Download P50 IMPROVEMENT IN WHEAT CARBON FLUX FOR INCREASED

Improvement in wheat carbon flux for increased yield and
Harvest Index
Palak Kathiria, Jeremie Diedhiou and Elizabeth-France Marillia
National Research Council-Saskatoon at AAFC Lethbridge
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With an increased global demand due to a growing world population, Canadian
farmers now request more productive and competitive varieties that can efficiently
metabolize more carbon fixed through photosynthesis.
Studies on Arabidopsis and Canola have shown that reduced activity of the
mitochondrial pyruvate dehydrogenase complex kinase (mtPDHK), the negative
regulator of the mt PDH, resulted in an increased carbon flux toward the seed and
higher yield. In addition, the strong correlation between increased mtPDH activity
(through reduced mtPDHK activity) and elevated CO2 fixation translated into a
higher photosynthetic rate and seed filling via increased cellular respiration rates.
We propose here to validate this concept in wheat and increase productivity by
exploiting the effects of reduced mtPDHK activity on enhanced respiration and
increased CO2 fixation for higher photosynthate production (source pull) and
consequent increases in seed size (sink push).
To that end, we designed a gene editing strategy (CRISPR/Cas9 or TALENs) to reduce
the expression of the PDHK gene by creating small mutations (insertion or deletion)
in the coding sequence. A breeding line amenable to microspore transformation &
regeneration of interest to the industry (AC Andrew) was selected for the production
of improved prototype lines with higher yield. This novel molecular, non-GMO,
approach combined with double haploid (DH) technology has the potential to
accelerate the production of genetically improved lines to be transferred into
existing commercial cultivars through breeding and agronomic R&D programs.
Recent developments
A. Increase in wheat somatic embryogenesis by modulation of
epigenetic status:
This project will use a somatic embryogenesis platform for genome editing in wheat.
To achieve a good induction of somatic embryogenesis from wheat microspores,
induction of genes responsible for embryogenesis is critical. Here, we tested
chemicals which can modify the epigenetic status of the genome and in turn
upregulate embryogenesis-related genes. Effect of two such chemicals, Trichostatin
A (TSA) and Sodium Butyrate (NaB) on plant regeneration through somatic
embryogenesis was studied.
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Introduction
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Preliminary results indicated a positive effect of epigenetic modifications on
induction of somatic embryogenesis. We observed an increased number of green
plants produced per unit (100,000 cells) of microspores in presence of TSA and NaB
(Fig. 1). An synergic effect was observed when both TSA and NaB were combined
together. We also obtained a dosage-dependent response when various amounts of
NaB were used with a same amount of TSA (Fig. 2). Finally, the ratio of green to
albino plants varied with various amounts of NaB.
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Our results indicated that the size of the DNA-peptide complex formed varies with
the amount of nanoparticles used. Also, differences in size and stability were
observed among the different classes of transfection agents used (Fig. 3 & 4).
C. Optimization of gene gun-mediated protein bombardment
The gene gun or particle bombardment approach has been successfully used for
delivery of DNA in various plant species. However, the approach has been limited to
delivery of DNA only. Here, we tested the possibility of delivering proteins using the
same approach. Gold particles were coated with mCherry protein and the particles
were retrieved post bombardment on a culture plate.
Results indicated that we successfully coated gold particles with protein (Fig. 5a).
Also, we were able to retrieve gold particles after bombardment (Fig. 5b). Feasibility
of bombarding nucleases as proteins will be further tested.
B. Characterization of nanocomplexes formed with different classes of
transfection agents
We are considering using a Cell Penetrating Peptides (CPP)-mediated transfection
approach for editing the wheat genome. To improve the efficiency of the process,
we analyzed the size of nanocomplexes formed with three different classes of
transfection agents at 0 and 24 hours after formation. A cationic peptide (R9), an
hydrophobic lipid (Lipofectamin2000) and a cationic-hydrophobic fusion peptide
(R3V6R9) were tested. DNA was successfully complexed with various amounts of
transfection agents and the size of nanoparticles were measured using a Zetasizer.
Fig. 5a
Acknowledgements: The authors wish to thank
Fig. 5b