基礎講座 電気泳動(Electrophoresis)

ISAAA Briefs brief 41 Global status of Commercialized biotech/GM Crops: 2009

The creation of transgenic plants has contributed extensively to the advancement of plant science. Establishing homozygous transgenic lines is time-consuming and laborious, and using antibiotics or herbicides to select transformed plants may adversely affect the growth of some transgenic plants. Here we describe a novel technology, which we have named FAST (fluorescence-accumulating seed technology), that overcomes these difficulties. Although this technology was designed for use in Arabidopsis thaliana, it may be adapted for use in other plants. The technology is based on the expression of a fluorescent co-dominant screenable marker FAST, under the control of a seed-specific promoter, on the oil body membrane. The FAST marker harbors a fusion gene encoding either GFP or RFP with an oil body membrane protein that is prominent in seeds. The marker protein was only expressed in a specific organ (i.e. in dry seeds) and at a specific time (i.e. during dormancy), which are desirable features of selectable and/or screenable markers. This technique provides an immediate and non-destructive method for identifying transformed dry seeds. It identified the heterozygous transformed seeds among the T(1) population and the homozygous seeds among the T(2) population with a false-discovery rate of <1%. The FAST marker reduces the length of time required to produce homozygous transgenic lines from 7.5 to 4 months. Furthermore, it does not require sterilization, clean-bench protocols or the handling of large numbers of plants. This technology should greatly facilitate the generation of transgenic Arabidopsis plants.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2009.04060.x

A rapid and non‐destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana

Statistical Methods in Agriculture and Experimental Biology.

http://www2.ufpel.edu.br/biotecnologia/gbiotec/site/content/paginadoprofessor/uploadsprofessor/096d8cf40f74d2aa8fea95d552b9e89f.pdf

Strategies for high-level recombinant protein expression in transgenic microalgae: A review

Hexaploid wheat, one of the world’s most important staple crops, remains a challenge for genetic transformation. We are developing a floral transformation protocol for wheat that does not require tissue culture. This paper presents three transformants in the hard red germplasm line Crocus that have been characterized thoroughly at the molecular level over three to six generations. Wheat spikes at the early boot stage, i.e. the early, mid or late uninucleate microspore stages, were immersed in an infiltration medium of strain C58C1 harboring pDs(Hyg)35S, or strain AGL1 harboring pBECKSred. pDs(Hyg)35S contains the NPTII and hph selectable markers, and transformants were detected using paromomycin spray at the whole plant level, NPTII ELISAs, or selection on medium with hygromycin. Strain AGL1, harboring pBECKSred, which contains the maize anthocyanin regulators, Lc and C1, and the NPTII gene, was also used to produce a Crocus transformant. T1 and T2 seeds with red embryos were selected; T1 and T2 plants were screened by sequential tests for paromomycin resistance and NPTII ELISAs. The transformants were low copy number and showed Mendelian segregation in the T2. Stable transmission of the transgenes over several generations has been demonstrated using Southern analysis. Gene expression in advanced progeny was shown using Reverse Transcriptase-PCR and ELISA assays for NPTII protein expression. This protocol has the potential to reduce the time and expense required for wheat transformation.

/pdf/evidence-for-stable-transformation-of-wheat-by-floral-dip-in-2djekfeavd.pdf

Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens.

Background
Grasses are relatively recalcitrant to genetic transformation in comparison to certain dicotyledons, yet they constitute some of the most important biofuel crops. Genetic transformation of switchgrass (Panicum virgatum L.) has previously been reported after cocultivation of explants with Agrobacterium and biolistics of embryogenic calli. Experiments to increase transient gene expression in planta may lead to stable transformation methods with increased efficiency.

/pdf/a-high-throughput-transient-gene-expression-system-for-1asz7xyvo7.pdf

A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings.

A method is described for the floral transformation of wheat using a protocol similar to the floral dip of Arabidopsis. This method does not employ tissue culture of dissected embryos, but instead pre-anthesis spikes with clipped florets at the early, mid to late uninucleate microspore stage are dipped in Agrobacterium infiltration media harboring a vector carrying anthocyanin reporters and the NPTII selectable marker. T1 seeds are examined for color changes induced in the embryo by the anthocyanin reporters. Putatively transformed seeds are germinated and the seedlings are screened for the presence of the NPTII gene based on resistance to paromomycin spray and assayed with NPTII ELISAs. Genomic DNA of putative transformants is digested and analyzed on Southern blots for copy number to determine whether the T-DNA has integrated into the nucleus and to show the number of insertions. The nonoptimized transformation efficiencies range from 0.3 to 0.6% (number of transformants/number of florets dipped) but the efficiencies are higher in terms of the number of transformants produced/number of seeds set ranging from 0.9 to 10%. Research is underway to maximize seed set and optimize the protocol by testing different Agrobacterium strains, visual reporters, vectors, and surfactants.

Floral transformation of wheat.

In the spring of 2007, switchgrass accessions and cultivars Alamo, Kanlow, SL-93-2001, and NSL 2001-1 (lowland), Blackwell (upland), and Grenville, Falcon, and Miami (unknown ploidy levels) were sown at the East Tennessee Research and Extension Center in Knoxville for evaluation and controlled hybridizations. In July and August of 2007, uredinia were observed primarily on the upper leaf surfaces, and to a lesser extent on the undersides of leaves, of switchgrass cvs. Alamo, Blackwell, Grenville, Falcon, Kanlow, and Miami. Uredinia were observed on all cultivars and accessions in 2008. Dimensions of spores are reported as mean ± standard deviation. Uredinia were epiphyllous, adaxial, caulicolous, oblong, and the color of cinnamon brown. Urediniospores were globose to broadly ellipsoid, 26.0 ± 3.0 × 23.2 ± 2.4 μm, with a wall that was cinnamon brown, 1.5 to 2.0 μm thick, finely echinulate with three to four equatorial pores, corresponding to Puccinia emaculata Schw. (3). Abundant teliospores were isolated from Grenville, Falcon, and Blackwell, with fewer teliospores isolated from Alamo. Telia were epiphyllous, adaxial, and caulicolous, densely crowded to scattered, oblong, and dark brown to black. Teliospores were dark brown, two-celled, ellipsoid to oblong, 33.6 ± 4.8 μm long with an apical cell width of 17.5 ± 1.2 μm and basal cell width of 15.9 ± 2.5 μm. Teliospore walls were 1.5 to 2.0 μm wide at the sides and 4 to 6 μm apically. Pedicels were brown or colorless and up to approximately one length of the teliospore, 28.5 ± 7.4 μm. Teliospore morphology confirmed the identification of this rust as P. emaculata (3), which has been reported to infect upland and lowland populations of switchgrass (2). A 2,109-bp fragment containing the internal transcribed spacer (ITS) 1, 5.8S, ITS 2, and D1/D2 region of the large subunit ribosomal DNA was sequenced for a specimen on 'Falcon' (GenBank Accession No. EU915294 and BPI No. 878722) from two overlapping PCR fragments amplified with primers PRITS1F (L. A. Castlebury, unpublished data) and ITS4B (1) for one fragment and Rust5.8SF (L. A. Castlebury, unpublished data) and LR7 (4) for the second fragment. No sequences of P. emaculata were available for comparison; however, BLAST searches of the ITS resulted in hits to P. asparagi DC (527 of 576, 91%) and P. andropogonis Schw. (523 of 568, 92%) placing this fungus in the genus Puccinia Pers. The alternate hosts of this rust are species of the Euphorbiaceae (2,3), which are ubiquitous in this area although the aecial stage has not been observed. To our knowledge, this is the first report of P. emaculata on switchgrass in Tennessee. Given the highly susceptible response of certain varieties of switchgrass to this rust in field plots, reduction in total biomass in large acreages is likely and long-standing fields of this perennial grass will compound the problem. References: (1) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (2) D. M. Gustafson et al. Crop Sci. 43:755, 2003. (3) P. Ramachar and G. Cummins. Mycopathol. Mycol. Appl. 25:7, 1965. (4) R. Vilgalys and M. Hester. J. Bacteriol. 172:4238, 1990.

First Report of Rust on Switchgrass (Panicum virgatum) Caused by Puccinia emaculata in Tennessee

We apply CE for high-throughput analysis of functional markers for marker-assisted selection in rice. The accuracy, throughput and reproducibility of CE analysis for sequence-tagged site (STS) and simple sequence repeat (SSR) markers for bacterial blight resistance and aroma genes are demonstrated by using a CE system. Multiplex PCR products displayed well-differentiated allelic variants using different STS and SSR markers for identification of xa13, Xa21 and fgr genes using the CE system compared to 1.2% agarose gel images. Moreover, consumption of PCR product is much less in the CE system compared to traditional agarose gel systems. Sample consumption is less than 0.1 μL per analysis, thereby conserving samples for further downstream analysis. Out of 29 genotypes in BC(1)F(3) generation, 16 plants were found homozygous for all the three genes, viz., xa13, Xa21 and fgr. These homozygous lines can be used as potential donors in rice breeding programmes.

Sujata Agarwal

Papers

Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens.

A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings.

Floral transformation of wheat.

First Report of Rust on Switchgrass (Panicum virgatum) Caused by Puccinia emaculata in Tennessee

High‐throughput functional marker assay for detection of Xa/xa and fgr genes in rice (Oryza sativa L.)