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

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.

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In planta genome editing with CRISPR/Cas9 ribonucleoproteins

10.1101/2021.06.24.449705 ◽

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.

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Origin of inverted vascular bundles in winged leaves of Scleria plusiophylla (Cyperaceae)

Botany ◽

10.1139/cjb-2015-0019 ◽

2015 ◽

Vol 93 (12) ◽

pp. 893-899

Author(s):

Kátia Arenhart Hoss ◽

Rafael Trevisan ◽

Ana C. Rodrigues

Keyword(s):

Transition Region ◽

Shoot Apical Meristem ◽

Leaf Blade ◽

Apical Meristem ◽

Normal Development ◽

Dorsal Surface ◽

Vascular Bundles ◽

Abaxial Surface ◽

Bulliform Cells ◽

Normal Differentiation

The genus Scleria P.J. Bergius is pantropical, occurs in various habitats such as forests, fields, and wetlands, and is morphologically diverse. Some species have a winged sheath, which remains on the leaf blade and forms the last pair of costae. Anatomical observations of leaf blades of Scleria plusiophylla Steud. revealed the presence of inverted vascular bundles in the region of the wing. During the development of the shoot apical meristem of S. plusiophylla, it was observed that the wing emerges through divisions and enlargement of protoderm cells of the abaxial surface and subsurface cells (ground meristem) of the sheath. The ground meristem, from both sides of the sheath, gives rise to the procambial strands, which have normal differentiation. Subsequently, in the transition region between the sheath and the leaf blade, the growth of the sheath ends and the development of the leaf blade continues with the wings remaining adnate to the blade and initially folded towards the dorsal surface. As the leaf blade develops, the wings gradually unfold. Bulliform cells on the abaxial surface of the epidermis, which marked the beginning of the wing, and inverted vascular bundles intercalated with normal vascular bundles, are also observed. It was concluded that all of the bundles in the wing of the leaf blade have normal development and follow the orientation of development related to the side of the sheath from which they were initially derived, resulting in vascular bundles with different phloem positions.

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Efficient knockout of phytoene desaturase gene using CRISPR/Cas9 in melon

Scientific Reports ◽

10.1038/s41598-019-53710-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Isidre Hooghvorst ◽

Camilo López-Cristoffanini ◽

Salvador Nogués

Keyword(s):

Genome Editing ◽

Cucumis Melo ◽

Phytoene Desaturase ◽

Desaturase Gene ◽

Transformed Plants ◽

Albino Plants ◽

Cotyledonary Explants ◽

Cas9 Protein ◽

Albino Phenotype ◽

Set Up

AbstractCRISPR/Cas9 system has been widely applied in many plant species to induce mutations in the genome for studying gene function and improving crops. However, to our knowledge, there is no report of CRISPR/Cas9-mediated genome editing in melon (Cucumis melo). In our study, phytoene desaturase gene of melon (CmPDS) was selected as target for the CRISPR/Cas9 system with two designed gRNAs, targeting exons 1 and 2. A construct (pHSE-CmPDS) carrying both gRNAs and the Cas9 protein was delivered by PEG-mediated transformation in protoplasts. Mutations were detected in protoplasts for both gRNAs. Subsequently, Agrobacterium-mediated transformation of cotyledonary explants was carried out, and fully albino and chimeric albino plants were successfully regenerated. A regeneration efficiency of 71% of transformed plants was achieved from cotyledonary explants, a 39% of genetic transformed plants were successful gene edited, and finally, a 42–45% of mutation rate was detected by Sanger analysis. In melon protoplasts and plants most mutations were substitutions (91%), followed by insertions (7%) and deletions (2%). We set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and feasible in melon, generating multi-allelic mutations in both genomic target sites of the CmPDS gene showing an albino phenotype easily detectable after only few weeks after Agrobacterium-mediated transformation.

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

Indian Journal of Genetics and Plant Breeding (The) ◽

10.31742/ijgpb.80.2.4 ◽

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.

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

PLoS Genetics ◽

10.1371/journal.pgen.1008661 ◽

2020 ◽

Vol 16 (4) ◽

pp. e1008661 ◽

Cited By ~ 2

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

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Sample preparation for laser-microdissection of soybean shoot apical meristem

International Journal of Plant Biology ◽

10.4081/pb.2012.e3 ◽

2012 ◽

Vol 3 (1) ◽

pp. 3 ◽

Cited By ~ 1

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.

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