Agrobacterium-Mediated Genetic Transformation of Withania coagulans (Dunal) with rol A Genes and Its Antioxidant Potential

Samiya Rehman; Amjad Hussain; Munzer Ullah; Enas Ali; Mariusz Mojzych; Syed Ali Raza Naqvi; Akbar Ali; Meher Ali; Ehab Gomaa; Sherif S M Ghoneim; Bushra Mirza; Ken Keefover Ring; Hidayat Hussain; Abdur Rauf; Najeeb Ur Rehman; Faisal Attique

doi:10.1021/acsomega.3c07069

Agrobacterium-Mediated Genetic Transformation of Withania coagulans (Dunal) with rol A Genes and Its Antioxidant Potential

ACS Omega. 2023 Oct 30;8(44):41918-41929. doi: 10.1021/acsomega.3c07069. eCollection 2023 Nov 7.

Authors

Samiya Rehman¹, Amjad Hussain², Munzer Ullah¹, Enas Ali³, Mariusz Mojzych^{4

5}, Syed Ali Raza Naqvi⁶, Akbar Ali⁶, Meher Ali⁷, Ehab Gomaa⁸, Sherif S M Ghoneim⁹, Bushra Mirza¹⁰, Ken Keefover Ring¹¹, Hidayat Hussain¹², Abdur Rauf¹³, Najeeb Ur Rehman¹⁴, Faisal Attique²

Affiliations

¹ Department of Biochemistry, University of Okara, Okara, Punjab 56300, Pakistan.
² Institiute of Chemistry, University of Okara, Okara, Punjab 56300, Pakistan.
³ Faculty of Engineering and Technology, Future University in Egypt, New Cairo 11835, Egypt.
⁴ Department of Chemistry, Siedlce University of Natural Sciences and Humanities ul. 3 Maja 54, Siedlce 08-110, Poland.
⁵ Faculty of Health Sciences, Collegium Medicum, The Mazovian Academy in Plock, Plac Dabrowskiego 2, 09-402 Płock, Poland.
⁶ Department of Chemistry, Government College University, Faisalabad 38040, Pakistan.
⁷ Department of Chemistry, Karakoram International University, Gilgit 15100, Pakistan.
⁸ Department of Civil Engineering, College of Engineering, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia.
⁹ Department of Electrical Engineering, College of Engineering, Taif University, P.O. BOX 11099, Taif 21944, Saudi Arabia.
¹⁰ Department of Biochemistry, Quaid-e-Azam University, Islamabad 44000, Pakistan.
¹¹ Department of Botany and Geography, University of Wisconsin, Madison, Madison, Wisconsin 53706, United States.
¹² Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, Halle (Saale) D-06120, Germany.
¹³ Department of Chemistry, University of Swabi, Anbar Khyber Pakhtunkhwa, Swabi 94640, Pakistan.
¹⁴ Natural & Medical Sciences Research Center, University of Nizwa, P.O Box 33, Postal Code Birkat Al Mauz, Nizwa 616, Sultanate of Oman.

Abstract

In ancient times, Withania coagulans Dunal was used as a therapeutic plant for the treatment of several diseases. This report aims to examine the effect of Agrobacterium tumefactions-mediated transformation of W. coagulans with the rolA gene to enhance secondary metabolite production, antioxidant activity, and anticancer activity of transformed tissues. Before transgenic plant production, the authors designed an efficient methodology for in vitro transformation. In this study, leaf explants were cultured on Murashage and Skoog (MS) media containing different amounts of naphthalene acetic acid (NAA) and benzyl adenine (BA). The best performance for inducing embryogenic callus was in MS medium containing 4 μM NAA and 6.0 μM BA, while the best results for shooting (100%) were obtained at 8 μM benzyl adenine. On the other hand, direct shooting was attained by subculturing leaves on MS medium supplemented with 8 μM benzyl adenine. Prolonged shoots showed excellent in vitro rooting results (80%) with 12 μM indole-3-butyric acid (IBA). The samples were precultivated for 3 days and were followed by 48 h infection with A. tumefaciens strain GV3101 having pCV002. Then, a vector expressed the rol A gene of strain Agrobacterium rhizogenes. Furthermore, three independent transgenic shoot lines and one callus line (T2) were produced and exhibited stable integration of transgene rol A genes, as revealed by PCR analysis. Transgenic strains showed a significant increase in antioxidant potential as compared to untransformed plants. Additionally, LC-MS analysis showed that the transformed strains have a higher withanolide content as compared to untransformed ones. Moreover, the reduced proliferation of prostate cancer cells was observed after treatment with extracts of transgenic plants. Furthermore, these transformed plants exhibited superior antioxidant capability and higher withanolide content than untransformed ones. In conclusion, the reported data can be used to select withanolide-rich germplasm from transformed cell cultures.