Peculiarities of <i>in vitro</i> parthenogenesis of unpollinated maize ovaries

The maize line AT-1 is characterized by a hereditary predisposition to parthenogenesis. The aim of this investigation is to study parthenogenetic embryo development in the culture of unpollinated ovaries in vitro . The unpollinated ovaries were explanted in 1, 3, 5, 7, 10, 15 days after the appearance of stigmas from ears. The nutrient medium included mineral components of MS, vitamins, sucrose (9,0%), 2,4-D (2,0 mg/l), agar-agar. The structure of megagametophytes at the time of inoculation of the ovaries and on the 3rd, 7th, 14th, 21th, 28th day of cultivation was studied. The first divisions of unfertilized egg cells were observed on the 5th-7th day after appearance of stigmas from ears, independently from whether all this time the ovaries were on the mother plant or they were inoculated into the nutrient medium. The formation of the autonomous abnormal endosperm in some cultivated ovaries was detected. The abnormal endosperm disturbed normal development of the proembryo. As a rule, the ovaries with embryo and endosperm degenerated. In the absence of endosperm, the morphogenesis of parthenogenetic proembryos was carried out in one of two directions in vitro : 1) development of plants by direct embryogenesis; 2) regeneration of plants from numerous embryoids, raised on the surface of globular proembryos. The second direction was prevailed. The culture of unpollinated ovaries can be a promising method of mass haploid regenerants not only in maize, but also in other types of agricultural plants.

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150 DEVELOPMENT OF ZONA-FREE AND WITH ZONA PARTHENOGENETIC GOAT EMBRYOS IN DIFFERENT MEDIA

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab150 ◽

2010 ◽

Vol 22 (1) ◽

pp. 233

Author(s):

M. K. Jena ◽

D. Malakar ◽

A. K. De ◽

S. Garg ◽

Y. S. Akshey

Keyword(s):

Embryo Development ◽

Chemical Activation ◽

Formation Rate ◽

Electrical Activation ◽

Cleavage Rate ◽

Maturation Medium ◽

Parthenogenetic Embryo ◽

Oviductal Fluid ◽

Phosphate Buffered Saline

The present study was carried out to see the developmental efficiency of zona-free and with zona parthenogenetic goat embryos cultured in Research Vitro Cleave from Cook Australia (RVCL), Embryo Development Media (EDM), modified synthetic oviductal fluid (mSOF), and modified Charles Rosenkrans media (mCR2a). Zona-free embryos were cultured in 4 media, whereas with zona embryos were cultured in 3 media except mCR2a. Ovaries were collected from slaughterhouse and oocytes were isolated by puncturing the follicles in medium containing Dulbecco’s phosphate-buffered saline, 3% BSA, and 50 μg mL-1 gentamicin. Oocytes were matured in maturation medium containing TCM-199 (HEPES modified), 0.05 mg mL-1 Na pyruvate, 0.003 mg mL-1 L-glutamine, 5.5 mg mL-1 glucose, 3 mg mL-1 BSA, 5 μg mL-1 FSH, 10 μg mL-1 LH, 1 μg mL-1 estradiol-17β, 50 μg mL-1 gentamicin, and 10% FBS in 5% CO2 in air at 38.5°C. The COC (15 to 20 oocytes) were placed in 100-μL droplets of maturation medium and incubated in a CO2 incubator (5% CO2 in air) with maximum humidity at 38.5°C for 27 h. Matured oocytes were made cumulus free by treatment with hyaluronidase (0.5 mg mL-1) and zona-free by pronase (2 mg mL-1) in zona-free parthenogenesis. Then the oocytes were activated by 5 μM Ca ionophore for 5 min in a CO2 incubator and then treated with 2 mM 6-DMAP for 4 h. Activation was also done by electrical activation with DC 1.78 kV cm-1, 20 μs, and 2 pulses. Then the zona-free oocytes were kept for in vitro culture in 4 types of media such as RVCL, EDM, mSOF, andm CR2a for 7 days in 5% CO2 in air at 38.5°C. The cleavage rate andmorulae formation were observed in RVCL 40.95%, 13.95%, in EDM 46.92%, 14.75%, in mCR2a 56.66%, 5.88%, and in mSOF 48.23%, 14.63%, respectively. The cleavage rate and morulae formation were also found 55.9%, 14.63% during chemical activation and 32%, 12.5% in electrical activation. Hence, better result was found in chemical activation than electrical activation. For with zona parthenogenesis, the matured oocytes were chemically activated by 5 μM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h. Then the oocytes were cultured in RVCL, EDM, and mSOF in 100-μL micro-drops media for 7 days. The cleavage, morulae, and early blastocyst production rate were as follows: cleavage rate 75.68%, 72.03%, and 57.11%; morulae 44.61%, 30.29%, and 40.22%; and early blastocyst 17.49%, 11.88%, and 25.01% in RVCL, EDM, and mSOF, respectively. Hatched blastocyst formation rate was 6.75%, 5.48%, and 1.15% in RVCL, EDM, and mSOF, respectively. It could be concluded that zona-free parthenogenetic embryos were produced better in EDM medium and with chemical activation. With zona parthenogenetic embryo development was significantly (P < 0.05) higher in RVCL and EDM media.

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21 EFFECT OF SEVERAL PARAMETERS ON PARTHENOGENETIC BOVINE EMBRYO DEVELOPMENT IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab21 ◽

2006 ◽

Vol 18 (2) ◽

pp. 119

Author(s):

S. Arat ◽

H. Bagis ◽

A. Tas ◽

T. Akkoc

Keyword(s):

Embryo Development ◽

Development Rate ◽

Electrical Pulse ◽

Bovine Embryo ◽

Cleavage Rate ◽

The Third ◽

Parthenogenetic Embryo ◽

Development In Vitro ◽

Parthenogenetic Embryos

The activation of oocytes is one of the most important steps for a successful cloning and has great importance on embryo development in vitro. The objective of this study was to examine the different parameters affecting parthenogenetic embryo development in vitro. In the first experiment, two activation protocols were compared to examine the effect of electrical pulse on activation. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM-199 supplemented with fetal bovine serum (FBS), sodium pyruvate, penicillin/streptomycin, rat insulin-like growth factor (rIGF-1), bovine follicle-stimulating hormone (bFSH), and bovine luteinizing hormone (bLH). A group of oocytes was exposed to a DC pulse of 133 V/500 �m for 25 �s, and then activated by calcium ionophore (5 �M) for 10 min, cytochalasin D (CD) (2.5 �g/mL) + cycloheximide (CHX, 10 �g/mL) for 1 h, and CHX alone for 5 h (Group 1). Another group of oocytes was activated only by chemicals without electrical pulse. Activated oocytes were cultured for 72 h in G1-3 and then 4-6 days in G2-3 medium. In the second experiment, oocytes activated by electrical pulse and chemicals were cultured in Barc medium for 7-9 days or 72 h in G1-3 and then 4-6 days in G2-3 medium. In the third experiment, oocytes activated by electrical pulse and chemicals were cultured for 48 h or 72 h in G1-3 and then 5-7 days or 4-6 days in G2-3 medium. The differences among groups were analyzed by one-way ANOVA after arcsin square transformation. In the first experiment, cleavage rate (75.6%), development rate (37.3%), and blastocyst cell number (78.4 � 3.2) of oocytes activated by electrical pulse was higher than for the group without electrical pulse (28.7%, 8.0%, 59.5 � 4.3, respectively; P < 0.05). This result showed that activation was started more effectively by electrical pulse than by chemicals. In the second experiment, there was no significant difference on cleavage rate between the two groups (66.6%, 65.0%, respectively), and the blastocyst development rate of parthenogenetic embryos cultured in G1-3/G2-3 (36.6%) was higher than in the Barc medium group (16.6%; P < 0.05). This result showed that G1-3/G2-3 medium was more effective for parthenogenetic embryo development than Barc medium. In the third experiment, although significant differences could not be found between the two groups in the development rate of parthenogenetic embryos cultured for a total of 7-9 days (30.8%, 39.2%, respectively), the development rate of embryos cultured for 72 h in G1-3 was higher (26.4%) than for the 48-h group (15%; P < 0.05) on Day 7. This result showed that embryos developed more slowly when cultured for a shorter time in G1-3 medium before transfer to G2-3 medium. This study was supported by a grant from TUBITAK, Turkey (VHAG-1022).

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332 EFFECT OF DIFFERENT PIG OOCYTE ACTIVATION PROTOCOLS ON EMBRYO DEVELOPMENT IN SOF AND NCSU-23

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab332 ◽

2007 ◽

Vol 19 (1) ◽

pp. 281 ◽

Cited By ~ 1

Author(s):

I. Lagutina ◽

G. Lazzari ◽

C. Galli

Keyword(s):

Embryo Development ◽

Embryo Culture ◽

Cell Number ◽

Additional Treatment ◽

Oocyte Activation ◽

Blastocyst Development ◽

Chi Square ◽

Parthenogenetic Embryo ◽

Pig Oocyte

Several factors affect nuclear transfer success. These include efficient parthenogenetic activation and embryo culture medium that should efficiently support pre-implantation development of good quality blastocysts. We investigated pig oocyte activation and embryo development in SOFaa in response to ionomycin (Io = 5 µM Io for 4 min; Io° = 15 µM Io for 20 min) and electric impulse (EL; one 30-µs pulse of DC 1.5 kV cm−1 in the presence of 50 µM Ca) in combination with 2 mM 6-DMAP or 10 µg mL−1 cycloheximide (CHX) +5 µg mL−1 cytochalasin B (CB) for 4 h. In addition, we studied the effect of elevated (1 mM) (Cheong et al. 2002 Mol. Reprod. Dev. 61, 488) in comparison with 50 µM Ca during EL activation on embryo development in SOFaa and NCSUaa-23. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon®, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng mL−1 long-EGF, 100 ng mL−1 long-IGF1, 5 ng mL−1 bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. The rates of cleavage, blastocyst formation (BL) and BL cell number on Day 7 (BL-D7) were recorded. All experiments were done with 3 replicates. The data were compared by chi-square test. There was no difference in the ability of Io (all groups) and EL + CB activated oocytes to cleave, whereas the additional treatment of EL-activated oocytes with DMAP and CHX + CB significantly increased cleavage. Io activation resulted in poor blastocyst development in comparison with all EL-activated groups (see Table 1). When calcium levels were elevated during EL activation, significantly more embryos developed in SOFaa (35.6%, n = 191 vs. 26%, n = 192; P < 0.05), but no differences were observed with culture in NCSUaa-23 (about 56%). The BL rate was significantly higher in NCSUaa-23 vs. SOFaa (55.9%, n = 68 vs. 34.8%, n = 69, respectively); however, the BL total cell number was significantly higher in SOFaa (58 ± 18, n = 40 vs. 86 ± 35, n = 56, respectively; P < 0.05). In conclusion, we have found that SOFaa and NCSUaa-23 differ in ability to support pig parthenogenetic embryo development. EL activation combined with elevated Ca significantly increased the embryo developmental capacity in SOFaa but not in NCSUaa-23. NCSUaa-23 was more efficient for embryo culture, whereas SOF produced BLs of higher quality. Table 1.Effect of activation protocol on the development of pig parthenogenetic embryos in SOFaa This work was supported by grants ISS-CS11 and Fondazione Cariplo.

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Exogenous hyaluronic acid enhances porcine parthenogenetic embryo development in vitro possibly mediated by CD44

Theriogenology ◽

10.1016/j.theriogenology.2004.12.005 ◽

2005 ◽

Vol 64 (2) ◽

pp. 378-392 ◽

Cited By ~ 15

Author(s):

K. Toyokawa ◽

H. Harayama ◽

M. Miyake

Keyword(s):

Hyaluronic Acid ◽

Embryo Development ◽

Parthenogenetic Embryo ◽

Development In Vitro

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Sorbents of Phenols As a Components of the Nutritional Medium in Microclonal Reproduction of Plants

Natural Systems and Resources ◽

10.15688/nsr.jvolsu.2021.3.6 ◽

2021 ◽

pp. 39-48

Author(s):

Anna Pugacheva ◽

◽

Kristina Bikmetova ◽

Yuliya Smirnova ◽

◽

...

Keyword(s):

Phenolic Compounds ◽

Silicon Dioxide ◽

Nutrient Medium ◽

Normal Development ◽

Expanded Graphite ◽

Mercury Chloride ◽

Negative Effects ◽

Carbon Compounds ◽

Nutritional Medium

In the process of microclonal reproduction, plants secrete various substances into the nutrient medium, for example, phenolic compounds, which act as inhibitors of growth processes and, accordingly, prevent the normal development of explants in vitro. Plant tissues are treated with stabilizing substances, and various sorbents are also used as components of the nutrient medium to neutralize the negative effects of phenols. This paper presents an overview of the approved methods for solving the problem of sorption of phenolic compounds during microclonal propagation of plants. Various studies are considering the addition of certain components to the nutrient medium that prevent the release of harmful growth-inhibiting substances. Most often, various carbon compounds, such as activated carbon, are used as an adsorbent. The authors, based on the analysis of domestic and foreign literature on this topic, conclude that the most effective and frequently used are carbon compounds and the polymer polyvinyl pyrrolidone, less common is the use of the following inhibitory substances: ascorbic and citric acids, silver nitrate and mercury chloride. According to the results of the conducted analytical studies, the prospects of using such substances as thermally expanded graphite (TEG) and colloidal silicon dioxide as sorbents in the composition of the drug “Polysorb” were revealed. Due to the inhomogeneous porous structure, including both micropores and meso- or macropores, TEG is able to adsorb pollutants both from the solution and from the water surface, which makes it a potential sorbent for phenolic compounds. The effect of silicon dioxide, in amorphous form, on plants in vitro has already been successfully tested by some researchers, which indicates the prospects of its study.

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ID: 1087 Effects of oocytes collection and electro-activation protocols on maturation and preimplantation development of parthenogenetic diploid porcine embryos

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.360 ◽

2017 ◽

Vol 4 (S) ◽

pp. 175

Author(s):

Le Thi Thu Thuy ◽

Nguyen Ba Tu ◽

Bui Hong Thuy ◽

Nguyen Van Thuan

Keyword(s):

Embryo Development ◽

In Vitro Maturation ◽

Blastocyst Stage ◽

Activation Condition ◽

Parthenogenetic Embryo ◽

Morula Stage ◽

Collection Methods ◽

Dissection Method

Advances in porcine embryos production have been impaired by the in vitro maturation (IVM) system and in vitro developments (IVD) are still not optimized. The present study was undertaken to determine the effects of different oocytes collection methods (aspiration and dissection) on oocytes maturation and combine with the different electro-activation protocols on in vitro pre-implantation development of parthenogenetic diploid porcine embryos. The results showed that dissection method was significantly higher both in quantity (87.23%, p <0.05) of matured oocytes and in the rate of oocytes with very good quality (53.00%) compared with aspiration method (62.00%, 30.22%; respectively). We further accessed the quality of matured oocytes competence via parthenogenetic embryo development. Using four electro-activation protocols, we found that the electro-activation condition had no effect on the development of embryos to the 2-cell, 4-cell and 8-cell stages. However, the oocytes activated by the 60V/cm amplitude of pulse group had less (41.33%; p <0.05) development at the morula stage than 90V/cm, 100V/cm and 150V/cm amplitude of pulse groups (73.99%, 67.62%, 72.55%; respectively). The embryos develop to blastocyst stage was highest in the group induced by 90V/cm electro pulse was 71.76% compared to 100V/cm, 150V/cm, and 60V/cm (62.86%, 62.05%, and 37.63%, p<0.001, respectively). We conclude that the combining of dissection method for porcine oocytes collection and electro-activation with the amplitude of pulses 90V/cm can enhance both the activation success and development competition in in vitro pre- implantation parthenogenetic diploid porcine embryos.

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Pyracantha Extract Acts as an Antioxidant Agent to Support Porcine Parthenogenetic Embryo Development In Vitro

Journal of Embryo Transfer ◽

10.12750/jet.2013.28.3.243 ◽

2013 ◽

Vol 28 (3) ◽

pp. 243-250

Author(s):

Sung-Hun Min ◽

◽

Ji-Yeong Yeon ◽

Jin-Woo Kim ◽

Soo-Yong Park ◽

...

Keyword(s):

Embryo Development ◽

Parthenogenetic Embryo ◽

Antioxidant Agent ◽

Development In Vitro

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Regeneration of Viburnum dentatum L. from Alginate-Encapsulated Shoot Explants after Short-Term Cold Storage and Assessment of Genetic Stability Using ISSR Analysis

Agronomy ◽

10.3390/agronomy10111660 ◽

2020 ◽

Vol 10 (11) ◽

pp. 1660

Author(s):

Stefanos Hatzilazarou ◽

Stefanos Kostas ◽

Maria Joachim ◽

Athanasios Economou

Keyword(s):

Calcium Chloride ◽

Cold Storage ◽

Genetic Stability ◽

Nutrient Medium ◽

Mother Plant ◽

Artificial Seeds ◽

Inter Simple Sequence Repeats ◽

Regeneration Response ◽

Regeneration Procedure

The present study demonstrates an efficient protocol for alginate encapsulation, interim cold storing of artificial seeds and conversion to genetically stable plants of Viburnum dentatum L. “Lucidum Aiton”. Explants of shoot tips and first-node segments, excised from in vitro-derived viburnum microshoots, were encapsulated in 2.5% sodium alginate mixed with liquid MS nutrient medium and hardened in 50 mM of calcium chloride producing solid, soft and uniform beads. These artificial seeds achieved 28.9% germination under light, forming 4.3 microshoots per bead. However, with 100 mM of calcium chloride for hardening, the beads were firm and of a uniform globular shape and suitable for handling and exhibited a germination response of 48.9%. Encapsulated shoot tip explants of viburnum, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in regeneration response (73.3, 62.2, 51.1%, respectively), while non-encapsulated explants, stored under same conditions, did not survive after the fourth week of cold storage. Microshoots from cold-stored encapsulated explants, which were rooted in solid MS nutrient medium with 0.5 μΜ of Indole-3-acetic acid (IAA) and transplanted to a substrate of peat-perlite (3:1, v/v), acclimatized successfully after application of 75 or 50% shading, which was gradually reduced, and were established with minimum losses in a greenhouse. For the genetic stability of the artificial seed-derived plantlets and compared with the mother plant, an assessment was conducted using Inter Simple Sequence Repeats (ISSRs) analysis. The ISSR profiles proved the genetic uniformity and clonal stability of the regenerated plantlets and their genetic resemblance to the mother plant. The present regeneration procedure could be used as an alternative method for the micropropagation of V. dentatum.

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First polar body morphology affects potential development of porcine parthenogenetic embryo in vitro

Zygote ◽

10.1017/s0967199414000252 ◽

2014 ◽

Vol 23 (4) ◽

pp. 615-621 ◽

Cited By ~ 2

Author(s):

Junhe Hu ◽

Chenzhong Jin ◽

Hui Zheng ◽

Qinyan Liu ◽

Wenbing Zhu ◽

...

Keyword(s):

Embryo Development ◽

Polar Body ◽

Cell Number ◽

Cortical Granules ◽

Potential Development ◽

First Polar Body ◽

Porcine Embryo ◽

Parthenogenetic Embryo ◽

The Mean

SummaryPrevious studies have reported that the first polar body (PB1) morphology reflects embryo development competence, but the effects of PB1 on porcine embryo development remain unknown. This study aims to determine whether the ability of porcine embryo development is related to oocytes’ PB1 in vitro. The distribution of type II cortical granules (CGs) of porcine matured oocytes in grade B PB1 is significantly greater compared with those in grades A and C PB1 (71.43% versus 52.46% and 50%; P < 0.05). The ratio of porcine parthenogenetic blastocysts and the mean cell number in each blastocyst in the group with grade B PB1 is significantly greater than that with grades A and C PB1 (30.81% vs. 19.02% and 15.15%; P < 0.05) and (36.67 versus 24.67, 28.67; P < 0.05), and no significant differences are found in the embryo cleavage for all groups (79.75%, 84.30%, and 78.18% in grades A, B, and C PB1; P > 0.05). The acetylation level of porcine embryos in the group with grade B PB1 is significantly greater compared with those in the other groups (P < 0.05), and is almost 2.5 times higher than that in grade A. Therefore, porcine oocytes with PB1 in grade B are more competitive in cytoplasmic maturation and further embryo development in vitro.

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