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- Author or Editor: Schuyler Korban x
Apple mosaic virus (ApMV) is transmitted by grafting to various Rosaceae plants including apple, rose, and plum, among others. ApMV reduces apple tree growth, yield, and fruit size; it causes mosaic symptoms on rose, and line patterns on plum and birch. ApMV is a member of ilarviruses, and it is characterized as isometric, labile, and producing ring spots. ApMV is a tripartite virus; RNA1 and RNA2 are monocistronic; whereas RNA3 contains two cistrons, one of which is the coat protein. The coat protein is translated from a subgenomic messenger, RNA4, which is derived completely from RNA3. We isolated and purified ApMV; virions containing RNA3 and RNA4 were separated from those containing RNA1 and RNA2. The sizes of RNA1 and RNA2 were estimated equal to 3 kb and 2.7 kb, respectively; the sizes of RNA3 and RNA4 were estimated equal to 2 kb and 0.9 kb, respectively. Inserts of clones from the 3′-end of RNA3 were sequenced and the 3′-primer was used to synthesize cDNA of RNA3. Overlapping cDNA clones were used to determine the nucleotide sequence of RNA4. Characterization and sequencing of the coat protein gene as well as transfer of this gene into apple will be discussed.
Peach shoots were grown in vitro for 0, 6, 12, 24, and 48 h on a basal medium containing one of several phytohormones or chemical elicitors, including abscisic acid (ABA), indolebutyric acid (IBA), indoleacetic acid (IAA), kinetin, gibberellic acid (GA3), aminocyclopropane-1-carboxylic acid (ACC), methyl jasmonate (MeJ), and salicylic acid (SA). Northern blot analysis was conducted using the 3' end of a peach-1,3-glucanase gene, PpGns1, used as a probe. Variations in levels of PpGns1 expression patterns were observed for each of the treatments. Shoots treated with ABA displayed high levels of transcripts at 12 and 24 h, followed by a sharp decline at 48 h. Shoots treated with ACC displayed a steady increase in transcripts throughout the 48 h period. The synthetic auxin IBA displayed a steady increase in mRNA accumulation for the first 24 h followed by a sharp decline at 48 h. Shoots treated with kinetin displayed low levels of transcripts after 24 h, while GA3 did not induce any accumulation of PpGns1 transcripts. Both SA and MeJA induced steady mRNA accumulation in peach shoots over the entire 48-h period. Induction of PpGns1 in response to SA, MeJ, and ACC also resulted in observed necrotic lesions on peach shoots, thus suggesting a different defense mechanism response.
Leaf segments of Prunus persica L. (peach) collected from greenhouse-grown plants and from micropropagated shoots were cultured on a basal medium containing half-strength Murashige and Skoog (MS), Staba vitamins, sucrose (30 g/1) and agar (6.5 g/l); medium adjusted to pH 5.6. The influence of 6 different growth regulators at 3 concentrations (5, 10, 15 μM) were investigated using leaf explants from proliferating shoots of 'Elberta Queen' peach. With thidiazuron (TDZ), compact and multiple green calli were obtained; with benzyladenine and zeatin, lower numbers of small sized calli were obtained; with kinetin, no callus development was observed. Among auxin treatments, both Dicamba and 2,4-D resulted in friable white and yellow calli. Most of the calli produced in all treatments were formed along the cut margins of the explants. In an another experiment, leaf explants of' Bellaire' (greenhouse) and `Elberta Queen' (in vitro shoots) were used to determine the influence of a large scale concentration of TDZ (3 to 23 |iM). Explants from greenhouse and in vitro leaves resulted in higher levels of callus development at TDZ concentrations of 8-13 μM. Higher TDZ levels resulted in necrosis of leaf explants. The-influence of different carbon sources on callogenesis was investigated. We observed more green and compact calli with glucose than with sucrose and fructose at 100 mM. The influence of the glucose at 10 different concentrations (30 to 300 mM) was also investigated.
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.
A high efficiency (up to 95%) shoot regeneration system for apple was developed. Explants consisted of young leaves harvested from in-vitro proliferating shoots cultures of several apple genotypes including `Dayton', `Gala', `Golden Delicious', `Malling 7a', `McIntosh', and `Royal Gala'. Leaf sections were cultured on a modified one-half strength Murashige and Skoog medium supplemented with thidiazuron (4.4 mg/L) and α-naphthaleneacetic acid (0.5 mg/L). Direct shoot organogenesis was often observed but sometimes was accompanied by callus formation. Regeneration frequency was genotype dependent. Other treatments investigated which enhanced regeneration included minced versus sectioned leaves and initial culture conditions of dark versus light. The possible applications of this regeneration system for Agrobacterium- and/or microprojectile-bombardment-mediated genetic transformation will be discussed.
Seeds of Antirrhinum majus (snapdragon), proprietary line OAK564, were treated with 0%, 0.10%, 0.25%, 0.5%, 0.75%, and 1.0% ethyl methanesulfonate (EMS) for 8, 10, and 12 h at room temperature. The experiment was replicated three times over time. Data were collected on percent seed germination, seedling survivability, and pollen viability to determine optimal conditions for induced mutagenesis in OAK564 seeds. In the pilot experiment, M1 seeds treated with 1.0% EMS for 12 h had the lowest seed germination rate among all 18 treatments. Based on this pilot experiment, a large-scale mutagenesis experiment was performed using three levels of EMS (0.5%, 0.75%, and 1.0%) for 10-h exposure period. Mutants were induced on all these treatments, and morphological changes in the M1 population were detected. These included dwarfism, chlorophyll deficiency, and leaf morphology abnormality. This indicated that the EMS treatments were successful in inducing mutations, and mutants were further characterized for morphological traits.
DNA was extracted from leaves of various Malus genotypes and used to screen synthetic decamer oligonucleotide primers. Samples from the following two groups were bulked: 1) seven scab-susceptible apple cultivars, and 2) 15 scab-resistant apple genotypes derived by introgressive hybridization from the previous group of cultivars. A third sample consisted of DNA extracted from Malus floribunda Sieb. clone 821, the original source of apple scab resistance for all genotypes in the second group. A total of 59 primers from kits A, L, and R (Operon Technologies) were screened. Amplified fragments were obtained for 93% of the primers tested, while random amplified polymorphic DNA (RAPD) fragments were detected among samples for 76% of the primers. One primer, A15, amplified a unique band in both M. floribunda clone 821 and the bulked scab-resistant sample. This RAPD marker, designated OA15900, identifies an amplified, introgressed fragment that likely corresponds to a region of the genome that may serve as a modifier for the scab resistance gene block V, derived from M. floribunda clone 821.
Phytochelatins (PCs) are heavy metal binding peptides that play important roles in sequestration and detoxification of heavy metals in plants. To develop transgenic plants with increased tolerance and/or accumulation of heavy metals from soil, an Arabidopsis thaliana FLAG–tagged AtPCS1 cDNA encoding phytochelatin synthase (PCS) under the control of a 35S promoter was expressed in Indian mustard (Brassica juncea). Four transgenic Indian mustard lines, designated pc lines, with different levels of AtPCS1 mRNA accumulation and correspondent AtPCS1 protein levels were selected and analyzed for tolerance to cadmium (Cd) and zinc (Zn). Heavy metal tolerance was assessed by measuring root length of 10-day-old seedlings grown on agar medium supplemented with different concentrations of Cd (0, 100, 150, and 200 μm CdCl2) and Zn (200, 400, 600, and 800 μm ZnCl2). All transgenic lines showed significantly longer roots when grown on a medium supplemented with 100 μm CdCl2. No significant differences were observed between transgenic lines and wild type when plants were grown on higher levels of Cd. This indicated that only partial tolerance to Cd was observed in these transgenic lines. Similarly, partial tolerance for Zn was also observed in these transgenic lines, but up to levels of 400 μm ZnCl2. Expression levels of AtPCS1 protein were not related to tolerance responses for either Cd or Zn stresses in transgenic lines.