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.

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.

Dicamba and Sugar Effects on Callus Induction and Plant Regeneration from Mature Embryo Culture of Wheat

To establish a highly efficient plant regeneration system for wheat genetic transformation, the effects of three different concentrations of dicamba and two different sugar types on callus induction and plant regeneration from mature embryo cultures were evaluated. Callus induction and plant regeneration were obtained from mature embryos of two commercial cultivars Zhoumai 18 and Yumai 34 （Triticum aestivum L.） cultured on L3 basal medium. The results showed that the efficiency of mature embryo culture was significantly influenced by the genotypes, sugar types and dicamba concentrations. 4 mg L^-1 dicamba proved the best effective for inducing embryogenic callus and also gave the highest proportion of plants regenerated across the two cultivars. Substitution of maltose by sucrose significantly improved the plant regeneration efficiency in both cultivars. There was a significant interaction between genotype-by-sugar types, and sugar types-bydicamba concentrations. Overall, Zhoumai 18 gave the highest frequency of plant regeneration （82.65%） when dicamba concentration was 4.0 mg L^-1 and with sucrose in initial callus induction. These results will facilitate genetic transformation work with elite wheat.

The Comparison in Tissue Culture Ability of Mature Embryo in Different Cultivars of Rice

In order to study the regeneration technology of mature embryos in different rice varieties,nine japonica,nine indica and eleven hybrid rice varieties of two line or three line or superiority combinations were selected as explants to study the callus induction,differentiation and regeneration rates on different media.The higher callus induction （61.7-89.2%） was observed in japonica rice,when cytokinin was added at lower concentration （0.3 mg L-1 6-BA） in M8 basal medium,supplemented with 30 g L-1 sucrose,8 g L-1 agar and 2 mg L-1 2,4-D.Further,the addition of two cytokinins （2 mg L-1 6-BA,0.5 mg L-1 KT） and 1 mg L-1 NAA in the M8 basal supplemented medium resulted in 9.1-100% of the callus induction in indica rice.The percent callus induction in hybrid rice varieties was 40-86.3% when addition of 1 mg L-1 6-BA and 1 mg L-1 KT was added,and the cytokinins was required by the japonica and indica rice varieties in the M8 basal supplemented medium.It was observed that when the 0.5 mg L-1 2,4-D and 1 mg L-1 6-BA were added in japonica rice,and 0.2 mg L-1 2,4-D and 0.5 mg L-1 6-BA were added in indica and hybrid rice in the MS different media,the regeneration rates were 9.2-59.5%,3.6-87.5% and 17.2-43.2% for japonica,indica and hybrid rice,respectively.Thus,the regeneration technology with higher output is established in the mature embryos of similar rice varieties.

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.

In vitro culture of immature embryos from Koelreuteria bipinnata var. integrifoliola

For the mass production of Koelreuteria bipinnata var. integrifoliola with selected, hybrid or genetically engineered genotypes, one potentially desirable propagation strategy is based on embryo culture. The immature embryo development in vitro from K. bipinnata var. integrifoliola was studied under different conditions of embryo age, basic culture media and plant growth regulators. The results show that： 1） germination rate of grade 3 embryos in immature seeds with 0.6-0.8 cm diameter was 98.9%. The germination rate of grade 2 embryos in immature seeds with 0.4-0.6 cm diameter was 77,8% and the germination rate of grade 1 embryos in immature seeds with 0.4 cm diameter was 15.6%. 2） The amounts of macroelements in MS medium had no clear effect on the germination rate of immature grade 3 embryos and had a modest effect on plantlet growth, where the best medium was MS or 1/2 MS. The rates were all greater than 90%. 3） The germination rate of grade 3 embryos was greater than 87% when the medium contained a low concentration of NAA or no plant growth regulators at all and decreased markedly when BAP alone or BAP and NAA together were added to the media. We suggest that in vitro culture of immature embryos from K. bipinnata vat. integrifoliola can be enhanced when a small amount of plant growth regulators is added. The addition of BAP has an adverse reaction to the germination and development of immature embryos.

Effects of different high temperatures and incubation time periods on embryogenesis in isolated microspore culture of Chinese cabbage (Brassica campestris L. ssp pekinensis)

Effects of five incubation temperatures (25℃ ,28℃ ,31℃ ,35℃ and 37℃ for 24hours) and four incubation time periods (0.4,16 and 24 hours at 35℃) on isolated microspore culture of Chinese cabbage were studied. The results showed that cultured microspores of Chinese cabbage developed into embryos at all the incubation temperatues from 28℃ to 37℃ ,but the best response to high temperature occured at 35℃. Among the four kinds of time periods, the highest yield of embryos was obtained at the 24h treatment. Therefore, the isolated microspore culture of Chinese cabbege ran be efficiently carried out at 35℃ for 24 hours.

Human Embryo Neuronal Culture <i>in Vitro</i>: A Model to Study Cellular Physiology, Receptors, Power and Toxicity of Cytostatic Drugs for Human Use

Neural cells cultures from human embryo brain of 9° - 11°W gestational age have been used to study ERα (Estrogens Receptor α) and to perform toxicity test for Mitomycin C and Methotrexate. Histochemical confirmation of cellular neuronal phenotype was based on histochemical evidence of NSE (Neuron Specific Enolase).The detection of ERα in neuronal cells was performed with a rabbit Monoclonal Antibody. ERα was absent both on neurons grown in vitro and on tissue brain specimens. This finding is apparently in contrast with the positive immunoreactivity of ERα and ERβ reported by other Authors on foetal and adult CNS (Central Nervous System). The absence of nuclear ERα on neurons in culture and in brain tissue specimens in our experiment is not in contrast with the relevant physiologic role of estrogens on nervous central system, but it could be correlated to the embryonic period of life and could represent a protection of male brain from an undue estrogens imprinting. The mitomycin C, alkylation agent, has shown in our experiment a major neurotoxic and cytostatic power in comparison with methotrexate. Our conclusion is that human embryo neuronal culture in vitro is a powerful instrument for physiology and human therapy for cancer and neurodegenerative diseases.

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

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.

A Preliminary Study on Immature Embryo Culture and Plant Regeneration of Lagerstroemia indica

[ Objective ] This study aimed to explore immature embryo culture of Lagerstroemia indica and investigate the appropriate conditions for growth and differentiation. [ Method] Immature embryos of L. indica were employed as the explants for germination induction to establish aseptic lines. Based on that, the effects of different hormone levels and culture conditions on immature embryo culture of L. indica were analyzed. [ Result ] Peeled immature embryos of L. indica were germinated easily, leading to a germination rate of 100%. The optimal initial medium was MS ＋ BA0.5 ＋ NAA0.1 ＋ sucrose 3.0% ＋ agar 0.7% ; the optimal shoot induction medium was MS ＋ BA0.5 ＋ NAA0.1 ＋ sucrose 3.0% ＋ agar 0.7% ＋ coconut milk 10% ; the optimal rooting medium was MS ＋ BA0.5 ＋ IBA0.1 ＋ sucrose 3.0% ＋ agar 0.7% ＋ coconut milk 10%. [ Conclusion] This study provided a technical reference for subsequent optimized breeding of L. indica.

Effect of L-carnitine supplementation during in vitro maturation and in vitro culture on oocyte quality and embryonic development rate of bovines

Objective:To assess the effect of L-carnitine supplementation during in vitro oocyte maturation and in vitro culture process of bovine oocytes.Methods:L-carnitine(3.8 mM)was added to maturation medium and the effect was assessed in the quality(Experiment 1)and in the cleavage and 4-cells stage(Experiment 2).Besides,the effect of L-carnitine addition on maturation medium(3.8 mM)and culture medium(1.5 mM)on embryo rate production was assessed.In Experiment 1,bovine oocytes from abattoir were randomly separated into two groups(the control group and L-carnitine group)forin vitro maturation.Matured oocytes were examined for cumulus cells expansion as an indicator of maturation,and the content of the mitochondrial activity,the presence of lipid droplets,the reduced glutathione,and the reactive oxygen species were measured by using specific fluorochromes.In Experiment 2,oocytes were matured as performed in Experiment 1,afterward fertilized and cultured until day 3,and cleavage rate and 4-cells stage rate were determinated.In Experiment 3,in vitro maturation and fertilization were done as performed in Experiment 2,but at day 3 of culture,each group of embryos was separated into two new groups,and L-carnitine(1.5 mM)was added in culture media until day 8.The cleavage and embryo development rate were determined on the basis with the oocytes put on maturation.Hatching rate was calculated from cleaved embryos.Results:The cumulus expansion rate at gradeⅢand mitochondrial activity were significantly higher in the L-carnitine group in comparison with the control group(P0.05).In addition,cleavage and the proportion of embryo development and hatching rate were similar for all groups(P>0.05).Conclusions:L-carnitine as a supplement in culture media improves the cumulus expansion and increases the mitochondrial activity during in vitro maturation process but has no apparent effect on the cleavage and development of bovine embryos.Further investigations of L-carnitine addition on in vitroculture are needed to test their effect on embryo quality.

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.

Co-Culture of Early Embryo with Human Decidual Stromal Cells in vitro by Improvement of Early Embryo Development

Summary: An early embryo co-culture system with human decidual stromal cells was established to study its effect on early embryonic cleavage and growth in vitro. Three hundred and eight 2-cell mouse embryos were co-cultured with human decidual stromal cell monolayer in MEM+0. 4 % bovine serum albumin (BSA) and 163 embryos cultured in MEM+15 % FCS alone as control. Among the mouse 2-cell embryos co-cultured with human decidual stromal cells, 72.73 % developed to the morula stage and 67.21 % cavitated to blastocysts with 59. 74 % hatching, as compared with 61. 34 % to morula stage, 48. 47 % to blastocysts and none hatching in the controls, respectively. Co-cultured embryos cleaved slightly faster than controls and showed no or less fragmentation than those in the control. These results suggested that human decidual stromal cells can support early embryonic development and yield a reasonable number of embryos with good quality up to blastocyst stage.

Effects of inter-culture, arabinogalactan proteins, and hydrogen peroxide on the plant regeneration of wheat immature embryos

The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins （AGPs） and hydrogen peroxide （H202） have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic calli and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199 （KN 199） and Xinchun 9 （XC9）, together with low-frequency regeneration wheat line Chinese Spring （CS）, presented striking increase in the induction of embryogenic calli and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L-1 AGP or 0.005 to 0.01‰ H2O2 to the callus induction medium, enhanced growth of embryogenic calli and plant regeneration rate in quite a few wheat genotypes. At 50 mg L-1 AGP application level in callus induction medium plant regeneration rates of 8.49,409.06 and 283.16% were achieved for Jimai 22 （JM22）, Jingdong 18 （JD18） and Yangmai 18 （YM18）, respectively; whereas at 100 mg L-1 AGP level, CS （105.44%）, Chuannong 16 （CN16） （80.60%） and Ningchun 4 （NC4） （62.87%） acted the best. Moreover CS （79.05%）, JM22 （7.55%）, CN16 （101.87%）, YM18 （365.56%）, Yangmai 20 （YM20） （10.48%）, and CB301 （187.40%） were more responsive to 0.005 %o of H2O2, and NC4 （35.37%） obtained the highest shoot regeneration rates at 0.01%o of H2O2. Overall, these two methods, inter-culture and AGP （or H2O2） application, can be further applied to wheat transgenic research.

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.

Effects of CuSO_4 and Uniconazole on Mature Embryo Culture in Japonica Rice

In order to establish the system of high frequency plant regeneration for japonica rice mature embryos, the effects of different concentrations of CuSO4 and uniconazole on in vitro culture of mature embryos were studied using three rice cultivars of Kongyu 131, Longjing 24, and Dongnong 425 as test materials. The results showed that callus induction and differentiation of japonica rice mature embryos were apparently improved on the medium with 10-15 μmol·L-1 CuSO4 and 0.50-1.00 mg·L-1 uniconazole. Induction and differentiation rates of different genotype rice mature embryos displayed different sensitivities to CuSO4 and uniconazole. For the callus induction frequency of three varieties, the optimal concentration of CuSO4 was 15.0 mol·L-1. When the concentration of CuSO4 was 15 μmol·L-1, the plantlet differentiation rates of Kongyu 131 and Dongnong 425 got to the highest, while the concentration of CuSO4 was 10 μmol·L-1 for Longjing 24. For the callus induction and plantlet differentiation rates of Kongyu 131 and Dongnong 425, the ideal concentration of uniconazole was 0.50 mg·L-1 and for Longjing 24 was 1.00 mg·L-1.

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

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.

Prolonged post-thaw culture of embryos does not improve outcomes of frozen human embryo transfer cycles: A prospective randomized study

Objective:To evaluate the impact of prolonged post-thaw embryos culture on pregnancy outcome during frozen embryo transfer cycles.Methods:This prospective cohort study evaluated 324 thaw transfer cycles with 819 embryos from 269 patients at the Center for Reproductive Endocrinology and Infertility of Hue University Hospital in Vietnam.These frozen embryo transfer cycles were divided into two groups at the time of thawing:the short culture group(2-hour post-thaw culture)and the overnight culture group(overnight culture for 18 h)before the embryo was transferred into the uterus.The rates of embryo intact,grade A embryo at frozen and transfer time and continuing cleavage were recorded.The clinical outcomes including serum beta-human chorionic gonadotropin,clinical pregnancy and implantation rate were evaluated after 14 days,4 weeks,6 weeks,respectively,after embryo transfer.Results:Human chorionic gonadotropin positive occurred in 39.5%of patients in the short culture group compared to 25.9%in the overnight culture group with risk difference(RD)=13.6%,relative risk(RR)=1.343,95%confidence interval(CI)1.085-1.663,P<0.01.Clinical pregnancy rate of the short culture group and overnight culture group was 33.3%and 24.1%,respectively(RD=9.2%,RR=1.242,95%CI 0.996-1.549,P=0.06)and the implantation rate in the short culture group and overnight culture group was 16.5%and 11.0%,respectively(RD=5.5%,RR=1.244,95%CI 1.046-1.479,P=0.01).In women of advanced age(≥35 years)and women who received 3 embryos,pregnancy outcomes were found to be significantly(P<0.05)higher in the short culture than in the overnight culture group.Conclusions:The prolonged post-thaw culture period does not increase pregnancy outcome in comparison with the short culture.

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.