Frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor

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

“…A major limitation of our transformation protocol is that the success rate might be highly dependent on the choice of the genotypes, which can vary in their callus induction rate (Table S1). A similar effect was also found in L. minor (Yang et al ., ), indicates that this might be a general pattern in species of the genus Lemna . To resolve this issue, two possible approaches can be applied.…”

“…Here, by optimizing the transformation protocol, we achieved a 94% transformation success rate, which is higher than previous reports on duckweed transformations. For example, in L. minor , a different species of the same genus, the efficiency obtained was approximately 10% (Chhabra et al ., ), 40% (Yang et al ., ) and 59% (Cantó‐Pastor et al ., ). Furthermore, the whole transformation only took 5–6 weeks, which is similar to the fastest protocol that was previously reported in L. minor (Cantó‐Pastor et al ., ), and the genetic transformation was stable in the regenerated plants for at least 70 days (~30 generations).…”

“…In comparison to previous Agrobacterium ‐mediated transformation protocols in L. minor (Cantó‐Pastor et al ., ; Chhabra et al ., ; Firsov et al ., , ; Ko et al ., ; Sun et al ., ; Yamamoto et al ., ; Yang et al ., , , ), the increased efficiency of our protocol is likely resulted from adopting sonication coupled with vacuum infiltration infection method. In several previous studies, such as banana (Subramanyam et al ., ), chickpea (Indurker et al ., ) and cowpea (Bakshi et al ., ), it has been shown that the combination of vacuum infiltration and sonication can greatly improve the efficiency of Agrobacterium infection.…”

“…To resolve this issue, two possible approaches can be applied. First, in addition to the conventional callus‐based transformation, previous studies showed that a direct transformation of fronds (Ko et al ., ; Yang et al ., ) or clusters (the specific structure for Wolffia ) (Heenatigala et al ., ; Khvatkov et al ., , ) can work in duckweeds. Therefore, for the genotypes in which callus induction rate is low, direct frond transformation, which worked for different tested genotypes of L .…”

“…The development of genetic manipulation tools requires both an efficient transformation protocol and an effective genome editing system. Among the 37 duckweed species that belong to five genera: Spirodela , Landoltia , Lemna , Wolffiella and Wolffia (Landolt, ; Sree et al ., ), the Agrobacterium‐ mediated transformation system has been developed in at least seven of them, including Lemna gibba (Cantó‐Pastor et al ., ; Yamamoto et al ., ), Lemna minor (Chhabra et al ., ; Firsov et al ., , ; Ko et al ., ; Sun et al ., ; Yamamoto et al ., ; Yang et al ., , ), Lemna turionifera (Yang et al ., ), Landoltia punctata (formerly Spirodela oligorrhiza ) (Rival et al ., ; Vunsh et al .,), Spirodela polyrhiza (Yang et al ., ), Wolffia arrhiza (Khvatkov et al ., , ) and Wolffia globosa (Heenatigala et al ., ). However, the efficiency of the transformation varied dramatically among species (from 0.14% to 82.5%).…”