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Rebecca Grumet

One of the first major successes in the genetic engineering of useful traits into plants has been the engineering of virus resistance. The first example of genetically-engineered virus resistance was published in 1986, since then there have been more than 50 reports of genetically engineered plant virus resistance. These examples span a range of virus types, a variety of plant species, and have utilized several different types of genes. A unique feature of the genetically-engineered virus resistance is that the resistance genes came from the virus itself, rather than the host plant. Most examples have utilized coat protein genes, but more recently, replicase-derived genes have proved highly effective. Other strategies include the use of antisense or sense-defective sequences, and satellite or defective interfering RNAs. This talk will provide an overview of the different approaches, possible mechanisms, the crops and viruses to which they have been applied, and progress toward commercial applications.

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David A. Somers

Genetic engineering offers numerous potentially useful genetic manipulations for the improvement of horticultural crops. Nevertheless, there are several impediments to the efficient integration of genetic engineering into plant improvement programs. The ability to regenerate plants from tissue cultures and, therefore, to genetically engineer most plant species is limited to specific genotypes. This may constrain introduction of genetically engineered traits into crops that have complex genetics, are highly heterozygous, or are propagated by asexual reproduction. Even in crops that are self-compatible, diploids, genotypic specificity of the transformation process often necessitates backcrossing for transfer of genetically engineered traits into elite lines and to reduce problems associated with tissue culture-induced genetic variation. This limits progress in improving other traits compared to other breeding strategies. Other challenges to applying genetic engineering exist. The major genomics initiatives currently underway for gene discovery from a broad range of organisms will provide plant improvers access to most genes. Yet there remains a dearth in tissue-specific, developmentally timed, and environmentally responsive promoters for appropriate expression of introduced genes (transgenes). Furthermore, transgene expression is not as controllable as desired due to transgene silencing. Transgene integration is a random process and further refinements of targeted DNA integration will likely enhance the stability of expression of transgenic traits. The availability of other tools, such as selectable marker genes, will become limiting as multiple transgenic traits are combined within a species. In addition to technical problems, there likely will be problems of access to proprietary technology and testing to meet federal regulations and public acceptance. While these various challenges and limitations currently may constrain progress in application of genetic engineering to horticultural crop improvement, it is foreseeable that with further improvements of genetic engineering technology and development of the appropriate molecular tools, genetic engineering will become a component of most plant improvement programs.

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Kurt D. Nolte, Andrew D. Hanson, and Douglas A. Gage

Proline and various betaines can function as osmoprotectants and cryoprotectants when accumulated in the cytoplasm of cells. Genetic engineering can raise levels of these compounds and thereby improve stress resistance; Citrus species are potential candidates for this. Before attempting such engineering, it is necessary to characterize the natural osmoprotectants of Citrus and related genera. We therefore surveyed 55 cultivated and wild species of the Aurantioideae, analyzing proline and betaines in leaves of mature trees. Some citrus relatives accumulated proline alone; others accumulated proline and proline betaine, as did all Citrus species studied. The levels of these two compounds ranged from about 20 to 100 μmol·g-1 dry mass, and were significantly inversely correlated. Proline betaine is known to be synthesized from proline and to be a better osmoprotectant. Because Citrus species all have more proline than proline betaine, there is scope for engineering more of the latter. Many species had small amounts of hydroxyproline betaine; other betaines were essentially absent. The lack of other betaines means that it would also be rational to engineer the accumulation of glycine betaine or similar compounds.

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Dane K. Fisher, Charles D. Boyer, and Mark Guiltinan

During plant starch biosynthesis, starch branching enzymes (SBE) catalyze a-1,6 branch point formation in starch, and thus are responsible for many properties of the starch polymer. Recently we have cloned cDNAs encoding the two major branching enzymes in developing maize endosperm, SBEI and SBEII. These genes are being used to alter starch biosynthesis via genetic engineering strategies. Transgenic tobacco plants with sense and antisense constructs of SBEI and SBEII have been produced. No major difference in the phenotypes of control and transgenic plants have been observed. Initial experiments demonstrated the transcription of the introduced genes. Enzyme levels and the molecular properties of the starch in the transgenic plants will be determined. These experiments will provide us with information as to the role of starch branching enzymes in starch biosynthesis, the feasibility of creating novel starch, and the effect altered starch has on plastid development and photosynthesis.

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Guowei Fang and Rebecca Grumet

Zucchini yellow mosaic virus (ZYMV), a potyvirus, can cause major losses in cucurbit crops. With the goal of genetically engineering resistance to this disease we have engineered the ZYMV coat protein gene into a plant expression vector. The complete coat protein coding sequence, or the conserved core portion of the capsid gene, was attached to the 5' untranslated region of tobacco etch virus (TEV) in the pTL37 vector (Carrington et al., 1987, Nucl. Acid Res. 15:10066) The capsid constructs were successfully expressed by in vitro transcription and translation systems as verified by SDS-PAGE and ZYMV coat protein antibody. The constructs were then subcloned using polymerase chain reaction and attached to the CaMV 35 S transcriptional promoter on the CIBA-GEIGY pCIB710 plasmid. The constructs containing the CaMV 35S promoter, the 5' untranslated leader of TEV, and ZYMV coat protein sequences were then put between the Agrobacterium tumefaciens left and right borders in the pCIB10 vector and transferred to A. tumefaciens strain LBA4404 by triparental mating. These vectors are now being used to transform muskmelon and cucumber; resultant transgenic plants will be tested for ZYMV coat protein expression.

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B.H. McCown, E.T. Jordan, C.H. Chen, D.D. Ellis, and R.D. Vierstra

Although the size of pot mums can be controlled with retardants, the use of such chemicals may become limited. Genetically dwarfing current cultivars may be an alternative. Using a construct including a chimeric oat phytochrome structural gene, tobacco phenotypes have been produced that strongly resemble retardant-treated plants. We wished to insert this construct in mum by using particle bombardment and determine the effects on plant size and flowering dynamics. A target system was developed using `Iridon' mum leaf sections regenerated on an IAA/BA medium. Shoots developed from surface cells principally at the cut edges. Regenerates were grown-on through flowering and no visual aberrations were apparent. Levels of 50 to 100 mg/l kanamycin were inhibitory to bud development. Sections were exposed to gene transfer and shoots recovered that appear resistant to kanamycin. Some appear chimeric while others appear to be escapes stimulated by a `feeder' effect from nearby transformed cells. Further analyses will determine whether some plants are stably transformed. (Supported by a Duffett Research Grant from Yoder Brothers, Inc.)

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Steven E. Lindow

Genes determining the ability of the bacterium Pseudomonas syringae to catalyze ice formation have been cloned and characterized. Ice nucleation active (Ice+) strains of this species are common on plants and the supercooling ability of frost sensitive plants is inversely proportional to the logarithm of the population size of Ice+ bacteria at temperatures above -5C. Recombinant Ice- derivatives off. syringae were produced by site-directed mutagenesis using deletion containing ice genes cloned form this species. The Ice- strains colonized potatoes well in field studies, reduced the population size of Ice+ bacterial strains by about 50-fold, and reduced the incidence of frost injury an average of 82% in several radiative frosts of temperatures in the range of -3 to -5 C. The ice gene has also been introduced into Solanum commersonii to determine its effect on increasing the tolerance of ice formation in this frost tolerant species. Transgenic plants exhibit a much higher threshold ice nucleation temperature than the parental plants.