Assessment of factors affecting Agrobacterium-mediated transformation of Citrus aurantifolia L. to increase transformation efficiency and reduce chimeric transgenic shoots

Soheilivand, Saeed; Mousavi, Amir; Safarnejad, Mohammadreza

doi:10.30473/cb.2021.57688.1832

Document Type : Research Paper

Authors

¹ Assistant Professor, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.

² Associate Professor, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

³ Associate Professor, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran.

https://doi.org/10.30473/cb.2021.57688.1832

Abstract

Sour lime (Citrus aurantifolia L.) is one of the most important woody plants is widely known for its recalcitrance to genetic transformation. We aimed herein to evaluate effective factors influencing the transformation efficiency and the reduction of chimeric transgenic shoots in sour lime. Epicotyl and internode explants were genetically transformed with different Agrobacterium tumefaciens strains e.g., LBA4404, GV3850, and GV3101, harboring the vectors pBI121 and pCAMBIA3301 containing β-glucuronidase (GUS) as a reporter gene. The effect of the following factors was evaluated: Agrobacterium concentration (OD600=0.3, 0.5 and 1), during inoculation (5 seconds, 10 minutes and 30 minutes), co-culture (2 and 3 days), and the selection regime (phosphinothricin at 1, 3, 5 and 10 mg/l and kanamycin at 25, 50, 75 and 100 mg/l). In following, transformation efficiency and the chimeric transgenic shoots rate were respectively confirmed by PCR and GUS assays. The results showed that Agrobacterium strain LBA4404, at the OD600 of 0.5, with 5 seconds (for epicotyl) and 10 minutes (for internode) inoculation at two-day co-culture period, were identified the most suitable treatments for both explants. The transformation frequencies ranged from 0.93% for internode on DKW medium containing 1.0 mg/l of phosphinothricin to 14.29% for epicotyl on DKW medium containing 50 mg/l of kanamycin. Inclusion of the high-level of selective treatments, improved the transformation rate through decreasing frequency escape and chimeric transgenic shoots. These findings provide novel insights into the appropriate procedure to constitute non-chimeric lime transgenic shoots.

Keywords

20.1001.1.22520783.1399.10.32.3.1

Main Subjects

Genetic Engineering and Gene Transformation

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Crop Biotechnology

Assessment of factors affecting Agrobacterium-mediated transformation of Citrus aurantifolia L. to increase transformation efficiency and reduce chimeric transgenic shoots

References

References

Volume 10, Issue 32 - Serial Number 2
March 2021
Pages 37-53

Assessment of factors affecting Agrobacterium-mediated transformation of Citrus aurantifolia L. to increase transformation efficiency and reduce chimeric transgenic shoots

References

References

Volume 10, Issue 32 - Serial Number 2March 2021Pages 37-53

Volume 10, Issue 32 - Serial Number 2
March 2021
Pages 37-53