Flower-visiting and pollen-carrying arthropods of Leucojum aestivum L. (Amaryllidaceae) in wild, reintroduced and ex situ populations

Leucojum aestivum L. is a wetland-dependent perennial geophyte occurring in Europe and western Asia. It is self-incompatible, with high level of fruit-set and seed-set. Yet, its pollinators are poorly known. Here, we present the most recent research on potential pollinators of L. aestivum. We collected data on flower-visiting and pollen-carrying arthropods in three populations of the species in the River Po plain (N-Italy), where L. aestivum occurs mainly in forests with Alnus glutinosa and Fraxinus sp. pl. close to rivers. We studied a wild population, a new reintroduced population and an ex situ population located at the Botanical Garden of the University of Pavia. Our study identified 18 arthropod taxa carrying L. aestivum pollen; the soft-winged flower beetle Dasytes plumbeus (Coleoptera: Melyridae) and the sac-spider Clubiona sp. pl. (Araneae: Clubionidae) were the most frequent visitors. Hymenoptera only occasionally visited L. aestivum flowers (e.g. the sweat bee Lasioglossum punctatissimum). D. plumbeus, the long-horned beetle Grammoptera ruficornis (Coleoptera: Cerambycidae), Clubiona sp. pl. and L. punctatissimum resulted in the taxa with the highest average abundance of pollen grains on their body suggesting a potential role in L. aestivum pollination. Differences in flower-visiting and pollen-carrying arthropods were observed between the three populations, with a decreasing taxonomic diversity of visiting species from the wild population to the ex situ population. Our results, based on direct observation in the field, are partially in contrast with a previous study on L. aestivum pollinators that suggested diurnal and nocturnal Lepidoptera and occasionally bees as main pollinators for the species.

In vitro regeneration of bulblet using two and four bulb-scales explants of summer snowflake (Leucojum aestivum L.)

Leucojum aestivum is a valuable and endangered plant species with bulb scales best suited as explants in micropropagation. In the current study, its micropropagation was investigated by using two different explants and various concentrations and combinations of plant growth regulators (PGRs). Bulbs were first disinfected with benomyl® for 5 hours. After meeting the chilling requirements, two-scale and four-scale explants were provided for direct and indirect organogenesis. Explants were exposed to hot water, 70% ethanol and 2.5% sodium hypochlorite for further disinfestation. Four-scale explants were treated with different concentrations and combinations of naphthaleneacetic acid (NAA), 6-benzyladenine (BA), and kinetin (Kin) for bulblet regeneration. For callogenesis, 0.5 mg L-1 of BA combined with 1, 2, 3, 4, 5 or 6 mg L-1 of 2,4-dichlorophenoxyacetic acid (2,4-D) were applied. Regarding two-scale explants, different combinations and concentrations of BA, Indole-3-butyric acid (IBA) and NAA were used for bulblet induction, and various combinations of Indoleacetic acid (IAA), NAA, 2, 4-D and BA were used for callus induction. None of the two-scale explants responded to the bulblet regeneration and callus induction media. Unlike, four-scale explants regenerated bulblets and roots in the control medium and MS media enriched with different PGRs. Callus was generated on MS medium supplemented with 2,4-D and BA, and indirect regeneration was observed in some cases. On the control medium, the regenerated roots had a natural form, but in PGRs-rich media, they were deformed. Regarding the regeneration percentage, bulblet number and length and root length, no significant differences were found between the control and the best PGR-treatment in each case. Therefore, it seems logical suggesting not to use PGRs, which will considerably reduce the costs at large-scale production.

Molecular Identification of Endophytic Bacteria in Leucojum aestivum In Vitro Culture, NMR-Based Metabolomics Study and LC-MS Analysis Leading to Potential Amaryllidaceae Alkaloid Production

In this study, endophytic bacteria belonging to the Bacillus genus were isolated from in vitro bulblets of Leucojum aestivum and their ability to produce Amaryllidaceae alkaloids was studied. Proton Nuclear Magnetic Resonance (1H NMR)-based metabolomics combined with multivariate data analysis was chosen to compare the metabolism of this plant (in vivo bulbs, in vitro bulblets) with those of the endophytic bacteria community. Primary metabolites were quantified by quantitative 1H NMR (qNMR) method. The results showed that tyrosine, one precursor of the Amaryllidaceae alkaloid biosynthesis pathway, was higher in endophytic extract compared to plant extract. In total, 22 compounds were identified including five molecules common to plant and endophyte extracts (tyrosine, isoleucine, valine, fatty acids and tyramine). In addition, endophytic extracts were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS) for the identification of compounds in very low concentrations. Five Amaryllidaceae alkaloids were detected in the extracts of endophytic bacteria. Lycorine, previously detected by 1H NMR, was confirmed with LC-MS analysis. Tazettine, pseudolycorine, acetylpseudolycorine, 1,2-dihydro-chlidanthine were also identified by LC-MS using the positive ionization mode or by GC-MS. In addition, 11 primary metabolites were identified in the endophytic extracts such as tyramine, which was obtained by decarboxylation of tyrosine. Thus, Bacillus sp. isolated from L. aestivum bulblets synthesized some primary and specialized metabolites in common with the L.aestivum plant. These endophytic bacteria are an interesting new approach for producing the Amaryllidaceae alkaloid such as lycorine.

МІКРОМОРФОЛОГІЯ ТА АНАТОМІЯ КВІТКИ LEUCOJUM AESTIVUM L. (AMARYLLIDACEAE J. ST.-HIL.)

In the gynoecium of L. aestivum there are synascidiate, hemisynascidiate, symplicate, and asymplicate vertical zones. The longest zone is the fertile hemisynascidiate zone and the shortest is the synascidiate zone in the ovary. It was discovered that in L. aestivum the peduncle consists of 12 vascular bundles, which are reorganized into two circles of bundles, the outer with massive leading bundles, departing as dorsal bundles of perianth, traces of perianth tepals and septal bundles of carpels and inner circle of bundles over the nests are divided into three groups of ventral carpel bundles are lined up on four, which are located in the center of the ovary and providing nutrition to the ovules. Dorsal carpels bundles are double. Above the locule, ventral bundles of the carpel, as well as the double septal bundles, merge with the dorsal bundles and form a dorsal vein. The outer tepals of the simple perianth have nine vascular traces, and the inner tepals of the perianth have eight vascular traces. Traces of stamens are single-bundle, formed from traces of perianth tepals. The ovary has features of the early stages of fruit morphogenesis and adaptation to disclosure, such as differentiation of mesocarp and endocarp cells, double dorsal bundles of carpels. Structural flower features related to pollen proposal as reward pollinators. Since ovary is a structural basis of the fruit, histological ovary wall differentiation reflects the features of the subsequent morphogenesis of the fruit.

A Csepel-sziget fehérnyár-ligetei (Senecioni sarracenici-Populetum albae Kevey in Borhidi & Kevey 1996)

Jelen tanulmány a Magyarország középső részén levő Duna-ártér fehérnyár-ligeteinek (Senecioni sarracenici-Populetum albae) társulási viszonyait mutatja be 25 cönológiai felvétel alapján. Laza öntéshomok alapkőzeten és nyers öntéstalajon kialakult állományaik az alacsony ártér viszonylag magasabb szintjeit foglalják el. Faji összetételükkel és fejlett cserjeszintjükkel jól elkülöníthetők a mintegy 1–1,5 m-rel mélyebben fekvő, kötött és iszapos talajú, cserjeszint nélküli fűzligetektől (Leucojo aestivi-Salicetum albae), valamint a magasabb ártéri szinten fejlődő tölgy-kőris-szil ligetektől (Scillo vindobonensis-Ulmetum). Aljnövényzetükben egyes – részben szubmontán jellegű – növények is megje­lenhetnek, amelyek az Alföld egyéb tájain ritkák, vagy teljesen hiányoznak: Anemone ranunculoides, Carex remota, Clematis recta, Crataegus × degenii, Crataegus nigra, Epipactis helleborine, Equisetum hyemale, Galanthus nivalis, Lathraea squamaria, Leucojum aestivum, Paris quadrifolia, Scilla vindobonensis, Vitis sylvestris. Az asszociáció a szüntaxonómiai rendszer „Populenion nigro-albae Kevey 2008” alcsoportjába helyezhető.

Carbohydrates stimulated Amaryllidaceae alkaloids biosynthesis in Leucojum aestivum L. plants cultured in RITA® bioreactor

Background Leucojum aestivum L. is an important medicinal plant which produces Amaryllidaceae alkaloids, especially galanthamine and lycorine. Research is currently exploring the possibility of producing these alkaloids using biotechnological methods, including in vitro cultures. The biosynthesis of alkaloids may be affected by the types and concentrations of carbohydrate sources used in the medium. In the present investigation we performed such studies on in vitro cultures of L. aestivum with a view to obtaining plant material of good quality, characterized, in particular, by a high content of valuable Amaryllidaceae alkaloids. Methods We examined the effects of various types of carbohydrate sources—sucrose, glucose, fructose and maltose—at different concentrations (30, 60 and 90 g/L)—on the quality of L. aestivum plants grown in the RITA® bioreactor. The plants’ quality was assessed by their biomass increments, as well by as analysing photosynthetic pigments, endogenous sugar, phenolics and Amaryllidaceae alkaloid content. We also investigated the effect of sugars on the activity of the antioxidant enzymes catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). Results The highest biomass increments were observed in plants cultivated in the medium containing 90 g/L sucrose. The highest CAT activity was noted in cultures growing in the medium supplemented with 90 g/L maltose, while the highest POD activity was observed in the presence of 90 g/L fructose and 60 g/L maltose. No differences in SOD activity were observed. Moreover, the sugars did not affect the contents of chlorophyll a and carotenoids, whereas the highest amount of chlorophyll b was recorded in plants growing in the medium with 60 g/L maltose. No statistically significant differences were observed in the contents of endogenous sugars and phenolics in any in vitro conditions. However, the addition of sugar had a decisive effect on the biosynthesis of the Amaryllidaceae alkaloids. The highest distribution of alkaloids occurred in plants cultured in the medium containing 60 g/L sucrose. Six Amaryllidaceae alkaloids were detected in the plant tissue. The addition of 30 g/L fructose in the medium resulted in the accumulation of five alkaloids, including ismine, which was not identified in other analysed tissues. The highest concentration of galanthamine was observed in plants cultured in the presence of 30 g/L fructose and 60 g/L sucrose (39.2 and 37.5 µg/g of dry weight (DW), respectively). The plants grown in the medium containing 60 g/L sucrose exhibited the highest lycorine content (1048 µg/g of DW). Conclusions The type and concentration of sugar used in the medium have an essential influence on the biosynthesis of Amaryllidaceae alkaloids in L. aestivum plants cultured in a RITA® bioreactor. The results point to an interesting approach for commercial production of galanthamine and lycorine.