Construction and Validation of Three Convenient and Practical Constructs for Plant Transformation
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    Abstract:

    To enrich tools for plant functional genomics and to develop marker-free transgenic plants, three plasmid vectors, pAH006, pWMB022 and pWMB025, ideal for cereal transformation, were constructed in this study by using some plasmids available publically. The vector pAH006 can be used to improve the Agrobacterium mediated transformation system on monocot plants and to evaluate the biolistic particle mediated transformation efficiency of linear transgene expression cassette on which both GUS and bar genes were controlled by the ubi promoters; the intact T-DNA region can be easily recovered by enzyme digestion. The vector pWMB022 carries maize pigment regulatory genes Lc and C1 under the control of double 35S promoters, which can be used to visually screen positive calli or shoots when co-bombarded with other expression vectors containing genes of interest. The vector pWMB025 carries the glyphosate-resistant gene EPSPS regulated by the ubi promoter; the vector can be used in Agrobacterium- or biolistic-mediated transformation of cereal plants to generate bio-safely transgenic materials. Genes of interest can be easily cloned into the multiple cloning site (MCS) between the ubi promoter and the nos terminator on pWMB025 by enzyme digestion. All three vectors were confirmed by enzyme digestion and then tested in Agrobacterium- or biolistic-mediated transformation by using wheat immature embryos derived calli or leaves as explants. It was shown that all three vectors were constructed successfully, and the selectable reporter/visual genes worked efficiently. Construction of these three expression vectors is important for the improvement of transformation efficiency of some recalcitrant cereals such as wheat, development of bio-safely transgenic crop varieties, and plant functional genomics.

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History
  • Received:June 28,2014
  • Revised:August 02,2014
  • Adopted:October 08,2014
  • Online: October 08,2014
  • Published:
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