The cellular localization and molecular interactions are indicative of functions of a protein. The development of a simple and efficient method for subcellular localization of a protein is indispensable to elucidate gene function in plants. In this study, we assessed the feasibility of Agrobacterium-mediated transformation (agroinfiltration) of tobacco and tomato leaf tissue to follow intracellular targeting of proteins from rice fused to green fluorescent protein (GFP). For this, a simple in planta assay for subcellular localization of rice proteins in the heterologous host systems of tobacco and tomato leaf via transient transformation was developed. We have tested the applicability of this method by expressing GFP fusions of the putative antiphagocytic protein 1 (APP1) (OsAPP, LOC_Os03g56930) and ZOS3-18-C2H2 zinc-finger protein (OsZF1, LOC_Os03g55540) from Oryza sativa L. subsp. japonica in tobacco and tomato leaf tissues. Our results demonstrate the suitability of GFP as a reporter in gene expression studies in tomato cv. MicroTom. The use of GFP-fused proteins from rice for subcellular targeting in the heterologous hosts of tobacco and tomato plant systems has been confirmed.Key words: agroinfiltration, confocal microscopy, GFP fusion protein, tomato cv, microtom相似文献
High efficient and repeatable in vitro regeneration protocol was established from embryo axis, half-seed, axillary meristem, and cotyledonary node explants of chickpea. Various concentrations and combinations of various plant growth regulators (PGRs) were employed to induce multiple shoots, shoot elongation and rooting of shoots to obtain complete plantlets of chickpea. The pretreatment of seeds with 6-benzyl aminopurine (BAP) at 1.0 mg l?1 was found to significantly increase the multiple shoot regeneration from the all explants tested. Among three PGRs such as BAP, kinetin (KIN) and thidiazuron (TDZ) tested for multiple shoot induction; BAP at 2.0 mg l?1 produced the maximum number of shoots in all tested explants. The maximum number of shoots (48.80 shoots/explant) was attained from the embryo axis explant followed by half-seed (32.76 shoots/explant), axillary meristem (28.34 shoots/explant) and cotyledonary node explant (18.47 shoots/explant) on medium augmented with 2.0 mg l?1 BAP along with 0.05 mg l?1 Indole-3-butyric acid (IBA). The optimum percentage of shoot elongation response was recorded (96.68%) on medium fortified with IAA (0.05 mg l?1), GA3 (1.0 mg l?1) and BAP (1.0 mg l?1) with an average shoot length of 8.82 cm. The elongated shoots were successfully rooted in medium augmented with 2.0 mg l?1 IBA. The complete plants were acclimatized in the greenhouse with a survival rate of 72%. The plantlets regenerated from four explants appeared to be morphologically similar to mother plants. The genetic fidelity of in vitro regenerated plants was evaluated using Start Codon Targeted and Inter simple sequence repeats molecular markers. The in vitro regenerated plants from all four explants were found to be the true to type with their mother plant. The in vitro protocol presented in the study should offer as a feasible system for chickpea genetic transformation.
High frequency of streptomycin resistant variants of Lycopersicon esculentum were isolated on selective shoot regeneration medium supplemented with IAA (0.5 mg/L), zeatin (1.5 mg/L) and streptomycin sulphate (500 mg/L). Nonmutagenized (controls) and NMU treated cotyledons were placed on shoot regeneration medium supplemented with antibiotic streptomycin. Resistant shoots appeared at a high frequency in mutagenized cotyledons, whereas in controls morphogenesis was suppressed, accompanied by bleaching. Shoot regeneration occurred from the nodular tissues developed at the cut ends of cotyledons. Resistant shoots developed into complete plantlets on rooting medium containing selective concentration of antibiotic. Stability of streptomycin resistance was confirmed by leaf assay and reciprocal crosses between streptomycin-resistant and sensitive plants. 相似文献
The effects of atrazine on cotyledon cultures of Solanum melongena were investigated with a view to establishing a system for in vitro selection of resistant mutants. At herbicide levels producing
little growth inhibition some chlorophyll loss occurred associated with the production of albino shoots. At 15 mg/l bleaching
was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated
with severe suppression of growth. Mutagenesis with EMS resulted in herbicide-resistant mutants based on the embryogenic ability
of mutagenised explants placed on medium containing selective levels of sucrose (0.2%) and atrazine (15 mg/l). Different morphogenetic
responses were observed when the levels of sucrose (0.2–5%) were altered. Somatic embryogenesis was observed at low sucrose
concentrations (0.2–0.5%). Both embryogenesis and shoot regeneration occurred in 1% sucrose. Shoot regeneration was maximum
in 2% sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3–5%). However, lowering
of sucrose concentration from 2% to 0.2% caused complete bleaching, permitting the selection of herbicide-resistant mutants.
Received: 26 November 1996 / Accepted: 20 December 1996 相似文献
Soil salinity is a major environmental stress limiting plant productivity. Vacuole Na+/H+ antiporters play important roles for the survival of plants under salt stress conditions. We have developed salt stress tolerant transgenic tomato plants (Solanum lycopersicum cv. PED) by overexpression of the wheat Na+/H+ antiporter gene TaNHX2 using Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBin438 that contains the TaNHX2 gene under the control of double CaMV 35S promoter and npt II as a selectable marker. PCR and Southern blot analysis confirmed that TaNHX2 gene has been integrated and expressed in the T1 generation transgenic tomato plants. When TaNHX2 expressing plants were exposed to 100 or 150 mM NaCl, they were found to be more tolerant to salt stress compared to wild type plants. Biochemical analyses also showed that transgenic plants have substantial amount of relative water content and chlorophyll content under salt stress conditions compared to wild type plants. The relative water content in transgenic and wild type plants ranged from 68 to 75 % and 46–73 % and chlorophyll content fall in between 1.8 to 2.4 mg/g fw and 1.0 to 2.4 mg/g fw, respectively, in all stress conditions. In the present study, we observed a better germination rate of T1 transgenic seeds under salt stress conditions compared with wild type plants. Our results indicated that TaNHX2-transgenic tomato plants coped better with salt stress than wild type plants. 相似文献
Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn4Ca cluster to the plastoquinone pool. It is a dimeric supramolecular complex comprising more than 30 subunits per monomer, of which 16 are bitopic or peripheral, low-molecular-weight components. Directed inactivation of the plastid gene encoding the low-molecular-weight peptide PsbTc in tobacco (Nicotiana tabacum) does not prevent photoautotrophic growth. Mutant plants appear normal green, and levels of PSII proteins are not affected. Yet, PSII-dependent electron transport, stability of PSII dimers, and assembly of PSII light-harvesting complexes (LHCII) are significantly impaired. PSII light sensitivity is moderately increased and recovery from photoinhibition is delayed, leading to faster D1 degradation in ΔpsbTc under high light. Thermoluminescence emission measurements revealed alterations of midpoint potentials of primary/secondary electron-accepting plastoquinone of PSII interaction. Only traces of CP43 and no D1/D2 proteins are phosphorylated, presumably due to structural changes of PSII in ΔpsbTc. In striking contrast to the wild type, LHCII in the mutant is phosphorylated in darkness, consistent with its association with PSI, indicating an increased pool of reduced plastoquinone in the dark. Finally, our data suggest that the secondary electron-accepting plastoquinone of PSII site, the properties of which are altered in ΔpsbTc, is required for oxidation of reduced plastoquinone in darkness in an oxygen-dependent manner. These data present novel aspects of plastoquinone redox regulation, chlororespiration, and redox control of LHCII phosphorylation. 相似文献