Lolium perenne engineered for elevated leaf lipids exhibits greater energy density in field canopies under defoliation

Beechey-Gradwell, Zac; Kadam, Suhas; Bryan, Gregory T.; Cooney, Luke J.; Nelson, Kelly A.; Richardson, Kim; Cookson, Ruth; Winichayakul, Somrutai; Reid, Michele; Anderson, Philip W.; Crowther, T. C.; Zou, Xiuying; Maher, Dorothy; Xue, Hong; Scott, Richard W.; Allan, Anne E.; Stewart, Alan V.; Nj, Roberts

doi:10.1016/j.fcr.2021.108340

Cited by 14 publications

(26 citation statements)

References 44 publications

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“…Comparing the efficiency of the transformation protocol presented here with those reported by others ( Supplementary Table S1 , [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ]) is challenging. Transformation efficiencies have been assessed using different methods.…”

Section: Discussionmentioning

confidence: 53%

“…Genetic transformation of crops (unlike the model species Arabidopsis thaliana (L.) Heyhn) requires an obligatory tissue culture step that represents a significant hurdle in many plant species, perennial ryegrass included [ 12 ]. Substantial efforts were invested in the genetic transformation of perennial ryegrass, as shown by the numerous publications reported by multiple independent research groups over the last three decades, which are summarized in Supplementary Table S1 [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ]. Unfortunately, despite the availability of such a large number of reports and steady progress in this field, the overarching problem remains that Lolium transformation is a technically complicated and time-consuming procedure.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)

Grogg

Rohner

Yates

et al. 2022

Plants

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Genetic transformation of perennial ryegrass (Lolium perenne L.) is critical for fundamental and translational research in this important grass species. It often relies on Agrobacterium-mediated transformation of callus tissue. However, callus induction is restricted to a few genotypes that respond well to tissue culture. Here, we report callus induction from different perennial ryegrass genotypes and explants, such as shoot tips, seeds, and anthers, which were transformed with several plasmids for functional genomics. β-glucuronidase (GUS) histochemical staining showed the LmdsRNAbp promoter sequence was active in stigmas, spikelets, anthers, and leaves. We also transformed calli with plasmids allowing gene silencing and gene knock-out using RNA interference and CRISPR/Cas9, respectively, for which genotypic and phenotypic investigations are ongoing. Using 19 different constructs, 262 transgenic events were regenerated. Moreover, the protocol regenerated a doubled haploid transgenic event from anther-derived calli. This work provides a proof-of-concept method for expanding the range of genotypes amenable to transformation, thus, serving research and breeding initiatives to improve this important grass crop for forage and recreation.

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Section: Discussionmentioning

confidence: 53%

Section: Introductionmentioning

confidence: 99%

Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)

Grogg

Rohner

Yates

et al. 2022

Plants

View full text Add to dashboard Cite

show abstract

“…Finally, the two-fold increase in leaf total lipid content that we achieve here, without inserting foreign DNA, is likely sufficient to significantly enhance livestock productivity and reduce enteric methane emissions in pasture-based ruminant farming systems, if translated to forage species. Evidence for this has been provided using a GMO approach to enhance oil content in perennial ryegrass ( Lolium perenne ) (7,8).…”

Section: Discussionmentioning

confidence: 99%

“…We engineered leaves to accumulate oil as a proof-of-concept. This is a synthetic trait with the potential to deliver a step change in crop oil yield (5,6), and to increase livestock productivity and suppress enteric methane emissions in pasture-based ruminant farming (7,8).…”

Section: Introductionmentioning

confidence: 99%

Oil accumulation in leaves driven by a native promoter-gene fusion created using CRISPR/Cas9 mediated genomic deletion

Bhunia

Ménard

Eastmond

2022

Preprint

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Achieving gain-of-function phenotypes without inserting foreign DNA is an important challenge for plant biotechnologists. Here we show that a gene can be brought under the control of a promoter from an upstream gene by deleting the intervening genomic sequence using dual-guide CRISPR/Cas9. We fuse the promoter of a non-essential photosynthesis-related gene to DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) to drive oil biosynthesis in leaves of a lipase deficient sugar-dependent 1 mutant. DGAT2 expression is enhanced more than twenty-fold and the triacylglycerol content increases by around thirty-fold. This strategy offers a transgene-free route to engineering gain-of-function traits such as high lipid forage to increase the productivity and sustainability of ruminant farming.

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“…This is a synthetic trait with the potential to deliver a step change in crop oil yield [7,8]. Even modest increases in leaf oil content, in the order of a few per cent of dry weight, can significantly increase livestock productivity and suppress enteric methane emissions in pasture-based ruminant farming [9,10].…”

mentioning

confidence: 99%

A native promoter–gene fusion created by CRISPR/Cas9‐mediated genomic deletion offers a transgene‐free method to drive oil accumulation in leaves

2022

View full text Add to dashboard Cite

Achieving gain‐of‐function phenotypes without inserting foreign DNA is an important challenge for plant biotechnologists. Here, we show that a gene can be brought under the control of a promoter from an upstream gene by deleting the intervening genomic sequence using dual‐guide CRISPR/Cas9. We fuse the promoter of a nonessential photosynthesis‐related gene to DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) in the lipase‐deficient sugar‐dependent 1 mutant of Arabidopsis thaliana to drive ectopic oil accumulation in leaves. DGAT2 expression is enhanced more than 20‐fold and the triacylglycerol content increases by around 30‐fold. This deletion strategy offers a transgene‐free route to engineering traits that rely on transcriptional gain‐of‐function, such as producing high lipid forage to increase the productivity and sustainability of ruminant farming.

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Lolium perenne engineered for elevated leaf lipids exhibits greater energy density in field canopies under defoliation

Cited by 14 publications

References 44 publications

Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)

Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)

Oil accumulation in leaves driven by a native promoter-gene fusion created using CRISPR/Cas9 mediated genomic deletion

A native promoter–gene fusion created by CRISPR/Cas9‐mediated genomic deletion offers a transgene‐free method to drive oil accumulation in leaves

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