High-quality Cleavage Embryo versus Low-quality Blastocyst in Frozen-thawed Cycles:Comparison of Clinical Outcomes

This study compared the clinical outcomes of the frozen-thawed cycles of high-quality cleavage embryos with low-quality blastocysts to provide a reference for the choice of frozen-thawed embryo transfer schemes and to improve clinical pregnancy rates.A retrospective analysis was performed on the clinical data of patients undergoing frozen-thawed embryo transfer at the Reproductive Medicine Center of Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology from 2016 to 2017.In total,845 cases were divided into a high-quality cleavage embryo group(group A)and a low-quality blastocyst group(group B).Each group was further divided into subgroups based on the number of transplants.Group A was categorized into two subgroups comprising of 94 cases in subgroup Al(1 high-quality 8-cell group)and 201 cases in subgroup A2(2 high-quality 8-cell group).Group B was divided into four subgroups consisting of 73 cases in subgroup B I(D53BC group),65 cases in subgroup B2(D54BC group),110 cases in subgroup B3(D63BC group),and 282 cases in subgroup B4(D64BC group).The pregnancy outcomes and neonatal outcomes between the groups were compared.The clinical pregnancy rates(56.72%and 60.00%)and live birth rates(47.76%and 46.15%)in subgroups A2 and B2 showed no significant differences,but these rates were significantly higher in subgroups A2 and B2 than in the rest subgroups(P<0.05).The multiple birth rate(26.32%)in the subgroup A2 was significantly higher than that in the rest subgroups(P<0.05).There were no statistically significant differences in the abortion rates among all groups(P>0.05).In terms of neonatal outcomes,there were no statistically significant differences in the proportion of premature births,sex ratios,and birth defects among the low-weight and gigantic infants(P>0.05).Transplanting two high-quality cleavage embryos during the frozen-thawed embryo transfer cycles could significantly increase clinical pregnancy rates and live birth rates,but at the same time,it also increased the risks of multiple births and complications to mothers and infants.The D54BC subgroup had the most significant advantages among all groups(P<0.05).The rest low-quality blastocysts had clinical outcomes similar to the single high-quality cleavage embryo group.

Pre-existing orthorhombic embryos-induced hexagonal-orthorhombic martensitic transformation in MnNiSi_(1-x)(CoNiGe)_x alloy

The thermal-elastic martensitic transformation from high-temperature Ni_(2)In-type hexagonal structure to low-temperature TiNiSi-type orthorhombic structure has been widely studied in MnMX(M=Ni or Co,and X=Ge or Si)alloys.However,the answer to how the orthorhombic martensite nucleates and grows within the hexagonal parent is still unclear.In this work,the hexagonal-orthorhombic martensitic transformation in a Co and Ge co-substituted MnNiSi is investigated.One can find some orthorhombic laths embedded in the hexagonal parent at a temperature above the martensitic transformation start temperature(M_(s)).With the the sample cooing to M_(s),the laths turn broader,indicating that the martensitic transformation starts from these pre-existing orthorhombic laths.Microstructure observation suggests that these pre-existing orthorhombic laths do not originate from the hexagonal-orthorhombic martensitic transformation because of the difference between atomic occupations of doping elements in the hexagonal parent and those in the preexisting orthorhombic laths.The phenomenological crystallographic theory and experimental investigations prove that the pre-existing orthorhombic lath and generated orthorhombic martensite have the same crystallography relationship to the hexagonal parent.Therefore,the orthorhombic martensite can take these pre-existing laths as embryos and grow up.This work implies that the martensitic transformation in MnNiSi_(1-x)(CoNiGe)_(x) alloy is initiated by orthorhombic embryos.

A New Micropropagation Technology of Tilia amurensis:In VitroMicropropagation of Mature Zygotic Embryos and the Establishment of a PlantRegeneration System

Tilia amurensis is an economically valuable broadleaf tree species in Northeast China.The production of highqualityT.amurensis varieties at commercial scales has been greatly limited by the low germination rates.Thereis thus a pressing need to develop an organogenesis protocol for in vitro propagation of T.amurensis to alleviate ashortage of high-quality T.amurensis seedlings.Here,we established a rapid in vitro propagation system forT.amurensis from mature zygotic embryos and analyzed the effects of plant growth regulators and culture mediain different stages.We found that Woody plant medium(WPM)was the optimal primary culture medium formature zygotic embryos.The highest callus induction percentage(68.76%)and number of axillary buds induced(3.2)were obtained in WPM+0.89μmol/L 6-benzyladenine(6-BA)+0.46μmol/L kinetin(KT)+0.25μmol/Lindole-3-butryic acid(IBA)+1.44μmol/L gibberellin A_(3)(GA_(3)).The multiple shoot bud development achievedthe highest percentage(83.32%)in the Murashige and Skoog(MS)+2.22μmol/L 6-BA+0.25μmol/L IBA+1.44μmol/L GA_(3).The rooting percentage(96.70%)was highest in 1/2 MS medium+1.48μmol/L IBA.Thesurvival percentage of transplanting plantlets was 82.22%in soil:vermiculite:perlite(5:3:1).Our study is the firstto establish an effective organogenesis protocol for T.amurensis using mature zygotic embryos.

Callus Induction from Mature Embryos of Maize

In this study we studied the factors influencing the callus induction from mature embryos of maize inbred lines Qi 319, Zhen 58, Chang 7 -2, Lx 9801 and 81162, such as genotype, combination of plant growth regulators, and low-temperature pretreatment. The results showed that the induction rate of Qi 319 was the highest among the four genotypes tested; combination of 4.0 mg/L 2,4-D ＋ 0.5 mg/L 6-BA was suitable for inducing callus from mature embryos; three days of 4℃ pretreatment can promote the callus induction significantly. The indices optimized in the present study are helpful for establishing genetic transformation system in maize without considering seasonal variation.

Effects of Genotypes and Basic Medium on Culture of Maize Mature Embryos

[Objective]This study was to screen out suitable genotypes and basic medium for the culture of maize mature embryos.[Method]Using mature embryos of nine maize genotypes as explants,the effects of genotypes and basic medium on callus induction and subculture were investigated.[Result]The genotypes performed better in callus induction and subculture were found in turn 853-35,853-209,Dan 34 and 81162.MS medium is better than N6 medium in the callus induction from maize embryos,while N6 medium is more suitable for callus subculture.[Conclusion]Our study further improved the tissue culture system in maize with mature embryos as explants.

Fluctuations of Endogenous Hormones in Isolated Rice Embryos During Embryogenesis and Early Stages of Germination

Fluctuations of levels of several endogenous plant hormones in isolated rice ( Oryza sativa ssp. japonica) embryos during early and mid-embryogenesis and early stages of germination were studied by enzyme-linked immunosorbent assay (ELISA). Embryos were collected at different days after pollination (DAP) and different days after imbibition (DAI) of mature seeds. The contents of gibberellin(1) (GA(1)), abscisic acid (ABA), isopentenyladenine and isopentenyladenosine ( iPAs), zeatin and zeatin riboside ( ZRs) were immunochemically assayed. The GA(1) level was the highest among all hormones tested. The variations of GA(1) levels were opposite with the ABA levels, with some exceptions. During early and mid-embryogenesis, the levels of GA(1) and ABA were the highest at 4 DAP. From 8 to 18 DAP, GA(1) level declined, whereas the ABA level increased. During germination, GA(1) level increased at 2 DAI whereas simultaneously the ABA content decreased. The highest ratio of GA(1)/ABA was observed at 2 DAI The levels of iPAs and ZRs were maxima in the embryos at 4 DAP, decreased to a very low level and maintained constant thereafter. Our results provide further evidence that GA(1) plays an important role in the early stages of embryo development and germination. The balance between GA(1) and ABA, rather than their absolute contents, controls these processes throughout the development, whereas iPAs and ZRs may play important roles in early embryogenesis. The use of isolated embryos as starting material avoids the usual interferences with other tissues such as the endosperm. In addition, this is the first report dealing with the hormonal balance of early-embryos in rice.

Using Isolated Embryo Sacs and Early Proembryos for Localization of Calmodulin mRNA Before and After Fertilization in Nicotiana

An in situ hybridization technique for localization of calmodulin(CaM) mRNA in isolated entire embryo sacs and proembryos in Nicotiana tabacum L.cv.W38 has been developed. This technique can be applied to small amounts of materials in which a whole view of CaM mRNA distribution can be obtained. The authors revealed that CaM mRNA expression changes dramatically before and after fertilization. Especially interesting is that a prominent CaM mRNA band appears between the egg apparatus and polar nuclei temporarily during the period of pollination and fertilization. The band disappears just prior to fertilization and expands to a fan_shaped region that occupies the micropylar portion of the embryo sac. After fertilization, CaM mRNA accumulates in the elongated zygotes with higher concentration in their chalazal portion than in the micropylar portion. Such an asymmetrical pattern continues to manifest in the early proembryos. It is supposed that CaM mRNA may be involved in the early events and signaling steps associated with double fertilization and zygote polarization in higher plants.

Studies on Enhancing Yield of Embryos in Microspore Culture of Brassica napus L.

In ecological zone of Chengdu, Sichuan, microspore culture was carried out in Brassica napus L. to study the influencing factors on microspore culture. The results showed that the temperature on microspore formation stage, day and night temperature, disinfection solution of buds, cultivation concentration on microspore and strain-age were both important influencing factors on microspore culture. At a temperature below 5 ℃ or above 20 ℃, the material had a much lower embryo producing rate or even could not produce any embryo, but at the optimum temperature of 10 -15 ℃ the embryo yield was up to 300 pieces per bud; the best cultivation effect appeared when 0. 1% HgCl2 was used for disin- fection; the best density of microspore was 3 -4 buds per dish; In 2009, while strain-age was from 125 d to 150 d, the microspore embryo yield increased as strain-age increased. When stain-age was 150 days, the microspore embryo yield was up to the highest, but the yield declined after 150 days.

Study on Culturing Prunus salicina cv.Zaoshi Embryos

[Objective]The aim was to study the effective factors on culturing Prunus salicina cv.Zaoshi embryos in vitro.[Method]Different age,culture medium and GA3 which affected culturing of prumssalicina embryos were discussed.[Result]The result showed that on the part of 26-41 d,after the blooming period,the embryos remained tiny and retained endosperms and showed no signs of change after having cultured for three generations.On the part of 48 d,after the blooming period,the endosperms had disappeared,the embryos kept growing until they filled the seed cavity;when they were planted on the MS culture medium,their survival rate reached 77%,in its first generation,the response of embryos was discernible.On the part of 65 d,after the blooming period,4.5% of their embryos grew into shoots on the MS culture medium;with the age of embryos growing,the survival rate of shoots increased until it reached 26% when the fruits went into ripeness;the embryos produced calli in their first generation of culturing.On the part of 65-83 d,after the blooming period,the embryos produced calli through more than 2-3 generations.On the part of 88 d,after the blooming period,the survival rate of shoots on the WPM culturing base doubled compared with that on the MS culturing base;on the same culture medium,the embryos were inhibited from growing into shoots when BA,KT or 2,4-D was added on to the culture medium.The survival rate of shoots was increased remarkably when the seeds were treated in 1 000 mg/L GA3.[Conclusion] This study provided experimental basis for the establishment of Prunus salicina cv.Zaoshi,embryos rescue techniques and cross breading.

Establishment of Plant Regeneration System from Immature Embryos of Maize(Zea mays L.) Inbred Lines

[Objective] The aim was to explore the conditions of high frequency induction of embryonic callus and plant regeneration of maize. [Method] Immature embryos of maize inbred lines were used as explants to study the effect of different 2,4-D concentrations on the induction of callus,the effect of different 6-BA concentrations on the differentiation of test-tube plantlet,as well as the effect of different IBA concentrations on the rooting of test-tube plantlet. [Result] 2,4-D showed obvious effect on the induction of inducement rate of maize,and the optimum induction medium was：N6 ＋ 2 mg/L of 2,4-D ＋ 500 mg/L of CH ＋ 500 mg/L of Pro ＋10 mg/L of AgNO3; the optimum differentiation medium was：N6 ＋ 0.5 mg/L of BA ＋ 500 mg/L of Pro; the optimum for the rooting of test-tube plantlet was 1/2 MS ＋ 0.5 mg/L of IBA. [Conclusion] The study had provided basis for the genetic transformation of maize.

A New Non-enzymatic Method to Isolate Immature Embryos of Capsella bursa-pastoris (L.) Medic.

[Objective] The aim of this study was to manually isolate the microstructures such as ovule and embryo of Capsella bursa-pastoris (L.) Medic. by the non-enzymatic method. [Method] With the gum block and dissecting microscope, its floral characteristics, ovule and embryo were manually isolated and observed by the non-enzymatic method. [Result] C. bursa-pastoris had four calyces, four petals, tetradynamous stamen and silicle, while its embryogenesis contained globular embryo, heart-shaped embryo and torpedo-shaped embryo. [Conclusion] The microstructures such as ovule and embryo of plants become easier to be isolated manually with the viscosity of gum block, which has small damage on embryo or disadvantages of keeping original shapes, and also can used in multi fields studies. This test can also be accomplished in labs with poor experiment facilities.

Effects of Alcohol and Liquid Paraffin on Development of Early Mouse Embryos in vitro

In culturing early mouse embryos in vitro,liquid paraffin and alcohol exert deleterious influence on the development of embryos. Some of light liquid paraffin produced by Chinese factories have proved harmful for early mouse embryos. As shown by our experiments, the nitronaphthalene contained and the specific gravity of liquid paraffin were not involved in the injurious effects.However,alcohol mingled in medium had harmful effects on the development of embryos. At the 0.1% concentration of alcohol in medium the proportion of embryos developing to blastocysts decreased to 73.9%. When the concentration of alcohol was increased to 0.8%, all embryos ceased developing. In our experiments, CO_2 which contained 0.13% alcohol had no visible effects on the development of embrvos in vitro.

Gene Transfer into Young Embryos via Electroporation and Regeneration of Plantlets in Rice

Green fluorescent protein ( GFP ) gene was expressed transiently in 2-3 d old rice embryos by electroporation with the aid of a specially designed loading net. Under suitable conditions (500 μF capacitance, 300 V/cm Voltage, 100 μg/mL plasmid DNA), the percentage of embryos expressing GFP was up to 35%. The highest electroporation efficiency (40%) was obtained at pH 5.8 of the electroporation buffer. The GFP gene driven by the Ubi promoter produced the highest efficiency. Thus, on the basis of optimizing electroporation conditions, a transformation system has been developed for young embryos in rice. The electroporated 4-6 d old embryos regenerated plantlets under the controlled cultural conditions. Fluorescence microscopic observations indicated that GFP gene expressed in their calli and R0 plantlets.

Plasmolysis treatment enhances the expression of callose synthase gene in zygotic embryos of Eleutherococcus senticosus

In previous study we reported that pretreatment with plasmolysis enhanced somatic embryo formation in hypocotyls of Eleutherococcus senticosus.In the present study,the expression level of callose synthase gene in embryos of E.senticosus in response to 2,4-D,sucrose and mannitol treatments was analyzed by RT-PCR.The results show that plasmolysis pretreatment using sucrose and mannitol significantly promoted the expression of callose synthase gene.Also,the thicker cell walls of explant plasmolyzed compared with controls were observed during the somatic embryogenesis.We suggest that the callose may make the cells in epidermis separate from neighboring cells and then develop into embryogenic potential cells.

The Study on the Gene Expression of Preimplantation IVF Bovine Embryos

The cattle different stage embryos obtained from in vitro was studied using the technology of single preimplantation embryo mRNA different display:single 8-cell and blastocyst stage embryos were studied using technology of mRNA different display and one different fragment was found. The result suggested that this fragment displayed high homology (99%) to cattle mRNA for ribosomal protein L31. Then to detect the expression of RPL31mRNA in 8 cell and blastocyst stage embryos by real-time quantitative PCR,the result showed the relative amount of 8 cells was 3.2 times of blastocyst's.

Improvement of Plant Regeneration from Immature Embryos of Wheat Infected by Agrobacterium tumefaciens

Wheat, one of the most important food crops, has been extensively studied with respects to plant regeneration and transformation employing the immature embryos as recipient tissues. However, the transformed tissues often become severely necrotic after co-cultivation with Agrobacterium, which is one of the major obstacles in gene delivery. In this study, wheat varieties CB037, Kenong 199, Xinchun 9, Lunxuan 987, and Shi 4185 showed desirable culture potential or high infection ability in Agrobacterium-mediated transformation. Similarly, optimal regeneration conditions were determined by testing their ability to inhibit the cell necrosis and cell death phenotype. Two auxins of 2,4-dichlorophenoxyacetic acid （2,4-D） and 3,6-dichloro-o-anisic acid （dicamba） were evaluated for highly significant effect on both callus and plantlet production, although they were genotype-dependent in wheat. Substitution of 2,4-D by dicamba enhanced the growth and regeneration ability of callus from the immature embryos of most genotypes tested. The callus growth state couldn’t be modified by adding antioxidants such as ascorbic acid, cysteine, and silver nitrate or organic additives such as thiamine HCl and asparagine to the media. On the contrary, the best tissue statement and plant regeneration was achieved by employing the media containing the simplest MS （Murashige and Skoog） components and dicamba without organic components and vitamins. Thereby, our results are thought to inhibit wheat cell necrosis effectively and suggested to be used for more wheat genotypes.

Effects of Environmental Temperature on the Regeneration Frequency of the Immature Embryos of Wheat(Triticum aestivum L.)

The immature embryos （IEs） of wheat are the most widely used tissues for in vitro culture and genetic transformation due to its high regeneration competency. However, this explant can only be maintained in 4℃ daily cooler for a short period time for its use in plant tissue culture or transformation experiments. This study aimed to investigate the effects of environmental temperature, cryopreservation storage temperature, and heat shock culture （HSC） temperature on the regeneration frequency of wheat IEs. Results indicated that environmental temperature significantly affected the induction of embryonic calli. The optimum total accumulated temperature （TAT） during the time of anthesis and sampling for regeneration of these tissues was around 280℃ for spring wheat type cv. CB037 and approximately 300℃ for winter wheat type cv. Kenong 199. Regeneration ability obviously declined when the highest environmental temperature was over 35℃ for 1 d or a high temperature between 30 and 33℃ lasted for 5 d during anthesis and sampling. This finding was verified by culturing the freshly isolated IEs under different temperatures from 29 to 37℃ in different controlled growth incubators for 5 d; the IEs almost completely lost regeneration ability when the temperature rose to 37℃. Cryopreservation of-20℃ caused the wheat samples lost ability of producing callus or embryonic callus in a few days, and cryopreservation of-10℃ more than 10 d made the regeneration potential of the tissues dramatically declined. Comparatively, the temperature that best maintained high regeneration ability was -5℃, at which the materials can be maintained for around 1 mon. In addition, the preservation of the immature samples at -5 or -10℃ inhibited the direct germination of the IEs, avoiding the embryo axis removing process. Our results are useful for ensuring that field collection and cryopreservation of the wheat IEs are done correctly to enable tissue culture and genetic transformation.

Study on Plant Regeneration of Wheat Mature Embryos Under Endosperm-Supported Culture

To reveal the suitability of using mature embryos as an explant source in wheat tissue culture, mature embryos from eight common wheat cultivars （Triticum aestivum L. cv.） were cultured with or without endosperm to test their efficiency of callus induction and plant regeneration. When embryos were cultured together with endosperm （endosperm-supported culture, ES）, the percentage of callus induction was significantly lower than that when embryos were cultured in the absence of endosperm （non-endosperm-supported culture, NES）. This pattern was evident in most genotypes, regardless of whether 2 or 8 mg L^-1 2,4-D was added in the NES culture. However, in ES culture, more induced calli were differentiated into distinct green spots and they further developed into plantlets. Thus, more plants were regenerated in ES culture than in the NES treatment. Most of the eight tested genotypes showed a significant difference in callus induction rate and plantlet regeneration in both ES and NES cultures. In addition, the enzymatic activity of oxalate oxidase in the callus of ES culture condition was obviously higher than that in the callus of NES culture condition, suggesting that the activity of oxalate oxidase may be a parameter for selection of calli with potential for plantlet regeneration. These results indicate that wheat mature embryos are valuable explants for highly efficient callus induction and plant regeneration, if proper treatment and medium are used.

Toxicity of Multi-Walled Carbon Nanotubes,Graphene Oxide,and Reduced Graphene Oxide to Zebrafish Embryos

Objective This study was aimed to investigate the toxic effects of 3 nanomaterials, i.e. multi-walled carbon nanotubes （MWCNTs）, graphene oxide （GO）, and reduced graphene oxide （RGO）, on zebrafish embryos. Methods The 2-h post-fertilization （hpf） zebrafish embryos were exposed to MWCNTs, GO, and RGO at different concentrations （1, 5, 10, 50, 100 mg/L） for 96 h. Afterwards, the effects of the 3 nanomateria on spontaneous movement, heart rate, hatching rate, length of larvae, mortality, and malformations Is were evaluated. Results Statistical analysis indicated that RGO significantly inhibited the hatching of zebrafish embryos. Furthermore, RGO and MWCNTs decreased the length of the hatched larvae at 96 hpf. No obvious morphological malformation or mortality was observed in the zebrafish embryos after exposure to the three nanomaterials. Conclusion MWCNTs, GO, and RGO were all toxic to zebrafish embryos to influence embryos hatching and larvae length. Although no obvious morphological malformation and mortality were observed in exposed zebrafish embryos, further studies on the toxicity of the three nanomaterials are still needed.

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L.‘Moldova’

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L^-1 6-BA and 2 mg·L^-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L^-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.