Agrobacterium-Mediated Genetic Transformation and Regeneration of Salt Tolerant Transgenic of Sour Orange Rootstock (Citrus au-rantium L.) | ||
| Journal of Plant Production | ||
| Article 2, Volume 12, Issue 11, November 2021, Pages 1145-1150 PDF (723.26 K) | ||
| Document Type: Original Article | ||
| DOI: 10.21608/jpp.2021.204911 | ||
| Authors | ||
| Doaa Mahmoud Abou Elyazid* 1; A. N. El-Banna2 | ||
| 1Department of Horticulture , Faculty of Agriculture, Kafrelsheikh university | ||
| 2Genetics Department, Faculty of Agriculture, Kafrelsheikh University, Kafr El-Sheikh 33516, Egypt | ||
| Abstract | ||
| Traditional breeding techniques of citrus are complicated and time intensive. In this study, an effective and simple method for the production of sour orange transgenic plants incorporating salt tolerance gene (Pr10a) has been established. Transgenic plants were produced by Agrobacterium tumefaciens -mediated gene transfer using hypocotyl explants under the selection pressure of phosphinothericin and NaCl as selective agents. Hypocotyl segments were excised from in vitro seedlings and incubated with Agrobacterium tumefaciens EHA105 strain harboring the transgene (Pr10a) under the control of mannopine synthase promoter (p-MAS). In addition, the used dicistronic binary vector contained luciferase (Luc) gene linked to internal ribosome entry site elements in the T-DNA region. MS medium containing 1.5 mg/l 6-benzylaminopurine (BA) and 0.2 mg/l indole butyric acid (IBA) proved optimal for shoot regeneration (97%) from sour orange hypocotyl. The number of marker gene (Luc) expressing plants increased as the inoculation time and bacterial density (OD600) increased up to20 min and 0.4, respectively. Under this condition, the transformation efficiency of hypocotyl segments was 24.2%. PCR analysis using Pr10a and Luc primers confirmed the existence of the transgenes in transgenic plants where the expected band size of 480 and 837 bp were obtained for both transgenes, respectively. The integration of T-DNA containing Pr10a and Luc genes in the transgenic sour orange was revealed by Southern blot analysis and verifiedthe results of luciferase enzyme activity and PCR analysis of Pr10a and Luc genes. A simple integration pattern for all the transgenes including the single copy integration was predominantly observed. | ||
| Keywords | ||
| Genetic transformation; Shoot organogenesis; Sour orange; Pr10a; Salinity | ||
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