Improvements of cyclic somatic embryogenesis of cassava (Manihot esculenta Crantz)

Summary In cassava a cyclic system of somatic embryogenesis was developed. Primary (torpedo shaped or germinated) embryos, originating from leaf lobes, could only be obtained after culture on solid medium. Cyclic embryos, originating from embryos, could be obtained in both liquid and on solid medium. The production of embryos in liquid medium was distinctly higher, faster and more synchronized than on solid medium. Lower densities and fragmentation of starting embryos improved the production significantly. The highest production found was 32.1 embryos per initial embryo. In all treatments the explants initiated multiple embryos. The production of single embryos was achieved by pressing starting embryos through a fine meshed sieve, indicating that embryos can be produced from a piece of tissue with a restricted number of cells. The shoot conversion rate of embryos from liquid medium was comparable with that of embryos from solid medium.Abbreviations BM Basal Medium - MIE medium volume per initial embryo - E/IE number of Embryos per Initial Embryo     

The effect of length of cryopreservation on the viability of bovine embryos in a commercial operation

A total of 2,232 bovine embryos was obtained from 294 flushings at a commercial embryo transfer operation. The embryos were frozen in groups from individual flushings using 0.25-cc straws and a conventional freezing procedure with glycerol as a cryoprotective agent. The embryos were stored in liquid nitrogen for up to 28 months. Sucrose was used for the removal of glycerol after the thawing of embryos. The thawed embryos were then examined morphologically, and 1,097 embryos (49%) with no apparent defects were used for subsequent transfer. The viability of the thawed embryos from the individual flushes was evaluated in relationship to the length of cryopreservation. No correlation (P > 0.1) was found between the two parameters in embryos from superovulations with above and below average yields. This finding was further confirmed in a proportion of the embryos by the evaluation of pregnancy rates. Thus, neither the typical length of embryo storage in a commercial operation nor the success of superovulation influenced the survival rate of embryos after thawing based on morphological criteria and pregnancy rates.     

Characteristics of embryos from repeat breeder and virgin heifers

The characteristics of 7-day-old embryos non-surgically collected from 35 repeat breeder heifers (RBH) and 24 virgin heifers (VH) were compared by repeated observations within each animal. A higher incidence of the embryos collected from the VH was classified as normal and had reached a more advanced developmental stage than embryos from the RBH. Nearly all VH yielded normal (N) embryos, but morphologically deviated (MD) or degenerated embryos (D) appeared occasionally in many VH. The RBH group contained three subgroups of animals. One group of RBH yielded a high percent of N embryos. A second group yielded mostly MD or D embryos with an occasional N embryo, and the third group only D embryos or no embryos. Heifers from which no embryos were recovered on day 7 yielded uncleaved ova, apparently retarded embryos or no embryos when slaughtered three days after insemination. It is concluded that retarded embryonic development may be a common factor for most RBH. The embryo morphology and the degree of retardation differ among animals and between oestrous periods in the same animal.     

花楸体细胞胚发生过程中抗氧化酶活性的变化

花楸体细胞胚发生过程中,胚性愈伤组织可溶性蛋白含量高于其他类型的愈伤组织,非胚性愈伤组织中超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均高于其他类型的愈伤组织;SOD、POD活性均在胚性细胞向球形胚转化时下降,球形胚向心形胚发育时下降,心形胚向鱼雷形胚和鱼雷形胚向子叶形胚发育时再升高;CAT活性变化规律与SOD和POD活性变化不同,从胚性细胞到鱼雷形胚的3个发育时间内表现为下降-升高-下降的趋势,鱼雷形胚向子叶胚发育时略有回升。据此认为,SOD酶活性降低似可作为花楸胚性细胞分化以及胚胎早期发育的一个判断指标。     

Regenerative ability of somatic single and multiple embryos from cotyledons of Korean ginseng on hormone-free medium

Cotyledon explants of Korean ginseng (Panax ginseng C. A. Meyer) produced somatic embryos directly on growth regulator-free medium. Somatic embryos developed as either multiple or single-state forms, depending on the degree of maturity of the cotyledons. Cotyledon explants from midmature zygotic embryos formed multiple embryos, while cotyledons from fully mature zygotic embryos formed single embryos. Somatic single embryos regenerated into normal plantlets with both roots and shoots, while multiple embryos did not produce roots but regenerated only into multiple shoots. In full-strength MS basal medium, the root growth of plantlets derived from single embryos was weak compared to that of shoots. Deletion of ammonium nitrate from the MS medium promoted the root growth of the plantlets. The ginseng plants with well-developed shoots and roots regenerated from single embryos were successfully acclimatized in a greenhouse when they were planted in soil. Received: 19 July 1997 / Revision received: 6 October 1997 / Accepted: 3 October 1997     

Development of single blastomeres from four- and eight-cell mouse embryos fused into the enucleated half of a two-cell embryo

Single blastomeres from four- and eight-cell mouse embryos were fused into the enucleated halves of two-cell embryos, and the ability of these reconstituted embryos to develop in vitro and in vivo was examined. The proportion of these reconstituted embryos developing to blastocysts was 74% (60/81) when four-cell embryo blastomeres were used as nuclei donors and 31% (57/182) when eight-cell embryo blastomeres were used. Eight complete sets of the quadruplet-reconstituted embryos developed to blastocysts, and five live young (9%, 5/57) were obtained after transfer; however, none of the live young were clones. Although when using blastomeres from eight-cell embryos no complete set of eight developed to blastocysts, sextuplets were obtained. The blastocysts, however, failed to produce live young after transfer. In assessing the outgrowths, it was found that 43% of those derived from reconstituted embryos using blastomeres from four-cell embryos had an inner cell mass (ICM); however, outgrowths derived from reconstituted embryos using blastomeres from eight-cell embryos lacked an ICM. These results suggest that the genomes of four- and eight-cell nuclei introduced into the enucleated halves of two-cell embryos are reversed to support the development of the reconstituted embryo.     

Development of in vivo derived diploid and tetraploid pig embryos in a modified medium NCSU 37

The aim of this study was to assess development of diploid and tetraploid in vivo derived pig embryos cultured in a modified medium NCSU 37 in an atmosphere with reduced concentration of oxygen. The tetraploid embryos were produced by electrofusion of two-cell embryos that had been cultured in vitro from the one-cell stage before fusion (cultured two-cell embryos) or by fusion of freshly recovered two-cell embryos. Development to blastocyst stage of tetraploid embryos, generated from the cultured two-cell embryos was significantly inferior to the development of control one-cell embryos (29.1 +/- 9.7% versus 66.8 +/- 9.7%; P < 0.05). However, development of tetraploid embryos produced from the freshly recovered two-cell embryos and control two-cell embryos was very similar (89.9 +/- 6.1% versus 81.3 +/- 3.4%). Detection of chromosomes 1 and 10 by in situ hybridization showed that more than 85% of the cultured control embryos were diploid while 15% of the embryos were mosaic. Among the fused embryos 50% were tetraploid, 29% mosaic and 21% diploid. These data indicate that the modified medium NCSU 37 provides optimum environment for pre-implantation development of pig diploid and tetraploid embryos.     

Partial rescue of dorsal, a maternal effect mutation affecting the dorso-ventral pattern of the Drosophila embryo, by the injection of wild-type cytoplasm

Mutant alleles at the maternal effect locus dorsal cause a dorsalization of the Drosophila embryo. In extreme mutants, the embryos develop exclusively structures which derive from the dorsal-most region in normal eggs, in less strong phenotypes in addition to dorsal structures, structures normally derived from a dorso-lateral to lateral egg region are formed. Injection of cytoplasm from wild-type embryos into mutant embryos partially restores the dorso-ventral pattern in that injected embryos develop additional structures never formed in uninjected control embryos or embryos injected with mutant cytoplasm. The phenotype of injected embryos resembles that of weaker alleles at the dorsal locus indicating that the wild-type cytoplasm partially rescues the mutant phenotype. The response of the mutant embryos is restricted to the site of injection and occurs only when cytoplasm is injected into the ventral and not into the dorsal side of mutant embryos. The rescuing activity appears to be equally distributed in cleavage stage wild-type embryos, whereas, in syncytial blastoderm embryos, cytoplasm from the ventral side is about twice as effective as that taken from the dorsal side.     

Development of receptors for alpha-bungarotoxin in chick embryo sympathetic ganglion neurons in vitro

The development of receptors for α-bungarotoxin was examined in neurons in dissociated cultures of cells derived from chick embryo sympathetic ganglia. Neurons from 12 day embryos showed a marked increase in receptor numbers per cell over 3–4 days in culture. The increase was less marked in neurons from 14 day embryos and absent in 19 day embryos. The incidence of cholinergic synapses in cultures from 12 day and 19 day embryos was also examined. Evidence for synapse formation was found only in cultures from older embryos.     

慢病毒载体介导的绿色荧光蛋白转基因标记法研究猪嵌合体制作

目的:通过建立慢病毒载体感染猪胚胎体系实现胚胎标记,进而研究不同发育阶段猪孤雌胚胎之间的嵌合能力,为进一步研究猪早期胚胎发育以及细胞分化奠定基础.方法:首先,通过显微注射的方法把2×109I.U./ml、2×108I.U./ml和2×107I.U./ml三个梯度的表达绿色荧光的慢病毒载体分别注射到猪1-细胞胚胎和2-细胞胚胎的透明带下,进行胚胎的GFP转基因标记,在荧光显微镜下观察比较卵裂率、阳性胚胎率、囊胚率、阳性囊胚率和囊胚细胞数.然后,采用凹窝聚合法对同步发育胚胎在不同阶段(2-细胞,4-细胞,8-细胞)进行嵌合,2-细胞胚胎与不同发育阶段(2-细胞、4-细胞、8-细胞)胚胎进行嵌合以及2-细胞胚胎卵裂球互换制作嵌合体胚胎,发育到囊胚时在荧光显微镜下检测胚胎的嵌合状态.结果:2×109I.U./ml的慢病毒感染猪2-细胞胚胎组中,体外受精和孤雌胚胎感染阳性率( 80.00％、76.36％)和阳性囊胚率(90.74％、89.56％)都显著高于其它滴度组(P＜0.05),另外,慢病毒感染的两种胚胎与对照组对卵裂率、囊胚率和囊胚细胞数三个指标没有显著影响(P＞0.05).2-细胞胚胎之间嵌合囊胚率和2-细胞卵裂球互换嵌合囊胚率( 53.85％、62.50％)显著高于2-细胞胚胎与4-细胞胚胎的嵌合率(18.60％,P＜0.05),在同步发育胚胎中8-细胞胚胎之间的嵌合率(75.00％)高于4-细胞胚胎之间和2-细胞胚胎之间的嵌合率( 65.00％、53.80％).结论:2×109I.U./ml的慢病毒感染2-细胞期胚胎效率最高,另外,慢病毒感染对猪胚胎发育没有明显影响.8-细胞间的嵌合率比较高;发育同步胚胎间的嵌合率高于发育非同步胚胎间的嵌合率.     

Embryo production in superovulated cows: Transferable embryos correlated with total embryos

Embryo production was studied in 1,263 donor cows. The number of transferable (good) embryos per collection was highly correlated with the total embryos and ova in a collection (r = 0.64). Total ova and embryos per collection averaged 10.1 with a range from 0-70; the number of good embryos averaged 4.5 with a range from 0-37. Of all collections, 15.3% failed to yield any embryos or ova, and 32.4% did not yield any good embryos. The percentage of good embryos averaged 46.1 while the ratio of good embryos produced (5,680) to total embryos and ova produced (12,699) was 0.45.     

Development of reconstituted mouse embryos produced from the cytoplast of bisected oocytes or pronuclear-stage embryos and single blastomeres of 2-cell stage embryos

The present study investigated the in vitro developmental potential of reconstituted mouse embryos produced from the cytoplast of pronuclear-stage embryos or oocytes and single blastomeres of 2-cell stage embryos by electrofusion. The cytoplast of pronuclear-stage embryos and oocytes were obtained by manual bisection with a fine glass needle under a dissecting microscope. The fusion rates of the reconstituted embryos produced from the cytoplast of oocytes and single blastomeres of 2-cell stage embryos (O-SB2: 38.1 and 41.5%) were significantly lower than those produced from the cytoplast of pronuclear-stage embryos and single blastomeres of 2-cell stage embryos (P-SB2: 91.2 and 97.6%; P<0.001). Reconstituted embryos were encapsulated in alginate gel and were cultured for 96 hours. Similarly, the cleavage and development rates to the blastocyst stage of O-SB2 (56.3, 61.2 and 2.0, 3.1%, respectively) were significantly lower than those of the P-SB2 (91.0, 91.2 and 18.6, 20.7%; respectively, P<0.05). The cleavage and development rates to the blastocyst stage (61.2 and 2.0%) of reconstituted embryos produced from single blastomeres of late 2-cell stage embryos and oocyte cytoplast improved after activation by ethanol treatment (76.1 and 21.7%). However, the use of single blastomeres of early 2-cell stage embryos as nuclear donors did not enhance the cleavage and development rates of the reconstituted embryos to the blastocyst stage.     

Karyotypically normal and abnormal human embryonic stem cell lines derived from PGD-analyzed embryos

Although a normal karyotype is generally a requirement for stem cell lines, new applications are likely to emerge for stem cells with defined chromosomal aneuploidies. We therefore investigated the use of embryos found to be aneuploid on biopsy followed by preimplantation genetic diagnosis (PGD) with fluorescent in situ hybridization (FISH), and developmentally arrested embryos for stem cell derivation. Eleven stem cell lines were obtained from 41 embryos in 36 cultures, with higher success rate achieved from PGD-analyzed, developmentally advanced embryos (45%) than from clinically unsuitable non-PGD embryos (13%). The resulting stem cell lines were karyotyped, and surprisingly, six of the nine lines from aneuploid embryos as well as both lines from non-PGD embryos were karyotypically normal. Three lines from PGD embryos were aneuploid exhibiting trisomy 5, trisomy 16, and an isochromosome 13, respectively. None of the aneuploid lines presented the same anomally as the original PGD analysis. Our study has three important implications. First, we confirm the ability to produce stem cell lines from PGD-tested embryos as well as developmentally abnormal embryos, offering specialty stem cell lines for research into the clinically important aneuploidies. Second, we observe that stem cell derivation from apparently aneuploid embryos is often thwarted by underlying mosaicism and emerging dominance of the stem cell line by karyotypically normal cells. The corollary, however, is that regular production of normal stem cell lines from developmentally abnormal embryos ordinarity discarded opens a new source of embryos for stem cells, whether for research or for eventual therapeutic use within the donating families.     

Embryo transfer as a means of controlling the transmission of viral infections. XI. The in vitro exposure of bovine and porcine embryos to vesicular stomatitis virus

Infectious virus was isolated from both porcine and bovine zona pellucida-intact embryos that had been exposed to the Indiana strain of vesicular stomatitis virus (VSV) and then washed. The amount of virus isolated from embryos depended on their initial exposure level. Porcine embryos always retained more virus than bovine embryos. When embryos were cultured for 24 h after viral exposure and washing, the number of embryos carrying VSV and the amount of virus on each of the embryos was reduced. Trypsin (0.25%) was also found to be effective in inactivating/removing the VSV from embryos, suggesting that most, if not all, of the virus was bound to the zona pellucida.     

Differences in protodermal cell wall structure in zygotic and somatic embryos of <Emphasis Type="Italic">Daucus carota</Emphasis> (L.) cultured on solid and in liquid media

The ultrastructure, cuticle, and distribution of pectic epitopes in outer periclinal walls of protodermal cells of Daucus carota zygotic and somatic embryos from solid and suspension culture were investigated. Lipid substances were present as a continuous layer in zygotic and somatic embryos cultured on solid medium. Somatic embryos from suspension cultures were devoid of cuticle. The ultrastructure of the outer walls of protodermis of embryos was similar in zygotic and somatic embryos from solid culture. Fibrillar material was observed on the surface of somatic embryos. In zygotic embryos, in cotyledons and root pectic epitopes recognised by the antibody JIM5 were observed in all cell walls. In hypocotyls of these embryos, these pectic epitopes were not present in the outer periclinal and anticlinal walls of the protodermis. In somatic embryos from solid media, distribution of pectic epitopes recognised by JIM5 was similar to that described for their zygotic counterparts. In somatic embryos from suspension culture, pectic epitopes recognised by JIM5 were detected in all cell walls. In the cotyledons and hypocotyls, a punctate signal was observed on the outside of the protodermis. Pectic epitopes recognised by JIM7 were present in all cell walls independent of embryo organs. In zygotic embryos, this signal was punctate; in somatic embryos from both cultures, this signal was uniformly distributed. In embryos from suspension cultures, a punctate signal was detected outside the surface of cotyledon and hypocotyl. These data are discussed in light of current models for embryogenesis and the influence of culture conditions on cell wall structure.     

Early development in utero of bovine nuclear transfer embryos using early G1 and G0 phase cells

Bovine somatic cell nuclear transfer (NT) embryos can develop to normal calves, but the success rates are still quite low. Recently, enhanced development of bovine NT embryos to full term has been achieved using fibroblasts at the early G1 phase instead of cells at the quiescent (G0) phase. In the present study, we examined the morphological development in utero of NT embryos using early G1 phase cells (eG1-NT embryos) and G0 phase cells (G0-NT embryos). We produced eG1- and G0-NT blastocysts, and then they were transferred to recipient heifers for transient development in utero up to day 14 of gestation. In vitro-fertilized (IVF), parthenogenetic and artificially inseminated (AI) embryos were used as controls. The rate of formation of embryonic disks of the recovered embryos was the same among the groups of eG1-NT, IVF, and AI embryos (p>0.05). The formation rate in eG1-NT embryos was significantly higher than that in G0-NT embryos (p<0.05). The lengths of eG1-NT embryos were the same as those of IVF, parthenogenetic, and AI embryos (p>0.05), but significantly shorter than those of G0-NT embryos (p<0.01). We conclude that the morphological development of day 14 embryos derived from eG1-NT embryos was mostly similar to that of AI embryos, but that the morphological development of G0-NT embryos was abnormally large and different from that of AI and eG1-NT embryos.     

An in vivo/in vitro evaluation of the teratogenic action of excess vitamin A

Pregnant rats of CFHB strain were injected 81/2 days postcoitum with a 1% suspension of retinoic acid (RA) in arachis oil to give 20 mg RA per kg body weight. Control rats were injected with arachis oil only. After 26 hours, one uterine horn was removed from each rat and the embryos cultured in serum from untreated rats. The embryos in the other horn were allowed to continue development in vivo. After a further 48 hours the cultures were terminated and the second uterine horn removed from each rat. This provided four groups of embryos for comparison: (1) embryos from RA-treated rats, (2) cultured embryos from RA-treated rats, (3) embryos from control rats, and (4) cultured embryos from control rats. The results showed that the effects of the teratogen on the cultured embryos were similar to those on the embryos allowed to continue development for the same period in the mother. In both groups RA reduced protein synthesis, inhibited somite and limb bud formation, and caused various neural tube defects, particularly microcephaly and abnormalities in the closure of the anterior and posterior neuropores.     

Time of embryo transport through the mare oviduct

The objectives of this study were 1) to determine the time of embryo transport through the mare oviduct, 2) to determine whether equine embryos increase in diameter prior to the time of oviductal transport, and 3) to assess the stage of equine embryonic development at the time of oviductal transport. The time of oviductal transport (interval from ovulation to uterine entry) was estimated by collecting embryos from the mare oviduct or uterus at 2-hour intervals from 120 to 168 h postovulation. The time of oviductal transport was 130 to 142 h, since 9 9 embryos were located in the oviduct from 120 to 128 h; 7 14 embryos were in the oviduct and 7 14 embryos were in the uterus from 130 to 142 h; and 13 14 embryos were in the uterus from 144 to 168 h postovulation. Embryos collected during the period of oviductal transport (130 h to 142 h) were not significantly larger (P>0.1) in diameter than embryos collected prior to the period of oviductal transport (162.5+/-3.7 vs 156.7+/-3.1 mum, respectively). During the period of oviductal transport, embryos collected from the uterus were not significantly larger (P>0.1) in diameter than embryos collected from the oviduct (160.7+/-3.2 vs 164.3+/-7.0 mum, respectively). During this same period 12 14 embryos were compact morulae, and 2 14 embryos were blastocysts.     

PCR sexing and developmental rate differences in preimplantation mouse embryos fertilized and cultured in vitro

A two-step polymerase chain reaction (PCR) assay was used to determine the sex of mouse preimplantation embryos obtained from oocytes fertilized and cultured in vitro, to investigate the differences in the developmental rates of mouse embryos according to the sex. All the in vitro developed embryos could be analyzed by this method. When the embryos were classified according to the time of morula to blastocyst transition as fast-intermediate- and slow-growing embryos, a significantly high percentage (78.0%) of the fast-developing embryos were identified as males; while a significantly lower percentage (42.5%) of slow-developing embryos were identified as males. The intermediate-developing embryos presented a sex ratio not significantly different from the total (57.5%). The deviation of sex ratio was further confirmed by embryo transfer experiment, where fast- and slow-developing embryos gave 76.2% and 25.7% male fetuses, respectively. We concluded that male mouse embryos fertilized and cultured in vitro develop faster than female embryos. © 1993 Wiley-Liss, Inc.