The master's thesis is focused on experiment that enables the selection of the most suitable culture medium for hop callus growth and transformation with bacteria Agrobacterium tumefaciens. From 15 hop genotypes, we selected 5 that had the highest rate of callus formation with high quality. We inoculated leaves, internodes and petioles from sterile hop plants on induction media. From 4 medium that had different compositions of hormones, we chose the most suitable one for growing callus. Transformation was performed using the plasmids pCAMBIA1302-ZsGreen, pCAMBIA1301-GUS and pGFPGUSplus. The LBA4044 bacteria strain was used. Transformed callus was first checked using epifluorescence microscope and a histochemical GUS test. In addition, the presence of the desired genes in the hop genome was verified by PCR analysis and agarose electrophoresis. The presence of ZsGreen transgene was confirmed in all analyzed samples. The entire gene construct together with hptII was present in 81 % of the calli transformed with plasmid pCAMBIA1302-ZsGreen. When using the plasmid pGFPGUSPlus, which contained EGFP, gusA, and hptII transgenes, the fragments were confirmed in 71 % of the samples. EGFP and gusA genes were absent in 5 samples. We found that the success of callus transformation in hops is mainly influenced by suitable callus induction, which strongly depends on the genotype, and the selected plasmid. With this protocol, we obtained a high transformation efficiency, especially with the plasmid pCAMBIA1302-ZsGreen. We can confirm that hop callus is suitable for transformation procedures.
|
