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Agrobacterium tumefaciens-mediated transformation system for the important medicinal plant Dendrobium catenatum Lindl

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Abstract

The orchid Dendrobium catenatum Lindl. has both ornamental and medicinal value. Studies on genetic transformation of other Dendrobium species have been reported; however, due to the heterogeneity among the Dendrobium species, an independent transformation system is required for optimum D. catenatum transformation. Agrobacterium tumefaciens-mediated transformation system for D. catenatum is reported herein. Three A. tumefaciens strains, GV3101, LBA4404, and EHA105, were tested for their ability to transform D. catenatum protocorms. It was found that infection with strain GV3101 at an OD600 of 0.6 yielded the highest transformation frequency of 56.5%. Addition of 0.001% (v/v) Triton™ X-100 or pretreatment with ultrasound (300 W, 40 kHz, 3 min) increased the transformation frequency by up to 2-fold. After 1 year, both green fluorescent protein (GFP) imaging and glucuronidase (GUS) histochemical analysis of transgenic plants demonstrated that DcObg-GFP and pDcObg-GUS construct DNA was transferred into the D. catenatum and expressed successfully. Functional and gene expression level analyses of transgenic plants containing a D. catenatum chloroplast-targeted homolog of Spo0B-associated GTP-binding protein (DcObgC) RNA interference construct (DcObgC-RNAi) further demonstrated that this transformation system was reliable and efficient for D. catenatum. The A. tumefaciens-based transformation protocol established and optimized in this current report could provide a powerful and efficient tool for academic research and genetic engineering-based breeding of D. catenatum.

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Funding

This work was supported by grants from The National Natural Science Foundation of China (No. 31501242) and The Education Department of Sichuan Province (No. 14ZB0013).

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Author notes

  1. Ji Chen, Li Wang, and Jianbin Chen equally contributed to this work.

Authors and Affiliations

  1. Agronomy College, Sichuan Agricultural University, Chengdu, 611130, China

    Ji Chen, Li Wang, Jianbin Chen, Jin Huang, Fan Liu, Rong Guo, Li Yang, Ke Zhao, Fanlei Kong & Chen Liu

  2. Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, TX, 77843, USA

    Aby Grabon

  3. Institute for New Rural Development, Sichuan Agricultural University, Ya’an, 625000, China

    Mengliang Tian

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  1. Ji Chen
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  2. Li Wang
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Correspondence to Mengliang Tian.

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Editor: Harold Trick

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Figure S1

Schematic map of the plasmid constructs used for the transformation in Dendrobium catenatum. Plasmids containing P600GUS construct were introduced into A. tumefaciens strains GV3101, LBA4404, and EHA105 for transformation and plasmids containing DcObgC-GFP, and DcObgC-RNAi constructs were introduced into Agrobacterium strain GV3101. LB left border; RB right border; hyg(r) hygromycin resistance gene; 35S cauliflower mosaic virus (CaMV) 35S promoter; gfp green fluorescent protein gene; DcObgC D. catenatum (Dc) chloroplast-targeted homolog of Spo0B-associated GTP-binding protein; P600 600 bp of DcObgC promoter; gus glucuronidase gene. (GIF 44 kb)

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Chen, J., Wang, L., Chen, J. et al. Agrobacterium tumefaciens-mediated transformation system for the important medicinal plant Dendrobium catenatum Lindl. In Vitro Cell.Dev.Biol.-Plant 54, 228–239 (2018). https://doi.org/10.1007/s11627-018-9903-4

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