Effects of Phloretin on Seedling Growth and Histochemical Distribution of Phenols, Polysaccharides and Lipids in Capsella bursa-pastoris (L.) Medik.

The present study evaluates the phytotoxic effects of phloretin, a prevalent secondary metabolite of apple trees, on the broadleaf weed Capsella bursa-pastoris (L.) Medik. known for its resistant myxospermous seeds that form a long-lasting soil bank. The results indicate a significant, dose-dependent inhibitory effect of phloretin on the growth and morphological parameters of weed seedlings grown in vitro. Although the applied phloretin concentrations (250-1000 µM) were not lethal to the C. bursa-pastoris seedlings after two weeks, the metabolism of the seedlings was impaired, resulting in an accumulation of lipid droplets in the root tips and root hairs. Histochemical analysis shows deposits of phenols in the root epidermal cells, which are probably aggregates of phloretin or its metabolic derivatives. The accumulation of pectin in the cell walls of root border cells in phloretin-treated seedlings indicates an attempt to reduce the uptake of phloretin and reduce its concentration in the cells. Inhibition of shoot growth associated with chlorosis and reduced photosynthetic pigment content is a consequence of seedling exposure to phloretin. This study provides a basis for further evaluation of phloretin as a new bioherbicidal compound and for elucidating the mechanism underlying its phytotoxic activity.

New Insights Into the Activity of Apple Dihydrochalcone Phloretin: Disturbance of Auxin Homeostasis as Physiological Basis of Phloretin Phytotoxic Action.

Apple species are the unique naturally rich source of dihydrochalcones, phenolic compounds with an elusive role in planta, but suggested auto-allelochemical features related to "apple replant disease" (ARD). Our aim was to elucidate the physiological basis of the phytotoxic action of dihydrochalcone phloretin in the model plant Arabidopsis and to promote phloretin as a new prospective eco-friendly phytotoxic compound. Phloretin treatment induced a significant dose-dependent growth retardation and severe morphological abnormalities and agravitropic behavior in Arabidopsis seedlings. Histological examination revealed a reduced starch content in the columella cells and a serious disturbance in root architecture, which resulted in the reduction in length of meristematic and elongation zones. Significantly disturbed auxin metabolome profile in roots with a particularly increased content of IAA accumulated in the lateral parts of the root apex, accompanied by changes in the expression of auxin biosynthetic and transport genes, especially PIN1, PIN3, PIN7, and ABCB1, indicates the role of auxin in physiological basis of phloretin-induced growth retardation. The results reveal a disturbance of auxin homeostasis as the main mechanism of phytotoxic action of phloretin. This mechanism makes phloretin a prospective candidate for an eco-friendly bioherbicide and paves the way for further research of phloretin role in ARD.

Germination and the Initial Seedling Growth of Lettuce, Celeriac and Wheat Cultivars after Micronutrient and a Biological Application Pre-Sowing Seed Treatment.

Seed treatments with zinc, boron, biostimulant Coveron and MIX (zinc + boron + Coveron) were applied to three lettuce and three celeriac cultivars. Seeds of three wheat cultivars were treated under laboratory conditions with Trichoderma harzianum and eight Bacillus spp. Seed germination, seedling growth, and the presence of the following pathogens were determined: Fusarium sp., Alternaria sp., Penicillium sp., and Mucor sp. The Coveron treatment was the most effective on lettuce seeds tested in the germination cabinet. Seed germination was higher by 4% than in the control. Alternatively, germination of seeds treated with boron in the greenhouse was higher by 12% than in the control. The Coveron treatment had the highest effect on the shoot length, which was greater by 0.7 and 2.1 cm in the germination cabinet and the greenhouse, respectively. This treatment was also the most effective on the root length. Zn, B, and MIX treatments increased celeriac seed germination by 14% in the germination cabinet. The Zn treatment was the most efficient on seeds tested in the greenhouse. The germination was higher by 15%. A significant cultivar × treatment interaction was determined in both observed species under both conditions. The maximum effect on wheat seed germination (8%) was achieved with the T. harzianum treatment in the Salazar cultivar. A significant interdependence (p ≤ 0.01 to p ≤ 0.001) was established between seed germination and the seedling growth. The interrelationship between seed germination and pathogens of all cultivars was negative.

Xanthones Production in Gentiana dinarica Beck Hairy Root Cultures Grown in Simple Bioreactors.

The hairy root clones of Gentiana dinarica cl-B, cl-D, cl-3, and cl-14 were cultivated in parallel in diverse simple bioreactors, including temporary immersion systems RITA® (TIS RITA®), bubble column bioreactors (BCB), and Erlenmeyer flasks (EF), and evaluated for biomass production and xanthone content. The obtained results showed that TIS RITA® and BCB containing ½ MS medium with 4% sucrose provided equally good growth conditions in which the majority of the clones displayed the higher percentage of dry matter (DM%), and xanthones norswertianin-1-O-primeveroside (nor-1-O-prim) and norswertianin production than those cultivated in EF. Thin and well branched hairy root clone cl-B grown in BCB for 7 weeks was superior regarding all growth parameters tested, including growth index (19.97), dry weight (2.88 g), and DM% (25.70%) compared to all other clones. Cl-B cultured in TIS RITA® contained the highest amount of nor-1-O-prim (56.82 mg per vessel). In BCB with constant aeration, cl-B accumulated the highest norswertianin content reaching 18.08 mg/vessel. The optimized conditions for cultivation of selected G. dinarica hairy root clones in highly aerated TIS RITA® and BCB systems contribute to the development of bioreactor technology designed for the large scale commercial production of xanthones nor-1-O-prim and norswertianin.

Gentianella lutescens subsp. carpatica J. Holub.: Shoot Propagation In Vitro and Effect of Sucrose and Elicitors on Xanthones Production.

In vitro shoot culture of the endangered medicinal plant Gentianella lutescens was established from epicotyl explants cultured on MS basal medium with 0.2 mg L-1 6-benzylaminopurine (BA) and evaluated for xanthones content for the first time. Five shoot lines were obtained and no significant variations in multiplication rate, shoot elongation, and xanthones profile were found among them. The highest rooting rate (33.3%) was achieved by shoots treated for 2 days with 5 mg L-1 indole-3-butyric acid (IBA) followed by cultivation in liquid PGR-free ½ MS medium for 60 days. HPLC analysis revealed the lower content of xanthones-mangiferin, bellidifolin, demethylbellidifolin, demethylbellidifolin-8-O-glucoside and bellidifolin-8-O-glucoside-in in vitro cultured shoots compared to wild growing plants. The increasing concentration of sucrose, sorbitol and abiotic elicitors salicylic acid (SA), jasmonic acid (JA) and methyl jasmonate (MeJA) altered shoot growth and xanthone production. Sucrose and sorbitol applied at the highest concentration of 233.6 mM increased dry matter percentage, while SA at 100 µM promoted shoot growth 2-fold. The increased sucrose concentration enhanced accumulation of xanthones in shoot cultures 2-3-fold compared to the control shoots. Elicitors at 100-300 µM increased the accumulation of mangiferin, demethylbellidifolin-8-O-glucoside, and bellidifolin-8-O-glucoside almost equally, while MeJA at the highest concentration of 500 µM enhanced amount of aglycones demethylbellidifolin and bellidifolin 7-fold compared to the control. The obtained results facilitate conservation of G. lutescens and pave the way for further research on large-scale shoot propagation and production of pharmacologically active xanthones.

Gentiana clusii Perr.&Song.: Enhanced production of secondary metabolites by in vitro propagation.

Shoot and root in vitro culture of endemic European species Gentiana clusii was established for the first time. The effects of different concentrations of benzyl adenine (BA), 6-phurphurylaminopurine (KIN), indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) on shoot propagation and rooting of G. clusii were investigated. The optimal in vitro conditions for shoot propagation and long-term maintenance were achieved using woody plant medium (WPM) supplemented with 0.5 mg l-1 KIN, and subsequent application of IBA at 0.5 mg l-1 significantly improved rooting of these shoots. Root culture was established from excised root tips cultured in ½ MS liquid media with increasing concentrations of IBA (0.1-1.0 mg l-1). A high root growth rate and considerable biomass yield were obtained by addition of 1.0 mg l-1 IBA. HPLC analysis revealed that in vitro culture considerably promoted the production of secondary metabolites in G. clusii. The selected protocol for shoot propagation (WPM + 0.5 mg l-1 KIN) increased the content of sweroside, gentiopicrin and norswertianin-1-O-primeveroside (N-1-P) for more than 2-fold compared with the wild plants. IBA promoted N-1-P and norswertianin production in root cultures; their contents were enhanced 6.4- and 18.6-fold, respectively, compared with the wild plants. The extract of these roots displayed the highest antioxidant capacity (IC50 = 66.57 µg ml-1). The established shoot and root propagation protocols facilitate in vitro conservation of G. clusii, and provides a promising tool for the large scale production of valuable secoiridoids and xanthones.

Beneficial implications of sugar beet proteinase inhibitor BvSTI on plant architecture and salt stress tolerance in Lotus corniculatus L.

Food demands of increasing human population dictate intensification of livestock production, however, environmental stresses could jeopardize producers' efforts. Forage legumes suffer from yield losses and poor nutritional status due to salinity increase of agricultural soils. As tools aimed to reduce negative impacts of biotic or abiotic stresses, proteinase inhibitors (PIs) have been promoted for biotechnological improvements. In order to increase tolerance of Lotus corniculatus L. to salt stress, serine PI, BvSTI, was introduced into this legume using Agrobacterium rhizogenes, with final transformation efficiency of 4.57%. PCR, DNA gel-blot, RT-PCR and in-gel protein activity assays confirmed the presence and activity of BvSTI products in transformed lines. Plants from three selected transgenic lines (21, 73 and 109) showed significant alterations in overall phenotypic appearance, corresponding to differences in BvSTI accumulation. Lines 73 and 109 showed up to 7.3-fold higher number of tillers and massive, up to 5.8-fold heavier roots than in nontransformed controls (NTC). Line 21 was phenotypically similar to NTC, accumulated less BvSTI transcripts and did not exhibit an additional band of recombinant trypsin inhibitor as seen in lines 73 and 109. Exposure of the transgenic lines to NaCl revealed different levels of salt stress susceptibility. The NaCl sensitivity index, based on morphological appearance and chlorophyll concentrations showed that lines 73 and 109 were significantly less affected by salinity than NTC or line 21. High level of BvSTI altered morphology and delayed salt stress related senescence, implicating BvSTI gene as a promising tool for salinity tolerance improvement trials in L. corniculatus.

Hairy root culture as a valuable tool for allelopathic studies in apple.

Allelopathic plants exploit their chemical 'weapons' to prevail over the competition, suppress neighboring plants and consequently use the available resources more efficiently. However, the investigation of plant allelopathic interactions in rhizosphere is difficult to perform because of its high complexity due to interactions of biotic and abiotic factors. Thus, autonomous, aseptic root cultures of apple (Malus × domestica Borkh.) could facilitate allelopathic studies. We report on the successful genetic transformation of apple cultivars Melrose, Golden Delicious, Cadel and Gloster using Agrobacterium rhizogenes (Riker et al. 1930) Conn 1942 strain 15834 and for the first time the establishment of apple autonomous and permanent in vitro hairy root cultures that could be used as a new tool for apple allelopathic assays. Molecular characterization of transgenic hairy root lines was conducted to elucidate the possible relationship between expression of T-DNA genes and root growth characteristics that include branching. Similar content of phenolic acids (chlorogenic, caffeic, syringic, p-coumaric and ferulic), glycosilated flavonoids (rutin, quercitrin, isoquercitrin, kaempferol-3-glucoside) and flavonoid aglycones (quercetin and naringenin), and dihydrochalcone phloridzin, was detected in untransformed and transgenic apple root tissue by ultra high-performance liquid chromatography with mass spectrometry (UHPLC/(+/-)HESI-MS/MS) analyses, confirming that genetic transformation did not disturb secondary metabolite production in apple. Chlorogenic and caffeic acids and dihydrochalcones phloridzin and phloretin were detected as putative allelochemicals exuded into the growth medium in which transgenic roots were maintained for 4 weeks. Apple hairy root exudates significantly affected shoot and root development and growth of test plant Arabidopsis thaliana (L.) Heynh. seedlings after 5 or 10 days of treatment. Additionally, core cell-cycle genes CDKA1;1, CDKB2;1, CYCA3;1 and CYCB2;4 were down regulated in Arabidopsis shoots suggesting, in part, their role in inhibition of shoot growth. The present work highlighted an autonomous and permanent in vitro hairy root culture system as a valuable tool for studying allelopathic potential of apple, offering new perspective for allelopathy background elucidation in this important fruit species.