Transferring the cultured anther to a medium without activated charcoal overcomes the recalcitrancy in pepper genotypes

2020 ◽  
Author(s):  
Hasan Pınar ◽  
Nedim Mutlu ◽  
Serhat Yildiz ◽  
Duran Simsek ◽  
Mostafakamal Shams
Keyword(s):  
Activated Charcoal ◽  
Plant Material ◽  
High Efficiency ◽  
In Vitro Growth ◽  
Vegetable Crops ◽  
Crop Breeding ◽  
Breeding Lines ◽  
Low Efficiency ◽  
Solanaceae Family

Anther culture is a relatively easy and high-efficiency technique, however, low efficiency in plant regeneration may restrict its use in crop breeding. Activated charcoal is often used in in-vitro culture, and it may ameliorate or hinder in-vitro growth, depending on genotype and tissue used. Pepper is one of the main vegetable crops of the Solanaceae family, but some pepper genotypes are known to be recalcitrant to androgenesis and formation of haploid regenerants. Therefore, this study was aimed to explore the effect of activated charcoal on response to androgenesis in pepper genotypes. The plant material including 34 Long Green (LG), 13 Bell pepper (BP), 13 Charleston (Ch), 6 California Wonder (CW), and 23 Capia (CP) advanced breeding lines. Initially, anthers were cultured in a medium with activated charcoal (WAC) for 25, 35, or 45 days, and then they were transferred to the same medium without activated charcoal (NAC). In the WAC medium, 15 lines of LG genotype showed the highest recalcitrancy while many lines of CW had the lowest recalcitrancy to androgenesis. However, after transferring the 35-day old anthers to a NAC medium, the androgenesis was observed in recalcitrant LG lines. The results indicated that transferring the cultured anthers from WAC medium, ideally after 35-day, to a NAC medium overcame the recalcitrancy to androgenesis in pepper.

In Vitro Growth and Sporulation of Blastomyces dermatitidis on Woody Plant Material

Mycologia ◽  
1977 ◽  
Vol 69 (6) ◽  
pp. 1193 ◽  
Author(s):  
D. M. Dixon ◽  
H. Jean Shadomy ◽  
S. Shadomy
Keyword(s):  
Plant Material ◽  
Woody Plant ◽  
Blastomyces Dermatitidis ◽  
In Vitro Growth

Metalloproteinase Regulation Determines Integrin αIIbβ3 Function and Enhances the Capacity of ES Cell-Derived Platelets.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 140-140
Author(s):  
Hidekazu Nishikii ◽  
Koji Eto ◽  
Taisuke Kanaji ◽  
Jerry Ware ◽  
Hiromitsu Nakauchi
Keyword(s):  
High Efficiency ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Egfp Expression ◽  
Transfusion Therapy ◽  
Integrin Αiibβ3 ◽  
Differentiated Cells ◽  
Low Efficiency

Abstract To resolve the lack of sources for products used in blood transfusion therapy, the utilization of embryonic stem cells (ESCs) has been proposed. However, the use of ESC-derived cells has potential drawbacks including, immunological rejection and teratoma formation. Being fully differentiated cells lacking a nucleus, platelets can be irradiated to eliminate passenger ESCs and other lineages before transfusion. We previously established in vitro differentiation system whereby proplatelet-producing megakaryocytes can be derived from murine ESCs on OP9 stroma, but the platelets were generated with a low efficiency and exhibited weak agonist-induced integrin αIIbβ3 activation and spreading on immobilized fibrinogen. Here, we describe an improved method to obtain ESC-derived platelets of potential use for hemostasis and thrombosis in vivo. We generated a novel murine ESC line wherein the GPIbα promoter regulates EGFP expression as a marker (GP-G ESCs). The combination of flow cytometry with GP-G-derived embryoid bodies and OP9 co-culture enabled us to enrich integrin αIIb + c-kit + CD9 + Sca-1 − progenitors on day 5–6 of culture as a major fraction capable of differentiating into GFP+ mature megakaryocytes on day 8–10 in the presence of TPO. This strategy increased platelet production by 10-fold (P<0.01) compared to our previous report (PNAS, 2002, 99:12819). Interestingly, αIIb+platelets included 35–40% GPIbα-negative population besides GPIbα-positive on day 13–15, which was caused by an increased activity of matrix metalloproteinase (MMP) and TACE (ADAM17) to shed GPIbα in culture. Inhibition of MMP and TACE with GM6001 or TAP1 delayed the clearance rate on post transfusion recovery in vivo as transfused into a platelet-depleted mouse model. Maintaining GPIbα expression in the presence of MMP/TACE inhibitors restored the specific fibrinogen-bound to integrin αIIbβ3 and platelet spreading on fibrinogen as evidenced by the generation of filopodia and lamellipodia. Collectively, these results suggest that 1) ESC-derived αIIb+ c-kit + CD9+ Sca-1 − progenitors may contribute to enrich megakaryocytopoiesis at high efficiency; 2) inhibition of metalloproteinase activity to retain GPIbα expression is required for αIIbβ 3 integrin inside-out and outside-in signaling in ESC-derived platelets; and 3) these platelets may be useful alternatives for future clinical application.

scholarly journals Stress conditions promote the mating competency of Leishmania promastigotes in vitro marked by expression of the ancestral gamete fusogen HAP2

2021 ◽  
Author(s):  
Isabelle Louradour ◽  
Tiago Rodrigues Ferreira ◽  
Emma Duge ◽  
Nadira Karunaweera ◽  
Andrea Paun ◽  
...  
Keyword(s):  
High Efficiency ◽  
Wide Spectrum ◽  
Sexual Cycle ◽  
Sand Fly ◽  
Large Numbers ◽  
Transcriptomic Signature ◽  
Total Dna ◽  
Low Efficiency ◽  
Transformative Approach

Leishmania are protozoan parasites transmitted by the bite of sand fly vectors producing a wide spectrum of diseases in their mammalian hosts. These diverse clinical outcomes are directly associated with parasite strain and species diversity. Although Leishmania reproduction is mainly clonal, a cryptic sexual cycle capable of producing hybrid genotypes has been inferred from population genetic studies, and directly demonstrated by laboratory crosses. Experimentally, mating competency has been largely confined to promastigotes developing in the sand fly midgut. The ability to hybridize culture promastigotes in vitro has been limited so far to low efficiency mating between two L. tropica strains, L747 and MA37, that mate with high efficiency in flies. Here, we show that exposure of promastigote cultures to DNA damage stress produces a remarkably enhanced efficiency of in vitro hybridization of the L. tropica strains, and extends to other species, including L. donovani, L. infantum, and L. braziliensis, a capacity to generate intra- and interspecific hybrids. Whole genome sequencing and total DNA content analyses indicate that the hybrids are in each case full genome, polyploid hybrids. Single-cell RNA sequencing of the L747 and MA37 parental lines highlights the transcriptome heterogeneity of culture promastigotes and reveals discrete clusters that emerge post-irradiation in which genes potentially involved in genetic exchange are expressed, including the ancestral gamete fusogen HAP2. By generating reporter constructs for HAP2, we could select for mating-competent and mating-incompetent promastigotes. Overall, this work reveals that there are specific populations involved in Leishmania mating associated with a discernible transcriptomic signature, and that stress facilitated in vitro hybridization can be a transformative approach to generate large numbers of hybrid genotypes between diverse species and strains.

scholarly journals Efficient and Rapid generation of neural stem cells by direct conversion Fibroblasts with A Single microRNA

2021 ◽  
Author(s):  
yuesi wang ◽  
Yuanyuan Li ◽  
Jing Sun ◽  
Tingting Xu ◽  
Xiaobin Weng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
High Efficiency ◽  
Disease Modeling ◽  
System Development ◽  
Direct Conversion ◽  
Tumor Formation ◽  
Low Efficiency

Neural stem cells (NSCs) have great potential in the application of neurodegenerative disease therapy, drug screening and disease modeling. NSC can be generated by reprogramming from terminally differentiated cells with transcription factors or small molecules. However, current methods for producing NSCs involve the danger of integrating foreign genes into the genome and the problem of low efficiency. Here, we report an efficient method to generate NSCs from human skin-derived fibroblasts with microRNA (mir-302a) in 2-3 days. The induced NSCs (iNSCs) have more than 90% of purity. Their morphology is similar to regular NSCs, expressing key markers including Nestin, Pax6 and Sox2, and can be expanded for more than 20 passages in vitro. They can also differentiate into functional neuron progeny, astrocytes and oligodendrocytes as well. Those cells can elicit action potential, can be xeno-transplanted into the brain of immune-deficient mice, and can survive and differentiate in vivo without tumor formation. This study shows that a single part of pluripotency-inducing mir-302 cluster can drive fibroblasts reprogramming, providing a general platform for high-efficiency generation of individual-specific human NSCs for studies of neuron system development and regenerative cell therapy.

In vitro zearalenone adsorption by a mixture of organic and inorganic adsorbents: application of the Box Behnken approach

2015 ◽  
Vol 8 (3) ◽  
pp. 291-299 ◽  
Author(s):  
J.G. Bordini ◽  
D. Borsato ◽  
A.S. Oliveira ◽  
M.A. Ono ◽  
T.H. Zaninelli ◽  
...  
Keyword(s):  
Cell Wall ◽  
Yeast Cell ◽  
Activated Charcoal ◽  
High Efficiency ◽  
Statistical Design ◽  
Yeast Cell Wall ◽  
Quadratic Model ◽  
Wide Range

Zearalenone (ZEA) adsorption by a mixture of organic (yeast cell wall) and inorganic (activated charcoal) adsorbents was evaluated by an incomplete Box Behnken (33) statistical design with a quintuplicate at the central point. The variables analysed were different ratios of adsorbents (yeast cell wall and activated charcoal) at 100:0, 87.5:12.5 and 75:25, pH (3.0, 4.5 and 6.0) and ZEA concentrations (300, 750 and 1,200 ng/ml). The adsorbent mixture at 75:25 showed higher efficiency for ZEA adsorption (≯96.1%) than the 87.5:12.5 ratio (81.3 to 93.7%) and with the pure yeast cell wall (78.1 to 55.7%). The significant variables were the ratio of adsorbent mixture and ZEA concentration. The effect of pH was not significant (P=0.05), indicating that the binding between ZEA and the adsorbent would be stable at different pH (3.0, 4.5 and 6.0). The quadratic model obtained by the Box Behnken (33) design can be used for predictive purposes, because it showed a non-significant deviation (P=49.54%) and a good correlation coefficient (R2=0.98), suggesting that the ZEA adsorption would be maximum (100%) when the adsorbent mixture is set at 75:25 and the ZEA concentration at 300 ng/ml. Although the predictive model showed that an increase in adsorption efficiency could occur in a smaller ZEA concentration (300 ng/ml), the mixture at the 75:25 ratio presented high efficiency (≯98%) in adsorption when high ZEA concentrations were used (1,200 ng/ml), indicating that these mixtures would be able to adsorb a wide range of ZEA concentrations. Therefore, this mixture of yeast cell wall and activated charcoal adsorbents at 75:25 might be a candidate for further in vivo testing.

scholarly journals The procedure providing enhanced Agrobacterium-mediated transformation of wheat

Biologia ◽  
2014 ◽  
Vol 69 (12) ◽  
Author(s):  
Nevena Mitić ◽  
Branka Vinterhalter ◽  
Slavica Ninković ◽  
Dejan Dodig
Keyword(s):  
Transformation Efficiency ◽  
Selection Procedure ◽  
Gus Expression ◽  
Marker Genes ◽  
Breeding Lines ◽  
Agrobacterium Mediated Transformation ◽  
Selectable Marker Genes ◽  
Low Efficiency ◽  
Whole Plants

AbstractThe examinations of conditions for establishing a variety independent Agrobacterium-mediated transformation procedure for wheat are preferable since many of cultivars and breeding lines remain recalcitrant for biotechnological manipulation, mainly due to low efficiency of plant regeneration in vitro, which is highly genotype specific. This paper describes and discusses an improved protocol for enhanced and low-genotype dependent Agrobacterium-mediated transformation using a super-binary vector LBA4404/pTOK233 carrying reporter gus-intron gene and hygromycin (hpt) and kanamicyn (nptII) selectable marker genes. The protocol was optimized on highly responsive common wheat cv. Vesna. Transient expression monitored by the gus-intron on explants after 3, 6 and 25 days of co-cultivation, followed by GUS expression and hygromycin resistance in whole plants indicated the protocol including a co-cultivation of freshly isolated immature embryos in the presence of ascorbic acid, and acetosyringone added only in the bacteria-containing infection medium combined with a delayed and stepwise increasing hygromycin B selection procedure significantly enhanced the transformation efficiency in cv. Vesna that exceed 7% of treated explants from previously 0.41%. Explant pre-cultivation did not additionally improve transformation efficiency. The optimized protocol was successful in evoking satisfactory transformation efficiencies from 3.6% to 10.8% in 5 less-responsive wheat genotypes. All 57 T0 hygromycin-resistant and GUS-positive lines were phenotypically normal and fertile. Therefore, the conditions employed in this study may serve as a base to facilitate the transformation in other, particularly recalcitrant wheat cultivars.

scholarly journals Differential regulation of OCT4 targets facilitates reacquisition of pluripotency

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Sudhir Thakurela ◽  
Camille Sindhu ◽  
Evgeny Yurkovsky ◽  
Christina Riemenschneider ◽  
Zachary D. Smith ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
High Efficiency ◽  
Experimental System ◽  
Regulatory Elements ◽  
Somatic Cell Reprogramming ◽  
Transient Period ◽  
Low Efficiency ◽  
Transcription Factor Expression

Abstract Ectopic transcription factor expression enables reprogramming of somatic cells to pluripotency, albeit with generally low efficiency. Despite steady progress in the field, the exact molecular mechanisms that coordinate this remarkable transition still remain largely elusive. To better characterize the final steps of pluripotency induction, we optimized an experimental system where pluripotent stem cells are differentiated for set intervals before being reintroduced to pluripotency-supporting conditions. Using this approach, we identify a transient period of high-efficiency reprogramming where ectopic transcription factors, but not serum/LIF alone, rapidly revert cells to pluripotency with near 100% efficiency. After this period, cells reprogram with somatic-like kinetics and efficiencies. We identify a set of OCT4 bound cis-regulatory elements that are dynamically regulated during this transient phase and appear central to facilitating reprogramming. Interestingly, these regions remain hypomethylated during in vitro and in vivo differentiation, which may allow them to act as primary targets of ectopically induced factors during somatic cell reprogramming.

scholarly journals Optimization of Activated Charcoal on in vitro Growth and Development of Potato (Solanum tuberosum L.)

2018 ◽  
Vol 7 (10) ◽  
pp. 3543-3548
Author(s):  
Tanuja Buckseth ◽  
R.K. Singh ◽  
Ashwani K. Sharma ◽  
Sumita Sharma ◽  
Vaishali Moudgil ◽  
...  
Keyword(s):  
Solanum Tuberosum ◽  
Activated Charcoal ◽  
Growth And Development ◽  
Solanum Tuberosum L ◽  
In Vitro Growth

scholarly journals Stress conditions promote Leishmania hybridization in vitro marked by expression of the ancestral gamete fusogen HAP2 as revealed by single-cell RNAseq

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Isabelle Louradour ◽  
Tiago Rodrigues Ferreira ◽  
Emma Duge ◽  
Nadira D Karunaweeera ◽  
Andrea Paun ◽  
...  
Keyword(s):  
Single Cell ◽  
High Efficiency ◽  
Wide Spectrum ◽  
Sexual Cycle ◽  
Sand Fly ◽  
Large Numbers ◽  
Transcriptomic Signature ◽  
Low Efficiency ◽  
Transformative Approach

Leishmania are protozoan parasites transmitted by the bite of sand fly vectors producing a wide spectrum of diseases in their mammalian hosts. These diverse clinical outcomes are directly associated with parasite strain and species diversity. Although Leishmania reproduction is mainly clonal, a cryptic sexual cycle capable of producing hybrid genotypes has been inferred from population genetic studies, and directly demonstrated by laboratory crosses. Experimentally, mating competence has been largely confined to promastigotes developing in the sand fly midgut. The ability to hybridize culture promastigotes in vitro has been limited so far to low efficiency crosses between two L. tropica strains, L747 and MA37, that mate with high efficiency in flies. Here, we show that exposure of promastigote cultures to DNA damage stress produces a remarkably enhanced efficiency of in vitro hybridization of the L. tropica strains, and extends to other species, including L. donovani, L. infantum, and L. braziliensis, a capacity to generate intra- and interspecific hybrids. Whole genome sequencing and total DNA content analyses indicate that the hybrids are in each case full genome, mostly tetraploid hybrids. Single-cell RNA sequencing of the L747 and MA37 parental lines highlights the transcriptome heterogeneity of culture promastigotes and reveals discrete clusters that emerge post-irradiation in which genes potentially involved in genetic exchange are expressed, including the ancestral gamete fusogen HAP2. By generating reporter constructs for HAP2, we could select for promastigotes that could either hybridize or not in vitro. Overall, this work reveals that there are specific populations involved in Leishmania hybridization associated with a discernible transcriptomic signature, and that stress facilitated in vitro hybridization can be a transformative approach to generate large numbers of hybrid genotypes between diverse species and strains.

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