Lilium Longiflorum Research Articles

LlbHLH87 interacts with LlSPT to modulate thermotolerance via activation of LlHSFA2 and LlEIN3 in lily.

Basic helix-loop-helix (bHLH) proteins comprise one of the largest families of transcription factors in plants, which play roles in plant development, secondary metabolism, and the response to biotic/abiotic stresses. However, the roles of bHLH proteins in thermotolerance are largely unknown. Herein, we identified a heat-inducible member of the bHLH family in lily (Lilium longiflorum), named LlbHLH87, which plays a role in thermotolerance. LlbHLH87 was rapidly induced by transient heat stress, and its encoded protein was localized to the nucleus, exhibiting transactivation activity in both yeast and plant cells. Overexpression of LlbHLH87 in Arabidopsis enhanced basal thermotolerance, while silencing of LlbHLH87 in lily reduced basal thermotolerance. Further analysis showed that LlbHLH87 bound to the promoters of HEAT STRESS TRANSCRIPTION FACTOR A2 (LlHSFA2) and ETHYLENE-INSENSITIVE 3 (LlEIN3) to directly activate their expression. In addition, LlbHLH87 interacted with itself and with SPATULA (LlSPT) protein. LlSPT was activated by extended heat stress and its protein competed for the homologous interaction of LlbHLH87, which reduced the transactivation ability of LlbHLH87 for target genes. Compared with that observed under LlbHLH87 overexpression alone, co-overexpression of LlbHLH87 and LlSPT reduced the basal thermotolerance of lily to sudden heat shock, but improved its thermosensitivity to prolonged heat stress treatment. Overall, our data demonstrated that LlbHLH87 regulates thermotolerance via activation of LlEIN3 and LlHSFA2, along with an antagonistic interaction with LlSPT.

An AP2/ERF member LlERF012 confers thermotolerance via activation of HSF pathway in lily.

Heat stress transcription factors (HSFs) are core factors of plants in response to heat stress (HS), but their regulatory network is complicated and remains elusive in a large part, especially HSFBs. In this study, we reported that the LlERF012-LlHSFA1 module participates in heat stress response (HSR) by directly regulating HSF pathway in lily (Lilium longiflorum). LlHSFB1 was confirmed as a positive regulator in lily thermotolerance and a heat-inducible AP2/ERF member LlERF012 (Ethylene Response Factor 012) was further identified to be a direct trans-activator of LlHSFB1. Overexpression of LlERF012 elevated the thermotolerance of transgenic Arabidopsis and lily, but silencing LlERF012 reduced thermotolerance in lily. Further analysis showed LlERF012 interacted with LlHSFA1, which led to enhanced transactivation activity and DNA-binding capability of LlERF012. In addition, LlERF012 also directly activated the expression of LlHSFA1 by binding its promoter. As expected, we found that LlERF012 bound the promoters of LlHSFA2, LlHSFA3A, and LlHSFA3B to stimulate their expression, and LlERF012-LlHSFA1 interaction enhanced these activation effects. Overall, our data suggested that LlERF012 was a key factor for lily thermotolerance and the LlERF012-LlHSFA1 interaction synergistically regulated the activity of the HSF pathway including the class A and B members, which might be of great significance for coordinating the functions of different HSFs.

Growth and Flowering of Lilium (Lilium longiflorum) cv. Eremo as Affected by CCC and GA3 under Naturally Ventilated Polyhouse Conditions of Prayagraj

The present investigation was carried out under naturally ventilated polyhouse conditions, Department of Horticulture, SHUATS, Prayagraj, during November, 2023 to March, 2024. The experiment was laid out in Randomized Block Design (RBD) with three replications. There were nine treatments comprising of Cycocel (CCC) and Gibberellic Acid (GA3) at different concentrations viz., GA3 (100, 200, 300 and 400 ppm) and CCC (100, 200, 300 and 400 ppm) along with Water spray. The results revealed foliar application of 300 ppm GA3 for Lilium significantly enhanced the number of leaves (71.83), plant height (76.47 cm), plant spread (15.44 cm), leaf area (9.73 cm2),number of buds (3.40),at 300 ppm of GA3 it takes lesser days taken for bud initiation(40.40),and also less days taken for first flower opening from planting (68.53), flower bud length (8.83 cm), flower stalk length (12.43 cm), stalk length (78.10 cm), self-life (12.97 days),flower yield per plant (3.40) and flower yield for 250 m2(6296.29) . Among the different treatments, the highest Gross return is 188888.7 Rs/250 m2, Net return is 120870.75 Rs/250 m2 and Benefit cost ratio is 2.77, obtained by the application of 300 ppm of GA3.Whereas in terms of only Bulb parameters, foliar application of 200 ppm of CCC for Lilium reported maximum number of bulblets per plant (4.11) and gives maximum weight of bulb per plant (74.07g).

Lily LlHSFC2 coordinates with HSFAs to balance heat stress response and improve thermotolerance.

Heat stress transcription factors (HSFs) are core regulators of plant heat stress response. Much research has focused on class A and B HSFs, leaving those of class C relatively understudied. Here, we reported a lily (Lilium longiflorum) heat-inducible HSFC2 homology involved in thermotolerance. LlHSFC2 was located in the nucleus and cytoplasm and exhibited a repression ability by binding heat stress element. Overexpression of LlHSFC2 in Arabidopsis, tobacco (Nicotiana benthamiana), and lily, all increased the thermotolerance. Conversely, silencing of LlHSFC2 in lily reduced its thermotolerance. LlHSFC2 could interact with itself, or interact with LlHSFA1, LlHSFA2, LlHSFA3A, and LlHSFA3B of lily, AtHSFA1e and AtHSFA2 of Arabidopsis, and NbHSFA2 of tobacco. LlHSFC2 interacted with HSFAs to accelerate their transactivation ability and act as a transcriptional coactivator. Notably, compared with the separate LlHSFA3A overexpression, co-overexpression of LlHSFC2/LlHSFA3A further enhanced thermotolerance of transgenic plants. In addition, after suffering HS, the homologous interaction of LlHSFC2 was repressed, but its heterologous interaction with the heat-inducible HSFAs was promoted, enabling it to exert its co-activation effect for thermotolerance establishment and maintenance. Taken together, we identified that LlHSFC2 plays an active role in the general balance and maintenance of heat stress response by cooperating with HSFAs, and provided an important candidate for the enhanced thermotolerance breeding of crops and horticulture plants.

A LlWRKY33-LlHSFA4-LlCAT2 module confers resistance to Botrytis cinerea in lily.

Gray mold caused by Botrytis cinerea is one of the major threats in lily production. However, limited information is available about the underlying defense mechanism against B. cinerea in lily. Here, we characterized a nuclear-localized class A heat stress transcription factor (HSF)-LlHSFA4 from lily (Lilium longiflorum), which positively regulated the response to B. cinerea infection. LlHSFA4 transcript and its promoter activity were increased by B. cinerea infection in lily, indicating its involvement in the response to B. cinerea. Virus-induced gene silencing (VIGS) of LlHSFA4 impaired the resistance of lily to B. cinerea. Consistent with its role in lily, overexpression of LlHSFA4 in Arabidopsis (Arabidopsis thaliana) enhanced the resistance of transgenic Arabidopsis to B. cinerea infection. Further analysis showed that LlWRKY33 directly activated LlHSFA4 expression. We also found that both LlHSFA4 and LlWRKY33 positively regulated plant response to B. cinerea through reducing cell death and H2O2 accumulation and activating the expression of the reactive oxygen species (ROS) scavenging enzyme gene LlCAT2 (Catalase 2) by binding its prompter, which might contribute to reducing H2O2 accumulation in the infected area. Taken together, our data suggested that there may be a LlWRKY33-LlHSFA4-LlCAT2 regulatory module which confers B. cinerea resistance via reducing cell death and the ROS accumulation.

HD-Zip I protein LlHOX6 antagonizes homeobox protein LlHB16 to attenuate basal thermotolerance in lily.

Homeodomain-leucine zipper (HD-Zip) I transcription factors are crucial for plant responses to drought, salt, and cold stresses. However, how they are associated with thermotolerance remains mostly unknown. We previously demonstrated that lily (Lilium longiflorum) LlHB16 (HOMEOBOX PROTEIN 16) promotes thermotolerance, whereas the roles of other HD-Zip I members are still unclear. Here, we conducted a transcriptomic analysis and identified a heat-responsive HD-Zip I gene, LlHOX6 (HOMEOBOX 6). We showed that LlHOX6 represses the establishment of basal thermotolerance in lily. LlHOX6 expression was rapidly activated by high temperature, and its protein localized to the nucleus. Heterologous expression of LlHOX6 in Arabidopsis (Arabidopsis thaliana) and overexpression in lily reduced their basal thermotolerance. In contrast, silencing LlHOX6 in lily elevated basal thermotolerance. Cooverexpressing or cosilencing LlHOX6 and LlHB16 in vivo compromised their functions in modulating basal thermotolerance. LlHOX6 interacted with itself and with LlHB16, although heterologous interactions were stronger than homologous ones. Notably, LlHOX6 directly bounds DNA elements to repress the expression of the LlHB16 target genes LlHSFA2 (HEAT STRESS TRANSCRIPTION FACTOR A2) and LlMBF1c (MULTIPROTEIN BRIDGING FACTOR 1C). Moreover, LlHB16 activated itself to form a positive feedback loop, while LlHOX6 repressed LlHB16 expression. The LlHOX6-LlHB16 heterooligomers exhibited stronger DNA binding to compete for LlHB16 homooligomers, thus weakening the transactivation ability of LlHB16 for LlHSFA2 and LlMBF1c and reducing its autoactivation. Altogether, our findings demonstrate that LlHOX6 interacts with LlHB16 to limit its transactivation, thereby impairing heat stress responses in lily.

Plant Hormone and Fatty Acid Screening of Nicotiana tabacum and Lilium longiflorum Stigma Exudates.

Pollen germination in vivo on wet stigmas is assisted by the receptive fluid-stigma exudate. Its exact composition is still unknown because only some components have been studied. For the first time, hormonal screening was carried out, and the fatty acid (FA) composition of lipid-rich (Nicotiana tabacum) and sugar-rich (Lilium longiflorum) exudates was studied. Screening of exudate for the presence of plant hormones using HPLC-MS revealed abscisic acid (ABA) in tobacco stigma exudate at the two stages of development, at pre-maturity and in mature stigmas awaiting pollination, increasing at the fertile stage. To assess physiological significance of ABA on stigma, we tested the effect of this hormone in vitro. ABA concentration found in the exudate strongly stimulated the germination of tobacco pollen, a lower concentration had a weaker effect, increasing the concentration did not increase the effect. GC-MS analysis showed that both types of exudate are characterized by a predominance of saturated FAs. The lipids of tobacco stigma exudate contain significantly more myristic, oleic, and linoleic acids, resulting in a higher unsaturation index relative to lily stigma exudate lipids. The latter, in turn, contain more 14-hexadecenoic and arachidic acids. Both exudates were found to contain significant amounts of squalene. The possible involvement of saturated FAs, ABA, and squalene in various exudate functions, as well as their potential relationship on the stigma, is discussed.

Enhancing heat stress tolerance in Lanzhou lily (Lilium davidii var. unicolor) with Trichokonins isolated from Trichoderma longibrachiatum SMF2.

Lanzhou lily (Lilium davidii var. unicolor) is a renowned edible crop produced in China and relatively sensitive to high temperature (HT). Trichokonins (TKs) are antimicrobial peptaibols secreted from Trichoderma longibrachiatum strain SMF2. Here, we report that TKs application improves the thermotolerance of Lanzhou lily. The activity of the antioxidant enzyme system (SOD, CAT, and POD), the level of heat-resistance-associated phytohormones (ABA, SA, and JA), the relative water content (RWC), the content of chlorophyll (Chl), and the net photosynthetic rate (P n) were promoted by TKs treatment in Lanzhou lily plants subjected to heat stress (HS). TKs treatment also mitigated cell injury as shown by a lower accumulation of malondialdehyde (MDA) and relative electrolyte leakage (REL) under HS conditions. RNA-seq data analysis showed that more than 4.5 times differentially expressed genes (DEGs) responded to TKs treatment under HS compared to non-HS, and TKs treatment reduced protein folding and enhanced cellular repair function under HS conditions. The analyses of DEGs involved in hormone (ABA, SA and JA) synthesis and signaling pathways suggested that TKs might improve Lanzhou lily heat tolerance by promoting ABA synthesis and signal transduction. TKs highly induced DEGs of the HSF-HSP pathway under HS, in which HSFA2 accounted for most of the HSF family. Furthermore, TKs treatment resulted in the upregulation of heat-protective genes LzDREB2B, LzHsfA2a, LzMBF1c, LzHsp90, and LzHsp70 involved in HSF-HSP signal pathway after long-term HS. LzHsfA2a-1 likely plays a key role in acquisition of TKs-induced thermotolerance of Lanzhou lily as evidenced by the sustained response to HS, the enhanced response to TKs treatment under long-term HS, and the high sequence similarity to LlHsfA2a which is a key regulator for the improvement of heat tolerance in Lilium longiflorum. Our results reveal the underlying mechanisms of TKs-mediated thermotolerance in Lanzhou lily and highlight an attractive approach to protecting crop plants from damage caused by HS in a global warming future.

Effect of Organic and Inorganic Fertilizer on Vegetative Growth Parameter of Lilium (Lilium Longiflorum) cv. Pavia under Shade Net Condition

The present investigation entitled, “Effect of Organic and Inorganic Fertilizer on Vegetative Growth Parameter of Lilium (Lilium Longiflorum) cv. Pavia Under Shade Net Condition” was under taken in the Department of Horticulture, Naini Agricultural Institute, Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj, (Allahabad). During winter season of 2020-2021, 2021-2022 and Pooled. The experiment was layout in Randomized Block Design (RBD) with 13 treatments and each treatment replicated thrice. The treatments consist of different combinations of inorganic fertilizers (N, P and K) and organic fertilizers (FYM, Vermicompost and Poultry Manure) and control (No fertilizers and manures). The treatment T5 50% RDF + FYM 14 t ha-1 was found the statistically significant compared to other treatment combination, which recorded highest plant height (23.68, 57.11 and 76.41 cm), spread area (8.19 cm2, 15.54 cm2 and 18.47 cm2), Number of leaves (36.63, 57.11 and 76.41), Leaf area (15.03 cm2, 15.63 cm2 and 15.33 cm2), Chlorophyll content (66.11 mg, 65.60 mg and 65.86 mg), followed by T9 25% RDF + Vermicompost 6.9 t ha-1 and lowest yield was obtained from T0 (control).

Field test of Easter lilies transformed with a rice cystatin gene for root lesion nematode resistance.

Easter lilies, Lilium longiflorum cv. Nellie White are a staple of the floral industry. In the U.S. most of the Easter lilies are grown in Oregon and California along the coast where there is a micro climate that is favorable to growth of lilies. The main pest when growing lilies in the field is Pratylenchus penetrans, the root lesion nematode. Easter lilies are one of the most expensive crops to produce because of the cost of chemicals used to control P. penetrans and other pathogens that infect the lilies. Our previous study had shown that transgenic Easter lilies containing a rice cystatin gene (Oc-IΔD86 that has a deleted Asp86) were resistant to P. penetrans in vitro. This study examined growth characteristics of five independently transformed lines of the cystatin Easter lilies compared to non-transformed Nellie White for three seasons in the field in Brookings, Oregon. Liles grown in three soil chemical treatments 1) preplant fumigation, 2) preplant fumigation plus at plant organophosphate, and 3) at plant organophosphate were compared to those grown in nontreated soil. Growth characteristics evaluated included: time of shoot emergence, survival of plants, size of plants, visual ratings of plant health, basal roots and stem roots, weight of foliage and roots, and number and size of bulblets that developed on stems. Nematodes were counted following their extraction from the roots. While not totally resistant, when planted in the field, transformed lines demonstrated and maintained a degree of resistance to lesion nematode over two growing seasons and displayed desirable growth and quality characteristics similar to non-transformed lilies.

Optimisation of the controlled atmosphere storage and shelf life of Lilium longiflorum

To prolong the storage period of fresh Lilium longiflorum, maintain its quality during storage, and optimise the controlled atmosphere (CA) storage parameters. In a single-factor experiment, the temperature, humidity, and O2 and CO2 concentrations were considered as primary factors affecting the CA storage of L. longiflorum. Then, a comprehensive score was optimised using a back propagation neural network combined with empirical data. Finally, experimental verification was undertaken. The optimal concentrations of O2 and CO2 were 4.5% and 3.8%, respectively. A storage temperature of 4.2°C and a relative humidity of 90% were ideal. Under these conditions, the comprehensive evaluation score for L. longiflorum was 0.8424 (P > 0.05), consistent with the predicted value of 0.8372. Compared to ordinary cold storage, the storage period of L. longiflorum under these CA storage conditions was effectively prolonged. This provided an experimental basis for the CA storage of L. longiflorum.

Bulb of Lilium longiflorum Thunb Extract Fermented with Lactobacillus acidophilus Reduces Inflammation in a Chronic Obstructive Pulmonary Disease Model.

Chronic obstructive pulmonary disease (COPD), one of the leading causes of death worldwide, is caused by repeated exposure to harmful matter, such as cigarette smoke. Although Lilium longiflorum Thunb (LLT) has anti-inflammatory effects, there is no report on the fermented LLT bulb extract regulating lung inflammation in COPD. Thus, we investigated the protective effect of LLT bulb extract fermented with Lactobacillus acidophilus 803 in COPD mouse models induced by cigarette smoke extract (CSE) and porcine pancreas elastase (PPE). Oral administration of the fermented product (LS803) suppressed the production of inflammatory mediators and the infiltration of immune cells involving neutrophils and macrophages, resulting in protective effects against lung damage. In addition, LS803 inhibited CSE- and LPS-induced IL-6 and IL-8 production in airway epithelial H292 cells as well as suppressed PMA-induced formation of neutrophil extracellular traps in HL-60 cells. In particular, LS803 significantly repressed the elevated IL-6 and MIP-2 production after CSE and LPS stimulation by suppressing the activity of the nuclear factor kappa-light-chain-enhancer of activated B (NFκB) in mouse peritoneal macrophages. Therefore, our results suggest that the fermented product LS803 is effective in preventing and alleviating lung inflammation.

Ethylene Response Factor LlERF110 Mediates Heat Stress Response via Regulation of LlHsfA3A Expression and Interaction with LlHsfA2 in Lilies (Lilium longiflorum).

Heat stress seriously affects the quality of cut lily flowers. The ethylene response factors (ERFs) participate in heat stress response in many plants. In this study, heat treatment increased the production of ethylene in lily leaves, and exogenous ethylene treatment enhanced the heat resistance of lilies. LlERF110, an important transcription factor in the ethylene signaling pathway, was found in the high-temperature transcriptome. The coding region of LlERF110 (969 bp) encodes 322 amino acids and LlERF110 contains an AP2/ERF typical domain belonging to the ERF subfamily group X. LlERF110 was induced by ethylene and was expressed constitutively in all tissues. LlERF110 is localized in the nucleus and has transactivation activity. Virus-induced gene silencing of LlERF110 in lilies reduced the basal thermotolerance phenotypes and significantly decreased the expression of genes involved in the HSF-HSP pathway, such as LlHsfA2, LlHsfA3A, and LlHsfA5, which may activate other heat stress response genes; and LlHsp17.6 and LlHsp22, which may protect proteins under heat stress. LlERF110 could directly bind to the promoter of LlHsfA3A and activate its expression according to the yeast one hybrid and dual-luciferase reporter assays. LlERF110 interacts with LlHsfA2 in the nucleus according to BiFC and the yeast two-hybrid assays. In conclusion, these results indicate that LlERF110 plays an important role in the basal thermotolerance of lilies via regulation of the HSF-HSP pathway, which could be the junction of the heat stress response pathway and the ethylene signaling pathway.

A lily membrane-associated NAC transcription factor LlNAC014 is involved in thermotolerance via activation of the DREB2-HSFA3 module.

The NTL (NAC with transmembrane motif 1-like) transcription factors with a conserved transmembrane motif are members of the NAC family and are important in plant development and in response to stress. However, knowledge of their regulatory pathways is scarce, especially under heat stress. Here, we cloned and identified a novel lily (Lilium longiflorum) NTL gene, LlNAC014, that increases thermotolerance. High temperature repressed LlNAC014 expression but activated its protein. LlNAC014 contained a typical transmembrane motif at its far C-terminus and was normally located on membranes, but under heat stress it entered the nucleus as a transcription factor. LlNAC014 also has a transactivation domain at its C-terminus, and its active form, LlNAC014ΔC, could function as a trans-activator in both yeast and plant cells. LlNAC014ΔC overexpression in lily and Arabidopsis increased thermotolerance, and also caused growth defects; silencing LlNAC014 in lily decreased thermotolerance. LlNAC014ΔC could constitutively activate the heat stress response by inducing the expression of heat-responsive genes, some of which were dependent on the HSF (heat stress transcription factor) pathway. Further analysis showed that LlNAC014 was a direct regulator of the DREB2-HSFA3 module, and bound to the CTT(N7)AAG element in the promoters of LlHSFA3A, LlHSFA3B, and LlDREB2B to activate their expression. Thus, LlNAC014 increased thermotolerance by sensing high temperature and translocating to the nucleus to activate the DREB2-HSFA3 module.

Taxonomy and Phylogenetic Appraisal of Dothideomycetous Fungi Associated with Magnolia, Lilium longiflorum and Hedychium coronarium.

This paper highlights the taxonomy of some interesting saprobic microfungi associated with dead plant materials of Hedychium coronarium, Lilium longiflorum, and Magnolia species. The taxa reported in this study belong to the orders Pleosporales and Kirschsteiniotheliales (Dothideomycetes). These taxa were identified based on multi-locus phylogeny of nuclear ribosomal DNA (rDNA) (LSU, SSU, and ITS) and protein-coding genes (tef1-α and rpb2), together with comprehensive morphological characterization. Two novel saprobic species, Leptoparies magnoliae sp. nov. and Neobambusicola magnoliae sp. nov., are introduced from Magnolia species in Thailand. Another new species, Asymmetrispora zingiberacearum sp. nov., is also described from dead stems of H. coronarium, which is the first asexual morph species of the genus Asymmetrispora. In addition, Ramusculicola thailandica and Kirschsteiniothelia thailandica are reported as new host records from dead twigs of Magnolia species. Sphaerellopsis paraphysata is reported as a new host record from L. longiflorum. Newly described taxa are compared with other similar species and detailed descriptions, micrographs, and phylogenetic trees to show the positions are provided.

Lily HD-Zip I Transcription Factor LlHB16 Promotes Thermotolerance by Activating LlHSFA2 and LlMBF1c.

HD-Zip I transcription factors play important roles in plant development and response to abiotic stresses; however, their roles in thermotolerance are largely unknown. Through transcriptome analysis in lily (Lilium longiflorum), we isolated and identified a HD-Zip I gene differentially expressed at high temperatures, LlHB16, which belongs to the β2 subgroup and positively regulates thermotolerance. The expression of LlHB16 was rapidly and continuously activated by heat stress. LlHB16 protein localized to the nucleus and exhibited transactivation activity in both plant and yeast cells, and its C-terminus contributed to its transcriptional activity. Overexpressing LlHB16 in Arabidopsis and lily improved thermotolerance and activated the expression of heat-related genes in both plants, especially that of HSFA2 and MBF1c. In addition, LlHB16 overexpression in Arabidopsis also caused growth defects, delayed flowering and abscisic acid (ABA) insensitivity. Further analysis revealed that LlHB16 directly binds to the promoters of LlHSFA2 and LlMBF1c and activates their expressions. Similarly, the expression of AtHSFA2 and AtMBF1c was also elevated in LlHB16 transgenic Arabidopsis lines. Together, our findings demonstrate that LlHB16 participates in the establishment of thermotolerance involved in activating LlHSFA2 and LlMBF1c, and LlHB16 overexpression resulted in ABA insensitivity in transgenic plants, suggesting that LlHB16 links the basal heat-responsive pathway and ABA signal to collaboratively regulate thermotolerance.

Chromosome characterization and physical mapping of 18S rDNA in Lilium longiflorum originated interspecific hybrids using combined genomic and fluorescent in situ hybridization

This study was aimed to differentiate parental genomes, examining intergenomic composition, and mapping mitotic metaphase chromosomes by localizing parental and 18S rDNA probes in seven interspecific hybrid progenies that originated from Lilium longiflorum. Since single method of in situ hybridization has been widely used in interspecific lily breeding, while in this study, flow cytometry was used in conjunction with genomic and fluorescent in situ hybridization to determine the genomic contribution of each parent to the interspecific progenies. A significant variation was observed in the DNA content, chromosome length, and 18S loci in F1 as compared to the female and male parents. L. longiflorum showed nearly two times higher DNA content than the male parents and L. longiflorum × Asiatic progenies, but eight times higher than L. longiflorum × L. hansonii. Genomic in situ hybridization results revealed that both female and male parents contributed an equal number of chromosomes to their interspecific F1 offspring. Fluorescent in situ hybridization mapping revealed that 18S rDNA had 8, 6 and 7 loci in L. longiflorum parents, i.e., White heaven, Bright tower, and White tower, respectively, whereas each Asiatic cultivar and L. hansonii used as male showed 8 and 12 loci respectively. Interspecific progenies showed 8 and 7 loci in LA, and 10–11 in LM hybrids. These cytogenetic results implied equal genetic and chromosomal contribution from both parents to their intergenomic progenies. Therefore, this combined cytogenetic method has the potential to be genome specific and time-saving approach in lily breeding that could determine the status of hybrids and their genomic origin while achieving physical mapping and detecting genes in different genomes.

Expression Patterns of Key Genes in the Photoperiod and Vernalization Flowering Pathways in Lilium longiflorum with Different Bulb Sizes.

Lilium longiflorum is a wild Lilium, and its flowering transition requires a long period of cold exposure to meet the demand of vernalization. The responses of different sized bulbs to cold exposure and photoperiod are different, and the floral transition pathways of small and large bulbs are different. In this study, small and large bulbs were placed in cold storage for different weeks and then cultured at a constant ambient temperature of 25 °C under long day (LD) and short day (SD) conditions. Then, the flowering characteristics and expression patterns of key genes related to the vernalization and photoperiod pathways in different groups were calculated and analyzed. The results showed that the floral transition of Lilium longiflorum was influenced by both vernalization and photoperiod, that vernalization and LD conditions can significantly improve the flowering rate of Lilium longiflorum, and that the time from planting to visible flowering buds’ appearance was decreased. The flowering time and rate of large bulbs were greatly influenced by cold exposure, and the vernalization pathway acted more actively at the floral transition stage. The floral transition of small bulbs was affected more by the photoperiod pathway. Moreover, it was speculated that cold exposure may promote greater sensitivity of the small bulbs to LD conditions. In addition, the expression of LlVRN1, LlFKF1, LlGI, LlCO5, LlCO7, LlCO16, LlFT1, LlFT3 and LlSOC1 was high during the process of floral transition, and LlCO13, LlCO14 and LlCO15 were highly expressed in the vegetative stage. The expression of LlCO13 and LlCO14 was different under different lighting conditions, and the flowering induction function of LlCO9 and LlFT3 was related to vernalization. Moreover, LlFKF1, LlGI, LlCO5, LlCO16, LlSOC1 and LlFT2 were involved in the entire growth process of plants, while LlCO6, LlCO16 and LlFT1 are involved in the differentiation and formation of small bulblets of plants after the inflorescence stage, and this process is also closely related to LD conditions. This study has great significance for understanding the molecular mechanisms of the vernalization and photoperiod flowering pathways of Lilium longiflorum.

Expression of anthocyanin biosynthesis-related genes during flower development in Lilium spp.

Sterility of hybrids produced from interspecific hybridization in lilies (Lilium, Liliaceae) is a great limitation in the breeding program, especially for Lilium longiflorum, which only has white-flowered cultivars. Because modification of flower colour in L. longiflorum by conventional breeding is limited by pre- and post-fertilization barriers, we think genetic modification could be used as an alternative in the future. For this, we need to understand what determines white colouration in L. longiflorum and other species and identify the molecular mechanisms regulating flower colour. In this study, we determined the accumulation of anthocyanins and related compounds in flower tissues during flower developmental stages in L. longiflorum cultivar ‘Lincoln’ and in the Oriental hybrid lily cultivars ‘Rialto’, ‘Perth’ and ‘Gran Tourismo’, respectively with white, pink and red flowers. Furthermore, the presence/absence and the expression of eight structural genes (CHSa, CHSb, CHIa, CHIb, F3H, F3’H, DFR, ANS) and three transcription factor genes (MYB12, MYB15, bHLH2) in flower tissues were investigated. Two structural genes (LlLinF3’H and LlLinDFR) and one transcription factor gene (LlLinbHLH2) were not detected in ‘Lincoln’ flowers. In ‘Rialto’, an amino acid substitution in the R2 repeat of LhRiaMYB12 which was previously reported to be responsible for the white flower colour is also found in the LhPerMYB12 of the pink ‘Perth’ flowers. Moreover, LhRiaDFR is present but not expressed in ‘Rialto’ flowers. Accumulation of cyanidin was observed in the flowers of ‘Perth’ and ‘Gran Tourismo’. High amounts of dihydrokaempferol accumulated in flowers of all four lily cultivars confirming the expression and functionality of early structural genes in the pathway. The elevated expression of the structural genes is strongly correlated with the expression of LhMYB12 and LhMYB15. This information can be used in the future to generate new L. longiflorum or Oriental lily hybrid cultivars with novel flower colours.

Garden Lupin (Lupinus polyphyllus) Is a New Natural Host of Lily Symptomless Virus in Korea.

Lupinus polyphyllus (Fabaceae), commonly known as garden lupin, is cultivated for commercial, private, and gardening purposes in many countries in Europe, North America, and Asia (Ramula and Kalske 2020). No previous studies have reported viruses infecting L. polyphyllus in the Republic of Korea. In May 2021, virus-like symptoms such as stunting, chlorotic striping, and mosaic were observed on the leaves of L. polyphyllus at the Suncheonman National Garden in Suncheon, South Korea; the disease incidence was estimated to be between 10 and 20%. These symptoms were similar to those observed in lily plants infected by lily symptomless virus (LSV) (Singh et al. 2005, Flores et al. 2019). Several different species of Lilium, including lilies, were grown in the same garden with L. polyphyllus on the adjacent block. Only carlavirus-like filamentous particles (approximately 610-700 nm in length and 12-15 nm in diameter) were observed using transmission electron microscopy in negatively stained extracts from five symptomatic samples. To further investigate the causative virus, a total of 20 symptomatic leaves were collected from 20 different L. polyphyllus plants and tested by DAS-ELISA using polyclonal antibodies against viruses infecting plants from the Lilium genus, including arabis mosaic virus, cucumber mosaic virus, LSV, lily mottle virus, and plantago asiatica mosaic virus (Agdia, Elkhart, IN, USA). Commercial positive and negative controls and extract from healthy L. polyphyllus tissue were included in each ELISA. The serological analyses showed that all symptomatic samples were positive only for LSV, but not for other viruses. To further confirm LSV infection, total RNA was extracted from 10 ELISA-positive plants and two healthy L. polyphyllus plants (negative control) using Clear-S Total RNA extraction kit (InVirusTech Co., Gwangju, Korea). RT-PCRs were performed using LSV coat protein (CP) specific primers, LSV1 (5'-CACATATGCAATCAAGACCAGCAC-3') and LSV2 (5'-GAGGATCCTCATCCATTATTTGCGTATC-3'), designed to amplify an 876-nt fragment of the CP gene (Lim et al. 2009). A DNA fragment of the expected size was amplified from only the ten symptomatic leaves. Three amplified RT-PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI) and sequenced. The sequences obtained from each amplicon showed 100% sequence identity; a single representative sequence was deposited to GenBank (LC649240). Pairwise nucleotide sequence analysis revealed that this LSV isolate LP2 had 99% nucleotide and 100% amino acid identity with Lilium longiflorum LSV isolate from South Korea (JX962776). To fulfill Koch's postulates, sap from an LSV-positive L. polyphyllus plant was inoculated to 10 young healthy L. polyphyllus plants. At 14 days post-inoculation, all the inoculated plants showed typical symptoms of LSV infection, such as mild vein-clearing and mosaic (Asjes 2000). The LSV infection in the inoculated plants was confirmed by RT-PCR using the aforementioned primers and sequencing of the 876-nt amplicons. To the best of our knowledge, this is the first report of LSV naturally infecting L. polyphyllus in the Republic of Korea. LSV could negatively impact the quality of L. polyphyllus and trade. Thus, it is important to investigate the extent of its distribution in South Korea and how it can be effectively managed.