Abstract
Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes.
Highlights
Modified plants are irreplaceable tools in plant science and have the potential to revolutionize agriculture
Transgenic plants can be produced by viral transformation [3], electroporation of protoplasts [4], particle bombardment [5] and Agrobacterium-mediated transformation [6], with the latter being the most popular method
The vector was made compatible for MultiSite Gateway by introducing the spectinomycin resistance gene (SpR) in the backbone of the plasmid and cloning of an attR4-attR3 cassette, containing a chloramphenicol resistance gene (CmR) and a ccdB gene, between the left and the right border sequences [31]
Summary
Modified plants are irreplaceable tools in plant science and have the potential to revolutionize agriculture. A long-standing goal is the transfer of the ability to establish a symbiosis with nitrogen-fixing rhizobia to non-legume crop species, making them independent of nitrogen fertilization [1,2] Most of such genetic modifications require transfer of more than one gene, up to the transfer of complete pathways. BIBAC and pYLTAC7, were designed for high stability of big constructs by employing low copy amplification in Escherichia coli (E. coli) as well as in Agrobacterium [8,9] Such vectors allow the transfer of large DNA fragments up to 150 kb into plant genomes. The synthetic R recombinase can be activated by dexametasone, by which it translocates from the cytoplasm into the nucleus of the plant cells It recombines the Rs sites thereby removing the marker genes from the genome [10,11]. Many vectors today are available with introduced gateway cassettes allowing easy cloning and thereby removing the need to work with rare cutting enzymes as well as enabling MultiRound Gateway technology to clone several genes in one binary vector [13,14,15]
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