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.
Hong Y. Yang and Schuyler S. Korban
Francisco A. A. Mourão Fo, Jude W. Grosser, and Frederick G. Gmitter Jr.
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.
Young Rog Yeoung and Dale O. Wilson
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
T.J. Montagno, P.S. Jourdan, and S. Z. Berry
Unilateral incompatibility has limited the direction of crossing between L. esculentum and L. hirsutum; the latter can only serve as the pollen parent. In an attempt to introduce the L. hirsutum cytoplasm into L. esculentum, thirty-three somatic hybrid plants have been regenerated following four separate fusions between leaf protoplasts of L. hirsutum PI 126445 and etiolated hypocotyl protoplasts of L. esculentum (`OH7870', `OH832', and `OH8245'). A 33% PEG solution supplemented with 10% DMSO was used as the fusogen. Selection of fusion products was based on treatment of L. hirsutum protoplasts with 1 mM iodoacetic acid and non-regenerability of the L. esculentum genotypes. Hybridity was initially confirmed by intermediate morphology, including leaf shape, type of trichomes, flower shape, stigma placement, and fruit size and color. Isozyme analysis for GOT, PGM, and 6-PDH verified hybridity. Six of the hybrids produced viable seed upon selfing. At least some of the hybrids contained chloroplast DNA from L. hirsutum, indicating that the wild species cytoplasm may be present in these plants.
E. Mergenthaler, Gy. Bisztray, and D.P. Coyne
As ancestors of higher plants, mosses offer advantages as simple model organisms in studying complex processes. The moss Physcomitrella patens became a powerful model system in the last few years (Cove and Knight, 1993). Adaptation of PEG-mediated DNA uptake procedure has permitted the establishment of efficient molecular genetic approaches. To study possible effects of a Type I phytochrome, the potato phyA gene was introduced into the moss P. patens. Stabile transformants exhibited a range of similar phenotypes (Schaefer et al., 1991). The aim was to differentiate the wild type from the transgenic moss plants with simple, quick measurements providing data suitable for analyzing offspring populations. Ten different morphological and biochemical methods were used to investigate the phenotype in order to choose the best phenotypical category to indicate the presence and the effect of the phytochrome transgene. Two selected strains were used with the most and the least intensive phenotypical features (3*, 29), along with their selfed progenies, as well as progenies from crosses with the nicotinic-acid auxotrophic mutant. The best methods to differentiate between wild type and transgenic plants were the statistical analysis of the number of gametophores, photometric measurement of pigment contents and composition under different light conditions, color evaluation by PC-based vision system, and visual observation of morphogenetic changes. Our investigations support that the potato phytochrome transgene has a pleiotropic effect in the moss P patens. The methods used would be applicable for the characterization of mosses with different transgenes.
Francisco A. A. Mourão Fo, Jude W. Grosser, and Frederick G. Gmitter Jr.
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.
Marietta Loehrlein and Dennis T. Ray
Triploid watermelon seed does not germinate in cold, wet soils as well as diploids; germination is slower due to reduced embryo size and thicker seed coat; fissures on the seed coat provide safe harbour for fungal spores; and triploid fruit set is later than most diploid cultivars. Because of these problems producers often transplant rather than direct-seed seedless watermelons. Seed priming has been shown to improve germination in other crops and would be an attractive method allowing for direct seeding of seedless watermelons. Seed from open-pollinated 4n × 2n crosses were primed in solutions of H2O, polyethylene glycol 8000, KNO3, or left untreated. Treatment times were 1, 3, or 6 days, and treated seed were subsequently dried for either 1 or 7 d. Seed were scored for germination in the laboratory and emergence under field conditions. Germination was better using H2O than KNO3 and PEG but not always better than the untreated control. Treatment time of 1 day was superior to 3 or 6 days, but length of drying time was insignificant. In the field trial, treatments did not differ in emergence.
William J. Carpenter
Priming permits seeds to slowly imbibe water at regulated rates and to begin the initial stages of germination. Hypertonic polyethylene glycol (PEG) 8000 solutions of 1.0 and 1.2 MPa at 15C improved seed germination of dusty miller (Senecio cineraria DC.). At 0.8 MPa, germination was promoted during priming. No differences in rates, span, or total germination were found among seeds primed for 1, 2, or 3 weeks with or without aeration during priming. Germination percentages of primed and nonprimed seeds were similar at 10, 15, 20, and 25C, but 42% to 81% higher for primed seed at 30 or 35C. Priming reduced days to 50% of total germination (T50) 23% to 61%, and germination spans in days 30% to 67%. Primed seeds germinated most rapidly and uniformly at 20 and 25C. No change in total germination, T50, or germination span resulted when moisture contents of primed seeds were lowered to 7.8% or seeds were held at –80C for 7 days. Primed seed performance was unchanged after storage at 5C and 52% RH for 16 weeks.
Lewis W. Jett, Gregory E. Welbaum, and Ronald D. Morse
Priming, a controlled-hydration treatment followed by redrying, improves the germination and emergence of seeds from many species. We compared osmotic and matric priming to determine which was the most effective treatment for improving broccoli seed germination and to gain a greater understanding of how seed vigor is enhanced by priming. Broccoli (Brassica oleracea L. var. italica) seeds were osmotically primed in polyethylene glycol (PEG 8000) at -1.1 MPa or matrically primed in a ratio of 1.0 g seed:0.8 g synthetic calcium silicate (Micro-Cel E):1.8 ml water at -1.2 MPa. In the laboratory, germination rates and root lengths were recorded from 5 to 42C and 10 to 35C, respectively. Broccoli seeds germinated poorly at >35C. Root growth after germination was more sensitive to temperatures >30C and <15C than radicle emergence. Matric and osmotic priming increased germination rate in the laboratory, greenhouse, and field. However, matric priming had a greater effect on germination and root growth rates from 15 to 30C. Neither priming treatment affected minimum or maximum germination or root growth temperatures. Both priming treatments decreased the mean thermal time for germination by >35%. The greater germination performance of matrically primed seeds was most likely the result of increased oxygen availability during priming, increased seed Ca content, or improved membrane integrity.
P.F. McCabe, L.J. Dunbar, A. Guri, and K.C. Sink
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.