Optimizing the Protein Fluorescence Reporting System for Somatic Embryogenesis Regeneration Screening and Visual Labeling of Functional Genes in Cotton

Protein fluorescence reporting systems are of crucial importance to in-depth life science research, providing systematic labeling tools for visualization of microscopic biological activities in vivo and revolutionizing basic research. Cotton somatic cell regeneration efficiency is low, causing difficulty in cotton transformation. It is conducive to screening transgenic somatic embryo using the fluorescence reporting system. However, available fluorescence labeling systems in cotton are currently limited. To optimize the fluorescence reporting system of cotton with an expanded range of available fluorescent proteins, we selected 11 fluorescent proteins covering red, green, yellow, and cyan fluorescence colors and expressed them in cotton. Besides mRuby2 and G3GFP, the other nine fluorescent proteins (mCherry, tdTomato, sfGFP, Clover, EYFP, YPet, mVenus, mCerulean, and ECFP) were stably and intensely expressed in transgenic callus and embryo, and inherited in different cotton organs derive from the screened embryo. In addition, transgenic cotton expressing tdTomato appears pink under white light, not only for callus and embryo tissues but also various organs of mature plants, providing a visual marker in the cotton genetic transformation process, accelerating the evaluation of transgenic events. Further, we constructed transgenic cotton expressing mCherry-labeled organelle markers in vivo that cover seven specific subcellular compartments: plasma membrane, endoplasmic reticulum, tonoplast, mitochondrion, plastid, Golgi apparatus, and peroxisome. We also provide a simple and highly efficient strategy to quickly determine the subcellular localization of uncharacterized proteins in cotton cells using organelle markers. Lastly, we built the first cotton stomatal fluorescence reporting system using stomata-specific expression promoters (ProKST1, ProGbSLSP, and ProGC1) to drive Clover expression. The optimized fluorescence labeling system for transgenic somatic embryo screening and functional gene labeling in this study offers the potential to accelerating somatic cell regeneration efficiency and the in vivo monitoring of diverse cellular processes in cotton.

Triticale Green Plant Regeneration Is Due to DNA Methylation and Sequence Changes Affecting Distinct Sequence Contexts in the Presence of Copper Ions in Induction Medium

Metal ions in the induction medium are essential ingredients allowing green plant regeneration. For instance, Cu(II) and Ag(I) ions may affect the mitochondrial electron transport chain, influencing the Yang cycle and synthesis of S-adenosyl-L-methionine, the prominent donor of the methylation group for all cellular compounds, including cytosines. If the ion concentrations are not balanced, they can interfere with the proper flow of electrons in the respiratory chain and ATP production. Under oxidative stress, methylated cytosines might be subjected to mutations impacting green plant regeneration efficiency. Varying Cu(II) and Ag(I) concentrations in the induction medium and time of anther culture, nine trials of anther culture-derived regenerants of triticale were derived. The methylation-sensitive AFLP approach quantitative characteristics of tissue culture-induced variation, including sequence variation, DNA demethylation, and DNA de novo methylation for all symmetric-CG, CHG, and asymmetric-CHH sequence contexts, were evaluated for all trials. In addition, the implementation of mediation analysis allowed evaluating relationships between factors influencing green plant regeneration efficiency. It was demonstrated that Cu(II) ions mediated relationships between: (1) de novo methylation in the CHH context and sequence variation in the CHH, (2) sequence variation in CHH and green plant regeneration efficiency, (3) de novo methylation in CHH sequences and green plant regeneration, (4) between sequence variation in the CHG context, and green plant regeneration efficiency. Cu(II) ions were not a mediator between de novo methylation in the CG context and green plant regeneration. The latter relationship was mediated by sequence variation in the CG context. On the other hand, we failed to identify any mediating action of Ag(I) ions or the moderating role of time. Furthermore, demethylation in any sequence context seems not to participate in any relationships leading to green plant regeneration, sequence variation, and the involvement of Cu(II) or Ag(I) as mediators.

Effect of Gamma Radiation on the Adsorption Capacity and Regeneration / Desorption of Sulphate and Phosphate Ions Using Smart Hydrogel.

Co-polymeric hydrogels containing poly (Acrylamide /Epichlorohydrine) P(AAm/EPI) with different acrylamide and Epichlorohydrine content were fabricated by gamma radiation at different irradiation doses as adsorbent materials for wastewater treatment. The mechanisms of radiation-induced crosslinking of hydrogel in aqueous solution has been evaluated. The gel contents and the swelling/diffusion kinetic parameters were evaluated at different irradiation doses, and the result confirm a non-fichian mechanism. The shape, surface morphology, and porosity of P (AAm/ Epi) hydrogel were characterized by scanning electron microscope (SEM). Adsorption experiments were carried out for the removal of sulphate and phosphate ions from wastewater using P(AAm/EPI) hydrogels as adsorbent materials. The isotherm data were analyzed by Freundlich equation. The equilibrium isotherm results show a better fitting (R2 > 0.9) to the Freundlich model for all anions. The calculated regeneration efficiency (%) values of sulphate and phosphate ions found to be ranged between 63.2 (%) and 46 (%).The relatively higher regeneration efficiency (%) and keeping the hydrogels its shape without any deformation promising to use the same hydrogels further times which decrease the economic cost.

Modifying the resin type of hybrid anion exchange nanotechnology (HAIX-Nano) to improve its regeneration and phosphate recovery efficiency

In order to avoid eutrophication of freshwater systems, regulations all around the world have become increasingly stringent toward the maximum phosphate concentration allowed in wastewater discharges. Traditional phosphate removal methods such as chemical precipitation and enhanced biological phosphorus removal struggle to lower phosphate levels to the new requirements. Hybrid anion exchange nanotechnology (HAIX-Nano) is composed of a selective adsorption material able to remove phosphate down to levels close to zero. Moreover, HAIX-Nano is not affected by intermittent flow and does not produce sludge making it an interesting alternative. The regeneration process of HAIX-Nano typically requires a chemical solution with a high concentration of sodium hydroxide (NaOH) and sodium chloride (NaCl) (2–5% w/w of each). To lower the environmental impact and the operational cost of the technology, this study aims to enhance the HAIX-Nano regeneration efficiency. Therefore, the backbone of HAIX-Nano, which is normally a strong base anionic (SBA) resin, was changed for a weak base anionic (WBA) resin. The resulting material (WBA-2) exhibited a higher adsorption capacity than the traditional version of HAIX-Nano (SBA-1) under the tested conditions, while also showing a much higher regeneration efficiency. For a desorption solution of only 0.4% NaOH and no NaCl, WBA-2 showed an average regeneration efficiency of 78 ± 1% compared to SBA-1 with 24 ± 1%.

Application of extraction steam graded heat storage in peak shaving of condensing units

In order to alleviate the peak shaving pressure of power grid and further improve the deep peak shaving capacity of coal-fired units, this paper applies staged heat storage to condensing units. Under the condition of constant boiler load, the heat of regenerative steam extraction is stored to reduce the electrical load output of the unit. Taking a 660MW ultra-supercritical unit as an example,30%THA,40%THA and 50%THA were taken as the initial conditions of heat storage respectively to discuss the peak shaving range and power regeneration efficiency of different extraction steam positions for heat storage. The results show that the “storage 1# HP heater extraction steam” scheme with “two-stage three tank” heat storage and release structure has the largest peak shaving range of 37.3%~56.9% under 50% initial conditions. When the “two-stage three tank” heat storage and release structure selects the “storage 1# HP heater extraction steam ”scheme under 40% initial working condition, the “power regeneration efficiency” reaches the highest——78.3%. And the “power regeneration efficiency” of “two stage three tank” storage structure are always higher than the “single-stage” storage structure.

Temporal Control of Morphogenic Factor Expression Determines Efficacy in Enhancing Regeneration

Background: Regeneration of fertile plants from tissue culture is a critical bottleneck in the application of new plant breeding technologies. Ectopic overexpression of morphogenic factors is a promising workaround for this hurdle. Methods: Conditional overexpression of WUS and ARF5Δ was used to study the effect of timing the overexpression of these morphogenic factors during shoot regeneration from root explants in Arabidopsis. In addition, their effect on auxin-signaling activation was examined by visualization and cytometric quantification of the DR5:GFP auxin-signaling reporter in roots and protoplasts, respectively. Results: The induced expression of both WUS and ARF5Δ led to an activation of auxin signaling in roots. Activation of auxin signaling by WUS and ARF5Δ was further quantified by transient transformation of protoplasts. Ectopic overexpression of both WUS and ARF5Δ enhanced regeneration efficiency, but only during the shoot-induction stage of regeneration and not during the callus-induction stage. Conclusions: The overexpression of WUS and ARF5Δ both lead to activation of auxin signaling. Expression during the shoot-induction stage is critical for the enhancement of shoot regeneration by WUS and ARF5Δ.

In Vitro Growth of Sunflower (Helianthus annuus) via Direct Organogenesis

Sunflower (Helianthus annuus) is a crop of increasing importance as a source of seed oil and proteins; nonetheless, the number of studies on sunflower tissue culture is somewhat limited. The development of a competent in vitro direct organogenesis protocol involves important basic steps of regeneration. In our study, chemically sterilized sunflower seeds were planted on induction media, and 52.54 % germination efficiency was found. While the seeds were subjected to regeneration containing 2 mg/L of cytokinin, Benzyl Adenopurine (BAP) as well as 1 mg/L of auxin, Naphthalene Acetic Acid (NAA); shoot growth was observed with41 % regeneration efficiency. Non-sterilized seeds germinated but showed fungal growth on the surface of media resulting in no regeneration of sunflower plantlet. On the other hand, sterile seeds germinated less with little or no fungal growth leading to successful regeneration. Frequent regeneration of sterile sunflower seeds through direct organogenesis can contribute to enhanced micro-propagation of this plant.

Characteristics of Ultrasonically Enhanced Low-Temperature Thermal Regeneration of Powdered Activated Carbon: A Case Study of Acetone and Aniline

Effective regeneration of powdered activated carbon (PAC) is the key to reduce the operating cost of the PAC in wastewater treatment processes. In this study, volatile acetone and semi-volatile aniline were selected to investigate the regeneration characteristics of ultrasonically enhanced low-temperature thermal process. The results showed that the regeneration efficiency of the PAC that had adsorbed aniline or acetone increased with the increase in ultrasonic power, and optimal value of frequency and regeneration times were determined. The concentration and properties of organic solvents had a significant influence on the ultrasonic regeneration process. With the increase in heating temperature and regeneration time, the regeneration efficiency increased, but the loss of mass of the saturated PAC increased noticeably. With the combination of ultrasonic treatment in a solvent with low temperature heating, the PAC regeneration efficiency was successfully improved, and the PAC mass loss rate was noticeably reduced. The microjet, shock wave, and cavitation effects produced by ultrasonic treatment restored the specific surface area of the PAC, expanded its mesopore volume, and increased the pore diameter. A reasonable selection of the regeneration solution and optimization of the ultrasonic treatment conditions could create favorable conditions for subsequent low temperature thermal regeneration.