As shown in Fig. 2a, using the binary plasmid pPK2BarGFPD, the maximum transformation efficiency (145 ± 10 transformants per plate) was obtained with AS at a concentration of 200 μM, and the number of transformants was reduced when the concentration was increased or decreased.
For example, at maturity, the transformants were much smaller than the wild type and their leaf trichomes were reduced in size and branching (Figure 10A).
Further studies demonstrated that by reducing the concentration of DNA used for transformation, the number of such double transformants could be reduced and also result in higher overall transformation efficiency (data not shown).
The LT50 of the transformant expressing CDEP1 Bbchit1 was reduced by 24.9%.
The percentage of Ura+ transformants that were Ade+ was reduced in the mph1 Δ mutant and template switching among the Ade+ events was also decreased, consistent with the need for Mph1 to dismantle the D-loop for template switching.
The percentage of Ade+ Ura+ transformants derived from pLAG6 was reduced by 3.9-fold in the mph1 Δ mutant as compared to wild type (0.68%; P < 0.0001; Figure 4A).
The percentage of Ade+ Ura+ transformants derived from pLAG6 was reduced by eightfold in the pif1 -m2 strain (0.31%; P < 0.0001), and even further decreased in the mph1 ∆ pif1 -m2 mutant (0.16%, P = 0.031 and P < 0.001 compared to pif1 -m2 and mph1 Δ strains, respectively; Figure 4A), suggesting an additive effect of MPH1 and PIF1.
This method was successful in silencing the gene, and the iron uptake of the transformants generated with this construct was reduced by roughly 50%%.
Filament elongation was reduced in the transformants, resulting in the heterostyly type of floral architecture.
The growth rate of the MstG transformant during the exponential growth phase was comparable in the three different conditions, while in case of the MstH transformant, at the highest glucose concentration the growth rate was reduced.
