Beeinflussung des Ertragspotentials von Gerste und Weizen durch Modifikation ährenarchitektonischer Eigenschaften mittels Cas-Endonuklease-vermittelter Mutagenese

authored by
Christian Wolfgang Hertig
supervised by
Jens Boch

This thesis describes the application of genome engineering using RNA-mediated Cas9 endonuclease in the cereal species wheat and barley. This new breeding tool makes it possible to induce specific changes in the genome of cereals within a short period of time, thereby specifically modifying plant characteristics. For this purpose, target motifs were selected in the wheat gene Branched head (Bh)/ Wheat frizzy panicle (Wfzp) and in the barley genes Squamosa-promoter binding protein-like 14 and 17 (Spl14/Spl17) and suitable guide RNA (gRNA)/cas9 constructs were cloned. The activity of the gRNAs was investigated in wheat and barley epidermal cells and in wheat protoplasts. Stable genetic transformation was carried out via ballistic gene transfer (wheat) or Agrobacterium-mediated introduction of T-DNA (barley) to immature embryos. The subsequently regenerated plants were examined for the presence of T-DNA and induced mutations. In wheat, regenerated mutant plants were generatively propagated by selfing, crossing and the application of haploid technology, and a collection of individuals carrying various mutated alleles including combinations of mutated homoeologous alleles was deployed. Resulting plants with one or two mutant Bh/Wfzp homoeoalleles were T-DNA-free and showed phenotypic modifications in ear phenotype, grain size, grain number and partially in roots. The presence of three mutant Bh/Wfzp homoeoalleles typically led to very drastic changes in the ears, which formed supernumerary spikelets and were also branching, which was associated with a great loss in fertility. Overall, the importance of the transcription factor BH/WFZP for the development of the spike and the grains as well as for the roots was further elucidated in the present investigation. The mutations in Spl14 induced in barley led to delayed generative development and in some cases to the reactivation of lateral spikelet formation. In addition, the ears were shortened and formed fewer grains. The mutations produced in Spl17 did not affect generative development, but did affect ear length and grain formation. Plants carrying double mutations in Spl14 and Spl17 showed a drastic aggravation of the spl14 phenotype, with generative development severely disrupted so that no ears could be formed. This suggests that SPL14 exerts a major function in early generative development and SPL17 acts as a co-factor.

Institute of Plant Genetics
Doctoral thesis
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