Ann Callahan

Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene

SummaryPlum hypocotyl slices were transformed with the coat protein (CP) gene of plum pox virus (PPV-CP) following cocultivation with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPVCP-33. This binary vector carries the PPV-CP gene construct, as well as the chimeric neomycin phosphotransferase and β-glucuronidase genes. Integration and expression of the transferred genes into regenerated plum plants was verified through kan resistance, GUS assays, and PCR amplification of the PPV-CP gene. Twenty-two transgenic clones were identified from approximately 1800 hypocotyl slices. DNA, mRNA, and protein analyses of five transgenic plants confirmed the integration of the engineered CP gene, the accumulation of CP mRNA and of PPV-CP-immunoreactive protein. CP mRNA levels ranged from high to undetectable levels, apparently correlated with gene structure, as indicated by DNA blot analysis. Western analysis showed that transgenic plants produced amounts of CP which generally correlated with amounts of detected mRNA.

Seasonal expression of a dehydrin gene in sibling deciduous and evergreen genotypes of peach (Prunus persica [L.] Batsch)

A cDNA library was created from cold-acclimated bark tissue of peach and selectively probed using an antibody directed against the lysine-rich consensus region of dehydrin proteins. Several clones were thus obtained which had a high degree of sequence similarity to other dehydrin genes. Northern analysis, using clone 5a, indicated that a 1.8 kb transcript was seasonally expressed in sibling deciduous and evergreen genotypes of peach, and also inducible by water deficit in cv. Rio Oso Gem. The evergreen and deciduous genotypes differ significantly in both their ability to cold-acclimate and in the seasonal expression of the dehydrin transcript and protein. In both genotypes, the transcript was maximally expressed during winter and undetectable in May-July. The evergreen genotype (less cold-tolerant), however, displayed transcript accumulation which lagged behind and declined sooner than in the deciduous genotype. Protein expression was similar to transcript expression, however, protein expression in the evergreen genotype lagged considerably behind transcript accumulation in the fall. This indicates that several levels of regulation of dehydrin proteins may exist during cold acclimation. A genomic clone (G10a) was isolated which contained the full-length dehydrin gene, designated ppdhn1. The peach dehydrin gene encodes 472 amino acids with a predicted size of 50 020 Da. The encoded protein (PCA60) contains nine of the lysine-rich repeats characteristic of dehydrins and two DEYGNP motifs at the amino acid terminus. A genomic blot, probed with clone 5a under stringent conditions, indicated that one or two highly homologous genes are present in peach, whereas an additional member was detected under low-stringency conditions. It is suggested that several members of the dehydrin gene family may exist in peach that vary in their relation to ppdhn1.

Development and Regulation of the Plum Pox Virus Resistant Transgenic Plum ‘HoneySweet’

Genetic engineering (GE) can target specific genetic improvements and allow for the development of novel, useful traits. In spite of the potential utility of GE for fruit tree improvement, the technology has not, to date, been widely exploited for variety development due, in part, to the reticence of researchers to become involved in the regulatory process. Over the past 20 years an intensive international research project focused on the development of GE resistance to Plum pox virus (PPV) the causative agent of Sharka, one of the most destructive diseases of plum and other stone fruits. This effort resulted in the development of ‘HoneySweet’ plum, a GE variety that has proven to be highly resistant to PPV, as demonstrated in over 15 years of field testing in the U.S. and Europe. In order to make this variety available to breeders and growers in the U.S., dossiers were submitted to the U.S. regulatory agencies. This process ultimately led to the regulatory approval of ‘HoneySweet’ in the U.S. The work with ‘HoneySweet’ demonstrates that the regulatory process, while a significant effort, can be successfully navigated by public institution researchers. Nevertheless, the few examples of such success demonstrate a need for public institutions to find ways to encourage, support and reward researchers who pursue deregulation efforts. The long-standing successes of virus control in squash and papaya, and the current work with plum demonstrate the power and the safety of GE for specialty crop improvement. The commitment of researchers, institutional support, clear, science-based regulatory frameworks that build upon a developing knowledge base, industry support, and public outreach are components that are now necessary to move this technology forward to improve agricultural production and its sustainability.

Auxin levels and MAX1–4 and TAC1 gene expression in different growth habits of peach

Branch orientation and distribution determine tree architecture that can influence orchard design and management. Peach [Prunus persica L. (Batch)] trees with three different branching genotypes were evaluated: fewer, nearly vertical branches (pillar), less vertical and more spreading branches (upright), and more abundant, least vertical branches (standard). Auxin concentrations and expression of genes that regulate branch development in herbaceous species, MAX1, 2, 3, 4 and TAC1 were determined. Shoots and roots of peach trees in the field and greenhouse were studied following pruning and during periods of growth when bud break and branch spatial orientation develop. Expression of MAX3 and MAX4 decreased in stems of field-grown peach trees that remained on the tree following pruning. In the greenhouse elevated auxin concentrations and higher gene expression of MAX3 in roots and MAX4 in stems were found in pillar rather than standard trees. Upright trees had auxin and MAX1–4 expression that was intermediate between pillar and standard trees. Temporal differences were found with MAX1–4 expression being greater in April or May but auxin concentrations were greater only in shoots in May. Expression of TAC1 was inversely related with auxin concentrations in shoots and was greatest in standard and least in pillar trees. The current work indicates that in stems, auxin, MAX3–4 genes, and TAC1 genes may influence regulatory processes that affect growth and development of peach trees with different growth habits. In addition to breeding, new plant growth regulators that affect the modes of action of root-originating signals may provide new cultural tools for managing tree growth and development.