Maize Transformation Using the Morphogenic Genes Baby Boom and Wuschel2

Jones, Todd J.; Lowe, Keith; Hoerster, George; Anand, Ajith; Wu, Emily; Wang, Ning; Arling, Maren; Lenderts, Brian; Gordon‐Kamm, William

doi:10.1007/978-1-4939-8778-8_6

Cited by 45 publications

(40 citation statements)

References 10 publications

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“…Culture media used for transformations and plant regeneration All media recipes are described by Lowe et al (2016) and Jones et al (2019), with minor modifications to media components as listed in Supplemental Table S-1, with all ingredients and sources listed in Table S-9. For selection, 0.1 mg L −1 imazapyr was present in the somatic embryo formation medium (13329) or 150 mg L −1 G418 was substituted for imazapyr.…”

Section: Methodsmentioning

confidence: 99%

Use of non-integrating Zm-Wus2 vectors to enhance maize transformation

Hoerster

Wang

Ryan

et al. 2020

In Vitro Cell.Dev.Biol.-Plant

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The use of Baby boom (Bbm) and Wuschel2 (Wus2) has made maize transformation more efficient across an increasingly wide range of inbreds. However, the benefits have come with the requirement of excising these transformation helper components to enable plant regeneration, which adds size to the T-DNA, and complexity to the transformation system. A new system with the advantages of smaller size and simplicity for the selectable marker gene-containing T-DNA is described. First, expression of Zm-Wus2 alone driven by the maize Pltp promoter (Zm-Pltp pro), was determined to be sufficient to induce rapid somatic embryo formation from the scutella of maize immature embryos. It was also demonstrated that co-infecting with two strains of Agrobacterium, one with a Wus2 expression cassette, and the other with a combination of both selectable and visual marker cassettes, produced transformed T0 plants that contained only a single copy of the selectable marker T-DNA, without the integration of Wus2. Furthermore, the process was optimized by varying the ratio of the two Agrobacterium strains, and by modulating Wus2 expression to enable high-frequency recovery of selectable marker-containing T0 plants that did not contain Wus2. Several factors may have contributed to this outcome. Wus2 expression in localized cell(s) appeared to stimulate somatic embryogenesis in neighboring cells, including those that had integrated the selectable marker. In addition, in cells in which the Wus2 T-DNA did not integrate but the selectable marker T-DNA did, transient Wus2 expression stimulated somatic embryo formation and regeneration of stable T0 plants that contained the selectable marker. In addition, augmenting the Pltp promoter with three viral enhancer elements to increase Wus2 expression stimulated embryogenesis while precluding their regeneration. The phenomenon has now been designated as "altruistic transformation."

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Section: Methodsmentioning

confidence: 99%

Use of non-integrating Zm-Wus2 vectors to enhance maize transformation

Hoerster

Wang

Ryan

et al. 2020

In Vitro Cell.Dev.Biol.-Plant

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“…As such, maize transformation is largely limited to a few service laboratories in the public sector, which are necessarily rather costly and can take up to 4–6 months to produce a transgenic line. It should be noted that new technologies promise to improve this situation (Jones et al, 2019), but they are not yet in the public domain. The recent availability of CRISPR/Cas9 technology in maize is revolutionizing the field by allowing us to make targeted genome edits (Svitashev et al, 2016; Liu et al, 2020) but is still limited by a relatively inefficient transformation system.…”

Section: Introductionmentioning

confidence: 99%

Use of virus‐induced gene silencing to characterize genes involved in modulating hypersensitive cell death in maize

Murphree

Kim

Karre

et al. 2020

Molecular Plant Pathology

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“…The donor template was positioned between the coding sequence of an Hra gene (selectable marker) and the upstream Sb-Als promoter (Supplementary Table 1), preventing Hra expression in this starting configuration. The construct also contained two morphogenic genes, maize phospholipid transferase gene promoter ( Pltp )-driven Bbm and maize auxin-inducible promoter ( Axig1 )-driven Wus2 34 .…”

Section: Resultsmentioning

confidence: 99%

“…Corteva Agriscience inbred line PH1V69 was grown in greenhouse growth conditions. Ear harvest, immature embryos isolation, Agrobacterium -mediated transformation, and plant regeneration were based on those described by Jones et al (2019) 34 .…”

Section: Methodsmentioning

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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Maize Transformation Using the Morphogenic Genes Baby Boom and Wuschel2

Cited by 45 publications

References 10 publications

Use of non-integrating Zm-Wus2 vectors to enhance maize transformation

Use of non-integrating Zm-Wus2 vectors to enhance maize transformation

Use of virus‐induced gene silencing to characterize genes involved in modulating hypersensitive cell death in maize

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

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