Development of partial abiotic stress tolerant Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck through Agrobacterium-mediated transformation method

Nazmul Hasan; Mohammad Kamruzzaman; Shariful Islam; Hammadul Hoque; Fahmid Hossain Bhuiyan; Shamsul H Prodhan

doi:10.1186/s43141-019-0014-3

Development of partial abiotic stress tolerant Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck through Agrobacterium-mediated transformation method

J Genet Eng Biotechnol. 2019 Dec 16;17(1):14. doi: 10.1186/s43141-019-0014-3.

Authors

Nazmul Hasan¹, Mohammad Kamruzzaman¹, Shariful Islam², Hammadul Hoque¹, Fahmid Hossain Bhuiyan¹, Shamsul H Prodhan³

Affiliations

¹ Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet, 3114, Bangladesh.
² Department of Molecular Biology and Genetic Engineering, Sylhet Agricultural University, Sylhet, 3100, Bangladesh.
³ Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet, 3114, Bangladesh. shamsulhp@yahoo.com.

Abstract

Background: Recent studies indicate that farmers are facing several challenges due to biotic and abiotic stresses like diseases, drought, cold, and soil salinity which are causing declined Citrus production. Thus, it is essential to improve these varieties which would be resistant against biotic and abiotic stresses as well as high yielding. The transformation of abiotic stress tolerant genes in Citrus species is essential for using areas affected by abiotic stresses. This study was aimed to improve resistance of Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck to abiotic stresses by transferring PsCBL and PsCIPK genes through Agrobacterium-mediated transformation.

Results: Abiotic stress tolerant PsCBL and PsCIPK genes isolated from Pisum sativum were transformed into two Citrus species, Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck, through Agrobacterium-mediated transformation method. Mature seed-derived calli of two Species were infected with Agrobacterium tumefaciens LBA4404 harboring PsCBL and PsCIPK genes. The infected calli were co-cultured in dark condition and later on washed with antibiotic solution and transferred to selection medium. Preliminary resistant calli were recovered and regenerated to plantlets. Maximum regeneration rate was 61.11 ± 1.35% and 55.55 ± 1.03%, respectively. The genetic transformation was confirmed by performing β glucuronidase (GUS) assays and subsequent PCR amplification of the GUS gene. The transformation rates of the two cultivated species were higher than previous reports. Maximum transformation frequencies were found when bacterial OD₆₀₀ was 0.5 and concentration of acetosyringone was 150 μM. In-vitro evaluation of drought and salt tolerance of transgenic plantlets were done, and transgenic plantlets showed better performance than the control plants.

Conclusions: The present study demonstrates that transformation of Citrus plants with PsCBL and PsCIPK genes result in improved abiotic stress tolerance.

Keywords: Abiotic stress; Agrobacterium; Citrus; PCR; Regeneration; Transformation.

Grants and funding

NIL/Ministry of Science and Technology, Government of the People's Republic of Bangladesh