A Unique Aegilops tauschii Genotype Needless to Immature Embryo Culture in Cross with Wheat

Common or bread wheat ( Triticum aestivum L., AABBDD, 2n=42) originated ca. 8 000 years ago from hybridization of tetraploid wheat ( Triticum turgidum L., AABB, 2n=28) and diploid Aegilops tauschii Coss. (DD, 2n=14). An essential prerequisite for this evolutionary step is that the natural hybrids between tetraploid wheat and diploid Aegilops tauschii can produce relatively many filled seeds which germinated well. In this study, without special techniques, e.g. immature embryo culture, out of 22 Ae. tauschii accessions, the genotype AS60 produced relatively many filled seeds which germinated well. The seed germination percentages in the crosses of Ae. tauschii ×tetraploid wheat, tetraploid wheat× Ae. tauschii and Ae. tauschii ×common wheat were, respectively, 50.0%, 57.1% and 45.5%. It seems that Ae. tauschii accession AS60 has a unique genotype which facilitate hybrid seed development and viability, and which meets with the prerequisite for wheat evolutionary. Furthermore, the significance of this finding for common wheat improvement and evolution was discussed.

Unisexual Pistillate Flower Regeneration in Immature Embryo Culture of Wheat

In this experiment, floral development from tissue culture of bread wheat (Triticum aestivum L.) was investigated. Immature embryos of 45 wheat cultivars were cultured, and 11.1% of the genotypes regenerated floral organs from the calli near the bases of the green buds or plantlets regenerated. The floral buds were morphologically incomplete with the appearances of unisexual pistillate flowers which were naked, clustered with normal ovaries and exuberant feather-like stigmas, but without stamens, paleas, lemmas and glumes. The histological examination showed that the pistils originated from the meristematic cells near the green buds or plantlets, and the clustered pistils were formed by secondary pistillate regeneration. The development of the feather-like structures was earlier than that of the ovules. Biovule developed from an ovary besides normal uniovule. Statistical analysis by X 2 test for independency demonstrated highly significant difference of flower regeneration among the tested genotypes. Wheat cultivar YA-1 revealed higher percentage (44.4%) than other genotypes, and the response could well be repeated in different years. It was indicated that the floral regeneration of immature embryo explants of YA-1 is relatively stable. The frequency of floral regeneration was mainly regulated by the components in the subculture media, compared with the response of the dedifferentiation media, despite the obviously different components involving basal medium type, inorganic Fe2+ concentration and plant growth regulators. The results suggested the combination of 6-benzylaminopurine, alpha-naphthalene acetic-acid and doubled inorganic Fe2+ might be more beneficial to inducing the floral development than that of 2,4-D and normal inorganic Fe2+ concentration in subculture medium. However, both immature inflorescence and mature embryo, as cultured explants of YA-1, did not regenerate any flower organs. It is believed that the immature embryo culture of YA-1 can be used to establish ideal experimental system for the study of floral developmental mechanism in wheat.

Establishment of 6VS Telocentric Lines of Haynaldia villosa Resistant to Powdery Mildew Induced by Immature Embryo Culture

The line of T240-6 was selected from 32 SC 2 lines of immature embryo culture of T240 (common wheat (Triticum aestivum L.)× Wheat-Haynaldia villosa (L.) Schur. 6D/6V substitution line) through powdery mildew characterization, glutamate oxaloacetate transaminase (GOT) enzyme and gliadin (Gli) analyses and in situ hybridization. All of the individual plants resistant to powdery mildew lacked the locus of GOT at 6VL arm (GOT-V 2) and had gliadin locus at 6VS arm (Gli-V 2) of Haynaldia villosa. All the plants resistant to powdery mildew had one or two telocentric chromosomes that did not pair with wheat chromosomes but paired between themselves. T240-6 was identified as a telocentric line through in situ hybridization.

Establishment of a Highly Efficient Regeneration System for the Mature Embryo Culture of Wheat

Establishment of a highly efficient regeneration system for the mature embryo of wheat will provide a convenient tool for wheat tissue culture and transformation, thereby facilitating the transformation of foreign genes into wheat. By using the mature embryos derived from 20 different wheat lines including Shi 4185, Yumai 66, Lunxuan 987, CB037, Yangmai 6, Xinchun 9, Bobwhite, Han 6172, Zheng 9023, Jimai 20, Ningchun 4, and Jing 411, the effects of some factors including inoculation methods, initiating culture media, organic additives, antioxidants, and auxins on the regeneration from the explants were evaluated. The results indicated that the scraping embryo culture was better than the whole embryo culture, the Aa medium was better than the SD2 medium and dicamba was better than 2,4-D in increasing the regeneration frequency. An Adi medium was established in this study by adding silver nitrate, cysteine, ascorbic acid, dicamba, glutamine into the Aa medium at the concentration of 4,40, 100, 2, and 5 mg L^-1, respectively. By using the Adi medium and the scraping technique, the regeneration frequencies of the mature embryos of CB037, Lunxuan 987, Hart 6172, Yangmai 6, Bobwhite, Zheng 9023, Shi 4 185, and Jimai 20 became 85.6, 60,1, 46.0, 42.1,42.0, 34.0, 33.0, and 32.0%, respectively, which were about 5-8 times higher than that obtained from the conventional culture mediums and techniques. This novel regeneration system could be helpful in wheat transformation.

Studies on Electrical Activation of Porcine Oocytes Matured in vitro and Embryo Culture Systems

Conditions for electrical parthenogenetic activation of porcine oocytes matured in vitro and in vitro culture systems of porcine embryo were studied. The best results were achieved under the conditions of electrical field strength and the pulse duration at 130Vmm-1/80 us, with a blastocyst development rate of (20.12 ± 8.18) % (P > 0.05). No significant difference was found between treatments of multiple pulses and a single pulse (P > 0.05). Parthenogenetic embryos were cultured with different methods and air conditions for 7 days in vitro, blastocyst development rate of embryos with changed culture media [ (26.44 ± 8.35) % ] or changed media with 10% fetal bovine serum (FBS) [ (17.68 ± 5.39)% ] on the fifth day showing no significant difference from that of embryos without change of culture media [ (25.30 ± 7.55) % , P > 0.05 ], while cell numbers of blastocysts from embryos with changed culture media (15.78 + 5.46 and 14.55 ± 4.81) were significantly lower than number of blastocysts from embryos without change of culture media (18.01 ± 6.79, P<0.01). Blastocyst development rate and blastocyst cell number of embryos cultured in lower O2(5%CO2: 7%O2:88%N2) also showed no significant difference from those in high O2(5%CO2 in air) [(20.78 ± 8. 80)% and 17.0016.12 vs. (25.30 ± 7.55)% and 18.0116.79, P>0.05]. It is concluded that change of culture media with the same new one or changing over to media with 10% fetal bovine serum (FBS) on the fifth day and low O2 environment are not necessary for porcine embryos development.

Analysis of Gene Effect on Four Characters of Immature Embryo Culture in Maize

This study was to find the regularity in the hereditary variation for the main culturing characters of the immature embryo culture in maize. Two kinds of inbred-line, R18-599 （red） with very excellent embryo culturing capacity and R15 with very poor embryo culturing capacity, were used as P1 and P2 for obtaining six generations. By culturing immature embryos of the six generations, four culturing characters, namely embryonic callus induction efficiency, nonembryonic callus induction efficiency, cloning ability of the embryonic callus, and number of regenerating plants, were analyzed using the general mean analysis and generation joint analysis. Results showed that the embryonic callus induction efficiency accorded with two major additive-dominance-epistatic genes and polygene-mixed additive-dominance-epistatic inheritance model. The induction efficiency of the nonembryonic callus accorded with two major additive-dominance-epistatic genes. The number of regenerating plants accorded with one major gene and polygene-mixed additive-dominance inheritance model. The cloning ability of the embryo callus accorded with two major genes and polygene-mixed inheritance model, whereas the effect of epistatic gene on this character was identified results of the two methods, generation joint analysis may genetic information. to be different using the two methods. By comparison of the not only raise experimental precision but also provide more

Embryo culture is an efficient way to conserve a medicinally important endangered forest tree species Strychnos potatorum

The present study reports a protocol for germination of Strychnos potatorum （ver. Tel. Chilla） using zygotic embryo culture as an embryo rescue method. A 100% germination rate was obtained by culturing the embryos on full-strength Murashige and Skoog＇s medium （MS） containing 20 g/L sucrose in comparison to McCown and Lloyd＇s Woody Plant Medium （WPM）. Germination rates decreased when the sucrose concentration was lower or higher than 20 g·L-1 . WPM/MS medium containing glucose at levels 30, 20, 15 g·L-1 showed a smaller percentage of germination and at quarter strength, WPM/MS medium with glucose did not respond. Multiple shoot formation was found at 1.0 2.0 mg/L BAP; 3.0 mg/L Kn; 2.0 mg/L TDZ on MS medium with 20 g·L-1 sucrose. Germination rates improved when the embryos were placed upright （vertically） in the medium. The in vitro germinated seedlings were acclimatized in a walk-in-chamber and maintained in the green house with the survival rate of 65% 75%. These plants were transferred to the field and were found to be phenotypically normal, healthy and similar to donor plants. This protocol will be useful to overcome seed dormancy and for rapid multiplication and conservation of S. potatorum using zygotic embryo culture.

Studies on Immature Embryo Culture In vitro in Wheat:Genetic stability analysis of somatic variation in main agronomic characteristics

The genetic stability of variations of main agronomic characteristics in progenies from the regenerated plants of immature embryo culture in vitro was studied. The results showed that the variations of earliness in maturity, high 1000-grain weight, plant height and grain weight per spike were heritable and tended to be stable in IE3 and completely stable in IE4 The wide variation of main agronomic characteristics induced by embryo culture in vitro provided the probability of selection in wheat improvement. Some somaclonal lines with useful variations could be used directly in wheat production.

Are Early Embryo Cleavage Kinetics Affected by Energy Substrates in Different Culture Media?

Objective This study aimed to investigate the influence of different culture media on early embryonic cleavage kinetics using time-lapse analysis and to determine the possible relationships between energy substrates in culture media and the cleavage kinetics.Methods A total of 10021 embryos from 1310 couples were cultured in time-lapse incubators.Embryos cultured in Vitrolife media were allocated to group I,and those in COOK media to group II.Embryo cleavage time points up to the 8-cell stage(t2–t8)were observed after pronuclei fading.Results The baseline demographic features,in vitro fertilization indications,ovarian stimulation protocol,oocyte-cumulus complexes,fertilization rate,together with pregnancy and perinatal outcomes were similar(P>0.05)between groups I and II.According to the time-lapse analysis,all embryos in group I showed significantly faster cleavage speed than those in group II(P<0.05).Furthermore,there was better synchrony in division(s3)and a longer length of the third cell cycle duration(cc3)in group II.Interestingly,implanted embryos in group II showed faster cleavage speed than those in group I,especially at t4 and t7.The glucose contents and multiple major amino acids were similar between the two groups.Lactic and pyruvic acid contents were generally higher in group I than those in group II.Conclusion Because different commercial culture media may influence cleavage kinetics of embryos,it is essential for embryologists to take culture media into consideration in selecting a potential embryo when using a time-lapse system before implantation.

Production of early monozygotic twin bovine embryos in vitro by the blastomere separation and coculture technique

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（b CCs）, intact embryos ＆ b CCs） 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 ＆ b CCs（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 ＆ b CCs 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.

Character of cell-free genomic DNA in embryo culture medium and the prospect of its clinical application in preimplantation genetic testing

There is increasing evidence that cell-free DNA (cfDNA) in spent culture media (SCM) can be amplified for genetic testing. Therefore, this paper reviews the characteristics of cfDNA, including its fragment size, amount, origin, as well as some factors affecting the success rate of its amplification, together to provide researchers with a more comprehensive perspective on embryonic cfDNA. The origin of cfDNA in SCM is complicated and poses challenges to the interpretation of genetic test results. Advanced molecular techniques should distinguish between embryonic and contaminated DNA to maximize the success rate of amplification and analysis. Recent data showed that the type of culture medium, assisted hatching or not, the type of amplification kit, and fresh or thawed embryos were not related to the success rate of amplification, but the length of culture time might affect the success rate. The longer culture time, the more cfDNA is available in the SCM. Then we focused on the concordance between trophectoderm (TE), inner cell mass, whole embryo, and embryonic cfDNA. Despite successful amplification, the concordance between TE and embryonic cfDNA was low. In summary, non-invasive genetic testing using SCM could represent a major advance in future single embryo selection, however, contamination and timing for media collection are key factors affecting the results, and current non-invasive cfDNA testing should not be directly applied to clinical practice. Further research is needed to improve the methods used for testing techniques and genetic analysis to achieve greater accuracy and trace its origins before it can be used in the clinics.

Manganese enhances the expression of the manganese superoxide dismutase in cultured primary chick embryonic myocardial cells

In the present study, the effect of manganese（Mn） on antioxidant status and the expression of the manganese superoxide dismutase（MnSOD） gene in cultured primary myocardial cells collected from the chick embryos was investigated. The hypothesis that Mn supplementation would enhance the expression of MnSOD in cultured primary myocardial cells of chick embryos was tested. Eggs collected from Mn-depleted Arbor Acres laying breeder hens were incubated for 10 days and then myocardial cells were isolated and cultivated for 8 days. The embryonic myocardial cells on day 6 were treated with Mn in the cell culture medium at different time points when the proportion of cells showing spontaneous contraction was over 95% after the 3-day primary culture. A completely randomized design involving a 3 Mn levels（0, 0.5 and 1.0 mmol L^（-1））×3 incubation time points（12, 24 and 48 h） factorial arrangement of treatments（n=6） was used in the current experiment. The results showed that MnSOD activity and m RNA expression level were induced by Mn and increased with incubation time, which supported the hypothesis that Mn would enhance the expression of the MnSOD gene, and thus might protect myocardial cells from oxidative stress during the chick embryonic development.

Early embryonic development and transplantation in tree shrews

As a novel experimental animal model, tree shrews have received increasing attention in recent years. Despite this, little is known in regards to the time phases of their embryonic development. In this study, surveillance systems were used to record the behavior and timing of copulations; embryos at different post-copulation stages were collected and cultured in vitro; and the developmental characteristics of both early-stage and in vitro cultured embryos were determined. A total of 163 females were collected following effective copulation, and 150 were used in either unilateral or bilateral oviduct embryo collections, with 307 embryos from 111 females obtained （conception rate=74%）. Among them, 237 embryos were collected from 78 females, bilaterally, i.e., the average embryo number per female was 3.04; 172 fertilized eggs collected from 55 females, bilaterally, were cultured for 24-108 h in vitro for developmental observations; finally, 65 embryos from 23 bilateral cases and 70 embryos from 33 unilateral cases were used in embryo transplantation.

Cytological Correlation Between Flower and Vegetative Shoot Development from Immature Embryo-Derived Calli in Wheat

In this paper, the traits of immature embryo culture from 45 genotypes of bread wheat were investigated in detail, and trait correlation analysis in combination with histomorphological examination was used to study the cytological origin of flower as well as the correlation between it and vegetative shoot development from the calli. The rate of floral development in the subculture was highly significantly and positively related to that of the differentiation culture, which reveals that the genetic system of floral development from immature embryo-derived calli in wheat was highly correspondent and stable. The rate of floral development was related neither to the traits in the dedifferentiation culture, nor to the vegetative shoot formation from the total induced calli, but significantly and positively related to that of the calli without leaf-like green spots in the differentiation culture. These findings indicate the partial correlation as well as differentiation between flower and vegetative shoot development. The floral development might only be associated with the genetic system controlling organ regeneration, while vegetative shoots formed from the two ways, including somatic embryogenesis and organ regeneration.

Hybridization between Gossypium herbaceum and Gossypium stocksii through embryo rescue

Nutrient media and culture conditions have been defined for ovules 3 and 5 d after pollination and embryos of Gossypium herbaceum respectively. The technique was then used to produce interspecific hybrids between a cultivated Gossypium herbaceum and a wild species, G. stocksii. The hybrid plants were transferred to field and they exhibited most of the characters of the pollen parent i.e.G. stocksii.

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.

Studies on Factors Affecting the in vitro Germination and Growth of Taxus cuspidata Embryos

Factors affecting the in vitro germination and growth of Taxus cuspidata embryos were examined. DCR medium was the best among 6 basal media tested; embryos from stage II seeds were the optimal developmental stage for in vitro germination; as the seeds approached maturity, germinability of embryos decrease. Embryos cultured in darkness tended to form callus and decrease the frequency of germination; inoculation method obviously affected frequency of embryo germination. Embryos cultured in optimal conditions developed into seedlings in 2 months.

Interspecific Cross Compatibility of Rhododendron in Changbai Mountain

In order to explore the interspecific cross compatibility of Rhododendron and the feasibility of interspecific cross in germplasrn innovation, reciprocal crosses among five species of Rhododendron in Changbai Mountain were made, In addition, the hybrid embryos were cultured in vitro. The results showed that the species of Rhododendron in Changbai Mountain had significant differences in reciprocal cross compatibility. When Rh. chrysanthum Pall. was used as female parent, it showed high cross compatibilities with Rh. mucronulatum Turcz. （92.55%）, Rh. mucronulamm Turcz. f, album Nakai （94.14%） and Yabuli Rhododendron （92.42%）, and no signifi- cant differences were found in fruit setting rate by serf-poUination. The germination rates of hybrid embryos were 13.60%, 11.59% and 4.12%, respectively. The hybrid seedlings had weak growth vigor and albino status and were trended to be aborted. After 2-3 generations of subculture, the growth vigor of hybrid seedlings were re- covered gradually, and their differentiation rate was also significantly higher than that of Rh. chrysanthum Pall. seedlings. But the hybrid seedlings were still yellowish green. In reciprocal crosses, the fruit setting rates were significantly reduced. The combination with the highest fruit setting rate （36.51%） was Yabuli Rhododendron x Rh chrysanthum Pall The fruit setting rates of reciprocal crosses between Rh chrysan- thum Pall and Rh. mucronulatum Turcz. and Rh.： chrysanthum Pall. and Rh. schlip- penbachii Maxim were 6.52% and 0, respectively. However, the hybrid embryo＇s germination rate （39,81%） of Yabuli Rhododendron x Rh. chrysanthum Pall. was significantly higher than that of their original cross. Moreover, the hybrid seedlings grew strongly and could be directly transplanted without subculture.

Cross Breeding of Populus and Its Hybrids for Cold Resistance

Populus tomentosa was crossed with P.tremuloidis, P.grandidentata, P.alba×P.grandidentata and P.alba×Ulmuspumila in order to maintain its rapid growth and high wood quality and improve its resistance to cold. Two methods were used to increase the germination rate from 1.5% to 41.1% and the remaining rate from 1.7% to 44.2%. Forty crossing combinations were conducted and 2744 hybrid seedlings were obtained. MX4×P.grandidentata （G-1-58）, MX3×P.tremuloidis （T-44-60）, MX2×P.tremuloidis （1-13-87-37） and MX2×（P.alba×P.grandidentata） were regarded as superior combinations after analysis and selection. Thirty seedlings of these combinations and 11 triploid seedlings identified by counting their chromosomes were selected as super plants.

Collection of superovulated mouse oocytes continuously by surgery and their development after activation

Objective:To establish a new way to collect superovulated oocytes or zygotes repeatedlyfrom an individual mouse.Methods:Superovulations were induced by injection PMSG and hCG in Kunming strainmice.The ampullaes of oviduct of all anaesthetised mouse were put in a specially designed'U'sink and released.The second and third times of PMSG injection were made on the sixth day andeleventh day after the first superovulation injection.The capacity of development was examinedby in vitro culture of parthenogenesis activation oocytes.Results:Development to blastocyst stage was not significantly different between the first andsecond time collection.The percentage of blastocyst stage in CD and Sr^(++) treatment was signifi-cantly higher(P<0.05)than the oocytes treated in CB and Sr^(++).Conclusion:This method enables us to collect oocytes or zygotes repeatedly from one individ-ual mouse in an interval as short as 5 days and without influence on the quality of oocytes.