Establishment of Efficient Genetic Transformation Systems and Application of CRISPR/Cas9 Genome Editing Technology in Lilium p umilum DC. Fisch. and Lilium l ongiflorum 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.

Keywords: CRISPR/Cas9; Lilium; Phytoene desaturase (PDS); genetic transformation; somatic embryogenesis.

Figure 1

Parameters affecting transformation efficiency. A and B Effects of pre-culture duration on transformation efficiency. C and D Effects of Agrobacterium concentration on transformation efficiency. E and F Effects of infection duration on transformation efficiency. G and H Effects of co-culture duration on transformation efficiency. Left results for Lilium pumilum DC. Fisch.; right results for ‘White Heaven’. All data are expressed as means ± standard deviation of triplicate samples. d: days.

Figure 2

Plant tissues at different stages of Agrobacterium-mediated transformation of Lilium. A–D Formation of transformed plants of Lilium pumilum DC. Fisch. A New embryogenic cells of resistant callus. B Resistant callus germination. C Resistant buds formed during germination. D Transformed plants. E–F Formation of transformed plants of ‘White Heaven’. E Resistant bud. F Transformed plants of ‘White Heaven’.

Figure 3

β-glucuronidase (GUS) histochemical assays of transgenic plants. A–H GUS histological staining results of Lilium pumilum DC. Fisch. A Untransformed embryogenic callus. B Transformed embryogenic callus. C–H show the scale, leaf, and root, respectively (C, E and G are untransformed plant; D, F and H are transformed plant). I–N GUS histological staining results for the scale, leaf, and root of ‘White Heaven’ (I, K and M are untransformed plant; J, L and N are transformed plant) (bars = 5 mm).

Figure 4

Polymerase chain reaction (PCR) analysis of transgenic plantlets using the GUS gene. A Only 22 Lilium pumilum DC. Fisch. lines are shown B Only 8 White Heaven’ lines are shown, and others no band was amplified. Lane M, DL2000 DNA marker; lane W, wild-type plant; lane P, plasmid control; lanes L1–22 are GUS-positive lines of Lilium pumilum DC. Fisch. and lanes W1–8, are GUS-positive lines of ‘White Heaven’. C, D Southern blot analysis of transformation. lane WT, wild-type plant; lane P, plasmid control; lanes L1-9, Only 9 PCR-positive transgenic lines of Lilium pumilum DC. Fisch. are shown. lanes W1, W3, W4 and W5, PCR-positive transgenic lines of ‘White Heaven’. W7 no band was amplified.

Figure 5

Phenotypes of LpPDS mutants after transformation with the CRISPR/Cas9 system. A–G show Lilium pumilum DC. Fisch. A Nontransgenic plant. B Yellowing mutant. C Resistant somatic embryos. D Mature embryo. E–F Dwarf mutants. G Completely albino plants. H–L show ‘White Heaven’. H Nontransgenic plant. I Yellowing mutant. J Resistant buds. K and L Mixed green and white leaves. M Completely albino plants (bars = 5 mm).

Figure 6

PCR analysis of plants with obvious albino phenotypes using the Bar gene and vector-specific primers. A, C and E PCR results for the Bar gene (433 bp) of Lilium pumilum DC. Fisch. G Bar gene (433 bp) of ‘White Heaven’. B, D and F PCR fragment of a partial sgRNA expression cassette (831 bp) of Lilium pumilum DC. Fisch. H PCR fragment of partial sgRNA expression cassette (831 bp) of ‘White Heaven’. Lane M, DL2000 DNA marker. lane WT, wild-type plant. lane P, plasmid control. A–F: Lanes 1–45, obvious albino phenotypes plants of Lilium pumilum DC. Fisch. G and H: Lanes W1–16, obvious albino phenotypes plants of ‘White Heaven’, Only 16 lines are shown, and others no band was amplified.

Figure 7

Different types of mutations detected in transgenic Lilium plants after CRISPR/Cas9-mediated gene editing. PAM, red. Bold, target sequence. r/green, replacements. i/blue, insertions. d/-, deletions. The purple font indicates the presence of mutant lines. Black fonts indicate lines that have not been mutated. #L, PCR-positive lines of Lilium pumilum DC. Fisch. #W, PCR-positive lines of ‘White Heaven’. A Sequencing results of Lilium pumilum DC. Fisch. B Sequencing results of ‘White Heaven’.