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. 2017 May 2;10:110.
doi: 10.1186/s13068-017-0795-z. eCollection 2017.

Impact of RAV1-engineering on Poplar Biomass Production: A Short-Rotation Coppice Field Trial

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Free PMC article

Impact of RAV1-engineering on Poplar Biomass Production: A Short-Rotation Coppice Field Trial

Alicia Moreno-Cortés et al. Biotechnol Biofuels. .
Free PMC article

Abstract

Background: Early branching or syllepsis has been positively correlated with high biomass yields in short-rotation coppice (SRC) poplar plantations, which could represent an important lignocellulosic feedstock for the production of second-generation bioenergy. In prior work, we generated hybrid poplars overexpressing the chestnut gene RELATED TO ABI3/VP1 1 (CsRAV1), which featured c. 80% more sylleptic branches than non-modified trees in growth chambers. Given the high plasticity of syllepsis, we established a field trial to monitor the performance of these trees under outdoor conditions and a SRC management.

Results: We examined two CsRAV1-overexpression poplar events for their ability to maintain syllepsis and their potential to enhance biomass production. Two poplar events with reduced expression of the CsRAV1 homologous poplar genes PtaRAV1 and PtaRAV2 were also included in the trial. Under our culture conditions, CsRAV1-overexpression poplars continued developing syllepsis over two cultivation cycles. Biomass production increased on completion of the first cycle for one of the overexpression events, showing unaltered structural, chemical, or combustion wood properties. On completion of the second cycle, aerial growth and biomass yields of both overexpression events were reduced as compared to the control.

Conclusions: These findings support the potential application of CsRAV1-overexpression to increase syllepsis in commercial elite trees without changing their wood quality. However, the syllepsis triggered by the introduction of this genetic modification appeared not to be sufficient to sustain and enhance biomass production.

Keywords: Field trial; Lignocellulosic biomass; Poplar; RAV1; Short-rotation coppice (SRC); Sylleptic branchiness; Sylleptic branching; TEM1; Tree biotechnology.

Figures

Fig. 1
Fig. 1
Field trial establishment, syllepsis of RAV1-engineered poplars and RAV1-protein abundances during the first cultivation cycle. a Image of the field trial once established (July 2012). b Sylleptic branches on the apical segment of the main stem in the representative event CsRAV1 OX#60 (white arrows), as opposed to wild-type (WT), and event PtaRAV1&2 KD#1 (November 2012); bar 10 cm. c Densities of sylleptic branches on the main stem of WT and CsRAV1-overexpression and PtaRAV1&2-knockdown transgenic poplars at the end of the establishment year (December 2012). Bars represent average values ± SE (CsRAV1 OX#60 n = 30, CsRAV1 OX#37 n = 30, WT n = 29, PtaRAV1&2 KD#22 n = 25, PtaRAV1&2 KD#1 n = 30). d Upper panel Western blot of the chestnut transgenic protein CsRAV1 tagged to 3xHA in both CsRAV1-overexpression events tested and the WT. Lower panel Western blot of the poplar endogenous protein PtaRAV1 in all four transgenics and the WT as control. Membranes were stained with Ponceau to ensure equal sample loading
Fig. 2
Fig. 2
Sylleptic branching and shoot resprouting phenotypes of RAV1-engineered poplars during the second cultivation cycle. a Densities of sylleptic branches on the dominant shoots of wild-type (WT) and CsRAV1-overexpression and PtaRAV1&2-knockdown transgenics. Measurements were made in December 2014 at the end of the first growing season after the first coppicing. b Shoot number growing from the remaining 10-cm-long stumps of WT and events CsRAV1 OX and PtaRAV1&2 KD. Scoring was made before a second harvest in December 2015. Bars represent average values ± SE (CsRAV1 OX#60 n = 30, CsRAV1 OX#37 n = 30, WT n = 29, PtaRAV1&2 KD#22 n = 30, PtaRAV1&2 KD#1 n = 30)
Fig. 3
Fig. 3
Wood structure and chemical wood composition of the RAV1-engineered poplars. a Wood histochemistry analyses of branch cross sections (5th internode) obtained from wild-type (WT) trees and representative events 3xHA:CsRAV1 OX#60 and PtaRAV1&2 KD#1. The sections, taken from the side of branches facing the main stem, were sampled after coppicing in December 2013. Left column cellulose detection by Calcofluor white staining. Right column detection of lignin autofluorescence. co cortex, xy xylem, * sclerenchyma bar 100 μm. bf Xylem composition of WT trees and representative events CsRAV1 OX#60 and PtaRAV1&2 KD#1 after coppicing in December 2013, including cP/cH ratio and percentage of levoglucosan, total extractives, Klason lignin content, S/G ratio. Bars represent average values ± SD (CsRAV1 OX#60 n = 4, WT n = 4, PtaRAV1&2 KD#1 n = 4). g Higher calorific values of coppiced biomass obtained from WT trees and events CsRAV1 OX#60 and PtaRAV1&2 KD#1. Bars represent average values ± SD (CsRAV1 OX#60 n = 3, WT n = 3, PtaRAV1&2 KD#1 n = 3)
Fig. 4
Fig. 4
Aboveground biomass yields of the RAV1-engineered poplars after two cultivation cycles. a Picture of the field trial after coppicing in December 2013, showing the 10-cm-long stumps. Dry aerial biomass yields of wild-type (WT) and CsRAV1-overexpression and PtaRAV1&2-knockdown transgenics, after b the first coppicing in December 2013, and c the second coppicing in December 2015. Bars represent average values ± SE (CsRAV1 OX#60 n = 30, CsRAV1 OX#37 n = 30, WT n = 29, PtaRAV1&2 KD#22 n = 30, PtaRAV1&2 KD#1 n = 30). Letters represent significant differences between genotypes (p < 0.05)

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