Establishment of Efficient Genetic Transformation Systems and Application of CRISPR/Cas9 Genome Editing Technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven

Abstract

<i>Lilium</i> spp. is a bulb flower with worldwide distribution and unique underground organs. The lack of an efficient genetic transformation system for <i>Lilium</i> has been an international obstacle. Because existing model plants lack bulbs, bulb-related gene function verification studies cannot be carried out in model plants. Here, two stable and efficient genetic transformation systems based on somatic embryogenesis and adventitious bud regeneration were established in two <i>Lilium</i> species. Transgenic plants and T-DNA insertion lines were confirmed by β<i>-</i><i>g</i><i>lucuronidase</i> <i>(</i><i>GUS</i><i>)</i> assay, polymerase chain reaction (PCR) and Southern blot. After condition optimization, transformation efficiencies were increased to 29.17% and 4% in <i>Lilium pumilum</i> DC. Fisch. and the <i>Lilium longiflorum</i> 'White Heaven', respectively. To further verify the validity of these transformation systems and apply the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9) technology in <i>Lilium</i>, the <i>LpPDS</i> gene in the two <i>Lilium</i> species was knocked out. Completely albino, pale yellow and albino-green chimeric mutants were observed. Sequence analysis in the transgenic lines revealed various mutation patterns, including base insertion, deletion and substitution. These results verified the feasibility and high efficiency of both transformation systems and the successful application of the CRISPR/Cas9 system to gene editing in <i>Lilium</i> for the first time. Overall, this study lays an important foundation for gene function research and germplasm improvement in <i>Lilium</i> spp<i>.</i>