&lt;em&gt;Agrobacterium&lt;/em&gt;-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Masters, Alicia; Kang, Minjeong; McCaw, Morgan; Zobrist, Jacob D.; Gordon‐Kamm, William; Jones, Todd J.; Wang, Kan

doi:10.3791/60782

Cited by 27 publications

(44 citation statements)

References 6 publications

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“…The model species Arabidopsis thaliana can be easily transformed via the floral dip method with Agrobacteria delivery of genetic material, a process which can generate stable transgenic seed within 3-5 weeks [12]. In contrast, maize transformation methods are more laborious and time consuming, taking up to 6 months after the transformation event to generate transgenic seed [13][14][15][16][17]. Typically, immature embryo-derived callus tissue is transformed either by particle bombardment or Agrobacterium-mediated delivery of transgenes and both processes have a transformation rate efficiency of about 12-30% [13].…”

Section: Introductionmentioning

confidence: 99%

A CRISPR/dCas9 toolkit for functional analysis of maize genes

et al. 2020

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Background The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9). Results In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression. Conclusions We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.

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Section: Introductionmentioning

confidence: 99%

A CRISPR/dCas9 toolkit for functional analysis of maize genes

et al. 2020

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“…The model species Arabidopsis thaliana can be easily transformed via the oral dip method with Agrobacteria delivery of genetic material, a process which can generate stable transgenic seed within 3-5 weeks [12]. In contrast, maize transformation methods are more laborious and time consuming, taking up to 6 months after the transformation event to generate transgenic seed [13][14][15][16][17] . Typically, immature embryo-derived callus tissue is transformed either by particle bombardment or Agrobacterium-mediated delivery of transgenes and both processes have a transformation rate e ciency of about 12-30% [13].…”

Section: Introductionmentioning

confidence: 99%

A CRISPR/dCas9 toolkit for functional analysis of maize genes

Gentzel

Park

Bellizzi

et al. 2020

Preprint

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Background: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9). Results: In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression. Conclusions: We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.

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“…FFMM-AT transformation experiments for delivery of the construct carrying CRISPR reagents were carried out using standard transformation protocol similar to Hi-II genotype described previously (Wang and Frame, 2004 ) with modifications. Briefly, the media of Wang and Frame ( 2004 ) were replaced with the following media ( Supplementary Material ): Liquid Infection Medium (700A), Cocultivation Medium (562V), Callus Development Medium (605J or 605T), Selection Media I and II, and Shoot Formation Medium (289O plus 3 mg/L bialaphos) per Lowe et al ( 2016 ), Jones et al ( 2019 ) and Masters et al ( 2020 ).…”

Section: Methodsmentioning

confidence: 99%

“…FFMM plants are short enough to grow on stackable shelves with a proper lighting system. The original FFMM lines are not capable of genetic transformation by traditional protocols, though they work very well with the QuickCorn Babyboom/Wuschel morphogenic genes technology (Lowe et al, 2016(Lowe et al, , 2018Jones et al, 2019;Masters et al, 2020). This morphogenic gene technology, while effective, does have some limitations such as large construct size and restrictive licensing options.…”

Section: Introductionmentioning

confidence: 99%

Development of a Transformable Fast-Flowering Mini-Maize as a Tool for Maize Gene Editing

et al. 2021

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Maize (Zea mays ssp. mays) is a popular genetic model due to its ease of crossing, well-established toolkits, and its status as a major global food crop. Recent technology developments for precise manipulation of the genome are further impacting both basic biological research and biotechnological application in agriculture. Crop gene editing often requires a process of genetic transformation in which the editing reagents are introduced into plant cells. In maize, this procedure is well-established for a limited number of public lines that are amenable for genetic transformation. Fast-Flowering Mini-Maize (FFMM) lines A and B were recently developed as an open-source tool for maize research by reducing the space requirements and the generation time. Neither line of FFMM were competent for genetic transformation using traditional protocols, a necessity to its status as a complete toolkit for public maize genetic research. Here we report the development of new lines of FFMM that have been bred for amenability to genetic transformation. By hybridizing a transformable maize genotype high Type-II callus parent A (Hi-II A) with line A of FFMM, we introgressed the ability to form embryogenic callus from Hi-II A into the FFMM-A genetic background. Through multiple generations of iterative self-hybridization or doubled-haploid method, we established maize lines that have a strong ability to produce embryogenic callus from immature embryos and maintain resemblance to FFMM-A in flowering time and stature. Using an Agrobacterium-mediated standard transformation method, we successfully introduced the CRISPR-Cas9 reagents into immature embryos and generated transgenic and mutant lines displaying the expected mutant phenotypes and genotypes. The transformation frequencies of the tested genotypes, defined as the numbers of transgenic event producing T1 seeds per 100 infected embryos, ranged from 0 to 17.1%. Approximately 80% of transgenic plants analyzed in this study showed various mutation patterns at the target site. The transformable FFMM line, FFMM-AT, can serve as a useful genetic and genomic resource for the maize community.

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<em>Agrobacterium</em>-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Cited by 27 publications

References 6 publications

A CRISPR/dCas9 toolkit for functional analysis of maize genes

A CRISPR/dCas9 toolkit for functional analysis of maize genes

A CRISPR/dCas9 toolkit for functional analysis of maize genes

Development of a Transformable Fast-Flowering Mini-Maize as a Tool for Maize Gene Editing

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