In vitro induction of tetraploids and their phenotypic and transcriptome analysis in Glehnia littoralis.

BACKGROUND: Glehnia littoralis is a medicinal and edible plant species having commercial value and has several hundred years of cultivation history. Polyploid breeding is one of the most important and fastest ways to generate novel varieties. To obtain tetraploids of G. littoralis in vitro, colchicine treatment was given to the seeds and then were screened based on morphology, flow cytometry, and root tip pressing assays. Furthermore, transcriptome analysis was performed to identity the differentially expressed genes associated with phenotypic changes in tetraploid G. littoralis. RESULTS: The results showed that 0.05% (w/v) colchicine treatment for 48 h was effective in inducing tetraploids in G. littoralis. The tetraploid G. littoralis (2n = 4x = 44) was superior in leaf area, leaf thickness, petiole diameter, SPAD value (Chl SPAD), stomatal size, epidermal tissues thickness, palisade tissues thickness, and spongy tissues thickness to the diploid ones, while the stomatal density of tetraploids was significantly lower. Transcriptome sequencing revealed, a total of 1336 differentially expressed genes (DEGs) between tetraploids and diploids. Chromosome doubling may lead to DNA content change and gene dosage effect, which directly affects changes in quantitative traits, with changes such as increased chlorophyll content, larger stomata and thicker tissue of leaves. Several up-regulated DEGs were found related to growth and development in tetraploid G. littoralis such as CKI, PPDK, hisD and MDP1. KEGG pathway enrichment analyses showed that most of DEGs were enriched in metabolic pathways. CONCLUSIONS: This is the first report of the successful induction of tetraploids in G. littoralis. The information presented in this study facilitate breeding programs and molecular breeding of G. littoralis varieties.

Sporogenesis, gametophyte development and embryogenesis in Glehnia littoralis.

BACKGROUND: Glehnia littoralis is an economic herb with both medicinal and edible uses. It also has important ecological value and special phylogenetic status as it is a monotypic genus species distributing around beach. Little information on its reproductive biology has been reported so far, which has hindered conservation and application of this species. In this study, we observed morphological changes from buds emergence to seeds formation and internal changes during sporogenesis, gametophyte development and embryo and endosperm development of G. littoralis using paraffin-embedded-sectioning and stereo microscope. RESULTS: The results showed that the stages of internal development events of G. littoralis corresponded to obvious external morphological changes, most of developmental features were consistent with other Apiaceae species. The development of male and female gametophytes was not synchronized in the same flower, however, exhibited temporal overlap. From mid-late April to mid-May, the anther primordial and ovule primordial developed into the trinucleate pollen grain and eight-nuclear embryo sac, respectively. From late-May to mid-July, the zygote developed into mature embryo. In addition, some defects in gynoecium or ovule development and abnormal embryo and endosperm development were found. We induced that the possible causes of abortion in G. littoralis were as follows: nutrient limitation, poor pollination and fertilization, and bad weather. CONCLUSIONS: This study revealed the whole process and morphological characteristics of the development of reproductive organ in G. littoralis, which not only provided important data for the study of systematic and conservation biology, but also provided a theoretical basis for cross breeding.

Complete chloroplast genome and phylogenetic analysis of Anemone shikokiana.

BACKGROUND: Anemone shikokiana (Makino) Makino, disjunctly distributed in Shandong Peninsula of China and Shikoku Island of Japan, is a rare and endangered species. To provide genetic information and understand its phylogeny, we conducted research on the chloroplast (cp) genome of A. shikokiana. METHODS AND RESULTS: The complete cp genome sequence of A. shikokiana was constructed in this study. The results showed that the cp genome of A. shikokiana has a typical quadripartite cyclic with a total length of 159,286 bp. In total, 111 unique genes were identified, including 78 protein-coding genes, 29 tRNA-coding genes and 4 rRNA-coding genes. A total of 37 long repeat sequences and 67 microsatellites were found in this cp genome. The cp genome of A. shikokiana was compared with eleven other Anemone cp genomes available from the Genbank database. We found some variations among the different genomes, especially in the LSC and SSC regions, and identified some regions as potential molecular markers such as ycf1, ndhE, ndhD, ndhF-trnL, ndhA and ndhF. The results of phylogenetic analysis suggested that A. narcissiflora was the closest relative of A. shikokiana. CONCLUSIONS: The results filled the gap of cp genome sequence information of A. shikokiana, laying the foundation to explore the evolutionary relationships of A. shikokiana in future studies. It provided a valuable genetic resource for the molecular identification and phylogenetic study of Anemone.

[C:N:P stoichiometry in plants and soils of Phragmites australis wetland under different water-salt habitats]. / 不同水盐生境下芦苇湿地植被及土壤碳氮磷生态化学计量特征.

We examined the effects of channel diversion of Yellow River on the content and stoichiometry of carbon (C), nitrogen (N) and phosphorus (P) in the organs of reeds (stem, leaf, rhizome and fibrous root) and soils in three typical Phragmites australis communities in the Yellow River Delta, including P. australis community in the former Yellow River course abandoned in 1996, P. australis community on the new Yellow River course and the P. australis communities on the intertidal area (far from the abandoned and current channel but affected by the tides). The results showed that foliar C, N and P contents of P. australis were highest in the communities of abandoned Yellow River course. Leaf N, stem C and rhizome P contents were highest in the communities of new Yellow River course. Leaf N and stem C and P contents were highest in the communities of intertidal area. The average leaf C (409.48 g·kg-1) and P (1.09 g·kg-1) contents in the three habitats were lower than national and global average levels, while leaf N content (21.71 g·kg-1) was higher than that of national and global average levels. The mean leaf N:P (20.22) was higher than 16 and the mean soil N:P (0.87) was lower than 14, indicating that the P. australis growth in the three habitats was limited by P. Correlation analysis showed that EC was one of the main factors affecting C:N:P stoichiometry in P. australis. In general, the C and P reserves in P. australis in the study area were low, and N reserve was high. The soil organic carbon content was low, the soil C reserves were large, while the N and P were relatively scarce.

Ethanol affects behavior and HPA axis activity during development in zebrafish larvae.

Recent studies have shown that long-term alcohol intake from food can lead to numerous mental disorders in humans, and the social and health effects of excessive intake of alcohol currently represent serious problems for governments and families worldwide. However, to date, it has not been determined how alcohol affects the hypothalamic-pituitary-adrenal (HPA) axis. The zebrafish offers a good model for studying the toxicology of food-grade ethanol. In the present study, using zebrafish larvae exposed to 1% ethanol, we performed zebrafish behavioral analysis. Samples were collected for enzyme-linked immunosorbent assay (ELISA) and quantitative real time-polymerase chain reaction (qRT-PCR) experiments, and statistical analysis was performed. We found that ethanol decreased the locomotor activity of zebrafish larvae, which showed a more intense reaction to external stimuli. Ethanol also increased the level of HPA axis hormones in zebrafish larvae, influenced the level of neurotransmitters, and altered the expression of key genes in neurotransmitter metabolism. Ethanol exposure affects zebrafish behavior, increases the level of HPA axis hormones in zebrafish larvae, affects the level of neurotransmitters, and affects the expression of key genes in dopamine and serotonin metabolism. These findings may help to elucidate the effects of ethanol on HPA axis activity.

[Cryopreservation of Cyclamen persicum Mill. Callus].

In this paper we studied cryopreservation of Cyclamen persicum Mill. callus to avoid variations produced by sub-culture. The callus in the logarithmic phase after sub-culture were used for experiments. Firstly, the callus were pre-cultured in culture-medium containing 4%, 6% or 8% sucrose for different time periods, transferred to different cryoprotectants to directly cryopreserve or incubated for 2 hours at -20 degrees C, then submersed in liquid nitrogen, lastly thawed rapidly in a waterbath at 37 degrees C, and washed with liquid culture-medium containing the corresponding concentration of sucrose. Cell survival rate was computed after stained by Neutral Red, and SPSS 13.0 software was used for statistical analysis. The results showed that sucrose concentration, pre-culture time, cryoprotectants had various impacts on cell survival rate. We have developed a simple but effective protocol for the cryopreservation of callus of C. persicum. Of the different protocols tested, 4%sucrose, pre-culturing for 3 days, No. 9 cryoprotectant and freezing directly after 30 minutes at 0 degrees C results in the highest cell survival rate.