Page 267 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

[P128] ESTABLISHING A NOVEL, AUTOMATED, MAGNETIC BEAD-BASED METHOD FOR
EXTRACTION OF SEQUENCING GRADE NUCLEIC ACID FROM DIFFERENT PLANT SAMPLES.
Sapna K., Ashwini J., Sneha T., Kushminda B., Somak C., Komal D., Radha H., Kavita Khadke, Rajas
Warke. HiMedia Laboratories Pvt. Ltd.
Correspondence to: kavita.khadke@himedialabs.com

Extraction of Nucleic acid from plant sample is essential for molecular biology, in identifying, isolating, and
extracting the desired gene to replicate in successive generations of plants. It is a crucial pre-analytic step
in the development and performance of any successful molecular diagnostic method and ensures reliable
result. Molecular techniques are mainly based on PCR assays, therefore a good quality of DNA and RNA
is prerequisite for any tools to be used. A variety of Nucleic acid extraction methods and kits are
available, however they are costly, low yield, and time consuming. The present study shows a Novel,
Automated, Magnetic bead-based platform and reagents for efficient extraction of Plant Nucleic Acid
(DNA & RNA) from different plant samples like Maize, Soyabean, Wheat, Tomato, Pigeon pea, Chick pea,
Tobacco, Mustard, Potato, Spinach, Scallions, Millets etc.

Hi-Media’s HiPurA pre-filled cartridge-based extraction kits, MB507PC16 & MB571PC16 for DNA
®
extraction and MB603PC16 for RNA extraction along with the Automated Magnetic extraction system,
Insta NX Mag16 Plus is found to be the most efficient nucleic acid extraction method, capable to provide
®
high yields with better quality, affordable cost and less time. The extracted high-quality nucleic acid is
suitable for PCR assays, library preparation followed by sequencing. We have recorded an average yield
of upto 100 ng/µl and purity (A260/ A280) in range of 1.7-1.9 for the extracted DNA and for RNA upto
500ng/µl of yield and purity (A260/A280) in range of 2.0-2.2. As compared to competitor kit used, the data
was found to be better than competitor.

®
Overall, Insta NX Mag16 Plus Automated extractor along with the pre-filled kits will be advantageous for
researchers working in this field. Using minimum amount of starting material (upto 200mg), good PCR
amplifiable, Sequencing Grade DNA can be extracted which can help in further downstream assays.

[P129] LACCASE GHLAC14-3 REGULATES CELL WALL DEFENSE TO CONFER RESISTANCE
AGAINST VERTICILLIUM WILT BY INTERACTING WITH GHMAPKKK2 IN COTTON. Yue Li , Guanfu
1,3
2
1
Cheng , Chuanzong Li , W. G. Dilantha Fernando , and Xiaofeng Su . College of Life Science, Xinjiang
2 1
3
Agricultural University, 311 Nongda East Road, Urumqi, China, 830052; Biotechnology Research
2
Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun South Street, Haidian District,
Beijing, China, 100081; and Department of Plant Sciences, University of Manitoba, Canada 66 Dafoe
3
Road, Winnipeg, Manitoba, Canada, R3T 2N2
Correspondence to: liyue6905@126.com

Verticillium wilt (VW), caused mainly by Verticillium dahliae, is one of the most important diseases of
cotton. Lignification of the cell wall, triggered by pathogen invasion, is an inherent defensive mechanism.
Laccases in plants are recognized for their function in lignifying secondary cell walls. However, their role
in cotton resistance to V. dahliae remains unclear. In our previous study, numerous differentially
expressed genes (DEGs) were identified in the transcriptome and metabolome of Arabidopsis thaliana
infected with V. dahliae. In the present study, we successfully characterized a laccase GhLAC14-3 and
identified it as a positive regulator associated with cotton resistance against V. dahliae. The expression of
GhLAC14-3 in cotton plants, significantly elevated at the early stage of V. dahliae invasion, was
transiently silenced, accompanied by a higher disease index, a reduction in lignin content, and the
expression of lignin-related genes, thereby obviously enhancing the VW susceptibility. In contrast, ectopic
expression of GhLAC14-3 in Arabidopsis conferred enhanced resistance to VW. The interaction between
GhLAC14-3 and GhMAPKKK2, a mitogen-activated protein kinase, was further determined in the cell
membrane using a yeast-two-hybrid screen and the bimolecular fluorescent complementation.
GhMAPKKK2 expression was significantly induced by V. dahliae, and its constitutive expression
enhanced the resistance of Arabidopsis. These results demonstrate that GhLAC14-3 interacted with
GhMAPKKK2 in the plant membrane, modulates cell wall defense, and contributes to cotton resistance
against V. dahliae, suggesting its potential applications in molecular breeding programs designed to
enhance VW resistance.
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