Agrobacterium- Mediated Genetic Transformation of Taiwanese Isolates of Lemna aequinoctialis

Abstract

Duckweed (Lemna aequinoctialis) is one of the smallest flowering plants in the world. Due to its high reproduction rate and biomass, duckweeds are used as biofactors and feedstuff additives for livestock. It is also an ideal system for basic biological research and various practical applications. In this study, we attempt to establish a micropropagation technique and Agrobacterium-mediated transformation in L. aequinoctialis. The plant-growth regulator type and concentration and Agrobacterium-mediated transformation were evaluated for their effects on duckweed callus induction, proliferation, regeneration, and gene transformation efficiency. Calli were successfully induced from 100% of explants on Murashige and Skoog (MS) medium containing 25.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μM thidiazuron (TDZ). MS medium containing 4.5 μM 2,4-D and 2.0 μM TDZ supported the long-lasting growth of calli. Fronds regenerated from 100% of calli on Schenk and Hildebrandt (SH) medium containing 1.0 μM 6-benzyladenine (6-BA). We also determined that 200 μM acetosyringone in the cocultivation medium for 1 day in the dark was crucial for transformation efficiency (up to 3 ± 1%). Additionally, we propose that both techniques will facilitate efficient high-throughput genetic manipulation in Lemnaceae.

Keywords: Agrobacterium tumefaciens; duckweed; tissue culture; transformation.

Figure 1

Lemna aequinoctialis. (A) Fronds of L. aequinoctialis; (B) fronds were cultured for 8 weeks on MS medium containing 3% sucrose, 25 μM 2,4-D, and 2.0 μM TDZ to induce callus formation. The arrow indicates the senescent frond; (C) browning phenomenon of callus cultured on previous CIM; (D) callus that was separated from the bleached frond explant and grown for an additional 4 weeks on MS medium containing 3% sucrose, 4.5 μM 2,4-D, and 2.0 μM TDZ for callus proliferation; (E,F) plants regenerating from callus incubated for 2 (E) and 4 (F) weeks on SH medium containing 1% sucrose and 1.0 μM 6-BA. Bar = 1.0 mm.

Figure 2

The effects of 2,4-D and TDZ on callus proliferation. Calli were cultured on the MS medium containing 3% sucrose and different plant growth regulators. Data (mean ± SD) with different letters among treatments significantly differ (p < 0.05).

Figure 3

Stable transformation and regeneration of L. aequinoctialis callus with a GFP expression construct. (A) Transformed nodular calli showed GFP expression under selection medium at day 9; (B) newly divided calli were observed after 10 weeks of selection; (C) newly divided calli were observed under bright-field and (D) GFP channel with long-pass filter; (E) transformed fronds were observed under bright-field and (F) GFP channel with long-pass filter.

Figure 4

Molecular analysis of putative transformants from DNA, mRNA, and protein. The atpF-atpH marker, LaActin, and coomassie brilliant blue (CBB) staining were used as internal controls for each molecular analysis, respectively.