The overall goal of our research is to develop an efficient transformation and regeneration system for `McIntosh' apple. The first objective was to determine the optimum combination of Gelrite (G) and agar (A) to maximize regeneration and minimize vitrification. Treatments included the following combinations of agar (in g–liter–1) and Gelrite (in g–liter–1): 1) 7 and 0; 2) 5.25 and 0.625; 3) 3.5 and 1.875; 4) 1.75 and 1.875; and 5) 0 and 2.5. There were 10 replications, and a single petri plate containing six leaf pieces was the unit of replication. Both 5.25(A) and 0.62(G) and 3.5(A) and 1.25(G) provided high regeneration of healthy, nonvitrified shoots. Since modification of media affects the concentration of antibiotics used in selection due to precipitation of antibiotics, the second objective was to determine the optimal concentration of antibiotic for the selection and regeneration of transformed `McIntosh' on gelrite–agar-based media. Kanamycin was tested at 0, 10, 25, 50, 75, and 100 μg–ml–1 and paromomycin was tested at 0, 50, 100, 150, 200, and 250 μg–ml–1. Antibiotic selection will be discussed relative to optimum concentration and efficiency of selection.
Jyothi Prakash Bolar, Susan K. Brown, John L. Norelli, and Herb S. Aldwinckle
Kim Patten, John Wang, Fred Katz, Don Riemer, Chuck Kusek, and Herb Hopen
Tolerance of cranberry (Vaccinium macrocarpon Ait.) at different phenological stages to the postemergent broadleaf herbicide clopyralid (0.21 or 0.42 kg a.i./ha) was evaluated in Washington, New Jersey, Massachusetts, and Wisconsin. Tolerance varied among states, rates, and application times. Applications made during early shoot growth, especially at the high rate, usually resulted in the most crop injury (leaf cupping and epinasty and reduced yield); while applications at the low rate made after vegetative development occurred usually resulted in less or no injury. No phytotoxicity occurred when applications were made before shoot growth (Washington and New Jersey). Chemical name used: 3,6-dichloro-2-pyridinecarboxylic acid (clopyralid).
Nicole L. Russo, Terence L. Robinson, Gennaro Fazio, and Herb S. Aldwinckle
In 2002, apple rootstock trials using three scion cultivars were established at Geneva, NY, to evaluate 64 apple (Malus ×domestica Borkh.) rootstocks for horticultural performance and fire blight resistance. Field trials compared several elite Geneva® apple rootstocks, which were bred for tolerance to fire blight and Phytophthora root rot, to both commercial standards and elite rootstock clones from around the world. Three rootstocks performed well with all scion cultivars: ‘B.9’, ‘Geneva® 935’, and ‘Geneva® 41’. All three rootstocks were similar in size to ‘M.9’ clones but with elevated yield efficiency and superior resistance to fire blight. ‘Geneva® 11’ also performed very well with ‘Golden Delicious’ and ‘Honeycrisp’ with regard to yield efficiency and disease resistance. Resistant rootstocks greatly enhanced the survival of young trees, particularly with the susceptible scion cultivars ‘Gala’ and ‘Honeycrisp’. Results demonstrate the ability of new rootstock clones to perform better than current commercial standards, reducing financial risk to producers while promoting orchard health with enhanced disease resistance.
Jyothi Prakash Bolar, John L. Norelli, Herb S. Aldwinckle, and Viola Hanke
To root tissue-cultured apple cultivars, shoots from proliferating cultures were first transferred to root induction medium with IBA for 1 week in the dark. Shoots were later transferred to the same medium without IBA and incubated under light for elongation of the roots. Rooted shoots were then transferred to Jiffy-7s supplemented with biological plant protectant and fertilizer, and incubated in plastic humidity trays. After 2 to 3 weeks, plants were transferred to pots and covered with plastic bags to facilitate acclimation. This technique has resulted in 70% to 100% of shoots selected in vitro producing vigorously growing, healthy plants in the greenhouse. Chemical name used: indolebutyric acid (IBA).
Minou Hemmat, Norman F. Weeden, Herb S. Aldwinckle, and Susan K. Brown
Bulked segregant analysis was used to identify RAPD markers that display tight linkage to the Vf gene in apple (Malus sp.) that confers resistance to five races of apple scab [Venturia inaequalis (Cke.) Wint.]. We identified several new RAPD markers linked to Vf. The most tightly linked marker in the test population, S52500, was cloned and sequenced. A linkage map of the Vf region was developed using these markers, RAPD markers previously described by other laboratories, and the isozyme locus Pgm-1. An assay was developed for Vf by multiplexing the two markers closely flanking the Vf locus. This assay has a theoretical `escape' value (discarding a resistant plant) of 3% and an error rate (selection of a susceptible plant) of 0.02%.
Kisung Ko, Susan K. Brown, John L. Norelli, and Herb S. Aldwinckle
Seven nptII and gus transgenic lines of the apple (Malus ×domestica Borkh.) rootstock Malling 7 (M.7) were examined by glucuronidase (GUS) histochemical testing and a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). These lines had different amounts of neomycin phosphotransferase II (NPTII). The amounts of NPTII among lines was positively correlated with the ability of the transgenic lines to regenerate in the presence of kanamycin, paromomycin, or geneticin. Regenerants derived from transgenic lines also varied greatly in GUS expression. The apical portion of regenerant stem tissues had stronger GUS staining than the basal portion of stem. All regenerated tissue of T1, a transgenic line originally classified as a uniform GUS staining line, showed non-GUS staining, while the regenerated tissues of chimeric transgenic lines showed nonstaining, chimeric staining, or uniform GUS staining, indicating the potential to select uniform GUS staining lines from chimeras. Polymerase chain reaction (PCR) indicated the gus gene was present in GUS negative (nonstaining) lines. Negative PCR results with primers derived from vir G of Agrobacterium tumefaciens, and failure to isolate A. tumefaciens from M.7 transgenics indicated that PCR and GUS staining results were not due to A. tumefaciens. A modified PCR methylation assay (MPMA) indicated that methylation of cytosines of the CCGG site in the gus gene, and in the border between the CaMV35S promoter and the gus gene, was positively correlated with complete gus gene silencing (nonstaining lines). However, the MPMA indicated that methylation was not always associated with variable GUS expression, suggesting that chimeric staining could be due to a mixture of transformed and nontransformed cells in some transgenic lines.
Kisung Ko, John L. Norelli, Jean-Paul Reynoird, Herb S. Aldwinckle, and Susan K. Brown
Genes encoding lysozyme (T4L) from T4 bacteriophage and attacin E (attE) from Hyalophora cecropia were used, either singly or in combination, to construct plant binary vectors, pLDB15, p35SAMVT4, and pPin2Att35SAMVT4, respectively, for Agrobacterium-mediated transformation of `Galaxy' apple, to enhance resistance to Erwinia amylovora. In these plasmids, the T4L gene was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream domain and the untranslated leader sequence of alfalfa mosaic virus RNA 4, and the attE gene was controlled by the potato proteinase inhibitor II (Pin2) promoter. All transgenic lines were screened by polymerase chain reaction (PCR) for T4L and attE genes, and a double-antibody sandwich enzyme-linked immunosorbent assay for neomycin phosphotransferase II. Amplification of T4L and attE genes was observed in reverse transcriptase-PCR, indicating that these genes were transcribed in all tested transgenic lines containing each gene. The attacin protein was detected in all attE transgenic lines. The expression of attE under the Pin2 promoter was constitutive but higher levels of expression were observed after mechanical wounding. Some T4L or attE transgenic lines had significant disease reduction compared to nontransgenic `Galaxy'. However, transgenic lines containing both attE and T4L genes were not significantly more resistant than nontransgenic `Galaxy', indicating that there was no in planta synergy between attE and T4L with respect to resistance to E. amylovora.
Stan C. Hokanson, Phil L. Forsline, James R. McFerson, Warren F. Lamboy, Herb S. Aldwinckle, and Aimak D. Djangaliev
Malus sieversii, the main progenitor of domesticated apple, is native to areas in Central Asia. To better represent Malus wild germplasm in the USDA–ARS germplasm collections, maintained in Geneva, N.Y., a cooperative project was initiated with the Republic if Kazakhstan to collect and assess that country's wild populations of M. sieversii and to develop more secure in situ reserves to complement ex situ holdings in the United States and Kazakhstan. To date, four exploration trips to the region have included participants from the United States, Kazakhstan, Canada, New Zealand, and South Africa. Four Kazkh scientists have toured USDA–ARS sites, exchanged information, and collected germplasm in the United States greenhouse screens of 1600 have revealed potentially new sources of resistance to apple scab, cedar apple rust, and fire blight. An isozyme analysis of maternal half-sib families from four regions suggests the populations of M. sieversii collected represent a single panmictic population, with over 85% of total genetic variation due to differences among families. The most recent collections in 1995 were directed towards more ecologically diverse regions, including a site (Tarbagatai) at the most northern limit for M. sieversii equivalent to northern Minnesota in the United States. Some trees in this region produced fruit nearly 70 mm in diameter with excellent aroma, firmness, and color. This germplasm is being systematically characterized for horticultural traits, pest and disease resistance, and molecular markers.
Gayle M. Volk, Christopher M. Richards, Ann A. Reilley, Adam D. Henk, Philip L. Forsline, and Herb S. Aldwinckle
Seeds and scionwood of Malus sieversii Lebed. have been collected from wild populations of apple trees in Kazakhstan. Seedlings and grafted trees were planted in the orchards at the U.S. Dept. of Agriculture Plant Genetic Resources Unit in Geneva, N.Y. We developed core collections to capture the genetic and phenotypic diversity represented in the trees from each of two of the Kazakhstan collection sites. These core collections capture more than 90% of the genetic diversity of the original populations, as determined using seven unlinked simple sequence repeat markers and 19 quantitative traits. Since phenotypic evaluations of these materials have been completed, the 35 trees within each population will be used as parents in crosses so that the genetic diversity in the orchard populations can be captured as seed for long-term ex situ conservation. This strategy of storing seeds, rather than maintaining costly field collections, could be applied to other collections of wild plant materials in the National Plant Germplasm System.
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.