Transient efflux inhibition improves plant regeneration by natural auxins

• Published in: The Plant journal : for cell and molecular biology 2024-04, Vol.118 (2), p.295-303
• Main Authors: Karami, Omid, Khadem, Azadeh, Rahimi, Arezoo, Zagari, Nicola, Aigner, Simon, Offringa, Remko
• Format: Article
• Language: English
• Subjects: 2,4‐dichlorophenoxyacetic acid; Acetic acid; Acids; Arabidopsis; auxin transport inhibition; Auxins; Brassica; carrot; Crop production; Dichlorophenoxyacetic acid; Efflux; Embryonic growth stage; Embryos; Explants; Genome editing; Genomes; Indoleacetic acid; indole‐3‐acetic acid (IAA); Plant breeding; Plant cells; Propagation; Regeneration; Seedlings; shoot regeneration; Shoots; Somatic embryogenesis
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/tpj.16682

SUMMARY Plant genome editing and propagation are important tools in crop breeding and production. Both rely heavily on the development of efficient in vitro plant regeneration systems. Two prominent regeneration systems that are widely employed in crop production are somatic embryogenesis (SE) and de novo shoot regeneration. In many of the protocols for SE or shoot regeneration, explants are treated with the synthetic auxin analog 2,4‐dichlorophenoxyacetic acid (2,4‐D), since natural auxins, such as indole‐3‐acetic acid (IAA) or 4‐chloroindole‐3‐acetic acid (4‐Cl‐IAA), are less effective or even fail to induce regeneration. Based on previous reports that 2,4‐D, compared to endogenous auxins, is not effectively exported from plant cells, we investigated whether efflux inhibition of endogenous auxins could convert these auxins into efficient inducers of SE in Arabidopsis immature zygotic embryos (IZEs). We show that natural auxins and synthetic analogs thereof become efficient inducers of SE when their efflux is transiently inhibited by co‐application of the auxin transport inhibitor naphthylphthalamic acid (NPA). Moreover, IZEs of auxin efflux mutants pin2 or abcb1 abcb19 show enhanced SE efficiency when treated with IAA or efflux‐inhibited IAA, confirming that auxin efflux reduces the efficiency of Arabidopsis SE. Importantly, in contrast to the 2,4‐D system, where only 50–60% of the embryos converted to seedlings, all SEs induced by transport‐inhibited natural auxins converted to seedlings. Efflux‐inhibited IAA, like 2,4‐D, also efficiently induced SE from carrot suspension cells, whereas IAA alone could not, and efflux‐inhibited 4‐Cl‐IAA significantly improved de novo shoot regeneration in Brassica napus. Our data provides new insights into the action of 2,4‐D as an efficient inducer of plant regeneration but also shows that replacing this synthetic auxin for efflux‐inhibited natural auxin significantly improves different types of plant regeneration, leading to a more synchronized and homogenous development of the regenerated plants. Significance Statement It has been a longstanding question why the synthetic auxin 2,4‐dichlorophenoxyacetic acid (2,4‐D) is so effective in inducing plant regeneration. Here we show that the secret lies in its inefficient export from plant cells and that using natural auxins can be as effective, even leading to improved regeneration, when their efflux is transiently inhibited.