# Developing functional genomics tools for sorghum, by transient or stable transformation

Caroline Calatayud, Frédérique Richaud, Angélique Berger, Maelle Rios, David Pot, Christophe Périn, Nancy Terrier

► To cite this version:

Caroline Calatayud, Frédérique Richaud, Angélique Berger, Maelle Rios, David Pot, et al. Developing functional genomics tools for sorghum, by transient or stable transformation. Sorghum in the 21st Century, Global Sorghum Conference, CIRAD; Kansas State University; Collaborative Research on Sorghum and Millet; SorghumID; IRD; CERAAS, Jun 2023, Montpellier, France. pp.216. hal-05181924

HAL Id: hal-05181924

https://hal.science/hal-05181924v1

Submitted on 23 Jul 2025

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# Developing functional genomics tools for sorghum,
by transient or stable transformation

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Calatayud C¹*, Richaud F¹*, Berger A¹, Rios M¹, Pot D¹, Périn C¹, Terrier N²

*The poster will be presented by both co-authors

¹ CIRAD, UMR AGAP Institut, F-34398 Montpellier, France

² UMR AGAP Institut, Univ Montpellier, CIRAD, INRAE, Institut Agro, Montpellier, France

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agAP

INSTITUT
Aménagement génétique et adaptation des
plantes méditerranéennes et tropicales

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## Context

Sorghum is the fifth most important cereal grown across the world. Grain and flour are used as a staple food in many low-income countries and therefore constitute a key crop for food security under severe environmental stresses. Grains and vegetative components can also be used for animal feed, energy production or engineering of biomaterials. However, improvements are still needed to adapt varieties to specific uses and various biotic and abiotic stresses. In order to identify molecular mechanisms that could be required for those improvements, numerous transcriptomic and genetic studies are performed and increasingly available in the literature and databases. Many candidate genes have been identified and need to be validated. However the low rate of genetic transformation efficiency for this recalcitrant species still remains a obstacle for functional genomics study.

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## Objectives

* To develop a method for direct gene transfer into protoplasts for transient transformation

* To optimize a protocol for stable transformation using immature embryos or leaf whorls

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## Development of a tool for transient transfection on protoplast

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Protoplast extraction
after enzymatic digestion of cell-wall

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Transfection

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ZmUBI::GFP

or/plus

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35S::mCherry

Image Analysis

Ex: 488/Em-507nm

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Ex: 587/Em-610nm

Transformation yield

with both vectors

with single vector

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✓ Around 60% of transformation yield with single vector

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✓ Same transformation yield (60%)

✓ Almost 90% of the cells are co-transformed (FACS analysis)

A performant transient transfection protocol on protoplast was developped

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## Application to over-expression factors (TF): example of VND7b et Myb60b involved in secondary wall formation

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35S::mCherry

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ZmUBI::
Transcription Factor

Co-transfection

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qPCR validation
after TF over-expression (VND7b et
Myb60b) on a putative target (4-CL)

RNA extraction

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RNAseq library

RNAseq analysis
after TF over-expression

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✓ qPCR and RNAseq results
are concordant

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✓ Identification of genes
regulated by Myb60b

Efficient tool to

✓ Validate TF of interest and identify their target genes

✓ Validate constructions before stable transformation

✓ Perform cellular localization of proteins

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However,

we need to develop a stable transformation tool to analyse phenotypic effect on plants

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## Stable transformation

We developed an effective regeneration system from sorghum explants (with Texas 430 genotype)

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Different stages of regeneration from immature embryos based on the modified portocol from Gurel et al., 2012. This plant material is the most commonly used in the literature for genetic transformation of sorghum. A. Callus initiation after 1 week on 2,4D 2 mg/l ; B. Shoot regeneration after 9 weeks ; C. Rooting ; D. Growth in greenhouse

Different stages of regeneration from leaf whorls of in vitro seedling based on the modified protocol from Gendy et al., 1996. This plant material allows experiments throughout the year. A. In vitro seedling. Immature leaf whorl cross-sections were sliced from the region above the apical meristem into 1-mm thick disks B. Callus initiation after 1 week ; C. Callus after 2 weeks D. Regeneration after 11 weeks

✓ The regeneration rate can reach up to 75%
but is highly dependent on the development stage of embryo

✓ The maximun regeneration rate can only reach 20%

The focus will be on Agrobacterium mediated transformation using immature embryos from sorghum genotype Texas 430 and reporter gene GFP

The experiments were performed with Agrobacterium strain EHA105 and p2301 plasmid with pCaMV35S::GFPs65Tintron::tNOS; pCaMV35S::nptII::tCaMV35S polyA

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Embryos or callus up to 14 days old have been infected
A. Immature embryos; B. 6 days old callus

Results obtained
after callus infection

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based on modified protocol from Gurel et al., 2012 ; Wu et al., 2014

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Transgenic shoots and roots expressing GFP using fluorescence microscopy. A. Negative control;
B. Transgenic shoots expressing GFP; C. Transgenic roots expressing GFP

✓ The transformation yield was less than 1%

## References:

Gendy et al., 1996. Plant cell reports 15,
900-904.

Gurel et al., 2012. In: Dunwell JM., In:
Wetten AC, eds. Transgenic Plants.
Totowa, NJ: Humana Press, 109-122.

Wu et al., 2014. In Vitro Cellular &
Developmental Biology - Plant 50, 9-18.

## Conclusions & Perspectives :

The transient transfection on protoplasts will allow to test plasmids intended to validate the function of candidate genes through overexpression or
knockout (through CRISPR/Cas9 technology for exemple) of the gene. Transcriptomic studies may be associated in the case of gene overexpression
(and in particular transcription factor).

We obtained transgenic sorghum plants using agrobacterium in two separate experiments. However the transformation frequency was very low and
should be increased. Different axes could be considered for the next transformation tests : to use more aggressive strains such as LBA4404 or AGL1, to
choice the Ubi promoter rather than the 35S promoter, to find a sensitive system to detect early transformation events on reporter gene. Once these
methods have been developed, different targets could be chosen to conduct functional analysis according to the priorities of the sorghum research
programs (biomass, grain quality...)