Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants

Che, Ping; Chang, Shujun; Simon, Marissa; Zhang, Zhifen; Shaharyar, Ahmed; Ourada, Jesse; O’Neill, Dennis; Torres‐Mendoza, Mijael; Guo, Yinping; Marasigan, Kathleen Monfero; Vielle‐Calzada, Jean‐Philippe; Ozias‐Akins, Peggy; Albertsen, Marc C.; Jones, Todd J.

doi:10.1111/tpj.15202

Cited by 56 publications

(43 citation statements)

References 52 publications

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“…Cowpea is known to be recalcitrant for transformation, hindering routine practices of CRISPR/Cas9. However, recent advances have demonstrated improved transformation efficiencies of 4.5–37% across nine cowpea genotypes (Che et al, 2021). In this work, Che et al used a CRISPR/Cas9 construct that expresses Cas9 under the soybean elongation factor (GmEF1A2) promoter and a gRNA under the VuU6 promoter to produce 35 T1 plants, of which 68.8% showed editing activity.…”

Section: Crispr/cas9 For Crop Improvement In Legumesmentioning

confidence: 99%

CRISPR/Cas9 gene editing in legume crops: Opportunities and challenges

et al. 2021

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Legumes are an excellent source of proteins and health‐promoting phytochemicals. Recognizing their importance in human nutrition and sustainable agricultural production, significant efforts are currently being made to accelerate genetic gain related to yield, stress resilience, and nutritional quality. Recent increases in genomic resources for multiple legume crops have laid a solid foundation for application of transformative breeding technologies such as genomic selection and genome editing for crop improvement. In this review, we focus on the recent plant‐specific advances in CRISPR/Cas9‐based gene editing technology and discuss the challenges and opportunities to harnessing this innovative technology for targeted improvement of traits in legume crops. Gene‐editing methods have been successfully established for soybean, cowpea, chickpea, and model legumes such as Medicago truncatula and Lotus japonicus. However, the recalcitrance of other legumes to in vitro gene transfer and regeneration has posed a serious challenge to application of gene editing. We discuss various modifications to in vitro culture methods, in terms of the choice of explant, media composition, and DNA delivery and gene‐editing detection methods that can potentially improve the rate of transformation and regeneration of whole plant in legume crops. Although gene‐editing technology can bring enormous benefits to legume breeding, regulatory hurdles are a cause for serious concern. We compare the regulatory environments existing in the European Union and the United States of America. A favorable regulatory framework and public acceptance are important factors in realizing CRISPR's potential benefits to global food security.

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Section: Crispr/cas9 For Crop Improvement In Legumesmentioning

confidence: 99%

CRISPR/Cas9 gene editing in legume crops: Opportunities and challenges

et al. 2021

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“…1c). The cassettes AtDD45 pro Cre and AtUbq10 pro lox were transferred respectively into the T-DNA region of the binary vector pAGM4673 (Weber et al 2011), together with a spectinomycin resistance cassette (Che et al 2021) (Table S1). Considering that the 35S enhancer in the spectinomycin resistance cassette might interact with the AtDD45 pro in the cassette AtDD45 pro Cre and affect its tissuespeci city, a 2 kb transformation booster sequence (TBS) (Singer et al 2011) was placed between the cassette AtDD45 pro Cre and the spectinomycin resistance cassette (Table S1).…”

Section: Cre and Lox-reporter Gene Constructsmentioning

confidence: 99%

“…The cassettes AtDD45 pro Cre and AtUbq10 pro lox were introduced into cowpea using Method 3 (Che et al 2021) given its higher e ciency in recovering transgenic plants. Plantlets derived from different cotyledonary-node explants were independent, while those from the same explants were considered as potentially the same lines.…”

Section: Agrobacterium Inoculum and Plant Transformationmentioning

confidence: 99%

“…The increase in cowpea production and consumption has been considerable (Singh 2014;FAOSTAT 2018) and improvement in cowpea varieties has been made in the last several decades (Boukar et al 2020;Singh 2014). Cowpea improvement likely will accelerate thanks to the availability of new genomic and transcriptomic resources (Huynh et al 2018;Lonardi et al 2019;Muñoz-Amatriaín et al 2017;Spriggs et al 2018;Yao et al 2016) as well as an e cient transformation method (Che et al 2021) that enables transgene introduction and genome editing. This study demonstrated the e cacy of the Cre/lox system for gene activation in cowpea with Cre controlled by two promoters AtRps5a pro and AtDD45 pro .…”

Section: Introductionmentioning

confidence: 99%

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Gene Activation via Cre/lox-mediated Excision in Cowpea (Vigna unguiculata)

Zhang¹,

Guo²,

Marasigan³

et al. 2021

Preprint

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Genetic engineering ideally results in precise spatiotemporal control of transgene expression. To activate transgenes exclusively in a hybrid upon fertilization, we evaluated a Cre/lox-mediated gene activation system with the Cre recombinase expressed by either AtRps5a or AtDD45 promoters that showed activity in egg cells and young embryos. In crosses between Cre recombinase lines and transgenic lines harboring a lox-excision reporter cassette with ZsGreen driven by the AtUbq3 promoter after Cre/lox-mediated recombination, we observed complete excision of the lox-flanked intervening DNA sequence between the AtUbq3pro and the ZsGreen coding sequence in F1 progeny upon genotyping but no ZsGreen expression in F1 seeds or seedlings. The incapability to observe ZsGreen fluorescence was attributed to the activity of the AtUbq3pro. Strong ZsGreen expression in F1 seeds was observed after recombination when ZsGreen was driven by the AtUbq10 promoter. Using the AtDD45pro to express Cre resulted in more variation in recombination frequencies between transgenic lines and crosses. Regardless of the promoter used to regulate Cre, mosaic F1 progeny were rare, suggesting gene activation at an early embryo-developmental stage. Observation of ZsGreen-expressing tobacco embryos at the globular stage from crosses with the AtRps5aproCre lines pollinated by the AtUbq3prolox line supported the early activation mode.

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“…e SE pathway is generally accepted to be the better route for plant regeneration since by its very nature, it tends to lead to the production of a greater number of regenerates compared to the organogenesis pathway [13,14]. Furthermore, in genetic engineering experiments, it is important to avoid the development of chimeric plants and any possible escapes [15]. Regeneration via SE can help to achieve this ideal state as the regenerated plantlets would have originated from a single cell [12].…”

Section: Introductionmentioning

confidence: 99%

Induction of Somatic Embryogenesis and Organogenesis in Zimbabwean Sweet Potato (cv Brondal)

Masekesa

Gasura

Kujeke

et al. 2021

Advances in Agriculture

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Somatic embryogenesis (SE) and organogenesis are crucial in the development of disease free plants and genetic engineering. An investigation was conducted on the ability of treatments containing a combination of 2,4-D and Kinetin to induce either SE or organogenesis from cultured sweet potato cv Brondal leaves. Ten treatments were evaluated and each treatment had an exclusive combination of 2,4-D (at 0.05, 0.1, 0.2, 0.5 or 1 mg/L) to kinetin (at either 0.1 or 0.5 mg/L). Callus initiation occurred earlier in treatments containing higher hormonal concentrations. The 2,4-D to Kinetin ratio had a highly significant ( p = 0.001 ) effect on callus growth and proliferation. Increasing 2,4-D to Kinetin ratio promoted profuse explant callusing while increasing Kinetin to 2,4-D ratio suppressed callusing but encouraged organogenesis, in particular shoot production (treatment containing 0.05 mg/L 2,4-D and 0.5 mg/L Kinetin). Embryogenic calli were formed seven weeks after leaf culture in the treatment containing 0.5 mg/L 2,4-D and 0.1 mg/L Kinetin. The embryogenic calli that developed from this treatment emerged from previously nonembryogenic calli. Plantlets produced via the SE pathway proved to be weak and unviable and died within four weeks of germination. In contrast, plantlets produced under organogenesis were strong, grew vigorously, and could be subcultured several times. This disparity may be accounted for by the fact that the cv Brondal embryos that developed under SE were not exposed to an embryo maturation staged before plantlet germination was initiated. The maturation stage would have assisted embryos to reach physiological maturity and a desired level of desiccation, both being critical elements in embryo to plantlet conversion. In this experiment, cv Brondal regeneration from leaf explants was successfully achieved via organogenesis using 0.05 mg/L 2,4-D and 0.5 mg/L Kinetin, and tentative steps towards development of SE based regeneration protocol were established using 0.5 mg/L 2,4-D and 0.1 mg/L Kinetin.

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Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants

Cited by 56 publications

References 52 publications

CRISPR/Cas9 gene editing in legume crops: Opportunities and challenges

CRISPR/Cas9 gene editing in legume crops: Opportunities and challenges

Gene Activation via Cre/lox-mediated Excision in Cowpea (Vigna unguiculata)

Induction of Somatic Embryogenesis and Organogenesis in Zimbabwean Sweet Potato (cv Brondal)

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