Evolution in vitro du contenu en ADN nucléaire et de la ploïdie des embryons polliniques du Datura innoxia

1981 ◽  
Vol 59 (4) ◽  
pp. 508-517 ◽  
Author(s):  
Brigitte S. Sangwan-Norreel
Keyword(s):  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Datura Innoxia ◽  
Cell Numbers ◽  
Irregular Structures ◽  
Three Stages ◽  
Androgenetic Embryos ◽  
Haploid Embryos ◽  
Vitro Development

Nuclear DNA content was estimated by densitometry in three stages of in vitro development of Datura innoxia Mill. embryos (3–25 cells) obtained from pollen. At the same time, the sizes, structure and arrangements of nuclei in young embryos were examined and chromosome numbers of adult embryos were determined. Results showed that: (i) Embryos originating from pollen were haploid, diploid, aneuploid, or myxoploid. Proportions of each ploidy level varied with cell numbers in embryos, and with ages of cultures, (ii) Young diploid embryos were very regular and had dense nuclei of one type only. Haploid embryos were less regular; they had one or two types of nuclei whose texture was often looser than that of diploid embryo nuclei. Aneuploid and myxoploid embryos had very irregular structures, (iii) Initiation of androgenetic embryos was spread out in time from the 2nd to the 12th day of culture. Embryos showed different nuclear features if they were initiated at the beginning, in the middle, or at the end of the culture period. Embryos initiated early were mostly haploid and the proportion of abnormal ploidy embryos was low during the 1st week of culture. In Datura innoxia, therefore, embryos must be taken after 1 week of culture to obtain the population which is most favourable to further genetic breeding. [Journal translation]

In vitro development of haploid mouse embryos produced by bisection of one-cell fertilized eggs

Development ◽  
1977 ◽  
Vol 38 (1) ◽  
pp. 187-202
Author(s):  
Andrzej K. Tarkowski
Keyword(s):  
X Chromosome ◽  
Culture Conditions ◽  
Extrinsic Factors ◽  
Cell Stage ◽  
Glass Needle ◽  
Androgenetic Embryos ◽  
Haploid Embryos ◽  
Vitro Development ◽  
Further Development

F1(CBA × C57BL'10) mouse eggs originating from spontaneous or induced ovulation and fertilized by CBA-T6T6 or PO spermatozoa were bisected with a glass needle into halves each containing a pronucleus. This technique offers a unique opportunity of producing both androgenetic and gynogenetic haploid embryos from one egg. Out of 600 operated eggs, in 406 (67·7%) both halves survived. During 96 h of culture in vitro the fragments were inspected once daily and finally examined in air-dried preparations. Eighty-seven per cent of halves underwent first cleavage but their further development was to a large extent affected by extrinsic factors connected with experimental procedure (mainly by suboptimal and variable culture conditions) and by the origin of eggs (those from spontaneous ovulation being superior). For this reason developmental capabilities of egg halves were assessed in a selected group of pairs in which at least one partner reached the stage of four or more blastomeres. The observed ratio between pairs with both or only one sister embryo developing successfully suggests that androgenetic embryos carrying Y rather than X chromosome can cleave twice but do not survive beyond 4-cell stage. None of the metaphase plates from older embryos contained a Y chromosome. These observations imply that the X chromosome is genetically active during early cleavage and that a full haploid set is required for preimplantation development to be completed. Formation of blastocysts varied from batch to batch, with an average of 12·8% and maximal incidence of 29·5% . In 34 pairs both fragments developed beyond the 4-cell stage but in only one case did both form blastocysts. Haploid blastocysts were composed of 27 cells on average which was about a half of the number of cells in control diploid zona-free whole eggs. Ten out of 51 embryos with metaphase plates proved to be haploid/diploid mosaics.

Assessment of the cellular DNA content of whole mounted mouse and human oocytes and of blastomeres containing single or multiple nuclei

Zygote ◽  
1993 ◽  
Vol 1 (1) ◽  
pp. 17-25 ◽  
Author(s):  
Nicola J. Winston ◽  
Martin H. Johnson ◽  
Peter R. Braude
Keyword(s):  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Structural Features ◽  
Chromosomal Dna ◽  
Individual Values ◽  
Mean Values ◽  
Mouse Oocytes ◽  
Cellular Dna

SummaryThe nuclear DNA content of intact, live or fixed, human and mouse oocytes and blastomeres has been measured rapidly and reliably. Chromosomal DNA has been stained with DAPI, the fluorescent emission from which has been measured photocytometrically.In vitrofertilised mouse oocytes and embryos at various stages of development were assessed for their DNA content. The mean values of 1C, 2C and 4C DNA content were clearly different, and it was possible to assign correctly individual values for DNA content to each class with 92%, 61% and 81% confidence respectively. Maintaining the cells as whole mounts allowed other morphological and structural features to be examined. When formation of multiple micronuclei was induced in mouse oocytes by their insemination in the presence of nocodazole, the additive signal from all the micronuclei in one zygote was equivalent to the expected DNA content. Application to early human blastomeres of this photocytometric technique for measurement of the total cellular DNA content revealed that multinucleated blastomeres contained 2C to 4C DNA levels, consistent with a diploid DNA content.

scholarly journals MICROPROPAGATION AND PLOIDY STABILITY OF Lippia lacunosa Mart. & Schauer: AN ENDANGERED BRAZILIAN MEDICINAL PLANT

2019 ◽  
Vol 6 (1) ◽  
pp. 1-7
Author(s):  
Diego Pandeló José ◽  
José Marcello Salabert De Campos ◽  
Lyderson Facio Viccini ◽  
Emilly Ruas Alkimim ◽  
Marcelo De Oliveira Santos
Keyword(s):  
Flow Cytometry ◽  
Medicinal Plant ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Naphthalene Acetic Acid ◽  
Flow Cytometry Analysis ◽  
Ploidy Levels

Lippia lacunosa is a Brazilian savanna plant that belongs to the Verbenaceae family. It has been used in folk medicine as a treatment for different diseases. This species represents an endangered Brazilian medicinal plant, and this is the first report documenting a reliable protocol for the in vitro propagation and regeneration of L. lacunosa. Axenic explants were cultivated in MS medium containing different concentrations of naphthalene acetic acid (NAA) to induce root growth. The mean shoot length and the number of roots were highest with 0.06 mg·L-1 NAA. The highest number of buds in shoot regeneration was induced with 2 mg·L-1 6-benzylaminopurine (BA). To obtain a long-term culture, the dwarf shoots were elongated on MS media containing 0.5 mg·L-1 BA alternated with MS containing 2 mg·L-1 BA every 40 days. In the present protocol, the long-term shoots retained the ability to root even after long periods of BA treatment. In addition, we evaluated the nuclear DNA content and ploidy levels, including the occurrence of endopolyploidy, in long-term micropropagated plant leaves using flow cytometry analysis. The plants propagated in vitro over several years possessed nuclear DNA contents ranging from 2.940 to 3.095 pg, and no differences in DNA content were found among in vitro plants or between these plants and the control (L. lacunosa from a greenhouse with a DNA content of 3.08 pg). The flow cytometry analysis also demonstrated that there was no polyploidization. The present study will be useful for biotechnological approaches and provides the first estimate of the nuclear DNA content of this species using flow cytometry.

scholarly journals Nuclear DNA content and ultrastructure of secretory cells of Vicia faba L. stigma

2014 ◽  
Vol 63 (2) ◽  
pp. 139-145 ◽  
Author(s):  
Bogdan Wróbel ◽  
Elżbieta Bednarska
Keyword(s):  
Vicia Faba ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Development Stage ◽  
Secretory Cells ◽  
Vicia Faba L ◽  
Three Stages ◽  
Object Of Study ◽  
Diploid Cells ◽  
Vacuolar System

The object of study was the level of nuclear DNA and the ultrastructural transformations in the secretory cells of the stigma in <i>Vicia faba</i> L. It has been found that the stigmal cells which are active in biogenesis and exudate secretion are diploid cells whose differentiation starts from 2C DNA level. The presence of a population of nuclei with an amount DNA of about 2.5 C suggests that the metabolic activity of those cells may be regulated through supplementary incomplete replication. The ultrastructural transformations of secretory cells point to three stages of biogenesis and secretion of exudate. Stage I, before the start of the cell's secretory functions, is characterized by the development of the protein synthesizing apparatus and the activity of dictyosomes. In development stage II vesicular electron-transparent exudate is secreted. Stage III of exudate biogenesis is production of lipids. They form mainly in the plastids and are secreted with the involvement of the cell's vacuolar system.

273 EFFECT OF IONOMYCIN ASSOCIATED WITH ROSCOVITINE, DEHYDROLEUCODINE, CYCLOHEXIMIDE, OR ETHANOL ON HAPLOID ACTIVATION OF BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 225
Author(s):  
M. Suvá ◽  
N. G. Canel ◽  
D. F. Salamone
Keyword(s):  
Chromosome Number ◽  
Polar Body ◽  
Routine Procedure ◽  
In Vitro Development ◽  
Second Polar Body ◽  
N 93 ◽  
Haploid Embryos ◽  
Vitro Development ◽  
Bovine Oocytes

Haploid activation of bovine oocytes after ICSI is a routine procedure. However, embryos frequently contain an abnormal chromosome set as a result of the drugs employed. We compared the efficiency of ionomycin (Io) followed by roscovitine (ROSC), cycloheximide (CHX), ethanol, and dehydroleucodine (DhL) to induce haploid parthenogenetic activation in bovine. Pronuclear (PN) formation, second polar body (2PB) extrusion, embryo development, and ploidy of blastocysts were evaluated. To this aim, COC were aspirated from slaughtered ovaries and IVM for 22 h. Oocytes were activated with 5 µM of Io for 4 min and then randomly allocated into 1 of the following treatments: 25 or 50 µM ROSC or 10 µg mL–1 of CHX for 5 h; 15 or 30 µM DhL for 3 h; or 5 min of exposure to 7% ethanol 4 h post-Io. Controls were Io followed by (1) 3 h in TCM-199 and 3 h in 1.9 mM 6-DMAP (Io-3h-DMAP) and (2) 3 h of exposure to 1.9 mM 6-DMAP (Io-DMAP). Oocytes were cultured in SOF medium. The PN formation and 2PB extrusion were assessed by 5 µg mL–1 of propidium iodide oocyte staining, 17 h after Io. Cleavage, morulae, and blastocyst stages were evaluated at Days 2, 5, and 8 of in vitro development, respectively. Chromosome number of blastocysts was evaluated by Giemsa staining. Data were analysed by Fisher's test (P < 0.05). Rates of 2PB extrusion were 75, 61.1, 60, 56.3, 54.6, and 42.9% for 15 µM DhL (n = 23), 50 µM ROSC (n = 22), Io-3h-DMAP (n = 9), CHX (n = 17), 25 µM ROSC (n = 22), and ethanol (n = 22), respectively, with no differences between groups. A PN was observed in over 81% of the oocytes activated with ethanol, 25 µM ROSC, CHX, 50 µM ROSC, and 15 µM DhL. Lower percentages of 2PB extrusion and PN formation were observed for 30 µM DhL (n = 22; 6.3 and 0%, respectively). The highest cleavage rates were 83.2% for 25 µM ROSC (n = 185), not differing from 78% in Io-DMAP (n = 159). Cleavage rates for 50 µM ROSC (n = 185), CHX (n = 143), and ethanol (n = 74; 80.5, 80.4 and 67.6%, respectively) were not different from Io-3h-DMAP (n = 78; 71.8%) and Io-DMAP. Cleavage rates for 15 µM DhL (n = 70) and 30 µM DhL (n = 93) were the lowest (48.6 and 25.8%). Blastocyst rates were the highest for CHX and 50 µM ROSC, not differing from Io-3h-DMAP (21.7 and 10.8 v. 18%). Very few or no blastocysts were obtained with ethanol, 25 µM ROSC, 30 µM DhL, and 15 µM DhL (4.1, 3.8, 1.1, and 0%, respectively), although ethanol was not different from Io-3h-DMAP. Chromosome number analysis showed that ethanol (n = 2) and CHX (n = 2) resulted in a higher percentage of haploid embryos (50% each), followed by 50 µM ROSC (n = 8), 25 µM ROSC (n = 3), and Io-3h-DMAP (n = 8; 37.5, 33.3% and 12.5%, respectively), although they were not different. Remaining embryos were diploid, aneuployd, or mixoployd. In conclusion, DhL and ROSC proved to be as effective as CHX or ethanol regarding 2PB extrusion and resulting ploidy, defining features when activating oocytes in ART, suggesting they could be efficiently used in bovine to assist ICSI.

scholarly journals Lycopene Improves In Vitro Development of Porcine Embryos by Reducing Oxidative Stress and Apoptosis

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 230
Author(s):  
Hyo-Gu Kang ◽  
Sanghoon Lee ◽  
Pil-Soo Jeong ◽  
Min Ju Kim ◽  
Soo-Hyun Park ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Caspase 3 ◽  
Developmental Competence ◽  
Oxygen Species ◽  
Cell Numbers ◽  
Porcine Embryo ◽  
Apoptosis Pathways ◽  
Vitro Development

In vitro culture (IVC) for porcine embryo development is inferior compared to in vivo development because oxidative stress can be induced by the production of excessive reactive oxygen species (ROS) under high oxygen tension in the in vitro environment. To overcome this problem, we investigated the effect of lycopene, an antioxidant carotenoid, on developmental competence and the mechanisms involved in mitochondria-dependent apoptosis pathways in porcine embryos. In vitro fertilized (IVF) embryos were cultured in IVC medium supplemented with 0, 0.02, 0.05, 0.1, or 0.2 μM lycopene. The results indicate that 0.1 μM lycopene significantly increased the rate of blastocyst formation and the total cell numbers, including trophectoderm cell numbers, on Day In terms of mitochondria-dependent apoptosis, IVF embryos treated with 0.1 μM lycopene exhibited significantly decreased levels of ROS, increased mitochondrial membrane potential, and decreased expression of cytochrome c on Days 2 and Furthermore, 0.1 μM lycopene significantly decreased the number and percentage of caspase 3-positive and apoptotic cells in Day-6 blastocysts. In addition, Day-2 embryos and Day-6 blastocysts treated with 0.1 μM lycopene showed significantly reduced mRNA expression related to antioxidant enzymes (SOD1, SOD2, CATALASE) and apoptosis (BAX/BCL2L1 ratio). These results indicate that lycopene supplementation during the entire period of IVC enhanced embryonic development in pigs by regulating oxidative stress and mitochondria-dependent apoptosis.

Nuclear DNA content and isozyme variation in relation to morphogenic potential of strawberry (Fragaria × ananassa) callus cultures

1991 ◽  
Vol 69 (2) ◽  
pp. 239-244 ◽  
Author(s):  
Narender S. Nehra ◽  
Kutty K. Kartha ◽  
Cecil Stushnoff
Keyword(s):  
Dna Content ◽  
Nuclear Dna ◽  
Flow Cytometric Analysis ◽  
Nuclear Dna Content ◽  
Leaf Explants ◽  
Callus Cultures ◽  
Fragaria Ananassa ◽  
Flow Cytometric ◽  
Ploidy Changes

Callus cultures of strawberry cv. Redcoat (2n = 8x = 56) initiated from greenhouse and in vitro leaf explants were examined at various culture periods for morphogenic response, changes in nuclear DNA content, and isozyme banding patterns of four enzymes. The flow cytometric analysis of nuclear DNA content revealed the occurrence of polyploid and aneuploid changes as a function of ageing of callus cultures. The calli initiated from in vitro leaf explants were more prone to such changes than those initiated from greenhouse leaf explants. The in vitro morphogenic ability of callus cultures was affected by the ploidy changes, but the latter were not the only cause for loss in regeneration potential of long-term callus cultures. The isozyme phenotypes of esterase, phosphoglucomutase, phosphoglucoisomerase, and leucine aminopeptidase did not change with the chromosomal variation in callus cultures. Key words: strawberry, Fragaria × ananassa, callus culture, flow cytometry, nuclear DNA content, isozyme.

A Sorghum bicolor × S. macrospermum hybrid recovered by embryo rescue and culture

2005 ◽  
Vol 53 (6) ◽  
pp. 579 ◽  
Author(s):  
H. James Price ◽  
George L. Hodnett ◽  
Byron L. Burson ◽  
Sally L. Dillon ◽  
William L. Rooney
Keyword(s):  
Nuclear Dna ◽  
Embryo Rescue ◽  
Nuclear Dna Content ◽  
Female Parent ◽  
Male Sterile ◽  
Leaf Pubescence ◽  
Culture Techniques ◽  
Close Phylogenetic Relationship ◽  
Improvement Programs

Although exotic germplasm is extensively used in sorghum improvement programs, Sorghum species classified in sections other than Eu-sorghum have not been utilised as germplasm because of strong reproductive barriers involving pollen–pistil incompatibilities. S. macrospermum is of particular interest to sorghum breeders because of its close phylogenetic relationship and cytogenetic similarities to S. bicolor and its resistance to important sorghum pests and pathogens, such as sorghum midge and sorghum downy mildew. A vegetatively vigorous interspecific hybrid was obtained from a cross between a cytoplasmic male-sterile S. bicolor plant and S. macrospermum by using embryo rescue and in vitro culture techniques. The hybrid was morphologically intermediate to S. bicolor and S. macrospermum in leaf width, leaf pubescence, plant height, inflorescence morphology, chromosome number and nuclear DNA content. It was male-sterile like its ATx623 parent. The hybrid produced no offspring when used as the female parent in a backcross with S. bicolor. This is the first confirmed hybrid between S. bicolor and S. macrospermum, and to our knowledge, it is the first reported hybrid between S. bicolor and any Sorghum species outside the Eu-sorghum section.

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