Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum

For many important crops including sorghum, the use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but more critically, by the ability to regenerate a fertile plant from the engineered cell through conventional tissue culture methods. Here, we report that Wuschel2 (Wus2)-enabled sorghum (Sorghum bicolor L.) transformation increases not only the efficiency of transgenic plant recovery, but also increases the observed efficiency of CRISPR/Cas-targeted genome editing within the population of T0 plants. Using Agrobacterium-mediated transformation, we have demonstrated Wus2-induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased regeneration capacity, resulting in higher transformation efficiency across different sorghum genotypes. Subsequently, advanced excision systems and "altruistic" transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrated up to an additional 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2-enabled, Agrobacterium-mediated transformation (compared to the control treatment with no Wus2), across various targeted loci in different sorghum genotypes. Wus2-enabled genome editing may be applicable to other crops and increasingly popular in planta transformation strategies.

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Optimization of protoplast regeneration in the model plant Arabidopsis thaliana

Plant Methods ◽

10.1186/s13007-021-00720-x ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Yeong Yeop Jeong ◽

Hun-Young Lee ◽

Suk Weon Kim ◽

Yoo-Sun Noh ◽

Pil Joon Seo

Keyword(s):

Tissue Culture ◽

Arabidopsis Thaliana ◽

Plant Regeneration ◽

Tissue Regeneration ◽

De Novo ◽

Callus Formation ◽

Protoplast Regeneration ◽

Culture Methods ◽

Cell And Tissue Culture

Abstract Background Plants have a remarkable reprogramming potential, which facilitates plant regeneration, especially from a single cell. Protoplasts have the ability to form a cell wall and undergo cell division, allowing whole plant regeneration. With the growing need for protoplast regeneration in genetic engineering and genome editing, fundamental studies that enhance our understanding of cell cycle re-entry, pluripotency acquisition, and de novo tissue regeneration are essential. To conduct these studies, a reproducible and efficient protoplast regeneration method using model plants is necessary. Results Here, we optimized cell and tissue culture methods for improving protoplast regeneration efficiency in Arabidopsis thaliana. Protoplasts were isolated from whole seedlings of four different Arabidopsis ecotypes including Columbia (Col-0), Wassilewskija (Ws-2), Nossen (No-0), and HR (HR-10). Among these ecotypes, Ws-2 showed the highest potential for protoplast regeneration. A modified thin alginate layer was applied to the protoplast culture at an optimal density of 1 × 106 protoplasts/mL. Following callus formation and de novo shoot regeneration, the regenerated inflorescence stems were used for de novo root organogenesis. The entire protoplast regeneration process was completed within 15 weeks. The in vitro regenerated plants were fertile and produced morphologically normal progenies. Conclusion The cell and tissue culture system optimized in this study for protoplast regeneration is efficient and reproducible. This method of Arabidopsis protoplast regeneration can be used for fundamental studies on pluripotency establishment and de novo tissue regeneration.

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Tissue culture-based Agrobacterium-mediated and in planta transformation methods

Czech Journal of Genetics and Plant Breeding ◽

10.17221/177/2016-cjgpb ◽

2017 ◽

Vol 53 (No. 4) ◽

pp. 133-143 ◽

Cited By ~ 8

Author(s):

M. Niazian ◽

S.A. Sadat Noori ◽

P. Galuszka ◽

S.M.M. Mortazavian

Keyword(s):

Tissue Culture ◽

Transformation Method ◽

Gene Transformation ◽

In Planta Transformation ◽

In Planta ◽

Agrobacterium Mediated Transformation ◽

Advantages And Disadvantages ◽

Culture Independent ◽

Transformation Methods ◽

Mutant Lines

Gene transformation can be done in direct and indirect (Agrobacterium-mediated) ways. The most efficient method of gene transformation to date is Agrobacterium-mediated method. The main problem of Agrobacterium-method is that some plant species and mutant lines are recalcitrant to regeneration. Requirements for sterile conditions for plant regeneration are another problem of Agrobacterium-mediated transformation. Development of genotype-independent gene transformation method is of great interest in many plants. Some tissue culture-independent Agrobacterium-mediated gene transformation methods are reported in individual plants and crops. Generally, these methods are called in planta gene transformation. In planta transformation methods are free from somaclonal variation and easier, quicker, and simpler than tissue culture-based transformation methods. Vacuum infiltration, injection of Agrobacterium culture to plant tissues, pollen-tube pathway, floral dip and floral spray are the main methods of in planta transformation. Each of these methods has its own advantages and disadvantages. Simplicity and reliability are the primary reasons for the popularity of the in planta methods. These methods are much quicker than regular tissue culture-based Agrobacterium-mediated gene transformation and success can be achieved by non-experts. In the present review, we highlight all methods of in planta transformation comparing them with regular tissue culture-based Agrobacterium-mediated transformation methods and then recently successful transformations using these methods are presented.

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A Rationally Designed Bovine IgA Fc Scaffold Enhances in planta Accumulation of a VHH-Fc Fusion Without Compromising Binding to Enterohemorrhagic E. coli

Frontiers in Plant Science ◽

10.3389/fpls.2021.651262 ◽

2021 ◽

Vol 12 ◽

Author(s):

Adam Chin-Fatt ◽

Reza Saberianfar ◽

Rima Menassa

Keyword(s):

Disulfide Bonds ◽

Rational Design ◽

De Novo ◽

Passive Immunization ◽

Fold Increase ◽

Secretory Component ◽

Secretory Iga ◽

In Planta ◽

Design Strategies ◽

Single Domain Antibody

We previously isolated a single domain antibody (VHH) that binds Enterohemorrhagic Escherichia coli (EHEC) with the end-goal being the enteromucosal passive immunization of cattle herds. To improve the yield of a chimeric fusion of the VHH with an IgA Fc, we employed two rational design strategies, supercharging and introducing de novo disulfide bonds, on the bovine IgA Fc component of the chimera. After mutagenizing the Fc, we screened for accumulation levels after transient transformation in Nicotiana benthamiana leaves. We identified and characterized five supercharging and one disulfide mutant, termed ‘(5 + 1)Fc’, that improve accumulation in comparison to the native Fc. Combining all these mutations is associated with a 32-fold increase of accumulation for the Fc alone, from 23.9 mg/kg fresh weight (FW) to 599.5 mg/kg FW, as well as a twenty-fold increase when fused to a VHH that binds EHEC, from 12.5 mg/kg FW tissue to 236.2 mg/kg FW. Co-expression of native or mutated VHH-Fc with bovine joining chain (JC) and bovine secretory component (SC) followed by co-immunoprecipitation suggests that the stabilizing mutations do not interfere with the capacity of VHH-Fc to assemble with JC and FC into a secretory IgA. Both the native and the mutated VHH-Fc similarly neutralized the ability of four of the seven most prevalent EHEC strains (O157:H7, O26:H11, O111:Hnm, O145:Hnm, O45:H2, O121:H19 and O103:H2), to adhere to HEp-2 cells as visualized by immunofluorescence microscopy and quantified by fluorometry. These results collectively suggest that supercharging and disulfide bond tethering on a Fc chain can effectively improve accumulation of a VHH-Fc fusion without impacting VHH functionality.

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New Technologies and Strategies for Grapevine Breeding Through Genetic Transformation

Frontiers in Plant Science ◽

10.3389/fpls.2021.767522 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gabriela Campos ◽

Constanza Chialva ◽

Silvana Miras ◽

Diego Lijavetzky

Keyword(s):

Tissue Culture ◽

Transgenic Plants ◽

Genetic Transformation ◽

New Technologies ◽

De Novo ◽

Culture Media ◽

Ectopic Expression ◽

Attractive Alternative ◽

Culture Methods ◽

Woody Perennials

Grapevine, as other woody perennials, has been considered a recalcitrant crop to produce transgenic plants. Since the production of transgenic and/or edited plants requires the ability to regenerate plants from transformed tissues, this step is often the biggest bottleneck in the process. The objective of this work is to review the state of the art technologies and strategies for the improvement of grapevine transformation and regeneration, focusing on three aspects: (i) problems associated with grapevine transformation; (ii) genes that promote grapevine regeneration; and (iii) vehicles for gene delivery. Concerning the first aspect, it is well documented that one of the main factors explaining the low success rate in obtaining transgenic plants is the regeneration process. After transgenic integration into receptor cells, tissue culture is required to regenerate transgenic seedlings from transformed cells. This process is time consuming and often requires the addition of environmentally damaging reagents (antibiotics and herbicides) to the culture medium to select transgenic plants. On the other hand, the expression of genes such as the so-called developmental regulators (DR), which induce specific development programs, can be used to avoid traditional tissue culture methods. The ectopic expression of specific combinations of DR in somatic cells has the potential to induce de novo meristems in diverse crops, including grapevine. Successful genome editing by de novo reprogramming of plant meristems in somatic tissues has been reported. Moreover, it has been shown that the expression of certain transcription factors can increase the regeneration efficiency in wheat, citrus, and rice. Finally, recent reports showed the use of nanoparticles, such as carbon dots (CDs), as an attractive alternative to Agrobacterium- and biolistic-mediated plant genetic transformation. In this way, the use of antibiotics in culture media is avoided, overcoming the loss of viability of plant tissues and accelerating the regeneration processes. It has been shown that CDs can act as a vehicle to transport plasmids to plant cells in transient transformation in several crops without negative impacts on photosynthesis or growth. Based on these advances, it is possible to combine these new available strategies and technologies to overcome the regeneration problems of species such as grapevine and other crops considered as recalcitrant.

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556 In Vitro Anther Culture of Cucumber (Cucumis sativus L.)

HortScience ◽

10.21273/hortsci.35.3.491e ◽

2000 ◽

Vol 35 (3) ◽

pp. 491E-491

Author(s):

M.H. Aboul-Nasr ◽

M.A. Ahmed

Keyword(s):

Tissue Culture ◽

Callus Formation ◽

Growth Substance ◽

Control Treatment ◽

Ms Medium ◽

Growth Substances ◽

Tetrad Stage ◽

Cucumis Sativus L ◽

Solid Media

This experiment was performed at the Tissue Culture Laboratory of the Horticulture Dept. of the Faculty of Agriculture at Assiut Univ., Egypt. After several attempts to determine the proper stage of buds for collection of pollen, we determined that the tetrad stage was most suitable. The pollen was cultured on either MS or B5 liquid or solid media (7% agar). Both media were used as basic salts or supplemented with growth regulators. The four growth substances were BA, NAA, K, and 2,4-D. Each growth substance was added to the medium separately as follow: BA, NAA at 15, 10, or 5 ppm; K at 0.1, 1, 2, or 5 ppm; and 2,4-D at 0.5, 1, or 5 ppm. The solidified medium was superior to the liquid medium at all the treatments that were used for callus formation. Using B5 medium did not result in any callus. The highest value of callus formation was obtained when MS medium supplemented with BA at 5 ppm. Moreover, the callus that was grown on the MS medium that had BA at 5 or 10 ppm developed a merstim tip. The control treatment produced calluses but did not develop any meristem tips. This process can be used to develop haploid plants.

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