An efficient genetic transformation method for glycerol producer Candida glycerinogenes

Abstract Background Upland cotton (Gossypium hirsutum), harboring a complex allotetraploid genome, consists of A and D sub-genomes. Every gene has multiple copies with high sequence similarity that makes genetic, genomic and functional analyses extremely challenging. The recent accessibility of CRISPR/Cas9 tool provides the ability to modify targeted locus efficiently in various complicated plant genomes. However, current cotton transformation method targeting one gene requires a complicated, long and laborious regeneration process. Hence, optimizing strategy that targeting multiple genes is of great value in cotton functional genomics and genetic engineering. Results To target multiple genes in a single experiment, 112 plant development-related genes were knocked out via optimized CRISPR/Cas9 system. We optimized the key steps of pooled sgRNAs assembly method by which 116 sgRNAs pooled together into 4 groups (each group consisted of 29 sgRNAs). Each group of sgRNAs was compiled in one PCR reaction which subsequently went through one round of vector construction, transformation, sgRNAs identification and also one round of genetic transformation. Through the genetic transformation mediated Agrobacterium, we successfully generated more than 800 plants. For mutants identification, Next Generation Sequencing technology has been used and results showed that all generated plants were positive and all targeted genes were covered. Interestingly, among all the transgenic plants, 85% harbored a single sgRNA insertion, 9% two insertions, 3% three different sgRNAs insertions, 2.5% mutated sgRNAs. These plants with different targeted sgRNAs exhibited numerous combinations of phenotypes in plant flowering tissues. Conclusion All targeted genes were successfully edited with high specificity. Our pooled sgRNAs assembly offers a simple, fast and efficient method/strategy to target multiple genes in one time and surely accelerated the study of genes function in cotton.

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Efficient Genetic Transformation of Rice for CRISPR/Cas9 Mediated Genome-Editing and Stable Overexpression Studies: A Case Study on Rice Lipase 1 and Galactinol Synthase Encoding Genes

Agronomy ◽

10.3390/agronomy12010179 ◽

2022 ◽

Vol 12 (1) ◽

pp. 179

Author(s):

Tanika Thakur ◽

Kshitija Sinha ◽

Tushpinder Kaur ◽

Ritu Kapoor ◽

Gulshan Kumar ◽

...

Keyword(s):

Genetic Transformation ◽

Crop Improvement ◽

Rice Cultivar ◽

Climatic Conditions ◽

Rice Transformation ◽

Transformation Protocol ◽

Galactinol Synthase ◽

Biotic Stresses ◽

Putative Transformants ◽

Efficient Genetic Transformation

Rice is a staple food crop for almost half of the world’s population, especially in the developing countries of Asia and Africa. It is widely grown in different climatic conditions, depending on the quality of the water, soil, and genetic makeup of the rice cultivar. Many (a)biotic stresses severely curtail rice growth and development, with an eventual reduction in crop yield. However, for molecular functional analysis, the availability of an efficient genetic transformation protocol is essential. To ensure food security and safety for the continuously increasing global population, the development of climate-resilient crops is crucial. Here, in this study, the rice transformation protocol has been effectively optimized for the efficient and rapid generation of rice transgenic plants. We also highlighted the critical steps and precautionary measures to be taken while performing the rice transformation. We further assess the efficacy of this protocol by transforming rice with two different transformation constructs for generating galactinol synthase (GolS) overexpression lines and CRISPR/Cas9-mediated edited lines of lipase (Lip) encoding the OsLip1 gene. The putative transformants were subjected to molecular analysis to confirm gene integration/editing, respectively. Collectively, the easy, efficient, and rapid rice transformation protocol used in this present study can be applied as a potential tool for gene(s) function studies in rice and eventually to the rice crop improvement.

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Efficient genetic transformation and CRISPR /Cas9‐mediated genome editing in Lemna aequinoctialis

Plant Biotechnology Journal ◽

10.1111/pbi.13128 ◽

2019 ◽

Vol 17 (11) ◽

pp. 2143-2152 ◽

Cited By ~ 3

Author(s):

Yu Liu ◽

Yu Wang ◽

Shuqing Xu ◽

Xianfeng Tang ◽

Jinshan Zhao ◽

...

Keyword(s):

Genetic Transformation ◽

Genome Editing ◽

Efficient Genetic Transformation

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Efficient genetic transformation of sour orange, Citrus aurantium L. using Agrobacterium tumefaciens containing the coat protein gene of Citrus tristeza virus

Plant Gene ◽

10.1016/j.plgene.2018.02.002 ◽

2018 ◽

Vol 14 ◽

pp. 7-11 ◽

Cited By ~ 2

Author(s):

Isa Ghaderi ◽

Mohammad Mehdi Sohani ◽

Ali Mahmoudi

Keyword(s):

Agrobacterium Tumefaciens ◽

Coat Protein ◽

Genetic Transformation ◽

Coat Protein Gene ◽

Citrus Tristeza Virus ◽

Protein Gene ◽

Sour Orange ◽

Citrus Aurantium ◽

Tristeza Virus ◽

Efficient Genetic Transformation

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Genetic Transformation of Tribonema minus, a Eukaryotic Filamentous Oleaginous Yellow-Green Alga

International Journal of Molecular Sciences ◽

10.3390/ijms21062106 ◽

2020 ◽

Vol 21 (6) ◽

pp. 2106

Author(s):

Yan Zhang ◽

Hui Wang ◽

Ruigang Yang ◽

Lihao Wang ◽

Guanpin Yang ◽

...

Keyword(s):

Fatty Acids ◽

Genetic Transformation ◽

Unsaturated Fatty Acids ◽

Value Added ◽

Green Fluorescence Protein ◽

Transformation Method ◽

Green Fluorescence ◽

Egfp Gene ◽

Tribonema Minus ◽

Fluorescence Protein

Eukaryotic filamentous yellow-green algae from the Tribonema genus are considered to be excellent candidates for biofuels and value-added products, owing to their ability to grow under autotrophic, mixotrophic, and heterotrophic conditions and synthesize large amounts of fatty acids, especially unsaturated fatty acids. To elucidate the molecular mechanism of fatty acids and/or establish the organism as a model strain, the development of genetic methods is important. Towards this goal, here, we constructed a genetic transformation method to introduce exogenous genes for the first time into the eukaryotic filamentous alga Tribonema minus via particle bombardment. In this study, we constructed pSimple-tub-eGFP and pEASY-tub-nptⅡ plasmids in which the green fluorescence protein (eGFP) gene and the neomycin phosphotransferase Ⅱ-encoding G418-resistant gene (nptⅡ) were flanked by the T. minus-derived tubulin gene (tub) promoter and terminator, respectively. The two plasmids were introduced into T. minus cells through particle-gun bombardment under various test conditions. By combining agar and liquid selecting methods to exclude the pseudotransformants under long-term antibiotic treatment, plasmids pSimple-tub-eGFP and pEASY-tub- nptⅡ were successfully transformed into the genome of T. minus, which was verified using green fluorescence detection and the polymerase chain reaction, respectively. These results suggest new possibilities for efficient genetic engineering of T. minus for future genetic improvement.

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