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H.S. Aldwinckle, P.L. Forsline, H.L. Gustafson, and S.C. Hokanson

Resistance to apple scab (Venturia inaequalis) in apple cultivar breeding has been derived mainly from the Vf gene from Malus floribunda 821, which introgresses horticulturally unfavorable characters. M. sieversii, now thought to be the primary progenitor of M. × domestica, grows wild in many diverse habitats in Central Asia and can have fruit quality comparable to commercial cultivars. Since 1989, four major collections of M. sieversii have been made in Central Asia, where scab is endemic. Some seed collections have been made from trees with superior fruit, that were not infected with scab. Over a 6-year period, 3000 seedlings from 220 wild M. sieversii trees representing 10 diverse ecosystems in Kazakstan, Uzbekistan, Kyrgyzstan, and Tajikistan have been inoculated with conidia of five races and two wild types of V. inaequalis. Suspensions (270,000 conidia/ml) were applied to 4- to 8-leaved seedlings, which were incubated for 48 h at 19°C with constant leaf wetness. Symptoms for three resistant reactions were assessed 2 to 4 weeks after inoculation: A = chlorosis with crinkling (Vf type reaction); B = stellate necrotic lesions (Vr type reaction), and N = large necrotic areas (uncharacterized resistant reaction). Results indicated that nearly 20% of the seedlings showed one or more of the resistant reactions. The range of resistance within seedling populations from each of the 220 single-tree sources ranged from 0% to 75%. Significant differences existed among seedlings from each of the ecosystems. Most resistance reactions appeared to be similar to those observed for Vr from “Russian seedling.” Resistant selections with superior horticultural traits may constitute a genepool for increased efficiency of breeding scab-resistant cvs. This genepool may also be useful to address the breakdown of resistance to V. inaequalis race 6.

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W.C. Johnson, H.S. Aldwinckle, J.L. Norelli, and H.T. Holleran

A primary focus of the apple rootstock breeding and evaluation program at USDA-ARS/Cornell Univ. has been to develop screening protocols to identify genotypes resistant to the fire blight bacterium (Erwinia amylovora). Direct inoculation is a simple technique, but does not represent the only mode by which rootstocks become infected in the orchard. Selection based on direct inoculation screens may, however, enrich the population for resistant genotypes. Large breeding populations from controlled crosses are shoot-tip inoculated with E. amylovora, and the fraction showing the highest levels of resistance are retained for further evaluations. These survivors are again screened through direct inoculation in the field, and the less-resistant genotypes are discarded. Following selection for other pathogen tolerance and horticultural characters, elite genotypes are multiplied through asexual propagation. Replicated tests using direct inoculation with multiple strains of E. amylovora are then used to estimate the level of fire blight resistance of elite genotypes. A final screen utilizes mature, grafted orchard trees to verify that the resistance of rootstock genotypes to fire blight is maintained under conditions simulating natural infection. Direct inoculation screening and selection have resulted in a high frequency of strong resistance to severe fire blight epidemics in recent orchard inoculation trials.

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A.G. Manganaris, F.H. Alston, N.F. Weeden, H.S. Aldwinckle, H.L. Gustafson, and S.K. Brown

Pgm-1, the gene responsible for variation in the most anodal isozyme of phosphoglucomutase in apple (Malus spp.), is shown to lie ≈8 centiMorgans from the gene Vf, which confers apple-scab resistance. The proximity of the marker and the ease by which allozymic forms can be resolved suggest that Pgm-1 will be useful for following the inheritance of scab resistance conferred by Vf.

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M.T. Momol, W.F. Lamboy, P.L. Forsline, and H.S. Aldwinckle

Malus sieversii is one of the primary progenitors of the cultivated apple. Since 1989, several collecting trips have been made to central Asia by personnel of the USDA and Cornell Univ. to collect seeds of wild Malus sieversii from many diverse ecosystems. In 1992, an ex situ plot in Geneva, N.Y., was established with trees grown from seed that was collected in three different habitats in Kazakstan, Tajikistan, and Uzbekistan in 1989. In 1995, trees grown from seed that was collected in five additional habitats in Kazakstan and Kyrgyzstan in 1993 were added to the ex situ plot. In the summers of 1995 and 1996, tips of vigorously growing shoots of 1135 seedlings from 79 different populations were inoculated by hypodermic syringe with 5 × 108 cfu/ml of Erwinia amylovora strain Ea273. Seedlings from the 1989 collection were in the fourth and fifth field-growing seasons, with some beginning to bear fruit. Seedlings from the 1993 collection were in first and second field-growing seasons. Results from both seasons indicated that individuals within each of the 79 populations of M. sieversii are resistant to fire blight (defined as ≤20% shoot length infected). Resistance differed among populations, with some populations having no resistant individuals and others having >80% of the seedlings resistant. The range of resistance is quite similar to that seen among apple cultivars from North America and Europe. In another test, some accessions from 1989 collection had sufficient bloom for inoculation in 1995 and 1996. At full bloom, blossoms on these trees were inoculated with the E. amylovora suspensions (5 × 107 cfu/ml) using a backpack sprayer. These also gave diverse resistant reactions.

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William C. Johnson, Phil L. Forsline, Herb S. Aldwinckle, William C. Johnson, Phil L. Forsline, H. Todd Holleran, Terence L. Robinson, and John J. Norelli

In 1998, the USDA-ARS and Cornell Univ. instituted a cooperative agreement that mobilized the resources for a jointly managed apple rootstock breeding and evaluation program. The program is a successor to the Cornell rootstock breeding program, formerly managed by Emeritus Professor of Horticultural Sciences James N. Cummins. The agreement broadens the scope of the program from a focus on regional concerns to address the constraints of all the U.S. apple production areas. In the future, the breeding program will continue to develop precocious and productive disease-resistant rootstock varieties with a range of vigor from fully dwarfing to near standard size, but there will be a renewed emphasis on nursery propagability, lodging resistance, tolerance to extreme temperatures, resistance to the soil pathogens of the sub-temperate regions of the U.S., and tolerance to apple replant disorder. The program draws on the expertise available at the Geneva campus through cooperation with plant pathologists, horticulturists, geneticists, biotechnologists, and the curator of the national apple germplasm repository. More than 1000 genotypes of apple rootstocks are currently under evaluation, and four fire blight- (Erwinia amylovora) resistant cultivars have been recently released from the program. As a service to U.S. apple producers, rootstock cultivars from other breeding programs will also be evaluated for productivity, size control, and tolerance to a range of biotic and abiotic stress events. The project will serve as an information source on all commercially available apple rootstock genotypes for nurseries and growers.

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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.