Potential of Picochlorum sp. AZL19 for natural casings waste remediation and biostimulant production for promoting the germination and early growth of dormant seeds of four Origanum species

Formation , germination and dormancy of seeds are important steps in the life cycle of higher plants. The seed is the generative dispersal unit, which enables plants to spread and survive through periods or seasons of less favourable conditions. In agriculture tomato is an important crop and seed companies go through big efforts to deliver uniformly germinating seed batches. Uniform germination of a seed lot does not often come naturally. Seed to seed variation in timing of germination and also dormancy cause non-uniform germination of seed batches. This variation and dormancy of tomato seeds is the subject of the experimental work presented in this thesis. Several molecular and biophysical techniques have been used to expand our knowledge of tomato seed physiology. The firefly luciferase-luciferin system has been used in two distinct techniques to study single tomato seeds. A reporter gene construct consisting of a CaMV 35S promoter and the luciferase gene was introduced in tomato by Agrobacterium mediated transformation (Chapter 2). Transgenic seeds were obtained and imbibed in 0.1 mM luciferin solutions. The expression of the luciferase gene was linked with photon emission from the seeds during germination. Luciferase was expressed in a developmental pattern during germination in all germinating seeds. Luciferase expression increased during germination. Although the expression pattern of luciferase was intrinsically linked with the completion of germination, the luciferase activity of a single seed could not be used as a prediction of the time point of visible germination or of the germination rate of a single seed. This was due to the combination of both a time component and an intrinsic variation in the level of expression. Both primarily and secondarily dormant tomato seeds did not show luciferase activity. This enabled us to distinguish, non destructively, dormant from germinating tomato seeds prior to radicle protrusion and, hence, separation of those seeds for future experiments. Luciferase was also used to visualize distribution of ATP in sections of tomato seeds during dormancy and germination (Chapter 3). It was shown that not the overall ATP level or concentration of a seed was related to germination or dormancy per se , but merely the localised increase of ATP levels in the radicle. Dormant tomato seeds did not show an increase in the level of ATP in the radicle. Germination of seeds starts with the uptake of water and finishes by water uptake by the radicle at the initiation of seedling growth. Water uptake by tomato seeds was studied with the use of NMR-imaging (Chapter 4) . Water uptake resulted in an uneven distribution of water over the seed tissues. The endosperm had higher water content during germination. Radicle protrusion was accompanied by an uptake of extra water, thereby stretching the endosperm outward which resulted in rupture of the endosperm cap, which marked the end of germination. In contrast with the commonly adopted model in which seeds take up extra water only after germination, tomato seeds showed this extra water uptake prior to germination. Linker histones play an important role in the regulation of gene expression by remodelling DNA architecture. Distinct linker histones are thereby under control of different developmental processes in plants. With this in mind we have studied the expression of two different linker histones in tomato, which were originally believed to be under control of either GA or ABA, by the use of reverse-transciptase PCR. ABA and GA are antagonists in the regulation of seed germination and this makes both linker histones excellent candidates to play a role in the regulation of germination and dormancy of tomato seeds (Chapter 5). It was shown that the two different linker histones were differentially expressed in seeds, in relation with dormancy or germination. The linker histones also appeared not to be necessarily under direct control of either GA or ABA. A model is presented in which dormancy and germination are controlled by the linker histones, which, on their turn, are under direct control of phytochrome signal transduction. Expression of the histones may be stimulated or accompanied by ABA or GA.

The use of chemical pesticides to protect agricultural products is a global concern because of their adverse effects on the environment and public health. To avoid the dangers of synthetic herbicides, research has turned to natural alternatives. This study was conducted to evaluate the allelopathic effect of essential oil (EO) extracted from Origanum syriacum, Origanum onites, and Origanum majorana. In addition, the chemical composition of the essential oil was elucidated by gas chromatography and mass spectrometry (GC–MS) analysis. A total of 11 different components of O.syriacum were identified, and the main components were carvacrol (88.49), p-Cymene (5.71), γ-Terpinene (1.63), β-Caryoplhyllene (1.48), and Terpinen-4-ol (0.65), respectively. For O. onites, 10 different compounds were identified, and the main components were carvacrol (58.65), Thymol (30.97), Linalool (4.17), p-Cymene (1.94), and β-Caryoplhyllene (0.98), respectively. Finally, for O. majorana, 14 different compounds were identified, and the main components were carvacrol (40.57), α-Terpineol (29.28), p-Cymene (9.02), γ-Terpinene (5.80), and carvacrol methyl ether (3.46). Finally, 14 compounds from the Origanum majorana species were identified, with carvacrol (40.57), -Terpineol (29.28), p-Cymene (9.02), and -Terpinene (5.80) as the parent compound (3.46). Oxygenated monoterpenes were the highest in all species’ EO content. EOs and plant extracts were tested at 5, 10, and 20 L/Petri concentrations against seed germination and seedling growth in four weed species (Thlaspi arvense, Amaranthus retroflexus, Rumex cripus, and Lactuca serriola). The concentrations of essential oil were set as 5, 10, and 20 µL/Petri dishes for seed germination. In the greenhouse experiment, the final concentration of solutions was set as 20 µL and the solutions were directly sprayed on the surface of the weeds, and the mortality rates were noted after 24 and 48 h of application. It was observed that increasing the application decreased seed germination. The phytotoxic effects on the seedling germination in the greenhouse were observed, resulting in 48.76–94% mortality rates. Consequently, the essential oil from Origanum species could be considered as an alternative bio-herbicide to tested weeds.

In order to investigate the effects of different exogenous hormones on the dormancy and germination of lettuce seeds, this paper soaks the lettuce seeds in ACC, GA, CaCl2 and SNP solutions with different concentrations, respectively, observes the quantity of lettuce seeds germinated, calculates the germination rate, and analyzes the optimum concentrations to break the period of dormancy and promote the germination of seeds to increase the germination rate of seeds. Through the observation of lettuce seeds soaked with ACC, GA, calcium chloride and SNP in different time periods, it is found that ACC, GA, calcium chloride and SNP solutions can all promote the germination of lettuce seeds. Lettuce falls within the Lactuca genus, the Asteraceae family. It likes cold climate and the suitable temperature for growth is 15-20°C. If the temperature exceeds 25°C, the high temperature in the leafy head will cause the necrosis and rot and poor growth of interior leaves (Song et al., 2013). The temperature for germination of seeds ranges between 15-20°C. If the temperature is higher than 25°C, the seed coat will not be able to absorb water, affecting the germination (Li et al., 2012). When lettuce is sowed in summer, it needs to be treated at a low temperature. After being soaked, it should be placed in the refrigerator freezer to accelerate germination and should not be sowed until the sprout turns white. Lettuce is rich in protein, sugar, vitamins, minerals and other nutrients. It is popular among consumers due to its crisp texture, fresh taste and fragrance and rich nutrition (Hu and Zhan, 2014). Few research has been conducted on the effects of exogenous hormones on the germination rate of lettuce seeds. This study takes lettuce seeds from the same source as the test material and conducts tests on the germination rate of lettuce seeds, trying to explore the effects of ACC, GA, CaCl2 and SNP solutions with different concentrations on the germination rate of lettuce seeds so as to improve the germination rate.

Handbook of seed science and technology

Seed dormancy and germination are regulated by complex mechanisms controlled by diverse hormones and environmental cues. Abscisic acid (ABA) promotes seed dormancy and inhibits seed germination and post-germination growth. Calmodulin (CaM) signals are involved with the inhibition of ABA during seed germination and seedling growth. In this study, we showed that Arabidopsis thaliana IQM4 could bind with calmodulin 5 (CaM5) both in vitro and in vivo, and that the interaction was the Ca2+-independent type. The IQM4 protein was localized in the chloroplast and the IQM4 gene was expressed in most tissues, especially the embryo and germinated seedlings. The T-DNA insertion mutants of IQM4 exhibited the reduced primary seed dormancy and lower ABA levels compared with wild type seeds. Moreover, IQM4 plays key roles in modulating the responses to ABA, salt, and osmotic stress during seed germination and post-germination growth. T-DNA insertion mutants exhibited ABA-insensitive and salt-hypersensitive phenotypes during seed germination and post-germination growth, whereas IQM4-overexpressing lines had ABA- and osmotic-hypersensitive, and salt-insensitive phenotypes. Gene expression analyses showed that mutation of IQM4 inhibited the expression of ABA biosynthetic genes NCED6 and NCED9, and seed maturation regulators LEC1, LEC2, ABI3, and ABI5 during the silique development, as well as promoted the expression of WRKY40 and inhibited that of ABI5 in ABA-regulated seed germination. These observations suggest that IQM4 is a novel Ca2+-independent CaM-binding protein, which is positively involved with seed dormancy and germination in Arabidopsis.

Seed Germination Ecophysiology of Meadow Salsify (<i>Tragopogon pratensis</i>) and Western Salsify (<i>T. dubius</i>)

Seed dormancy and germination are regulated by endogenous gene expression as well as hormonal and environmental conditions, such as salinity, which greatly inhibits seed germination. MOTHER OF FT AND TFL1 (MFT), which encodes a phosphatidylethanolamine-binding protein, is a key regulator of seed germination in Arabidopsis thaliana. There are two orthologous genes of AtMFT in rice (Oryza sativa), namely, OsMFT1 and OsMFT2. However, the functions of these two genes in regulating rice seed germination under salt stress remain unknown. In this study, we found that seeds of loss-of-function osmft1 mutants germinated faster than wild-type (WT) seeds under salt stress, but this was not the case for loss-of-function osmft2 mutants. Overexpression of OsMFT1 (OsMFT1OE) or OsMFT2 increased the sensitivity to salt stress during seed germination. Transcriptome comparisons of osmft1 vs WT in the absence and presence of salt stress yielded several differentially expressed genes, which were associated with salt stress, plant hormone metabolism and signaling pathways, such as B-BOX ZINC FINGER 6, O. sativa bZIP PROTEIN 8 and GIBBERELLIN (GA) 20-oxidase 1. In addition, the sensitivity of OsMFT1OE seeds to GA and osmft1 seeds to abscisic acid (ABA) during seed germination increased under salt stress. Overall, our results indicate that ABA and GA metabolism and their signaling pathways are regulated by OsMFT1, modulating seed germination in rice under salt stress.

Seed dormancy and germination are important physiological processes during the life cycle of a seed plant. Recently, auxin has been characterized as a positive regulator that functions during seed dormancy and as a negative regulator during germination. Through chemical genetic screenings, we have identified a small molecule, germostatin (GS), which effectively inhibits seed germination in Arabidopsis. GSR1 (germostatin resistance locus 1) encodes a tandem plant homeodomain (PHD) finger protein, identified by screening GS-resistant mutants. Certain PHD fingers of GSR1 are capable of binding unmethylated H3K4, which has been reported as an epigenetic mark of gene transcriptional repression. Biochemical studies show that GSR1 physically interacts with the transcriptional repressor ARF16 and attenuates the intensity of interaction of IAA17/ARF16 by directly interacting with IAA17 to release ARF16. Further results show that axr3-1, arf10 arf16 are hyposensitive to GS, and gsr1 not only resists auxin-mediated inhibition of seed germination but also displays decreased dormancy. We therefore propose that GSR1 may form a co-repressor with ARF16 to regulate seed germination. Besides promoting auxin biosynthesis via upregulating expression of YUCCA1, GS also enhances auxin responses by inducing degradation of DΙΙ-VENUS and upregulating expression of DR5-GFP. In summary, we identified GSR1 as a member of the auxin-mediated seed germination genetic network, and GS, a small non-auxin molecule that specifically acts on auxin-mediated seed germination.

Seed dormancy and germination traits of Campanulaceae species in relation to ecological factors remains unclear. Hence, we clarified the seed germination characteristics of five Campanulaceae species (Adenophora triphylla (Thunb.) A.DC., Asyneuma japonicum (Miq.) Briq., Campanula punctata Lam., Codonopsis pilosula (Franch.) Nannf., and Lobelia sessilifolia Lamb.) native to Korea. Seeds were subjected to varying temperatures, cold stratification (CS) durations, and gibberellic acid (GA3) concentrations. Seeds of all species imbibed water readily, suggesting the absence of physical dormancy. For A. triphylla, A. japonicum, and L. sessilifolia, suitable seed germination occurred under elevated temperature conditions: 74.0 ± 6.2%, 37.0 ± 1.0%, and 26.0 ± 3.5% germination, respectively, at 25 °C, and 79.0 ± 3.8%, 38.0 ± 3.5%, and 62.0 ± 8.4% germination, respectively, at 25/15 °C (day/night) after 7 weeks after incubation. Germination of C. punctata and C. pilosula was consistently low across all temperatures. CS treatment resulted in significant final germination improvement to ~70.0% in four species, excluding C. pilosula. GA3 application significantly enhanced seed germination by ~60.0% across all species, with the most notable effects observed at 1000 mg∙L−1. Overall, Campanulaceae species seeds are permeable, and pre-treatment with CS and GA3 is required for effective seed germination.

Seed dormancy and germination, two physiological processes unique to seed-bearing plants, are critical for plant growth and crop production. The phytohormone brassinosteroid (BR) regulates many aspects of plant growth and development, including seed germination. The molecular mechanisms underlying BR control of rice (Oryza sativa) seed germination are mostly unknown. We investigated the molecular regulatory cascade of BR in promoting rice seed germination and post-germination growth. Physiological assays indicated that blocking BR signaling, including introducing defects into the BR-insensitive 1 (BRI1) receptor or overexpressing the glycogen synthase kinase 2 (GSK2) kinase delayed seed germination and suppressed embryo growth. Our results also indicated that brassinazole-resistant 1 (BZR1) is the key downstream transcription factor that mediates BR regulation of seed germination by binding to the alpha-Amylase 3D (RAmy3D) promoter, which affects α-amylase expression and activity and the degradation of starch in the endosperm. The BZR1-RAmy3D module functions independently from the established Gibberellin MYB-alpha-amylase 1A (RAmy1A) module of the gibberellin (GA) pathway. We demonstrate that the BZR1-RAmy3D module also functions in embryo-related tissues. Moreover, RNA-sequencing (RNA-seq) analysis identified more potential BZR1-responsive genes, including those involved in starch and sucrose metabolism. Our study successfully identified the role of the BZR1-RAmy3D transcriptional module in regulating rice seed germination.

Seed dormancy and germination are the two important traits related to plant survival, reproduction and crop yield. To understand the regulatory mechanisms of these traits, it is crucial to clarify which genes or pathways participate in the regulation of these processes. However, little information is available on seed dormancy and germination in peanut. In this study, seeds of the variety Luhua No.14, which undergoes nondeep dormancy, were selected, and their transcriptional changes at three different developmental stages, the freshly harvested seed (FS), the after-ripening seed (DS) and the newly germinated seed (GS) stages, were investigated by comparative transcriptomic analysis. The results showed that genes with increased transcription in the DS vs FS comparison were overrepresented for oxidative phosphorylation, the glycolysis pathway and the tricarboxylic acid (TCA) cycle, suggesting that after a period of dry storage, the intermediates stored in the dry seeds were rapidly mobilized by glycolysis, the TCA cycle, the glyoxylate cycle, etc.; the electron transport chain accompanied by respiration was reactivated to provide ATP for the mobilization of other reserves and for seed germination. In the GS vs DS pairwise comparison, dozens of the upregulated genes were related to plant hormone biosynthesis and signal transduction, including the majority of components involved in the auxin signal pathway, brassinosteroid biosynthesis and signal transduction as well as some GA and ABA signal transduction genes. During seed germination, the expression of some EXPANSIN and XYLOGLUCAN ENDOTRANSGLYCOSYLASE genes was also significantly enhanced. To investigate the effects of different hormones during seed germination, the contents and differential distribution of ABA, GAs, BRs and IAA in the cotyledons, hypocotyls and radicles, and plumules of three seed sections at different developmental stages were also investigated. Combined with previous data in other species, it was suggested that the coordination of multiple hormone signal transduction nets plays a key role in radicle protrusion and seed germination.

Seeds are designed to permit the mature embryo, surrounded by its seed coat, to survive the period between detachment from the parental plant and its establishment as a new seedling. Seeds have a number of characteristics related to their survival strategy. They are resistant to desiccation; they possess food reserves, assumed to be required for proper growth as a seedling; and they exhibit seed dormancy, a mechanism that prevents germination in periods unfavorable for seedling growth but allows germination at the proper time in the right environment. A well-adapted species only germinates at the beginning of the season that benefits seedling growth, which allows the completion of the life cycle. Thus, proper timing of germination is essential for survival of the species. Arabidopsis is an annual distributed over a large part of the northern, mainly temperate, parts of the world. Since in many sites it flowers before the onset of a warm and dry summer, it can survive that summer period only in the seed stage. In general, conditions become favorable for germination during the fall when seeds germinate, and then the plants survive winter as seedlings or young nonflowering plants (Baskin and Baskin 1972). This is the typical behavior of a winter annual. In some cases, seed germination is postponed until the following spring, where-after the plant completes its life cycle in the same year (Ratcliffe 1976). Arabidopsis is well suited for the study of the physiological mechanisms of dormancy and germination, because as a “wild” plant, it still...

Light is one of the most important environmental factors regulating seed germination. It is known that light inhibits seed germination of some monocotyledonous species and that it is mostly related to the blue wavelength of the spectrum received by cryptochromes (cry). Research has also found that the red light (R) stimulates germination of dicotyledonous seeds and that this reaction involves mainly phytochromes (phy). Surprisingly, up to date, the role and the mechanism of action of blue light (BL) in seed biology of dicot plants is still very poorly understood and some questions are unexplained, e.g., whether BL plays a role in regulation of dicot seeds dormancy and/or germination? If, so what particular elements of light signaling pathway are involved in modulation of this(ese) process(es)? Also, is the BL action in regulation of dicot seeds dormancy and/or germination maybe due to changes of expression of genes related to metabolism and/or signaling of two phytohormones controlling seed-related events, such as gibberellins (GA) and abscisic acid (ABA)? To answer these intriguing questions, the combination of biological, transcriptomic, and genetic approaches was performed in this particular study. The germination tests show that freshly harvested wild type (WT) Arabidopsis thaliana Col-0 seeds are dormant and do not germinate in darkness (at 25 °C), while nondormant (after-ripened) seeds germinate well in these conditions. It is also proven that dormancy of seeds of this species is released in the presence of white and/or BL (λ = 447 nm) when placed at 25 °C. Presented here, novel results emphasize the role of BL in dormancy alleviation of dicot seeds, indicating that this wavelength of light spectrum received by phyB induces this process and that the sensitivity to this stimulus depends on the depth of seed dormancy. In addition, it is demonstrated that various elements of phy-mediated pathway can be used in response to the signal induced by BL in germinating dormant seeds of Arabidopsis. The quantitative real time PCR analysis supported by results of germination tests of WT, T-DNA insertion mutants (i.e., hy5, hfr1, and laf1) and overexpression transformants of Arabidopsis seeds (i.e., 35S:OE:HY5, 35S:OE:HYH, 35S:OE:HFR1, and 35S:OE:LAF1) revealed that the HY5 gene coding transcription factor is most probably responsible for the control of expression of genes involved in GA/ABA metabolism and/or signaling pathways during BL-dependent dormancy alleviation of Arabidopsis seeds, while biological functions of HYH and HFR1 are associated with regulation of germination. The model of BL action in regulation of dormancy alleviation and germination potential of Arabidopsis seeds is proposed.

Influence of shrub cover on germination, dormancy and viability of buried and unburied seeds of Piptochaetium napostaense (Speg.) Hackel

Freshly harvested empress tree ( Paulownia elongata ) seeds have physiologic dormancy. The aim of this study was to investigate the effects of exogenous and endogenous nitric oxide (NO) on the dormancy and germination of empress tree seeds. After treatment with different concentrations of sodium nitroprusside (an NO-releasing compound) solution, the germination percentage of seeds under 12 h of continuous light was significantly greater. Seed germination percentage was promoted significantly by 10 –4 M sodium nitroprusside plus cold stratification compared with seeds treated with cold stratification only. At different hours during imbibition, empress tree seeds treated with 2-(4-carboxyphenyl)-4, 4, 5, 5- tetramethylimidazoline -1-oxyl-3-oxide potassium salt (c-PTIO), NG-nitro-L-arginine methyl ester (L-NAME), and sodium tungstate showed reduced seed germination percentages. During the early hours of imbibition, c-PTIO or sodium tungstate treatment inhibited seed germination significantly. The results showed that both exogenous and endogenous NO can release empress tree seed dormancy. Endogenous NO oxide was involved in dormancy release and germination of seeds during the early stages of imbibition. Wider application of NO may be used for breaking seed dormancy in other species.