Rapid and hormone-free Agrobacterium rhizogenes-mediated transformation in rubber producing dandelions Taraxacum kok-saghyz and T. brevicorniculatum

Abstract

Taraxacum kok-saghyz (TK) and Taraxacum brevicorniculatum (TB) are rubber-producing dandelion species under development as potential crops and model systems of rubber biosynthesis. The former is of industrial interest, as it produces a high percentage of high quality rubber in its roots; the latter is an apomictic cousin of TK and is of interest as a model system for rubber biosynthesis and a source of vigor in breeding efforts. Accordingly, there is interest in developing genetic transformation protocols for applied research, such as metabolic engineering. A rapid and hormone-free transformation system was developed for these two species. Dandelions can naturally regenerate from root fragments, and can be vegetatively propagated using root cuttings. Here, we show that root fragments can regenerate entire plants on half strength Murashige and Skoog medium without iterative hormone treatments or manual manipulations. Regeneration efficiency was increased from 36.6% to 65.3% for TK and from 95.2% to 152.3% for TB by inoculation with Agrobacterium rhizogenes wild type strain K599. After root fragments were inoculated with A. rhizogenes harboring kanamycin resistance genes encoding neomycin phosphotransferase II (nptII), as well as green fluorescent protein or cyan fluorescent protein, non-composite transgenic plants were obtained within 8 weeks. A root diameter of at least 1mm was required for efficient regeneration and transformation. Expression of fluorescent proteins in all cells was validated using confocal microscopy. On average, transformation efficiency (number of transgenic plants/number of root fragments) was 24.7% and 15.7% for TK and TB, respectively; about seven independent transgenic events were generated per starting plant for TK and four for TB. Overall, this high efficiency transformation method provides a rapid and simple system for these two dandelions to yield viable transgenic seeds in as little as 20 weeks. Protocols developed in this study allow introduction of genes of interest to facilitate the improvement of these rubber producing plants into domestic crops and provide an avenue to explore rubber biosynthesis and gene functions.