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.
Highlights
Lilium is one of the most important horticultural crops, with high ornamental value and market demand [1,2]
The results showed that 40 mg·L−1 Hyg could strongly inhibit the growth of cells around the embryogenic calli of L. pumilum, resulting in almost entirely browned and dead calli and a very low proliferation rate, while some adventitious buds were still produced on the scales of ‘White Heaven’ (Table 1)
The results showed that hybridization signals were observed in 15 lines out of the 35 lines (42.86%) of L. pumilum, 2 lines out of the 5 lines (40%) of ‘White Heaven’ and the plasmid control plants, while hybridization signals were not observed in the nontransgenic control plants (Figure 4C,D), confirming that the GUS gene had been integrated into the Lilium genome
Summary
Lilium is one of the most important horticultural crops, with high ornamental value and market demand [1,2]. Lilium trait improvement and new variety creation have depended on long periods of hybridization and selection, while the application of more efficient genetic engineering and molecular breeding has not been possible. With recent developments in molecular biology, especially the gene-editing technology represented by CRISPR/Cas, rapid and targeted genetic improvement has been achieved in almost all grain crops and most horticultural crops, launching a new wave of molecular breeding [5]. This powerful and universal technology requires a highly efficient and stable genetic transformation system [6,7]. There is an urgent need to establish a more efficient, stable, and universal genetic transformation system in Lilium
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