Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies.

Sharma, Rita; Liang, Yan; Lee, Mi Yeon; Pidatala, Venkataramana R.; Mortimer, Jenny C.; Scheller, Henrik Vibe

doi:10.21203/rs.2.11984/v1

Cited by 4 publications

(7 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Epicuticular wax and high silica content are the biggest bottlenecks of Agrobacterium -based infiltration methods in rice plants as they prevent the Agrobacterium from attaching and penetrating the plant surface ( Sharma et al, 2020 ). The source of silica for plants is the water and soil in which the plant grows.…”

Section: Discussionmentioning

confidence: 99%

“…The thick waxy cuticle of rice leaves is known to scatter light, thereby, impeding high resolution fluorescent protein visualization ( Luginbuehl et al, 2020 ). The presence of epicuticular wax on rice leaves also makes transient expression of genes using Agrobacterium difficult ( Sharma et al, 2020 ). Here, we present a highly efficient and rapid protocol for transient expression of genes in rice seedlings without the requirement of high-end equipment.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A Rapid and Highly Efficient Method for Transient Gene Expression in Rice Plants

2020

View full text Add to dashboard Cite

Rice is the model plant system for monocots and the sequencing of its genome has led to the identification of a vast array of genes for characterization. The tedious and time-consuming effort of raising rice transgenics has significantly delayed the pace of rice research. The lack of highly efficient transient assay protocol for rice has only added to the woes which could have otherwise helped in rapid deciphering of the functions of genes. Here, we describe a technique for efficient transient gene expression in rice seedlings. It makes use of co-cultivation of 6-day-old rice seedlings with Agrobacterium in the presence of a medium containing Silwet R L-77, acetosyringone and glucose. Seedlings can be visualized 9 days after co-cultivation for transient expression. The use of young seedlings helps in significantly reducing the duration of the experiment and facilitates the visualization of rice cells under the microscope as young leaves are thinner than mature rice leaves. Further, growth of seedlings at low temperature, and the use of surfactant along with wounding and vacuum infiltration steps significantly increases the efficiency of this protocol and helps in bypassing the natural barriers in rice leaves, which hinders Agrobacterium-based transformation in this plant. This technique, therefore, provides a shorter, efficient and cost-effective way to study transient gene function in intact rice seedling without the need for a specialized device like particle gun.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Rapid and Highly Efficient Method for Transient Gene Expression in Rice Plants

2020

View full text Add to dashboard Cite

show abstract

Section: Transient Crispr/cas9 Assaysmentioning

confidence: 99%

“…In sorghum, both protoplast expression and agroinfiltration have recently been successfully used to evaluate genome editing reagents (Meng et al 2020;Sharma et al 2020). Sharma et al (2020) demonstrated agroinfiltration on 3-4wk-old Tx430 sorghum plants at the three-leaf stage. Similar to the approach taken by Jiang et al (2013), a binary vector was used which contained an intentionally frameshifted green fluorescence protein (GFP) gene under the control of a CaMV 35S promoter, a plant codon-optimized SpCas9 under control of the maize ubiquitin 1 promoter, a sgRNA under control of the rice U3 promoter, and a red fluorescence protein (DsRED) gene under control of nopaline synthase promoter.…”

Section: Transient Crispr/cas9 Assaysmentioning

confidence: 99%

CRISPR/Cas-mediated genome editing in sorghum — recent progress, challenges and prospects

Parikh

Brant

Baloğlu

et al. 2021

In Vitro Cell.Dev.Biol.-Plant

View full text Add to dashboard Cite

Sorghum is a versatile crop with great potential as a sustainable food, feed, and bioenergy source. To mitigate the severely negative impact of climate change and population growth on food and energy security, further elevation of the crops stress tolerance is urgently needed. Genome editing technologies such as CRISPR/Cas have great potential to accelerate functional genomics and crop improvement by supporting targeted modification of almost any crop gene sequence. We describe the recent progress in genome editing of sorghum. In addition, we review remaining challenges and prospects of emerging gene editing technologies for rapid precision breeding of this crop.

show abstract

“…Transient gene expression can provide insights into gene function and success in this approach has recently been achieved in sorghum (Sharma et al 2020). However, given the complications of this approach (Ueki et al 2013), especially under field-relevant conditions, other methods are needed to fully understand gene function in the context of the whole plant.…”

Section: Introductionmentioning

confidence: 99%

Pathway to Validate Gene Function in Key Bioenergy Crop,Sorghum bicolor

Aregawi

Shen

Pierroz

et al. 2020

Preprint

View full text Add to dashboard Cite

Determining gene function is an essential goal for the key bioenergy crop, Sorghum bicolor (L.) Moench - particularly for genes associated with its notable abiotic stress tolerances. However, detailed molecular understanding of the genes associated with those traits is limited. This was made clear in our in-depth transcriptome studies in sorghum, which indicated nearly 50% of its transcriptome is not annotated. In this report, we describe a full spectrum of tools needed to transform sorghum in order to validate and annotate genes. Efforts began with modifying a transformation method that uses the morphogenic genes Baby Boom and Wuschel2 (Ovule Development Protein2) to accelerate transformation speed and expand amenable genotypes. In our experience, transforming RTx430 without morphogenic genes requires ~18 to 21 weeks, compared with ~10 to 12 weeks to generate T0 plants using methods with morphogenic genes. Utilizing morphogenic genes also allowed for the transformation of several sorghum genotypes not previously transformed or historically recalcitrant to transformation, i.e., rapid cycling SC187, stay-green BTx642, BTx623 and sweet sorghum Ramada. In order to validate candidate genes via engineering, while simultaneously introducing the morphogenic genes, a co-transformation strategy, termed altruistic transformation, was developed. To accomplish editing of the target gene, phytoene desaturase, novel constructs were created that also included morphogenic genes. To enable full characterization of transformed plants, we adapted techniques to determine copy number and independence of events at high-throughput levels. Through these efforts, we created a complete pathway from Agrobacterium infection to high-throughput molecular genotyping that can be used to ascertain gene function and expedite basic genetic research in this widely-grown bioenergy crop plant.

show abstract

Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies.

Cited by 4 publications

References 18 publications

A Rapid and Highly Efficient Method for Transient Gene Expression in Rice Plants

A Rapid and Highly Efficient Method for Transient Gene Expression in Rice Plants

CRISPR/Cas-mediated genome editing in sorghum — recent progress, challenges and prospects

Pathway to Validate Gene Function in Key Bioenergy Crop,Sorghum bicolor

Contact Info

Product

Resources

About