Efficient genetic transformation and CRISPR/Cas9‐mediated genome editing in Lemna aequinoctialis

Abstract

Summary The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently available transformation protocols, we reduced the duration time of Agrobacterium‐mediated transformation to 5–6 weeks with a success rate of over 94%. Based on the optimized transformation protocol, we generated 15 (14.3% success rate) biallelic LaPDS mutants that showed albino phenotype using a CRISPR/Cas9 system. Investigations on CRISPR/Cas9‐mediated mutation spectrum among mutated L. aequinoctialis showed that most of mutations were short insertions and deletions. This study presents the first example of CRISPR/Cas9‐mediated genome editing in duckweeds, which will open new research avenues in using duckweeds for both basic and applied research.

A submerged duckweed mutant with abundant starch accumulation for bioethanol production

“…The duckweed used in this study was previously identified and named L. aequinoctialis 6002 (Liu, Wang, et al, 2019). L. aequinoctialis 6002 was cultured in Schenk & Hildebrandt (SH) liquid medium supplemented with 10 g/L sucrose, and maintained in a growth chamber at 24°C under a light intensity of 110 μmol m −2 s −1 and a photoperiod of 16 hr (light):8 hr (dark).…”

“…As a supplement to the screening of natural germplasms, the transgenic technology and DNA mutagenesis technology are efficient and frequently used methods to obtain better genetic traits. Genetic manipulation has already been performed in some species in the duckweed family (Chhabra, Chaudhary, Sainger, & Jaiwal, 2011; Vunsh et al., 2007; Yang et al., 2018) including gene overexpressing, artificial microRNA‐induced posttranscriptional gene silencing, and CRISPR/Cas9‐mediated genome editing (Cantó‐Pastor et al., 2015; Liu, Wang, et al, 2019; Yang et al., 2017). It can be anticipated that these approaches will definitely help us to uncover the mechanisms underlying starch accumulation in duckweed.…”

Research Progress of a Potential Bioreactor: Duckweed

“…They found that conjunction of N6-(2-Isopentenyl) adenine (2IP) (1 mg/L) and 2,4-dichlorophenoxyacetic (2,4-D) (10 mg/L) effectively induced callus formation, and the addition of indole acetic acid (4 mg/L) and kinetin (1 mg/L) successfully induced frond regeneration. Subsequently, the tissue culture of a wide range of Lemna was established, including Lemna aequinotialis [ 53 ], Lemna minor [ 54 , 55 ], and Lemna turionifera [ 56 ]. Li et al [ 57 ] established an efficient tissue culture cycle for Spirodela oligorrhiza , Landoltia punctata , and Lemna gibba .…”

Multifaceted roles of duckweed in aquatic phytoremediation and bioproducts synthesis

“…The transformation efficiency varies, depending on the frond, callus, and hormone. The transform efficiency of Lemna reaches 10%–94% (Chhabra, Chaudhary, Sainger, & Jaiwal, 2011; Liu, Wang, et al, 2019; Yang, Fang, et al, 2018), while it is 5%–13% in Spirodela (Rival et al., 2008; Vunsh et al., 2007; Yang, Li, et al, 2018). By contrast, Wolffia has the lowest transform efficiency of about 0.14%–0.4% (Heenatigala et al., 2018; Pavel et al., 2018).…”