Stan C. Hokanson, James R. McFerson, Philip L. Forsline, Warren F. Lamboy, James J. Luby, Aimak D. Djangaliev, and Herb S. Aldwinckle
Maureen M.M. Fitch, Terryl C.W. Leong, Xiaoling He, Heather R.K. McCafferty, Yun J. Zhu, Paul H. Moore, Dennis Gonsalves, Herb S. Aldwinckle, and Howard J. Atkinson
Methods to increase transformation efficiency and yields of transgenic Anthurium andraeanum Linden ex. André hybrids were sought while effecting gene transfer for resistance to the two most important pests, bacterial blight (Xanthomonas axonopodis pv. dieffenbachiae) and nematodes (Radopholus similis and Meloidogyne javanica). Differentiated explant tissues, embryogenic calli, and comingled mixtures of the two were transformed with binary DNA plasmid constructs that contained a neomycin phosphotransferase II (nptII) selection gene with a nos promoter and terminator. Explants included ≈1-cm long laminae, petioles, internodes, nodes, and root sections from light- and dark-grown in vitro plants. Bacterial blight resistance genes were NPR1 from Arabidopsis, attacin from Hyalophora cecropia, and T4 lysozyme from the T4 bacteriophage. For nematode resistance, rice cystatin and cowpea trypsin inhibitor genes were used. Cocultivation with Agrobacterium tumefaciens strains EHA105, AGLØ, and LBA4404 ranged from 2 to 14 days. Over 700 independent, putatively transformed lines were selected with 5 and 20 mg·L−1 geneticin (G418) for cultivars Midori and Marian Seefurth, respectively. Putative transgenic lines were selected 1 to 11.5 months, but on average 5.2 to 8.4 months, after cocultivation depending on the tissue type transformed. Significantly more embryogenic calli (one line per 5 mg calli) produced transgenic lines than did explants (one line per 143 mg explants) (P < 0.004) from ≈30 mg of tissue. Calli grew selectively from all explant types, but the type of explant from which each selection was made was not recorded because root, internode, and petiole explants were difficult to discern by the time calli developed. Shoots formed 3 months after calli were transferred to light. Non-transgenic control and transgenic ‘Marian Seefurth’ formed flower buds in the greenhouse ≈28 months after cocultivation. The plants resembled commercially grown plants from a private nursery. No non-transformed escapes were detected among the selections screened for NPTII by enzyme-linked immunosorbent assay and polymerase chain reaction (PCR). The selections were positive for transgenes as assayed by PCR and Southern hybridizations. Southern blots showed single-copy insertions of the NPR1 regulatory gene. The ability to produce large quantities of independent transgenic lines from embryogenic calli in a relatively short time period should enable researchers to evaluate the effectiveness of any transgene by screening numerous anthurium lines for improved performance.
Gayle M. Volk, Christopher M. Richards, Adam D. Henk, Ann A. Reilley, Patrick A. Reeves, Philip L. Forsline, and Herb S. Aldwinckle
Seeds from wild Malus orientalis trees were collected during explorations to Armenia (2001, 2002), Georgia (2004), Turkey (1999), and Russia (1998). Seedling orchards with between eight and 171 individuals from each collection location were established at the U.S. Department of Agriculture–Agricultural Research Service Plant Genetic Resources Unit (PGRU) in Geneva, NY. Genotypic (seven microsatellite markers) and disease resistance data were collected for the 776 M. orientalis trees. The genetic diversity of the 280 individuals from Armenia and Georgia was compared with data previously published for the M. orientalis individuals from Russia and Turkey. A total of 106 alleles were identified in the trees from Georgia and Armenia and the average gene diversity ranged from 0.47 to 0.85 per locus. The genetic differentiation among sampling locations was greater than that found between the two countries. Six individuals from Armenia exhibited resistance to fire blight (Erwinia amylovora), apple scab (Venturia inaequalis), and cedar apple rust (Gymnosporangium juniperi-virginianae). The allelic richness across all loci in the individuals from Armenia and Georgia was statistically the same as that across all loci in the individuals from Russia and Turkey. A core set of 27 trees was selected to capture 93% of the alleles represented by the entire PGRU collection of 776 M. orientalis trees. This core set representing all four countries was selected based on genotypic data using a modified maximization algorithm. The trees selected for the M. orientalis core collection will be added to the main field collection at the PGRU.
Christopher M. Richards, Gayle M. Volk, Patrick A. Reeves, Ann A. Reilley, Adam D. Henk, Philip L. Forsline, and Herb S. Aldwinckle
We estimate the minimum core size necessary to maximally represent a portion of the U.S. Department of Agriculture's National Plant Germplasm System apple (Malus) collection. We have identified a subset of Malus sieversii individuals that complements the previously published core subsets for two collection sites within Kazakhstan. We compared the size and composition of this complementary subset with a core set composed without restrictions. Because the genetic structure of this species has been previously determined, we were able to identify the origin of individuals within this core set with respect to their geographic location and genetic lineage. In addition, this core set is structured in a way that samples all of the major genetic lineages identified in this collection. The resulting panel of genotypes captures a broad range of phenotypic and molecular variation throughout Kazakhstan. These samples will provide a manageable entry point into the larger collection and will be critical in developing a long-term strategy for ex situ wild Malus conservation.
Gayle M. Volk, Christopher M. Richards, Ann A. Reilley, Adam D. Henk, Patrick A. Reeves, Philip L. Forsline, and Herb S. Aldwinckle
Genetic diversity and disease resistance are described for 496 seedlings from wild populations of Malus orientalis Uglitzh. collected in southern Russia and Turkey in 1998 and 1999. Eighty-five half-sib families were genotyped using seven microsatellite markers, and disease resistance was determined for apple scab (Venturia inaequalis Cooke), cedar apple rust (Gymnosporangium juniperi-virginianae Schwein), and fire blight (Erwinia amylovora Burrill). Individuals from the two Russian Caucasus collection locations were homogeneous compared with populations from the four Turkish collection locations. Within three of the Turkish collection locations, some half-sib families were highly diverse and several of these families had unusually high levels of disease resistance. In all, twenty individuals exhibited resistance to all three diseases. Bayesian analyses of the population structure revealed six distinct clusters. Most of the individuals segregated into two clusters, one containing individuals primarily from southern Russia and the other containing individuals from both Russia and northern Turkey. Individuals in the four small clusters were specific to Turkish collection locations. These data suggest wild populations of M. orientalis from regions around the Black Sea are genetically distinguishable and show high levels of diversity.