Effect of antibiotics on Agrobacterium-mediated transformation from anther derived embryos of Eriobotrya japonica (Thunb.) Lindl. cv.‘Dawuxing’

ABSTRACT

A stable transformation protocol using Agrobacterium tumefaciens strain EHA 52 harbouring the recombinant plasmid pBI121-CycD₂ for anther derived embryos of Eriobotrya japonica (Thunb.) Lindl. cv. ‘Dawuxing’ was established. Parameters optimised were the concentration of antibiotics and their combinations. Results showed that strain EHA 52 is completely not sensitive to carbenicillin at any concentration. On the contrary, ampicillin (Amp), cephalothin (Cef), and gentamicin (Gt) have the poisonous action to both anther derived embryos and strain EHA 52. Amp is the optimal antibiotic for the highest survival rate (42.00%) of the anther derived embryos. Gt shows the strongest inhibitory effect on the survival of anther derived embryos. Thus, both Carb and Gt were not suitable for using in the further transformation protocol. The combination of 800 mg·L⁻¹ Amp and 100 mg·L⁻¹ Kan is suggested to use for selection transgenic secondary embryos. Molecular analysis (PCR) confirms that transformation efficiency of the secondary embryos is ranged from up to 22.00%. Histological observation shows that the cell volume of the secondary embryos is smaller than that of on-transformants. Moreover, the cell division rate in transformed secondary embryos is much more rapid than that in non-transformants. Finally, nine Kan^R plantlets from two anther derived embryos had the expected amplified fragment (0.53 Kb) by PCR analysis.

KEYWORDS:

• Eriobotrya japonica
• Agrobacterium-mediated transformation
• CycD₂ gene
• anther derived embryos; loquat

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work is supported by the National Natural Science Foundation of China [No. 31971716]. Research was supported by A. tumefaciens EHA52 from the Professor Ren Zhenglong’s laboratory.