The shoot regeneration capacity of excised Arabidopsis cotyledons is established during the initial hours after injury and is modulated by a complex genetic network of light signalling
Article first published online: 3 JUL 2012
© 2012 Blackwell Publishing Ltd
Plant, Cell & Environment
Volume 36, Issue 1, pages 68–86, January 2013
How to Cite
NAMETH, B., DINKA, S. J., CHATFIELD, S. P., MORRIS, A., ENGLISH, J., LEWIS, D., ORO, R. and RAIZADA, M. N. (2013), The shoot regeneration capacity of excised Arabidopsis cotyledons is established during the initial hours after injury and is modulated by a complex genetic network of light signalling. Plant, Cell & Environment, 36: 68–86. doi: 10.1111/j.1365-3040.2012.02554.x
- Issue published online: 3 DEC 2012
- Article first published online: 3 JUL 2012
- Accepted manuscript online: 8 JUN 2012 12:31PM EST
- Received 12 April 2012; received in revised form 25 May 2012; accepted for publication 27 May 2012
Figure S1. Representative images of regenerating explants of the unusual ecotype Est-1. Shown are explants regenerating shoots when exposed to: (a) continuous low light (20–25 μmol m−2 s−1), (b) continuous medium light (44–55 μmol m−2 s−1), (c) continuous high light (80–94 μmol m−2 s−1), (d) 5 days darkness followed by continuous low light, (e) 5 days darkness followed by continuous medium light, (f) 5 days darkness followed by continuous high light. Though significant differences in shoot regeneration were not observed at these light levels when regeneration was quantified as percent explants with shoots (Fig. 1), these scans, which show the total number of regenerated shoots per explant, demonstrate that shoot regeneration from Est-1 explants was promoted by increasing light intensity following excision.
Figure S2. Exposure of Arabidopsis cotyledon explants to early high light, or high light followed by darkness, promotes callus failure. (a) Shown is the experimental design, including media (CIM, SIM) and light–dark shifting treatments. (b-e) Shown are the percentages of cotyledon explants that failed to regenerate any visible callus 5 weeks after excision for ecotypes: (b) Ler-0, (c) DijG, (d) No-0 and (e) Est-1. One particular treatment (high light, followed by darkness, followed by high light) promoted the highest rates of callus failure. Each histogram represents 78 pooled cotyledons. (f-j) Pictures of ecotype DijG showing the callus failure response, where reduced green callus can be observed on selected treatments. The treatments shown are: (f) constant low light, (g) constant high light, (h) 10 days of darkness followed by high light, (i) 5 days of high light, followed by 10 days of darkness, followed by high light, (j) 5 days of low light, followed by 10 days of darkness, followed by low light. Early high light as in (g) and (i) caused considerable callus failure, two examples of which are boxed in yellow. All ecotypes shown were from Lehle Seeds: DijG (WT-10), Est-1 (WT-6A), Ler-0 (WT-4) and No-0 (WT-9). For all experiments, explants (6–7 days post-germination) were placed on CIM plates for 5 days, then SIM for 10 days followed by fresh SIM for the remaining ∼3 weeks.
Figure S3. Effect of the quadruple blue/UVA light photoreceptor mutant cry1cry2phot1phot2 (quadblue) on shoot regeneration. Shown are the two wild-type parents of quadblue (Ohgishi et al. 2004), ecotypes Ler-0 (CS20) and Ws2 (CS2360/CS22659). Graphed are the (a) average number of regenerated shoots/explant and (b) percent explants with shoots, scored 4 weeks following excision. For (a,b), cotyledons were exposed to either darkness (black bars) or high light (∼100 μmol m−2 s−1, white bars) for 24 h after excision, then treated for another 4 weeks with continuous high light. For all experiments, explants (6–7 days post-germination) were placed on CIM plates for 5 days, then SIM for 10 days followed by fresh SIM for the remaining ∼3 weeks. See the legend for Figure 3 for explanations of percentages, asterisks and error bars, and Supporting Information Table S2 for statistical analysis. Each histogram is the mean of 5–18 replicates (26 cotyledons per replicate).
Figure S4. Effect of mutations on visible anthocyanin accumulation in cotyledon explants four weeks after excision. Explants (6–7 days post-germination) were placed on CIM plates for 5 days, then SIM for 10 days followed by fresh SIM for the remaining ∼3 weeks. Explants were exposed to constant high light (∼100 μmol m−2 s−1) for ∼4 weeks, constant low light (∼20–30 μmol m−2 s−1) for ∼4 weeks, 1 or 5 days of darkness followed by constant high light for ∼4 weeks. Pictures of representative explants were taken at the end of the experiments. Shown are explants of the following ecotypes: (a-d) wild-type Ler-0 (CS20), (e-h) phyA-203 (CS6221), (i-l) phyB-1 (CS6211), (m-p) hy1-1 (CS67), and (q-t) cry1 (hy4-1, CS70). The asterisks denote treatment/genotype combinations that resulted in extremely low visible anthocyanin accumulation (n = 72).
Figure S5. The effect of a cytokinin-overexpressing transgene (CaMV35S-IPT161) on shoot regeneration and anthocyanin accumulation. (a) Shoot regeneration of explants containing the CaMV35S-IPT161 transgene (pCYT::IPT, CS117) compared to wild-type ecotype C24 (CS906). Four light treatments were used: continuous high light (white, ∼100 μmol m−2 s−1) for ∼4 weeks, continuous low light (light grey, ∼20–30 μmol m−2 s−1) for ∼4 weeks, five days of high light followed by 10 days of darkness followed by continuous high light (white with diagonal lines), and five days of darkness post-excision then continuous high light (black). (b-e) Transgene effects on anthocyanin accumulation. The two treatments were: (b,d) extended high light (100 μmol m−2 s−1), followed by extended darkness, followed by high light (double shift), or (c,e) 5 days of darkness after excision and 4 more weeks of high light. For all experiments, explants (6–7 days post-germination) were placed on CIM plates for 5 days, then SIM for 10 days followed by fresh SIM for the remaining ∼3 weeks. The error bar is the standard error of the mean (SEM). Total experimental n = 732 cotyledons.
Figure S6. Pigment absorbance spectra and light emission spectra. Absorbance spectra of (a) chlorophylls and carotenoids (Buchanan, Gruissem & Jones 2000) and (b) anthocyanins (cyanidin-3-glucoside) (Strack, 1997; Gould et al. 2002b) as well as emission spectra of (c) cool white fluorescent light emitted from Sylvania F72T12CW/VHO bulbs and (d) incandescent Sylvania light bulbs (http://www.sylvania.com).
Table S1. Statistical testing for significant differences between mean shoot regeneration rates and regenerative tissue biomass, for initial light/dark treatments.
Table S2. Statistical testing for significant differences between mean shoot regeneration rates for all mutant, filter or pharmacological treatments presented.
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