An inexpensive, rapid, and nontoxic DNA extraction method for rice production

Abstract 
Genotyping efforts can be significantly hindered by suboptimal DNA quality. Traditional methodologies, which involve the use of mortar and pestle-based homogenization techniques along with hazardous chemicals such as phenol or chloroform, are effective in extracting substantial quantities of high-quality DNA. Nevertheless, these traditional approaches are not only time-intensive but also environmentally detrimental, rendering them unsuitable for high-throughput processing or educational laboratory settings.In this context, we present a protocol that relies solely on standard laboratory instruments and non-toxic reagents, significantly reducing the processing time while still yielding genomic DNA. This protocol represents an improved alternative that addresses the limitations of traditional methods.

Background
The acquisition of high-quality DNA is of paramount importance for the investigation of molecular genetics. In the realm of rice biology, the initial and fundamental step in the process of isolating DNA necessitates the breakdown of cell walls, thereby facilitating the liberation of nuclei. A frequently employed homogenization methodology involves the grinding of frozen tissue in liquid nitrogen within a chilled mortar and pestle(Allen et al., 2006). This guarantees complete pulverization of tissue and optimizes the quantity of DNA that can be extracted(Allen et al., 2006). However, for the purpose of genotyping, this approach is relatively time-consuming and consequently less efficient, as it permits the processing of only a limited number of samples simultaneously. In contrast, bead beater homogenizers exhibit superior throughput capabilities in comparison to mortar and pestle techniques, owing to their ability to significantly reduce the time required for sample homogenization, thereby enabling the processing of a larger number of samples concurrently(Leach et al., 2016; Lunde, 2018; Guo, 2022).With the BBs in the protocol of Guo et al(Guo, 2022)，After homogenization, cetyltrimethylammonium bromide (CTAB) based protocols are commonly used for DNA extraction (Abdel-Latif and Osman, 2017; Allen et al., 2006).CTAB is a surfactant that effectively facilitates the segregation of protein from nucleic acids, albeit its relatively hazardous nature necessitates cautious handling(Healey Adam, 2014). Protocol uses phenol and chloroform to purify nucleic acids. Both solvents are volatile and toxic, unsuitable for bulk samples, trainees, or classrooms(Guo, 2022).
Wide variety of non-toxic plant DNA purification kits available. Expensive, impractical for large-scale sample processing in resource-limited labs/schools, not necessary for genotyping(Guo, 2022).
Guo propose a modified sodium lauryl sulfate(SDS) and potassium acetate-based protein kalium aceticum(KOAc) with less tissue and bead-based homogenization ,Homogenized samples are submerged in polyvinyl pyrrolidone(PVP-40) buffer to remove polyphenolic compounds.(Dellaporta Stephen, 1983; Edwards K., 1991; Guo, 2022) .However, due to the cost issues of isopropanol and PVP-40, here we introduce a cost-effective, rapid, and non-toxic method for DNA extraction from rice,Named "HDG Extraction buffer", suitable for the extraction of genomic DNA from rice, which can be used for PCR gene amplification, genotyping, and other experiments. It is applicable to various tissues and organs of rice (leaves, roots, panicles, seeds), and does not require toxic organic solvents such as chloroform and phenol, nor does it require controlled substances in specific countries such as potassium chloride (KCl) and isopropanol. The efficiency of the formulation is also demonstrated by the fact that no pH adjustment is needed.

Materials and reagents 
1. Collect different tissues and organs from rice (leaves, roots, panicles, seeds).
2.  BBs (Daisy, catalog number: 980060-446)
3.  Eppendorf tubes (Dot Scientific, catalog number: 609-GMT; RA2000-GMT)
4.  Tris (hydroxymethyl) aminomethane(Sangon Biotech, catalog number: A100193-0500)
5.  EDTA disodium salt dihydrate(EDTA·2H2O) (Sangon Biotech, catalog number: A610185-0500)
6.  Sodium chloride (NaCl) (Sangon Biotech, catalog number: A610476-0001)
7.  Sodium acetate, anhydrate(CH3COONa) (Fisher scientific, catalog number: A16321.36)
8.  100% Ethanol (Fisher scientific, catalog number: T038181000)
9.  Pipettes and tips

HDG Extraction bufferr (1L)
12.1 g Tris
3.7 g EDTA·2H2O
58.5 g NaCl
24.6 g CH3COONa
to 40 mL Water

Procedure
1.Collecting fresh rice tissue about 0.2 g, place it in a 2mL centrifuge tube, and add two BBs (2 beads) and 1mL of HDG Extraction buffer. Homogenize using a Bead Beater homogenizer with the program "55Hz, 45 seconds".
2.After homogenization, place the obtained tissue homogenate in an oven or water bath at a temperature above 55°C for 5 minutes, shake well, then centrifuge at 13,400 rpm for 1 minute. Transfer 650-700 µL of the supernatant to a new 1.5mL centrifuge tube.
3.To the obtained supernatant fluid, add 600 µL precool -80℃ anhydrous ethanol, mix well by inverting the tube,and puting at then centrifuge at 13,400 rpm for 1 minute. Carefully decant the supernatant.
4.Add 1 mL of 70% ethanol to the centrifuge tube, and carefully decant the supernatant again. Invert the centrifuge tube onto a piece of paper to absorb any remaining liquid, then place the tube in an oven set at 55-70℃ for 5 minutes to completely dry the remaining liquid.
5.Add 100 µL of ddH2O or TE buffer to the centrifuge tube, vortex to mix, then centrifuge briefly at 5000 rpm for 10 seconds to pellet any debris. Transfer the entire supernatant to a new centrifuge tube. For short-term storage, keep the sample at 4 ℃ in a refrigerator. For long-term storage, store the sample at -20 ℃ in a freezer
 

Conclusion
We demonstrate a DNA extraction protocol that works well with multiple plant systems, and we highlight the use of inexpensive standard equipment and non-toxic chemicals that balances time, cost, and efficiency that is useful for both high-quality genotyping ，PCR gene amplification, genotyping, and other experiments.
Competing interests
The authors declare that there is no conflict of interest.
References
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Guo, W. , Binstock, B. , Cannon , A. and Lisch, D. (2022). An inexpensive, fast, and robust DNA extraction method for high-quality DNA for use in genotyping and next-generation sequencing applications in plants. Bio-protocol Preprint.
Abdel-Latif, A., and Osman, G. (2017). Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize Plant Methods 13, 1
Allen, G.C., Flores-Vergara, M.A., Krasynanski, S., Kumar, S., and Thompson, W.F. (2006). A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide Nat Protoc 1, 2320-2325.
Healey Adam, F.A., Cooper Tal, Henry Robert (2014). Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species Plant Methods 10
Dellaporta Stephen, W.J., Hicks James (1983). A plant DNA minipreparation: Version II Plant Mol Biol Rep 1, 19-21.
Edwards K., J.C., Thompson C. (1991). A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19, 1349.

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