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All Journal HAYATI Journal of Biosciences
Chatchawan Jantasuriyarat
Department of Genetics, Faculty of Science, Kasetsart University, Bangkok, Thailand
Agriculture, Biological Sciences & Forestry Earth & Planetary Sciences

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Carbon-nanotube for Transient Expression in Rice Calli Paphawarin Pinyokham; Kamolwan Khianchaikhan; Pongsakorn Sunvittayakul; Supachai Vuttipongchaikij; Piyama Tasanasuwan; Chatchawan Jantasuriyarat
HAYATI Journal of Biosciences Vol. 29 No. 6 (2022): November 2022
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.29.6.845-850

Abstract

Transient gene expression is an important technique in gene functional analysis, protein production and in plants. However, traditional transient expression methods using Agrobacterium are time-consuming with low efficiency. In this study, we demonstrated the use of a single-walled carbon nanotube (SWCNT) to deliver 35S:mCherry:pCXSN plasmid into rice calli. This transient expression protocol used a plastic medical syringe to create the physical pressure to help the delivery of plasmid DNA into plant cells. This protocol is relatively easy to perform and low cost. The transient expression was observed under fluorescence microscopy, and the mCherry fluorescence signal was quantified. The plasmid DNA was delivered into the rice cell using a 3:1 ratio (plasmid: carbon nanotube). The result showed that the mCherry signal of carbon nanotube + plasmid DNA treatment was the highest signal at 3 days post-transformation and decreased to a lower signal at 6 days post-transformation. Moreover, the quantitative analysis of mCherry mean intensity revealed that the signal intensity of carbon nanotube + plasmid DNA treatment was the highest level, and significantly higher than the control treatments at 3 days post-transformation. Plasmid DNA can be transported easily into plant calli using this carbon nanotube transient expression protocol.
Detection of Avirulence Gene AvrPi9 in Magnaporthe oryzae, a Rice Blast Fungus, Using a Combination of RPA and CRISPR-Cas12a Techniques Piyawan Puanprapai; Pattavipha Songkumarn; Theerayut Toojinda; Chatchawan Jantasuriyarat
HAYATI Journal of Biosciences Vol. 30 No. 5 (2023): September 2023
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.30.5.885-894

Abstract

Rice blast disease is one of the most devastating diseases of rice production worldwide, which causes by an ascomycete fungus, Magnaporthe oryzae. The virulence of the rice blast fungus is determined by avirulence genes (Avr genes). Therefore, the identification of Avr genes is important for rice resistance variety improvement. Avr genes are currently identified using the pathogenicity assay with rice near-isogenic lines (NILs) or PCR amplification and gene sequencing, both of which are time-consuming and labor-intensive methods. This study aims to develop a simple method for Avr gene identification using AvrPi9 as a model. A recombinase polymerase amplification (RPA) technique was carried out to amplify AvrPi9 by incubating rice blast fungus genomic DNA with gene-specific primers at 37°C for 20 min. Cas12a-based AvrPi9 detection was performed by incubating at 37°C for 5 min. The fluorescence signal was visualized by the naked eye under an LED transilluminator. The study found that AvrPi9 can be amplified and detected using RPA and a Cas12a-based method. AvrPi9_crRNA2 has a higher efficiency than AvrPi9_crRNA1. The sensitivity of the method was 3.8 ng of DNA target for AvrPi9_crRNA1 and 1.9 ng of DNA target for AvrPi9_crRNA2. This RPA and Cas12a combination technique is a newer method for Avr gene detection in plants and has several advantages over traditional methods. It is considered easier to use and more efficient in terms of time and labor, making it a potentially useful tool for plant breeders and pathologists.
Co-Authors Kamolwan Khianchaikhan Paphawarin Pinyokham Pattavipha Songkumarn Piyama Tasanasuwan Piyawan Puanprapai Pongsakorn Sunvittayakul Supachai Vuttipongchaikij Theerayut Toojinda