Secondary metabolites are closely related to the nutritional quality and health functions of plants.  We investigated the secondary metabolites of both wild (n=23) and cultivated (n=27) pomegranate plants (Punica granatum L.) growing in China.  The total flavonoid (TF) and tannin (TT) contents from the peel and juice were determined and the secondary metabolites in the peel (ZLP) and juice (ZLZ) of ‘Zela 4’ were identified using liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS).  Analysis of variance (P<0.05) showed that there were significant differences in the TF content of peel (TF (P)) and juice (TF (J)), and the TT content of peel (TT (P)) and juice (TT (J)) among different pomegranate accessions.  Pearson correlation analysis showed that latitude and altitude might be the main environmental factors affecting TF and TT contents in pomegranates.  In this study, 279 secondary metabolites were identified in the ZLP and ZLZ.  In addition, we report for the first time 227 secondary metabolites in pomegranates.  Using orthogonal partial least squares discriminant analysis, 90 differential metabolites were identified in ZLP and ZLZ.  In addition, we screened eight specific germplasms (high-TF (P), ‘Junyong 3’; low-TF (P), ‘Yanzhihong’; high-TF(J), ‘Zela 4’; low-TF (J), ‘Yudazi’, high-TT (P), ‘Junyong 4’; low-TT (P), ‘Anba 1’; high-TT(J), ‘Yeba 1’; and low-TT (J), ‘Baihuayushizi’).  The results of our study provide a reference for the development and utilization of wild pomegranate resources and pomegranate breeding in China.

    The antioxidant of reduced glutathione (GSH) is the most abundant thiol in cells for the maintenance of the intracellular redox balance. The study aimed to assay the effect of sperm treatment with GSH before incubation with oocytes on the development potential of embryos obtained by in vitro fertilization (IVF). Also the mitochondrial membrane potential (ΔΨm), plasma membrane integrity (viability), DNA fragmentation, reactive oxygen species (ROS) content, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities, methane dicarboxylic aldehyde (MDA) level as indices of lipid peroxidation in sex-sorted and unsorted sperm from three bulls were investigated using flow cytometry and enzyme-labeled instrument individually. The results showed that 2 mmol L^–1 GSH increased significantly the cleavage rate (86.68% vs. 82.78%), 4- to 8-cell rate (82.30% vs. 73.43%) and blastocyst rate (43.15% vs. 35.24%) of IVF embryos compared with untreated group. Furthermore, addition of GSH increased significantly the ΔΨm and viability, decreased the ratio of DNA fragmentation in sex-sorted or unsorted semen (P<0.05), except the sex-sorted semen from bull 019. Similarly, activities of SOD, CAT and GPx were increased significantly. However, the contents of MDA were decreased significantly both in sex-sorted and unsorted semen treated with GSH (P<0.05). These results suggest that sperm pretreatment with GSH during IVF can maintain better the viability and fertility of sperm through reducing apoptosis and increasing the antioxidant capacity, which improves the IVF embryos development.

The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres (groups of at least 30 at the eight-cell stage) were separated into eight segments (to regard an eight-cell embryo as a tangerine, a blastomere as one segment) and one, two and four segments (blastomeres) were cultured respectively in microwells on the bottom of the four-well dish (Nunc, Denmark) with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics (cocultured with nothing, intact embryos, bovine cumulus cells (bCCs), intact embryos & bCCs) were applied to the group of four segments (blastomeres). Finally, diameter and inner cell mass (ICM):trophectoderm (TE) cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group (P<0.05). In addition, rate of blastocyst formation was significantly increased when the four segments were cocultured with intact embryo & bCCs (P<0.05). Although the ICM, TE and total cells of blastocysts derived from separated blastomeres was less than the control group from intact embryo (P<0.05), more important quality indicator of the blastocyst diameter and ICM:TE cell ratio was similar between our experimental group and the control group (P>0.05). Thus, these results suggest that combined with intact embryos & bCCs coculture system, culturing four isolated segments (blastomeres) per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.

Wheat (Triticum aestivum L.) often experiences photoinhibition due to strong light during the grain filling stage. As such, increasing the tolerance of wheat to photoinhibition is very desirable in breeding efforts focused on increasing grain yields. Previous reports have suggested that PROTON GRADIENT REGULATION 5 (PGR5) plays a central role in the generation of a proton gradient across the thylakoid membrane (DpH) and in acclimation to high light intensity conditions. Three PGR5 homoeologues were isolated from wheat, and mapped onto chromosomes 7A, 7B and 7D, respectively. The TaPGR5s shared highly similar genomic sequences and gene structures. The transcripts of TaPGR5s were found to be abundantly expressed in the flag leaves, and were transiently up-regulated by treatment with high light. High light treatment inhibited the net photosynthetic rate (Pn) and the maximal quantum yield of photosystem II (Fv/Fm). Further, these inhibitions were more evident in the leaves with reduced expression of TaPGR5s achieved using virus-induced gene silencing methods. Moreover, reducing TaPGR5 expression impaired the induction of non-photochemical quenching (NPQ), which caused more severe cell membrane damage and lipid peroxidation in high light. Additionally, we observed that TaPGR5s transcripts were more abundantly expressed in the wheat genotypes with higher ms-delayed light emission (ms-DLE), a value reflecting transthylakoid DpH. These results suggested that TaPGR5s play important roles in the tolerance of wheat to photoinhibition.

EST-derived SSR marker has been developed in many species, but few methods of high efficiency have been reported for the exploitation of EST-SSR markers. Thus, a high efficiency method for mining millions of redundant EST data is needed. A modified method for the EST-SSR development with high efficiency was established based on the redundant EST data of soybean in this study. The method achieved its function through classifying ESTs according to the same SSR motif and detected candidate loci with redundant sequences. In this study, a total of 80 polymorphic EST-SSR markers of soybean were developed, 50 of them were exploited by this modified method which proved the higher speed and efficiency of this method. All the 80 polymorphic EST-SSRs were mapped on soybean physical map through in silico mapping and 15 markers were integrated on a genetic map constructed in previous study. A software named hpSSR (high polymorphic SSR) was programmed based on the concept of the up-built method for EST-SSR development. This method is not only pragmatic for EST-SSR exploitation in soybean, but also effective for the development of the marker in other species if the redundancy EST data is available.

To our knowledge, no single study has systemically compared cryopreservation efficiencies of bovine blastocysts derived from in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT) by controlled freezing and vitrification. This experiment, therefore, was designed to compare the cryopreservation of these blastocysts with controlled freezing and OPS vitrification. Adenosine-5´-triphosphate (ATP) content and reactive oxygen species (ROS) level in blastocysts were also analyzed. Firstly, for each type of blastocyst (IVF, ICSI or SCNT), significant differences were observed between the survival rates of the controlled freezing ((81.56±2.33), (68.18±4.72) or (47.89±5.83)%) and OPS vitrification groups ((92.24±4.54), (82.40±3.76) or (78.71±5.91)%; P<0.05). Secondly, for each type of blastocyst (IVF, ICSI or SCNT), ATP content was significantly decreased after controlled freezing or vitrification, and the ATP content in the controlled freezing group (0.43±0.06), (0.35±0.05) or (0.21±0.02) pmol) was significantly lower than that found in the OPS vitrification group (0.62±0.04), (0.46±0.03) or (0.30±0.01) pmol; P<0.05). Thirdly, ROS level in fresh IVF ((47.33±3.56) c.p.s (counted photons per second), ICSI ((36.51±2.58) c.p.s) or SCNT blastocysts ((26.44±1.49) c.p.s) was significantly lower than that found in the OPS vitrification group ((72.14±4.31), (58.89±3.89) or (40.11±5.73) c.p.s; P<0.05), but higher than that of the controlled freezing group (34.41±3.32), (23.13±1.26) or (15.46±2.45) c.p.s; P<0.05). The present study indicated that vitrification is more efficient in the cryopreservation of bovine blastocysts derived from IVF, ICSI or SCNT than controlled freezing. Furthermore, both vitrification and controlled freezing significantly altered the ATP content and ROS level in those blastocysts.