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. 2018 Nov 29;19(12):3811.
doi: 10.3390/ijms19123811.

Cell Wall Epitopes and Endoploidy as Reporters of Embryogenic Potential in Brachypodium Distachyon Callus Culture

Alexander Betekhtin  1 , Magdalena Rojek  2 , Katarzyna Nowak  3 , Artur Pinski  4 , Anna Milewska-Hendel  5 , Ewa Kurczynska  6 , John H Doonan  7 , Robert Hasterok  8
Affiliations

Cell Wall Epitopes and Endoploidy as Reporters of Embryogenic Potential in Brachypodium Distachyon Callus Culture

Alexander Betekhtin et al. Int J Mol Sci. .

Abstract

Effective regeneration of callus tissue into embryos and then into whole plants is essential for plant biotechnology. The embryonic potential is often low and can further decrease with time in culture, which limits the utilisation of calli for transformation procedures and in vitro propagation. In this study, we show that the loss of embryogenic potential in callus cultures of Brachypodium distachyon is progressive over time. Flow cytometry analyses indicated endoploidy levels increased in 60- and 90-day-old calli with effective loss of the 2C DNA content peak in the latter. Analysis of indolic compounds content revealed a decrease in 60- and 90-day-old calli compared to either freshly isolated explants or 30-day-old calli. Immunohistochemical analysis revealed a decrease in arabinogalactan proteins (AGP) signal with the time of culture, but extensin (EXT) epitopes either increased (JIM12 epitopes) or decreased (JIM11 epitopes). The transcript accumulation levels of AGPs and EXTs confirmed these results, with most of AGP and EXT transcripts gradually decreasing. Some chimeric EXT transcripts significantly increased on the 30th day of culture, perhaps because of an increased embryogenic potential. Selected somatic embryogenesis-related genes and cyclins demonstrated a gradual decrease of transcript accumulation for YUCCA (YUC), AINTEGUMENTA-LIKE (AIL), BABY BOOM (BBM), and CLAVATA (CLV3) genes, as well as for most of the cyclins, starting from the 30th day of culture. Notably, WUSCHEL (WUS) transcript was detectable only on the 30th and 60th day and was not detectable in the zygotic embryos and in 90-day-old calli.

Keywords: Brachypodium distachyon; arabinogalactan proteins; cell wall; cyclins; extensins; pectins; ploidy instability; somaclonal variation; somatic embryogenesis; transcript accumulation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure A1
Figure A1
Immunolocalisation of JIM13 (A–F) and JIM16 (G–L) in B. distachyon callus. (A–C, G–I) 30th day of cultivation; (D–F, J–L) 90th day of cultivation. FB: fluorescent brightener. Scale bars, 10 μm.
Figure A2
Figure A2
Immunolocalisation of LM2 (A–F) in B. distachyon callus. (A–C) 30th day of cultivation; (D–F) 90th day of cultivation. Scale bars, 10 μm.
Figure A3
Figure A3
Immunolocalisation of JIM11 (A–F) and JIM12 (G–L) in B. distachyon callus. (A–C, G–I) 30th day of cultivation; (D–F, J–L) 90th day of cultivation. The red arrow indicates a very weak signal for JIM12. The greenish background on the photomicrographs is due to autofluorescence. Scale bars, 10 μm.
Figure A4
Figure A4
Immunolocalisation of LM16 (A–F) and LM19 (G–L) in B. distachyon callus. (A–C, G–I): 30th day of cultivation; (D–F, J–L) 90th day of cultivation. The greenish background on these photomicrographs is due to autofluorescence. Scale bars, 10 μm.
Figure A5
Figure A5
Immunolocalisation of LM6 (A-F) and LM20 (G–L) in B. distachyon callus. (A–C, G–I) 30th day of cultivation; (D–F, J–L) 90th day of cultivation. The red arrows indicate the presence of the epitope on the callus surface. Scale bars, 10 μm.
Figure A6
Figure A6
Immunolocalisation of LM25 (A–F) in B. distachyon callus. (A–C) 30th day of cultivation; (D–F) 90th day of cultivation. Scale bars, 10 μm.
Figure 1
Figure 1
Morphology and histology of representative structures in the in vitro culture of Brachypodium distachyon callus. (A–C) General morphology and (D–E) histological section of the callus on the 30th (A,D), 60th (B,E), and 90th (C,F) day of cultivation. The red arrows point at brownish parts of the callus. EM: embryogenic masses, PC: parenchymatous cells. Scale bars, (A–C)—1 mm, (D)—10 μm, (E)—50 μm, (F)—10 μm.
Figure 2
Figure 2
Relative DNA content determined by flow cytometry in zygotic embryos (A) and in B. distachyon callus on the 30th (B–C, embryogenic and non-embryogenic parts, respectively), 60th (D–E, embryogenic and non-embryogenic parts, respectively), and 90th (F) day of cultivation. The red arrow demonstrates the absence of 2C DNA on the 90th day of cultivation.
Figure 3
Figure 3
Indolic compounds content on the 30th, 60th, and 90th day of cultivation. The asterisks * indicate values that are significantly different from the immature zygotic embryo control (Student’s t-test, p < 0.05; n = 3 ± SD).
Figure 4
Figure 4
Relative transcript accumulation levels of genes associated with cell wall architecture, i.e., AGPs (Bradi3g39740, Bradi2g60270, Bradi2g31980, and Bradi5g18950), EXTs (Bradi2g05080, Bradi1g22980, Bradi2g00900, Bradi3g12902, and Bradi2g57740), and PECTIN METHYLESTERASE (Bradi3g24750)), on the 30th, 60th, and 90th day of cultivation. Relative transcript accumulation levels of (A) AGPs, (B) EXTs, (C) PECTIN METHYLESTERASE, (D) chimeric EXT Bradi3g12902. The relative transcript accumulation levels were normalised to an internal control (AK437296, gene encoding for ubiquitin) and calibrated to the control (explants, zygotic embryos); *: value is significantly different from the control (Student’s t-test, p < 0.05; n = 3 ± SD).
Figure 5
Figure 5
Relative transcript accumulation levels of (A) YUC (Bradi5g01327), AIL (Bradi1g72890), SERK (Bradi3g46747 and Bradi5g12227), LEA (Bradi5g26600), BBM (Bradi2g577747 and Bradi4g14960), CLV3 (Bradi1g05010), and (B) WUS (Bradi5g25113) and YUC (Bradi1g72500) genes on the 30th, 60th, and 90th day of cultivation. The relative transcript accumulation levels were normalised to an internal control (AK437296, a gene encoding ubiquitin) and calibrated to the control (explants, zygotic embryos); *: value is significantly different from the control (Student’s t-test, p < 0.05; n = 3 ± SD).
Figure 6
Figure 6
Relative transcript accumulation levels of (A) CYCD3 (Bradi3g58300), CYCA3 (Bradi1g14820), CYCB1 (Bradi2g52760), CDKPK (Bradi1g54570), CDKA (Bradi3g02270), CDKB2 (Bradi3g40200), CDKD (Bradi2g26510), WEE (Bradi3g03112), and CYCD4 (Bradi4g32556), and (B) CDKB1 (Bradi4g25980) on the 30th, 60th and 90th day of culture. The relative transcript accumulation levels were normalised to an internal control (AK437296, gene encoding ubiquitin) and calibrated to the control (explants, zygotic embryos); *: value is significantly different from the control (Student’s t-test, p < 0.05; n = 3 ± SD).
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