Establishing and Optimization of Tissue Culture work with Erigeron canadensis

Glyphosate resistance among weed species has been an increasing problem in crop production over the past two decades. Glyphosate resistant Erigeron canadensis (horseweed) has been found on multiple continents and 20 U.S. states. It appears that the resistance is of non-target type, with mechanisms of action that have yet to be elucidated at the gene level. For the first portion of the study, I focused on reproducing the original indirect organogenesis protocol but discovered biotype variability can influence regeneration success. An improved indirect organogenesis method was then developed using the previously published methods that could be used across biotypes. A DBI (DeVerna et al. 1984) based solid medium (I Macro/I Micro Murashige and Skoog (MS) salts/vitamins, 3% sucrose, 0.2% gelzan, 2 mg/L kinetin, 0.438 mg/L indole acetic acid (IAA), pH 5.8) called DBIX was statistically significant (p<0.001) over the several media designed and had a 77% regeneration rate in wild-type tissue. The second portion of the study was to establish a new liquid callus culture protocol that was adaptable to different biotypes. I evaluated three different published liquid media types for their effects on callus and cell growth in liquid culture using three different horseweed biotypes. With a statistical difference (p<0.001) in all three media, Medium 2 (MS basal salts, 3% sucrose, 1 mg/L 6-benzylaminopurine (BAP) and 0.02 mg/L indole-3-butyric acid (IBA), pH 5.8) produced healthy viable cells/callus pieces and a faster regeneration protocol over the other media types. Two new methods were developed for tissue culture using horseweed and provide a foundation for genetic engineering and gene editing.