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.

Preliminary Study on Transgenesis by Injecting Exogenous DNA into Zygote Cytoplasm of Buffalo

[Objective] This study aimed to investigate the feasibility of transgenesis by injecting exogenous DNA into zygote cytoplasm of Buffalo. [Method] Buffalo oocytes were randomly divided into two groups 20-22 h after in vitro maturation. One group of oocytes was introduced with about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection 7-10 h or 18-20 h after in vitro fertilization （IVF）; the other group of oocytes was introduced with mixture of a single buffalo sperm and about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection （generally called ICSI-Mediated Gene Transfer, ICSI-Tr）. Expression of exogenous DNA was observed and recorded during the process of embryonic development. [Result] Early embryonic gene expression efficiency and blastocyst gene expression efficiency in IVF injection group showed no significant difference compared with that in ICSI-Tr group （P0.05）. In addition, the cleavage rate and early embryonic gene expression efficiency in IVF injection group were significantly higher with injection at 7-10 h post IVF than that at 18-20 h post IVF （P0.05）. [Conclusion] These results indicate that transgenic buffalo embryos can be generated by injecting exogenous DNA into cytoplasm of IVF oocytes, and the optimal injection time is 7-10 h post IVF.

High Frequency of GFP Gene Transient Expression in Electroporated Zygotes and Early Proembryos of Wheat

Green fluorescent protein (GFP) gene was successfully transferred into the isolated zygotes and early proembryos of wheat (Triticum aestivum L.) by electroporation. A frequency, as high as 46.7% of GFP gene transient expression in early proembryos, was achieved under 150 V/cm electric field strength, 25 muF capacitor, 200 mug/mL of linear plasmid DNA and an electroporation buffer at pH 7.2. Compared with five-day-old proembryos, the zygotes and early proembryos needed lower optimum strength of electric field. After culturing in KM8p medium, the electroporated early proembryos divided and GFP gene expression was observed in daughter cells and subsequent divisions. There was no mosaicism of gene expression in the zygotes and 2-, 4- and 8-celled proembryos.

The role of Dby mRNA in early development of male mouse zygotes

Ejaculated mammalian spermatozoa contain a complex yet specific population of mRNA. However, the possible roles that mRNA has in early zygotic and embryonic development remain unclear. We found that Dby mRNA is selectively retained in capacitated mouse spermatozoa, and is transferred into the oocyte during fertilization by reverse transcription-polymerase chain reaction even though no DBY protein expression is detected. The cellular location ofDby mRNA is seen in the post-acrosome region, and it comprises nearly half of the mouse spermatozoa in in situ hybridization. In contrast, transcripts of the control gene, Smcy, are not detected in capacitated mouse spermatozoa, although the H-Y antigen encoded by Smcy is expressed on the surface of the spermatozoa. In our microinjection experiment, the zygotic development rate of the as-Dby male pronucleus injection group was significantly lower than that of the as-Smcy male pronucleus injection group （35.9% vs. 95%, P = 0.001） and the as-Dby female pronucleus injection group （35.9% vs. 93.8%, P = 0.001）. The rate of male-developed zygotes was also lower than that of the as-Smcy male pronucleus injection group （17.4% vs. 57.9%, P = 0.002） and the as-Dby female pronucleus injection group （17.4% vs. 54.1%, P = 0.002）. Thus, we conclude that Dby mRNA is selectively retained in capaci- tated mouse spermatozoa, and it has an important role in the early zygotic development of male mouse zygotes. This might imply that spermatozoa mRNA is involved in early zygotic and embryonic stages of reproduction.

Improvement of the zygote utilization and reduction of the seedling loss in the early stage of seedling production of Sargassum thunbergii (Fucales, Phaeophyta)

Artificial seedling production of Sargassum thunbergii is an effective way to relieve pressure on natural resources.In order to improve the utilization of zygotes and reduce the loss of seedlings,studies on the characteristic of the zygotes release,the development of rhizoids,the attachment of germlings,and the influence of jet washing were conducted.Results show that the percent of zygotes released was increased with time in the first 60 h.The capacity of germlings attached to the substratum was significantly increased,especially coincident with the time of the new rhizoids emerged and elongated.The detachment rate of germlings significantly decreased with the delay of starting time of jet washing or the reduction of jet washing velocity.However,the jet washing at any level applied in the experiment could cause considerable loss of germlings within the 20 days after the attachment.Our study provided some parameters to optimize the operation in the early stage of seedling production.

Transcriptomic Analysis of Pacific Oyster(Crassostrea gigas) Zygotes Under Hypotonic Triploid Induction

Polyploid breeding is widely used in various marine species. Low salinity treatment is an effective method of inducing triploid of bivalve mollusks. In this study, RNA-seq was performed to determine genes and pathways involved in hyposaline adaption and cell division of Pacific oyster(Crassostrea gigas) zygotes, trying to better understand the possible molecular mechanism of hypo-osmotic induction. A total of 26965 unigenes were generated in the de novo assembly of clean Illumina reads with an average length of 934 bp and N50 of 1721 bp. Of 3024 differentially expressed genes(DEGs), 2501 were up-regulated and 523 were downregulated. GO(Gene Ontology) annotation and KEGG(Kyoto Encyclopedia of Genes and Genomes) pathway analysis of these DEGs revealed that these DEGs participate a variety of biological processes including osmoregulation, cytoskeleton organization, cell survival and death, and substantially modulate cell proliferation and embryonic development. In summery, RNA-seq methodology was applied for the first time to demonstrate hypotonic-induced transcriptomic alteration in oyster zygotes. Our findings not only interpreted the relatively high mortality of induced larvae, but also provided a valuable reference for further investigations on the mechanism of hyposaline induction, thus should aid to the application of low salinity in triploid induction in large scale aquaculture in future.

Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection

Background： Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i） how much linear DNA to inject and ii） when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein （EGFP） plasmid into buffalo zygotes. Results： First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/pL DNA was significantly higher than from those injected with 5 pg/mL. However, 50 ng/pL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/pL DNA. Second, zygotes injected early （7-8 h post-insemination [hpi]） developed better than those injected at mid （12-13 hpi） or late （18-19 hpi） time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections （16.4 % vs 7.7 %）. With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions： Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method.

哺乳动物合子基因激活时期染色体的表观遗传学变化

哺乳动物受精后,胚胎由母源因子调控逐渐转变为合子型调控,表达合子转录本,即合子基因激活(zygotic gene activation,ZGA)。其间发生了剧烈的表观遗传学变化,如组蛋白修饰、DNA甲基化重编程以及染色质重塑等。表观遗传学重塑的异常可导致严重的发育缺陷,甚至胚胎致死。因此,了解ZGA过程中的表观遗传学变化对于明确其致病机制至关重要。本文综述了哺乳动物合子基因激活时期DNA甲基化重编程、组蛋白修饰以及染色质重塑3个方面的最新研究进展,以期为深入探究哺乳动物合子基因激活时期染色体的表观遗传变化提供参考。

SIRT2在小鼠1-细胞期受精卵中的表达和定位研究

目的:探讨沉默信息调节蛋白2(SIRT2)在小鼠1-细胞期受精卵的表达和定位。方法:利用qRT-PCR、Western blotting分别检测SIRT2在小鼠1-细胞期受精卵发育全过程中mRNA和蛋白表达谱;采用细胞免疫荧光法观察SIRT2和α-tubulin的共定位情况。结果:小鼠1-细胞期受精卵SIRT2 mRNA和蛋白表达变化趋势一致,均由G 1向G 2期过渡时表达水平升高;由G 2期向M期过渡时表达水平下降(P<0.05)。在G 2期向M期过渡过程中,SIRT2的定位由细胞质向细胞核转移,于M期中期进入细胞核并定位于纺锤体,在M期后期重新定位于细胞皮质。结论:SIRT2蛋白在小鼠1-细胞期受精卵中的表达呈细胞周期时相依赖性,在1-细胞期小鼠受精卵有丝分裂间期(G 1、S、G 2)中细胞质内表达增加,积累的SIRT2在某种条件下进入细胞核调节纺锤体微管动力学,在G 2/M过渡期发挥作用。

早期胚胎发育合子基因组激活调控

动物早期胚胎发育始于分化成熟的雌雄配子经受精后重编程为全能性合子。在胚胎发育的初期,合子基因组的转录水平处于静默状态,母源物质调控占据主导地位。随着胚胎发育的进行,母源物质会经历分阶段的降解,合子基因组开始逐渐激活转录,标志着早期胚胎发育从母源性调控向合子基因组调控的转变,也称为母源-合子转换(maternal-zygotic transition,MZT)。其中一个关键的转折性事件就是合子基因组激活(zygotic genome activation,ZGA),ZGA的正确发生对于早期胚胎发育和细胞命运决定至关重要。然而,目前对于ZGA的调控因子和具体的分子机制仍知之甚少。研究表明,ZGA在不同物种中存在较大差异,可能受到DNA甲基化、组蛋白修饰、非编码RNA、染色质重塑以及ZGA相关因子等多种调控因素的影响。本文探讨了上述几种调控因素影响合子基因组激活的研究进展,对进一步研究早期胚胎ZGA的相关机制具有借鉴意义。

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.

Regeneration of <i>in Vitro</i>Shoot and Root Structure through Hormone Manipulation of Coconut (MATAG F2) Zygotic Embryos

Zygotic embryos tissues have found to be more responsive explants for clonal propagation of coconut. In the present study, the feasibility of using zygotic embryos explants for clonal propagation of a local coconut variety MATAG F2 was assessed. Callus was induced by incorporating of cytokinin and auxin into the medium. The sliced embryos explants were immersed in 1 M maltose for 60 mins, then with 0.05 M maltose for 1 min and followed by 0.01 M maltose for 5 mins was the best for prevention of phenolic compounds excretion. The callus formation depended on the concentration of 2,4-D in the media and the best effect was observed with the high level (2,4-D and BAP) tested. Attempts at inducing multiple shoot from the zygotic embryos callus were unsuccessful. No multiple shoots was present;most of the callus became root structure.

Zygotic combinatorial process in plants

Experimental data that prove the existence of the zygotic combinatorial process occurring in an embryogenesis-entering zygote are presented in the paper. The zygotic combinatorial process is found when analyzing F1 hybrid plants obtained from crossing homozygous forms different, minimum, in two marker enzymes, and it is found in that hybrid plant which, with one marker enzyme heterozygous spectrum, has a homozygous spectrum of the other. The zygotic combinatorial process leads to F1 hybrids uniformity aberration. The zygotic combinatory process revealed in the study is supposed to be conditioned by chromosome polyteny in mother plant cells and diminution of chromatin excess from the embryogenesisentering zygote. An obligatory condition for combinatorial process is the presence of free exchange of chromatides among homological chromosomes in an embryogenesis-entering cell, i.e. the presence of crossing-over analogous to the one proceeding at meiosis.

Plantlet regeneration from mature zygotic embryos andembryonic explants of masson pine (Pinus massoniana Lamb.)

Excised zygotic embryos, cotyledons and hypocotyls of juvenile seedlings of masson pine were grown on DCR medium supplemented with several concentrations of various plant phytohormones. BA (1.0 mg/ L) in combination with NAA (0.05 mg/L)in DCR medium was found to increase the formation of adventitious buds from mature zygotic embryos, but most of them were formed at the tips of embryonic cotyledons. Adventitious buds were obtained from cotyledons and hypocotyls from juvenile seedlings when they were cultured on DCR medium containing BA 3-5 mg/L and NAA 0.1-0.2 mg/L. Elongation of buds were observed on hormone-free DCR medium with or without activated charcoal (0.5%). Root initiation was achieved with full or half strength DCR inedium supplemented with IBA 1.0 mg/L and NAA 0.25-0.5 mg/L. Approximately 11-20 axillary buds formed on each explant when juvenile seedling explants were treated (3-20h) with BA 50-100 mg/L, followed by transfer to hormone-free DCR medium. The maximum number of shoots obtained per explant within six months was 33.

Cryopreservation by Vitrification of Vitis vinifera cv. ＂Red Globe＂ Zygotic Embryos and Effect on the Expression of DNA Methyltransferase Genes

Mexico is a large producer of table grape （Vitis vinifera L.） and therefore it is important to develop protocols to store the grape varieties germplasm. The objective of the present work was to design a protocol for the cryopreservation by vitrification of zygotic embryos of V. vinifera cv. ＂Red Globe＂ and evaluate possible epigenetics changes. The plant vitrification solution 2 （PVS2） was utilized before the utilization of liquid nitrogen （LN）. The effect of this protocol on embryo viability was tested by the triphenyl-tetrazolium chloride solution, as well as by the in vitro development of grape embryos into plantlet. A cDNA expression library of grape zygotic embryos was created to isolate expressed sequence tags of several DNA methyltrasferases. Gene expression of domains rearranged methyltransferase type 1 （DMR1） and DNA （cytosine-5）-methyltransferase 1 （MET1-2） isozymes was analyzed by quantitative reverse transcriptase PCR. The optimal conditions for vitrification were 10 min in 50% PVS2, followed by I0 min in 100% PVS2. Under these conditions, about 30% of plantlet was obtained from embryos after cryopreservation. It was recorded a reduction in the MET1-2 gene expression, which plays a role in the maintenance of DNA methylation. It is possible to cryopreserve viable grape zygotic embryos, although the treatment seems to induce alterations in the normal DNA methylation pattern of the zygotic embryo genome.

超低温保存处理对水曲柳胚胎生理生化特征的影响

以合子胚作为保存材料,进一步分析超低温保存处理前后的水曲柳合子胚细胞各生理生化指标的变化情况,明确冷冻方式和脱水时间对生理生化指标的影响,为建立水曲柳优良遗传材料长期保存方法提供生理数据支撑。在脱水120 min条件下,合子胚经快冻法保存后,细胞内脱氢酶活性最高,同时过氧化氢酶活性、过氧化物酶活性、脯氨酸质量分数均达到最大值,分别为1168.85 U·g^(-1)·min^(-1)、338.33 U·g^(-1)·min^(-1)、394.99μg·g^(-1);经玻璃化法保存后,合子胚细胞内脱氢酶活性、脯氨酸含量、过氧化物酶活性、过氧化氢酶活性均为最小值;经慢冻法保存后,合子胚细胞内可溶性蛋白质量分数和超氧化物歧化酶活性均达到最大值,分别为18.82 mg·g^(-1)和361.97 U·g^(-1),显著高于其他冷冻方法。干燥脱水120 min结合快冻法处理条件下,合子胚的活力达到最高值。研究结果表明,不同冷冻方式、不同脱水时间及二者的交互作用均会影响水曲柳合子胚的存活率和各项生理指标。除丙二醛质量分数降低外,水曲柳合子胚细胞内脱氢酶活性、抗氧化酶活性、脯氨酸质量分数、可溶性蛋白质量分数在经不同冷冻方式保存后,均随脱水时间增加而增加。超低温保存处理后的合子胚及体胚的存活率可达62.26%和51.68%。因此认为,干燥脱水120 min结合快冻法适合水曲柳胚胎的超低温保存。

一种高效的成年小鼠同步诱导多细胞胚胎及受精卵的双排卵实验方案

目的:探讨二次超排方案对提高成年小鼠超排效果和胚胎发育潜能的可行性。方法:首先比较常规双排卵方案与自然排卵、随机超排实验方案所获总卵数的差异,然后在初步研究外源孕马血清促性腺激素(pregnant mare serum gonadotropin,PMSG)对自然受孕一次胚胎发育影响的基础上,进一步对常规双排卵实验方案进行优化。结果:优化后的双排卵实验组单只鼠平均总卵数高达61.7枚,与自然排卵组、随机超排组和常规双排卵组差异显著,提升了成年小鼠的超排卵效果。结论:优化后的超数双排卵方案为同步大量获得小鼠多细胞胚胎及受精卵提供了一种新的、高效的技术方法。

ZSCAN4在早期胚胎及多能干细胞中的作用及调节机制

含有锌指和SCAN结构域的蛋白质4(zinc finger and SCAN domain containing 4,ZSCAN4)在2细胞期胚胎以及胚胎干细胞中作为DNA结合蛋白质特异性表达。ZSCAN4能够调控早期胚胎发育过程,在合子基因组激活(zygotic genome activation,ZGA)期间通过促进DNA损伤修复和纠正染色体异常,以维持植入前胚胎的基因组和染色体完整性。在小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)向2细胞样细胞转换期间,ZSCAN4与ATP依赖性染色质重塑因子相互作用,调节鼠内源性逆转录病毒L(murine endogenous retrovirus L,MERVL)增强子的活性,激活周边的2细胞期基因的表达,促进胚胎干细胞向2细胞样细胞的转变。ZSCAN4还能通过降低DNA甲基化水平同时介导异染色质沉默,并促使端粒重组和端粒延伸,保持基因组稳定性,进一步维持多能干细胞的无限自我更新能力和多能性,促进mESCs向胚胎2细胞样细胞转变。此外,ZSCAN4还能在重编程中重新激活早期胚胎基因,显著提高诱导多能干细胞(induced pluripotent stem cells,iPSCs)的产生效率,降低iPSCs形成过程中的DNA损伤,并通过延长端粒保持基因组稳定性,从而促进无遗传缺陷和高质量iPSCs的生成。该文围绕ZSCAN4调控早期胚胎发育过程、介导多能干细胞的端粒延长以及在体细胞重编程中的作用等生物学功能展开论述,以期为早期胚胎发育、多能干细胞的维持以及重编程技术的优化提供参考。

粘附剂与附着基质对马尾藻受精卵附着的影响

初选6种粘附剂与马尾藻受精卵混合后,培养观察其附着萌发情况,筛选使受精卵成活率最高的粘附剂;使用4%海藻酸钠、3%CaCl_(2)混合受精卵涂覆在3种粗糙度不同的附着基质上,观察受精卵附着密度,筛选对受精卵附着最有利的粘附剂与基质组合。结果表明:(1)涂抹海藻酸钠的受精卵附着密度达259 ind.·cm^(-2),成活率9.4%;超支化聚合物胶粘剂(HBPA)包裹的受精卵附着密度虽低,但材料黏度大、粘附性强、溶解慢,是潜在的室外移植粘附材料;(2)砖块、水泥板、PVC基质上喷涂3%CaCl_(2)的附着密度达86、48、8 ind.·cm^(-2),成活率分别为52%、29%、5%;实验说明基面越粗糙,受精卵附着数量越多。因此,在大型海藻修复时可投放基面粗糙度大、有颗粒凹点的藻礁与人工粘附剂相结合的方法移植大型海藻。

受精卵原核面积差及合子胞质平均直径对单囊胚移植活产的影响

目的探讨利用激光破膜软件测量计算受精卵原核面积差及合子胞质平均直径,寻找一种客观评价两原核(2PN)受精卵的方法,从而提高胚胎选择准确率,有助于实行单囊胚移植。方法回顾性分析接受助孕治疗患者的194个改良长方案新鲜第5天(D5)单囊胚移植周期,应用激光破膜软件测量所移植胚胎的合子期2PN直径、合子胞质平均直径,计算合子期2PN面积差。通过形态学参数的变化,评估胚胎发育潜能。结果194个新鲜D5单囊胚移植周期,活产组116个周期,未活产组78个周期。两组女方年龄、不孕年限、体质量指数、基础卵泡刺激素(bFSH)、基础雌二醇(bE2)、窦卵泡计数(AFC)、基础促黄体生成素(bLH)、基础抗缪勒氏管激素(bAMH)、促性腺激素(Gn)启动总剂量、Gn启动总天数、人绒毛膜促性腺激素(HCG)日内膜厚度、HCG日血雌二醇(E2)、HCG日血促黄体生成素(LH)、HCG日血孕酮(P)、穿卵数、获卵数、MⅡ卵数、2PN面积差比较差异均无统计学意义(P>0.05);活产组移植优胚数(1.0±0.2)个多于未活产组的(0.9±0.3)个,合子胞质平均直径(111.3±7.2)μm长于未活产组的(109.1±5.8)μm,差异具有统计学意义(P<0.05);根据合子胞质平均直径不同分为A组(<115.0μm,158个移植周期)和B组(≥115.0μm,36个移植周期)。B组女方年龄(32.1±4.3)岁、Gn启动总剂量(2510.4±859.8)IU、活产率77.8%均高于A组的(30.4±4.0)岁、(2162.7±1321.4)IU、55.7%,Gn启动总天数(12.3±1.9)d长于A组的(11.0±2.0)d,差异具有统计学意义(P<0.05)。结论合子胞质平均直径可纳入胚胎评估标准的辅助指标中,以期增加患者活产的机会。