Endophytic Bacillus and Pseudomonas spp. Modulate Apple Shoot Growth, Cellular Redox Balance, and Protein Expression Under in Vitro Conditions

Interactions between host plants and endophytic microorganisms play an important role in plant responses to pathogens and environmental stresses and have potential applications for plant stress management under in vitro conditions. We assessed the effect of endophytic bacteria on the growth and proliferation of domestic apple cv. Gala shoots in vitro. Further, a model apple cell suspension system was used to examine molecular events and protein expression patterns at an early stage of plant–endophyte interaction. Among the seven strains used in the study, Bacillus spp. strains Da_1, Da_4, and Da_5 and the Pseudomonas fluorescens strain Ga_1 promoted shoot growth and auxiliary shoot proliferation. In contrast, Bacillus sp. strain Oa_4, P. fluorescens strain Ga_3 and P. orientalis strain G_12 inhibited shoot development. In the cell suspension, the effects of the association between endophytic bacteria and plant cells were specific to each strain. Modulation of the cellular redox balance was monitored in the apple cells using a 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) probe, and strain-specific effects were observed that correlated with the in vitro shoot development results. Proteomic analysis revealed differences in protein expressions in apple cells co-cultivated with different Bacillus spp. strains that had contrasting effects on cellular redox balance and shoot development. The Bacillus sp. strain Da_4, which enhanced shoot development and oxidation of H2DCFDA, induced differential expression of proteins that are mainly involved in the defense response and regulation of oxidative stress. Meanwhile, treatment with Bacillus sp. strain Oa_4 led to strong upregulation of PLAT1, HSC70-1 and several other proteins involved in protein metabolism and cell development. Taken together, the results suggest that different cell signaling and response events at the early stage of the plant–endophyte interaction may be important for strain-dependent regulation of cellular redox balance and development of shoot phenotype

Cold hardiness identification and selection of strawberry varieties In Vivo

Straipsnyje aptariami augalų atrankos modeliavimo, kūrimo metodai siekiant augalus diferencijuoti pagal jų ištvermingumą žiemą. Siekiant iškelto tikslo buvo analizuota tiesioginė sąsaja augalų būklės, jų užsigrūdinimo lygio galimybės in vivo, esant atitinkamoms klimatinėms sąlygoms, tiriamuoju laikotarpiu. Straipsnyje pateikiama skirtingo atsparumo šalčiui braškių veislių užsigrūdinimo greičio svarba atrenkant, diferencijuojant augalus pagal šį požymįThe article discusses the modelling of plant selection and origination methods in order to differentiate plants according to their resistance in winter. For the purpose of research, a direct link between plant health and possibilities of their cold acclimation level in vivo in appropriate climatic conditions during the investigation period was analysed. In this article, the importance of cold acclimation speed of strawberry varieties with different cold hardiness in the selection and differentiation of plants in accordance with this feature is discussedBotanikos sodasSodininkystės ir daržininkystės institutasVytauto Didžiojo universiteta

Investigations of resistance to cold and hardening in vitro of Rosaceae family plants

Dehydrin-like proteins, which are responsible for resistance to cold, were investigated in model plants, the genes of dehydrins were identified in many herbaceous and woody plants, however we know very little about the hardening and the specificity of the resistance to cold of the most part of the garden plants - the Rosaceae family plants. Therefore, the study of dehydrins of the Rosaceae family plants was performed in The Institute of Horticulture of the Lithuanian Research Center for Agriculture and Forestry. The efficacy of the hardening of strawberry (Fragaria ananassa ) , wild strawberry (F. vesca), cherry (Prunus cerasus), sweet cherry (P. avium), apple (Malus domestica) and pear (Pyrus communis) cultivars was investigated under controled cooling conditions in vitro, the ion yield of cells was measured and critical temperature of the tissue damage was calculated. It was found that the maximum hardening is reached in 4 weeks and the critical temperature of the hardened microshoots has increased approximately 1,2°C, compared to non-hardened microshoots. Protein electrophoresis and immunochemical analysis showed that the expression of the two to five dehydrin-like proteins of the different molecular mass is characteristic to investigated cultivars and the significant increase of the amount of dehydrins in hardened microshootsGamtos mokslų fakultetasSodininkystės ir daržininkystės institutasVytauto Didžiojo universiteta

Influence of different exogenous saccharose and proline of strawberrymicroshoots in vitro

LAMMC Sodininkystės ir daržininkystės institute, Sodo augalų genetikos ir biotechnologijos skyriuje, buvo tiriamas įvairių braškių veislių atsparumas šalčiui po 14 dienų grūdinimo ir šaldymo in vitro –10 o C temperatūroje. Buvo įvertintas braškių atsparumas šalčiui, priklausomai nuo sacharozės, rafinozės ir prolino priedų, įdėtų į maitinamąją terpę (MS). T.Murashige’as ir F. Skoogas (1962) maitinamąją terpę papildė 3 ir 6% sacharozės, 5 % sacharozės + 1 % rafinozės, 6 % sacharozės + 0,001 % prolino priedais. Pašaldžius buvo vertinamas išgyvenusių mikroūglių skaičius (%) ir pažeidimas šalčiu (balais). Išryškėjo tiesioginė priklausomybė tarp grūdinimo trukmės, sacharozės, rafinozės ir prolino kiekio maitinamojoje terpėje ir mikroūglių būklės. Analizuojant cukrų priedų maitinamojoje terpėje įtaką jų kaupimuisi braškių mikroūgliuose, MS terpė buvo papildyta įvairiu kiekiu sacharozės (0,5%, 2% ir 6%). Nustatyta, kad tirpių angliavandenių kiekis braškių mikroūgliuose, auginant juos MS terpėje, pasižyminčioje skirtinga sacharozės koncentracija, didėja nepriklausomai nuo veislėsWinter and cold hardiness remain among most important traits for strawberry and other fruit crops. Biotechnological, in vitro methods make it possible to investigate separate factors of those complex traits in detail and give recommendations for improving cold hardiness by genetic engineering, marker assisted selection or breeding. The study was performed at Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry Effect of sucrose, raffinose and proline additions to growing media for cold acclimation efficiency, duration and cold hardiness were investigated in vitro. Plants of strawberry cultivars ‘Melody’, ‘Venta’ were planted on MS media with addition of different variants of 3%, 6% sucrose, 5% sucrose +1 % raffinose and 6% sucrose + 0.01% proline. After one week plants were transferred for acclimation at +4°C temperature for 14 days, frozen at -10°C temperature. Survival rate and cold injury score (0-5) were evaluated 3 weeks after freezing. It was shown direct correlation between acclimation duration, sucrose, raffinose concentration and cold hardiness of investigated strawberry plants. It was found that the soluble carbohydrate content of strawberries microshoots, growing them in MS medium with different sucrose concentrations increased regardless of the varietyBotanikos sodasSodininkystės ir daržininkystės institutasVytauto Didžiojo universiteta

Production of recombinant extracllular matrix mimicking peptides in tobacco plant

Application of the plant cultivation in the closed system (in vitro) technology for production of recombinant proteins provides a cost and product quality effective alternative as compared to traditional sources and it could be easily adapted for the large-scale industrial production [1]. Tobacco is one of the most popular plant-based expression systems, and it is widely used in producing antibodies, pharmaceutical and industrial proteins [2]. Composite mixtures of polymers and animal tissue specific extracellular matrix proteins – such as collagen, fibronectin, laminin – are being used for biomaterial production. However, specific functions of the extracellular matrix proteins could be readily reproduced using relatively short segments of their structure – peptides mimicking extracellular matrix. Therefore, aim of our research was to develop a closed type plant tissue cultivation system using tabaco plants (Nicotiana tabacum) dedicated for production for production of the recombinant peptide mimicking fibronectin typeIII domain 9-10 segments (FN9-10). DNA construct with constant p35S promotor and t35S terminator was prepared and pDGB3_alpha1 based plasmid vector was developed using the GoldenBraid 2.0 cloning system. The construct included a green fluorescent protein (avGFP) marker for the recombinant protein expression analysis, polyhistidine (6xHis) tag for protein affinity purification and a cleavage site for the WELQut protease for the separation of target peptide (Fig. 1). Tobacco leave tissues were transformed using Agrobacterium tumefaciens. Selection of tabaco transformants was carried out using different concentrations of antibiotic. It was established that concentration of kanamycin and geneticin suitable for transformed callus tissue selection was 150mg/L and 40 mg/L, respectively. To eliminate agrobacteria, thimetin was used at 300 mg/L with no adverse effect on tabaco callus viability. Further, selection of transformed tabaco callus [...]Gamtos mokslų fakultetasLietuvos agrarinių ir miškų mokslų centro filialas Sodininkystės ir daržininkystės institutasVytauto Didžiojo universiteta

Characteristics of Fragaria vesca yield parameters and anthocyanin accumulation under water deficit stress

Plants exposed to drought stress conditions often increase the synthesis of anthocyanins—natural plant pigments and antioxidants. However, water deficit (WD) often causes significant yield loss. The aim of our study was to evaluate the productivity as well as the anthocyanin content and composition of berries from cultivated Fragaria vesca “Rojan” and hybrid No. 17 plants (seedlings) grown under WD. The plants were grown in an unheated greenhouse and fully irrigated (control) or irrigated at 50% and 25%. The number of berries per plant and the berry weight were evaluated every 4 days. The anthocyanin content and composition of berries were evaluated with the same periodicity using HPLC. The effect of WD on the yield parameters of two evaluated F. vesca genotypes differed depending on the harvest time. The cumulative yield of plants under WD was not less than that of the control plants for 20–24 days after the start of the experiment. Additionally, berries accumulated 36–56% (1.5–2.3 times, depending on the harvest time) more anthocyanins compared with fully irrigated plants. Our data show that slight or moderate WD at a stable air temperature of about 20 °C positively affected the biosynthesis of anthocyanins and the yield of F. vesca berries

Enduring Effect of Antibiotic Timentin Treatment on Tobacco In Vitro Shoot Growth and Microbiome Diversity

Plant in vitro cultures initiated from surface-sterilized explants often harbor complex microbial communities. Antibiotics are commonly used to decontaminate plant tissue culture or during genetic transformation; however, the effect of antibiotic treatment on the diversity of indigenous microbial populations and the consequences on the performance of tissue culture is not completely understood. Therefore, the aim of this study was to assess the effect of antibiotic treatment on the growth and stress level of tobacco (Nicotiana tabacum L.) shoots in vitro as well as the composition of the plant-associated microbiome. The study revealed that shoot cultivation on a medium supplemented with 250 mg L−1 timentin resulted in 29 ± 4% reduced biomass accumulation and a 1.2–1.6-fold higher level of oxidative stress injury compared to the control samples. Moreover, the growth properties of shoots were only partially restored after transfer to a medium without the antibiotic. Microbiome analysis of the shoot samples using multivariable region-based 16S rRNA gene sequencing revealed a diverse microbial community in the control tobacco shoots, including 59 bacterial families; however, it was largely dominated by Mycobacteriaceae. Antibiotic treatment resulted in a decline in microbial diversity (the number of families was reduced 4.5-fold) and increased domination by the Mycobacteriaceae family. These results imply that the diversity of the plant-associated microbiome might represent a significant factor contributing to the efficient propagation of in vitro tissue culture

Brassica napus L. ūglių regeneracija in vitro iš hipokotilių ir stiebo segmentų kultūrų

Augalų genetinės transformacijos metodus siekiant panaudoti rapso genofondui gausinti, būtina optimizuoti ūglių regeneracijos procesą in vitro. Tirtas žieminio rapso 10 veislių organogenezės procesas hipokotilių ir stiebo segmentų kultūrose in vitro. Nustatyta, kad tirtų eksplantų ūglių regeneravimo dažnis priklausė nuo augalo genotipo. Iš tirtų veislių vidutiniškai didžiausiu dažniu ūglius regeneravo veislės ‛Valesca’ hipokotiliai. Vidutiniškai didžiausias ūglių formavimosi dažnis iš stiebo segmentų gautas auginant veislių ‛Insider’, ‛Siska’ ir ‛Kazimir H’ eksplantus. Nustatytas ūglių regeneracijos dažnio priklausomumas nuo eksplanto tipo: stiebo segmentai ūglius regeneravo nuo 25,78 % (‛Casino’) iki 85,11 % (‛Libea’) didesniu dažniu nei hipokotiliai. Egzogeniniai augimo reguliatoriai skatino ūglių formavimąsi iš hipokotilių ir stiebo segmentų in vitro kultūroje. Tačiau augimo reguliatorių poveikis priklausė nuo genotipo, todėl jų sudėtis maitinamojoje terpėje turi būti parenkama konkrečiam Brassica napus L. genotipui ir eksplanto tipuiIn order to use genetic transformation to enrich Brassica napus L. genetic resources, an efficient method of shoot regeneration in vitro is required. In this assay ten commercially important winter rapeseed cultivars were tested for the regeneration capacity through shoot organogenesis from hypocotyls and stem segments. Results show that shoot regeneration intensity depends on the genotype used. Hypocotyls of cultivar ‛Valesca’ show the highest overall capacity to produce shoots among all genotypes tested, while the higher overall regeneration frequency from stem segments was obtained for the cultivars ‛Insider’, ‛Siska’ and ‛Kazimir H’. Shoot regeneration efficiency strongly correlated with the explant type used: regeneration frequencies from stem segments were by 25.78% (‛Casino’) to 85.11% (‛Libea’) higher than those from hypocotyls. Exogenous growth regulators promoted shoot formation from both hypocotyls and stem segments, but they had variable effect on different genotypes. Thus, the composition of growth regulators must be optimized for every Brassica napus L. genotype and explant typeVytauto Didžiojo universitetasŽemės ūkio akademij

Anthocyanin content and stability in Ribes species and interspecific hybrids

Anthocyanins are widely distributed plant pigments. One of the richest sources of anthocyanins is blackcurrant. Our aim was to establish anthocyanin content in various Ribes species and interspecific hybrids, and to evaluate the stability of anthocyanins in berry extracts depending on light and temperature. Anthocyanins from berries of R. nigrum ‘Ben Tirran’, R. uva-crispa ‘Čiornyj negus’, R. aureum ‘Corona’, R. petraeum ‘Jonkher van Tets’ cultivars, and asymmetric interspecific hybrids of R. nigrum x R. petraeum, R. nigrum x R. uva-crispa and R. nigrum x R. aureum were studied using HPLC. Delphinidins constitute 64 %, cyanidins 31 % and minor anthocyanins up to 5 % of total anthocyanin amount in berries of blackcurrant ‘Ben Tirran’. Cyanidins dominate in berries of gooseberry ‘Čiornyj Negus’ (91.5 %) and golden currant ‘Corona’ (81 %). 6.2 percent of peonidin 3-O-rutinoside was identified in ‘Corona’. Only cyanidins were identified in berries of red currant ‘Jonkher van Tets’. Anthocyanin composition in berries of asymmetric interspecific hybrids was similar to the berries of blackcurrant. Anthocyanin stability in Ribes berry extracts depends on its composition and storage conditions. Red currant berry extract anthocyanins were the most stable, stability of anthocyanin extracts from berries of golden currant and gooseberry was lower, and extracts from blackcurrant were the most degraded. After storage of anthocyanin extracts in dark and cold (+4 oC) conditions up to 90 % of red currant, up to 80 % of gooseberry and golden currant and up to 50 % of black currant cyanidins remained stable. Cyanidins in Ribes berry extracts were more stable than delphinidins in all storage conditionsLietuvos agrarinių ir miškų mokslų centro filialas Sodininkystės ir daržininkystės institutasVytauto Didžiojo universitetasŽemės ūkio akademij

Potential Markers for Selecting Self-Eliminating Apple Genotypes

The heavy blooming of apple trees results in the inefficient usage of energy and nutritional material, and additional expenditure on fruitlet thinning is required to maintain fruit quality. A possible solution for controlling the fruit load on trees is the development of new cultivars that self-eliminate excess fruitlets, thus controlling yield. The aim of our study was to identify biological differences in apple cultivars in terms of blooming intensity and fruitlet load self-regulation. In total, 19 apple cultivars were studied in the years 2015–2017. The dynamics of fruitlet self-elimination, seed development in fruitlets and fruits, photosynthetic parameters, carbohydrates, and plant hormones were evaluated. We established that apple cultivars self-eliminating a small number of fruitlets need a lower number of well-developed seeds in fruit, and their number of leaves and area per fruit on a bearing branch are larger, compared to cultivars, self-eliminating large numbers of fruitlets. A higher carbohydrate amount in the leaves may be related to smaller fruitlet self-elimination. The amount of auxin and a high indole-3-acetic acid/zeatin ratio between leaves of cultivar groups with heavy blooming were higher than in cultivars with moderate blooming. A lower amount of abscisic acid was found in heavy-blooming cultivars during drought stress. All these parameters may be used as markers for the selection of different apple genotypes that self-eliminate fruitlets