The pCLEAN dual binary vector system for Agrobacterium-mediated plant transformation

Abstract

The development of novel transformation vectors is essential to the improvement of plant transformation technologies. Here, we report the construction and testing of a new multifunctional dual binary vector system, pCLEAN, for Agrobacterium-mediated plant transformation. The pCLEAN vectors are based on the widely used pGreen/pSoup system and the pCLEAN-G/pCLEAN-S plasmids are fully compatible with the existing pGreen/pSoup vectors. A single Agrobacterium can harbor (1) pCLEAN-G and pSoup, (2) pGreen and pCLEAN-S, or (3) pCLEAN-G and pCLEAN-S vector combination. pCLEAN vectors have been designed to enable the delivery of multiple transgenes from distinct T-DNAs and/or vector backbone sequences while minimizing the insertion of superfluous DNA sequences into the plant nuclear genome as well as facilitating the production of marker-free plants. pCLEAN vectors contain a minimal T-DNA (102 nucleotides) consisting of direct border repeats surrounding a 52-nucleotide-long multiple cloning site, an optimized left-border sequence, a double left-border sequence, restriction sites outside the borders, and two independent T-DNAs. In addition, selectable and/or reporter genes have been inserted into the vector backbone sequence to allow either the counter-screening of backbone transfer or its exploitation for the production of marker-free plants. The efficiency of the different pCLEAN vectors has been assessed using transient and stable transformation assays in Nicotiana benthamiana and/or Oryza sativa.

Figure 1.

Schematic illustration of T-DNA regions constructed for the pCLEAN vector series. A, Six types of T-DNA regions (nos. 1–6) were developed for pCLEAN-G vectors and two types of T-DNA regions (nos. 7 and 8) for pCLEAN-S vectors. T-DNA regions numbers 1 and 2 contain the original large inner T-DNA of 728 nt from pGreenII (black box), and regions numbers 3 to 8 contain the minimal pCLEAN inner T-DNA of 52 nt (red box). Unique restriction endonuclease recognition sites within and surrounding the T-DNA regions are displayed. Brackets indicate restriction sites that are not single cutters. T-DNA regions numbers 1 and 3 contain the pGreenII original suboptimal 24-nt-long LB (yellow striped box), regions numbers 2 and 4 to 8 a 25-nt-long nopaline-type consensus LB (yellow textured box), and regions numbers 5 and 6 harbor a double LB. The RB is represented as a light yellow-colored box and its adjacent overdrive sequence (24 nt long) is shown as a light blue box. T-DNA regions numbers 6 and 8 contain a wild-type virG gene (pink-colored box) outside the RB/overdrive. For each type of T-DNA region, the corresponding basic pCLEAN vector plasmid is specified. B, Sequence details of the pCLEAN minimal T-DNA regions numbers 4 and 5. T-DNA region number 5 includes a double LB separated by a 26-nt-long spacer sequence. Additional sequence data for pCLEAN plasmids and their T-DNA regions are available at http://www.jic.ac.uk/staff/philippe-vain/vectors.htm and GenBank, respectively.

Figure 2.

Schematic illustration of the dual binary pCLEAN vector series A, Basic pCLEAN vectors (i.e. 12 pCLEAN-G and two pCLEAN-S vectors containing the T-DNA regions nos. 1–6 and nos. 7 and 8, respectively; Fig. 1). B, pCLEAN vectors containing selectable marker and/or reporter genes within their T-DNA. The T-DNA region into which the transgene(s) was inserted is indicated. C, Components constituting the different pCLEAN vectors.

Figure 3.

Evaluation of pCLEAN vectors using transient expression assays in N. benthamiana. A, Effect of the number of consensus LB repeats and the presence of a transgene in the T-DNA on the level of backbone transfer into plant cells. Fluorescence levels (average ± se) indicate delivery and expression of the GFP gene located in the backbone sequence. Values followed by different letters are significantly different (Kruskal-Wallis test, P < 0.05). B, Expression of a reporter gene (GUS) in large and minimal T-DNAs. “n” represents the total number of leaves analyzed. For further details of the vector components, refer to Figure 2C.

Figure 4.

Stable transformation of rice plants using either a T-DNA or the vector backbone to deliver transgenes. “n” indicates the total number of rice embryogenic calli used for Agrobacterium-mediated transformation. Transgenic plants are GFP+ and resistant to hygromycin. Values followed by different letters are significantly different (χ² test, P < 0.05). For further details of the vector components, see Figure 2C.