A novel dominant selection system for plant transgenics based on phosphite metabolism catalyzed by bacterial alkaline phosphatase

Selective markers are generally indispensable in plant genetic transformation, of which the frequently used are of antibiotic or herbicide resistance. However, the increasing concerns on transgenic biosafety have encouraged many new and safe selective markers emerging, with an eminent representative as phosphite (Phi) in combination to its dehydrogenase (PTDH, e.g. PtxD). As bacterial alkaline phosphatase (BAP) can resemble PtxD to oxidatively convert toxic Phi into metabolizable phosphate (Pi), herein we harnessed it as the substitute of PtxD to develop an alternative Phi-based selection system. We first validated the Escherichia coli BAP (EcBAP) did own an extra enzymatic activity of oxidizing Phi to Pi. We further revealed EcBAP could be used as a dominant selective marker for Agrobacterium-mediated tobacco transformation. Although the involved Phi selection for transformed tobacco cells surprisingly required the presence of Pi, it showed a considerable transformation efficiency and dramatically accelerated transformation procedure, as compared to the routine kanamycin selection and the well-known PtxD/Phi system. Moreover, the EcBAP transgenic tobaccos could metabolize toxic Phi as a phosphorus (P) fertilizer thus underlying Phi-resistance, and competitively possess a dominant growth over wild-type tobacco and weeds under Phi stress. Therefore, this novel BAP/Phi-coupled system, integrating multiple advantages covering biosafe dominant selective marker, plant P utilization and weed management, can provide a PTDH-bypass technological choice to engineer transgenic plant species, especially those of great importance for sustainable agriculture.

A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii

ABSTRACTCaldicellulosiruptor bescii, an anaerobic Gram-positive bacterium with an optimal growth temperature of 78°C, is the most thermophilic cellulose degrader known. It is of great biotechnological interest, as it efficiently deconstructs nonpretreated lignocellulosic plant biomass. Currently, its genetic manipulation relies on a mutant uracil auxotrophic background strain that contains a random deletion in thepyrFgenome region. ThepyrFgene serves as a genetic marker to select for uracil prototrophy, and it can also be counterselected for loss via resistance to the compound 5-fluoroorotic acid (5-FOA). To expand theC. besciigenetic tool kit, kanamycin resistance was developed as a selection for genetic manipulation. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs inC. bescii. These strains showed resistance to kanamycin at concentrations >50 μg · ml−1, whereas wild-typeC. besciiwas sensitive to kanamycin at 10 μg · ml−1. In addition, placement of the Cbhtkmarker between homologous recombination regions in an integrating vector allowed direct selection of a chromosomal mutation using both kanamycin and 5-FOA. Furthermore, the use of kanamycin selection enabled the targeted deletion of thepyrEgene in wild-typeC. bescii, generating a uracil auxotrophic genetic background strain resistant to 5-FOA. ThepyrEgene functioned as a counterselectable marker, likepyrF, and was used together with Cbhtkin the ΔpyrEbackground strain to delete genes encoding lactate dehydrogenase and the CbeI restriction enzyme.IMPORTANCECaldicellulosiruptor besciiis a thermophilic anaerobic bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass. It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel production. The current genetic system used withC. besciiis based upon only a single selection strategy, and this uses the gene involved in a primary biosynthetic pathway. There are many advantages with an additional genetic selection using an antibiotic. This presents a challenge for thermophilic microorganisms, as only a limited number of antibiotics are stable above 50°C, and a thermostable version of the enzyme conferring antibiotic resistance must be obtained. In this work, we have developed a selection system forC. besciiusing the antibiotic kanamycin and have shown that, in combination with the biosynthetic gene marker, it can be used to efficiently delete genes in this organism.

StableAgrobacterium-Mediated Transformation of Maritime Pine Based on Kanamycin Selection

An efficient transformation protocol based on kanamycin selection was developed forAgrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which containedneomycin phosphotransferase II(nptII) as a selectable marker gene andβ-glucuronidase(uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL−1kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated withAgrobacteriumstrain AGL1 at the optical density (OD600 nm) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for thenptIIanduidAgenes and expression of theuidAgene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth.

Transient expression of uidA gene in grapevine protoplasts after PEG-mediated transformation

<p style="text-align: justify;">Leaf protoplasts, isolated from <em>Vitis vinifera</em> «Sakasly» and «Muscat d’Alexandrie» and protoplasts from embryogenic tissue of <em>Vitis</em> sp. «Seyval blanc» were incubated in the presence of PEG in a transformation solution containing the plasmid pBI426 which carries the β-glucuronidase (<em>gus</em>) and the neomycin phosphotransferase II (<em>npt</em>II) genes. The treated protoplasts were cultivated in CPW13 medium without kanamycin selection. 48h after the PEG transformation, transient expression of gus gene was detected histochemically and fluorimetrically in the protoplast cultures.</p>

Transformation of `Beurre Bosc' Pear with the rolC Gene

`Beurre Bosc' pear (Pyrus communis L.) was transformed with Agrobacterium tumefaciens (E.F. Smith & Townsend) Conn strain EHA101 containing the binary vector pGA-GUSGF into which the rolC gene had been inserted. Leaf explants from in vitro shoot tip cultures were wounded, Agrobacterium-inoculated, and cultured on kanamycin selection medium. Regenerating shoots were transferred to proliferation medium without antibiotics. Three clones tested positive for GUS and nptII enzyme activity. Transformation with the rolC gene was confirmed by DNA, RNA, and protein blot analyses. The number of copies of the rolC transgene varied from one to three. Plantlets of the three transgenic clones were acclimated and transferred to the greenhouse. Preliminary observations of phenotype indicate that the rolC gene reduced height, number of nodes, and leaf area of transgenic `Beurre Bosc'.

Transformation of an Australian Cotton Cultivar: Prospects for Cotton Improvement Through Genetic Engineering

Somatic embryogenesis and regeneration of whole plants is a highly genotype-dependent process in cotton. We have identified at least one highly regenerable Australian cultivar, Siokra 1-3, which is a sister line to the current major variety being grown in Australia. A number of plants have been regenerated and although some are showing abnormal pollen development, most can produce fertile seed when selfed or crossed with a normal pollen donor. Agrobacterium tumefaciens has been used to efficiently produce fertile transgenic Siokra 1-3 plants expressing novel genes such as the bacterial neomycin phosphotransferase or the β-glucuronidase. This is the first example of the transformation of an elite commercial cultivar. Critical factors in the transformation are the use of a supervirulent disarmed Ti-plasmid with a binary transformation vector, and a highly regenerable genotype of cotton. Bacterial concentration at the time of infection, tissue age, kanamycin selection regime, and co-cultivation support and media composition all have an influence on transformation efficiency and were optimised in our protocol. The ability to transform an elite Australian cultivar of cotton paves the way for agronomic improvements through genetic engineering. We have concentrated on increasing the tolerance of Australian cotton to the herbicide 2,4-D (to protect it from spray drift damage from adjacent cereal crops), and increasing its tolerance to insect pests, such as Helicoverpa armigera, using BT-toxin genes, protease inhibitors and other novel insect resistance genes.