Demonstrate
Demonstration of permanent transformation
The present of an antibiotic marker in our plasmid (kanamycin) allowed us to identify transformed from untransformed seedlings. When a kanamycin-resistant seedling grow on medium containing this antibiotic we can detect green, open and expanded cotyledons and leaves.
Selection of kanamycin-resistant Arabidopsis seedlings
Transgenic Arabidopsis plants were transferred to soil to produce totally developed rosettes. After that, we confirmed the presence of GFP bound to starch granule by using confocal microscopy. When we analyzed WT plants we could observe the chlorophyll fluoresce of the chloroplast (red color) and dark spots that correspond to the presence of starch granules. However, in the transgenic plants we could observe the chlorophyll fluoresce of the chloroplast and GFP signal in the starch granules (green color). These data confirmed that our transgenic plants carried out the coding sequence GBSS-Xa-GFP.
Protein Yield
To determine the efficiency of our proposed system we quantified, as described in measurements, the amount of starch and protein (GFP) recovered.
Using 1 Kg of leaves from Arabidopsis plants, we purified 12 g of starch. Using 1 g of starch, we was able to recover 5.4 μg of GFP. The resulting protein yield was 65 μg of GFP per Kg of leaves.
Protein Yield Improvement
To improve the protein yield we grew the plants in the presence/absence of adjacent cultures of Alternaria alternate (see “Improvement of protein production” section). In that conditions we were able to improve starch content in leaves 8 times.
Starch content of Arabidopsis treated for one day with Alternaria alternata VCs
In consequence we could improve the protein yield 8 times obtaining a final yield of half milligram of GFP per Kg of leaves.
