Marker-free apple plants expressed the supersweet protein thaumatin II gene driven by plant promoter

Using the pMF1 vector, which combines inducible site-specific recombinase and bifunctional selectable gene, we have obtained marker-free apple plants with the supersweet thaumatin II protein under the control of tomato genetic regulatory elements. Exploitation of this gene in our laboratory allowed enhancing of sweetness, as well as improving the taste characteristics of fruits and vegetables of plants such as strawberry, carrot, tomato, pear and apple. In the early works we used a constitutive virus 35S promoter. GM plants with viral and bacterial genes are adopted by consumers with concerns; in addition, constitutive promoters have a number of disadvantages in the industrial cultivation of agricultures. In this paper, we used a plant mainly fruit-specific promoter, along with a selective gene removal system, which prevented the presence of any foreign DNA in the apple genome. And we were faced with the task of evaluating the of transferred gene expression level in 39 derived apple sublines. Despite the predominant fruit specificity of the promoter, the mRNA of the thaumatin II gene was detected in the leaves. Two methods for analyzing the expression of thauII at the RNA level were used - semiquantitative RT-PCR and real-time PCR. Six sets of primers were designed - 3 for the housekeeping genes and 3 for the thaumatin II gene. Both methods showed high repeatability and similar results, but one highly productive line was identified only by real-time PCR. All apple sublines were divided into groups according to the level of thaumatin II gene expression, the plants with the highest level were chosen for further experiments.