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Wheat (Triticum aestivum) is one of the most important crops grown globally together with maize and rice. Many crop species, wheat included, contain undesired traits or traits that could be better adapted to the changing environment. Biotechnological modifications to alter such traits in wheat are much less studied than in many other crops and there are several obstacles to overcome such as lack of efficient standard protocols for the transformation step. One part of the problem is the large size of its genome which makes gene integration more difficult. One problematic trait is that wheat can take up and store large amounts of cadmium which is a toxic heavy metal that should not be consumed in large quantities. Cadmium is transported into the root cells through different transporters such as NRAMP6. The cadmium accumulates in the grains which are consumed by humans and animals and can cause health problems. Development of biotechnological methods for wheat transformation is therefore desired. With such methods, the undesired cadmium uptake and storage in wheat could potentially be decreased. The aim of this MSc project was to develop an improved transformation method to faciliate gene editing in wheat, and the target was the NRAMP6 transporter. Additionally, a new plasmid for this purpose was designed and successfully cloned in silico using SnapGene. 

Two new transformation methods were tested in this project: floral dipping in an Agrobacterium solution and Agrobacterium pipetting. They both utilize Agrobacterium tumefaciens' natural ability to transfer part of its DNA into the genome of a plant cell. A plasmid with a transfer-DNA (T-DNA) containing a CRISPR/Cas9 construct targeting the NRAMP6 transporter was transformed into A. tumefaciens. The Agrobacterium was thereafter added to an infiltration solution which was used to treat pre-anthesis wheat spikes with either floral dipping or Agrobacterium pipetting with the purpose of transferring the T-DNA into them. Both methods managed to produce surviving spikes and the results showed that spikes treated with the dipping method had a higher survival rate. The surviving spikes were furthermore able to produce seeds, but the seed set was low for both the treated spikes and the controls. A hygromycin selection was then used on the mature seeds to test for T-DNA insertion. In total, 135 treated seeds were harvested but only 46 of them were placed on the hygromycin selection medium. None of the 46 seeds germinated on the hygromycin in the set timeframe of the project and the number of potentially successful transformants could therefore not be determined. Despite inconclusive results, this MSc project have contributed to increased knowledge of genetic modifications in wheat and have laid a foundation for continued progress within the important area of gene transformation of agricultural crops.