A protocol for efficient callus induction and stable transformation of Spirodela polyrhiza (L.) Schleiden using Agrobacterium tumefaciens

Abstract

Spirodela polyrhiza represents the largest specie with the smallest genome of all the members of the Lemnoideae. Its genome features have been delineated, revealing its fewest predicted genes of any known plant genome. It is also ideal system for basic biological researches and various practical applications including toxicity testing, bioreactor, biomonitoring and biofuel. In this study, we reported the successful induction of S. polyrhiza callus coupled with the efficient stable transformation using the Agrobacterium tumefaciens strain LBA4404 by optimizing each step of the process. We found that the highest callus induction efficiency was achieved with 22.62 μM 2,4-D and 8.88 μM 6-BA, with above 90% of fronds forming calli. We also determined that 100 μM acetosyringone in the co-cultivation medium and the maintenance of pH value at 5.2 were crucial for high transformation efficiency (up to 13 ± 1.5%). As proof of concept, we transformed S. polyrhiza with the DR5 and TCS synthetic reporters, which have previously been used to report cytokinin and auxin signaling output in the model plant Arabidopsis thaliana. The cytokinin showed highest accumulation at the initial stage of bud formation and the frond apex of S. polyrhiza whilst the expression of auxin was observed highest at frond with middle size. These transformed lines provide an effective way to investigate the development of S. polyrhiza and may shed light on the interesting way in which this specie reproduces. This is the first report of highly efficient callus induction and Agrobacterium tumefaciens-mediated transformation in S. polyrhiza.