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Meiru Li - One of the best experts on this subject based on the ideXlab platform.

  • establishment of a highly efficient agrobacterium tumefaciens mediated Leaf Disc transformation method for broussonetia papyrifera
    Plant Cell Tissue and Organ Culture, 2008
    Co-Authors: Meiru Li, Hongqing Li, Huawu Jiang, Guojiang Wu
    Abstract:

    Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both Leaf and petiole explants. Shoots formed on these calli with a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively. For genetic transformation, Leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, Leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473, 1962) (MS) medium supplemented with 1.5 mg l−1 benzyladenine (BA) and 0.05 mg l−1 indole-3-butyric acid (IBA) (CI medium) containing 5 mg l−1 hygromycin and 500 mg l−1 cefotaxime, in the dark. Hygromycin-resistant calli were induced from Leaf explants 3 weeks thereafter. Regenerating shoots were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l−1 BA, 0.05 mg l−1 IBA, and 0.5 mg l−1 gibberellic acid (GA3) (SI medium), 5 mg l−1 hygromycin and 250 mg l−1 cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l−1 hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay. Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as 44%.

  • establishment of an efficient agrobacterium tumefaciens mediated Leaf Disc transformation of thellungiella halophila
    Plant Cell Reports, 2007
    Co-Authors: Hongqing Li, Jie Xu, Lei Chen, Meiru Li
    Abstract:

    Thellungiella halophila is a salt-tolerant close relative of Arabidopsis, which is adopted as a halophytic model for stress tolerance research. We established an Agrobacterium tumefaciens-mediated transformation procedure for T. halophila. Leaf explants of T. halophila were incubated with A. tumefaciens strain EHA105 containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following co-cultivation, Leaf explants were cultured on selective medium containing 10 mg l−1 hygromycin and 500 mg l−1 cefotaxime. Hygromycin-resistant calluses were induced from the Leaf explants after 3 weeks. Shoot regeneration was achieved after transferring the calluses onto fresh medium of the same composition. Finally, the shoots were rooted on half strength MS basal medium supplemented with 10 mg l−1 hygromycin. Incorporation and expression of the transgenes were confirmed by PCR, Southern blot analysis and GUS histochemical assay. Using this protocol, transgenic T. halophila plants can be obtained in approximately 2 months with a high transformation frequency of 26%.

Hansjoerg Jacobsen - One of the best experts on this subject based on the ideXlab platform.

  • development of a simple and effective protocol for agrobacterium tumefaciens mediated Leaf Disc transformation of commercial tomato cultivars
    GM crops & food, 2010
    Co-Authors: Noel Ferro, Hansjoerg Jacobsen
    Abstract:

    The transformation of tomato (Solanum lycopersicum) through Agrobacterium tumefaciens is still far from being routine, particularly when it comes to commercial varieties. In the present paper, we present an efficient and simple protocol for Leaf Disc transformation of three Vietnamese tomato cultivars (DM8, MTS, FM372C) by comparing shoot regeneration media for expanding leaves and examining different parameters of inoculation, co-culture and selection conditions. The present transformation method requires neither feeder layers of cell suspension cultures nor pre-culture. The data clearly show that appropriate cytokinin- and auxin combinations and concentrations provide competent tissues for transformation. Supplementing of 8µM trans-zeatin and 5 µM indoleacetic acid (IAA) into pre-treatment, inoculation and co-culture media resulted in higher frequency of transformation and stronger GUS-expression than that of media supplemented with 4 µM trans-zeatin and 2 µM IAA. The experiments also exhibited that tom...

Miguel Lara - One of the best experts on this subject based on the ideXlab platform.

  • protoplast isolation transient transformation of Leaf mesophyll protoplasts and improved agrobacterium mediated Leaf Disc infiltration of phaseolus vulgaris tools for rapid gene expression analysis
    BMC Biotechnology, 2016
    Co-Authors: Kalpana Nanjareddy, Manojkumar Arthikala, Lourdes Blanco, Elizabeth S Arellano, Miguel Lara
    Abstract:

    Phaseolus vulgaris is one of the most extensively studied model legumes in the world. The P. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization of P. vulgaris genes is impeded chiefly due to the non-amenable nature of Phaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for protoplast isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legume P. vulgaris. Herein, we provide methodologies for the high-throughput isolation of Leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from P. vulgaris. The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of Leaf mesophyll protoplasts was optimized using a GUS reporter gene. We used the P. vulgaris SNF1-related protein kinase 1 (PvSnRK1) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of protoplasts that had been transformed with PvSnRK1-RNAi and PvSnRK1-OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the PvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assisted Agrobacterium-mediated transformation (SAAT), for the Leaf Disc infiltration of P. vulgaris. Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60–85 % of the cells in a given area of the Leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies. We present simple and efficient methodologies for protoplast isolation from multiple P. vulgaris tissues. We also provide a high-efficiency and amenable method for Leaf mesophyll transformation for rapid gene functional characterization studies. Furthermore, a modified SAAT Leaf Disc infiltration approach aids in validating genes and their functions. Together, these methods help to rapidly unravel novel gene functions and are promising tools for P. vulgaris research.

Lowell D. Owens - One of the best experts on this subject based on the ideXlab platform.

  • Use of beta-glucuronidase (GUS) as a marker for transformation in sugarbeet
    Journal of Sugarbeet Research, 1993
    Co-Authors: Chris A. Wozniak, Lowell D. Owens
    Abstract:

    Accurate detection of a genetic or biochemical marker introduced into sugarbeet (Beta vulgarjs L.) is based on the absence of native sequences or activities in the plant that could confound the analysis of expression of the introduced marke r. During the course of experiments designed to optimize DNA transfer from Agrobacterjum tumef aciens to sugarbeet Leaf Disc cells, an endogenous enzyme activity was Discovered which utilizes all the common substrates recognized by the marker enzyme l3,-glucuronidase (GUS) from E. coli. This native sugarbeet enzyme (SB-GUS) was characterized immunologically and biochemically. GUS and SB-GUS were found to be distinct with regard to pH optima, thermal inactivation, reaction to denaturants and protein modifying reagents, inhibition by metals and saccharo-Iactone, and molecular mass. The two activities are not immunologically related, as judged by Western blot and immunoprecipitation analyses. A protocol was developed to accurately quantitate introduced GUS in the presence of SB-GUS, by utilizing selective inhibition of GUS at pH 7.0 by saccharic acid l,4-lactone. Under these conditions GUS activity is completely eliminated, while SB­ GUS activity was unaffected.

  • sugarbeet Leaf Disc culture an improved procedure for inducing morphogenesis
    Plant Cell Tissue and Organ Culture, 1992
    Co-Authors: Lowell D. Owens, Debra R Eberts
    Abstract:

    In preparation for gene transfer experiments we investigated factors that might affect the production of shoots and somatic embryos from the wound callus of cultured sugarbeet Leaf Discs. A complex interaction was found between the Leaf Disc plating density, the Disc culture medium, the source-shoot culture medium and the frequency of Disc transfer to fresh medium. The most productive protocol utilized: source shoots maintained on MS medium containing 0.25 mg 1-1 BA; multiple Leaf Discs (ten 4-mm Discs/plate) plated onto an enriched modification of MS medium (RV) containing 1.0 mg 1-1 BA and solidified with 0.3% Gelrite (not permitted to dry during hardening); and transfer of the Discs to fresh medium every two weeks during the first month. This standard protocol produced more callus per plate and higher rates of morphogenesis per unit dry weight of callus than did the one-step method of Saunders and Doley. Water availability considerations were found to be critical to obtaining high morphogenic rates. Root induction frequency and quality was superior on shoots transplanted to MS medium containing 1 mg 1-1 NAA as the sole growth regulator compared to IAA at the same concentration.

  • Measurement and effects of gel matric potential and expressibility on production of morphogenic callus by cultured sugarbeet Leaf Discs
    Plant Cell Tissue and Organ Culture, 1991
    Co-Authors: Lowell D. Owens, Chris A. Wozniak
    Abstract:

    During studies to optimize production of morphogenic callus from cultured Leaf Discs of sugarbeet (Beta vulgaris L.) large differences were observed associated with the gelling agent employed. Water availability, as determined mainly by gel matric potential, was found to be the dominant factor. A simple method was devised to measure the relative matric potential of different gels. A precisely moistened filter-paper Disc was placed on the gel surface, allowed to equilibrate, removed and weighed. The relative gain or loss of water from the paper Disc was a measure of the matric potential of the gel and varied with both gel type and concentration. Leaf Disc expansion and production of callus-derived embryos and shoots were shown to be directly proportional to gel matric potential. Water availability may also be affected by the ease with which liquid is expressed from gels in response to localized pressure caused by explant expansion and contortion. This property, called gel expressibility, was easily measured with a weight and capillary pipette and shown also to vary with gel type and concentration. Validity of the technique for measuring relative matric potential was verified physiologically by culturing Leaf Discs on filter-paper overlays to eliminate expressibility differences among gels. Additionally, comparison of Leaf Disc growth on uncovered gel surfaces versus filter-paper overlays demonstrated the contribution of liquid expression to overall water availability. Expression of liquid by explants on uncovered gel surfaces greatly enhanced the production of morphogenic callus.

Ji Hyun Park - One of the best experts on this subject based on the ideXlab platform.

  • screening of lethal genes for feeding rnai by Leaf Disc mediated systematic delivery of dsrna in tetranychus urticae
    Pesticide Biochemistry and Physiology, 2013
    Co-Authors: Deok Ho Kwon, Ji Hyun Park
    Abstract:

    Abstract To identify genes that kill Tetranychus urticae when knocked down via RNA interference (RNAi), several lethal genes were screened by the systemic delivery of dsRNA via Leaf Disc feeding. Four candidate genes (β subunit of coatomer protein complex, T-COPB2; M1 metalloprotease, T-M1MP; Ribosomal protein S4, T-RPS4; A subunit of V-ATPase, T-VATPase) and a control gene (EGFP) were tested for RNAi. All dsRNAs that permeated the Leaf Disc (ca. 15-mm diameter) were detected at 12 h post-treatment, indicating that dsRNA could move through vascular tissues. To evaluate RNAi toxicity, mortalities were assessed for 120 h following treatment with dsRNA. Treatment with T-COPB2, T-M1MP, T-RPS4 and T-VATPase dsRNAs caused 65.4%, 15.9%, 36.1% and 21.1% mortalities at 120 h post-treatment, respectively. Reduction of all target gene transcripts following dsRNA treatment was confirmed by quantitative PCR, demonstrating that dsRNA feeding-based RNAi could indeed kill T. urticae. In summary, dsRNA delivery via Leaf Disc is an effective system to screen for lethal genes. Furthermore, some genes, such as T-COPB2, T-M1MP, T-RPS4 and T-VATPase, can be used to establish an RNAi-based control system against T. urticae.