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Manfredo J. Seufferheld, Cecil Stushnoff and Philip Forsline

Cryopreservation of mature dormant vegetative buds is a useful method to preserve germplasm of a large number of cold hardy apple cultivars. However, cold tender cultivars have proven to be much more difficult to cryopreserve. Eight cultivars were harvested in September 1993 at Geneva, NY before developing cold hardiness naturally. The twigs were encapsulated with 5% alginate and treated with stepwise imbibition of 0.5 to 1.0 M sucrose. The samples were desiccated over glycerol at 0C. Half of the samples were plunged directly into liquid nitrogen (IN) and the other half were first cooled slowly to -30C. The twigs that had been exposed to prefreezing conditions showed the highest survival (20 to 100%). The samples that were plunged directly in LN survived poorly (0 to 20 %). Samples without encapsulation and no sucrose imbibition had 0% survival. We conclude that this protocol opens up the possibility to expand cryopreservation of cold tender apple cultivars, presently grown only in field gene banks, at great expense and inconvenience.

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Xuetong Fan, J.P. Mattheis, M.E. Patterson and J.K. Fellman

Several strains of Fuji apples were harvested weekly from September through October in 1990 and 1991, and evaluated for maturation and quality after 1 and 7 days at 20 °C following harvest and storage in atmospheres of 0.5%, 1.0%, 2.0% O2 and air. Results showed that Fuji apples have very low ethylene production rates and little firmness loss during maturation. A change in the postharvest respiration pattern preceded the increase ethylene synthesis. Oxygen concentration during storage directly affected apple respiration rate after removal from storage. Ethylene production rates and internal ethylene concentrations indicated that the apples were still in the preclimacteric stage after 7 to 9 months storage at 0.5%, 1.0%, or 2% O2. Fuji apples develop watercore and tend to have a particular type of corebrowing during maturation on the tree, or during and after storage. The cause is unknown.

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Jacques J. Crabbe

The flushing behavior of shoot growth and its consequences on shoot differentiation are important features in fruit tree development, with regard to flowering ability. In this respect, two different approaches were applied to young `M26' apple trees. First, poorly branched 2-year-old trees were headed back, either in the second-year or in the first-year wood, at different times from right before to 6 weeks after budbreak. Early pruning resulted in rapid and prolonged regrowth, with a final very similar shaping of the tree to that of the intact controls. Late pruning, in contrast, leads to a two-step reaction (late spring and summer flushes), sometimes stronger on 2-year-old than on 1-year-old wood. In a second experiment, buds and young shoots were sampled on pruned trees in locations where they could be supposed to remain short shoots or grow long, with one or two flushes. They were weighed, their leaves and internodes measured, and the plastochron evaluated. During budbreak and the first month afterwards, shoot differentiation appears achieved. The primary difference between long and short shoot types does not consist in faster internode elongation but, rather, in faster production (reduced plastochron) of larger leaves.

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Chang-Hoo Lee, N. I. Hyung and S. E. Kim

Experiments were conducted to investigate the factors influencing mesophyll protoplast isolation in `Fuji' apple. Half an hour pretreatment in 0.6M mannitol gave the highest protoplast yield.The enzyme solution containing 2% Cellulase Onozuka R-10 and 0.5% macrozyme R-10 with CPW 0.6M mannitol at pH 5.5 was most effective for protoplast isolation from leaf. Effective incubation time for the enzyme treatment was found to be 15-20 hrs at 25°C in the dark. Use of 1.0-2.0% PVP and 0.5mM MES was essential for higher yield and viability of protoplast. Supplementation of BA and IBA to the shoot culture media gave the higher yield of viable protoplast. From these protoplast, new cell walls were regenerated and 4 cell structures developed from one protoplast by cell division in K8P medium supplemented with 3A and NAA. Planting density higher than 10 protoplasts/ml was required for cell division from protoplast in liquid or 0.5% agarose culture.

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Frank Cheng, Norman Weeden and Susan Brown

The ability to pre-screen apple populations for fruit color at an early seedling stage would be advantageous. In progeny of the cross `Rome Beauty' × `White Angel' red/yellow color variation was found to be highly correlated with the genotype at Idh-2, an isozyme locus that was heterozygous in both parents. We postulate that the red/yellow color variation was produced by a single gene linked to I&-2 and also heterozygous in both parents. This population was also screened with over 400 primers to detect randomly amplified polymorphic (RAPD) markers for fruit color. DNA extraction procedures were developed for bark, and DNA was extracted from bark samples and leaves. Red and yellow fruited individuals were examined in bulk. Several markers have been found that are linked to red color. A high density map is being constructed in this region. These markers are being examined in other crosses segregating for fruit color. The application of these markers will be discussed in relation to the inheritance and manipulation of fruit color.

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Giorgio Bargioni, Giorgio Baroni, Pietro Tonutti, Andrea Pitacco and Angelo Ramina

Effects of scion inclination on root growth and distribution were studied on INRA GF 677 (Prunus persica × Prunus amygdalus) and apple/M.9 trees. At planting, central leaders were positioned vertically (0°) or inclined 45° or 60° to the north and south. Three years after planting, root total dry weight of inclined trees was lower than that of the control (0°, vertical central leader). Five years after planting, the isotropic distribution of the normal root systems was distorted by inclination in both species. Roots were more numerous and more elongated in the direction of inclination. Statistical analysis of root density data, using a polar coordinate system, confirmed that the trunk inclination reduced root development and redirected root distribution. The major effect was induced on GF 677 by 60° inclinations. Tree orientation did not seem to influence root distribution.

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Shiow Y. Wang and Miklos Faust

The ability of low and high temperatures and S-containing compounds to overcome endo- and paradormancy along with the possible mechanisms involved in these treatments for breaking `Anna' apple bud dormancy were studied. All three treatments induced budbreak in paradormant (July) and endodormant (October) buds. Cold, heat, and allyl disulfide increased ascorbic acid, the reduced form of glutathione (GSH), total glutathione, total nonprotein thiol (NPSH), and nonglutathione thiol (RSH), whereas dehydroascorbic acid and oxidized glutathione (GSSG) decreased. The treatments also increased the ratios of ascorbic acid: dehydroascorbate and GSH: GSSG and the activities of ascorbate free-radical reductase (AFR, EC, ascorbate peroxidase (EC, dehydroascorbate reductase (DHAR, EC, ascorbate oxidase (AAO, EC, and glutathione reductase (GR, EC in the buds. These results indicate that budbreak induced by cold, heat, and allyl disulfide is associated with the removal of free radicals through activated peroxide-scavenging systems.

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Shiow Y. Wang, Miklos Faust and Michael J. Line

The effect of IAA on apical dominance in apple buds was examined in relation to changes in proton density (free water) and membrane lipid composition in lateral buds. Decapitation induced budbreak and enhanced lateral bud growth. IAA replaced apical control of lateral buds and maintained paradormancy. Maximal inhibition was obtained when IAA was applied immediately after the apical bud was removed; delaying application reduced the effect of IAA. An increase in proton density in lateral buds was observed 2 days after decapitation, whereas the change in membrane lipid composition occurred 4 days later. Removing the terminal bud increased membrane galacto- and phospholipids and the ratio of unsaturated to corresponding saturated fatty acids. Decapitation also decreased the ratio of free sterols to phospholipids in lateral buds. Applying thidiazuron to lateral buds of decapitated shoots enhanced these effects, whereas applying IAA to the terminal end of decapitated shoots inhibited the increase of proton density and prevented changes in membrane lipid composition in lateral buds. These results suggest that change in water movement alters membrane lipid composition and then induces lateral bud growth. IAA, presumably produced by the terminal bud, restricts the movement of water to lateral buds and inhibits their growth in apple.

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James F. Harbage, Dennis P. Stimart and Ray F. Evert

Anatomical events of adventitious root formation in response to root induction medium, observing changes during induction and post-induction stages, were made with microcuttings of `Gala' apples. Shoot explants on root induction medium containing water, 1.5 μm IBA, 44 mm sucrose, or 1.5 μm IBA + 44 mm sucrose after 4 days of treatment averaged 0, 0.2, 2.2, and 11.9 meristemoids per microcutting, respectively. Meristemoids formed in response to sucrose were confined to leaf gaps and traces. Time-course analysis of root induction with 1.5 μm IBA + 44 mm sucrose over 4 days revealed that some phloem parenchyma cells became densely cytoplasmic, having nuclei with prominent nucleoli within 1 day; meristematic activity in the phloem was widespread by 2 days; continued division of phloem parenchyma cells advanced into the cortex by 3 days; and that identifiable root primordia were present by 4 days. Cell division of pith, vascular cambium, and cortex did not lead to primordia formation. Meristematic activity was confined to the basal 1 mm of microcuttings. Time-course analysis of post-induction treatment revealed differentiation of distinct cell layers at the distal end of primordia by 1 day; primordia with a conical shape and several cell layers at the distal end by 2 to 3 days; roots with organized tissue systems emerging from the stem by 4 days; and numerous emerged roots by 6 days. Root initiation was detectable within 24 hours and completed by day 4 of the root induction treatment and involved only phloem parenchyma cells. Chemical names used: 1 H -indole3-butryic acid (IBA).

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Patrick J. Conner, Susan K. Brown and Norman F. Weeden

Two half-sib populations (cross 1 = `Wijcik McIntosh' (WM) × NY 75441-67, and cross 2 = WM × NY 75441-58) were used to create maps for the parents and to find RAPD or isozyme markers for qualitative and quantitative traits. WM is a sport of `McIntosh' and is heterozygous for the dominant columnar (Co) gene for reduced branching. WM is of great interest in breeding because of the tremendous effect of the Co gene on many aspects of plant form. NY 75441-67 and NY 75441-58 are advanced selections with commercial fruit quality and resistance to scab (Vf resistance from M. floribunda). Traits examined included both tree (plant height, stem diameter, suckering, branching habit, leaf break, burr knot production) and fruit (size, shape, color, stem length, seed number) characters and fruit quality traits (pH, acid content, Brix). The conservation of RAPD markers in these closely related crosses will be examined and the usefulness of molecular markers to preselect for components of plant form and fruit quality will be discussed. Molecular markers will increase the efficiency of the apple breeding program by aiding the understanding and manipulation of complex genetic traits.