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

1
Key Laboratory of Protected Horticulture of Education Ministry and Liaoning Province, College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China
2
National and Local Joint Engineering Research Center of Northern Horticultural Facilities Design and Application Technology, Shenyang 110866, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(12), 2920; https://doi.org/10.3390/ijms20122920
Received: 20 May 2019 / Revised: 8 June 2019 / Accepted: 11 June 2019 / Published: 14 June 2019
(This article belongs to the Section Molecular Pharmacology)
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. View Full-Text
Keywords: Lilium; genetic transformation; CRISPR/Cas9; somatic embryogenesis; phytoene desaturase (PDS) Lilium; genetic transformation; CRISPR/Cas9; somatic embryogenesis; phytoene desaturase (PDS)
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Yan, R.; Wang, Z.; Ren, Y.; Li, H.; Liu, N.; Sun, H. Establishment of Efficient Genetic Transformation Systems and Application of CRISPR/Cas9 Genome Editing Technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven. Int. J. Mol. Sci. 2019, 20, 2920.

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