Direct shoot regeneration from Arabidopsis thaliana shoot apical meristems

Abstract

In Arabidopsis thaliana in vitro culture, shoots were induced from the shoot apical meristem (SAM) of germinating seeds in the presence of 2,4-dichlorophenoxyacetic acid. Primary shoot primordia developed leaf-like structures, from which secondary shoot primordia were produced. Regenerated shoots were recovered when the material was transferred to a medium lacking auxin. Adventitious roots formed from a callusing basal region of the secondary shoots. The CUC1 transcription factor was expressed at the apex of the primary shoot primordium and at the boundary between the regenerated SAM and the developing leaf primordia. The DR5::GUS transgene was used to localize sites of maximum auxin occurrence. Auxin was firstly detected in the dividing cells beneath the SAM epidermis, which coincided with sites where primary shoot primordia were initiated. In the regenerated shoots, auxin response was not detected in the basal region of the stem, suggesting that the regenerating structures were shoots rather than somatic embryos. Direct shoot regeneration from the A. thaliana SAM requires a localized accumulation of auxin.Additional key words: auxin accumulation, 2,4-dichlorophenoxyacetic acid, shoot apical meristem, transcription factors.

Cytosine methylation of the FWA promoter promotes direct in vitro shoot regeneration in Arabidopsis thaliana

“…We also examined the epigenetic mutants fwa-1 and fwa-2, which have reduced DNA methylation in the FWA promoter and show higher expression of FWA compared to the WT (Koornneef et al, 1991). Using our previously described culture system for direct in vitro shoot regeneration (Xin et al, 2012;Liu et al, 2013), we detected no differences between acc1-1, max3-9, tfl1-1, and grp3 and the WT after 14 d in vitro (Figure S5). After 7 d of culture, smooth-surfaced protuberances were produced from the SAMs of the explants, but slightly fewer fwa-1 and fwa-2 explants developed such protuberances compared to WT explants (Figure 3A-D).…”

“…T he ability to regenerate shoots or roots from in vitrocultured explants is a property shared by most plant species (Che et al, 2007;Ikeuchi et al, 2019;Lardon et al, 2020). In Arabidopsis thaliana, shoot regeneration can be induced directly or indirectly (Che et al, 2007;Xin et al, 2012). In indirect shoot regeneration, the explant is cultured…”

“…By contrast, in direct shoot regeneration systems, shoot formation is induced from the shoot apical meristems (SAMs) of germinating seeds in the presence of auxin (2,4-dichlorophenoxyacetic acid [2,4-D]). Primary shoot primordia develop leaf-like structures (Xin et al, 2012;Liu et al, 2013), from which secondary shoot primordia are produced. Regenerated shoots are recovered when the material is transferred to medium lacking auxin (Xin et al, 2012).…”

“…Primary shoot primordia develop leaf-like structures (Xin et al, 2012;Liu et al, 2013), from which secondary shoot primordia are produced. Regenerated shoots are recovered when the material is transferred to medium lacking auxin (Xin et al, 2012).…”

Comparison of Sample Preparation Techniques for Inspection of Leaf Epidermises Using Light Microscopy and Scanning Electronic Microscopy

“…It was reported that t-butanol is equivalent or sometimes superior to CPD (Inoué and Osatake, 1988;Baskin et al, 2014). The t-butanol freeze-drying method is commonly used to prepare biological SEM samples in Japan (Kaneko et al, 1990;Tuji, 2000;Watanabe et al, 2015), China (Xin et al, 2012), and other regions (El Sharaby et al, 2012). Methanol is reported to be a better fixative than glutaraldehyde and FAA when preparing SEM samples (Neinhuis and Edelmann, 1996;Pathan et al, 2010;Talbot and White, 2013b).…”