Micropropagation of Encyclia cordigera (Kunth) Dressler in Ebb-and-Flow Bioreactor.

The use of new technologies for micropropagation such as temporary immersion systems (TISs) is important, because it reduces costs by 40% lowering labor, agar and containers. TISs are containers designed for large-scale, semiautomatic production of plants in a liquid medium, which has been used in propagation of commercial orchids. This tool has high potential for application in micropropagation of medicinal and endangered orchids for conservation and commercial purposes. In this chapter, we describe a detailed protocol for propagation and development of Encyclia cordigera to be used in research projects for small-scale production. This protocol comprises all steps from explant preparation to the establishment orchids plantlets.

Resistant and Susceptible Pinus thunbergii ParL. Show Highly Divergent Patterns of Differentially Expressed Genes during the Process of Infection by Bursaphelenchus xylophilus.

Pine wilt disease (PWD) is a devastating disease that threatens pine forests worldwide, and breeding resistant pines is an important management strategy used to reduce its impact. A batch of resistant seeds of P. thunbergii was introduced from Japan. Based on the resistant materials, we obtained somatic plants through somatic embryogenesis. In this study, we performed transcriptome analysis to further understand the defense response of resistant somatic plants of P. thunbergii to PWD. The results showed that, after pine wood nematode (PWN) infection, resistant P. thunbergii stimulated more differential expression genes (DEGs) and involved more regulatory pathways than did susceptible P. thunbergii. For the first time, the alpha-linolenic acid metabolism and linoleic acid metabolism were intensively observed in pines resisting PWN infection. The related genes disease resistance protein RPS2 (SUMM2) and pathogenesis-related genes (PR1), as well as reactive oxygen species (ROS)-related genes were significantly up-expressed in order to contribute to protection against PWN inoculation in P. thunbergii. In addition, the diterpenoid biosynthesis pathway was significantly enriched only in resistant P. thunbergii. These findings provided valuable genetic information for future breeding of resistant conifers, and could contribute to the development of new diagnostic tools for early screening of resistant pine seedlings based on specific PWN-tolerance-related markers.

Confocal Microscopy Investigations of Biopolymeric PLGA Nanoparticle Uptake in Arabidopsis thaliana L. Cultured Cells and Plantlet Roots.

To date, most endocytosis studies in plant cells have focused on clathrin-dependent endocytosis, while limited evidence is available on clathrin-independent pathways. Since dynamin a is a key protein both in clathrin-mediated endocytosis and in clathrin-independent endocytic processes, this study investigated its role in the uptake of poly-(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs). The experiments were performed on cultured cells and roots of Arabidopsis thaliana. Dynasore was used to inhibit the activity of dynamin-like proteins to investigate whether PLGA NPs enter plant cells through a dynamin-like-dependent or dynamin-like-independent endocytic pathway. Observations were performed by confocal microscopy using a fluorescent probe, coumarin 6, loaded in PLGA NPs. The results showed that both cells and roots of A. thaliana rapidly take up PLGA NPs. Dynasore was administered at different concentrations and exposure times in order to identify the effective ones for inhibitory activity. Treatments with dynasore did not prevent the NPs uptake, as revealed by the presence of fluorescence emission detected in the cytoplasm. At the highest concentration and the longest exposure time to dynasore, the fluorescence of NPs was not visible due to cell death. Thus, the results suggest that, because the NPs' uptake is unaffected by dynasore exposure, NPs can enter cells and roots by following a dynamin-like-independent endocytic pathway.

Bio-priming of banana tissue culture plantlets with endophytic Bacillus velezensis EB1 to improve Fusarium wilt resistance.

Tissue culture techniques have been routinely used for banana propagation and offered rapid production of planting materials with favorable genotypes and free of pathogenic microorganisms in the banana industry. Meanwhile, extensive scientific work suggests that micropropagated plantlets are more susceptible to Fusarium oxysporum f. sp. cubense (Foc), the deadly strain that causes Fusarium wilt of bananas than conventional planting material due to the loss of indigenous endophytes. In this study, an endophytic bacterium Bacillus velezensis EB1 was isolated and characterized. EB1 shows remarkable in vitro antagonistic activity against Foc with an inhibition rate of 75.43% and induces significant morphological and ultrastructural changes and alterations in the hyphae of Foc. Colony-forming unit (c.f.u.) counting and scanning electron microscopy (SEM) revealed that EB1 could colonize both the surface and inner tissues of banana tissue culture plantlets. Banana tissue culture plantlets of late rooting stage bioprimed with EB1 could efficiently ward off the invasive of Foc. The bio-priming effect could maintain in the acclimatized banana plants and significantly decrease the disease severity of Fusarium wilt and induce strong disease resistance by manipulating plant defense signaling pathways in a pot experiment. Our results provide the adaptability and potential of native endophyte EB1 in protecting plants from pathogens and infer that banana tissue culture plantlets bio-priming with endophytic microbiota could be a promising biological solution in the fight against the Fusarium wilt of banana.

Inoculation with Oidiodendron maius BP Improves Nitrogen Absorption from Fertilizer and Growth of Vaccinium corymbosum during the Early Nursery Stage.

Blueberry roots are inefficient in taking up water and nutrients, a fact partially related to their scarcity of root hairs, but they improve nutrient uptake by associating with ericoid mycorrhizal and endophytic fungi. However, the benefits of this association are both cultivar- and fungus-dependent. Our objective was to assess the effect of inoculation with three native fungal strains (Oidiodendron maius A, O. maius BP, and Acanthomyces lecanii BC) on plantlet growth, plantlet survival, and nitrogen (N) absorption of the southern highbush blueberry (SHB) cultivars Biloxi and Misty. The fungal strains were inoculated into the peat-based substrate for growing blueberry cultivars, and plantlets produced by micropropagation were transplanted and grown for four months. The three inoculated strains positively affected the survival percentage in at least one of the cultivars tested, whereas O. maius BP positively affected plant biomass, N derived from fertilizer absorption, N content, and plant N recovery (%) in both Biloxi and Misty. Our results show that the O. maius BP strain may prove useful as a bio-inoculant to improve blueberry production during the nursery stage.

Influence of Light of Different Spectral Compositions on Growth Parameters, Photosynthetic Pigment Contents and Gene Expression in Scots Pine Plantlets.

The photoreceptors of red light (phytochromes) and blue light (cryptochromes) impact plant growth and metabolism. However, their action has been barely studied, especially in coniferous plants. Therefore, the influence of blue (maximum 450 nm), red (maximum 660 nm), white light (maxima 450 nm + 575 nm), far-red light (maximum 730 nm), white fluorescent light and dark on seed germination, growth, chlorophyll and carotenoid contents, as well as the transcript levels of genes involved in reception, photosynthesis, light and hormonal signaling of Scots pine plantlets, was investigated. The highest values of dry weight, root length and photosynthetic pigment contents were characteristic of 9-day-old plantlets grown under red light, whereas in the dark plantlet length, seed vigor, seed germination, dry weight and pigment contents were decreased. Under blue and white lights, the main studied morphological parameters were decreased or close to red light. The cotyledons were undeveloped under dark conditions, likely due to the reduced content of photosynthetic pigments, which agrees with the low transcript levels of genes encoding protochlorophyllide oxidoreductase (PORA) and phytoene synthase (PSY). The transcript levels of a number of genes involved in phytohormone biosynthesis and signaling, such as GA3ox, RRa, KAO and JazA, were enhanced under red light, unlike under dark conditions. We suggest that the observed phenomena of red light are the most important for the germination of the plantlets and may be based on earlier and enhanced expression of auxin, cytokinin, gibberellin and jasmonate signaling genes activated by corresponding photoreceptors. The obtained results may help to improve reforestation technology; however, this problem needs further study.

Comparative Transcriptome Analysis Reveals the Interaction of Sugar and Hormone Metabolism Involved in the Root Hair Morphogenesis of the Endangered Fir Abies beshanzuensis.

Abies beshanzuensis, an extremely rare and critically endangered plant with only three wild adult trees globally, is strongly mycorrhizal-dependent, leading to difficulties in protection and artificial breeding without symbiosis. Root hair morphogenesis plays an important role in the survival of mycorrhizal symbionts. Due to the lack of an effective genome and transcriptome of A. beshanzuensis, the molecular signals involved in the root hair development remain unknown, which hinders its endangered mechanism analysis and protection. Herein, transcriptomes of radicles with root hair (RH1) and without root hair (RH0) from A. beshanzuensis in vitro plantlets were primarily established. Functional annotation and differentially expressed gene (DEG) analysis showed that the two phenotypes have highly differentially expressed gene clusters. Transcriptome divergence identified hormone and sugar signaling primarily involved in root hair morphogenesis of A. beshanzuensis. Weighted correlation network analysis (WGCNA) coupled with quantitative real-time PCR (qRT-PCR) found that two hormone-sucrose-root hair modules were linked by IAA17, and SUS was positioned in the center of the regulation network, co-expressed with SRK2E in hormone transduction and key genes related to root hair morphogenesis. Our results contribute to better understanding of the molecular mechanisms of root hair development and offer new insights into deciphering the survival mechanism of A. beshanzuensis and other endangered species, utilizing root hair as a compensatory strategy instead of poor mycorrhizal growth.

Integrating transcriptome and metabolome to explore the growth-promoting mechanisms of GABA in blueberry plantlets.

Tissue culture technology is the main method for the commercial propagation of blueberry plants, but blueberry plantlets grow slowly and have long growth cycles under in vitro propagation, resulting in low propagation efficiency. In addition, the long culturing time can also result in reduced nutrient content in the culture medium, and the accumulation of toxic and harmful substances that can lead to weak growth for the plantlets or browning and vitrification, which ultimately can seriously reduce the quality of the plantlets. Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid that can improve plant resistance to various stresses and promote plant growth, but the effects of its application and mechanism in tissue culture are still unclear. In this study, the effects of GABA on the growth of in vitro blueberry plantlets were analyzed following the treatment of the plantlets with GABA. In addition, the GABA-treated plantlets were also subjected to a comparative transcriptomic and metabolomic analysis. The exogenous application of GABA significantly promoted growth and improved the quality of the blueberry plantlets. In total, 2,626 differentially expressed genes (DEGs) and 377 differentially accumulated metabolites (DAMs) were detected by comparison of the control and GABA-treated plantlets. Most of the DEGs and DAMs were involved in carbohydrate metabolism and biosynthesis of secondary metabolites. The comprehensive analysis results indicated that GABA may promote the growth of blueberry plantlets by promoting carbon metabolism and nitrogen assimilation, as well as increasing the accumulation of secondary metabolites such as flavonoids, steroids and terpenes.

In Vitro Sprouted Plantlets of Citrullus colocynthis (L.) Schrad Shown to Possess Interesting Levels of Cucurbitacins and Other Bioactives against Pathogenic Fungi.

Cucurbitacins, structurally different triterpenes mainly found in the members of Cucurbitaceae, possess a vast pharmacological potential. Genus Cucurbita, Cucumis, and Citrullus are affluent in these bioactive compounds, and, amongst them, Citrullus colocynthis (L.) Schrad. is widely exploited in folk medicine, since a huge number of diseases are successfully treated with organic and aqueous extracts obtained from different organs and tissues of the plant. The well-known pharmacological activities of such species have been attributed to its peculiar composition, which includes cucurbitacins and other bioactive molecules; thus, owing to its high importance as a valuable natural resource for pharmaceuticals and nutraceuticals, C. colocynthis propagation and multiplication protocols are considered significant, but the exploitation of its phytochemical potential is limited by the restricted cultivation conditions and the low rate of seed germination in the natural environment; in fact, the assessment of accumulation rate of specific phytochemicals under controlled conditions is still missing. Axenically sprouted plantlets obtained without the use of culture media or the addition of hormones have been evaluated here for the production of bioactive compounds and relevant bioactive features. Our results proved that derived organic extracts contain cucurbitacins and other bioactives, show antioxidant potential, and exert activity against some pathogenic fungi (Candida krusei, C. albicans, C. parapsilosis, C. glabrata, and Aspergillus flavus), supporting the feasibility of a methodology intended to scale-up cultivation of this species as a source of pharmaceutically interesting compounds, achievable from plantlets cultivated under laboratory conditions.

Between Light and Shading: Morphological, Biochemical, and Metabolomics Insights Into the Influence of Blue Photoselective Shading on Vegetable Seedlings.

High nursery densities reduce the seedling quality due to the competition for light. High light intensity, shading, and blue light depletion activate morphophysiological and metabolomic responses in plants, resulting in size modification to gain an advantage over neighboring plants. Our research aimed to unravel the effects of light intensity and quality on nursery seedlings at the morphological and biochemical levels. To this aim, the effect of black shading and blue photoselective shading nets were investigated in terms of morphometric, ionomic, and untargeted metabolomics signatures in Cucurbita pepo L., Citrullus lanatus L., Solanum lycopersicum L., and Solanum melongena L. seedlings. Plant height, diameter, sturdiness index, leaf area, specific leaf area, shoot/root ratio, and mineral content (by ion chromatography-IC) were evaluated. In C. pepo L and C. lanatus L., the blue net reduced the shoot/root and chlorophyll a/b ratios and increased stem diameter and total chlorophyll content. The black net increased plant height, stem diameter, and sturdiness index in Solanum lycopersicum L. and Solanum melongena L. At the same time, unshading conditions reduced leaf area, specific leaf area, shoot/root ratio, and total chlorophyll content. The blue net improved the sturdiness index and quality of C. pepo L. and C. lanatus L. Such impact on morphological parameters induced by the different shading conditions was corroborated by a significant modulation at the metabolomics level. Untargeted metabolomic phytochemical signatures of the selected plants, and the subsequent multivariate analysis coupled to pathway analysis, allowed highlighting a broad and diverse biochemical modulation. Metabolomics revealed that both primary and secondary metabolism were largely affected by the different shading conditions, regardless of the species considered. A common pattern arose to point at the activation of plant energy metabolism and lipid biosynthesis, together with a generalized down accumulation of several secondary metabolites, particularly phenylpropanoids. Our findings indicate an intriguing scientific interest in the effects of selective shading and its application to other species and different phenological stages.

Somatic embryogenesis from flower tepals of Hippeastrum aiming regeneration of virus-free plants.

Hippeastrum hybridum is an important bulbous flower plant in world floriculture, which are propagated conventionally by the technique known as double or twin scales to obtain plants with clonal origin. However, this technique promotes the propagation of systemic diseases, particularly mosaic-inducing viruses. The aim of this paper was to evaluate the somatic embryogenesis (SE) from tepals as an alternative to provide a technique for SE induction and to obtaining virus-free plantlets of Hippeastrum from infected plants. The concentrations of 2,4-Dichlorofenoxiacetic Acid (2,4-D) and thidiazuron (TDZ) was evaluated in SE induction pathway. The monitoring of viruses during the assays with tepals was performed by Reverse Transcription-Polymerase Chain Reaction. SE induction was obtained, for the first time, in tepal segments from flower buds of Hippeastrum. The 2,4-D was the main factor for embryogenic callus induction, and TDZ increased the SE induction rate. However, conversion of somatic embryos into plantlets were only developed in free-2,4-D media, replaced by 1.0 mg L-1 6-Benziladenine. Out of five virus species monitored during the experiment, Cucumber mosaic virus was detected in tepals and Hippeastrum mosaic virus in leaves of donor plants. The SE-derived plantlets that germinated in vitro were acclimatized and tested negative for all viruses assayed.

Development and application of reverse transcription droplet digital PCR assay for sensitive detection of apple scar skin viroid during in vitro propagation of apple plantlets.

Apple scar skin viroid (ASSVd), of the genus Apscaviroid, causes serious pome fruit diseases, such as apple scar skin, dapple apple, pear rusty skin, pear fruit crinkle, and pear dimple fruit. This study aimed at establishing a sensitive and accurate method for quantification of ASSVd in apple leaves and plantlets using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The specificity was analyzed using other apple viruses, and the negative amplification of the cross-reaction assay demonstrated the high specificity of RT-ddPCR. The detection limit of ASSVd by RT-ddPCR was 1.75 × 102 copies/µL (0.14 concentration), and the sensitivity was ten-fold higher than that of RT-qPCR. Similarly, positive detection in apple plantlet samples by RT-ddPCR was higher than that by RT-qPCR. The RT-ddPCR assay represents a promising alternative for accurate quantitative detection and diagnosis of ASSVd infection in ASSVd-free certification programs.

Methods for a Quantitative Comparison of Gravitropism and Posture Control Over a Wide Range of Herbaceous and Woody Species.

Quantitative measurements of plant gravitropic response are challenging. Differences in growth rates between species and environmental conditions make it difficult to compare the intrinsic gravitropic responses of different plants. In addition, the bending movement associated with gravitropism is competing with the tendency of plants to grow straight, through a mechanism called proprioception (ability to sense its own shape). Disentangling these two tendencies is not trivial. Here, we use a combination of modeling, experiment and image analysis to estimate the intrinsic gravitropic and proprioceptive sensitivities of stems, using Arabidopsis as an example.

Short term UV-B radiation mediated modulation of physiological traits and withanolides production in Withania coagulans (L.) Dunal under in-vitro condition.

Accumulation of secondary metabolites is a key process in the growth and development of plants under different biotic/abiotic constraints. Many studies highlighted the regulatory potential of UV-B treatment towards the secondary metabolism of plants. In the present study, we examined the impact of UV-B on the physiology and secondary metabolism of Withania coagulans, which is an important ayurvedic plant with high anti-diabetic potential. Results showed that in-vitro UV-B exposure negatively influenced chlorophyll content and photosynthetic machinery. However, Fv/Fm ratio was found non-significantly altered up to 3 h UV-B exposure. The maximum lipid peroxidation level was recorded with 46.8% higher malondialdehyde content in the plants supplemented with 5 h UV-B radiation, that was indicated the oxidative stress in W. coagulans. Conversely, UV-B treatment significantly increased the plant's stress protective compounds like carotenoids, anthocyanin, phenol and proline, in W. coagulans. Free radical scavenging activity was also significantly increased ~ 18% than the control with 3 h UV-B treatment. The maximum antioxidative enzymes activities were observed with the short-term (up to 3 h) UV-B treatment. Specifically, UV-B radiation exposure significantly increased the content of withaferin A and withanolide A in W. coagulans with maximum 1.38 and 3.42-folds, respectively. Additionally, withanolides biosynthesis related genes transcript levels were found over-expressed under the response of UV-B elicitation. The acquired results suggested that short-term UV-B supplementation triggers secondary metabolism along with combating oxidative stress via improving the antioxidative defense system in W. coagulans. Also, UV-B can be used as an efficient abiotic elicitor to increase pharmaceutical compounds (withanolides) production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01046-7.

In vitro Regeneration of Clematis Plants in the Nikita Botanical Garden via Somatic Embryogenesis and Organogenesis.

The effects of growth regulators, namely, 6-benzylaminopurine (BAP) and thidiazuron (TDZ), on the morphogenic capacity of 13 cultivars of clematis plants, in terms of their morphological structure formation, shoot regeneration, and somatic embryo development, are presented. The clematis cultivars 'Alpinist,' 'Ay-Nor,' 'Bal Tsvetov,' 'Crimson Star,' 'Crystal Fountain,' 'Kosmicheskaya Melodiya,' 'Lesnaya Opera,' 'Madame Julia Correvon,' 'Nevesta,' 'Nikitsky Rosovyi,' 'Nikolay Rubtsov,' 'Serenada Kryma,' and 'Vechniy Zov' were taken in collection plots of the Nikita Botanical Gardens for use in study. After explant sterilization with 70% ethanol (1 min), 0.3-0.4% Cl2 (15 min), and 1% thimerosal (10 min), 1-cm long segments with a single node were introduced to an in vitro culture. The explants were established on the basal MS medium supplemented with BAP (2.20-8.90 µM) and 0.049 µM NAA, or TDZ (3.0; 6.0, and 9.0 µM) with 30 g/L sucrose and 9 g/L agar. The medium with 0.89 µM BAP served as the control. Culture vessels and test tubes with the explants were maintained in plant growth chamber-controlled conditions: with a 16-h photoperiod, under cool-white light fluorescent lamps with a light intensity of 37.5 µmol m-2 s-1, at a temperature of 24 ± 1°C. Histological analysis demonstrated that adventitious bud and somatic embryo formation in studied clematis cultivars occurred at numerous areas of active meristematic cell zones. The main role of plant growth regulators and its concentrations were demonstrated. It was determined that maximum adventitious microshoot regeneration without any morphological abnormalities formed on the media supplemented with BAP or TDZ. 4.40 µM BAP, or 6.0 µM TDZ were optimal cytokinin concentrations for micropropagation. The explants of 'Alpinist,' 'Ay-Nor,' 'Crimson Star,' 'Crystal Fountain,' 'Nevesta,' and 'Serenada Kryma' cultivars displayed high morphogenetic capacity under in vitro culturing. During indirect somatic embryogenesis, light intensity 37.5 µmol m-2 s-1 stimulated a higher-number somatic embryo formation and a temperature of 26°C affected somatic embryo development. Active formation of primary and secondary somatic embryos was also demonstrated. 2.20 µM BAP with 0.09 µM IBA affected the high-number somatic embryo formation for eight cultivars. Secondary somatic embryogenesis by the same concentration of BAP was induced. The frequency of secondary somatic embryogenesis was higher in 'Crystal Fountain' (100%), 'Crimson Star' (100%), 'Nevesta' (97%), and 'Ay-Nor' (92%) cultivars. Based on these results, the methodology for direct somatic embryogenesis and organogenesis of studied clematis cultivars has been developed.

The Effect of Zinc, Copper, and Silver Ions on Oat (Avena sativa L.) Androgenesis.

Oat (Avena sativa L.) cultivars 'Bingo' and 'Chwat' were used to compare the embryogenesis competence of another culture. Despite the embryo-like structures obtained from both tested cultivars, only 'Chwat' produced green plantlets, which confirmed the cultivar dependency. 'Chwat' produced the highest number of embryo-like structures and green plantlets (0.7/100 anthers and 0.1/100 anthers, respectively). The embryo-like structure formation also depended on cold pretreatment combined with Cu2+, Zn2+, or Ag+ ion supplementation, which was applied during the tiller pretreatment or added to the induction media. The highest number of embryo-like structures (2.1/100 anthers) were observed on anthers derived from the tillers kept in a 50% Hoagland medium with the addition of 10 µM of CuSO4. In turn, the induction media supplemented with the ions Cu2+, Zn2+, or Ag+ increased neither the number of embryo-like structures nor the green plantlet production compared to the control conditions. However, such ion applications turned out to be most effective when the induction medium was enriched with 25 µM of AgNO3 and left to obtain the highest number of embryo-like structures and green plantlets (0.8/100 anthers and 0.2/100 anthers, respectively). Therefore, more attention should be paid to the possibilities of adjusting the media nutrient composition, as this may be the only way to significantly increase the efficiency of this method.

Melatonin promotes adventitious root formation in apple by promoting the function of MdWOX11.

BACKGROUND: Melatonin (MT) is important for plant growth and development; however, it is not known whether MT is involved in apple adventitious root (AR) development. In this study, we treated Malus prunifolia (MP) at four different stages of AR development, and analyzed the level of the endogenous hormones MT, auxin (IAA), zeatin-riboside (ZR), abscisic acid (ABA), and gibberellins (GA1 + 3) in all four treatment groups and the untreated control group. The expression of MT, IAA biosynthesis, transport and signal transduction, the cell cycle, and root development related genes were quantified by RT-qPCR. The function of MdWOX11 was analyzed in transgenic apple plants. RESULTS: The promotion of AR development by MT was dependent on the stage of AR induction between 0 and 2 d in apple rootstocks. MT-treatment increased the level of IAA and crosstalk existed between MT and IAA during AR formation. The expression of MdWOX11 was induced by MT treatment and positively regulated AR formation in apple. Furthermore, transgenic lines that overexpressed MdWOX11 lines produced more ARs than 'GL3'. Phenotypic analysis indicated that MdWOX11 overexpression lines were more sensitive to exogenous MT treatment than 'GL3', suggesting that MdWOX11 regulates AR formation in response to MT in apple rootstock. CONCLUSIONS: MT promotes AR formation mainly during the AR induction stage by inducing IAA levels and upregulating MdWOX11.

Influence of Continuous Spectrum Light on Morphological Traits and Leaf Anatomy of Hazelnut Plantlets.

Light spectra influence growth, development, and quality of plants and seedlings, that is one of the main aspects engaging the interests of private and public researchers and nursery industries. Propagation of hazelnut (Corylus avellana L.), which in the past has been held in low consideration because of the widespread use of rooted suckers directly collected in the field, today is taking on increasing interest due to the strong expansion of hazelnut cultivation. In order to improve the quality of plants and seedlings in greenhouse acclimatization, the effects of light emitting diodes (LED) lights during the ex vitro growth of two hazelnut cultivars (Tonda di Giffoni and Tonda Gentile Romana) were investigated. Plantlets were maintained in a growth chamber and exposed to three different continuous spectrum LED systems as a primary source of illumination and to fluorescent lamps used as control. LEDs differed in the percentage of some wavelength ranges in the spectrum, being AP673L rich in green and red wavelengths, NS1 in blue and green light, G2 in red and far red wavelengths. After a 4-week experimental period, morphometric, biochemical, and histological analyses were carried out. Shoot and leaf growths were influenced by LEDs more than by fluorescent lamps in both cultivars. G2 positively affected biomass increment more than the other LEDs, by inducing not only cell elongation (increase in shoot length, new internodes length, leaf area) but also cell proliferation (increase in new node number). G2 exposure had negative effects on total chlorophyll content but positively affected synthesis of flavonoids in both varieties; therefore, plants grown under this LED showed the lowest nitrogen balance index. Leaf morpho-anatomical analyzed traits (thickness, palisade cell height, number of chloroplasts, number of palisade cells), were influenced especially by G2 and, to a less extent, by NS1 light. Significant differences in some parameters were observed between the two cultivars in response to a same light source. The results obtained underline the importance of light modulation for hazelnut, providing useful information for ex vitro growth of hazelnut plantlets.

Green Bio-Assisted Synthesis, Characterization and Biological Evaluation of Biocompatible ZnO NPs Synthesized from Different Tissues of Milk Thistle (Silybum marianum).

The purpose of the current study was green synthesis of ZnO-nanoparticles (NPs) from different tissues of Silybum marianum (L.) Gaernt. (i.e., seeds, wild plant, in vitro derived plantlets and callus cultures) followed by extensive characterization and evaluation of their biological potency. ZnO-NPs thus synthesized were subjected to characterization using standard techniques such as XRD, FTIR and SEM. Thermal stability of synthesized NPs was also evaluated using thermo-gravimetric analysis. Highly stable crystalline NPs with size ranging between 30.8 and 46.0 nm were obtained from different tissues of S. marianum. These NPs have revealed a wide range of biological applications showing antioxidant, moderate α-amylase inhibitor, antibacterial and cytotoxic potencies. The highest antibacterial activity (20 ± 0.98 mm) was shown by seed extract-mediated ZnO NPs against Staphylococcus aureus (ATCC-6538). Seed extract-mediated ZnO NPs also showed the most potent antioxidant activity (27.7 ± 0.9 µgAAE/mg, 23.8 ± 0.7 µgAAE/mg and 12.7 ± 1.9% total antioxidant capacity (TAC), total reducing power (TRP) and DPPH-free radical scavenging assay (FRSA), respectively). All of the synthesized ZnO NPs also showed cytotoxic activity against the hepato-cellular carcinoma (HepG2) human cells. Interestingly, these ZnO NPs were also highly biocompatible, as evidenced by the brine shrimp lethality and human red blood cells hemolytic assays. Among all of the NPs synthesized and used, the effect of seed extract-mediated NPs was found to be most promising for future applications.

De novo in vitro shoot morphogenesis from shoot tip-induced callus cultures of Gymnema sylvestre (Retz.) R.Br. ex Sm.

BACKGROUND: Gymnema sylvestre is a medicinal woody perennial vine known for its sweetening properties and anti-diabetic therapeutic uses in the modern and traditional medicines. Its over-exploitation for the therapeutic uses and to meet the demand of pharmaceutical industry in raw materials supply for the production of anti-diabetic drugs has led to considerable decline in its natural population. RESULTS: An efficient system of shoot bud sprouting from nodal segment explants and indirect plant regeneration from apical meristem-induced callus cultures of G. sylvestre have been developed on Murashige and Skoog (MS) medium amended with concentrations of cytokinins. Of the three growth regulators tested, N6-benzylaminopurine (BAP) was the most efficient and 2.0 mg L-1 gave the best shoot formation efficiency. This was followed by thidiazuron (TDZ) and kinetin (Kin) but, most of the TDZ-induced micro shoots showed stunted growth. Multiple shoot formation was observed on medium amended with BAP or TDZ at higher concentrations. The produced micro shoots were rooted on half strength MS medium amended with auxins and rooted plantlets acclimatized with 87% survival of the regenerates. CONCLUSIONS: The developed regeneration system can be exploited for genetic transformation studies, particularly when aimed at producing its high yielding cell lines for the anti-diabetic phytochemicals. It also offers opportunities for exploring the expression of totipotency in the anti-diabetic perennial vine.