Engineering the plant metabolic system by exploiting metabolic regulation.

Plants are the most sophisticated biofactories and sources of food and biofuels present in nature. By engineering plant metabolism, the production of desired compounds can be increased and the nutritional or commercial value of the plant species can be improved. However, this can be challenging because of the complexity of the regulation of multiple genes and the involvement of different protein interactions. To improve metabolic engineering (ME) capabilities, different tools and strategies for rerouting the metabolic pathways have been developed, including genome editing and transcriptional regulation approaches. In addition, cutting-edge technologies have provided new methods for understanding uncharacterized biosynthetic pathways, protein degradation mechanisms, protein-protein interactions, or allosteric feedback, enabling the design of novel ME approaches.

The factors affecting the development of medicinal plants from a value chain perspective.

MAIN CONCLUSION: From a value chain perspective, this paper examines the important factors from the selection of planting areas to storage, which restrict the development of medicinal plants. The purpose of this paper is to provide theoretical basis for the sustainable development of medicinal plants. Medicinal plants have significant economic and medicinal value. Due to the gradual depletion of wild medicinal plant resources, cultivators of medicinal plants must resort to artificial cultivation to cope. However, there are still many problems in the production process of medicinal plants, resulting in decreases in both yield and quality, thus hindering sustainable development. To date, research on the value chain of medicinal plants is still limited. Therefore, this paper analyzes the factors affecting the development of medicinal plants from the perspective of the value chain, including the selection of growing areas to the storage process of medicinal plants, and summarizes the challenges faced in the production process of medicinal plants. The purpose of this paper is to provide theoretical basis for the sustainable development of medicinal plants.

Stomatal evolution and plant adaptation to future climate.

Global climate change is affecting plant photosynthesis and transpiration processes, as well as increasing weather extremes impacting socio-political and environmental events and decisions for decades to come. One major research challenge in plant biology and ecology is the interaction of photosynthesis with the environment. Stomata control plant gas exchange and their evolution was a crucial innovation that facilitated the earliest land plants to colonize terrestrial environments. Stomata couple homoiohydry, together with cuticles, intercellular gas space, with the endohydric water-conducting system, enabling plants to adapt and diversify across the planet. Plants control stomatal movement in response to environmental change through regulating guard cell turgor mediated by membrane transporters and signaling transduction. However, the origin, evolution, and active control of stomata remain controversial topics. We first review stomatal evolution and diversity, providing fossil and phylogenetic evidence of their origins. We summarize functional evolution of guard cell membrane transporters in the context of climate changes and environmental stresses. Our analyses show that the core signaling elements of stomatal movement are more ancient than stomata, while genes involved in stomatal development co-evolved de novo with the earliest stomata. These results suggest that novel stomatal development-specific genes were acquired during plant evolution, whereas genes regulating stomatal movement, especially cell signaling pathways, were inherited ancestrally and co-opted by dynamic functional differentiation. These two processes reflect the different adaptation strategies during land plant evolution.

Interplay between Livestock Grazing and Aridity on the Ecological and Nutritional Value of Forage in Semi-arid Mediterranean Rangelands (NE Spain).

Rangeland-based livestock production constitutes a primary source of livelihood for many inhabitants of dryland regions. Their subsistence relies heavily on maintaining the productivity, biodiversity and services of these ecosystems. Harsh environmental conditions (e.g., drought) combined with land use intensification (e.g., overgrazing) make dryland ecosystems vulnerable and prone to degradation. However, the interplay between livestock grazing intensity and aridity conditions in driving the conservation and nutritional value of forage in arid and semi-arid rangelands is still not fully understood. In this study, we performed structural equation models (SEM) to assess the simultaneous direct and indirect effects of livestock grazing intensity and aridity level on community structure, diversity, biomass, forage production, forage C:N ratio and forage fiber composition in two semi-arid Mediterranean rangelands, NE Spain. Not surprisingly, we found that higher livestock grazing intensity led to lower community plant cover, especially when combined with higher aridity. However, both increasing grazing intensity and aridity were associated with higher forage production after one year of grazing exclusion. We did not find any adverse effect of livestock grazing on plant diversity, although plant species composition differed among grazing intensity levels. On the other hand, we found an aridity-driven trade-off in regard of the nutritional value of forage. Specifically, higher aridity was associated with a decrease in the least digestible fiber fraction (i.e., lignin) and an increase in forage C:N ratio. More interestingly, we found that livestock grazing modulated this trade-off by improving the overall forage nutritional value. Altogether, our results provide further insights into the management of semi-arid Mediterranean rangelands, pointing out that maintaining traditional rangeland-based livestock production may be a sustainable option as long as rangeland conservation (e.g., community plant cover) is not severely compromised.

Enhancing soil health and nutrient availability for Carrizo citrange (X Citroncirus sp.) through bokashi and biochar amendments: An exploration into indoor sustainable soil ecosystem management.

This study investigated the efficacy of organic soil amendments: bokashi (Bok), biochar (BC), and their combination (Bok_BC) in promoting soil health, nutrient availability, and growth of Carrizo citrange (X Citroncirus sp. Rutaceae, Parentage Citrus sinensis × Poncirus trifoliata) under indoor greenhouse settings. Results indicate significant alterations in soil parameters like total carbon (C), total nitrogen (N), and C:N ratio due to Bok, BC, and Bok_BC treatments. BC treatments boosted total C, while Bok increased total N, compared to controls. A note-worthy 25 % average decrease in C:N ratio was observed with Bok and Bok_BC, nearing the optimal 24:1 C:N for microbial growth. This highlights the potential of waste by-products in balancing nutrient release to benefit soil health and plant development. Analysis of nitrite (NO2-), nitrate (NO3-), and ammonium (NH4-N) levels revealed a dynamic relationship between soil treatments and time. Bok and Bok_BC amendments combined with both fertilizer doses [700 and 1400 Electrical Conductivity, EC] showed an initial NH4-N spike (averaging 1513 and 1288 µg N/g dry, respectively), outperforming control soils (average 503 µg N/g dry). Other key elements like phosphorus, potassium, calcium, and chlorine also experienced initial surges in Bok and Bok_BC soils before declining, suggesting a gradual nutrient release. The concentration of potentially toxic elements remained mostly stable or inconclusive, warranting further exploration. Bok, BC, and Bok_BC treatments considerably influenced germination rate and plant growth. The germination rate averaged 24.2 %, 23 %, and 22.5 % for Bok, BC, and Bok_BC, compared to the 15.9 % control. Plant height increased with Bok, BC, and Bok_BC to 18.4 cm, 18.7 cm, and 16.4 cm, respectively, from the 14.8 cm control. The results remained consistent across fertilizer doses, emphasizing the soil amendments' role in bolstering soil and plant health. In summary, the research underscores the potential of carbon-based amendments like bokashi and biochar in enhancing soil health, reducing reliance on synthetic fertilizers, and fostering sustainable soil ecosystems. The insights are pivotal for advancing sustainable agriculture in indoor greenhouse settings for nursery plant production.

Litter and soil biodiversity jointly drive ecosystem functions.

The decomposition of litter and the supply of nutrients into and from the soil are two fundamental processes through which the above- and belowground world interact. Microbial biodiversity, and especially that of decomposers, plays a key role in these processes by helping litter decomposition. Yet the relative contribution of litter diversity and soil biodiversity in supporting multiple ecosystem services remains virtually unknown. Here we conducted a mesocosm experiment where leaf litter and soil biodiversity were manipulated to investigate their influence on plant productivity, litter decomposition, soil respiration, and enzymatic activity in the littersphere. We showed that both leaf litter diversity and soil microbial diversity (richness and community composition) independently contributed to explain multiple ecosystem functions. Fungal saprobes community composition was especially important for supporting ecosystem multifunctionality (EMF), plant production, litter decomposition, and activity of soil phosphatase when compared with bacteria or other fungal functional groups and litter species richness. Moreover, leaf litter diversity and soil microbial diversity exerted previously undescribed and significantly interactive effects on EMF and multiple individual ecosystem functions, such as litter decomposition and plant production. Together, our work provides experimental evidence supporting the independent and interactive roles of litter and belowground soil biodiversity to maintain ecosystem functions and multiple services.

Generation of unequal nuclear genotype proportions in Rhizophagus irregularis progeny causes allelic imbalance in gene transcription.

Arbuscular mycorrhizal fungi (AMF) form mutualisms with most plant species. The model AMF Rhizophagus irregularis is common in many ecosystems and naturally forms homokaryons and dikaryons. Quantitative variation in allele frequencies in clonally dikaryon offspring suggests they disproportionately inherit two distinct nuclear genotypes from their parent. This is interesting, because such progeny strongly and differentially affect plant growth. Neither the frequency and magnitude of this occurrence nor its effect on gene transcription are known. Using reduced representation genome sequencing, transcriptomics, and quantitative analysis tools, we show that progeny of homokaryons and dikaryons are qualitatively genetically identical to the parent. However, dikaryon progeny differ quantitatively due to unequal inheritance of nuclear genotypes. Allele frequencies of actively transcribed biallelic genes resembled the frequencies of the two nuclear genotypes. More biallelic genes showed transcription of both alleles than monoallelic transcription, but biallelic transcription was less likely with greater allelic divergence. Monoallelic transcription levels of biallelic genes were reduced compared with biallelic gene transcription, a finding consistent with genomic conflict. Given that genetic variation in R. irregularis is associated with plant growth, our results establish quantitative genetic variation as a future consideration when selecting AMF lines to improve plant production.

Promotion of Zinc Tolerance, Acquisition and Translocation of Phosphorus in Mimosa pudica L. Mediated by Arbuscular Mycorrhizal Fungi.

Heavy metal contamination of soil is of increasing concern because of its potential risk to human health. In this study, two AMFs (Rhizophagus intraradices and Funneliformis mosseae) substantially increased the biomass of bashfulgrass in Zn-contaminated soil, even at Zn levels of up to 600 mg kg-1. Zn uptake in R. intraradices- and F. mosseae-mycorrhizal bashfulgrass was increased by 40-fold and 7-fold, respectively, when plants grown in Zn-contaminated (400 mg kg-1) soil. Elemental analysis showed that neither AMF had an effect on Zn concentration in plant tissues, including the roots and shoots. However, a significant increase of phosphorus (P) concentration was observed, suggesting the increased is from the improved use efficiency of soil nutrients by AMFs. Comparing the two AMFs, better growth performance with more biomass occurred with R. intraradices-inoculated bashfulgrass in Zn-contaminated soil. This is consistent with R. intraradices being more tolerant to Zn than F. mosseae, indicated by a higher colonization percentage in bashfulgrass roots. Taken together, our data indicate that AMFs possibly improve acquisition and translocation of P to promote increased biomass. Moreover, mycorrhiza did not enhance Zn accumulation in shoots and roots of bashfulgrass at the same Zn level. In the future, developing AMF (especially R. intraradices) inoculation of plants might be a desirable means of safe production of ornamental plants in metal-polluted soil.

Metabolic engineering for the production of fat-soluble vitamins: advances and perspectives.

Fat-soluble vitamins are vitamins that are insoluble in water, soluble in fat, and organic solvents; they are found in minute amount in various foods. Fat-soluble vitamins, including vitamins A, D, E, and K, have been widely used in food, cosmetics, health care products, and pharmaceutical industries. Fat-soluble vitamins are currently produced via biological and chemical synthesis. In recent years, fat-soluble vitamin production by biotechnological routes has been regarded as a very promising approach. Based on biosynthetic pathways, considerable advances of α-tocopherol and ß-carotenes have been achieved in transgenic plants and microalgae. Microbial fermentation, as an alternative method for the production of vitamin K and ß-carotenes, is attracting considerable attention because it is an environment friendly process. In this review, we address the function and applications of fat-soluble vitamins, and an overview of current developments in the production of fat-soluble vitamins in transgenic plants, microalgae, and microorganisms. We focus on the metabolic and process engineering strategies for improving production of fat-soluble vitamins, and we hope this review can be useful for the people who are interested in the production of fat-soluble vitamins by biotechnological routes.

Optimized production of a biologically active Clostridium perfringens glycosyl hydrolase phage endolysin PlyCP41 in plants using virus-based systemic expression.

BACKGROUND: Clostridium perfringens, a gram-positive, anaerobic, rod-shaped bacterium, is the third leading cause of human foodborne bacterial disease and a cause of necrotic enteritis in poultry. It is controlled using antibiotics, widespread use of which may lead to development of drug-resistant bacteria. Bacteriophage-encoded endolysins that degrade peptidoglycans in the bacterial cell wall are potential replacements for antibiotics. Phage endolysins have been identified that exhibit antibacterial activities against several Clostridium strains. RESULTS: An Escherichia coli codon-optimized gene encoding the glycosyl hydrolase endolysin (PlyCP41) containing a polyhistidine tag was expressed in E. coli. In addition, The E. coli optimized endolysin gene was engineered for expression in plants (PlyCP41p) and a plant codon-optimized gene (PlyCP41pc), both containing a polyhistidine tag, were expressed in Nicotiana benthamiana plants using a potato virus X (PVX)-based transient expression vector. PlyCP41p accumulated to ~ 1% total soluble protein (100µg/gm f. wt. leaf tissue) without any obvious toxic effects on plant cells, and both the purified protein and plant sap containing the protein lysed C. perfringens strain Cp39 in a plate lysis assay. Optimal systemic expression of PlyCP41p was achieved at 2 weeks-post-infection. PlyCP41pc did not accumulate to higher levels than PlyCP41p in infected tissue. CONCLUSION: We demonstrated that functionally active bacteriophage PlyCP41 endolysin can be produced in systemically infected plant tissue with potential for use of crude plant sap as an effective antimicrobial agent against C. perfringens.

Regulation of antioxidant production, ion uptake and productivity in potato (Solanum tuberosum L.) plant inoculated with growth promoting salt tolerant Bacillus strains.

The exchangeable sodium (Na+) in salt affected soils is a major constraint in potassium (K+) availability to plants that disturb ion transport and inhibit plant growth, adversely. Salt tolerant plant growth promoting rhizobacteria (PGPR) may regulate the Na+/K+ efflux and increase K+ uptake by the plant from the soil. Therefore, a pot study was performed to examine the effect of salt tolerant PGPR Bacillus sp. alone and in consortium, on antioxidant enzyme activity, ion uptake and potato (Solanum tuberosum L.) tuber yield in normal and salt affected soils. We observed that Bacillus sp. (strains SR-2-1 and SR-2-1/1) solubilized insoluble phosphorous and produced indole-3-acetic acid while only SR-2-1/1 produced ACC deaminase in culture medium supplemented with various concentrations of NaCl (0-6%). In the pot experiment, the consortium treatment of strains was found to increase relative leaf water contents whereas decreased the electrolyte leakage and antioxidant enzyme activity both in normal and salt affected soils. Similarly, consortium treatment decreased Na+ whereas increased K+, Ca+2, K+/Na+ and Ca+2/Na+ in plant dry matter in both soils. It has been investigated that inoculation of PGPR significantly (p < 0.05) increased plant biomass, number of tubers per plant and tuber weight as compared to un-inoculated plants in both soils. In addition, PGPR inoculation enhanced auxin production in root exudates of young potato plants and bacterial population dynamics in both soils. Na+ ion regulation (R2 = 0.95) and tuber weight (R2 = 0.90) in salt affected soil were significantly correlated with auxin production in the rhizosphere. Results of this study conferred that consortium of Bacillus strains (SR-2-1, SR-2-1/1) enhanced auxin production in the rhizosphere of potato plants and that ultimately regulated antioxidant enzyme production and uptake of Na+, K+ and Ca+2 in potato plants resulted into a higher tuber yield in both normal and salt affected soils.

Characterization of biomass sorghum for copper phytoremediation: photosynthetic response and possibility as a bioenergy feedstock from contaminated land.

In order to decrease the concentration of toxic metals in contaminated lands, phytoextraction can be suitable considering the use of plant species with high potential for biomass production, such as biomass sorghum (Sorghum bicolor L.). We assessed a biomass sorghum (BRS716) potential as a copper phytoextractor as well as the physiological stability under this stressful condition. A completely randomized experimental design was used for a greenhouse experiment in which sorghum plants were submitted to a range of Cu2+ concentrations: 2.3, 10.9, 19.6, 30.5, 37.6 and 55.6 mg dm-3. The plant growth was not affected by increasing Cu2+ concentrations, suggesting that this species is tolerant to copper. There was a decrease in photosynthetic rate according to the increase in Cu2+ concentration, but not at a level that could disturb plant metabolism and eventual death. The values obtained for transfer index ranged from 0.62 to 0.11 which evidenced the restriction of Cu2+ transport to the aerial parts. The more Cu2+ available in soil, the smaller the amount of Cu2+ transported to aerial parts of sorghum. So, our results show that biomass sorghum has potential to be used for Cu2+ phytoextraction in concentration of up to 20 mg dm-3. Also, in heavily Cu2+ polluted sites, it can be used to produce biomass for bioenergy purpose, promoting a low rate of Cu2+ extraction.

Expression of Rift Valley fever virus N-protein in Nicotiana benthamiana for use as a diagnostic antigen.

BACKGROUND: Rift Valley fever virus (RVFV), the causative agent of Rift Valley fever, is an enveloped single-stranded negative-sense RNA virus in the genus Phlebovirus, family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing abortion storms in pregnant ruminants, high neonatal mortality in animals, and morbidity and occasional fatalities in humans. The disease is endemic in parts of Africa and the Arabian Peninsula, but is described as emerging due to the wide range of mosquitoes that could spread the disease into non-endemic regions. There are different tests for determining whether animals are infected with or have been exposed to RVFV. The most common serological test is antibody ELISA, which detects host immunoglobulins M or G produced specifically in response to infection with RVFV. The presence of antibodies to RVFV nucleocapsid protein (N-protein) is among the best indicators of RVFV exposure in animals. This work describes an investigation of the feasibility of producing a recombinant N-protein in Nicotiana benthamiana and using it in an ELISA. RESULTS: The human-codon optimised RVFV N-protein was successfully expressed in N. benthamiana via Agrobacterium-mediated infiltration of leaves. The recombinant protein was detected as monomers and dimers with maximum protein yields calculated to be 500-558 mg/kg of fresh plant leaves. The identity of the protein was confirmed by liquid chromatography-mass spectrometry (LC-MS) resulting in 87.35% coverage, with 264 unique peptides. Transmission electron microscopy revealed that the protein forms ring structures of ~ 10 nm in diameter. Preliminary data revealed that the protein could successfully differentiate between sera of RVFV-infected sheep and from sera of those not infected with the virus. CONCLUSIONS: To the best of our knowledge this is the first study demonstrating the successful production of RVFV N-protein as a diagnostic reagent by Agrobacterium-mediated transient heterologous expression in N. benthamiana. Preliminary testing of the antigen showed its ability to distinguish RVFV-positive animal sera from RVFV negative animal sera when used in an enzyme linked immunosorbent assay (ELISA). The cost-effective, scalable and simple production method has great potential for use in developing countries where rapid diagnosis of RVFV is necessary.

Potential use and perspectives of nitric oxide donors in agriculture.

Nitric oxide (NO) has emerged in the last 30 years as a key molecule involved in many physiological processes in plants, animals and bacteria. Current research has shown that NO can be delivered via donor molecules. In such cases, the NO release rate is dependent on the chemical structure of the donor itself and on the chemical environment. Despite NO's powerful signaling effect in plants and animals, the application of NO donors in agriculture is currently not implemented and research remains mainly at the experimental level. Technological development in the field of NO donors is rapidly expanding in scope to include controlling seed germination, plant development, ripening and increasing shelf-life of produce. Potential applications in animal production have also been identified. This concise review focuses on the use of donors that have shown potential biotechnological applications in agriculture. Insights are provided into (i) the role of donors in plant production, (ii) the potential use of donors in animal production and (iii) future approaches to explore the use and applications of donors for the benefit of agriculture. © 2016 Society of Chemical Industry.

[Nutrient transfer and growth of Pinus greggii Engelm. inoculated with edible ectomycorrhizal mushrooms in two substrates]. / Transferencia de nutrientes y crecimiento de Pinus greggii Engelm. inoculado con hongos comestibles ectomicorrícicos en dos sustratos.

An ectomycorrhiza is a mutualistic symbiosis of paramount importance in forestry and tree production. One of the selection criteria of ectomycorrhizal fungi that has currently gained importance is their edibility due to the economic, ecological and cultural relevance of edible ectomycorrhizal mushrooms as a non-timber forest product. The effect of the inoculation with three edible ectomycorrhizal mushrooms: Laccaria laccata, Laccaria bicolor y Hebeloma leucosarx, which are widely sold in Mexico, on the growth and nutrient contents of Pinus greggii grown in an experimental substrate and a commercial substrate enriched with a slow-release fertilizer, was evaluated. Two years after sowing, differences in terms of shoot and root biomass and macro and micronutrient contents between inoculated and non-inoculated plants, were recorded independently of the fungal species and the substrate. Despite the fact that plants grown in the commercial substrate had higher growth and nutrient contents, their ectomycorrhizal colonization percentages were smaller than those of the plants grown in the experimental substrate. The differences in the nutrient transfer to the inoculated plant shoots among the evaluated fungal species were recorded. Ca mobilization by L. laccata, Na by L. bicolor and Mn by H. leucosarx were observed in the plants growing in the experimental substrate. It has been demonstrated that the selection of substrates constitutes an important factor in the production of ectomycorrhizal plants and that the three evaluated species of edible ectomycorrhizal mushrooms have an enormous potential in the controlled mycorrhization of P. greggii.

Alteration of the phenology of leaf senescence and fall in winter deciduous species by climate change: effects on nutrient proficiency.

Leaf senescence in winter deciduous species signals the transition from the active to the dormant stage. The purpose of leaf senescence is the recovery of nutrients before the leaves fall. Photoperiod and temperature are the main cues controlling leaf senescence in winter deciduous species, with water stress imposing an additional influence. Photoperiod exerts a strict control on leaf senescence at latitudes where winters are severe and temperature gains importance in the regulation as winters become less severe. On average, climatic warming will delay and drought will advance leaf senescence, but at varying degrees depending on the species. Warming and drought thus have opposite effects on the phenology of leaf senescence, and the impact of climate change will therefore depend on the relative importance of each factor in specific regions. Warming is not expected to have a strong impact on nutrient proficiency although a slower speed of leaf senescence induced by warming could facilitate a more efficient nutrient resorption. Nutrient resorption is less efficient when the leaves senesce prematurely as a consequence of water stress. The overall effects of climate change on nutrient resorption will depend on the contrasting effects of warming and drought. Changes in nutrient resorption and proficiency will impact production in the following year, at least in early spring, because the construction of new foliage relies almost exclusively on nutrients resorbed from foliage during the preceding leaf fall. Changes in the phenology of leaf senescence will thus impact carbon uptake, but also ecosystem nutrient cycling, especially if the changes are consequence of water stress.

Target and non-target effects of insecticide use during ornamental milkweed production.

There are widespread public efforts to conserve wildlife in urbanized landscapes via the installation of nursery-grown plants that support Lepidoptera taxa. Insecticides are commonly used during nursery production to suppress key plant pests, and many products have extended periods of toxicity and affect a wide range of herbivore taxa. While there are plentiful toxicological data on bee species, predominantly the Western honey bee (Apis mellifera L.), little is known about how insecticides affect nonpest lepidopterans. Lepidoptera has different modes of exposure (e.g., leaf-feeding) and differences in susceptibility to insecticide target sites compared to bees. Consequently, many products compatible with bee conservation pose an uncertain risk to nonpest lepidopterans and thus may represent an under-recognized conflict with conservation efforts. Using the monarch butterfly (Danaus plexippus, L.), tropical milkweed (Asclepias curassavica, L.), and oleander aphid (Aphis nerii, Fonscolombe, 1841) system, we conducted leaf and whole-plant feeding assays to evaluate effects of acute and chronic monarch exposure to industry standard and alternative reduced-risk insecticides used during nursery production. We also evaluated the efficacy of these insecticides against their target pest, the oleander aphid. Our results indicate that insecticides used to control pests on ornamental milkweed can cause monarch larval mortality up to 4 wk after treatment application. Furthermore, the duration of aphid suppression is often shorter than the duration of adverse effects on monarchs. This study demonstrates a conflict between insect pest management and Lepidoptera conservation during ornamental plant production and has implications for the conservation value of ornamentals after retail sale.

Diversity of Treatments in Overcoming Morphophysiological Dormancy of Paeonia peregrina Mill. Seeds.

Paeonia peregrina Mill. is a protected, herbaceous species native to Southeastern Europe and Turkey. Due to its vulnerability, it has to be protected both in its natural habitats and through cultivation. Peonies are known to have a low potential for natural regeneration due to their seed dormancy, low germination rate, and long germination period. In this study, treatments with gibberellic acid (GA3 150, 200, 250, 300, and 350 mg L-1 GA3) and warm (at 20/16 °C day/night regime) and cold stratification (at 4 °C) were used to accelerate dormancy release and increase the germination rate. The seeds of P. peregrina from four natural habitats in Serbia and the Institute's collection were collected and analyzed. They showed an underdeveloped embryo that needs to grow inside the seed before it can germinate. The application of GA3 accelerated each stage of germination (seed coat rapturing, hypocotyl dormancy release, and epicotyl dormancy release) for approximately 10 days compared to the control. It was also found that two-day imbibition with 200 mg L-1 GA3 significantly accelerated and equalized germination. Higher GA3 concentrations had a more pronounced impact on each stage but also resulted in greater seed infection after the seed coat rupture, elongated and weak seedlings, while lower concentrations did not result in obtaining uniform seedlings. There were no significant differences observed between localities. Restoring P. peregrina through seeds and nursery-produced plants is crucial for conserving the genetic diversity of the tested species.

Responses of Pea (Pisum sativum L.) to Single and Consortium Bio-Fertilizers in Clay and Newly Reclaimed Soils.

The huge development of climatic change highly affects our crop production and soil fertility. Also, the rise in the uncontrolled, excessive use of chemical fertilizers diminishes the soil prosperity and generates pollutants, threatening all environmental life forms, including us. Replacement of these chemical fertilizers with natural ones is becoming an inevitable environmental strategy. In our study, we evaluated the responses of Pisum sativum L. to the action of single species and consortiums of plant growth-promoting bacteria (Azotobacter chroococcum, Bacillus megaterium, and Bacillus cerkularice) in clay and new reclaimed soil types in terms of phenotype, yield components, and physiological and biochemical responses. Data analysis showed single or consortium microbial inoculation significantly increased the measured traits under clay and calcareous sandy soils compared to the control. Shoot physiological and biochemical activities, and seed biochemical activities were significantly enhanced with the inoculation of pea seeds with three types of bacteria in both soil types. The bud numbers, fresh weight, and seeds' dry weight increased in seeds treated with A. chroococcum and B. megaterium in the sandy soil. Taken together, these findings suggested that the inoculation of plants with PGP bacteria could be used to diminish the implementation of chemical fertilizer and improve the goodness of agricultural products. These findings expand the understanding of the responsive mechanism of microbial inoculation under different soil types, especially at physiological and biochemical levels.