(245) Oryzalin Use on Buddleja to Facilitate Interspecific Hybridization

Ploidy level and fertility status are often the two biggest barriers a breeder must overcome when trying to incorporate novel characteristics among related taxa. This study was aimed at developing an efficient chromosome doubling method for Buddleja L., commonly known as butterfly bush, with the goal of equalizing the ploidy level and restoring the fertility of a diploid (2n=38) F1 interspecific hybrid that has a unique orange color but happens to be sterile. This method would ease the crossing of the hybrid to the tetraploid (2n=76) B. davidii Franch. cultivars commonly found in the industry. An antimitotic treatment of oryzalin was tested on 02-25-142 (B. madagascarensis Lam. × B. crispa Benth.) in vitro using nodal sections. A factorial of varying concentrations [3, 5, and 7 μM (micromolar)] by different exposure times (1, 2, and 3 day) plus controls was set up. Oryzalin appeared to be an efficient agent for chromosome doubling in Buddleja. Significant differences in the number of polyploids were not seen between chemical concentrations and exposure times. However, higher chemical concentrations and exposure times did have a significant effect on the number of nodes that survived tissue culture. Increased leaf size and color, stem thickness, shortened internode length, and upright growth habit were all good early phenotypic indicators of polyploidy induction as later confirmed by flow cytometry. Significant increases in pollen viability accompanied chromosome doubling as crosses between 02-25-142 × B. davidii cultivars produced viable seedlings.

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Initial segmentation patterns of microspores and pollen viability in soybean cultured anthers: indication of chromosome doubling

Brazilian Archives of Biology and Technology ◽

10.1590/s1516-89132004000500005 ◽

2004 ◽

Vol 47 (5) ◽

pp. 703-712 ◽

Cited By ~ 10

Author(s):

Milena Barcelos Cardoso ◽

Eliane Kaltchuk-Santos ◽

Elsa Cristina de Mundstock ◽

Maria Helena Bodanese-Zanettini

Keyword(s):

Chromosome Doubling ◽

Culture Media ◽

Pollen Viability ◽

Pollen Grains ◽

Chromosome Counts ◽

Viable Pollen ◽

Stage Of Development ◽

Initial Segmentation ◽

Bud Size

Anthers obtained from flowers buds of soybean cultivar IAS-5 were cultured in two basal culture media (B5 and B5 long). Cytological examinations of the in vitro anthers were performed during the first 20 days of culture to assay the viability (by propionic-carmine and fluorescein diacetate tests) and the stage of development of pollen grains. The frequencies of viable pollen grains varied significantly between bud sizes on the propionic-carmine analysis. The basal culture media and bud size had no clear effect on the frequencies of binucleate symmetrical and multinucleate pollen grains. Chromosome counts of metaphasic microspores throughout the culture period showed microspores with higher ploidy level in addition to normal chromosome number (n=20).

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Genetic variability in high temperature effects on seed-set in sorghum

Functional Plant Biology ◽

10.1071/fp12264 ◽

2013 ◽

Vol 40 (5) ◽

pp. 439 ◽

Cited By ~ 33

Author(s):

Chuc T. Nguyen ◽

Vijaya Singh ◽

Erik J. van Oosterom ◽

Scott C. Chapman ◽

David R. Jordan ◽

...

Keyword(s):

High Temperature ◽

Genetic Variability ◽

Pollen Germination ◽

Seed Set ◽

Pollen Viability ◽

Leaf Size ◽

Tropical Environments ◽

Development In Vitro ◽

Sorghum Genotypes

Sorghum (Sorghum bicolor (L.) Moench) is grown as a dryland crop in semiarid subtropical and tropical environments where it is often exposed to high temperatures around flowering. Projected climate change is likely to increase the incidence of exposure to high temperature, with potential adverse effects on growth, development and grain yield. The objectives of this study were to explore genetic variability for the effects of high temperature on crop growth and development, in vitro pollen germination and seed-set. Eighteen diverse sorghum genotypes were grown at day : night temperatures of 32 : 21°C (optimum temperature, OT) and 38 : 21°C (high temperature, HT during the middle of the day) in controlled environment chambers. HT significantly accelerated development, and reduced plant height and individual leaf size. However, there was no consistent effect on leaf area per plant. HT significantly reduced pollen germination and seed-set percentage of all genotypes; under HT, genotypes differed significantly in pollen viability percentage (17–63%) and seed-set percentage (7–65%). The two traits were strongly and positively associated (R2 = 0.93, n = 36, P < 0.001), suggesting a causal association. The observed genetic variation in pollen and seed-set traits should be able to be exploited through breeding to develop heat-tolerant varieties for future climates.

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SOMACLONAL VARIATION IN CULTURED IN VITRO TOBACCO PLANTS FROM THE HYBRID NICOTIANA GOSSEY DOMIN. X N. TABACUM L.

Israel Journal of Plant Sciences ◽

10.1080/07929978.1998.10676705 ◽

1998 ◽

Vol 46 (1) ◽

pp. 35-40 ◽

Cited By ~ 2

Author(s):

Violeta Nikova ◽

Maria Palakarcheva ◽

Roumiana Pundeva ◽

Dobrinka Krusteva

Keyword(s):

Somaclonal Variation ◽

Pollen Viability ◽

Basal Medium ◽

Leaf Size ◽

Culture Method ◽

Stem Length ◽

Naphthaleneacetic Acid ◽

In Vitro Techniques ◽

Plant Forms

Incompatibility between the speciesNicotiana gosseyDomin. andN. tabacumL., resulting in complete sterility of F1hybrid, was overcome using in vitro techniques. Explants of stem parenchyma were put in culture and were regularly subcultured on Murashige and Skoog basal medium, supplemented with different compounds: α-naphthaleneacetic acid (2 mg L−1) and kinetin (0.5 mg L−1) for callus induction, kinetin (2 mg L−1) and indule-3-acetic acid (0.5 mg L−1) for organ formation, and ferulic acid (2 mg L−1) for rooting. The regenerants obtained showed significant morphological and cytological variability. They differed from each other as well as from the initial material in stem length, leaf size, and flower morphology. Most of the plants examined were mixoploids. A great number of aberrations were established during the meiotic division of the regenerants. Pollen viability varied from 0 to 92%. The results of our investigations showed that the tissue culture method could be successfully applied not only for overcoming species incompatibility but also for inducing somaclonal variation and creation of a large variety of plant forms.

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Oryzalin-induced Chromosome Doubling in Buddleja to Facilitate Interspecific Hybridization

HortScience ◽

10.21273/hortsci.42.6.1326 ◽

2007 ◽

Vol 42 (6) ◽

pp. 1326-1328 ◽

Cited By ~ 15

Author(s):

Bruce L. Dunn ◽

Jon T. Lindstrom

Keyword(s):

Interspecific Hybridization ◽

Untreated Control ◽

Treatment Duration ◽

Chromosome Doubling ◽

Survival Rates ◽

Leaf Size ◽

Fruit Characteristics ◽

Ploidy Levels

A protocol for producing fertile tetraploid forms of the hybrid Buddleja madagascarensis Lam. × B. crispa Benth. would enable introgression of orange flower, pubescence, and nondehiscent fruit characteristics found in section Nicodemia (Tenore) Leeuw. into B. davidii Franchet section Buddleja. Excised nodal sections of a single sterile diploid selection from that cross were treated in vitro with 3, 5, or 7 μm oryzalin concentrations for 1, 2, or 3 days or were left as an untreated control. A population of plants was generated from these cultures and transferred to the greenhouse. Treated plants were initially screened phenotypically for higher ploidy levels on the basis of stem thickness and leaf size. Those selected based on polyploidy characteristics were subjected to cytometric analysis, confirming that six tetraploid plants were generated. Nodal survival rates were dependent on oryzalin concentration and treatment duration. Significant increases in fertility accompanied polyploidy induction, because crosses between the newly developed tetraploids and B. davidii cultivars produced viable fertile plants. Chemical name used: 3,5-dinitro-N 4,N 4-dipropylsulfanilamide (oryzalin).

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An in vitro chromosome doubling method for clovers (Trifolium spp.)

Genome ◽

10.1139/g91-001 ◽

1991 ◽

Vol 34 (1) ◽

pp. 1-5 ◽

Cited By ~ 17

Author(s):

J. A. Anderson ◽

C. Mousset-Déclas ◽

E. G. Williams ◽

N. L. Taylor

Keyword(s):

Interspecific Hybrids ◽

Chromosome Doubling ◽

Shoot Proliferation ◽

Red Clover ◽

Colchicine Treatment ◽

Root Tips ◽

Axillary Meristems ◽

Doubling Method ◽

A New Technique

This research reports a new technique for chromosome doubling of clover (Trifolium sp.) axillary meristems via in vitro colchicine application. Plant material utilized included T. pratense (red clover) cv. Kenstar clones, and three interspecific hybrids: T. ambiguum (kura clover) × T. repens (white clover); T. alpestre × T. pratense; and T. sarosiense × T. pratense. Vegetative axillary meristems were excised from plants, surface sterilized, and trimmed to a length of 0.5–1 mm. Meristems were placed on the surface of a shoot proliferation medium (ML8) containing colchicine (0.1%) for 48 or 72 h and then transferred back to ML8. Alternative treatments were to preculture meristems on ML8 for 7 days prior to colchicine treatment. Plantlets with two or three trifoliolate leaves were induced to root on CR2 or RL rooting media. Preculturing of meristems on ML8 prior to colchicine exposure resulted in the highest chromosome doubling frequencies among the different genotypes, although there was apparent genotype × treatment interaction. Chromosome doubling frequencies were as high as 81 and 44% for initial root tips and mature shoots, respectively. To make rapid assessments of ploidy level of flowering plants, pollen shape was examined. Chromosome doubling increased the pollen stainability of the T. ambiguum × T. repens hybrid from 2.5 to 33.6%, but did not result in fertility in the other two interspecific hybrids.Key words: Trifolium, colchicine, chromosome doubling, interspecific hybrids.

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A Heparin-coated Circuit Maintains Platelet Aggregability in Response to Shear Stress in an In Vitro Model of Cardiopulmonary Bypass

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615226 ◽

1998 ◽

Vol 80 (09) ◽

pp. 437-442 ◽

Cited By ~ 2

Author(s):

I. Hioki ◽

K. Onoda ◽

T. Shimono ◽

H. Shimpo ◽

K. Tanaka ◽

...

Keyword(s):

Shear Stress ◽

Cardiopulmonary Bypass ◽

Membrane Skeleton ◽

Platelet Glycoprotein ◽

Platelet Surface ◽

Platelet Aggregability ◽

Vitro Model ◽

Platelet Defects ◽

Set Up

SummaryAlterations in platelet aggregability may play a role in the pathogenesis of qualitative platelet defects associated with cardiopulmonary bypass (CPB). We circulated fresh heparinized whole blood through tubing sets coated with heparin (C group, n = 10) and through non-coated sets (N group, n = 10) as a simulated CPB circuit. Shear stress (108 dyne/cm2)-induced platelet aggregation (hSIPA), plasma von Willebrand factor (vWF) activity and platelet glycoprotein (GP) Ib expression were measured, before, during, and after this in vitro set up of circulation. In the two groups, the extent of hSIPA significantly decreased during circulation and was partially restored after circulation. Decreases in the extent of hSIPA were significantly less with use of heparin-coated circuits. There was an equivalent reduction in plasma vWF activity, in the two groups. Expression of platelet surface GP Ib decreased significantly during circulation and recovered after circulation. Reduction of surface GP Ib expression during circulation was significantly less in the C group than that in the N group. Decrease in surface GP Ib expression correlated (r = 0.88 in either group) with the magnitude of hSIPA, in the two groups. The progressive removal of surface GP Ib was mainly attributed to redistribution of GP Ib from the membrane skeleton into the cytoskeleton. Our observations suggest that use of heparin-coated circuits partly blocks the reduction of hSIPA, as a result of a lesser degree of redistribution of GP Ib.

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Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

10.26434/chemrxiv.7725002.v1 ◽

2019 ◽

Author(s):

Priya Prakash ◽

Travis Lantz ◽

Krupal P. Jethava ◽

Gaurav Chopra

Keyword(s):

Amyloid Beta ◽

Western Blots ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Set Up ◽

Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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Plant Biotechnology Workshop for High School Students

HortScience ◽

10.21273/hortsci.33.3.504e ◽

1998 ◽

Vol 33 (3) ◽

pp. 504e-504

Author(s):

Erika Szendrak ◽

Paul E. Read ◽

Jon S. Miller

Keyword(s):

High School ◽

High School Students ◽

Medical Science ◽

Plant Biotechnology ◽

Plant Science ◽

High School Biology ◽

School Students ◽

Subsequent Transformation ◽

Set Up

Modern aspects of many subjects (e.g., computer science and some aspects of medical science) are now taught in many high schools, but the plant sciences are often given short shrift. A collaboration was therefore established with a high school biology program in which pilot workshops could be developed to enable advanced students to gain insights into modern plant science techniques. A successful example is the workshop on plant biotechnology presented in this report. This workshop is simple and flexible, taking into account that most high school biology laboratories and classrooms are not set up for sophisticated plant science/biotechnology projects. It is suitable for from 10 to 30 students, depending upon space and facilities available. Students work in pairs or trios, and learn simple disinfestation and transfer techniques for micropropagation and potential subsequent transformation treatments. Students gain insights into: sterile technique and hygiene; plant hormones and their physiological effects; plant cell, tissue and organ culture; the influence of environmental factors on response of cells and tissues cultured in vitro; and an understanding of the phenomenon of organogenesis and resulting plant growth and development. This workshop has been tested on several classes of students and following analysis, several refinements were included in subsequent iterations. Results of the students' experiments have been positive and instructive, with student learning outcomes above expectations. Further details of the workshop techniques and approach will be presented.

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Multiple screening approaches reveal HDAC6 as a novel regulator of glycolytic metabolism in triple-negative breast cancer

Science Advances ◽

10.1126/sciadv.abc4897 ◽

2021 ◽

Vol 7 (3) ◽

pp. eabc4897

Author(s):

Catríona M. Dowling ◽

Kate E. R. Hollinshead ◽

Alessandra Di Grande ◽

Justin Pritchard ◽

Hua Zhang ◽

...

Keyword(s):

Breast Cancer ◽

Triple Negative Breast Cancer ◽

Histone Deacetylase Inhibitors ◽

Triple Negative ◽

Mitochondrial Apoptosis ◽

Glycolytic Metabolism ◽

Set Up ◽

Screening Approaches

Triple-negative breast cancer (TNBC) is a subtype of breast cancer without a targeted form of therapy. Unfortunately, up to 70% of patients with TNBC develop resistance to treatment. A known contributor to chemoresistance is dysfunctional mitochondrial apoptosis signaling. We set up a phenotypic small-molecule screen to reveal vulnerabilities in TNBC cells that were independent of mitochondrial apoptosis. Using a functional genetic approach, we identified that a “hit” compound, BAS-2, had a potentially similar mechanism of action to histone deacetylase inhibitors (HDAC). An in vitro HDAC inhibitor assay confirmed that the compound selectively inhibited HDAC6. Using state-of-the-art acetylome mass spectrometry, we identified glycolytic substrates of HDAC6 in TNBC cells. We confirmed that inhibition or knockout of HDAC6 reduced glycolytic metabolism both in vitro and in vivo. Through a series of unbiased screening approaches, we have identified a previously unidentified role for HDAC6 in regulating glycolytic metabolism.

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Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa315 ◽

2020 ◽

Author(s):

Lina Y Alkaissi ◽

Martin E Winberg ◽

Stéphanie DS Heil ◽

Staffan Haapaniemi ◽

Pär Myrelid ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Ex Vivo ◽

Ussing Chambers ◽

E Coli ◽

Follicle Associated Epithelium ◽

Vitro Model ◽

Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

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