Regeneration from apple (Malus × domestica Borkh.) M.26 leaf tissue was completely inhibited by (μg·ml-1) 1 geneticin, 5 kanamycin, 10 to 25 paromomycin, and 100 neomycin. nptII-transgenic M.26 had an increased tolerance to all four of the antibiotics tested, with inhibition of regeneration occurring at (μg·ml-l) 2.5 geneticin, 100 kanamycin, 375 paromomycin, and 375 neomycin. Paromomycin (100 to 250 μg·ml-l) and neomycin (250 μg·ml-1) significantly increased the amount of regeneration from nptII-transgenic M.26 apple leaf tissue. p35SGUS-INT, a plasmid with a chimeric b -glucuronidase gene containing a plant intron, was useful for studying the early events of apple transformation by eliminating GUS expression from Agrobacterium tumefaciens. It was used to determine that the optimal aminoglycoside concentrations for the selection of nptII-transgenic M.26 cells were (μg·ml-1) 2.5 to 16 kanamycin, 63 to 100 neomycin, and 25 to 63 paromomycin. Geneticin was unsuitable as a selective agent.
John L. Norelli and Herb S. Aldwinckle
Gennaro Fazio, Herb S. Aldwinckle, Terence L. Robinson and James Cummins
The Geneva® Apple Rootstock Breeding program initiated in 1968 by Cummins and Aldwinckle of Cornell University and continued as a joint breeding program with the USDA-ARS since 1998, has released a new dwarf apple rootstock named Geneva® 41 or G.41. G.41 (a progeny from a 1975 cross of `Malling 27' × `Robusta 5') is a selection that has been tested at the N.Y. State Agricultural Experiment Station, in commercial orchards in the United States, and at research stations across the United States, Canada, and France. G.41 is a fully dwarfing rootstock with vigor similar to M.9 T337, but with less vigor than M.9 Pajam2. It is highly resistant to fire blight and Phytophthora with no tree death from these diseases in field trials or inoculated experiments. G.41 has also shown tolerance to replant disease. Its precocity and productivity have been exceptional, equaling M.9 in all trials and surpassing M.9 in some trials. It also confers excellent fruit size and induces wide crotch angles in the scion. It appears to be very winter hardy and showed no damage following the test winter of 1994 in New York. Propagation by layering in the stool bed G.41 is not consistent and may require higher layering planting densities or tissue culture mother plants to improve its rooting. G.41 also produces some side shoots in the stool bed. The nursery liners of G.41 produce a smaller tree than G.16 liners, but similar to M.9, which is very acceptable. Unlike G.16, G.41 is not sensitive to latent viruses. G.41 has similar graft union strength to M.9 and requires a trellis or individual tree stake when planted in the orchard. Suggested orchards planting densities with this rootstock are 2,000-4,000 trees/ha. This rootstock has been released for propagation and commercial sale by licensed nurseries.
Gennaro Fazio, Herb S. Aldwinckle, Terence L. Robinson and James Cummins
The Geneva® Apple Rootstock Breeding program, which was initiated in 1968 by Dr. James Cummins and Dr. Herb Aldwinckle of Cornell University and which has been continued as a joint breeding program with the U.S. Dept. of Agriculture Agricultural Research Service (USDA-ARS) since 1998, has released a new semi-dwarfing apple rootstock which is named Geneva® 935 or G.935. G.935 (a progeny from a 1976 cross of `Ottawa 3' × `Robusta 5') is a selection that has been widely tested at the New York State Agricultural Experiment Station in Geneva, N.Y., in commercial orchards in the United States and at research stations across the United States and Canada. G.935 is a semi-dwarfing rootstock that produces a tree slightly larger than M.26. G.935 is the most precocious and productive semi-dwarf rootstock we have released. It has had similar yield efficiency to M.9 along with excellent fruit size and wide crotch angles. It showed no symptoms of winter damage during the 1994 test winter in N.Y. G.935 is resistant to fire blight and Phytophthora; however. it is susceptible to infestations by woolly apple aphids. G.935 has shown tolerance to replant disease complex in several trials. It has good propagation characteristics in the stool bed and produces a large tree in the nursery. G.935 has better graft union strength than M.9, but will require a trellis or individual tree stake in the orchard to support the large crops when the tree is young. G.935 will be a possible replacement for M.26. Suggested orchards planting densities with this rootstock are 1,500-2,500 trees/ha. It has been released for propagation and sale by licensed nurseries. Liners will be available in the near future.
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.
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%.
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).
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.
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.
Jyothi Prakash Bolar, Susan K. Brown, John L. Norelli and Herb S. Aldwinckle
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.
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.