scholarly journals Establishment of Efficient Genetic Transformation Systems and Application of CRISPR/Cas9 Genome Editing Technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven

2019 ◽  
Vol 20 (12) ◽  
pp. 2920 ◽  
Author(s):  
Rui Yan ◽  
Zhiping Wang ◽  
Yamin Ren ◽  
Hongyu Li ◽  
Na Liu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Gene Function ◽  
High Efficiency ◽  
Lilium Longiflorum ◽  
Adventitious Bud ◽  
Worldwide Distribution ◽  
Condition Optimization ◽  
Lilium Species ◽  
Transformation Systems ◽  
Efficient Genetic Transformation

Lilium spp. is a bulb flower with worldwide distribution and unique underground organs. The lack of an efficient genetic transformation system for Lilium has been an international obstacle. Because existing model plants lack bulbs, bulb-related gene function verification studies cannot be carried out in model plants. Here, two stable and efficient genetic transformation systems based on somatic embryogenesis and adventitious bud regeneration were established in two Lilium species. Transgenic plants and T-DNA insertion lines were confirmed by β-glucuronidase (GUS) assay, polymerase chain reaction (PCR) and Southern blot. After condition optimization, transformation efficiencies were increased to 29.17% and 4% in Lilium pumilum DC. Fisch. and the Lilium longiflorum ‘White Heaven’, respectively. To further verify the validity of these transformation systems and apply the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9) technology in Lilium, the LpPDS gene in the two Lilium species was knocked out. Completely albino, pale yellow and albino–green chimeric mutants were observed. Sequence analysis in the transgenic lines revealed various mutation patterns, including base insertion, deletion and substitution. These results verified the feasibility and high efficiency of both transformation systems and the successful application of the CRISPR/Cas9 system to gene editing in Lilium for the first time. Overall, this study lays an important foundation for gene function research and germplasm improvement in Lilium spp.

scholarly journals A Highly Efficient Protocol To Establish Regeneration And Genetic Transformation Systems And Create Targeted Mutations Using CRISPR/Cas9 in Lycium Ruthenicum

2021 ◽  
Author(s):  
Wang Wang ◽  
Hai Wang ◽  
Jiangmiao Liu ◽  
Tong Li ◽  
Huien Zhao
Keyword(s):  
Genetic Transformation ◽  
Gene Editing ◽  
High Efficiency ◽  
Fruit Size ◽  
Fruit Weight ◽  
Lycium Ruthenicum ◽  
Genetic Transformation System ◽  
Biallelic Mutations ◽  
Transformation Systems ◽  
First Time

Abstract Background: The CRISPR/Cas9 system has rapidly become the preferred tool for various biological sequencing projects due to its high efficiency, specificity, simplicity and versatility, and it has been utilized for targeted genomic alternations in several important plants of Solanaceae, including tomato, tobacco, potato, petunia and groundcherry. Wolfberry is the sixth most important solanaceous crop in China following potato, tomato, eggplant, pepper and tobacco. To date, there has been no report on CRISPR/Cas9 technology to improve Lycium ruthenicum due to the unknown genome and the lack of efficient regeneration and genetic transformation systems.Results: In this study, we established a suitable regeneration and genetic transformation system of Lycium ruthenicum, the fw2.2 gene was identified, which was the first fruit weight gene identifified from tomato and accounted for 30% of the variation in fruit size. The gene editing of black wolfberry were carried out by CRISPR/ Cas9 for the first time here with a very high editing efficiency (95.45%) in fw2.2. Four homozygous mutations and nine biallelic mutations were obtained from T0 generation plants. Conclusions: These results suggest that the CRISPR/Cas9 system is effective for gene editing study of black wolfberry, and we expect this approach to be routinely applied to this important economic fruit.

scholarly journals 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 ◽  
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.

scholarly journals Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens

2018 ◽  
Vol 150 ◽  
pp. 9-17 ◽  
Author(s):  
Claudia D. Norzagaray-Valenzuela ◽  
Lourdes J. Germán-Báez ◽  
Marco A. Valdez-Flores ◽  
Sergio Hernández-Verdugo ◽  
Luke M. Shelton ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Transformation Method ◽  
Dunaliella Tertiolecta ◽  
Efficient Genetic Transformation

scholarly journals Current status of tissue culture and genetic transformation systems in oilseed rape plants (Brassica napus L.)

2010 ◽  
Vol 37 (4) ◽  
pp. 379-387 ◽  
Author(s):  
Sang-Il Lee ◽  
Yun-Hye Kim ◽  
Dong-Hee Lee ◽  
Yu-Mi Lee ◽  
Seo-Jun Park ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Brassica Napus ◽  
Genetic Transformation ◽  
Oilseed Rape ◽  
Current Status ◽  
Brassica Napus L ◽  
Transformation Systems

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

2019 ◽  
Vol 17 (11) ◽  
pp. 2143-2152 ◽  
Author(s):  
Yu Liu ◽  
Yu Wang ◽  
Shuqing Xu ◽  
Xianfeng Tang ◽  
Jinshan Zhao ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Genome Editing ◽  
Efficient Genetic Transformation

scholarly journals Efficient Delivery of dsRNA and DNA in Cultured Silkworm Cells for Gene Function Analysis Using PAMAM Dendrimers System

Insects ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 12
Author(s):  
Chenchen Lu ◽  
Zhiqing Li ◽  
Li Chang ◽  
Zhaoming Dong ◽  
Pengchao Guo ◽  
...  
Keyword(s):  
Gene Function ◽  
Culture Medium ◽  
Target Genes ◽  
High Efficiency ◽  
Function Analysis ◽  
Binding Capacity ◽  
Pamam Dendrimers ◽  
Binding Property ◽  
Gene Function Analysis

Polyamidoamine (PAMAM) dendrimers are emerging as intriguing nanovectors for nucleic acid delivery because of their unique well-defined architecture and high binding capacity, which have been broadly applied in DNA- and RNA-based therapeutics. The low-cost and high-efficiency of PAMAM dendrimers relative to traditional liposomal transfection reagents also promote their application in gene function analysis. In this study, we first investigated the potential use of a PAMAM system in the silkworm model insect. We determined the binding property of G5-PAMAM using dsRNA and DNA in vitro, and substantially achieved the delivery of dsRNA and DNA from culture medium to both silkworm BmN and BmE cells, thus leading to efficient knockdown and expression of target genes. Under treatments with different concentrations of G5-PAMAM, we evaluated its cellular cytotoxicity on silkworm cells, and the results show that G5-PAMAM had no obvious toxicity to cells. The presence of serum in the culture medium did not affect the delivery performance of DNA and dsRNA by G5-PAMAM, revealing its convenient use for various purposes. In conclusion, our data demonstrate that the PAMAM system provides a promising strategy for delivering dsRNA and DNA in cultured silkworm cells and promote its further application in individuals.

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