Genomic DNA extraction from the medicinal plant Crocus sativus : Optimization of Standard Methods

Soumaya EL MERZOUGUI; Imane  BOUDADI; Houda EL FISSI; Mohamed LACHHEB; Khadija LACHGUER; Khalid LAGRAM; Mohamed BEN EL CAID; Rachida EL BOULLANI; Mohammed AMINE  SERGHINI

doi:10.18006/2023.11(6).940.946

Authors

Soumaya EL MERZOUGUI Laboratory of Biotechnology and Valorization of Natural Resources, Sciences Faculty, Ibn Zohr University, Agadir, Morocco
Imane BOUDADI Laboratory of Biotechnology and Valorization of Natural Resources, Sciences Faculty, Ibn Zohr University, Agadir, Morocco
Houda EL FISSI Laboratory of Biotechnology and Valorization of Natural Resources, Sciences Faculty, Ibn Zohr University, Agadir, Morocco
Mohamed LACHHEB Laboratory of Biotechnology and Valorization of Natural Resources, Sciences Faculty, Ibn Zohr University, Agadir, Morocco
Khadija LACHGUER Laboratory of Biotechnology and Valorization of Natural Resources, Sciences Faculty, Ibn Zohr University, Agadir, Morocco
Khalid LAGRAM Laboratory of Biotechnology and Valorization of Natural Resources, Sciences Faculty, Ibn Zohr University, Agadir, Morocco
Mohamed BEN EL CAID Laboratory of Biotechnology and Valorization of Natural Resources, Sciences Faculty, Ibn Zohr University, Agadir, Morocco
Rachida EL BOULLANI Laboratory of Biotechnology and Valorization of Natural Resources, Sciences Faculty, Ibn Zohr University, Agadir, Morocco
Mohammed AMINE SERGHINI Laboratory of Biotechnology and Valorization of Natural Resources, Sciences Faculty, Ibn Zohr University, Agadir, Morocco

DOI:

https://doi.org/10.18006/2023.11(6).940.946

Keywords:

Genomic DNA extraction, Cetyltrimethylammonium bromide, CTAB, SDS, Crocus sativus

Abstract

High-quality genomic DNA is essential for genomic and molecular investigations such as next-generation sequencing. However, DNA extraction from medicinal plants like Crocus sativus can be challenging due to their high secondary metabolite content, which can interact with nucleic acids and affect the quality and yield of extraction. This study aimed to optimize the quality and yield of DNA using the cetyltrimethylammonium bromide (CTAB) extraction method from the leaves, stigma, and saffron corm. This new method is easy to use and can be performed using standard equipment and inexpensive reagents. The modifications made to the CTAB lysis buffer in this study, with the addition of SDS, resulted in a yield of 4233 ng/µl of DNA per sample of saffron corm (100 mg). This protocol is efficient and cost-effective for DNA extraction for studies with large samples and limited resources. This method is expected to be widely used for large-scale plant extraction and has a broad application in PCR-based sequencing studies.

References

Aboul-Maaty, N. AF, & Oraby, H. AS (2019). Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bulletin of the National Research Centre, 43(1), 25. https://doi.org/10.1186/s42269-019-0066-1. DOI: https://doi.org/10.1186/s42269-019-0066-1

Alavi-Siney, S. M., Saba, J., & Nasiri, J. (2022). Genetic variability and population genetic structure in autotriploid saffron using allelic phenotypes of microsatellite markers. Scientia Horticulturae, 299, 111043. DOI: https://doi.org/10.1016/j.scienta.2022.111043

Anabat, M. M., Riahi, H., Sheidai, M., & Koohdar, F. (2020). Population genetic study and barcoding in Iran saffron (Crocus sativus L.). Industrial Crops and Products, 143, 111915. DOI: https://doi.org/10.1016/j.indcrop.2019.111915

Bansal, S., Thakur, S., Mangal, M., Mangal, A. K., & Gupta, R. (2019). Identification of Suitable Locus for Specific Detection of Biological Adulterants of Saffron Food Analytical Methods, 12(11), 2509-2517. DOI: https://doi.org/10.1007/s12161-019-01604-6

Boskabady, M.H., Gholamnezhad, Z., Khazdair, M.R., & Tavakol-Afshari, J. (2020). Anti-inflammatory and immunomodulatory effects of saffron and its derivatives. In A. Koocheki, & M. Khajeh-Hosseini (Eds.) Saffron (pp. 405-421), Woodhead Publishing, Elsevier. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00026-5

Doyle, J. (1991). DNA Protocols for Plants. In: G.M. Hewitt, A.W.B. Johnston, & J.P.W.Young, (eds) Molecular Techniques in Taxonomy. NATO ASI Series, vol 57. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-83962-7_18. DOI: https://doi.org/10.1007/978-3-642-83962-7_18

El Caid, M. B., Salaka, L., El Merzougui, S., Lachguer, K., Lagram, K., El Mousadik A., & Serghini M. A. (2020). Multi-site evaluation of the productivity among saffron (Crocus sativus L.) for clonal selection purposes. Journal of Applied Research on Medicinal and Aromatic Plants, 17, 100248. https://doi.org/10.1016/j.jarmap.2020.100248. DOI: https://doi.org/10.1016/j.jarmap.2020.100248

Esmaeilian, Y., Amiri, M. B., Tavassoli, A., Caballero-Calvo, A., & Rodrigo-Comino, J. (2022). Replacing chemical fertilizers with organic and biological ones in transition to organic farming systems in saffron (Crocus sativus) cultivation. Chemosphere, 307 (Pt 1), 135537. https://doi.org/10.1016/j.chemosphere.2022.135537. DOI: https://doi.org/10.1016/j.chemosphere.2022.135537

Ghanbari, J., Khajoei-Nejad, G., & van Ruth, S. M. (2019a). Effect of saffron (Crocus sativus L.) corm provenance on its agro-morphological traits and bioactive compounds. Scientia Horticulturae, 256, 108605. https://doi.org/10.1016/ j.scienta.2019.108605 DOI: https://doi.org/10.1016/j.scienta.2019.108605

Ghanbari, J., Khajoei-Nejad, G., van Ruth, S. M., Aghighi, S. (2019b). The possibility for improvement of flowering, corm properties, bioactive compounds, and antioxidant activity in saffron (Crocus sativus L.) by different nutritional regimes. Industrial Crops and Products, 135, 301-310. https://doi.org/10.1016/j.indcrop.2019.04.064 DOI: https://doi.org/10.1016/j.indcrop.2019.04.064

Heikrujam, J., Kishor R., & Mazumder, P. B. (2020). The chemistry behind plant DNA isolation protocols. In O. M. Boldura, C. Balta, & N. S. Awwad (Eds.) Biochemical Analysis Tools - Methods for Bio-Molecules Studies. IntechOpen. doi: 10.5772/intechopen.92206. DOI: https://doi.org/10.5772/intechopen.92206

Jiang, C., Cao, L., Yuan, Y., Chen, M., Jin, Y., & Huang, L. (2014). Barcoding melting curve analysis for rapid, sensitive, and discriminating authentication of saffron (Crocus sativus L.) from its adulterants. BioMed research international, 2014, 809037. https://doi.org/10.1155/2014/809037 DOI: https://doi.org/10.1155/2014/809037

Li, H., Li, J., Cong, X. H., Duan, Y. B., Li, L., Wei, P. C., Lu, X. Z., & Yang, J. B. (2013). A high-throughput, high-quality plant genomic DNA extraction protocol. Genetics and molecular research : Genetic and Molecular Research, 12(4), 4526–4539. https://doi.org/10.4238/2013.October.15.1. DOI: https://doi.org/10.4238/2013.October.15.1

Mançano, A.P., de Oliveira, C.A., Cauz-Santos, L.A., da Cruz, D.D., Dequigiovanni, G., et al. (2022). A reliable DNA extraction protocol for the medicinal plant Chrysobalanus icaco (Chrysobalanaceae), a recalcitrant species. Brazilian Journal of Botany, 45(2), 619-624. DOI: https://doi.org/10.1007/s40415-022-00797-1

Moratalla-López, N., Bagur, M. J., Lorenzo, C., Salinas, M. E. M. R., & Alonso, G. L. (2019). Bioactivity and Bioavailability of the Major Metabolites of Crocus sativus L. Flower. Molecules (Basel, Switzerland), 24(15), 2827. https://doi.org/10.3390/ molecules24152827. DOI: https://doi.org/10.3390/molecules24152827

Nath, O., Fletcher, S. J., Hayward, A., Shaw, L. M., Agarwal, R., et al. (2022). A Comprehensive High-Quality DNA and RNA Extraction Protocol for a Range of Cultivars and Tissue Types of the Woody Crop Avocado. Plants (Basel, Switzerland), 11(3), 242. https://doi.org/10.3390/plants11030242. DOI: https://doi.org/10.3390/plants11030242

Rezadoost, M. H., Kordrostami, M., & Kumleh, H. H. (2016). An efficient protocol for isolation of inhibitor-free nucleic acids even from recalcitrant plants. 3 Biotech, 6(1), 61. https://doi.org/10.1007/s13205-016-0375-0. DOI: https://doi.org/10.1007/s13205-016-0375-0

Sabriu-Haxhijaha, A., Ilievska, G., Stojkovski, V., & Blagoevska, K. (2020). A modified SDS–based method applied for extraction of high-quality DNA from raw corn and roasted soybean. Macedonian Veterinary Review, 43(1), 61-67. DOI: https://doi.org/10.2478/macvetrev-2020-0017

Schenk, J. J., Becklund, L. E., Carey, S. J., & Fabre, P. P. (2023). What is the "modified" CTAB protocol? Characterizing modifications to the CTAB DNA extraction protocol. Applications in plant sciences, 11(3), e11517. https://doi.org/10.1002/aps3.11517. DOI: https://doi.org/10.1002/aps3.11517

Shahrajabian, M. H., Sun, W., Soleymani, A., & Cheng, Q. (2021). Traditional herbal medicines to overcome stress, anxiety and improve mental health in outbreaks of human

coronaviruses. Phytotherapy research : PTR, 35(3), 1237–1247. https://doi.org/10.1002/ptr.6888 DOI: https://doi.org/10.1002/ptr.6888

Sika, K. C., Kefela, T., Adoukonou-Sagbadja, H., Ahoton, L., Saidou, A., et al. (2015). A simple and efficient genomic DNA extraction protocol for large scale genetic analyses of plant biological systems. Plant Gene, 1, 43-45. DOI: https://doi.org/10.1016/j.plgene.2015.03.001

Singh, A., Ginwal, H.S., Kumar, J., & R. K. Meena (2023). Improved DNA isolation protocol from leaves and embryos of Terminalia bellirica: a tree species of high-medicinal importance. Brazilian Journal of Botany, 1-6. DOI:10.1007/s40415-023-00920-w. DOI: https://doi.org/10.1007/s40415-023-00920-w

Villa, C., Costa, J., Meira, L., Oliveira, M. B. P., & Mafra, I. (2016). Exploiting DNA mini-barcodes as molecular markers to authenticate saffron (Crocus sativus L.)." Food Control, 65, 21-31. DOI: https://doi.org/10.1016/j.foodcont.2016.01.008

Wang, X., Teng, D., Tian, F., Guan, Q., & Wang, J. (2012). Comparison of three DNA extraction methods for feed products and four amplification methods for the 5'-junction fragment of Roundup Ready soybean. Journal of agricultural and food chemistry, 60(18), 4586–4595. https://doi.org/10.1021/jf300827q. DOI: https://doi.org/10.1021/jf300827q

Yu, D., Zhang, J., Tan, G., Yu, N., Wang, Q., et al. (2019). An easily-performed high-throughput method for plant genomic DNA extraction. Analytical biochemistry, 569, 28–30. https://doi.org/10.1016/j.ab.2019.01.007. DOI: https://doi.org/10.1016/j.ab.2019.01.007

Zhao, M., Wang, B., Xiang, L., Xiong, C., Shi, Y., et al. (2019). A novel onsite and visual molecular technique to authenticate saffron (Crocus sativus) and its adulterants based on recombinase polymerase amplification. Food control, 100, 117-121. DOI: https://doi.org/10.1016/j.foodcont.2019.01.011