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Posted by David on October 8th, 2014  ⟩  0 comments

In part two of our series of blogs highlighting recently published research involving the use of bialaphos and PPT, we now present a summary of exciting research utilizing Gold Biotechnology’s bialaphos and phosphinothricin (PPT) to demonstrate dramatically improved Agrobacterium-mediated ryegrass transformation.

Perennial ryegrass, or Lolium perenne, stands as the preferred cool-weather pasture and forage grass in many regions throughout the world and it serves as an important groundcover. Although perennial ryegrass is suitable for all grazing livestock, it is primarily used as the main source of nutrition for lactating dairy cows, due to the superior quality and digestibility of this species. Ryegrass is also widely utilized as a turf grass and southern state `overseed’, comprising about 40% of the nearly 50 million acres of US lawns, and it is a favorite for use in sporting fields, such as the Kansas City Royals’ Kauffman Stadium, the Seattle Mariners’ Safeco Field and golf courses worldwide.

Despite the broad cultivation of this diploid monocot, it has been quite recalcitrant to genetic modification methods, save traditional breeding, with previous transformation efficiencies reaching only about 1%.  In a recent publication, North Carolina State scientists working under a grant from Bayer Crop Science and using GoldBio’s bialaphos and PPT, reported a dramatic improvement in transformation of Lolium perenne. The researchers achieved a level of 20% transformation efficiency via cocultivation of ryegrass callus with an Agrobacterium tumefaciens strain harboring a binary vector. This plasmid contained the bar gene, which provides resistance to bialaphos and PPT, and a gene encoding green fluorescent protein (GFP).

To take this significant step forward, the researchers at North Carolina State developed a variety of modifications to traditional methods of transforming perennial ryegrass. Transformation efficiency during each experiment was measured by counting the number of GFP-expressing callus and dividing this sum by the total number of callus cocultivated with the Agrobacterium strain.

The group first demonstrated a two-fold improvement in transformation efficiency via a change in infection culture medium. The scientists employed a Murashigie and Skoog (MS)-type media, instead of the more traditional YEP infection medium, when culturing the Agrobacterium strain. The strain was then used to infect two-to-six month old ryegrass callus tissue.

Next, the team determined that a three minute 42 °C heating treatment during the initial infection with the Agrobacterium strain allowed them to achieve a four-fold increase in GFP-expressing callus selected on GoldBio’s bialaphos or PPT (Figure 2 below). Further experiments (Figure 3) showed that increasing the maltose concentration from 3% to 6% in the cocultivation medium and switching to NS6 medium (similar to MS) for that step gave an additional four-fold increase in efficiency.

Catching GoldBio Ryegrass Fig 2-3

When transgenic bialaphos-resistant GFP-expressing plants were eventually grown from callus tissue transformed using a combination of the medium modifications and heating step described above, the efficiency was shown to be more than 20%, marking a 20-fold improvement over previous efficiency in this agriculturally important plant species. This equates to an average of 20 GFP-expressing plants, like those pictured in Figure 7, per 100 callus transformed. The authors proceeded to apply this learning to transformation of rice, Oryza sativa, and observed significant improvements in efficiency with that critical crop as well.

Catching GoldBio Ryegrass Fig 7

Our third blog entry in this series will discuss the generation of bialaphos-resistant soybean, using GoldBio products, that was developed at Nanjing University. GoldBio is excited to be the choice of researchers seeking to further understanding of the biology of crop plants important to populations throughout the world.

Patel, M., Dewey, R. E., & Qu, R. (2013). Enhancing Agrobacterium tumefaciens-mediated transformation efficiency of perennial ryegrass and rice using heat and high maltose treatments during bacterial infection. Plant Cell, Tissue and Organ Culture (PCTOC), 114(1), 19-29. Plant Cell Tiss Organ Cult (2013) 114:19–29 DOI 10.1007/s11240-013-0301-7

Zhang, W. J., Dewey, R. E., Boss, W., Phillippy, B. Q., & Qu, R. (2013). Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses. Plant molecular biology, 81(3), 273-286. Plant Mol Biol (2013) 81:273–286

Category Code: 88241

Posted by David on September 16th, 2014  ⟩  0 comments

Farmers are constantly in need of a more diversified herbicide treatment program. Though RoundUp has held the position of the world’s top selling herbicide for quite some time, scientists and farmers have been hard at work testing alternatives, such as bialaphos, to better handle the near-certainty and complication of herbicide tolerance. This natural herbicide, along with its metabolite phosphinothricin, marketed in its ammonium salt form as Liberty or Basta, appears to be gaining popularity. Scientists have been successfully producing bialaphos-resistant crop plants through current transfection technologies. The development of these new crops will offer farmers the flexibility of using bialaphos and phosphinothricin as effective alternate herbicides.

Briefly, as discussed in a 2012 product spotlight, bialaphos becomes phosphinothricin (PPT) in the plant cell, which inhibits glutamine biosynthesis unless transgenic enzymes encoded by the pat or bar genes are present in the plant to break it down. We will now begin a three-part series of blogs highlighting recently published research involving the use of this herbicide.

Recently, employing Gold Biotechnology’s bialaphos and PPT for selection, North Carolina State scientists, working under a grant from Bayer Crop Science, demonstrated improved Agrobacterium-mediated ryegrass crop transfection using heat and maltose treatment. Half a world away in Taiwan, GoldBio’s bialaphos was also chosen by a group of Nanjing University (PRC) scientists for use in research published this year reporting the creation of a bialaphos-resistant soybean line via Agrobacterium tumefaciens-mediated transfection. Finally, a group at Shimane University in Japan recently constructed new Gateway® binary vectors employing the bialaphos resistance (bar) gene for use in studying plant promoters. Their research focuses on powerful promoter:reporter analysis techniques to study tissue and cell-specific gene expression.

In the case of the Japanese research team’s work, R4L1 Ti plasmids for Agrobacterium-mediated transfection were developed with multiple reporter genes, such as G3GFP, G3 green fluorescent protein and GUS, B-glucoronidase in addition to selectable markers like bar, which conveys bialaphos resistance. The bar gene is under the control of the constitutive nopaline synthase (nos) promoter and terminator, while the reporter genes: G3GFP, TagRFP, GUS, etc., often linked together to be polycistronic, depend upon the host promoter adjacent to the plant chromosomal insertion site for expression. Tissue-specific promoter activity can be interrogated in robust fashion using this method. In the image below, panel F shows selection on phosphinothricin ammonium, while panel J shows imaging of GFP (green) and TagRFP (red) expression in leaf tissue. The key advantage of employing binary vectors for transfection via Agrobacterium lies in the ability to generate plant lines from plants already containing separate genetic modifications, which remain homozygous in the progeny. Previous methods involved crossing promoter:reporter plants with existing lines to stack the desired modifications. This older methodology required laborious analysis of progeny to find the desired genotypes.

Future blog entries will further discuss the generation of bialaphos-resistant ryegrass achieved at North Carolina State University and the bialaphos-resistant soybean developed at Nanjing University. GoldBio is excited to be the choice of researchers seeking to further understanding of the biology of crop plants important to populations throughout the world.

Category Code: 88241

Posted by Patrick on November 1st, 2012  ⟩  0 comments

Bialaphos is a tripeptide antibiotic naturally produced by a few species of the soil bacteria, Streptomyces. It is made up of two alanine residues and the glutamic acid analog phosphinothricin, also called glufosinate. Inside the cell, peptidases release the phosphinothricin compound, enabling it to inhibit normal nitrogen metabolism and causing an accumulation of intracellular ammonia.  The pat gene from Streptomyces viridochromogenes or the bar gene from Streptomyces hygroscopicus encode enzymes that modify phosphinothricin and detoxify the compound, conferring cellular resistance to bialaphos as well as phosphinothricin or glufosinate(1).

Bialaphos has most commonly been used in plant molecular biology to select for genetically engineered cell lines containing one of the resistance genes.  Bialaphos has increasingly become an important drug selection for yeast genetics as well.  Yeast cells carrying the pat gene grow in the presence of bialaphos and resistance is a dominant trait. Bialaphos resistance can be used in combination with auxotrophic yeast markers as well as other dominant selectable drugs like hygromycin B or kanamycin.  Dominant drug resistant markers like bialaphos resistance are especially useful in studies involving wild or industrial yeast strains that are often prototrophic for the most common yeast nutritional markers.  Resistance to bialaphos is also an efficient way to identify diploid progeny from crosses between haploids lacking compatible auxotrophic markers(2).  Cells resistant to bialaphos have no detectable growth disadvantage, and therefore can be used in competition experiments or for monitoring other processes dependant on metabolic state(3). Finally, the pat gene is heterologous to the yeast genome, therefore inappropriate integration or recombination is unlikely.

1. Tan, S., Evans, R. & Singh, B. Herbicidal inhibitors of amino acid biosynthesis and herbicide- tolerant crops. Amino Acids 30, 195-204(2006).

2. Sadowski, I., Lourenco, P. & Parent, J. Dominant marker vectors for selecting yeast mating products. Yeast 25, 595-599(2008).

3. Goldstein, A.L. & McCusker, J.H. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541-1553(1999)

Category Code: 88221 88251