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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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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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Amy K. Szewc-McFadden, Warren F. Lamboy and James R. McFerson

To comprehend genetic identity and relatedness in Malus germplasm held in situ and ex situ, we are employing simple sequence repeat (SSR) DNA fragment information in combination with passport and horticultural data. SSRs offer certain advantages for characterizing large arrays of germplasm efficiently. They are abundantly dispersed throughout plant genomes and are exceedingly polymorphic. In addition, they can be PCR-amplified and detected by automated fluorescence-based technology. A size-fractionated DNA library of M. ×domestica cv Golden Delicious was screened to identify SSR loci. Eight loci were found to be reliably informative and were used to prepare locus-specific primer pairs. Characterization of the 75 M. ×domestica accessions included in the core subset of the USDA-ARS Malus germplasm collection revealed six of the eight loci were polymorphic within the array. The number of alleles per locus ranged from two to 21. Throughput was enhanced by multiplexing, allowing simultaneous use of two or three primer pairs. With improved genetic characterization of Malus germplasm, we intend to better develop and relate the core subset to the rest of the collection and to in situ Malus genetic resources. SSR markers appear to be an efficient and reliable tool to expedite this process.

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Minou Hemmat, Norman F. Weeden and Susan K. Brown

We mapped DNA polymorphisms generated by 41 sets of Simple Sequence Repeat (SSR) primers, developed independently in four laboratories. All primer sets gave polymorphisms that could be located on our `White Angel' x `Rome Beauty' map for apple [Malus sylvestris (L.) Mill. Var. domestica (Borkh.) Mansf.]. The SSR primers were used to identify homologous linkage groups in `Wijcik McIntosh', NY 75441-58, `Golden Delicious', and `Liberty' cultivars for which relatively complete linkage maps have been constructed from isozyme and Random Amplified Polymorphic DNA (RAPD) markers. In several instances, two or more SSRs were syntenic, and except for an apparent translocation involving linkage group (LG) 6, these linkages were conserved throughout the six maps. Twenty-four SSR primers were consistently polymorphic, and these are recommended as standard anchor markers for apple maps. Experiments on a pear (Pyrus communis L.) population indicated that many of the apple SSRs would be useful for mapping in pear. However some of the primers produced fragments in pear significantly different in size than those in apple.

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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 1.6.5.4), ascorbate peroxidase (EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1), ascorbate oxidase (AAO, EC 1.10.3.3), and glutathione reductase (GR, EC 1.6.4.2) 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.

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Lidia Lozano, Ignasi Iglesias, Diego Micheletti, Michela Troggio, Satish Kumar, Richard K. Volz, Andrew C. Allan, David Chagné and Susan E. Gardiner

in apple ( Malus × domestica Borkh.) breeding programmes: Prospects, challenges and strategies Tree Genet. Genomes 8 1 14 Kumar, S. Garrick, D.J. Bink, M.C. Whitworth, C. Chagné, D. Volz, R.K. 2013 Novel genomic approaches unravel genetic

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Keiko Sekido, Yusaku Hayashi, Kunio Yamada, Katsuhiro Shiratake, Shogo Matsumoto, Tsutomu Maejima and Hiromitsu Komatsu

Apples ( Malus × domestica Borkh.) are produced commercially in most temperate countries not only for fresh use, but also for processed goods such as juice and in slices as an ingredient for cakes, pies, and tarts. ‘Fuji’, ‘Shinano Sweet’, ‘Orin