scholarly journals Introduction into in vitro culture and cytogenetic analysis of Iris attica Boiss. & Heldr. and Iris pseudopumila Tineo plants

2019 ◽  
Vol 16 (2) ◽  
pp. 203-211 ◽  
Author(s):  
M. O. Twardovska ◽  
I. O. Andreev ◽  
V. A. Kunakh
Keyword(s):  
Chromosome Number ◽  
In Vitro Culture ◽  
Cytogenetic Analysis ◽  
Root Meristem ◽  
Chromosomal Rearrangements ◽  
High Survival ◽  
High Survival Rate ◽  
Survival Level

Aim. The work was aimed at the development of conditions for introduction into in vitro culture of two species of irises, Iris attica and I. pseudopumila to obtain aseptic seedlings with subsequent reintroduction into natural environment, as well as at cytogenetic analysis of the obtained plants. Methods. In vitro seed germination and seedling cultivation. Cytogenetic analysis of cells of root meristem, determination of chromosome number and morphology in mitotic metaphase plates, anaphase analysis. Results. The plants of I. attica and I. pseudopumila were introduced in vitro. Aseptic seedlings were obtained, which were actively growing on MS/2 medium without phytohormones. The experiments on the adaptation of the plants to greenhouse conditions revealed the high survival rate for both species. The chromosome number 2n = 16 was established for the obtained plants of both I. attica and I. pseudopumila. Mixoploidy was detected in root meristem of some of the plants, the incidence of which was 10.9 % for I. pseudopumila and 30.4 % for I. attica. The frequency of cells with chromosomal rearrangements revealed by anaphase analysis in root meristem of I. pseudopumila seedlings was 2.6 %; in I. attica plants, chromosome aberrations were not detected. Conclusions. The plants of I. attica and I. pseudopumila were introduced into in vitro culture, aseptic seedlings were obtained, which showed a high survival level when adapted to greenhouse conditions. Chromosome number 2n = 16 was established for the obtained plants of both species. The root apical meristems of the seedlings were found to be mixoploid, with the incidence of mixoploidy in I. attica identified as three times higher than in I. pseudopumila plants.Keywords: I. attica Boiss. & Heldr., I. pseudopumila Tineo, aseptic seedlings, mixoploidy, anaphase aberration.

Period of harvest, sprouting ability of cuttings, and in vitro plant regeneration in Fraxinus excelsior

1994 ◽  
Vol 72 (2) ◽  
pp. 261-267 ◽  
Author(s):  
Conceição Eneida Silveira ◽  
Alain Cottignies
Keyword(s):  
Chromosome Number ◽  
In Vitro Culture ◽  
Adventitious Roots ◽  
Woody Plant ◽  
Fraxinus Excelsior ◽  
Stem Cuttings ◽  
Natural Conditions ◽  
Apical Bud ◽  
Woody Plant Medium

Propagation by stem cuttings and in vitro culture of apical bud explants were studied on Fraxinus excelsior L. Stem cuttings from 4- to 7-year-old trees growing under natural conditions sprouted only when cuttings were taken from dormant material. Only 6% of those that had sprouted developed roots by the 7th month of culture. Similarly, only apical bud explants harvested during the dormant period sprouted in vitro. Up to 87% of these sprouts developed two to four branching adventitious roots after 5 months of culture. During the initial phase of in vitro culture, the Quoirin and Lepoivre medium and the woody plant medium favoured sprout lengthening. During the phase of multiplication, up to three sprouts per explant developed with the woody plant medium in the presence of a combination of high 6-benzylaminopurine (3.0–4.0 mg∙L−1) and low indole-3-butyric acid (0.01–0.03 mg∙L−1) concentrations. Rooting was obtained in a medium without any growth regulators. Microscopic analysis showed a direct connection between the vascular elements of adventitious roots and stem of plantlet. Chromosome number in root apices of ash plantlets and ash trees grown under natural conditions was 2n = 46. Key words: chromosome number, Fraxinus excelsior L., in vitro plants, micropropagation, stem cuttings.

scholarly journals Influence of Indole acetic acid and Tryptophan on production of Vinblastine and Vincristine of Catharanthus roseous callus cells in the accumulation media of In Vitro tissue culture

2010 ◽  
Vol 7 (3) ◽  
pp. 1113-1119
Author(s):  
Baghdad Science Journal
Keyword(s):  
Tissue Culture ◽  
High Performance Liquid Chromatography ◽  
Acetic Acid ◽  
Liquid Chromatography ◽  
In Vitro Culture ◽  
High Performance ◽  
Indole Acetic Acid ◽  
Culture Technique

This study on the plant of Ain –AL Bason Catharanthus roseous showed the ability of callus cells that is produced by In Vitro culture technique and transformed to the accumulated media (MS 40gm/L sucrose ,2gm/L IAA Indole acetic acid , 0.5gm/L Tryptophan) to produce Vinblastine and Vincristine compounds. Extraction, purification and quantitive determination of Vinblastine and Vincristine compounds using High performance liquid chromatography technique (HPLC)were carried out. The results showed that the highest concentration of Vinblastine and Vincristine compounds were ( 4.653,12.5 (ppm /0.5 dry Wight respectively from transformed callus cells from MS 40 gm /L sucrose , 2 gm / L NAA Naphthaline acetic acid .

302 REPLACEMENT OF FETAL BOVINE SERUM WITH KNOCKOUT SERUM REPLACER AND STEM CELLS CONDITIONED MEDIUM DURING IN VITRO CULTURE OF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 307
Author(s):  
M. M. Souza ◽  
N. Z. Saraiva ◽  
C. S. Oliveira ◽  
T. A. D. Tetzner-Nanzeri ◽  
R. Vantini ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Protein Supplementation ◽  
Control Group ◽  
Bovine Embryos ◽  
Fetal Bovine

The use of fetal bovine serum (FBS) as protein supplementation in IVP of bovine embryos has presented difficulties because it can introduce a number of pathogenic components in culture systems, can be related to the birth of calf with abnormal growth and development, and precludes the establishment of the actual nutritional needs of the embryo, because it contains an unlimited variety of substances. This study evaluated the replacement of the FBS in the medium of in vitro culture (IVC) of bovine embryos, using the knockout serum replacer (KSR) as protein supplementation and culture medium conditioned with stem cells. Therefore, bovine oocytes from ovaries of slaughterhouse were selected and matured in vitro in TCM-199 medium supplemented with 10% FBS (Crypion), 1.0 μg mL-1 FSH (Pluset®, Calier, Barcelona, Spain), 50 μg mL-1 hCG (Profasi®, Serono, Geneva, Switzerland), 1.0 μg mL-1 estradiol (Sigma E-2758, Sigma Chemical, St. Louis, MO, USA), 0.2 mM sodium pyruvate, and 83.4 μg mL-1 amikacin for 24 h. After that, 1144 oocytes were fertilized in IVF-TALP medium containing 6 mg mL-1 of BSA. After 18 to 22 h, the zygotes were cultured in SOF + 5% FBS (group 2); SOF + 5% KSR (group 3); SOF (5% FBS) + 10% SOF (5% FBS) conditioned by stem cells (group 4); or SOF (5% KSR) + 10% SOF (5% KSR) conditioned by stem cells (group 5), in an atmosphere of 5% O2 at 38.5°C for 8 days. A control group outside the controlled atmosphere was added, supplemented with 5% FBS (group 1). The SOF medium supplemented with 5% FBS or KSR was conditioned by stem cells and added to SOF medium for the culture of embryo at a concentration of 10%. The rates of cleavage and production of blastocysts were assessed 48 hours and 7 days after IVF, respectively, and analyzed by chi-square test, with a significance level of 5% in the statistical program Minitab® (release 14.1, Minitab, State College, PA, USA). On the eighth day, the TUNEL test for determination of the percentage of apoptosis and the differential staining technique for determination of inner cell mass (ICM) and trophoblast (TF) were performed. The results were submitted to ANOVA, followed by comparing the means by Tukey’s test using the program GraphPad Prism (GraphPad, San Diego, CA, USA). The treatments did not differ in the production of embryos, being similar to the control group: G1 = 31.75% (74/233), G2 = 35.26% (79/224), G3 = 32.70% (74/226), G4 = 28.76% (63/219), and G5 = 26.85% (65/242). With regard to the assessment of embryonic quality, the treatments showed similar results to the control groups. No differences were observed among groups both in color and ICM/TF ratio (G1 = 0.60, G2 = 0.62, G3 =0.65, G4 = 0.60, and G5 = 0.60). Furthermore, the TUNEL showed no significant difference in the percentage of apoptosis among groups (G1 = 7.10%, G2 = 3.76%, G3 = 5.58%, G4 = 4.50%, and G5 = 4.11%). The data obtained so far indicate that it is possible to produce embryos in vitro by replacing the FBS in the culture, achieving results similar to those obtained with serum. Financial support: FAPESP 2007/58506-6.

53 IMPACTS OF USING PROCAINE AS A DNA-DEMETHYLATING AGENT IN IN VITRO CULTURE OF BOVINE CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 132
Author(s):  
V. A. Michalczechen-Lacerda ◽  
F. C. Rodrigues ◽  
R. V. de Sousa ◽  
R. Rumpf ◽  
M. M. Franco
Keyword(s):  
Dna Methylation ◽  
Cell Viability ◽  
In Vitro Culture ◽  
Cytogenetic Analysis ◽  
Imprinted Genes ◽  
Demethylating Agent ◽  
Chromosome Integrity ◽  
Methylation Patterns ◽  
Number Of Cells

Euchromatin and heterochromatin organisation define the specificity of each cell type. This structure is controlled by epigenetic modifications and the DNA methylation is one of the best known for inducing transcriptional repression. Recently, procaine was uncovered as a DNA-demethylating agent, but there are few reports about its dynamic epigenetic action on somatic cells. Mono-allelic expression of imprinted genes is controlled by DNA methylation and inherited to somatic tissues of a sex-specific manner. The aim was to investigate the effects of using procaine, a DNA-demethylating agent, in in vitro culture of bovine (Bos taurus indicus) fibroblast for 72 h (passage 4). We have evaluated cell viability, chromosome integrity, and DNA methylation patterns. To evaluate cell viability, we have used trypan blue 0.4%. To evaluate chromosome integrity, we have used conventional cytogenetic analysis. To investigate DNA methylation patterns, we have analysed 2 differentially methylated regions (DMR) located into the exon 10 of IGF2 and exon 1 of XIST imprinted genes, using the bisulfite sequencing method (EZ DNA methylation kit, Zymo Research, Orange, CA, USA). After bisulfite treatment and nested-PCR, the amplicons were separated in agarose gel electrophoresis, purified with GenClean III kit (MP Biomedicals, Irvine, CA, USA), cloned in a pGEM-T easy vector system (Promega, Madison, WI), and sequenced. The DNA sequences were analysed using the BiQ Analyzer v. 2.0 (2008) software. The cell viability data were analysed using ANOVA and Tukey or Kruskal-Wallis and Mann-Whitney tests, and the methylation status were analysed using Student’s t-test or Mann-Whitney tests in the Prophet software (BBN Systems and Technologies). Cell culture using 0.1 mM or 0.5 mM of procaine were viable and the number of cells with intact membrane was higher than the control and 2.0 mM of procaine groups (P ≤ 0.05). The total number of cells was lower in the group with 2.0 mM of procaine (P ≤ 0.01). Cytogenetic analysis showed no differences among the groups, with no chromosome abnormalities detected. The methylation pattern was not different for both DMR evaluated among the groups. We have observed that there was a beneficial effect to the cells that have received supplementation with 0.1 mM or 0.5 mM of procaine, because there was an increase in the number of viable cells without chromosomal abnormalities. We cannot ignore that a global DNA demethylation may have occurred, which was not detected in the specific analysed regions. The results obtained here may contribute to improving the efficiency of animal cloning, transgenic animal production, and the knowledge about stem cells. Supported by Embrapa Genetic Resources and Biotechnology and CAPES.

108 CALYCULIN-A INDUCES CHROMOSOME CONDENSATION FOR CYTOGENETIC ANALYSIS IN BOVINE AND MURINE BLASTOMERES

2008 ◽  
Vol 20 (1) ◽  
pp. 134
Author(s):  
J. M. Kramer ◽  
A. Evans ◽  
K. Drury ◽  
K. Moore
Keyword(s):  
Cytogenetic Analysis ◽  
Chromatin Condensation ◽  
Chromosomal Rearrangements ◽  
Chromosome Condensation ◽  
Preimplantation Embryos ◽  
Hypotonic Solution ◽  
Calyculin A ◽  
Chi Square ◽  
Degree Of Condensation

Cytogenetic studies of preimplantation embryos have traditionally used interphase fluorescence in situ hybridization (FISH) to examine chromosome copy number, chromosomal rearrangements, or sex determination. Comprehensive analysis of chromosomes is hindered by the low mitotic index of blastomeres, combined with the technical limitations of interphase FISH. Efforts to overcome these limitations include inducing premature chromosome condensation (PCC) for metaphase FISH, by fusing blastomeres with metaphase II-stage oocytes, or with time-consuming incubations with okadaic acid or vinblastine. Our objective was to evaluate Calyculin-A (CA) as an alternative to induce PCC in blastomeres. In vitro-produced bovine 8-cell embryos and frozen–thawed mouse 8-cell embryos were cultured with CA and examined for changes in morphology before fixation. Colcemid (0.1 mg mL–1), the standard for cytogenetic analysis, and vehicle served as positive and negative controls. Blastomeres were washed in hypotonic solution, loaded onto slides, and fixed in methanol:acetic acid (3:1). The degree of chromatin condensation and quality of chromosome spreads were determined by 42,6-diamidino-2-phenylindole staining and visualization on an epifluorescence microscope (100�). Experiment 1 (1 rep, 136 cells) tested dose of CA on bovine blastomeres at 50, 100, 150, 250, 500, and 750 nm. Experiments 2 and 3 tested culture duration of CA (50 nm) at 60, 120, and 180 min in bovine blastomeres and 60, 90, and 120 min in mouse blastomeres (2 reps each, 132 and 207 cells, respectively). Data were analyzed by chi-square with significance deemed P < 0.05. Cell lysis and blebbing was observed in bovine blastomeres treated with greater than 150 nm CA. Duration of CA treatment affected the frequency of bovine and mouse blastomeres undergoing PCC. Fewer bovine blastomeres treated for 60 min underwent chromatin condensation compared to blastomeres treated for at least 120 min with 50 nm CA (25% v. 100%; P < 0.05). In mice, the frequency of blastomeres undergoing PCC was lower (43%) for 50 nm CA at 60 min than at 90 and 120 min (90 and 100%, respectively; P < 0.05). Chromatin condensation suitable for cytogenetic analysis was 20, 34, and 20%, respectively, but did not differ (P > 0.10). Further examination (Exp. 4, 5 reps, 293 cells) comparing the degree of condensation revealed 55% of the total bovine blastomeres treated with 50 nm CA for 120 min were suitable for cytogenetic analysis, as compared to 30% treated with colcemid for 16 h (P < 0.05). Bovine and mouse blastomeres treated with vehicle had lower PCC than all other treatments averaging 2 and 7%, respectively (P < 0.05). These results suggest that CA can rapidly induce PCC in blastomeres from pre-implantation bovine and murine embryos, but the degree of chromatin condensation may not always be suitable for detailed cytogenetic analysis from a single blastomere.

Viable rabbits derived from oocytes by intracytoplasmic injection of spermatozoa from an infertile male

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 157-162 ◽  
Author(s):  
Qiuyan Li ◽  
Jian Hou ◽  
Sheng Wang ◽  
Hong Guan ◽  
Nan Zhang ◽  
...  
Keyword(s):  
Early Stage ◽  
White Male ◽  
High Survival ◽  
High Survival Rate ◽  
Infertile Male ◽  
Intracytoplasmic Injection ◽  
Limited Success ◽  
Single Living ◽  
Assisted Fertilization

SummaryIntracytoplasmic sperm injection (ICSI) is an assisted fertilization technique and has been widely applied in human medicine to overcome some obstacles of infertility. However, this technology has not yet been used as a mainstream technique for animal production, including the rabbit, due to its limited success. The aim of this study was to improve ICSI techniques and establish an efficient ICSI method for rabbits. Spermatozoa used for ICSI were collected from mature New Zealand white male rabbits. They were washed two to three times with HEPES-buffered TCM199 containing 10% FBS and then mixed with 10% polyvinylpyrrolidone (PVP) prior to microinjection. Oocytes were harvested from superovulated donor rabbits after 14–15 h hCG treatment and were fertilized by microinjection of a single living spermatozoon into the ooplasm of each oocyte without additional activation treatment. A total of 317 injected oocytes resulted in the high survival rate of 86.1%. Among the surviving oocytes, 273 were placed into culture dishes for in vitro development. The fertilization, cleavage and blastocyst rates were 59.0%, 88.2% and 45.3% respectively. Furthermore, ICSI embryos were produced with spermatozoa from an infertile male rabbit, and 21 early-stage embryos (2-cell and 4-cell) were surgically transferred into the oviducts of two adult female rabbits. On day 31 after transfer, one out of the two recipients gave birth to two normal and healthy young rabbits. These results demonstrate that rabbit oocytes can be successfully activated and fertilized by the new ICSI protocol. Spermatozoa derived from infertile rabbits can successful fertilize oocytes and produce offspring by the simple ICSI technique.

scholarly journals Rapid, sensitive, and validated UPLC/Q-TOF-MS method for quantitative determination of vasicine in Adhatoda vasica and its in vitro culture

2014 ◽  
Vol 10 (37) ◽  
pp. 198 ◽  
Author(s):  
Garg Madhukar ◽  
EnnusTajuddin Tamboli ◽  
Parveen Rabea ◽  
SH Ansari ◽  
MZ Abdin ◽  
...  
Keyword(s):  
Quantitative Determination ◽  
In Vitro Culture ◽  
Adhatoda Vasica ◽  
Tof Ms

scholarly journals Identification of putative origin of Iris pumila L. karyotype

2019 ◽  
Vol 25 ◽  
pp. 20-25
Author(s):  
M. O. Twardovska ◽  
I. O. Andreev ◽  
V. A. Kunakh
Keyword(s):  
Chromosome Number ◽  
Chromosomal Aberrations ◽  
Cytogenetic Analysis ◽  
Apical Meristem ◽  
Root Apical Meristem ◽  
Mitotic Metaphase ◽  
Putative Origin ◽  
Iris Pumila ◽  
High Level

Aim. The study was aimed at cytogenetic analysis of Iris pumila, I. attica, and I. pseudopumila, comparative study of the karyotypes of these species, as well as identification of putative origin of I. pumila karyotype. Methods. Cytogenetic analysis of root apical meristem, determination of chromosome number in mitotic metaphase plates, anaphase analysis. Results. The chromosome numbers observed were 2n=32 for I. pumila plants from different localities in Ukraine and 2n=16 for I. attica and I. pseudopumila plants from Greece and Italy, respectively. Some of the plants were mixoploids, the smallest proportion of mixoploids was in I. pseudopumila (10.9%) and the largest in I.pumila from all studied populations (60-80%). Anaphase analysis showed the presence of chromosomal aberrations in 2.6% of cells in roots of I. pseudopumila seedlings. The highest level of structural chromosomal aberrations (9.2%) was found in root apical meristem cells of I. pumila seedlings. Conclusions. The chromosome number was established as 2n=32 for I.pumila plants and 2n=16 for I. attica and I. pseudopumila plants. The high level of mixoploidy (60–80% of mixoploid plants) and anaphase chromosomal aberrations (up to 9.2%) was found in apical meristem of I. pumila seedlings. The amphidipiloid nature of I. pumila was established; the karyotype of the species could be formed as a result of a combination of chromosome sets from hypothetical ancestral species I. attica and I. pseudopumila. Keywords: Iris pumila L., Iris attica Boiss. & Heldr., Iris pseudopumila Tineo, chromosome number, amphidiploid, mixoploidy.

scholarly journals Studies on in Vitro Culture of the Australian Fan Flower, Scaevola

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 548B-548
Author(s):  
Prem L. Bhalla ◽  
Katherine Tozer
Keyword(s):  
In Vitro Culture ◽  
Stem Explants ◽  
High Survival ◽  
Shoot Induction ◽  
Free Medium ◽  
Direct Shoot Regeneration ◽  
Important Species ◽  
Survival Percentage ◽  
Regeneration Response

Plants of genus Scaevola (family, Goodeniaceae), commonly known as “fan flowers,” are mostly endemic to Australia. Commercially popular species are Scaevola aemula, S. albida, S. striata, and S. phlebopetala. These plants are used as ground covers in Australia and as hanging baskets, window boxes, and garden bed plants in Europe and America. Two aspects of in vitro culture of Scaevola are reported here; micropropagation and direct shoot regeneration. A number of commercially available cultivars of S. aemula, S. phlebopetala, S. striata and wild-collected S. phlebopetala, S. glandulifera, S. hookeri, and S. ramonissima were used for micropropagation experiments. Micropropagation medium contained salts, vitamins, L-cysteine, sucrose, and agar. Tissue-cultured shoots were rooted in hormone-free medium. A high survival percentage (>95%) was obtained when plants were transferred to soil under glasshouse conditions. Results on in vitro shoot induction and regeneration response of leaf, stem, root, node, and flower explants of two horticulturally important species of the Australian fan flower, Scaevola aemula and Scaevola striata arealso presented. Of all the explants tested, node explants of these species were the first to respond in tissue culture. Maximum number of shoot induction and regeneration was achieved from node explants of Scaevola aemula and node and stem explants of Scaevola striata. More than 95% of the regenerated shoots were rooted on the medium supplemented with 4 mg/L of IBA. The significance of above findings in assisting breeding program for new horticultural desirable cultivars of Australian fan flowers will be discussed.

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