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.

Melatonin has been confirmed to relieve drought stress in various plant species. However, the mechanism in garden chrysanthemums remains to be elucidated. Here, we examined the putative role of exogenous melatonin application in improving drought-stress tolerance in Chrysanthemum seedlings. Treatment with melatonin (100 µM) significantly mitigated the negative effects of drought stress on the seedling photosynthetic parameters and physiological and biochemical indexes. The increased tolerance conferred by exogenous melatonin was attributable to increased photosynthetic activity, which alleviates drought-induced oxidative damage by reducing malondialdehyde accumulation. In other words, the combined application of MT and PEG was better than their individual applications, as it prevented heat-induced membrane damages by declining the hydrogen peroxide (28%), malondialdehyde contents (38%), and relative electrical conductivity (19%) as compared to PEG. Interestingly, the melatonin application stimulated the activity of major antioxidant enzymes such as superoxide dismutase (21%), catalase (26%) and polyphenol oxidase (35%). In addition, exogenous melatonin promoted photosynthesis. Compared with the PEG drought-stress treatment, the melatonin treatment significantly increased the net photosynthetic rate (39%), stomatal conductance (27%), and transpiration rates (18%), while decreasing the intercellular CO₂ concentration (10%). The photochemical quenching coefficient (46%), photosystem II (PSII) maximum photochemical quantum yield (22%), apparent electron transfer rate (37%), and PSII potential photochemical efficiency (18%) showed an upward trend. In summary, exogenous spraying of MT can effectively improve drought tolerance and mitigate the damage caused by drought stress in the “Xuanqiu ninghong” Chrysanthemum.

Chrysanthemum (Chrysanthemum morifolium (Ramat.) Hemsl.) is an important species in China’s flower industry, and drought stress seriously affects the growth, quality, yield, and geographical distribution of this species. Melatonin (MT) plays a key role in regulating plant abiotic stress responses and stress resistance, but the mechanism through which exogenous MT regulates drought resistance in chrysanthemum remains unclear. This study explored the protective effect of MT on chrysanthemum drought tolerance and its key regulatory pathways. Exogenous MT application increased the photosynthetic capacity (Tr increased by 18.07%; Pn increased by 38.46%; and Gs increased by 26.52%) of chrysanthemum and attenuated decreases in its chlorophyll (19.89%) and relative water contents (26.94%). Moreover, MT increased the levels of osmolarity-related compounds such as soluble sugars (43.60%) and soluble protein (9.86%) under drought stress and increased antioxidant enzyme activity (SOD increased by 20.98%; POD increased by 35.04%; and CAT increased by 26.21%). Additionally, MT increased the endogenous MT (597.96%), growth hormone (45.31% and 92.09%), gibberellic acid (75.92% and 3.79%), salicylic acid (33.02%), and cytokinin contents (1400.00%) under drought stress while decreasing the abscisic acid (50.69% and 56.79%), jasmonate contents (62.57% and 28.31%), and ethylene contents (9.28%). RNA-seq analysis revealed 17,389, 1466, and 9359 differentially expressed genes (DEGs) under three treatments (PEG, MT, and MT _ PEG, respectively) compared with the control. Enrichment analyses of the DEGs identified more than 10 GO terms and 34 KEGG pathways. Nitrogen metabolism, sulfur metabolism, and alanine, aspartate, and glutamate metabolism were significantly increased under all three treatments. The DEGs included many transcription factors, such as MYB, WRKY, and NAC proteins. Our results preliminarily classify candidate genes and metabolic pathways with active roles in the interaction between MT and drought stress and advance the understanding of the molecular mechanism of the response to drought stress under MT conditions, thereby providing a theoretical basis for the breeding of drought-resistant chrysanthemum.

Auxin transport mediates the asymmetric distribution of auxin that determines the fate of cell development. Agrobacterium-mediated genetic transformation is an important technical means to study gene function. Our previous study showed that the expression levels of LpABCB21 and LpPILS7 are significantly up-regulated in the somatic embryogenesis (SE) of Lilium pumilum DC. Fisch. (L. pumilum), but the functions of both genes remain unclear. Here, the genetic transformation technology previously developed by our team based on the L.pumilum system was improved, and the genetic transformation efficiency increased by 5.7–13.0%. Use of overexpression and CRISPR/Cas9 technology produced three overexpression and seven mutant lines of LpABCB21, and seven overexpression and six mutant lines of LpPILS7. Analysis of the differences in somatic embryo induction of transgenic lines confirmed that LpABCB21 regulates the early formation of the somatic embryo; however, excessive expression level of LpABCB21 inhibits somatic embryo induction efficiency. LpPILS7 mainly regulates somatic embryo induction efficiency. This study provides a more efficient method of genetic transformation of L. pumilum. LpABCB21 and LpPILS7 are confirmed to have important regulatory roles in L. pumilum SE thus laying the foundation for subsequent studies of the molecular mechanism of Lilium SE.