SEARCH

SEARCH BY CITATION

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.

FilenameFormatSizeDescription
pce2554_sm_FigureS1.tif2931KSupporting info item
pce2554_sm_FigureS2.tif6728KSupporting info item
pce2554_sm_FigureS3.tif1383KSupporting info item
pce2554_sm_FigureS4.tif5898KSupporting info item
pce2554_sm_FigureS5.jpg484KSupporting info item
pce2554_sm_FigureS6.jpg386KSupporting info item
pce2554_sm_summary.doc23KSupporting info item
pce2554_sm_TableS1.pdf88KSupporting info item
pce2554_sm_TableS2.pdf81KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.