Key role of auxin cellular accumulation in totipotency and pluripotency acquisition

Genome editing and in vitro based-plant propagation require efficient plant regeneration system. Somatic embryogenesis (SE) or de novo shoot regeneration are two major systems that widely used for plant in vitro regeneration. Most SE or shoot regeneration protocols rely on the exogenous application of the synthetic auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthylene acetic acid (NAA), whereas the natural auxins indole-3-acetic acid (IAA), 4-chloroindole-3-acetic acid (4-Cl-IAA) or indole-3-butyric acid (IBA) are not or less effective for plant regeneration. Although these synthetic auxins mimics the physiological activity of the main natural auxin IAA in many aspects, there are also clear differences that have been attributed to differences in stability or to different affinities for certain TIR1/AFB-Aux/IAA auxin co-receptor pairs. Here we show that the success of 2,4-D in inducing SE from Arabidopsis is related to ineffectiveness as substrate for auxin efflux, resulting in its intracellular 2,4-D accumulation. Reducing auxin efflux by addition of the auxin transport inhibitor naphthylphthalamic acid (NPA) also allowed natural auxins and other synthetic analogs to induce SE in Arabidopsis with similar efficiencies as 2,4-D. The PIN-FORMED auxin efflux carriers PIN1, PIN2 and the ATP-binding cassette-B auxin transporters ABCB1 and ABCB19 were shown to be partially responsible for the efflux of natural auxins during SE induction. Importantly, all somatic embryos induced in Arabidopsis by IAA in the presence of NPA showed a normal embryo to seedling conversion and subsequent plant development, whereas for the 2,4-D system this was limited to 50-60% of the embryos. We showed that the auxin transport inhibition promotes de novo shoot regeneration capacity from callus induced by 4-Cl-IAA in Brassica napus. In addition, we observed a obvious acceleration in shoot bud emerging from callus induced by 4-Cl-IAA than 2,4-D. Based on our data we conclude, that the efficiency of plant propagation can be significantly improved by applying the natural auxins in the presence of the auxin transport inhibitor NPA.