Since Mtz treatment is mutagenic [26] and these transformants had been selected on Mtz-containing agar, we tested if their fur alleles did indeed cause increased Mtz resistance by transforming them into strain M1.5 Δfur-aphA, with selection only for the linked chloramphenicol resistance marker.
Genomic DNA was isolated from the 38 transformants about 100 generations after the transformants had been generated.
As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies.
Briefly, after subcloning the variable domain repertoire in the pMECS phage display vector, which adds an HA (haemagglutinin) and a His tag, a library of 1.2×10 transformants, which has been panned on recombinant GAK, was generated, of which 78% had correctly sized inserts.
Primary plant transformant DNA has been extracted using the standard MATAB protocol (Mieulet et al. 2013).
A comparable value of 1.1 – 6.6 × 10-7 transformants per cell has been reported for the related G. pectorale species [ 17].
It is worth mentioning that variation of polyamine levels in the genetic transformants overexpressing polyamine biosynthetic genes has been reported in earlier studies [ 32, 40].
Another category consisting in regenerating transformants from established embryogenic cultures has been described in several species including grapevine [ 21, 37], Citrus [ 38], rubber tree [ 22], Prunus subhirtella [ 18, 19] and American chestnut [ 23].
We also demonstrate that Rubisco LSU protein derived solely from the fusion protein construct is completely functional, as evidenced by normal photosynthesis in transformants where the endogenous rbcL gene has been eliminated.
