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Developing an efficient gene transfer system for apple (Malus ×domestica L.) remains a major objective in genetic engineering efforts of this fruit crop. Transient expression of the uidA gene coding for β-glucuronidase (GUS) and driven by the cauliflower mosaic virus 35S promoter (CaMV35S) has been induced in apple cotyledonary explants of mature seeds by tungsten particle bombardment using the Particle Inflow Gun (PIG). Several factors that affect transient expression of the GUS gene in apple cotyledons were investigated. The gene transfer efficiency was monitored by recording the number of blue spots observed on explants two days following bombardment. Precultivation of cotyledons for 18 hours before bombardment significantly increased the number of blue foci. Of the three different precipitation methods tested including water, 25% PEG, and 60% glycerol, the latter was the most effective for coating DNA onto tungsten particles. Washing DNA-coated tungsten particles with 70% ethanol and resuspending in 100% ethanol significantly enhanced gene delivery to cotyledons. The amount of particles used for each bombardment also influenced GUS expression. About 0.5 mg of particles per shot resulted in the highest number of blue foci. Using larger quantity of particles (i.e., 2 mg) drastically decreased GUS expression probably due to the toxicity of tungsten particles.

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Pregermination techniques of osmotic priming and hydropriming have been used to enhance seed performance on planting Osmotic priming and hydropriming method were compared on the basis of germination performance O2, N2 and air were supplied to 500 ml vessels containing seeds with distilled water or -1.31 MPa PEG solution for 10 days On removing seeds from vessels, seeds were dried back to original water content. There were no differences in total germination between osmotic priming and hydropriming treatments t50 was reduced dramatically from 112 to 32 hours, using hydropriming with air and N2supply for 1 day, compared to 70 hours of osmotic priming. Solute leakage from O2supply of both methods was higher than air or N treatment, Indicating the loss of membrane integrity Hydropriming with O2induced radicle emergence and loss of desiccation tolerance around 28 hours after treatment LEA protein levels were not changed in both treatments except for hydropriming with O2. The timing of desiccation tolerance loss was correlated with that of degradation of LEA protein. O2supply caused the adverse effects on seed performance from both methods 1 day after treatment

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Sexual hybrid plants of Lycopersicon esculentum × L. pennellii (E × P) have been transformed and the T-DNA inserts genetically mapped. Donor protoplasts of E × P were isolated from leaves, and subsequently irradiated with 0, 5, 10 and 20 krad of a 60Co. They were then fused with suspension-derived protoplasts of S. lycopersicoides using the PEG-CA++-high pH technique. The protoplasts were cultured in medium 8E at 1.5 × 106 protoplasm/ml. Selection of heterokaryon-derived macrocalli was facilitated by the inability of irradiated donor protoplasts to divide and by plating p-calli on regeneration medium containing kanamycin - an antibiotic for which the resistance gene NPTII is part of the T-DNA. Morphological characteristics of the resulting asymmetric somatic hybrid plants indicate that 10 and 20 krad irradiation eliminates a larger port ion of the genome than does 5 krad. This has been confirmed with isozyme analysis and chromosome counts which generally show the 5 krad asymmetric somatic hybrid plants to differ little from symmetric hybrids although they contain some significant exceptions. Isozyme data reveals the 10 and 20 krad plants to have received much less of the donor genome. Exact quantification is continuing using isozyme markers, chromosome counts and cDNA probes.

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Protoplast culture following polyethylene glycol (PEG)-induced fusion resulted in the regeneration of somatic hybrid plants from the following combinations: `Succari' sweet orange (C. sinensis L. Osbeck) + `Ponkan' mandarin (C. reticulata Blanco), `Succari' sweet orange + `Dancy' mandarin (C. reticulata), `Succari' sweet orange + `Page' tangelo [a sexual hybrid between `Minneola' tangelo (C. reticulata × C. paradisi Mcf.) × `Clementine' mandarin (C. reticulata)], `Valencia' sweet orange (C. sinensis) + `Page' tangelo. `Succari' and `Valencia' protoplasts were isolated -from ovule-derived embryogenic cell suspension cultures and from seedling leaves for the other parents. Somatic hybrid plants were Identified on the basis of leaf morphology and electrophoretic analysis of isozyme banding patterns. Root tip cell chromosome counting is being performed on all plants. Other putative somatic hybrids Include: `Succari' sweet orange + `Minneola' tangelo; `Succari' sweet orange + `Murcott' tangos (C. sinensis × C. reticulata); `Valencia' sweet orange + `Murcott' tangor; and `Valencia' sweet orange + `Dancy' mandarin. These plants may have direct cultivar potential, but there primary use will be for interploid hybridization with selected monoembryonic scions to produce improved seedless triploids.

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Protoplast culture following polyethylene glycol (PEG)-induced fusion resulted in the regeneration of somatic hybrid plants from the following combinations: `Succari' sweet orange (C. sinensis L. Osbeck) + Severinia disticha; `Hamlin' sweet orange (C. sinensisj + S. disticha: `Valencia' sweet orange (C. sinesis) + S. disticha; `Nova' tangelo (C. reticulata hybrid) + S. disticha; `Succari' sweet orange + S. buxifolia; `Nova' tangelo + Citropsis gilletiana; and `Succari' sweet orange + Atlantia ceylanica. `Succari', `Hamlin', `Valencia', and `Nova' protoplasts were Isolated from ovule-derived embryogenic callus and/or suspension cultures whereas protoplasts of S. disticha, S. buxifolia, C. gilletiana, and A. ceylanica were isolated from leaves of potted trees in a greenhouse. Plants were regenerated via somatic embryogenesis and somatic hybrids were identified on the basis of leaf morphology. Electrophoretic analysis of isozyme banding patterns and root tip chromosome counts are being performed. Somatic hybrids with S. disticha are apparently weak whereas the other somatic hybrid plants with S. buxifolia, C. gilletiana, and A. ceylanica exhibit adequate vigor. These are more examples that the the techique of protoplast fusion can be an important tool in overcoming barriers to hybridization of sexually incompatible species.

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was placed in either H 2 O 2 or polyethylene glycol (PEG) (5%; 1, 3, or 5 h). Proteins were extracted as previously described ( Ahn et al., 2004 ) using an extraction buffer [0.3% sodium dodecyl sulfate (SDS), 200 m m dithiothreitol, 28 m m Tris

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Using an aqueous polymer two-phase [polyethylene glycol (PEG) 3400/dextran T500, 6.2%: 6.2%, w/w] partitioning procedure combined with isopycnic fractionation, plasma membranes derived from muskmelon (Cucumis melo L. var. reticulates Naud.) leaf blades have been isolated and examined for marker enzyme activity, density, and molecular composition. After aqueous polymer partitioning, plasma membranes were centrifuged on a linear sucrose density gradient, and a single band was found at the 31% (w/w) sucrose (1.13 g-cm-3). Identification of plasma membranes was performed by the combination of K+-stimulated ATPase, pH 6.5, vanadate inhibition of ATPase and KNO3-insensitive ATPase activity. Plasma membranes from seedling leaves grown for 5 days at 15C had the highest concentration of total phospholipids, the lowest concentration of proteins, and a total sterol concentration not significantly different from leaves grown at 30C. The total sterol to total phospholipid ratio of the plasma membrane from leaves grown for 5 days at 15C was ≈1:1; from leaves grown for 10 days at 15C or 5 days at 30C the ratio was ≈2:1; and from leaves grown for 10 days at 30C the ratio was ≈3:1. The plasma membrane phospholipid saturated to unsaturated fatty acid ratio from leaves grown for 5 days at 15C was ≈0.8:1.0; from leaves grown for 10 days at 15C or 5 days at 30C the ratio was ≈1.0:1.0; and from leaves grown for 10 days at 30C it was 1.4:1.0.

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Among the factors affecting germinability of a seed lot are the environmental conditions under which the seeds are produced. The objective of this study was to determine the effects of temperature during seed development on seed quality of two Asteraceae species. Seeds of lettuce cv. Tango and Helianthus debilis cv. Vanilla Ice and sp. cucumerifolius were produced in a greenhouse under one of two treatments: i) hot (27, 40, and 20 °C temperatures average, max, and min, respectively), and ii) cool (23, 33, and 18 °C temperatures average, max, and min, respectively). In both species, heavier seeds were produced under the cool conditions and no differences were observed in standard germination. In lettuce, germination percentage and rate were both affected by increased levels of exogenous ABA concentrations and reduced water potential (PEG solutions), and, in both cases, seeds from cool treatments were more affected. Germination at 30 °C and constant light was higher for seeds from the hot treatment. Lettuce seed showed a strong light requirement for germination. However, seeds from the hot treatment gave better dark germination at 13 and 19 °C. Seeds of H. debilis did not required light for germination, and the germination percentage and rates were evaluated at 13, 21, and 29 °C. For both lines, seeds from each treatment behave similarly; however, the germination of H. debilis cv. Vanilla Ice at 29 °C was higher when seeds were produced in the hot conditions. The results showed that temperature during seed development affected aspects of seed quality that are not detectable by the standard germination, but by germination at suboptimal conditions. Within the Asteraceae family, differences varied among and within species.

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The effects of chemical or physical factors during pregermination imbibition phase, or on dry seeds, on embryo growth potential (EGP) was studied in lettuce (Grand Rapids and Mesa 659) and tomato (H-9889) seeds in relation to dormancy, invigoration, and vigor loss. Embryos were excised from treated seeds (washed if imbibed in chemical solutions) and their growth rate (a measure of EGP) followed at 25°C at high magnification (X55). Treated seeds were also germinated at 25°C. In lettuce seeds, dormancy inducing treatments, i.e., a 2-day dark soak at 25°C with 50–100 μM tetcyclacis (TCY) or a 2-day dark soak in water at 35°C, reduced the subsequent embryo growth and germination rate at 25°C. The reduction was prevented by 1 mM GA4+7 or irradiation applied during dormancy induction. A -d osmoconditioning (OC) at 15C with -1.2 MPa PEG-8000 solution in light or in dark with added GA4+7 enhanced the EGP; addition of TCY reduced the EGP and the TCY inhibition reversed by GA4+7. A progressive reduction in EGP occurred with increase in vigor loss. In tomato seeds, a soak with 100 μM TCY in light or dark for 2 days at 30°C induced a dormancy, but had little effect on EGP. Application of GA4+7 plus TCY prevented dormancy induction without affecting EGP. A 4-day matriconditioning (MC) at 25°C in light or dark with moist Micro-Cel E enhanced the EGP; TCY and/or GA added during MC, had little effect on EGP. EGP progressively decreased as the aging period increased. Thus, in lettuce, the EGP is coupled with the reversible –GA/+GA or phytochrome-controlled dormancy induction/release process, enabling germination, its inhibition, or its enhancement. In tomato, the EGP is not subject to light or GA control. Reduction in EGP, accompanying vigor loss in both seeds, is independent of light or GA action.

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Aleppo Pine (Pinus halepensis Mill.) is known to be the most drought-resistant Mediterranean Pine. This species is widely distributed throughout the Mediterranean region and displays a high intraspecific variability, with respect to its physiological and morphological response to environmental conditions. In this experiment we evaluated the response of Pinus halepensis seedlings to drought. Sixty germinated seeds (accession A6, Shaharia, Israel) were grown in soil for 8 weeks and then transferred to black plexiglass tanks containing half-strength air-sparged Hoagland solution. After 6 weeks of acclimation to hydroponics, the osmotic potential of the solution was lowered by adding polyethylene-glycol (PEG) 8000. Water potential was lowered in 0.2 MPa increments every 4 days, until a final value of –0.8 was reached. The seedlings were then maintained at –0.8 MPa for a further 8 days. Ultrasonic acoustic emissions, pressure–volume (P–V) curves, shoot and root growth, leaf area, xylem diameter, root apex mitotic index and cell length were measured on control and stressed seedlings. Seedlings were then transferred to normal Hoagland solution for 24 hours to simulate rewatering, and P–V curves and ultrasonic emissions measurements were repeated. Results showed that the final root growth is maintained in the stressed seedlings at the same rate as controls, whereas shoot growth was significantly reduced. The leaf area was reduced by stress to 36%, but the xylem diameter only to 10%, leading to a lower leaf area:xylem section ratio in the stressed plants. Ultrasonic emissions in the stressed plants were 365% of the control, and 182%, after rewatering. Specific details of the growth and physiology data are presented.

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