Creating a novel petal regeneration system for function identification of colour gene of grape hyacinth

Background Grape hyacinth (Muscari spp.) is one of the most important ornamental bulbous plants. However, its lengthy juvenile period and time-consuming transformation approaches under the available protocols impedes the functional characterisation of its genes in flower tissues. In vitro flower organogenesis has long been used to hasten the breeding cycle of plants but has not been exploited for shortening the period of gene transformation and characterisation in flowers. Results A petal regeneration system was established for stable transformation and function identification of colour gene in grape hyacinth. By culturing on Murashige and Skoog medium (MS) with 0.45 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 8.88 μM 6-benzyladenine (6-BA), during the colour-changing period, the flower bud explants gave rise to regeneration petals in less than 3 months, instead of the 3 years required in field-grown plants. By combining this system with Agrobacterium-mediated transformation, a glucuronidase reporter gene (GUS) was delivered into grape hyacinth petals. Ultimately, 214 transgenic petals were regenerated from 24 resistant explants. PCR and GUS quantitative analyses confirmed that these putative transgenic petals have stably overexpressed GUS genes. Furthermore, an RNAi vector of the anthocyanidin 3-O-glucosyltransferase gene (MaGT) was integrated into grape hyacinth petals using the same strategy. Compared with the non-transgenic controls, reduced expression of the MaGT occurred in all transgenic petals, which caused pigmentation loss by repressing anthocyanin accumulation. Conclusion The Agrobacterium transformation method via petal organogenesis of grape hyacinth took only 3–4 months to implement, and was faster and easier to perform than other gene-overexpressing or -silencing techniques that are currently available.

Identification and characterization of a new potyvirus infecting Muscari in Hungary

Grape hyacinths (Muscari spp.) are popular spring flowering bulbs in Europe and also in Hungary. In the spring of 2017, we came across grape hyacinth plants showing mosaic symptoms, which indicated viral infection. Currently Hyacinth mosaic virus (genus Potyvirus), a proposed member of the genus named Muscari mosaic virus, Arabis mosaic virus (genus Nepovirus), Cucumber mosaic virus (genus Cucumovirus) and Tobacco rattle virus (genus Tobravirus) are known to infect grape hyacinth. Leaf samples of symptomatic grape hyacinths were observed, collected and the presence of potyviruses was proved with potyvirus specific monoclonal antibody by ELISA and by potyvirus specific RT-PCR. Laboratory host plants and seed grown Muscari plants were inoculated with leaf sap and symptoms were recorded. Nucleotide sequences of the cloned fragments were compared to GenBank data. In the case of Muscari ‘Helena’ the highest nucleotide identity in the coat protein coding region was found with two Muscari mosaic virus isolates (95.51% and 95.79%). In the case of the clones derived from Muscari ‘Pink Sunrise’ plant, the highest identity was recorded with Muscari mosaic virus isolates (57.65% and 57.80%) and with a Tulip breaking virus strain (55.13%) indicating the existence of a novel potyvirus species, tentatively named Muscari chlorotic mottle virus. The coat protein sequences were aligned and Maximum Likelihood trees were built to analyze phylogenetic relationships.

Anthocyanins Profile Characterization of Common Grape Hyacinth (Muscari neglectum Guss. ex Ten.) Flowers Growing in Hungary by High-performance Liquid Chromatography Coupled to Diode Array Detector and Mass Spectrometry using the Electrospray Ionization Interface

Objective: Anthocyanins are one of the biologically active substances group playing an important role in the state of physiological functions referring to human health. This research aimed to investigate the anthocyanins profiles in the grape hyacinth (Muscari neglectum Guss. ex Ten.). Materials and methods: The identification of individual anthocyanins was carried using the method of high-performance liquid chromatography with diode array detection and mass spectrometry with electrospray ionization (DAD-ESI-MS) analysis. Chromatographic separation and tandem mass spectrometric analyses were performed on an Agilent 1100 HPLC system and Agilent 6410 triple quadrupole system equipped with an electrospray ion source (ESI) in positive ion mode. Results: In the M. neglectum flowers (tepals), nine individual anthocyanins, containing delphinidin, petunidin, malvidine, pelargonidin aglycones were found. In this research, we report anthocyanin profiles for the M. neglectum flowers for the first time. Conclusion: The obtained results concerning anthocyanins composition may be very useful for researchers in the field of the standardization and activity evaluation of extracts produced from M. neglectum.

Cloning and Functional Characterization of Dihydroflavonol 4-Reductase Gene Involved in Anthocyanidin Biosynthesis of Grape Hyacinth

Grape hyacinth (Muscari spp.) is a popular ornamental plant with bulbous flowers noted for their rich blue color. Muscari species have been thought to accumulate delphinidin and cyanidin rather than pelargonidin-type anthocyanins because their dihydroflavonol 4-reductase (DFR) does not efficiently reduce dihydrokaempferol. In our study, we clone a novel DFR gene from blue flowers of Muscari. aucheri. Quantitative real-time PCR (qRT-PCR) and anthocyanin analysis showed that the expression pattern of MaDFR had strong correlations with the accumulation of delphinidin, relatively weak correlations with cyanidin, and no correations with pelargonidin. However, in vitro enzymatic analysis revealed that the MaDFR enzyme can reduce all the three types of dihydroflavonols (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), although it most preferred dihydromyricetin as a substrate to produce leucodelphinidin, the precursor of blue-hued delphinidin. This indicated that there may be other functional genes responsible for the loss of red pelargonidin-based pigments in Muscari. To further verify the substrate-specific selection domains of MaDFR, an assay of amino acid substitutions was conducted. The activity of MaDFR was not affected whenever the N135 or E146 site was mutated. However, when both of them were mutated, the catalytic activity of MaDFR was lost completely. The results suggest that both the N135 and E146 sites are essential for the activity of MaDFR. Additionally, the heterologous expression of MaDFR in tobacco (Nicotiana tabacum) resulted in increasing anthocyanin accumulation, leading to a darker flower color, which suggested that MaDFR was involved in color development in flowers. In summary, MaDFR has a high preference for dihydromyricetin, and it could be a powerful candidate gene for genetic engineering for blue flower colour modification. Our results also make a valuable contribution to understanding the basis of color variation in the genus Muscari.

Grape hyacinth [Muscari botryoides (L.) Mill] control in a wheat-soybean rotation

Grape hyacinth is a perennial bulbous species in the Liliaceae. It is commonly grown as an ornamental plant, but it can spread into agricultural fields and become weedy, potentially interfering with harvest and fall-planted crops. There has been limited research on controlling grape hyacinth in cropping systems. Fall and spring applied field-research studies were conducted to determine grape hyacinth control with herbicides labeled for use in wheat or winter fallow before planting soybean. Among fall-applied herbicides, paraquat resulted in the greatest initial grape hyacinth control (90% to 100%). Grape hyacinth control, 16 months after application (MAA), was variable, but the top-performing treatments were glyphosate and metsulfuron plus paraquat, resulting in 65% and 50% control, respectively. After spring applications, grape hyacinth control in November (7 MAA) was variable, but top-performing treatments were glyphosate and metsulfuron, which resulted in at least 26% control. Spring-applied paraquat, carfentrazone, metsulfuron, and sulfosulfuron resulted in 73%, 68%, 69%, and 60% reductions in grape hyacinth bulb counts, compared with the nontreated control 7 MAA, and were the top-performing treatments. Despite product-label prohibitions on rotation to soybeans, no soybean yield reductions were observed from any treatment in either study. Single applications of certain herbicides in the fall or spring can result in good control (>80%) of grape hyacinth initially, but long-term control is poor, and additional research is required.

Identification and Functional Analysis of a Flavonol Synthase Gene from Grape Hyacinth

Flavonols are important copigments that affect flower petal coloration. Flavonol synthase (FLS) catalyzes the conversion of dihydroflavonols to flavonols. In this study, we identified a FLS gene, MaFLS, expressed in petals of the ornamental monocot Muscari aucheri (grape hyacinth) and analyzed its spatial and temporal expression patterns. qRT-PCR analysis showed that MaFLS was predominantly expressed in the early stages of flower development. We next analyzed the in planta functions of MaFLS. Heterologous expression of MaFLS in Nicotiana tabacum (tobacco) resulted in a reduction in pigmentation in the petals, substantially inhibiting the expression of endogenous tobacco genes involved in anthocyanin biosynthesis (i.e., NtDFR, NtANS, and NtAN2) and upregulating the expression of NtFLS. The total anthocyanin content in the petals of the transformed tobacco plants was dramatically reduced, whereas the total flavonol content was increased. Our study suggests that MaFLS plays a key role in flavonol biosynthesis and flower coloration in grape hyacinth. Moreover, MaFLS may represent a new potential gene for molecular breeding of flower color modification and provide a basis for analyzing the effects of copigmentation on flower coloration in grape hyacinth.