Asiatic Hybrid Research Articles

Breeding of Asiatic hybrid lily cultivars to flower with increased number of flowers in one year from bulbils

Breeding of Asiatic hybrid lily cultivars to flower with increased number of flowers in one year from bulbils

Multifaceted roles of LhWRKY44 in promoting anthocyanin accumulation in Asiatic hybrid lilies (Lilium spp.).

The Asiatic hybrid lily (Lilium spp.) is a horticultural crop with high commercial value and diverse anthocyanin pigmentation patterns. However, the regulatory mechanism underlying lily flower color has been largely unexplored. Here, we identified a WRKY transcription factor from lily tepals, LhWRKY44, whose expression was closely associated with anthocyanin accumulation. Functional verification indicated that LhWRKY44 positively regulated anthocyanin accumulation. LhWRKY44 physically interacted with LhMYBSPLATTER and directly bound to the LhMYBSPLATTER promoter, which enhanced the effect of the LhMYBSPLATTER-LhbHLH2 MBW complex activator on anthocyanin accumulation. Moreover, EMSA and dual-luciferase assays revealed that LhWRKY44 activated and bound to the promoters of gene LhF3H and the intracellular anthocyanin-related glutathione S-transferase gene LhGST. Interestingly, our further results showed that LhWRKY44 participated in light and drought-induced anthocyanin accumulation, and improved the drought tolerance in lily via activating stress-related genes. These results generated a multifaceted regulatory mechanism for the LhWRKY44-meditaed enhancement by the environmental signal pathway of anthocyanin accumulation and expanded our understanding of the WRKY-mediated transcriptional regulatory hierarchy modulating anthocyanin accumulation in Asiatic hybrid lilies.

Production of Asiatic Hybrid Lily ‘Beni no Mai’ with More than Two Flowers within One Year from Bulbils Influenced by Development Stage, Flower Bud Removal, and Temperature Manipulation

Background and objective: The objective of this study was to produce Asiatic hybrid lily with more than two flowers in one year starting from bulbils. This study was to force 'Beni no Mai' bulbils influenced by harvesting date, flower bud removal, and temperature manipulation and to evaluate dormancy and maturity by nuclear magnetic resonance imaging (NMRI) and carbohydrates analysis.Methods: In experiment 1, bulbils were harvested at 0-30 days after anthesis (DAA), and were divided into 7 stages. And, soluble monosaccharides and monosaccharide constituents of bulbils were analyzed. In experiment 2, flower buds of plants that flowered on 21 April were not removed or removed on 1, 13, and 25 April. Bulbils harvested were divided into four groups and planted after cold treatment. In experiment 3, bulbils received a sequential temperature (SEQ CD) treatments for three weeks at 2.5, 5.0, and 7.5°C, 1 week at 10, 12.5, and 15°C, and 3 weeks at 2.5, 5.0, and 7.5°C. In experiment 4, bulbils were harvested 0-70 DAA, and were subjected to spin-spin lattice relaxation time by NMRI.Results: The bulbils harvested at 30 DAA produced two flowers within 282 days, and were better in growth. The bulbils harvested at 30 DAA showed higher concentration of soluble glucose and lower concentration of non-cellulosic neutral glucose. Growth of bulbils removed flower buds before flowering were better than those of bulbils that flower bud was removed at anthesis and 13 Apr. However, the number of flowers of bulbils harvested from plants removed flower buds on 1 Apr. were less than 2. The SEQ CD treatment of 7.5°C/3W-15°C/1W-5°C/3W in bulbils could produce the plants with 2.2 flowers within 300 days after planting. When bulbils were harvested at 0-28 DAA, yellow color corresponding to T2 relaxation time of 15-20 ms was dominant, and bulbils harvested at 56-70 DAA showed T2 relaxation time of 35-50 ms and 50-100 ms.Conclusion: Considering the results of soluble carbohydrate content and T2 relaxation time of bulbils, it was found that the bulbils had a shallower dormancy and were more mature at 30-42 DAA than before 28 DAA. Also, when these bulbils were planted after SEQ CD treatment at 7.5°C/3W-15°C/1W-5°C/3W, plants with two or more flowers could be produced within one year.

Development of EST-SSR markers based on transcriptome sequencing for germplasm evaluation of 65 lilies (Lilium).

Lilium genus consists of approximately 100 species and numerous varieties, many of which are interspecific hybrids, which result in a complicated genetic background. The germplasm identification, genetic relationship analysis, and breeding of Lilium rely on exploiting genetic information among different accessions. Hence, an attempt was made to develop new EST-SSR markers and study the molecular divergence among 65 genotypes of Lilium. A total of 5509 EST-SSRs were identified from the high-throughput sequencing database of L. 'Elodie'. After primer screening, six primer pairs with the most abundant polymorphic bands were selected from 100 primer pairs. Combined with the other 10 reported SSR primers, a total of 16 pairs detected genetic information with an average PIC value of 0.7583. The number of alleles per locus varied from four to 33, the expected heterozygosity varied from 0.3289 to 0.9231, and the observed heterozygosity varied from 0.2857 to 0.5122. Based on the phylogenic results, 22 Asiatic hybrids (A), seven Longiflorum × Asiatic hybrids (LA), as well as two native species were grouped. Eighteen Oriental hybrids (O) and nine Oriental × Trumpet (OT) hybrids, four native species, one LO, and one LL (L. pardalinum × L. longiflorum) variety were grouped. Two major clusters were reported and a large number of genotypes were grouped based on UPGMA and STRUCTURE analysis methods. The PIC value as well as other parameters revealed that the EST-SSR markers selected were informative. In addition, the clustering pattern displayed better agreement with the cultivar's pedigree. The newly identified SSRs in this study provides molecular markers for germplasm characterization and genetic diversity for Lilium.

Introduction of Floral Scent Traits into Asiatic Hybrid Lilies (Unscented) by Crossbreeding with <i>Lilium cernuum</i> (Scented)

Although a strong floral scent is typical of hybrid lilies, Asiatic hybrid lilies (Lilium spp.) have only a weak scent. Therefore, developing new cultivars with pleasant floral scents is an important objective in Asiatic hybrid lily breeding programs. Among the wild species contributing to Asiatic hybrid lily establishment, L. cernuum has a characteristic scent that the nose can perceive. We crossed Asiatic hybrid lily cultivars twice with L. cernuum to introduce floral scent traits into Asiatic hybrid lilies, and scent emission in the F1 plants and their progeny lines was evaluated instrumentally. Flowers of L. cernuum emitted benzenoids/phenylpropanoids (mainly 2-phenylethanol and benzaldehyde), monoterpenes (predominantly linalool and linalool oxides), and a fatty acid (iso-valeric acid). Lilium cernuum gave off unpleasant odors mainly because of iso-valeric acid emission. The F1 flowers also emitted these three chemical classes. Among the lines derived from the crosses between Asiatic hybrid lily cultivars and the F1 plant, lines that emitted all three chemical classes and lines that released one or two of the three chemical classes were segregated, and some lines emitted benzenoids/phenylpropanoids or terpenoids without emitting fatty acids. We successfully selected elite breeding lines that predominantly emitted pleasant scents. Therefore, this study demonstrated the introduction of pleasant scent production capability into Asiatic hybrid lilies using interspecific hybridization with L. cernuum.

Cytokinins influence bulblet formation by modulating sugar metabolism and endogenous hormones in Asiatic hybrid lily

Cytokinins influence bulblet formation by modulating sugar metabolism and endogenous hormones in Asiatic hybrid lily

The Composition of Anthocyanins and Carotenoids Influenced the Flower Color Heredity in Asiatic Hybrid Lilies

Flower color, including color hues and spot patterns, is an extremely important trait in Asiatic hybrid lilies. Investigating flower color variation across different cross combinations may help us select breeding parents efficiently. Thus, three hybridizations with different compositions of anthocyanins and carotenoids were performed, and 65, 289 and 125 offspring were obtained, respectively. For the parents and progenies, flower color hues were quantified by the CIELAB system, and the total number of raised spots on tepals was counted. Then, a cluster analysis and a statistical analysis were used to explore the inheritance patterns of flower color. The results showed that an orange flower color with high levels of carotenoids was highly heritable and that the progenies were less segregated, showing orange, orange-red, and yellow flowers. Parents showing red and purple-red flowers with differing levels of carotenoids and anthocyanins produced offspring with extensive segregation, including pink-white, pink, orange-pink, orange-red, yellow, and orange flowers. Meanwhile, different contents of anthocyanins and carotenoids resulted in variable proportions in color groups. Additionally, for F1 generation, the number of raised spots was continuously separated, demonstrating distinct quantitative genetic characteristics. For parents with few or intermediate spots, the hybrid plants showed both prominent negative and positive heterosis. For parents with many spots that covered almost the full tepals, the hybrid seedlings all exhibited raised spots.

The complete chloroplast genome sequence of Lilium concolor var. pulchellum

Lilium concolor var. pulchellum is a perennial herbaceous plant with high ornamental and edible value; it is a critical breeding parent of Asiatic hybrids. In this study, we reported the complete chloroplast genome of L. concolor var. pulchellum. The total size of the genome is 152,126 bp with a GC content of 37.0%. It has a conserved quadripartite structure comprising 136 genes, including 38 tRNA genes, 8 rRNA genes, 83 protein-coding genes, and 7 pseudogenes. Phylogenetic analysis strongly supported a close relation between L. concolor var. pulchellum and L. callosum. The complete plastome sequence of L. concolor var. pulchellum could provide useful information for phyletic evolution of the genus Lilium.

Phylogenetic Analysis of Wild Species and the Maternal Origin of Cultivars in the Genus Lilium Using 114 Plastid Genomes.

Lilies are one of the most important ornamental flowers worldwide with approximately 100 wild species and numerous cultivars, but the phylogenetic relationships among wild species and their contributions to these cultivars are poorly resolved. We collected the major Lilium species and cultivars and assembled their plastome sequences. Our phylogenetic reconstruction using 114 plastid genomes, including 70 wild species representing all sections and 42 cultivars representing six hybrid divisions and two outgroups, uncovered well-supported genetic relationships within Lilium. The wild species were separated into two distinct groups (groups A and B) associated with geographical distribution, which further diversified into eight different clades that were phylogenetically well supported. Additional support was provided by the distributions of indels and single-nucleotide variants, which were consistent with the topology. The species of sections Archelirion, Sinomartagon III, and Leucolirion 6a and 6b were the maternal donors for Oriental hybrids, Asiatic hybrids, Trumpet hybrids, and Longiflorum hybrids, respectively. The maternal donors of the OT hybrids originated from the two sections Archelirion and Leucolirion 6a, and LA hybrids were derived from the two sections Leucolirion 6b and Sinomartagon. Our study provides an important basis for clarifying the infrageneric classification and the maternal origin of cultivars in Lilium.

Comparative metabolome analysis unravels a close association between dormancy release and metabolic alteration induced by low temperature in lily bulbs.

The correlation between dormancy release and metabolic metabolic changes in lily bulbs during low temperature storage was investigated. Low temperature is a major environmental factor required for dormancy release in lily bulbs. Although great advances in plant metabolomics have been achieved, knowledge about the molecular basis of lily bulb metabolomes at different developmental stages in response to low temperature is still limited. In this work, the dormancy release, vegetative growth, flowering, metabolic profile and gene expression in the less dormant cultivar Lilium longiforum × Oriental hybrid 'Triumphator' (T) and the more dormant cultivar Lilium Asiatic hybrid 'Honesty' (H) were compared. Exposure to low temperature (LT) successfully promoted stem elongation, floral transition and flowering of both T and H bulbs. However, exposure to room temperature (RT) restricted stalk elongation of both T and H bulbs, and prohibited floral transition and flowering of H bulbs. Correspondingly, higher antioxidant enzyme activity and total primary metabolite contents were observed in the apical bud of T bulbs. Gene expression analysis revealed that expressions of LiFT, LiFLK, LiSOC1 and LiCBF were decreased, whereas the expression of LiSVP and LiFLC were increased, in the apical bud of H bulbs under RT storage condition. Our findings reveal that the growth and dormancy breaking of lily bulbs are closely associated with the metabolic changes in the apical buds during postharvest storage.

High temperature enhances anthocyanin coloration in Asiatic hybrid lily flowers via upregulation of the MYB12 positive regulator

Although high atmospheric temperatures suppress anthocyanin accumulation in most plant species, we show that high temperatures accelerate anthocyanin pigmentation in flower tepals of Asiatic hybrid lilies (Lilium spp.). After incubation at high temperatures (35 °C) for two days, anthocyanin color became deeper in the most of tepal parts of two Asiatic hybrid lily cultivars, although the basal parts of ‘Montreux’ tepals and top parts of 'Toronto' outer tepals were whitened. Environmental stimuli often affect the expression of R2R3-MYB positive regulators that control anthocyanin biosynthesis. Although their expression is often suppressed by hot temperatures in other species, the expression of lily MYB12 in tepals was upregulated by high temperatures. MicroRNA828 (miR828), which suppresses the action of MYB12 post-transcriptionally, exhibited reduced accumulation levels under high temperature, indicating that miR828 regulation is involved in MYB12 upregulation. In addition, transcription levels of MYB12, estimated by unspliced MYB12 transcript accumulation, were also activated by high temperatures. Thus, both suppressed miR828 accumulation and increased MYB12 transcription are likely to be involved in MYB12 activation at high temperatures. In the whitened basal parts of ‘Montreux’ tepals at 35 °C, expression of bHLH2 was severely suppressed while that of MYB12 was not affected. The present results demonstrate that plants display diverse responses to hot climatic conditions and shed new light on anthocyanin regulation under various environmental conditions.

Morphological characteristics and DNA barcoding in bach hop (Lilium poilanei Gagnep) in Vietnam

The Lilium genus is a member of the Liliaceae family and is comprised of approximately 110 to 120 species. This genus is considered the largest genome with a haploid DNA content in the plant kingdom and is currently receiving much attention for its great commercial and trading prospects. Among them, Lilium poilanei belongs to the Sinomartagon group, plays an important role in the breeding of Asiatic hybrids. L. poilanei, widely known as an endemic plant in mountainous areas of Sapa, Vietnam and has been intensively exploited due to great commercial significance. In this study, the morphological characteristics and DNA sequence data including the nuclear ribosomal DNA segments of ITS, ITS2, and plastid matK, psbA-trnH and rbcL, rpoC1 were applied to identify L. poilanei collected. The results showed that some morphological details of the lily were specifically analyzed and both markers ITS2 and rpoC1 disclosed superiority in discrimination of L. polinanei with 100% similarity score by the one of ours reported in NCBI Genebank with the accession number of KR632775.1 and KR632777.1, respectively, while the remaining regions have been neither unamplified nor unsuccessfully discriminated and identified. Our findings in morphology and barcodes were reliable, effective and powerful for distinguishing L. poilanei and may identify other species of the family of Liliaceae.

Transcriptome profiling during double-flower development provides insight into stamen petaloid in cultivated <i>Lilium</i>

Asiatic hybrid lilies (<italic>Lilium</italic> spp.), as the biggest cultivar groups of the lily variety are an ornamental plant with elegant floral patterns and bright colors. We discovered a double-flower Asiatic hybrid cultivar ‘Annemarie’s Dream’ whose stamens convert into petaloid stamens in varying degrees. Double-flower is a significant ornamental trait of the flower organ. However, the molecular mechanism of stamen petaloid formation has not been widely studied in Asiatic hybrid lily. Therefore, we used RNA-seq to contrast transcriptomes of stamen, petaloid stamen and inner tepal at two developmental stages. In total, 190,488 unigenes were obtained and 37,549 differentially expressed genes (DEGs) were identified. We focused on DEGs involved in phytohormone signaling and transcription factor regulation, especially the MADS-box genes (A-class gene<italic> LiAP1</italic>; B-class gene <italic>LiPI</italic>; C-class gene <italic>LiAG</italic>; E-class gene <italic>LiAGL6</italic>, <italic>LiSEP3</italic>). Furthermore, we performed weight gene co-expression network analysis (WGCNA) and identified two co-expressed MADS-box homeotic genes (<italic>LiAG</italic>, CL14315.Contig2_All; <italic>LiAP1</italic>, CL10060.Contig3_All) as hubs. We also found that CL3014.Contig2_All (<italic>PYL</italic>) and CL5627.Contig1_All (<italic>GID2</italic>) as phytohormone-related genes may participate in the regulation of the stamen petaloid during double-flower development. In summary, our findings provide an insight into the molecular regulatory network underlying stamen petaloid and thereby offering a theoretical basis for double-flower breeding in <italic>Lilium</italic>.

Evaluation, Breeding, and Forcing Asiatic Hybrid Lilies Starting from Bulbils to Maximize the Number of Flowers in One Year

Evaluation, Breeding, and Forcing Asiatic Hybrid Lilies Starting from Bulbils to Maximize the Number of Flowers in One Year

Construction of yeast one-hybrid library and screening of transcription factors regulating LhMYBSPLATTER expression in Asiatic hybrid lilies (Lilium spp.)

BackgroundAnthocyanins, which belong to flavonoids, are widely colored among red-purple pigments in the Asiatic hybrid lilies (Lilium spp.). Transcription factor (TF) LhMYBSPLATTER (formerly known as LhMYB12-Lat), identified as the major kernel protein, regulating the anthocyanin biosynthesis pathway in ‘Tiny Padhye’ of Tango Series cultivars, which the pigmentation density is high in the lower half of tepals and this patterning is of exceptional ornamental value. However, the research on mechanism of regulating the spatial and temporal expression differences of LhMYBSPLATTER, which belongs to the R2R3-MYB subfamily, is still not well established. To explore the molecular mechanism of directly related regulatory proteins of LhMYBSPLATTER in the anthocyanin pigmentation, the yeast one-hybrid (Y1H) cDNA library was constructed and characterized.ResultsIn this study, we describe a yeast one-hybrid library to screen transcription factors that regulate LhMYBSPLATTER gene expression in Lilium, with the library recombinant efficiency of over 98%. The lengths of inserted fragments ranged from 400 to 2000 bp, and the library capacity reached 1.6 × 106 CFU of cDNA insert, which is suitable to fulfill subsequent screening. Finally, seven prey proteins, including BTF3, MYB4, IAA6-like, ERF4, ARR1, ERF WIN1-like, and ERF061 were screened by the recombinant bait plasmid and verified by interaction with the LhMYBSPLATTER promoter. Among them, ERFs, AUX/IAA, and BTF3 may participate in the negative regulation of the anthocyanin biosynthesis pathway in Lilium.ConclusionA yeast one-hybrid library of lily was successfully constructed in the tepals for the first time. Seven candidate TFs of LhMYBSPLATTER were screened, which may provide a theoretical basis for the study of floral pigmentation.

The introduction study of new lily cultivars from Section I. Asiatic hybrids in Altai

The introduction study of new lily cultivars from Section I. Asiatic hybrids in Altai

Salt solution treatment plays an important role in overcoming pre-fertilization barriers during Asiatic and Oriental lily crossbreeding

Incompatibility between the stigma and pollen is the key factor limiting distant cross-breeding to produce Lilium Asiatic × Oriental hybrids (AO hybrids). To date, AO hybrids have not been available on the flower market. To produce AO hybrids, three pollination methods were applied to perform distant crosses between the Asiatic (A) hybrid ‘Pollyanna’ and the Oriental (O) hybrid ‘Siberia’ in this study. The seed-setting rate was significantly improved after a salt solution treatment. To clarify the mechanism by which the salt solution treatment allowed cross-incompatibility to be overcome, the effects of NaCl and KCl on the papilla cell morphology, pollen tube growth and seed setting rate were studied in the ‘Pollyanna’ × ‘Siberia’ crosses. After a salt treatment, the papilla cells at each stage swelled significantly, and the viscid exudates covering the papilla cell surface were diluted or dissolved. Furthermore, the pollen germination percentage increased, and the pollen tubes grew rapidly, causing more pollen tubes to enter the treated styles than the control styles. Hybridity of seedlings of ‘Pollyanna’ × ‘Siberia’ were confirmed by simple sequence repeat analysis, and their leaves showed intermediate traits between both the parents. These results suggest that salt solution treatments could be utilized for overcoming cross-incompatibility between A and O lilies to achieve AO hybrids, and that they provide insights into achieving other distant hybridizations in lilies.

High promoter sequence variation in subgroup 6 members of R2R3-MYB genes is involved in different floral anthocyanin color patterns in Lilium spp.

The spatially and temporally distinct expression of R2R3-MYB positive regulators is among the major mechanisms that create various anthocyanin color patterns in many flowers. However, we do not know how these positive regulators have gained different expression profiles. In the Asiatic hybrid lily 'Lollypop' (derived from the crosses of species belonging to Sinomartagon/Daurolirion section), MYB12 and MYB19S regulate the pigmentation at whole tepals and raised tepal spots, respectively. In the Oriental hybrid lily 'Sorbonne' (derived from the crosses of species belonging to the Archelirion section), MYB12 regulates both whole tepal and raised spot pigmentation. The genes have similar amino acid sequences with similar protein functions but exhibit different expression profiles in lily flowers. As promoters are among the most significant factors affecting gene expression profiles, their promoter sequences were determined in this study. The three genes had very different promoter sequences, and putative cis-regulatory elements were not conserved in numbers or order. To further confirm the promoter functions, tobacco plants were transformed with native promoter-driven MYB12 or MYB19S genes of 'Lollypop.' Expression levels of MYB12 were higher in corolla tubes than in lobes, while those of MYB19S were higher in corolla lobes than in tubes. Thus, the diverse promoter functions were likely to be the leading causes of their different expression profiles and generation of unique color patterns. Finally, the history of R2R3-MYB gene establishment during lily evolution was estimated using sequence data.

Anti-Inflammatory Activity of Benzyl Glycosides from the Flowers of Lilium Asiatic Hybrids

Repeated column chromatography of solvent fractions extracted from the flowers of Lilium Asiatic hybrids ensured purification of three benzyl glycosides I – III, which were reliably identified based on the analysis of various spectroscopic data. Compounds I – III (20 μg/mL) reduced nuclear factor-kappa B (NF-kB) (p-p65/p-65) by 9.64 ± 0.66%, 30.98 ± 1.08%, and 54.04 ± 1.12%, respectively. Compounds II and III markedly inhibited the expression of vascular cell adhesion molecule 1 (VCAM-1) in tumor necrosis factor (TNF)-α-stimulated human aortic smooth muscle cells (HASMCs) by 38.68 ± 2.52% and 37.9 ± 2.76%, respectively. Compound III significantly suppressed U937 monocytes attachment to the TNF-α-stimulated HASMCs.

LhGST is an anthocyanin-related glutathione S-transferase gene in Asiatic hybrid lilies (Lilium spp.).

LhGST, an anthocyanin-related GST gene, was identified from Asiatic hybrid lilies. Expression and functional analyses demonstrated that LhGST might be involved in anthocyanin sequestration in lily tepals. Anthocyanins are responsible for the pink, red and purple pigmentation of flowers in Asiatic hybrid lilies, synthesized at the cytoplasmic surface of the endoplasmic reticulum (ER) and then transported to the vacuole. To date, the mechanism involved in the intracellular transport of anthocyanins in lilies has not been well elucidated. Here, full-length glutathione S-transferase gene (LhGST) was identified from lilies. Expression analysis revealed that LhGST was positively correlated with anthocyanin accumulation. Phylogenetic tree analysis showed that LhGST clustered with other anthocyanin-related GSTs in the same phi clade. Moreover, functional complementation of an Arabidopsis tt19 mutant demonstrated that LhGST might be involved in anthocyanin accumulation in lily tepals. Additionally, according to phenotype analysis, LhGST was found to be correlated with the transport of anthocyanin in lilies by virus-induced gene silencing (VIGS) assay. In addition, cis-element analysis of the LhGST promoter showed the presence of ABA-, auxin-, MeJA-, gibberellin-, light-, and stress-responsive elements and an MYB recognition site (MRS, CCGTTG). Yeast one-hybrid and dual-luciferase report assays revealed that the promoter of LhGST was activated by LhMYB12-lat, which is a key R2R3-MYB transcription factor that regulates anthocyanin biosynthesis in lilies. In conclusion, our results revealed that LhGST plays a key role in anthocyanin transport and accumulation in the tepals of lilies.