scholarly journals Using Morphogenic Genes to Improve Recovery and Regeneration of Transgenic Plants

Plants ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 38 ◽  
Author(s):  
Bill Gordon-Kamm ◽  
Nagesh Sardesai ◽  
Maren Arling ◽  
Keith Lowe ◽  
George Hoerster ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tissue Culture ◽  
Transgenic Plants ◽  
Plant Genome ◽  
Inducible Promoters ◽  
Genome Modification ◽  
Meristem Development ◽  
Ectopic Overexpression ◽  
Transgenic Cells ◽  
Culture Strategies

Efficient transformation of numerous important crops remains a challenge, due predominantly to our inability to stimulate growth of transgenic cells capable of producing plants. For years, this difficulty has been partially addressed by tissue culture strategies that improve regeneration either through somatic embryogenesis or meristem formation. Identification of genes involved in these developmental processes, designated here as morphogenic genes, provides useful tools in transformation research. In species from eudicots and cereals to gymnosperms, ectopic overexpression of genes involved in either embryo or meristem development has been used to stimulate growth of transgenic plants. However, many of these genes produce pleiotropic deleterious phenotypes. To mitigate this, research has been focusing on ways to take advantage of growth-stimulating morphogenic genes while later restricting or eliminating their expression in the plant. Methods of controlling ectopic overexpression include the use of transient expression, inducible promoters, tissue-specific promoters, and excision of the morphogenic genes. These methods of controlling morphogenic gene expression have been demonstrated in a variety of important crops. Here, we provide a review that highlights how ectopic overexpression of genes involved in morphogenesis has been used to improve transformation efficiencies, which is facilitating transformation of numerous recalcitrant crops. The use of morphogenic genes may help to alleviate one of the bottlenecks currently slowing progress in plant genome modification.

scholarly journals Expression of Human Lactoferrin cDNA Confers Resistance to Ralstonia solanacearum in Transgenic Tobacco Plants

1998 ◽  
Vol 88 (7) ◽  
pp. 730-734 ◽  
Author(s):  
Zhanyuan Zhang ◽  
Dermot P. Coyne ◽  
Anne K. Vidaver ◽  
Amitava Mitra
Keyword(s):  
Gene Expression ◽  
Transgenic Plants ◽  
Ralstonia Solanacearum ◽  
Plant Genome ◽  
Human Lactoferrin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blots ◽  
Expression Levels ◽  
Lactoferrin Gene ◽  
Gene Expression Levels

A construct containing a human lactoferrin cDNA was used to transform tobacco (Nicotiana tabacum) using an Agrobacterium-mediated DNA-transfer system to express this human protein in transgenic plants. Transformants were analyzed by Southern, Northern, and Western blots to determine integration of the cDNA into the plant genome and lactoferrin gene expression levels. Most transgenic plants demonstrated significant delays of bacterial wilt symptoms when inoculated with the bacterial pathogen Ralstonia solanacearum. Quantification of the expressed lactoferrin protein by enzyme-linked immunosorbent assay in transgenic plants indicated a significant positive relationship between lactoferrin gene expression levels and levels of disease resistance. Incorporation of the lactoferrin gene into crop plants may enhance resistance to other phytopathogenic bacteria as well.

scholarly journals GhKWL1 Upregulates GhERF105 but Its Function Is Impaired by Binding with VdISC1, a Pathogenic Effector of Verticillium dahliae

2021 ◽  
Vol 22 (14) ◽  
pp. 7328
Author(s):  
Yang Chen ◽  
Mi Zhang ◽  
Lei Wang ◽  
Xiaohan Yu ◽  
Xianbi Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Plants ◽  
Gossypium Hirsutum ◽  
Verticillium Wilt ◽  
Verticillium Dahliae ◽  
Defense Response ◽  
Upland Cotton ◽  
Effector Protein ◽  
Positive Regulator ◽  
Its Gene

Verticillium wilt, caused by Verticillium dahliae, is a devastating disease for many important crops, including cotton. Kiwellins (KWLs), a group of cysteine-rich proteins synthesized in many plants, have been shown to be involved in response to various phytopathogens. To evaluate genes for their function in resistance to Verticillium wilt, we investigated KWL homologs in cotton. Thirty-five KWL genes (GhKWLs) were identified from the genome of upland cotton (Gossypium hirsutum). Among them, GhKWL1 was shown to be localized in nucleus and cytosol, and its gene expression is induced by the infection of V. dahliae. We revealed that GhKWL1 was a positive regulator of GhERF105. Silencing of GhKWL1 resulted in a decrease, whereas overexpression led to an increase in resistance of transgenic plants to Verticillium wilt. Interestingly, through binding to GhKWL1, the pathogenic effector protein VdISC1 produced by V. dahliae could impair the defense response mediated by GhKWL1. Therefore, our study suggests there is a GhKWL1-mediated defense response in cotton, which can be hijacked by V. dahliae through the interaction of VdISC1 with GhKWL1.

scholarly journals Inter-species functional compatibility of the Theobroma cacao and Arabidopsis FT orthologs: 90 million years of functional conservation of meristem identity genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
S. F. Prewitt ◽  
A. Shalit-Kaneh ◽  
S. N. Maximova ◽  
M. J. Guiltinan
Keyword(s):  
Gene Expression ◽  
Tissue Culture ◽  
Gene Family ◽  
Flowering Time ◽  
Molecular Mechanisms ◽  
Regulatory Pathways ◽  
Late Flowering ◽  
Precocious Flowering ◽  
Transition To Flowering

Abstract Background In angiosperms the transition to flowering is controlled by a complex set of interacting networks integrating a range of developmental, physiological, and environmental factors optimizing transition time for maximal reproductive efficiency. The molecular mechanisms comprising these networks have been partially characterized and include both transcriptional and post-transcriptional regulatory pathways. Florigen, encoded by FLOWERING LOCUS T (FT) orthologs, is a conserved central integrator of several flowering time regulatory pathways. To characterize the molecular mechanisms involved in controlling cacao flowering time, we have characterized a cacao candidate florigen gene, TcFLOWERING LOCUS T (TcFT). Understanding how this conserved flowering time regulator affects cacao plant’s transition to flowering could lead to strategies to accelerate cacao breeding. Results BLAST searches of cacao genome reference assemblies identified seven candidate members of the CENTRORADIALIS/TERMINAL FLOWER1/SELF PRUNING gene family including a single florigen candidate. cDNA encoding the predicted cacao florigen was cloned and functionally tested by transgenic genetic complementation in the Arabidopsis ft-10 mutant. Transgenic expression of the candidate TcFT cDNA in late flowering Arabidopsis ft-10 partially rescues the mutant to wild-type flowering time. Gene expression studies reveal that TcFT is spatially and temporally expressed in a manner similar to that found in Arabidopsis, specifically, TcFT mRNA is shown to be both developmentally and diurnally regulated in leaves and is most abundant in floral tissues. Finally, to test interspecies compatibility of florigens, we transformed cacao tissues with AtFT resulting in the remarkable formation of flowers in tissue culture. The morphology of these in vitro flowers is normal, and they produce pollen that germinates in vitro with high rates. Conclusion We have identified the cacao CETS gene family, central to developmental regulation in angiosperms. The role of the cacao’s single FT-like gene (TcFT) as a general regulator of determinate growth in cacao was demonstrated by functional complementation of Arabidopsis ft-10 late-flowering mutant and through gene expression analysis. In addition, overexpression of AtFT in cacao resulted in precocious flowering in cacao tissue culture demonstrating the highly conserved function of FT and the mechanisms controlling flowering in cacao.

scholarly journals Development of Beet necrotic yellow vein virus ‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

2019 ◽  
Vol 17 (7) ◽  
pp. 1302-1315 ◽  
Author(s):  
Ning Jiang ◽  
Chao Zhang ◽  
Jun‐Ying Liu ◽  
Zhi‐Hong Guo ◽  
Zong‐Ying Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genome Editing ◽  
Plant Genome ◽  
Yellow Vein ◽  
Multiple Gene ◽  
Guide Rna

scholarly journals Optimization of Agrobacterium-mediated transformation conditions of prospective genotypes of winter bread wheat by in planta method

2021 ◽  
Vol 28 ◽  
pp. 66-71
Author(s):  
O. V. Dubrovna ◽  
L. V. Slivka
Keyword(s):  
Transgenic Plants ◽  
Bread Wheat ◽  
Optical Density ◽  
Dna Transfer ◽  
Plant Genome ◽  
In Planta ◽  
Agrobacterium Mediated Transformation ◽  
The Third ◽  
Winter Bread Wheat ◽  
Inoculation Medium

Aim. Optimization of conditions for genetic transformation of new promising genotypes of winter bread wheat (T. aestivum L.) by in planta method. Methods. Agrobacterium-mediated transformation by in planta method using the strain AGL0 and vector construct pBi2E. Results. The influence of air temperature, optical density of cells of agrobacterial suspension, inoculation day and composition of inoculation medium on the frequency of obtaining transgenic plants of new winter wheat genotypes was studied. The dependence of the frequency obtaining of transgenic plants from environmental conditions, in particular temperature, has been established. It was found that the temperature regime of 20-22°C provided the largest number (4.8%) of wheat transformants, and when the temperature is reduced to 16-18°C there is a decrease in the efficiency of T-DNA transfer into the plant genome and the lowest frequency of transformation (0.7%). Conclusions. The largest number of transformants was obtained using a inoculation medium without sucrose, the optical density of cells of the agrobacterial suspension of 0.6 op.od. and inoculation on the third day after castration of ears. Keywords: T. aestivum, Agrobacterium-mediated transformation in planta, optimization of conditions.

scholarly journals Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

2020 ◽  
Author(s):  
Daniel M. Sapozhnikov ◽  
Moshe Szyf
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Promoter Region ◽  
Dna Methyltransferase ◽  
Dna Demethylation ◽  
New Method ◽  
Inducible Promoters ◽  
Specific Targeting ◽  
Main Effect

AbstractAlthough associations between DNA methylation and gene expression were established four decades ago, the causal role of DNA methylation in gene expression remains unresolved. Different strategies to address this question were developed; however, all are confounded and fail to disentangle cause and effect. We developed here a highly effective new method using only deltaCas9(dCas9):gRNA site-specific targeting to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzymatic activity, enabling examination of the role of DNA methylation per se in living cells. We show that the extensive induction of gene expression achieved by TET/dCas9-based targeting vectors is confounded by DNA methylation-independent activities, inflating the role of DNA methylation in the promoter region. Using our new method, we show that in several inducible promoters, the main effect of DNA methylation is silencing basal promoter activity. Thus, the effect of demethylation of the promoter region in these genes is small, while induction of gene expression by different inducers is large and DNA methylation independent. In contrast, targeting demethylation to the pathologically silenced FMR1 gene targets robust induction of gene expression. We also found that standard CRISPR/Cas9 knockout generates a broad unmethylated region around the deletion, which might confound interpretation of CRISPR/Cas9 gene depletion studies. In summary, this new method could be used to reveal the true extent, nature, and diverse contribution to gene regulation of DNA methylation at different regions.

scholarly journals Promoter architecture determines co-translational regulation of mRNA

2017 ◽  
Author(s):  
Lorena Espinar ◽  
Miquel Àngel Schikora Tamarit ◽  
Júlia Domingo ◽  
Lucas B. Carey
Keyword(s):  
Gene Expression ◽  
Translational Regulation ◽  
Open Reading Frames ◽  
Mrna Levels ◽  
Rna Seq ◽  
Linear Interaction ◽  
Information Encoding ◽  
Inducible Promoters ◽  
Promoter Architecture ◽  
Inducible Genes

AbstractInformation that regulates gene expression is encoded throughout each gene but if different regulatory regions can be understood in isolation, or if they interact, is unknown. Here we measure mRNA levels for 10,000 open reading frames (ORFs) transcribed from either an inducible or constitutive promoter. We find that the strength of co-translational regulation on mRNA levels is determined by promoter architecture. Using a novel computational-genetic screen of 6402 RNA-seq experiments we identify the RNA helicase Dbp2 as the mechanism by which co-translational regulation is reduced specifically for inducible promoters. Finally, we find that for constitutive genes, but not inducible genes, most of the information encoding regulation of mRNA levels in response to changes in growth rate is encoded in the ORF and not in the promoter. Thus the ORF sequence is a major regulator of gene expression, and a non-linear interaction between promoters and ORFs determines mRNA levels.

scholarly journals Inactivating Gene Expression with Antisense Modified Oligonucleotides

Acta Naturae ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 101-105
Author(s):  
Sidney Altman ◽  
Carlos Angele-Martinez
Keyword(s):  
Gene Expression ◽  
Tissue Culture ◽  
Plasmodium Falciparum ◽  
Human Tissue ◽  
Target Gene ◽  
Modified Oligonucleotides ◽  
Modified Nucleotides ◽  
Culture Cells ◽  
Tissue Culture Cells

Modified nucleotides, including phosphoramidates and mesyl nucleotides, are very effective in inactivating gene expression in bacteria. Gyr A is the target gene in several organisms, including Plasmodium falciparum. Antisense reactions with bacteria infecting citrus plants are promising but incomplete. Human tissue culture cells assayed with a different target are also susceptible to the presence of mesyl oligonucleotides.

scholarly journals Altered Patterns of Gene Expression in Arabidopsis Elicited by Cauliflower Mosaic Virus (CaMV) Infection and by a CaMV Gene VI Transgene

1999 ◽  
Vol 12 (5) ◽  
pp. 377-384 ◽  
Author(s):  
Chiara Geri ◽  
Edi Cecchini ◽  
Maria E. Giannakou ◽  
Simon N. Covey ◽  
Joel J. Milner
Keyword(s):  
Gene Expression ◽  
Transgenic Plants ◽  
Mosaic Virus ◽  
Rna Binding ◽  
Important Determinant ◽  
Blot Hybridization ◽  
Cauliflower Mosaic Virus ◽  
Slot Blot Hybridization ◽  
Pcr Products ◽  
Polymerase Chain

Cauliflower mosaic virus (CaMV) gene VI protein (P6) is an important determinant of symptom expression. Differential display polymerase chain reaction (PCR) was used to identify changes in gene expression in Arabidopsis elicited by a P6 transgene that causes a symptomatic phenotype. We used slot blot hybridization to measure the abundance of mRNAs complementary to 66 candidate PCR products in transgenic, CaMV-infected, and uninfected Arabidopsis plants. CaMV-infected and P6 transgenic plants showed broadly similar changes in abundance of mRNA species. In P6 transgenic plants we detected 18 PCR products that showed unambiguous changes in abundance plus another 15 that showed more limited changes (approximately twofold). CaMV-infected plants showed 17 unambiguous and 13 limited changes. Down-regulated species include those encoding a novel, phenol-like sulfotransferase, and a glycine-rich, RNA-binding protein. Up-regulated species included ones encoding an myb protein, glycine-rich and stress-inducible proteins, and a member of a previously unreported gene family. CaMV infection causes alterations in expression of many Arabidopsis genes. Transgene-mediated expression of P6 mimics virus infection in its effect on host gene expression, providing a potential mechanism for this process.

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