Poplar Transformation

Brügmann, Tobias; Polak, Olaf; Deecke, Khira; Nietsch, Julia; Fladung, Matthias

doi:10.1007/978-1-4939-8778-8_12

Cited by 10 publications

(8 citation statements)

References 29 publications

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“…Agrobacterium -mediated genetic transformations of poplar plants were performed by applying a leaf-disc transformation protocol [58] or with an advanced leaf disc method as described in Bruegmann et al [59]. In summary, for the latter, plant tissue was infected with Agrobacterium and washed with Cefotaxime (500 mg/L, Duchefa Biochemie) and cultivated on WPM medium as described in Fladung et al [58] and Bruegmann et al [59], with thidiazuron added to a final concentration of 0.0022 mg/L ([60], Duchefa Biochemie). For selection, the regeneration medium was supplemented with Cefotaxime (500 mg/L, Duchefa Biochemie) and kanamycin (50 mg/L, Duchefa Biochemie) or hygromycin (20 mg/L, Duchefa Biochemie), depending on the selection marker gene.…”

Section: Methodsmentioning

confidence: 99%

Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

Brügmann¹,

Deecke²,

Fladung³

2019

IJMS

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CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.

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Section: Methodsmentioning

confidence: 99%

Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

Brügmann¹,

Deecke²,

Fladung³

2019

IJMS

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“…Plant transformation was carried out by applying the Agrobacterium-mediated leaf-disc co-cultivation method as described in [28,29,61]. Leaf-discs of in vitro grown P1-plants were harvested and soaked with Agrobacterium tumefaciens strain GV3101::pMP90RK (chromosomal background C58) carrying the CRISPR/Cas9 system (35S-driven Cas9 nuclease and gRNAs: C672p9ioR-35sCasWT-Nuc, Supplementary Materials Figure S1).…”

Section: Guide Rna (Grna) Design and Agrobacterium-mediated Poplar Tr...mentioning

confidence: 99%

Targeted CRISPR/Cas9-Based Knock-Out of the Rice Orthologs TILLER ANGLE CONTROL 1 (TAC1) in Poplar Induces Erect Leaf Habit and Shoot Growth

Fladung

2021

Forests

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Pyramidal-, erect- or upright-growing plant forms are characterized by narrow branch angles of shoots and leaves. The putative advantage of upright-leaf and shoot habit could be a more efficient penetration of light into lower canopy layers. Pyramidal genotypes have already been reported for various tree genotypes including peach. The paralogous rice ortholog TILLER ANGLE CONTROL 1 (TAC1) has been proposed to be the responsible gene for upright growth. However, it has not really been demonstrated for any of the pyramidal tree genotypes that a knock-out mutation of the TAC1 gene is causing pyramidal plant growth. By in silico analyses, we have identified a putative rice TAC1 ortholog (Potri.014G102600, “TAC-14”) and its paralog (Potri.002G175300, “TAC-2”) in the genome of P. trichocarpa. Two putative PcTAC1 orthologs in the P. × canescens clone INRA 717-1B4 were successfully knocked-out by applying a transgenic CRISPR/Cas9-approach. The mutants were molecularly analyzed and phenotyped over a period of three years in a glasshouse. Our results indicate that the homozygous knock-out of “TAC-14” is sufficient to induce pyramidal plant growth in P. × canescens. If up to twice as many pyramidal individuals were planted on short rotation coppices (SRCs), this could lead to higher wood yield, without any breeding, simply by increasing the number of trees on a default field size.

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“…The leaves from 40-day-old tissue-cultured ‘shanxin’ seedlings were used as the transformation acceptor plantlets. The 35S::lncWOX11a plasmid was introduced into EHA105 and used for the genetic transformation of hybrid poplar ‘Shanxin’ using the Agrobacterium -based leaf disc method [ 57 , 58 ]. The OE lines were obtained after screening in MS medium with kanamycin.…”

Section: Methodsmentioning

confidence: 99%

Long Non-Coding RNA lncWOX11a Suppresses Adventitious Root Formation of Poplar by Regulating the Expression of PeWOX11a

Ran

Liu

et al. 2023

IJMS

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Long non-coding RNAs (lncRNAs), a class of poorly conserved transcripts without protein-encoding ability, are widely involved in plant organogenesis and stress responses by mediating the transmission and expression of genetic information at the transcriptional, posttranscriptional, and epigenetic levels. Here, we cloned and characterized a novel lncRNA molecule through sequence alignment, Sanger sequencing, transient expression in protoplasts, and genetic transformation in poplar. lncWOX11a is a 215 bp transcript located on poplar chromosome 13, ~50 kbp upstream of PeWOX11a on the reverse strand, and the lncRNA may fold into a series of complex stem–loop structures. Despite the small open reading frame (sORF) of 51 bp within lncWOX11a, bioinformatics analysis and protoplast transfection revealed that lncWOX11a has no protein-coding ability. The overexpression of lncWOX11a led to a decrease in the quantity of adventitious roots on the cuttings of transgenic poplars. Further, cis-regulatory module prediction and CRISPR/Cas9 knockout experiments with poplar protoplasts demonstrated that lncWOX11a acts as a negative regulator of adventitious rooting by downregulating the WUSCHEL-related homeobox gene WOX11, which is supposed to activate adventitious root development in plants. Collectively, our findings imply that lncWOX11a is essential for modulating the formation and development of adventitious roots.

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Poplar Transformation

Cited by 10 publications

References 29 publications

Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

Targeted CRISPR/Cas9-Based Knock-Out of the Rice Orthologs TILLER ANGLE CONTROL 1 (TAC1) in Poplar Induces Erect Leaf Habit and Shoot Growth

Long Non-Coding RNA lncWOX11a Suppresses Adventitious Root Formation of Poplar by Regulating the Expression of PeWOX11a

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