produced plants that survived at a significantly higher rate (two times) in the greenhouse than when embryos were cultured without abscisic acid. The highest rate of plant regeneration occurred when embryogenic callus of ‘Kardinal’ was maintained on MS medium supplemented with dicamba and kinetin, maturation of embryos occurred on MS maturation medium without hormones, and conversion of cotyledonary-stage embryos occurred on MS conversion medium supplemented with abscisic acid.

Information Loss: Potential for Accelerating Natural Genetic Attenuation of RNA Viruses

Loss of information is not always bad. In this paper, we investigate the potential for accelerating the genetic degeneration of RNA viruses as a means for slowing/containing pandemics. It has previously been shown that RNA viruses are vulnerable to lethal mutagenesis (the concept of inducing mutational degeneration in a given pathogen). This has led to the use of lethal mutagenesis as a clinical treatment for eradicating RNA virus from a given infected patient. The present study uses numerical simulation to explore the concept of accelerated mutagenesis as a way to enhance natural genetic attenuation of RNA viral strains at the epidemiological level. This concept is potentially relevant to improved management of pandemics, and may be applicable in certain instances where eradication of certain diseases is sought. We propose that mutation accumulation is a major factor in the natural attenuation of pathogenic strains of RNA viruses, and that this may contribute to the disappearance of old pathogenic strains and natural cessation of pandemics. We use a numerical simulation program, Mendel’s Accountant, to support this model and determine the primary factors that can enhance such degeneration. Our experiments suggest that natural genetic attenuation can be greatly enhanced by implementing three practices. (1) Strategic use of antiviral pharmaceuticals that increase RNA mutagenesis. (2) Improved hygiene to reduce inoculum levels and hence increase genetic bottlenecking. (3) Strategic use of broad-spectrum vaccines that induce partial immunity. In combination, these three practices should profoundly accelerate loss of biological information (attenuation) in RNA viruses.

Genetic Transformation of Gladiolus

Gladiolus is a monocotyledonous floral bulb crop which ranked fifth in 1993 for the number of stems (79 663) shipped worldwide (USDA 1994). In Florida alone, about 3600 acres are devoted to cut flower production, and about 120 acres to the production of stock corms of Gladiolus (Wilfret 1980). Its production areas are severely plagued by viral, microbial, and fungal pathogens, so that a typical cultivar lasts only a few years before it succumbs to disease and is removed from production. It has not been possible to effectively breed pathogen resistance into Gladiolus using conventional breeding methods so this plant represents a commercially important floral crop that would benefit from disease resistance mediated by genetic engineering.