Electrotransfection of protoplasts from tomato, wild tomato, barley and chrysanthemum with tobacco mosaic virus RNA

Matsunaga, Rino; Sawamura, Kimiko; Kok, Marieke; Makino, Toshiaki; Miki, Keizaburo; Kojima, Makoto; Tsuchizaki, Tsuneo; Hibi, Tadaaki

doi:10.1099/0022-1317-73-4-763

Cited by 3 publications

(2 citation statements)

References 17 publications

(8 reference statements)

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“…In general, electrotransfection of the same amount of viral RNA into rapeseed protoplasts gave rise to Northern blot signals approximately 1.5-to twofold the intensity of those obtained using Chinese cabbage protoplasts. It has already been observed that the efficiency with which a virus replicates in a plant does not necessarily correlate with the efficiency at the protoplast level (discussed in Matsunaga et al, 1992). Indeed, Chinese cabbage rather than rapeseed plants are more commonly used as propagating hosts for TYMV, although it remains to be determined whether this could be correlated with higher efficiency of viral replication in host cells.…”

Section: Resultsmentioning

confidence: 99%

Electrotransfection of turnip yellow mosaic virus RNA into Brassica leaf protoplasts and detection of viral RNA products with a non-radioactive probe

Boyer

Zaccomer²,

Haenni

1993

Journal of General Virology

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We describe here a convenient and efficient system for studying turnip yellow mosaic virus (TYMV) replication in leaf protoplasts. Inoculation of rapeseed (Brassica napus) or Chinese cabbage (B. sinensis) protoplasts was achieved via electroporation, and sensitive detection of viral RNA products was performed by Northern blot analyses using a non-radioactive digoxigenin-labelled cDNA probe. Virus replication was detected when 1"5 z 106 rapeseed protoplasts were inoculated with 20 ng of TYMV RNA. Electrotransfection of TYMV RNA was more efficient in rapeseed than in Chinese cabbage protoplasts, and gave somewhat higher signals than those of TYMV virions. TYMV RNA appeared to replicate equally well whether the protoplasts were incubated in the dark or under constant light.

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Section: Resultsmentioning

confidence: 99%

Electrotransfection of turnip yellow mosaic virus RNA into Brassica leaf protoplasts and detection of viral RNA products with a non-radioactive probe

Boyer

Zaccomer²,

Haenni

1993

Journal of General Virology

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“…Experiments assessing replication of HaSV in protoplasts were done similarly except as noted. Pulse-label experiments were conducted by transferring the protoplasts after electroporation with 1.5 g of HaSV (5 ϫ 10 10 virions), 1.5 g of HaSV RNA, 1.5 g of TMV, and 0.16 g of TMV RNA (Matsunaga et al, 1992) in a six-well tray with medium containing 150 Ci of [ 32 P]orthophosphate (Amersham). After 3 days growth, the protoplasts were collected by roughly pipetting them from the tray, pelleted, and freeze-thawed to lyse them.…”

Section: Preparation and Electroporation Of Protoplastsmentioning

confidence: 99%

Replication-Independent Assembly of an Insect Virus (Tetraviridae) in Plant Cells

et al. 2001

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Infectious virions of the insect RNA virus Helicoverpa armigera stunt virus (HaSV; Omegatetravirus, Tetraviridae) were assembled in cultured plant protoplasts of Nicotiana plumbaginifolia in the absence of detectable replication. Assembly of the virus, which has not been grown in cell culture, required cotransfection of a DNA plasmid expressing the HaSV capsid gene in combination with either genomic RNA or with DNA plasmids carrying the complete cDNAs to the two HaSV genomic RNAs. Each cDNA was placed under the control of the cauliflower mosaic virus 35S promoter and followed by a cis-acting ribozyme so that the resultant transcripts corresponded precisely to the two genomic RNAs. Protoplast assembly of infectious particles was confirmed by EM and bioassay of host insect larvae, which became diseased and produced virus particles confirmed as HaSV. Variant transcripts carrying nonviral sequences at either or both termini of the RNAs showed no infectivity, except for RNA2 carrying only a 3' terminal extension. No replication of HaSV in protoplasts was detected in pulse-labeling and blotting experiments. Insects showed less severe disease symptoms when fed protoplasts transfected with only the RNA1 and coat protein plasmids. The symptomatic larvae contained only RNA1 and failed to yield infectious progeny virus, suggesting that RNA1 is capable of self-replication. This novel plasmid-based system confirms that the reported sequence of HaSV represents an infective genome and establishes a procedure for the reverse genetics of a tetravirus.

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Optimized protocols for protoplast isolation, transfection, and regeneration in the Solanum genus for the CRISPR/Cas-mediated transgene-free genome editing

Yang,

Kim,

Lee

et al. 2024

Appl Biol Chem

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The Solanaceae family includes the largest flowering crops such as tomatoes, potatoes, and eggplants. Consumer demand has led to massive development of plants in the Solanum genus, and many different Solanum varieties are now available on the market. The recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based genome editing have allowed laboratories and smaller crop production companies to utilize the technology in various crops. The traditional transformation method in crops involves the use of Agrobacterium, which is considered the most efficient method for introducing exogenous genetic materials in target plants. The Agrobacterium-mediated transformation method has been also established in the Solanaceae family, enabling CRISPR/Cas-based genome editing in crops like tomatoes, potatoes, and eggplants. However, the Agrobacterium-mediated approach inevitably accompanies the insertion of exogenous DNA into the plant genome and often causes the formation of chimera that require further propagation steps. Alternatively, the CRISPR/Cas components can be introduced into protoplasts in the form of DNA for transient expression or a mixture of protein and RNA to avoid genomic insertion of foreign materials. The protoplast transformation approach involves processes including protoplast preparation, transfection, and regeneration, which require a comprehensive understanding and greater technical mastery of the tissue culture phase. Here we highlight the current research advances in protoplast transformation and discuss how to optimize the procedures of protoplast isolation, transfection, and regeneration for efficient and reproducible CRISPR/Cas-based genome editing in the genus Solanum.

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Electrotransfection of protoplasts from tomato, wild tomato, barley and chrysanthemum with tobacco mosaic virus RNA

Cited by 3 publications

References 17 publications

Electrotransfection of turnip yellow mosaic virus RNA into Brassica leaf protoplasts and detection of viral RNA products with a non-radioactive probe

Electrotransfection of turnip yellow mosaic virus RNA into Brassica leaf protoplasts and detection of viral RNA products with a non-radioactive probe

Replication-Independent Assembly of an Insect Virus (Tetraviridae) in Plant Cells

Optimized protocols for protoplast isolation, transfection, and regeneration in the Solanum genus for the CRISPR/Cas-mediated transgene-free genome editing

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