scholarly journals Optimization of Cucumber Doubled Haploid Line Production Using In Vitro Rescue of In Vivo Induced Parthenogenic Embryos

2005 ◽  
Vol 130 (4) ◽  
pp. 555-560 ◽  
Author(s):  
Elisabet Claveria ◽  
Jordi Garcia-Mas ◽  
Ramon Dolcet-Sanjuan
Keyword(s):  
Flow Cytometry ◽  
Doubled Haploid ◽  
Chromosome Doubling ◽  
Doubled Haploid Line ◽  
High Rate ◽  
Limiting Factors ◽  
Line Production ◽  
Haploid Plants

Homozygous doubled haploid lines (DHLs) from new cucumber (Cucumis sativus L.) accessions could be useful to accelerate breeding for resistant varieties. DHLs have been generated by in vitro rescue of in vivo induced parthenogenic embryos. The protocol developed involves the following: 1) induction of parthenogenic embryos by pollinating with pollen irradiated with a Co60 γ-ray source at 500 Gy; 2) in vitro rescue of putative parthenogenic embryos identified by their morphology and localized using a dissecting scope or X-ray radiography; 3) discrimination of undesirable zygotic individuals from the homozygous plants using cucumber and melon SSR markers; 4) determination of ploidy level from homozygous plants by flow cytometry; 5) in vitro chromosome doubling of haploids; and 6) acclimation and selfing of selected lines. Codominant markers and flow cytometry confirmed the gametophytic origin of plants regenerated by parthenogenesis, since all homozygous lines were haploids. No spontaneous doubled haploid plants were rescued. Chromosome doubling of haploid plants was accomplished by an in vitro treatment with 500 μm colchicine. Rescue of diploid or chimeric plants was shown by flow cytometry, prior to their acclimation and planting in the greenhouse. Selfing of colchicine-treated haploid plants allowed for the perpetuation by seed of homozygous lines. The high rate of seed set, 90% of the lines produced seed, facilitated the recovery of inbred lines. Despite some limiting factors, parthenogenesis is routinely used in a cucumber-breeding program to achieve complete homozygosity in one generation. Breeding for new commercial hybrid cultivars will be accelerated. DHLs are ideal resources for genomic analyses.

scholarly journals Doubled haploid technology for line development in maize: technical advances and prospects

2019 ◽  
Vol 132 (12) ◽  
pp. 3227-3243 ◽  
Author(s):  
Vijay Chaikam ◽  
Willem Molenaar ◽  
Albrecht E. Melchinger ◽  
Prasanna M. Boddupalli
Keyword(s):  
Doubled Haploid ◽  
Chromosome Doubling ◽  
Production Costs ◽  
Great Promise ◽  
Success Rates ◽  
Haploid Induction ◽  
Maize Breeding ◽  
Breeding Programs ◽  
Line Production

Key Message Increased efficiencies achieved in different steps of DH line production offer greater benefits to maize breeding programs. Abstract Doubled haploid (DH) technology has become an integral part of many commercial maize breeding programs as DH lines offer several economic, logistic and genetic benefits over conventional inbred lines. Further, new advances in DH technology continue to improve the efficiency of DH line development and fuel its increased adoption in breeding programs worldwide. The established method for maize DH production covered in this review involves in vivo induction of maternal haploids by a male haploid inducer genotype, identification of haploids from diploids at the seed or seedling stage, chromosome doubling of haploid (D0) seedlings and finally, selfing of fertile D0 plants. Development of haploid inducers with high haploid induction rates and adaptation to different target environments have facilitated increased adoption of DH technology in the tropics. New marker systems for haploid identification, such as the red root marker and high oil marker, are being increasingly integrated into new haploid inducers and have the potential to make DH technology accessible in germplasm such as some Flint, landrace, or tropical material, where the standard R1-nj marker is inhibited. Automation holds great promise to further reduce the cost and time in haploid identification. Increasing success rates in chromosome doubling protocols and/or reducing environmental and human toxicity of chromosome doubling protocols, including research on genetic improvement in spontaneous chromosome doubling, have the potential to greatly reduce the production costs per DH line.

scholarly journals Induction of Parthenogenetic Haploid Embryos after Pollination by Irradiated Pollen in Watermelon

HortScience ◽  
1994 ◽  
Vol 29 (10) ◽  
pp. 1189-1190 ◽  
Author(s):  
N. Sari ◽  
K. Abak ◽  
M. Pitrat ◽  
J.C. Rode ◽  
R. Dumas de Vaulx
Keyword(s):  
In Vitro Culture ◽  
Doubled Haploid ◽  
Chromosome Doubling ◽  
Citrullus Lanatus ◽  
Doubled Haploid Lines ◽  
Irradiated Pollen ◽  
Haploid Embryos ◽  
Haploid Plants

Parthenogenetic haploid embryos of `Crimson Sweet', `Halep Karasi', `Sugar Baby' and `Panonia F1' watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] were obtained after pollination with γ-irradiated (200 or 300 Gy) pollen. Some globular and heart-shaped embryos were observed in fruit harvested 2 to 5 weeks after pollination. The number of embryos per 100 seeds was highest for `Halep Karasi'. After in vitro culture, 17 haploid plants were obtained and doubled haploid lines were generated after chromosome doubling using colchicine.

Strategy for improvement of doubled haploid production in maize

2005 ◽  
Vol 53 (2) ◽  
pp. 177-182
Author(s):  
B. Barnabás ◽  
T. Spitkó ◽  
K. Jäger ◽  
J. Pintér ◽  
L. C. Marton
Keyword(s):  
Doubled Haploid ◽  
Doubled Haploid Line ◽  
Breeding Strategy ◽  
Breeding Value ◽  
Line Production ◽  
Hybrid Plants ◽  
Heterosis Effect ◽  
Anther Cultures ◽  
Chinese Germplasm

In the present study the applicability of a self-constructed doubled haploid line (DH 105) in the in vitro breeding of maize was evaluated. This line, which contained only 50% exotic (Chinese) germplasm, could be used to transmit in vitro androgenic ability into non-responsive breeding materials. F1 hybrids resulting from single crosses between the moderately responsive line DH 105 and recalcitrant genotypes with high breeding value showed a considerable heterosis effect in their androgenic responses. Most of the hybrids had favourable morphological and agronomic characters on the basis of “per se” evaluation. The data of the experiments showed that these F1 hybrid plants could be successfully used as anther donors, since numerous fertile DH plants were developed from their anther cultures. By the use of this in vitro breeding strategy the genetic variability can be widened and the effectiveness of inbred line production might be improved.

scholarly journals Chromosome doubling effects of selected antimitotic agents in Brassica napus microspore culture

2008 ◽  
Vol 44 (No. 1) ◽  
pp. 30-36 ◽  
Author(s):  
M. Klíma ◽  
M. Vyvadilová ◽  
V. Kučera
Keyword(s):  
Embryo Development ◽  
Doubled Haploid ◽  
Chromosome Doubling ◽  
Microspore Culture ◽  
Antimitotic Drugs ◽  
Winter Rapeseed ◽  
The Mean ◽  
Whole Plants

Effects of microspore culture treatment with antimitotic agents colchicine, trifluralin and oryzalin on the frequency of embryo formation, embryo development, plant regeneration and diploidization rate in three F<sub>1</sub> hybrids of winter rapeseed cultivars were compared. The ploidy level analysis of 1709 flowering microspore-derived plants showed that in vitro applications of all antimitotic drugs increased the rate of doubled haploid (DH) plants significantly. The mean rate of DH plants from the trifluralin treatment was 85.7%, from colchicine 74.1% and 66.5% in the case of oryzalin, while only 42.3% in the untreated control variant whereas in vivo additional application of colchicine at the plantlet stage did not significantly increase the mean rate of DH plants (55.6%). Although there were no significant differences in diploidization efficiency between the in vitro applications of particular antimitotic agents, trifluralin showed to be the most suitable because of its positive effect on embryo development and conversion into whole plants. In addition, the diploidization rate was sufficient and stable in all genotypes tested. The results indicate that the trifluralin treatment of microspore cultures could provide efficient chromosome doubling for the production of doubled haploid lines from winter oilseed rape breeding materials.

Doubled haploid production in maize under Sub-montane Himalayan conditions using R1-nj-based haploid inducer TAILP1

2020 ◽  
Vol 80 (03) ◽  
Author(s):  
R. K Khulbe ◽  
A. Pattanayak ◽  
Lakshmi Kant ◽  
G. S. Bisht ◽  
M. C. Pant ◽  
...  
Keyword(s):  
Doubled Haploid ◽  
Sweet Corn ◽  
Haploid Induction ◽  
Maize Breeding ◽  
Line Production ◽  
Haploid Inducer ◽  
Breeding Programmes ◽  
Source Populations ◽  
Developmental Phases

The use of in vivo haploid induction system makes the doubled haploid (DH) technology easier to adopt for the conventional maize breeders. However, despite having played an important role in the initial developmental phases of DH technology, Indian maize research has yet to harvest its benefits. Haploid Inducer Lines (HILs) developed by CIMMYT are being widely used in maize breeding programmes in many countries including India. There, however, is no published information on the efficiency of DH line production using CIMMYT HILs in Indian maize breeding programmes. In the present study, the efficiency of DH production using CIMMYT’s tropically adapted inducer line TAILP1 was investigated with eight source populations including two of sweet corn. The average haploid induction rate (HIR) of TAILP1 was 5.48% with a range of 2.01 to 10.03%. Efficiency of DH production ranged from 0.14 to 1.87% for different source populations with an average of 1.07%. The information generated will be useful for maize breeders intending to use DH technology for accelerated development of completely homozygous lines.

A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

2019 ◽  
Vol 19 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Yang Liu ◽  
Jingyin Zhang ◽  
Shuyun Feng ◽  
Tingli Zhao ◽  
Zhengzheng Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Cyclin D ◽  
Dna Relaxation ◽  
Cycle Arrest ◽  
H460 Cell ◽  
H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

scholarly journals STEM-26. SMALL MOLECULE DRUG CBL0137 INHIBITS THE FACT COMPLEX AND SENSITIZES GLIOBLASTOMA CANCER STEM CELLS TO RADIOTHERAPY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii201-ii202
Author(s):  
Miranda Tallman ◽  
Abigail Zalenski ◽  
Amanda Deighen ◽  
Morgan Schrock ◽  
Sherry Mortach ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
High Rate ◽  
Orthotopic Model ◽  
In Vivo Models ◽  
Specific Cytotoxicity ◽  
Treatment Paradigm ◽  
Cancer Agent

Abstract Glioblastoma (GBM) is a malignant brain tumor with nearly universal recurrence. GBM cancer stem cells (CSCs), a subpopulation of radio- and chemo-resistant cancer cells capable of self-renewal, contribute to the high rate of recurrence. The anti-cancer agent, CBL0137, inhibits the FACT (facilitates chromatin transcription) complex leading to cancer cell specific cytotoxicity. Here, we show that CBL0137 sensitized GBM CSCs to radiotherapy using both in vitro and in vivo models. Treatment of CBL0137 combined with radiotherapy led to increased DNA damage in GBM patient specimens and failure to resolve the damage led to decreased cell viability. Using clonogenic assays, we confirmed that CBL0137 radiosensitized the CSCs. To validate that combination therapy impacted CSCs, we used an in vivo subcutaneous model and showed a decrease in the frequency of cancer stem cells present in tumors as well as decreased tumor volume. Using an orthotopic model of GBM, we confirmed that treatment with CBL0137 followed by radiotherapy led to significantly increased survival compared to either treatment alone. Radiotherapy remains a critical component of patient care for GBM, even though there exists a resistant subpopulation. Radio-sensitizing agents, including CBL0137, pose an exciting treatment paradigm to increase the efficacy of irradiation, especially by inclusively targeting CSCs.

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

scholarly journals A novel phosphoramide compound, DCZ0805, shows potent anti-myeloma activity via the NF-κB pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xuejie Gao ◽  
Bo Li ◽  
Anqi Ye ◽  
Houcai Wang ◽  
Yongsheng Xie ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Burden ◽  
High Rate ◽  
Cell Counting ◽  
Tumor Xenograft ◽  
Luciferase Reporter ◽  
Therapeutic Effects ◽  
Xenograft Mouse Model

Abstract Background Multiple myeloma (MM) is a highly aggressive and incurable clonal plasma cell disease with a high rate of recurrence. Thus, the development of new therapies is urgently needed. DCZ0805, a novel compound synthesized from osalmide and pterostilbene, has few observed side effects. In the current study, we intend to investigate the therapeutic effects of DCZ0805 in MM cells and elucidate the molecular mechanism underlying its anti-myeloma activity. Methods We used the Cell Counting Kit-8 assay, immunofluorescence staining, cell cycle assessment, apoptosis assay, western blot analysis, dual-luciferase reporter assay and a tumor xenograft mouse model to investigate the effect of DCZ0805 treatment both in vivo and in vitro. Results The results showed that DCZ0805 treatment arrested the cell at the G0/G1 phase and suppressed MM cells survival by inducing apoptosis via extrinsic and intrinsic pathways. DCZ0805 suppressed the NF-κB signaling pathway activation, which may have contributed to the inhibition of cell proliferation. DCZ0805 treatment remarkably reduced the tumor burden in the immunocompromised xenograft mouse model, with no obvious toxicity observed. Conclusion The findings of this study indicate that DCZ0805 can serve as a novel therapeutic agent for the treatment of MM.

scholarly journals Inhibition of Glycolysis Suppresses Cell Proliferation and Tumor Progression In Vivo: Perspectives for Chronotherapy

2021 ◽  
Vol 22 (9) ◽  
pp. 4390
Author(s):  
Jana Horváthová ◽  
Roman Moravčík ◽  
Miroslava Matúšková ◽  
Vladimír Šišovský ◽  
Andrej Boháč ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Progression ◽  
Umbilical Vein ◽  
Cell Metabolism ◽  
High Rate ◽  
Ki 67 ◽  
Immunodeficient Mice ◽  
Inhibition Of Glycolysis

A high rate of glycolysis is considered a hallmark of tumor progression and is caused by overexpression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Therefore, we analyzed the possibility of inhibiting tumor and endothelial cell metabolism through the inhibition of PFKFB3 by a small molecule, (E)-1-(pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one (PFK15), as a promising therapy. The effects of PFK15 on cell proliferation and apoptosis were analyzed on human umbilical vein endothelial cells (HUVEC) and the human colorectal adenocarcinoma cell line DLD1 through cytotoxicity and proliferation assays, flow cytometry, and western blotting. The results showed that PFK15 inhibited the proliferation of both cell types and induced apoptosis with decreasing the Bcl-2/Bax ratio. On the basis of the results obtained from in vitro experiments, we performed a study on immunodeficient mice implanted with DLD1 cells. We found a reduced tumor mass after morning PFK15 treatment but not after evening treatment, suggesting circadian control of underlying processes. The reduction in tumor size was related to decreased expression of Ki-67, a marker of cell proliferation. We conclude that inhibition of glycolysis can represent a promising therapeutic strategy for cancer treatment and its efficiency is circadian dependent.

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