scholarly journals In planta genome editing with CRISPR/Cas9 ribonucleoproteins

2021 ◽  
Author(s):  
Yuya Kumagai ◽  
Yuelin Liu ◽  
Haruyasu Hamada ◽  
Weifeng Luo ◽  
Jianghui Zhu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Particle Bombardment ◽  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Wheat Variety ◽  
In Planta ◽  
Culm Length ◽  
Direct Delivery ◽  
Transformation Methods

In planta genome editing represents an attractive approach to engineering crops/varieties that are recalcitrant to culture-based transformation methods. Here, we report the direct delivery of CRISPR/Cas9 ribonucleoproteins into the shoot apical meristem using in planta particle bombardment and introduction of a semidwarf1 (sd1)-orthologous mutation into wheat. The triple knockout tasd1 mutant of an elite wheat variety reduced culm length by 10% without a reduction in yield.

scholarly journals In planta test system for targeted cellular mutagenesis by injection of oligonucleotides to apical meristem of maize seedlings

2021 ◽  
Vol 43 (5) ◽  
Author(s):  
Feríz Rádi ◽  
Bettina Nagy ◽  
Györgyi Ferenc ◽  
Katalin Török ◽  
István Nagy ◽  
...  
Keyword(s):  
Genome Editing ◽  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Stop Codon ◽  
Test System ◽  
Phytoene Desaturase ◽  
Vascular Bundles ◽  
Maize Seedlings ◽  
In Planta ◽  
Desaturase Gene

AbstractGenome-editing tools from Oligonucleotide-Directed Mutagenesis (ODM) to CRISPR system use synthetic oligonucleotides for targeted exchange of nucleotides. Presently, majority of genome-editing protocols are dependent on the in vitro cell or tissue culture systems with somaclonal variation, and limitations in plant regeneration. Therefore, here, we report an alternative in planta cellular test system for optimization of the ODM, based on the injection of oligonucleotide solution into the apical meristematic region of haploid maize seedlings. Using 5′-fluorescein-labeled oligonucleotides, we detected accumulation of synthetic DNA molecules in cells of the shoot apical meristem and of the vascular bundles of leaf primordia. For silencing or knocking down of the phytoene desaturase gene in somatic cells, 41-mer long single-stranded oligonucleotides with TAG stop codon were injected into maize seedlings. We detected out-growing M1 plantlets that developed leaves with white stripes or pale-green color. Confocal microscopy of white stripes showed that in addition to the chlorophyll fluorescence-deficient tissue region, chlorophyll containing cells are present in white stripes. The Ion Torrent sequencing of DNA samples from the white stripes indicated 0.13–1.50% read frequency for the TAG stop codon in the phytoene desaturase gene. Appearance of chlorotic abnormalities supports the mutagenic nature of oligonucleotide molecules after injection into the shoot apical meristem region of maize seedling. The described protocol provides basis for early seedling stage characterization of functionality of a mutagenic oligonucleotide with different chemistry and testing efficiency of various treatment combinations at plant level.

scholarly journals In planta particle bombardment (iPB): A new method for plant transformation and genome editing

2020 ◽  
Vol 37 (2) ◽  
pp. 171-176 ◽  
Author(s):  
Ryozo Imai ◽  
Haruyasu Hamada ◽  
Yuelin Liu ◽  
Qianyan Linghu ◽  
Yuya Kumagai ◽  
...  
Keyword(s):  
Genome Editing ◽  
Particle Bombardment ◽  
Plant Transformation ◽  
New Method ◽  
In Planta

scholarly journals Plant viral vectors: Expanding the Possibilities of Precise Gene Editing in Plant Genomes

2021 ◽  
Author(s):  
Stuti Kujur ◽  
Muthappa Senthil-Kumar ◽  
Rahul Kumar
Keyword(s):  
Genome Editing ◽  
Plant Transformation ◽  
Viral Vectors ◽  
Gene Editing ◽  
Genome Engineering ◽  
Plant Cells ◽  
In Planta ◽  
Plant Genomes ◽  
Efficient Gene ◽  
Transformation Methods

Abstract The lack of a highly efficient method for delivering reagents for genome engineering to plant cells remains a bottleneck in achieving efficient gene-editing in plant genomes. A suite of recent reports uncovers the newly emerged roles of viral vectors, which can introduce gene-edits in plants with high mutation frequencies through in planta delivery. Here, we focus on the emerging protocols that utilized different approaches for virus-mediated genome editing in model plants. Testing of these protocols and the newly identified hypercompact Casɸ systems is needed to broaden the scope of genome-editing in most plant species, including crops, with minimized reliance on conventional plant transformation methods in the future.

scholarly journals Optimization of genetic transformation method for an indica rice (Oryza sativa L.) variety Ratnagiri-711

2020 ◽  
Vol 80 (02) ◽  
Author(s):  
G. B. Sawant ◽  
S. G. Bhave ◽  
S. V. Sawardekar ◽  
M. M. Burondkar ◽  
N. B. Gokhale ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Transformation Method ◽  
Transformation Frequency ◽  
Vector System ◽  
Nptii Gene ◽  
Cry Genes ◽  
In Planta ◽  
Germinating Seeds

A study for method optimization of Agrobacterium-mediated genetic transformation for insect resistance was carried out for a rice variety Ratnagiri-711 showing better regenerability. Three different cry genes viz. cry1Aabc, cry1Fa1 and cry2Aa with a vector system consisting of the disarmed hyper virulent Agrobacterium tumefaciens strain EHA-105 harboring pBinAR or BinBt3 were used. The respective genes were linked to CaMV35S promoter and nptII gene under control of nos promoter and terminator. Scutellum-derived callus bits and embryonic shoot apical meristem of germinating seeds were used as target tissues for callus-mediated and in planta transformation, respectively. Kanamycin screening and PCR analysis was employed for confirmation of presence of transgene. Among five methods of colonization and co-cultivation tried with three cry genes, a callus-mediated transformation method consisting of 20 minutes colonization and 3 days co-cultivation with cry2Aa gene recorded highest transformation frequency (13.79%) but minimum survival (5.27%). On the contrary, considerable transformation frequency (6.35%) with highest survival (79.42%) was observed in an in planta method employing mild injury to embryonic shoot apical meristem of germinating seeds followed by injection of Agrobacterium having cry2Aa gene followed by 15 minutes colonization and then directly sowing in pots. Among three cry genes used, the gene cry2Aa was found most effective showing more transformation frequency.

scholarly journals An integrated analysis of cell-type specific gene expression reveals genes regulated by REVOLUTA and KANADI1 in the Arabidopsis shoot apical meristem

PLoS Genetics ◽  
2020 ◽  
Vol 16 (4) ◽  
pp. e1008661 ◽  
Author(s):  
Hasthi Ram ◽  
Sudeep Sahadevan ◽  
Nittaya Gale ◽  
Monica Pia Caggiano ◽  
Xiulian Yu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Integrated Analysis ◽  
Specific Gene ◽  
Cell Type ◽  
Specific Gene Expression ◽  
Cell Type Specific

scholarly journals Sample preparation for laser-microdissection of soybean shoot apical meristem

2012 ◽  
Vol 3 (1) ◽  
pp. 3 ◽  
Author(s):  
Chui E. Wong ◽  
Mohan B. Singh ◽  
Prem L. Bhalla
Keyword(s):  
Sample Preparation ◽  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Laser Microdissection ◽  
Specific Cell ◽  
Legume Crop ◽  
Continuous Formation ◽  
Molecular Events ◽  
Depth Analysis ◽  
Specialized Equipment

The shoot apical meristem houses stem cells responsible for the continuous formation of aerial plant organs including leaves and stems throughout the life of plants. Laser-microdissection in combination with high-throughput technology such as next generation sequencing permits an in-depth analysis of molecular events associated with specific cell type of interest. Sample preparation is the most critical step in ensuring good quality RNA to be extracted from samples following laser-microdissection. Here, we optimized the sample preparation for a major legume crop, soybean. We used Farmer’s solution as a fixative and paraffin as the embedding medium for soybean shoot apical meristem tissue without the use of any specialized equipment. Shorter time for tissue fixation (two days) was found to be critical for the preservation of RNA in soybean shoot apical meristem. We further demonstrated the utility of this method for different tissues derived from soybean and rice. The method outlined here shall facilitate studies on crop plants involving laser-microdissection.

Somatic embryogenesis from Arabidopsis shoot apical meristem mutants

Planta ◽  
2002 ◽  
Vol 214 (6) ◽  
pp. 829-836 ◽  
Author(s):  
Andreas Mordhorst ◽  
Marijke Hartog ◽  
Mazen El Tamer ◽  
Thomas Laux ◽  
Sacco de Vries
Keyword(s):  
Somatic Embryogenesis ◽  
Shoot Apical Meristem ◽  
Apical Meristem

scholarly journals Arabidopsis Argonaute10 Specifically Sequesters miR166/165 to Regulate Shoot Apical Meristem Development

Cell ◽  
2011 ◽  
Vol 145 (2) ◽  
pp. 242-256 ◽  
Author(s):  
Hongliang Zhu ◽  
Fuqu Hu ◽  
Ronghui Wang ◽  
Xin Zhou ◽  
Sing-Hoi Sze ◽  
...  
Keyword(s):  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Meristem Development

scholarly journals Spatiotemporal Sequestration of miR165/166 by Arabidopsis Argonaute10 Promotes Shoot Apical Meristem Maintenance

Cell Reports ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. 1819-1827 ◽  
Author(s):  
Yuyi Zhou ◽  
Minami Honda ◽  
Hongliang Zhu ◽  
Zhonghui Zhang ◽  
Xinwei Guo ◽  
...  
Keyword(s):  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Meristem Maintenance
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