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.

Glucose Induces Thylakoid Formation and Upregulates Green Pigment Contents in Complete Dark Culture of the Angiosperm Pachiramacrocarpa

In addition to angiosperms, most plants are able to synthesize chlorophyll (Chl)-generating green tissues in total darkness. In this study, 140 plants of the angiosperm Pachira macrocarpa were divided into five groups. Among them, one group was grown for 2 weeks under natural light conditions, whereas the others were grown in complete darkness (0 μmol m−2 s−1). Dark-grown plants were then treated with 0~6% glucose for another 8 weeks. The budding and greening ratios, ultrastructure of chloroplasts (ChlPs) of newly developed leaves, and green pigment contents of pre-illuminated mature and young leaves, and totally dark-grown newly developed leaves were measured. Results showed that glucose inhibited the budding and promoted the greening of newly developed leaves. Pre-illuminated mature and young leaves were able to synthesize green pigments during the 2 weeks of dark adaption. Dark-grown newly developed leaves contained high levels of green pigments at 2 and 3 weeks after budding. Green pigments of glucose-fed newly developed leaves had increased, whereas they had decreased in control leaves. In addition, ChlPs of dark-grown glucose-fed newly developed leaves contained both giant grana and prolamellar bodies (PLBs), usually found in shade plants and etiolated seedlings, respectively. The higher the glucose concentration was, the greater the numbers of grana, thylakoids, and PLBs. Glucose increased the green pigment contents and grana formation in newly developed leaves in a dark condition, and the mechanisms are discussed.

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.

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.

Regeneration of double haploid plants from unpollinated ovary cultures of watermelon

Background: Watermelon (Citrullus lanatus), a major fresh fruit, is planted worldwide. Because double haploid plants may be used as parents to shorten watermelon breeding cycle, the present study optimized conditions for regenerating haploid plants from ovaries without pollination.Results: The results revealed that, the 10% sodium hypochlorite sterilized for 10 min is best for ovary enlargement. In addition, a dark culture period of 14 days promoted the ovary enlargement. The MS medium containing 0.5 mg/L NAA, 1.0 mg/L 6-BA and 0.5mg/L KT promoted the embryoid differentiation. The M2 medium containing 0.02 mg/L TDZ, 0.5 mg/L NAA, 0.5 mg/L 6-BA could be used for producing complete plants. The different genotypes affected the embryoid induction. The present study obtained regenerated plants that were established in field. Flow cytometry analyses revealed that the regenerated plants were haploid, diploid or tetraploid plant. The seedlings which were treated with culture medium can increase the chance of chromosome doubling. The SSR marker analyses showed that the diploid and tetraploid plants were homozygous at all six loci tested, indicating that these regenerated plants were double- or tetra-haploid plants.Conclusions: Haploid and homozygous diploid can be obtained through the culture of unpollinated ovary of watermelon, which is an effective way to innovate watermelon germplasm. The present study provides homozygous plants for future watermelon breeding.

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.

Effects of light on in vitro fiber development and flavonoid biosynthesis in green cotton (Gossypium hirsutum)

As an environmental factor, light influences the physiological functions and secondary metabolism of plants. However, the role of light in cotton fiber development and pigment biosynthesis has not yet been thoroughly explored. In this study, ovules of green cotton were cultured in vitro under dark and light conditions, and fiber and ovule growth parameters as well as fiber carbohydrate and cellulose contents and the expression of genes related to fiber development were investigated to elucidate the effect of light on fiber development. In addition, to investigate the effect of light on fiber pigment biosynthesis, the fiber flavonoid content and related gene expression were determined. The results demonstrated that the fiber length and the expression levels of fiber elongation genes under light culture were significantly lower than under dark culture, however, the ovule and fiber weight were significantly higher than under dark culture. The fiber developed under light culture had higher carbohydrate concentrations and carbohydrate transformation rate than under dark culture. Additionally, light culture exhibited higher cellulose contents and expression levels of cellulose biosynthesis genes compared with dark culture. In contrast, the pattern of the effect of light on flavonoid biosynthesis differed from that for cellulose biosynthesis. At 10 DAC (days after culture) and 20 DAC, the flavonoid contents and the expression levels of genes related to flavonoid biosynthesis were lower than under dark culture. However, the flavonoid contents and gene expression levels observed at 30 DAC and 40 DAC were higher in the light culture than in the dark culture. These results suggested that light hindered fiber elongation, but promoted carbohydrate accumulation and carbohydrate transformation, which resulted in fiber weight gain and increased cellulose accumulation in fibers. In addition, light inhibited flavonoid biosynthesis at early stage of fiber development, but promoted it at later stages. These findings provide the basis for intensive study of fiber development and flavonoids biosynthesis in green cotton.

In Vitro Culture and Rooting of Diospyros virginiana L.

The hard, strong, very close-grained wood of common persimmon (Diospyros virginiana L.; Ebenaceae) is desirable for specialty products such as golf club heads, percussion sticks, billiard cues, and for wood turnery. The edible fruit of cultivated varieties is sold as pulp for use in puddings, cookies, cakes, and custards. Persimmon is usually propagated by grafting. Own-rooted clonal persimmon could offer several advantages to specialty fruit growers such as elimination of grafting, graft incompatibility issues, and improved rootstocks for variety testing. Four mature, grafted (male and female) persimmon genotypes and one hybrid were used for nodal explant culture. Nodal stem explants were cultured on Murashige and Skoog (MS) medium containing 10 μM zeatin, 3% (w/v) sucrose, and 0.7% (w/v) Bacto agar. Explants were routinely transferred to fresh medium every 3 weeks until shoot cultures were established. All nodal explants excised from grafted greenhouse plants produced at least one viable shoot. For in vitro rooting of microshoots, half-strength MS medium with 0, 5, 10, or 15 μM indole-3-butyric acid (IBA), 0.1 g·L−1 phloroglucinol, 3% (w/v) sucrose, and 0.7% (w/v) Bacto agar were tested with a 10-day dark culture treatment followed by culture in the light. Best rooting (14% to 87%) was achieved on medium containing 5 μM IBA for the common persimmon genotypes with means averaging from 0.5 to 3.9 roots per shoot. Ninety-one percent rooting with 5.3 ± 2.6 roots per shoot was achieved for the hybrid persimmon. Rooted plants were successfully acclimatized to the greenhouse.

Production of Synseed for Hybrid Cymbidium Using Protocorm-Like Bodies

Synthetic seed were produced from protocorm-like bodies (PLBs) of hybrid Cymbidium Twilight Moon ‘Day Light’ after culture on a new medium, Teixeira Cymbidium (TC) medium. This new medium contained, in addition to a unique selection of macro- and micronutrients, 0.1 mg/l α-naphthaleneacetic acid and 0.1 mg/l kinetin, 2 g/l tryptone and 20 g/l sucrose, and was solidified with 8 g/l Bacto agar. Several explant types and sizes (intact PLBs, half-PLBs, PLB longitudinal thin cell layers) were tested. In addition, pretreatment of PLB-synseeds with 200 mM KNO3 solution, the addition of activated charcoal or coconut water to synseeds, light vs dark culture, short-term (1 month) and long-term (6 and 12 months) low-temperature (4°C) storage, as well as cryostorage were also tested. All treatments resulted in less PLBs than the control treatment. Among all these treatments, only the use of TC medium or incorporation of coconut water into synseeds resulted in “germination” while lowtemperature storage (1-6 months) was only possible under liquid TC. These results would allow for the short-term preservation of Cymbidium germplasm but not for effective cryopreservation.