Dark Culture Research Articles

Enhanced growth of Chromochloris zofingiensis through the transition of nutritional modes

The green microalga Chromochloris zofingiensis is an emerging alternative to commercial astaxanthin due to its high productivity and metabolic flexibility with regard to growing in different nutritional modes, namely autotrophic, heterotrophic, and mixotrophic. Although the transition from one nutritional mode to another in each culture step (stock, preculture, and biomass production) can be easily realized and may trigger various metabolic functions, there have only been a few studies on its effects on growth and cell characteristics. Therefore, in this study, the effects of the transition of nutritional modes were investigated by transferring cells precultured in the three above-mentioned nutritional modes to the biomass production phase in the three modes. The heterotrophically precultured cells showed decreases in growth and biomass productivity when cultivated in prolonged dark cultures. Conversely, the heterotrophic cells showed a higher growth rate when irradiated in the autotrophic and mixotrophic biomass production phases, probably due to the accumulation of growth-promoting factors in the heterotrophic preculture. The mixotrophic biomass production phase showed higher maximum biomass productivity (1.28 g L−1 d−1) compared with the autotrophic (0.065 g L−1 d−1) and heterotrophic (0.448 g L−1 d−1) modes. The optimum conditions for stock, preculture and biomass production phase were autotrophic in order to maintain the following heterotrophic culture condition, heterotrophic to store growth-promoting factors, and mixotrophic to achieve high productivity, respectively.

Evaluation of Euglena gracilis 815 as a New Candidate for Biodiesel Production.

Euglena comprises over 200 species, of which Euglena gracilis is a model organism with a relatively high fatty acid content, making it an excellent potential source of biodiesel. This study isolated and characterized a new strain named E. gracilis 815. E. gracilis 815 cells were cultivated under light and dark conditions, with either ethanol or glucose as an external carbon source and an autotrophic medium as control. To achieve maximum active substances within a short period i.e., 6 days, the effects of the light condition and carbon source on the accumulation of bioactive ingredients of E. gracilis 815 were explored, especially fatty acids. In comparison with the industrially used E. gracilis Z strain, E. gracilis 815 exhibited high adaptability to different carbon sources and light conditions, with a comparable biomass and lipid yield. The content and composition of fatty acids of E. gracilis 815 were further determined to assess its potential for biodiesel use. Results suggested that E. gracilis 815 has biodiesel potential under glucose addition in dark culture conditions and could be a promising source for producing unsaturated fatty acids. Therefore, E. gracilis 815 is a candidate for short-chain jet fuel, with prospects for a wide variety of applications.

Establishment of an efficient regeneration and genetic transformation system for Malus prunifolia Borkh. ‘Fupingqiuzi’

Malus prunifolia Borkh. ‘Fupingqiuzi’ has significant ecological and economic value and plays a key role in germplasm development and resistance research. However, its long juvenile phase and high heterozygosity are barriers to the identification of ‘Fupingqiuzi’ progeny with excellent traits. In-vitro regeneration techniques and Agrobacterium-mediated genetic transformation systems can efficiently produce complete plants and thus enable studies of gene function. However, optimal regeneration and genetic transformation systems for ‘Fupingqiuzi’ have not yet been developed. Here, we evaluated the factors that affect the in-vitro regeneration and transformation of ‘Fupingqiuzi’. The best results were obtained when transverse leaf sections were used as explants, and they were grown in dark culture for three weeks with their adaxial sides contacting the culture medium (MS basal salts, 30 g L−1 sucrose, 8 g L−1 agar, 5 mg L−1 6-benzylaminopurine (6-BA), 2 mg L−1 thidiazuron (TDZ), and 1 mg L−1 1-naphthlcetic acid (NAA), pH 5.8). A genetic transformation system based on this regeneration system was optimized: after inoculation with A. tumefaciens solution for 8 min, 4 days of co-culture, and 3 days of delayed culture, the cultures were screened with cefotaxime (150 mg L−1) and kanamycin (15 mg L−1). We thus established an efficient regeneration and genetic transformation system for ‘Fupingqiuzi’, enabling the rapid production of transgenic material. These findings make a significant contribution to apple biology research.

Effects of an environmental endocrine disruptor, para-nonylphenol on the cell growth of Euglena gracilis: association with the cellular oxidative stress.

Effects of an environmental endocrine disruptor, para-nonylphenol (NP) on the cell growth of a photosynthetic eukaryotic microorganism, Euglena gracilis were analysed under different cell culture conditions. Although NP did not show significant inhibitory effects on the cell growth of E. gracilis (Z and SM strains) under light culture condition, NP exhibited significant suppressive effects under dark culture condition. Exogenous supplementation with lipophilic antioxidants (α-tocopherol, β-carotene or 6-O-palmitoyl-ascorbic acid) to E. gracilis caused strong preventive effects against NP-induced cell growth inhibition under dark culture condition, but hydrophilic antioxidants [ascorbic acid, glutathione and epigallocatechin gallate (EGCG)] did not show significant preventive effects. NP caused significant generation of reactive oxygen species (ROS) in E. gracilis under dark culture condition, but E. gracilis under light culture condition did not show significant increase in ROS generation. Supplementation with lipophilic antioxidants to E. gracilis caused significant suppressive effects against NP-induced cellular ROS generation under dark culture condition, but hydrophilic antioxidants did not show significant suppressive effects. Furthermore, the productivities of typical cellular antioxidants (α-tocopherol, β-carotene and ascorbic acid) in E. gracilis under light culture conditions were much higher than those under dark culture conditions.

The Effect of Daminozide, Dark/Light Schedule and Copper Sulphate in Tissue Culture of Triticum timopheevii.

Triticum timopheevii Zhuk. is a tetraploid wheat that is utilized worldwide as a valuable breeding source for wheat improvement. Gene-based biotechnologies can contribute to this field; however, T. timopheevii exhibits recalcitrance and albinism in tissue cultures, making this species of little use for manipulation through genetic engineering and genome editing. This study tested various approaches to increasing in vitro somatic embryogenesis and plant regeneration, while reducing the portion of albinos in cultures derived from immature embryos (IEs) of T. timopheevii. They included (i) adjusting the balance between 2,4-D and daminozide in callus induction medium; (ii) cultivation using various darkness/illumination schedules; and (iii) inclusion of additional concentrations of copper ions in the tissue culture medium. We achieved a 2.5-fold increase in somatic embryogenesis (up to 80%) when 50 mg L−1 daminozide was included in the callus induction medium together with 3 mg L−1 2,4-D. It was found that the dark cultivation for 20–30 days was superior in terms of achieving maximum culture efficiency; moreover, switching to light in under 2 weeks from culture initiation significantly increased the number of albino plants, suppressed somatic embryogenesis, and decreased the regeneration of green plants. Media containing higher levels of copper ions did not have a positive effect on the regeneration of green plants; contrarily, the elevated concentrations caused albinism in plantlets. The results and relevant conclusions of the present study might be valuable for establishing an improved protocol for the regeneration of green plants in tissue cultures of T. timopheevii.

Light Irradiation Coupled with Exogenous Metal Ions to Enhance Exopolysaccharide Synthesis from Agaricus sinodeliciosus ZJU-TP-08 in Liquid Fermentation.

To promote Agaricus sinodeliciosus var. Chaidam ZJU-TP-08 growth and metabolites accumulation, a novel integrated strategy was developed by adopting high levels of metal ions coupled with light treatment. The results revealed that yellow and blue light could significantly promote biomass and exopolysaccharides production, respectively. Furthermore, the yellow–blue light shift strategy could stimulate exopolysaccharides formation. Ca2+ ions coupled with blue light mostly promoted exopolysaccharides production related to oxidative stress, which was 42.00% and 58.26% higher than that of Ca2+ ions coupled with the non-light and dark cultivation without Ca2+ ions in 5-L bioreactor. RNA-seq was performed to uncover the underlined molecular mechanism regulated by light-induced gene expressions in exopolysaccharides biosynthesis and oxidative stress. The findings of this work provide valuable insights into adopting metal ions coupled with the light-assisted method for the macrofungus submerged fermentation for exopolysaccharides production.

Profiles of Cytokines Secreted by ARPE-19 Cells Exposed to Light and Incubated with Anti-VEGF Antibody.

The retinal pigment epithelium (RPE) is the major source of cytokines in the retina regulating the intraocular immune environment, and a primary target of photodamage. Here, we examined 27 types of cytokines secreted by ARPE-19 cells exposed to visible light and incubated with aflibercept or ranibizumab, which are two anti-vascular endothelial growth factor (VEGF) antibodies. The cells were cultured for 24 h in the dark or under 2000 lux irradiation from a daylight-colored fluorescent lamp, and cytokine levels in the culture supernatant were measured. In the light-irradiated culture, the levels of IL-9, IL-17A and bFGF were higher, and the levels of IL-6, IL-7, IL-8 and MCP-1 were lower than those in the dark culture, while there was no significant difference with the VEGF-A level. In subgroup analyses of the light-irradiated culture, the bFGF level under 250 to 2000 lux irradiation was elevated in a light intensity-dependent manner. In culture exposed to blue, green or red light, the bFGF level was elevated by blue light and was high compared to that by green or red light. In culture with aflibercept or ranibizumab in the dark, the levels of IL-6, IL-8, bFGF and MCP-1 were increased, and the IL-12 level decreased synchronously with a reduction in the VEGF-A level. Our findings indicate that continuous irradiation of visible light and VEGF suppression may be an influential factor in expression patterns of inflammatory cytokines secreted by human RPE cells.

Autotrophic vs. Heterotrophic Cultivation of the Marine Diatom Cyclotella cryptica for EPA Production.

Recently, the marketable value of ω-3 fatty acid, particularly eicosapentaenoic acid (EPA), increased considering their health effects for human consumption. Microalgae are considered a valuable and “green” source of EPA alternative to fish oils, but considerable efforts are necessary for their exploitation at an industrial level. Due to the high operation costs of photoautotrophic microalgae cultivation, heterotrophic growth represents a promising economic solution. Marine diatoms are the major ecological producers of ω-3 fatty acids. Few species of diatoms are capable to grow in the dark using organic carbon sources. The marine diatom Cyclotella cryptica was cultivated for 14 days under photoautotrophic and heterotrophic conditions to define the effects on growth parameters, lipid production, total fatty acids and EPA content. Photoautotrophic conditions led to a total EPA production of 1.6% of dry weight, 12.2 mg L−1 culture and productivity of 0.9 mg L−1 day−1. The heterotrophy cultures reported a total EPA production of 2.7% of dry cell weight, 18 mg L−1 culture, a productivity of 1.3 mg L−1 day−1, which are promising values in the prospective of improving culture parameters for the biotechnological exploitation of dark cultivation. C. cryptica could be a potential candidate for the heterotrophic production of EPA, also considering its robustness, capacity to resist to bacterial contaminations and plasticity of lipid metabolism.

Effects of Light Regulation on Proteome Expression in Rhodobacter sphaeroides 2.4.1.

Light plays an important role in the transcriptional regulation of photosynthetic apparatus. The influence of oxygen and light conditions on the protein expression of Rhodobacter sphaeroides was investigated using a proteomic approach. The R. sphaeroides was grown aerobically under dark cultivation (D24) and light cultivation (L24) for 24h. An average of 950 distinguishable spots were obtained on 2-D analytic gel for D24 and L24 conditions, of which 48 proteins exhibited significant changes in protein expression levels. Among the 48, 31 proteins were upregulated and 17 proteins were downregulated in L24 when compared with D24. The results depict the comparative protein expression in R. sphaeroides mediated through growth under light or dark conditions. The data suggest that the overexpressed proteins, phosphoribosyl-ATP pyrophosphatase (HisE), in the D24/aerobic culture are involved in the positive regulation of PAC production can be functionally applied in metabolic engineering and industrial processes.

Isolation and molecular confirmation of leptospires from dogs in Himachal Pradesh, India

Leptospirosis is a common but largely underreported zoonotic disease in India. It has been recognized as a re-emerging infectious disease causing deaths in dogs as well as humans. Leptospires have been isolated from both clinically ill and apparently healthy animals from different geographical regions but there is no information on the presence of leptospires in various clinical conditions of animals in the northern state of Himachal Pradesh, India. Here, using dark field microscopy, culture isolation and molecular biology-based tests, we confirm leptospirosis in three clinically affected dogs with chronic renal involvement. The study reports the first isolation and molecular confirmation of leptospires from clinically affected dogs in Himachal Pradesh, India.

A descriptive study of the clinical and etiological profile of balanoposthitis.

Background:Balanoposthitis is defined as an inflammatory condition of glans penis and prepuce. There are wide variety of etiologies including both infectious and noninfectious conditions. This study attempts to throw light on information regarding clinical and microbiological aspects of balanoposthitis.Objectives:To study various clinical patterns, etiologies, and predisposing factors of balanoposthitis.Methodology:A descriptive study was undertaken on 106 cases who presented to sexually transmitted disease (STD) clinic with balanoposthitis between November 2017 and April 2019. A detailed history, physical examination, and investigations like KOH mount, leishman staining, gram staining, dark field microscopy, cultures, and other investigations were done wherever indicated. The data collected was tabulated and analyzed.Results:In our study, infectious etiology was the most common and was found in 77.36% cases. About 13.41% of cases with infectious balanoposthitis had multiple etiological agents. Noninfectious etiology was found in 22.64% cases. The most common infectious cause of balanoposthitis was candida, noted in 59.76% cases, followed by herpes simplex virus (19.51%), human papilloma virus (13.41%), and scabies (8.54%). Among noninfectious etiologies, adverse drug reaction (4.72% of total cases) was the most common, followed by lichen planus (3.77%) and psoriasis (3.77%). There was significantly higher incidence of phimosis in diabetic patients with candidal balanoposthitis.Conclusion:Identifying the etiology facilitates early treatment and hence reduces the infectivity and transmission of disease and also the disease complications like phimosis. In addition, multiple infectious etiologies should always be kept in mind while evaluating STDs.

Sustained photobiological hydrogen production by Chlorella vulgaris without nutrient starvation

This article describes the ability of the Chlorella vulgaris BEIJ strain G-120 to produce hydrogen (H2) via both direct and indirect pathways without the use of nutrient starvation. Photobiological H2 production reached a maximum rate of 12 mL H2 L−1 h−1, corresponding to a light conversion efficiency (light to H2) of 7.7% (average 3.2%, over the 8-day period) of PAR, (photosynthetically active irradiance). Cells presented a maximum in vivo hydrogenase activity of 25.5 ± 0.2 nmoles H2 μgChl−1 h−1 and the calculated in vitro hydrogenase activity was 830 ± 61 nmoles H2 μgChl−1 h−1. The strain is able to grow either heterotrophically or photo autotrophically. The total output of 896 mL of H2 was attained for illuminated culture and 405 mL for dark cultures. The average H2 production rate was 4.98 mL L−1 h−1 for the illuminated culture and 2.08 mL L−1 h−1 for the one maintained in the dark.

Maternal haploid induction in African marigold (Tagetes erecta L.) through in vitro culture of un-fertilized ovules

A protocol for production of haploids from two heterozygous male sterile lines of Tagetes erecta using un-pollinated ovules as explant was standardized. Various factors affecting the in vitro gynogenic response viz., growth regulators, ovule developmental stage, basal media, sucrose concentration, photoperiod and cold shock duration were assessed in Tagetes erecta. Direct induction of parthenogenic embryos occurred in cultivar ‘Local Orange’ when the un-pollinated ovules were cultured in EMS (MS medium enriched with coconut water, AgNO3, PVP etc.) medium supplemented with 2 mg l−1 BAP and 0.5 mg l−1 NAA or 2 mg l−1 BAP along with 2 mg l−1 2,4-D. The developmental stage of the flower buds was vital in embryo induction from the excised ovules. The ovules collected from half-open flower buds were more responsive to gynogenesis as compared to ovules collected from un-open and fully open flower buds. EMS basal medium was found best for gynogenesis over the other commercially available basal media. Cold pre-treatment of flower buds at 4 oC had no stimulatory effect and negatively affected the gynogenesis. The dark culture condition was imperative for direct parthenogenic embryo induction than 16 h light duration. The ploidy levels of 17 regenerants were determined by cytological studies, revealed 47.06% as diploids, 41.18% as haploids and 11.76% as mixoploids. These results were reconfirmed with flow cytometry analysis. The determination of ploidy level by counting the number of chloroplasts in stomatal guard cells of marigold was also established for rapid screening of haploids. The resultant haploids were successfully diploidised and are being utilized in the hybrid breeding programme at our institute. The standardized protocol for doubled haploids (DHs) production by un-pollinated ovule culture paved the way for Tagetes F1 hybrid breeding. This is the first successful report of in vitro gynogenesis in Tagetes spp for production of doubled haploids (DHs). The protocol for obtaining high frequency parthenogenic embryo induction from unpollinated ovules was standardized and validated for rapid production of homozygous lines.

Detection of a pheomelanin-like pigment by EPR spectroscopy in the mycelium of Plenodomus biglobosus.

Melanin occurrence in Plenodomus biglobosus was investigated using electron paramagnetic (spin) resonance (EPR, ESR) spectroscopy. The fungus was isolated from living and dead leaves of European ash (Fraxinus excelsior L.). Dark pigmentation of P. biglobosus mycelium in vitro, especially on the reverse, was observed. The black coloration intensified with the age of the culture and inspired us to check if the analyzed fungus species synthesizes melanin. Melanin contains unpaired electrons, thus, EPR spectroscopy was applied, as a specific technique, to verify its presence in P. biglobosus. The EPR spectrum of the mycelium showed a very strong melanin signal, revealing pheomelanin-like features. Thus, the black pigment of P. biglobosus was clearly identified as melanin. However, no melanin was detected in the apparently dark culture medium even when zinc (II) acetate was added to increase the sensitivity of detection. Pheomelanin has many unusual biological functions but it is not commonly found in fungi. Detection of this type of melanin in P. biglobosus, which can be both endophytic or pathogenic, suggests a closer examination of the potential role of this melanin in host-parasite interaction.

High-efficiency somatic embryogenesis techniques for different hybrids of cut lilies

Somatic embryogenesis (SE) is a unique method of in vitro regeneration for plant reproduction, germplasm resource preservation, and molecular-assisted breeding. Although lily is an internationally valued ornamental plant, its SE technology is still incomplete. Here, the effects of different explants, picloram (PIC) concentrations, and light conditions on somatic embryo induction, development, germination and seedling formation were studied in five Lilium varieties belonging to different hybrids. The embryo histomorphology, development and origins were observed, and a broadly efficient embryogenesis system was firstly established in Lilium. With leaf explants, all varieties tested underwent indirect somatic embryogenesis (SE). With scale explants, the Lilium Asiatic ‘Strawberry and Cream’, Lilium Oriental ‘Double Surprise’, and Lilium longiflorum Thunb. ‘White Heaven’ underwent indirect SE and the Lilium longiflorum × Asiatic hybrid ‘Eyeliner’ and Lilium longiflorum × Oriental ‘Triumphator’ underwent direct SE. Somatic embryogenesis in the above varieties involved four stages: globular, heart-shaped, torpedo-shaped, and cotyledon embryos. Two original SE pathways (endogenous and exogenous) were observed, and their appearance times differed. In indirect SE, exogenous embryos appeared first, followed by endogenous embryos; whereas in direct SE, the order was reversed. The different somatic embryo origins and the proportion of internal and external origins were closely related to the occurrence of somatic embryos by direct or indirect pathways and significantly affected the induction cycle of somatic embryos. Dark culture promoted lily somatic embryo induction, while 16 h/day light culture promoted germination, and the germination rates of all five lily varieties exceeded 90%. Murashige Skoog (MS) medium + (1.5–2.5 mg L−1) PIC + 0.2 mg L−1 1-Naphthaleneacetic acid (NAA) was suitable for SE from lily leaves and scales with an induction efficiency of 88–96%. Our research developed a widely applicable regeneration procedure for different hybrids of lily via somatic embryogenesis. Morphohistological analysis confirmed the origins, developmental stages and dynamic changes of cells during in vitro morphogenesis. This study will provide a valuable reference for the propagation of Lilium.

Clinical profile of leptospirosis and role of various diagnostic methods, a hospital based prospective observational study

Leptospirosis is a potentially life-threatening zoonotic disease of worldwide distribution. Accurate diagnosis and prompt treatment are essential to minimize morbidity and mortality. The current study was conducted to analyse the clinical profile of leptospirosis and the diagnostic yield of various diagnostic methods. The present study was a cross-sectional study. A total of 60 patients who were suspected of leptospirosis were enrolled in the study. Direct examination of blood was done using dark-ground microscopy, the culture was done by inoculation of the blood sample into the EMJH medium, antibodies against Leptospira was demonstrated using Panbio IgM ELISA kit, and antigen products were demonstrated using polymerase reaction (PCR) with primers G1and G2.The study population included 63% of males and 37% females. A majority of 38% of the study subjects were farmers. Pallor and icterus are the predominant clinical signs found among 96% of the study population. IgM ELISA has labelled the highest number i.e. 55 (91.66%) of subjects as positive. The number of subjects diagnosed positive by PCR, culture and Dark ground microscopy (DGM) were 33 (55.00%), 22 (36.66%) and 12 (20%) subjects respectively.Leptospirosis proves to be an important health concern. Prominent clinical conditions were observed. IgM-ELISA proved superior to Dark ground microscopy (DGM), Culture and Polymerase chain reaction (PCR) and suitable for early diagnosis of leptospirosis.

Effects of dihydro-epi-deoxyarteannuin B on artemisinin biosynthesis, transcriptional profile and associated gene expression in suspension-cultured cells of Artemisia annua

Biotechnological methods producing artemisinin (ART) directly are limited because the final stages of the ART biosynthesis pathway remain elusive. Dihydro-epi-deoxyarteannuin B (DHEDB) is synthesized from dihydroartemisinic acid (DHAA) and used as a substrate at the present research. DHEDB was fed to cultured cells of Artemisia annua 10 days post subculture under light and dark conditions. The structures of the biotransformed products were elucidated by HR-ESI-MS and NMR analysis. Three hydroxylated products, 3α-hydroxy-dihydro-epi-deoxyarteannuin B (1), 9β-hydroxy-dihydro-epi-deoxyarteannuin B (2) and 14-hydroxy-dihydro-epi-deoxyarteannuin B (3), were obtained. Among them, compound 3 is a new compound. The autoxidation experiment of DHEDB indicated that three compounds were generated by enzymatic catalysis. The content of ART increased by 23.9 % compared to the control group under dark culture. Effects of DHEDB on the transcriptome of A. annua were determined under the optimal conditions. The genes HMGR, FPS, ADS, CYP71AV1, CPR, DBR2 and ALDH1 were involved in the process of biotransformation. qRT-PCR results showed that the increase in ART content was correlated with the increasing expression of the related genes. However, DHEDB and its biotransformed products are unlikely to be the intermediates of ART production because they showed no similarities in structure. Otherwise, the increase of ART content may due to the elicitor like effect of DHEDB as exogenous substance in A. annua cells.

Transcriptome analysis of the edible mushroom Lentinula edodes in response to blue light.

Lentinula edodes is one of the most popular edible mushrooms worldwide and contains important medicinal components such as lentinan, ergosterol, and eritadenine. Mushroom metabolism is regulated by the mycelia and fruit body using light; however, in mushrooms, the underlying molecular mechanisms controlling this process as well as light-induced gene expression remain unclear. Therefore, in this study, we compared morphological changes and gene expression in the fruit bodies of L. edodes cultivated under blue light and continuous darkness. Our results showed that blue light primarily induced pileus growth (diameter and thickness) compared to dark cultivation. Alternatively, stipe length development was promoted by dark cultivation. We also performed RNAseq on L. edodes under the blue light/dark cultivation conditions. A total of 12,051 genes were used for aligning the Illumina raw reads and 762 genes that showed fold change cut-offs of >|2| and significance p-values of <0.05 were selected under blue light condition. Among the genes which showed two-fold changed genes, 221 were upregulated and 541 were downregulated. In order to identify blue light induced candidate genes, differentially expressed genes (DEGs) were selected according to 4-fold changes and validated by RT-PCR. We identified 8 upregulated genes under blue light condition, such as DDR48-heat shock protein, Fasciclin-domain-containing protein and carbohydrate esterase family 4 protein, FAD NAD-binding domain-containing protein that are involved in morphological development of primordium and embryonic muscle development, cell adhesion and affect the structure of cellulosic and non-cellulosic cell walls of fruit body development, and photoreceptor of blue light signaling for fruit body and pigment development, respectively. This study provides valuable insights into the molecular mechanisms underlying the role of blue light in mushroom growth and development and can thus contribute to breeding programs to improve mushroom cultivation.

Reinvigoration of diploid strawberry (Fragaria vesca) during adventitious shoot regeneration

Diploid strawberry (Fragaria vesca ‘Baiguo’) is a model plant for studying functional genomics in Rosaceae. Adventitious shoot regeneration is essential for functional genomics by Agrobacterium tumefaciens-mediated transformation. An efficient shoot regeneration method using diploid strawberry leaf explants was conducted on 1/2MS + 1/2B5 medium that contained 2.0 mg L−1 TDZ over 14 days of dark culture; this induced the maximum percentage of shoot regeneration (96.44 ± 1.60%) and the highest number of shoots per explant (23.46 ± 2.14) after 11 weeks of culture. The explants considerably enlarged after 12 days; then, turned greenish brown after 30 days, yellowish brown after 36 days, and completely brown and necrotic after 48 days. Large numbers of adventitious shoots were produced from 48 to 66 days, and the shoots elongated from 66 to 78 days; this represents a critical period of reinvigoration, which included 30 days for leaf explant chlorosis, 36 days for adventitious shoot appearance, and 48 days for generation of numerous shoots. During the reinvigoration process, higher expressions of the hormone synthesis-related genes Ciszog1, CKX2, CKX3, CKX7, YUC2, YUC6, YUC10, YUC9, and GA2ox were detected from 30 to 48 days. Our results indicate that these genes may regulate reinvigoration of shoot regeneration.

Characteristics of wastewater treatment by Chlorella sorokiniana and comparison with activated sludge.

Characteristics of Chlorella sorokiniana treating wastewater with consideration of HRT (6 d, 16 h, 8 h), hydraulic conditions, light or dark culture were evaluated and compared with activated sludge. Results showed that optimal HRT was 8 h; if longer, effluent chemical oxygen demand (COD) and NH4 +-N in the dark began to rebound. Mixing was beneficial to COD removal of algae, while aeration was suitable for nutrient removal. Growth of C. sorokiniana in the light was mixotrophic growth and 1.3-1.7 times more than that of dark heterotrophic growth. The maximum specific growth rate (µmax), productivity, and biomass yields on COD (YCOD), N (YNH4), P (YP) of algae were higher in the light than that in the dark. COD assimilation capacity of algae was similar to activated sludge but with different dynamics. N and P assimilation capacity of algae was 1.4, 1.2-2.5 times more than activated sludge; N and P removal efficiency of algae was 5%-10%, 10%-55% respectively higher than activated sludge. This study confirmed the advantage of algae over activated sludge and reveal why algae could assist the activated sludge process.