Abstract

SOUZA CANADA, Eduardo Daniel; FETTIG, Sebastian; ZIEGLER, Paul  and  BECK, Erwin. Transformation of an Argentine spring wheat genotype: optimization of the protocols for particle bombardment of excised immature embryos and rapid isolation of transgenic plants. BAG, J. basic appl. genet. [online]. 2015, vol.26, n.1, pp.18-37. ISSN 1852-6233.

Klein Brujo was found to be a promising Argentine spring wheat genotype for transformation studies. In the present work we optimized the biolistic transformation of embryogenic scutellar calli from this genotype. We first identified scutellar callus induction media (SCIM) most promising for in vitro embryogenic plant regeneration with Klein Brujo. We then co-bombarded embryos of Klein Brujo and, for comparison, of Bobwhite, a highly transformable wheat line, on these media with 1 μm gold particles coated with two plasmids. One of these contained the marker gene gfp (linked to the CaMV35S-promoter) and the selection gene bar (resistance to phosphinothricin: PPT, linked to the maize Ubi1-promoter), whereas the other contained ipt (encoding isopentenyltransferase) as a candidate gene under the control of the HvS40- or SAG12- promoter. Transformation efficiencies of up to 16.4% with Klein Brujo and 6% with Bobwhite were obtained with embryos pre-cultured on SCIM for 96 h and subjected to pre- and post-bombardment osmotic treatment for 4-5 and 16 h, respectively. Transgenic calli and plants regenerating in vitro were identified by screening for GFP expression and PPT resistance. One hundred and three transgenic lines of Klein Brujo - far more than of Bobwhite (12 lines) - were established on soil, often within only nine weeks. The frequency of co-transformation of gfp and/or bar and ipt exceeded 97% for both genotypes, and the three genes were shown to co-segregate with selected individuals. The ipt-gene was structurally stable up to the T3 generation, whereas gfp and bar were susceptible to silencing.

Keywords : Particle bombardment parameters; Co-transformation; Green fluorescent protein; Phosphinothricin resistance; Transgene segregation.

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