We have developed a successful method for the elimination of cannabis seed infections (Cannabis sativa L.) and at the same time the successful transformation of hypocotyls, with which we introduced the hptII selection gene and the ZsGreen reporter gene into plant cells, which expresses green fluorescence via protein. Gene transformations were performed by three-day cocultivation of hypocotyls with Agrobacterium tumefaciens. Adventitious regeneration of callus was achieved by growing explants on MS medium supplemented with vitamins (Duchefa, M0222), 30 g/l sucrose, 8 g/l agar, 0.2 mg/l TDZ, 0.5 mg/l NAA, 200µM acetosiringone and pH 5.8 (the KON3 medium). The success of the transformation was confirmed using an epifluorescent macroscope, as the regenerated hypocotyls glowed fluorescent green. In another experiment, where sliced hypocotyls were placed on KON3 medium, we examined whether the position of explants on the hypocotyl influenced adventitious regeneration. It was found that there were no differences in regeneration. For the success of the experiment, we found that the key is to choose undamaged seeds with a characteristically marbled seed coat pattern. Otherwise, a larger number of bacterial and fungal infections, mainly of endogenous origin, occur, as these cannot be eliminated by classical sterilization.
|
