Abstract
Virus-induced gene silencing (VIGS) is an effective reverse genetics method used to study the gene function by analysis of plants at phenotype and molecular levels. Here, the standardization of the protocol for VIGS through Agrobacterium-mediated transformation was successfully achieved. Stem cuttings with roots were established in Coleus forskohlii. Phytoene desaturase gene from Coleus forskohlii (CfPDS) was isolated and cloned in TRV2 vector to construct CfPDS:TRV2. CfPDS:TRV2 construct was transformed in Agrobacterium tumefaciens. Agro-infiltration was carried out using leaf infiltration, agrodrench and submerged methods. Submerged method gave prominent results as compared to others. The expression of CfPDS gene was significantly decreased in plants agro-infiltrated with CfPDS:TRV2 construct resulting in photobleached areas of the affected parts of leaves. CfPDS:TRV2 treated plants showed lower chlorophyll a concentration and production of reactive oxygen species (ROS) as compared to TRV1 + 2 treated control plants. This study resulted in the establishment of VIGS protocol for Coleus forskohlii that can be further used to engineer the genes involved in the secondary metabolite pathway.
Key message
Standardization of Virus Induced Gene Silencing (VIGS) in Coleus forskohlii using CfPDS gene as marker using submerged method for agroinfiltration.
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Data availability
All data supporting the findings of this study are available within the paper and its Supplementary Information.
Abbreviations
- TRV:
-
Tobacco rattle virus
- PTGS:
-
Posttranscriptional gene silencing
- VIGS:
-
Virus-induced gene silencing
- PDS:
-
Phytoene desaturase
- LB:
-
Luria-Bertani
- MES:
-
4-Morpholineethanesulfonic acid
- qRT-PCR:
-
Quantitative real-time PCR
- ROS:
-
Reactive oxygen species
- NBT:
-
Nitroblue tetrazolium
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Acknowledgements
AB is thankful to Ms. Rekha Chauhan and Mr. Sajad Ahmed for their technical support.
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This study has been supported by the CSIR funded project MLP-110006.
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SGG and AB designed the experiment. AB performed the experiment, analysed the data, and wrote the manuscript. SS helped AB in carrying out some of the experiments. SGG helped in analysis of data, proofread the manuscript, provided resources and supervised AB and SS.
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Bhat, A., Sharma, S. & Gandhi, S.G. Optimization of Virus-induced Phytoene Desaturase (PDS) gene silencing using Tobacco Rattle Virus in Coleus forskohlii. Plant Cell Tiss Organ Cult 156, 103 (2024). https://doi.org/10.1007/s11240-024-02697-6
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DOI: https://doi.org/10.1007/s11240-024-02697-6