Rapid genotype “independent” Zea mays L. (maize) transformation via direct somatic embryogenesis

Lowe, Keith; Rota, Mauricio La; Hoerster, George; Hastings, Craig; Wang, Ning; Chamberlin, Mark A.; Wu, Emily; Jones, Todd J.; Gordon‐Kamm, William

doi:10.1007/s11627-018-9905-2

Cited by 207 publications

(269 citation statements)

References 38 publications

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“…Based on our transient expression assays, the FBP can function in a broad range of plant species. With the recent development of rapid morphogen mediated plant transgenesis protocols 24 , we believe FBPbased biosensors and reporters will be easy to extend across a range of plants for long term visualization of gene expression. This extensibility to a broad range of plants makes this approach to autoluminescence superior to the existing transplastomic approach 11 , as plastid transformation is challenging and largely limited to tobacco and a few related Solanaceae 25 .…”

Section: Discussionmentioning

confidence: 99%

Building customizable auto-luminescent luciferase-based reporters in plants

Khakhar

Starker

Chamness

et al. 2019

Preprint

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Bioluminescence is a powerful biological signal that scientists have repurposed to design reporters for gene expression in plants and animals. However, there are some downsides associated with the need to provide a substrate to these reporters, such as its high cost and non-uniform tissue penetration. In this work we reconstitute a fungal bioluminescence pathway (FBP) in planta using an easily composable toolbox of parts. We demonstrate that the FBP can create luminescence across various tissues in a broad range of plants without external substrate addition. We also show how our toolbox can be used to deploy the FBP in planta to build auto-luminescent reporters for the study of gene-expression and hormone fluxes. A low-cost imaging platform for gene expression profiling is also described. These experiments lay the groundwork for the future construction of programmable auto-luminescent plant traits, such as creating light driven plant-pollinator interactions or light emitting plant-based sensors.

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Section: Discussionmentioning

confidence: 99%

Building customizable auto-luminescent luciferase-based reporters in plants

Khakhar

Starker

Chamness

et al. 2019

Preprint

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“…Plant tissue culture system is an efficient in vitro based technique used for genetic modification and plant regeneration, and reduces yield loss [15][16][17]. Development of plants with potential improved traits such as superior quality, high yield, better adaptability, disease tolerance and stress resistance is mainly based on micropropagation technique of tissue culture system [18][19][20]. Relatively very poor progress has been accomplished on plant regeneration system in legumes [21], due to the unsuccessful in vitro generation tissue culture systems.…”

Section: Introductionmentioning

confidence: 99%

In Vitro Regeneration Potential of White Lupin (Lupinus albus) from Cotyledonary Nodes

Aslam

Karanja

Zhang

et al. 2020

Plants

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The tissue culture regeneration system of Lupinus albus has always been considered as recalcitrant material due to its genotype-dependent response and low regeneration efficiency that hamper the use of genetic engineering. Establishment of repeatable plant regeneration protocol is a prerequisite tool for successful application of genetic engineering. This aim of this study was to develop standardized, efficient protocol for successful shoot induction from cotyledonary node of white lupin. In this study, 5 day old aseptically cultured seedlings were used to prepare three explants (half cotyledonary node, HCN; whole cotyledonary node, WCN; and traditional cotyledonary node, TCN), cultured on four concentrations of M519 medium (M519, ½ M519, 1/3 M519, and ¼ M519), containing four carbohydrate sources (sucrose, fructose, maltose, and glucose), and stimulated with various combinations of KT (kinetin), and NAA (naphthalene acetic acid) for direct shoot regeneration. High frequency of 80% shoot regeneration was obtained on ½ M519 medium (KT 4.0 mg L−1 + NAA 0.1 mg L−1) by using HCN as an explant. Interestingly, combinations of (KT 4.0 mg L−1 + NAA 0.1 mg L−1 + BAP 1.67 mg L−1), and (KT 2.0 mg L−1 + NAA 0.1 mg L−1) showed similar shoot regeneration frequency of 60%. Augmentation of 0.25 g L−1 activated charcoal (AC) not only reduced browning effect but also improved shoot elongation. Among the all carbohydrate sources, sucrose showed the highest regeneration frequency with HCN. Additionally, 80% rooting frequency was recorded on ½ M519 containing IAA 1.0 mg L−1 + KT 0.1 mg L−1 (indole acetic acid) after 28 days of culturing. The present study describes establishment of an efficient and successful protocol for direct plant regeneration of white lupin from different cotyledonary nodes.

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“…Intra-genomic homologous recombination has previously been demonstrated in whole plants 22,28–30 . Recent improvements in GT frequency have used either different CRISPR systems 44 , an egg cell-specific promoter driving nuclease expression 45 , or sequential transformation 46 . These recent developments have been demonstrated mainly in Arabidopsis .…”

Section: Discussionmentioning

confidence: 99%

“…Based on these observations, we speculate that GT frequency could be increased by extending resting period post-Cas9 induction in the HS11 treatment. Compared to HS4 treatment, explants in HS11 group were enriched for cells containing Cas9 reagents and donor template (potentially 500-1000-fold) 45 due to the additional 7 days of growth prior to Cas9 induction. Refinement of the time interval of Cas9 induction and donor template release followed by stringent selection regime for plant regeneration could further improve GT.…”

Section: Discussionmentioning

confidence: 99%

Efficient Gene Targeting in Maize using Inducible CRISPR-Cas9 and Marker-Free Donor Template

Barone

Lenderts

et al. 2020

Preprint

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10CRISPR-Cas9 is a powerful tool for generating targeted mutations and genomic 11 deletions. However, precise gene insertion or sequence replacement remains a major 12 hurdle before application of CRISPR-Cas9 technology is fully realized in plant 13 breeding. Here we report high frequency, selectable marker-free intra-genomic gene 14 targeting (GT) in maize. Heat shock-inducible Cas9 was used for generating targeted 15 double-strand breaks (DSBs) and simultaneous mobilization of the donor template 16 from pre-integrated T-DNA. The construct was designed such that release of the 17 donor template and subsequent DNA repair activated expression of the selectable 18 marker gene within the donor locus. This approach generated up to 4.7% targeted 19 insertion of the donor sequence into the target locus in T0 plants, with up to 86% 20 detected donor template release and 99% mutation rate were observed at the donor 21 loci and the genomic target site, respectively. Unlike previous in planta or intra-22 genomic homologous recombination reports, that required multiple generations and 23 extensive screening, our method provides non-chimeric, heritable GT in the T0 24 generation. 25 26 involves ligation of non-homologous sequences or sequences with micro-homologies, 34 often resulting in small insertions or deletions (indels) at the DSB site. HR requires a 35 DNA repair template with sequences homologous to those flanking the genomic DSB 36 site, leading to precise repair of the DSB. NHEJ is the primary DNA repair pathway 37 in somatic cells, while HR is observed with much lower frequencies and 38 predominantly occurs during S and G2 phases of the cell cycle 7 . 39 CRISPR-Cas9-mediated genome editing can be used to improve agronomic 40 characteristics, introduce targeted disease resistance [8][9][10][11][12] , and speed-up domestication 41 for development of new beneficial plant varieties 13 . Some traits that depend on 42 endogenous gene mutation can be generated through NHEJ-mediated DNA repair 14-43 18 . Other traits require precise genome editing and need gene targeting (GT) via HR to 44 make large insertions or allele replacements 19,20 . While high frequency NHEJ-45 mediated gene mutations (30-100%) have been obtained in plants 21 , precise GT via 46 HR remains exceedingly low and a major improvement is needed for routine 47 application. Targeted mutagenesis relies on NHEJ-mediated repair of DSBs without 48 any repair template, and therefore has become routine in plants that are amenable to 49 transformation and regeneration. HR-mediated GT requires simultaneous creation of 50 targeted DSB(s) and supply of a donor DNA repair template containing flanking 51 regions of homology (also referred to as homology arms) 22 . While targeted DSBs can 52 render a desired genomic site accessible to a donor repair template, the DSB does not 53 prevent ectopic integration of a donor sequence elsewhere in the genome. Therefore, 54 high frequency random integration of donor template exacerbated by limitations in 55 transformatio...

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Rapid genotype “independent” Zea mays L. (maize) transformation via direct somatic embryogenesis

Cited by 207 publications

References 38 publications

Building customizable auto-luminescent luciferase-based reporters in plants

Building customizable auto-luminescent luciferase-based reporters in plants

In Vitro Regeneration Potential of White Lupin (Lupinus albus) from Cotyledonary Nodes

Efficient Gene Targeting in Maize using Inducible CRISPR-Cas9 and Marker-Free Donor Template

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