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

Lilium spp. is a bulb flower with worldwide distribution and unique underground organs. The lack of an efficient genetic transformation system for Lilium 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 Lilium species. Transgenic plants and T-DNA insertion lines were confirmed by β-glucuronidase (GUS) assay, polymerase chain reaction (PCR) and Southern blot. After condition optimization, transformation efficiencies were increased to 29.17% and 4% in Lilium pumilum DC. Fisch. and the Lilium longiflorum ‘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 Lilium, the LpPDS gene in the two Lilium 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 Lilium for the first time. Overall, this study lays an important foundation for gene function research and germplasm improvement in Lilium spp.

Expression optimization, purification and in vitro characterization of human epidermal growth factor produced in Nicotiana benthamiana

“…Transformation conditions are key elements affecting the transformation efficiency which in turn affect the protein expression. Agrobacterium concentration or cell density is one of such factors that affect the efficiency of plant transformation; low concentration may result in insufficient Agrobacterium infection, whereas high concentration can cause the plant cell damage or trigger hypersensitive response leading to plant tissue necrosis [ 41 , 42 ]. We found that Agrobacterium cell density at the OD 600 of 0.2 was found to be optimal for plant transformation.…”

Anthocyanins in Floral Colors: Biosynthesis and Regulation in Chrysanthemum Flowers

“…Petunia has been edited with CRISPR-Cas9 targeting various traits, including an albino phenotype [ 155 ], flower longevity [ 156 ], deficiency in nitrate assimilation [ 157 ], and self-incompatibility [ 158 ]. The LpPDS gene in two Lilium species was successfully edited resulting in albino, pale yellow, and albino-green chimeric mutants [ 159 ]. Multiple MADS genes have been targeted in Phalaenopsis for flower initiation and development [ 160 ].…”

Progress and Challenges in the Improvement of Ornamental Plants by Genome Editing

“…Two genetic transformation systems, such as somatic embryogenesis and adventitious bud regeneration, have been established for the application of CRISPR-Cas9 technology in two Lilium species, L. longiflorum and L. pumilum. By targeting the phytoene desaturase (PDS) gene encoding a key enzyme of carotenoid synthesis for induced mutagenesis, mutant Lilium species of the albino, pale-yellow, and albino-green chimeric types were produced with various patterns of mutation, including base insertion, deletion, and substitution [58]. In addition, some results from orchid studies using CRISPR-Cas systems for genome editing have been recently released.…”