Agrobacterium rhizogenes–induced Altered Morphology and Physiology in Rubber Dandelion after Genetic Transformation

Abstract

Agrobacterium rhizogenes transformation is a more rapid method of obtaining transgenic and edited rubber dandelion (Taraxacum kok-saghyz) plants than Agrobacterium tumefaciens. The hairy root rol genes are present alongside transgenes after transformation, and they change the morphology of rubber dandelion significantly. Although these rol genes are useful visual markers indicating successful transformation of rubber dandelion, they modify the phenotype induced by the target transgenes and are ultimately detrimental to agronomic traits. Fortunately, the rol genes can be removed by conventional plant breeding because they segregate in progeny separately from the targeted transgenes. However, it is preferable to have preliminary identification of promising effects induced by transgenes or gene edits before rol gene removal so that only the best plants are used for breeding. Therefore, the goal of this research was to characterize rol– and rol+ plant morphology so that, in the future, rol+ transgene+ plants can be easily distinguished from rol+ transgene– plants. This requires that rol gene–induced morphological changes and simply assayed physiological traits are first characterized thoroughly so that transgene changes may be observed. Taproot formation is reduced or eliminated in rubber dandelion by rol genes, and rol-induced hairy roots are identifiable easily because they grow shallowly in potting soil, so only partial unearthing is needed. Both leaf and flower numbers are increased by rol genes, but leaves and flowers are smaller than in rubber dandelion wild type with longer stalks. The rosette doming phenotype caused by the induction of a large number of leaf primordia is obvious in rooted plants as young as 1 month old. Photosynthetic rates are reduced significantly in rol+ plants, although growth is not. An accurate description of the morphology of rubber dandelion after A. rhizogenes transformation may allow for initial selection of promising transformed plants before confirmation with polymerase chain reaction, by phenotypic comparison of plants expressing transgenes and the rol gene, with those only expressing the rol gene.

CRISPR/Cas9-mediated Targeted Mutagenesis of Inulin Biosynthesis in Rubber Dandelion

“…Agrobacterium rhizogenes has higher transformation efficiency in Taraxacum spp. than A. tumefaciens (Bae et al 2005;Lankitus et al 2023;Lee et al 2004). In Taraxacum platycarpum leaf discs, the transformation efficiency using A. tumefaciens was 1% to 5% (Bae et al 2005), whereas the transformation efficiency in root fragments using A. rhizogenes was 76.5% (Lee et al 2004).…”

“…Then, these knockouts can be fully characterized without the confounding influence of rol genes. This process can be time-consuming and laborious, and some effort has been made to distinguish transgene efforts in the presence of rol genes to reduce the number of PCR reactions needed to confirm the presence of transgenes (Lankitus et al 2023).…”

“…It is well known that root locus (rol) genes transferred to the plant genome with the gene(s) of interest in A. rhizogenes-mediated plant transformation alter endogenous plant hormone concentrations and the production of secondary metabolites (Bulgakov et al 2011). Characterization of phenotypic changes induced by wildtype A. rhizogenes K599 strain (without accompanying transgenes or edited genes) found no rol-induced changes in the root rubber concentration (Lankitus et al 2023). Thus, the increased rubber levels detected in K599-FFT plants are solely caused by the redirection of excess carbon from the inhibited inulin pathway.…”

“…Root samples from plants confirmed to contain a mutated 1-FFT gene were analyzed to determine their inulin and rubber contents. Roots from 3-month-old rubber dandelion plants were harvested and dried as previously described (Lankitus et al 2023). Briefly, plants were removed from their tree pots, and excess dirt from the roots was shaken loose.…”

Molecular Genetic Research and Genetic Engineering of Taraxacum kok-saghyz L.E. Rodin

“…CRISPR/Cas TKS mutants for the TkRALFL1-like1 gene were also created using A. tumefaciens [74]. Genetic transformation of dandelions appeared to be more efficient with A. rhizogenes than with A. tumefaciens [83]. However, it should be borne in mind that, during the transformation of A. rhizogenes into plants, in addition to the target genes, rol-genes are also transferred, and in the future, it will become necessary to select plants without these genes.…”