Key messages

• Text advanced materials with increased yield potential, nitrogen use efficiency and coleoptile length have been developed and can be transformed into new profitable varieties.
• New investments are being sought for product commercialisation and adopting the technology for the targeted trait improvement in other crops.

Aims

• Develop advanced materials with increased yield potential, nitrogen use efficiency and coleoptile length for profit and environmental adaptation.
• Improve crop traits through efficient and targeted gene editing technology.

Introduction

The ‘Green Revolution’ in cereal crops is attributed to the introduction of semi-dwarf genes that reduce plant height and thus increase lodging resistance and yield potential. Such semi-dwarf alleles are mainly induced by artificial mutagenesis and are now pervasive in current commercial crop varieties (Daba et al 2020). Notably, multiple genes are responsible for GA metabolism, and they often have distinct tissue and developmental specific expression patterns (Pearce et al 2015). Also, gene pleiotropic effects have to be well documented, given that GA is not only regulating plant height but also several other traits such as flowering time, tillering, seed germination and coleoptile length. Hence, caution should be shown when introducing semi-dwarf alleles into genetic backgrounds that possess diverse combinations of GA-related genes. Identifying novel semi-dwarf alleles with minimum trade-off effects would be of enormous value for crop breeding.

In barley, semi-dwarf genes have been identified within the loci semi-dwarf 1 (sdw1; Jia et al 2015) and breviaristatum-e (ari-e; Wendt et al 2016) located on chromosomes 3H and 5H respectively, which have been used to develop high yielding varieties such as RGT Planet and La Trobe. The mutations of most dwarfing genes were reported linked with pleiotropic effects on key agronomic traits. For instance, sdw1.d with 7bp deletion in exon1 of HvGA20ox2 affects GA biosynthesis in different tissues,causing several unwanted phenotypes such as late flowering by 3-5 days and shorter coleoptiles (Teplyakova et al 2017). Therefore, creating novel semi-dwarf genes with less adverse impact on other key agronomic traits is still essential for barley breeding (Cheng et al 2023).

Precision breeding allows breeders to target specific parts of the genome and develop new crop lines with desirable traits. New biotechnologies including CRISPR that mediate genome editing hold great potential to speed up crop innovation. Recently, the U.S. Department of Agriculture approved some CRISPR-edited crops such as browning-resistant mushrooms, drought-tolerant soybean, and omega-3 enhanced oilseed Camelina sativa. The Australian government will not regulate the use of gene-editing techniques in plants, animals and human cell lines that do not introduce new genetic material. The Office of the Gene Technology Regulator (OGTR) clarify that genetic edits made without templates are no different from changes that occur in nature, and therefore do not pose an additional risk to the environment and human health.

Recently, we successfully established a highly efficient and genotype-independent barley gene editing platform that allows gene targeting in Australian commercial barley varieties (Han et al 2021). The platform has been employed for the improvement of key agronomic traits, including nitrogen use efficiency (Karunarathne et al 2022), phenology (Nejat et al 2022), plant stature and coleoptile length (Cheng et al 2023). The enhanced yield potential and climate resilience of such material will be invaluable for future variety breeding and commercialisation and will increase the profitability and sustainability of grain production systems.

Method

Plant materials

Barley varieties including Golden Promise, RGT Planet and Vlamingh were used as donor plants for gene editing. Golden Promise is the genetic transformation reference while RGT Planet and Vlamingh are commercial Australian varieties.

Plant growth, transformation and gene editing

Donor plants for anther culture or embryo transformation were grown in a controlled environment room at 18/13°C (day/night) with a 12h photoperiod. Preliminary target sequences were identified using http://cbc.gdcb.iastate.edu/cgat/ and CRISPOR (http://crispor.tefor.net/), both of which have the barley reference genome for off-target evaluation. Potential targets were further filtered for a 23bp GN19NGG sequence since sgRNA would be driven by a U6 promoter and coupled with the Cas9 nuclease derived from S. pyogenes that recognises a protospacer adjacent motif (PAM) sequence of NGG (Han et al 2021). The barley anther culture protocol was described in Broughton et al (2014), with modifications for Agrobacterium infection and genetic transformation. The immature embryo transformation followed the protocol by Wang et al (2017).

Plant genotyping and phenotyping

Barley genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. Primer design was based on the barley reference genome sequence using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast). PCR amplification and Sanger sequencing were carried out to identify gene mutations in progenies.

For phenotyping of plant height, phenology, nitrogen use efficiency and coleoptile length, seeds were germinated and plants then grown in controlled Physical Containment 2 environments. Seeds of wild-type or null transgenic lines were used as controls in independent experiments. Traits were measured and statistics were performed using IBM SPSS Statistics Package (Version 24) for the significance test.

Results

A range of mutants with novel sdw1 alleles were created (Figure 1). Representative lines were selected, which developed about three times more tillers and thus greatly increased yield potential. The mutants displayed similar, medium-late or extremely late maturity compared with the wild-type, together with little or only a slight reduction in plant height. Some mutants (the 2nd and 3rd plants in Figure1) could be transformed into new high-yielding varieties, with minor changes in plant height and phenology.

Figure 1. Barley mutants with novel sdw1 alleles created through gene editing

We also successfully edited the barley abnormal cytokinin response1 repressor 1 (HvARE1) gene, a candidate for involvement in nitrogen use efficiency previously identified in a genome‐wide association study (Karunarathne et al 2022). The mutants displayed a 1.5 to 2.8‐fold increase in total chlorophyll content in the flag leaf at the grain filling stage (Figure 2). Delayed senescence by 10–14d was also observed in mutant lines. Barley are1 mutants had a higher nitrogen content in shoots under low nitrogen conditions so could maintain biomass and grain yield with reduced nitrogen fertilisation, not only reducing cropping inputs but also greenhouse gas emissions.

Figure 2. Improving barley nitrogen use efficiency through editing the HvARE1 gene (modified from Karunarathne et al 2022). (A) delayed leaf senescence of are1 mutants compared with wild type (WT). (B) increased grain yield of mutants. (C) total nitrogen content (%) in the wild type (WT) and are1 mutants grown under limited nitrogen supply (50mg/kg).

Coleoptile length is an important agronomic trait in barley and other cereal crops such as wheat, as it determines maximum seeding depth. Developing cereal crop varieties with long coleoptiles, allowing deeper sowing to take advantage of stored soil water, has been advocated as one of the key adaptation strategies for production improvement under climate change with unreliable rainfall and increased drought stress (Cheng et al 2023). We edited HvGA3ox1 and the mutants all had delayed flowering time and reduced plant height (Figure 3). More importantly, we measured root length and coleoptile length after germination and found there was no significant change in root length but both mutants had increased coleoptile length by an average of 8mm. The developed mutants pave the way for selecting HvGA3ox1 alleles to develop barley varieties with reduced plant height and longer coleoptiles.

Figure 3. New semi-dwarfing alleles with increased coleoptile length by gene editing of gibberellin 3-oxidase 1 (modified from Chen et al 2023). (A) comparison of plant height of ga3ox1 mutants and null transgenic line. (B) comparison of coleoptile lengths of ga3ox1 mutants and null transgenic line.

Conclusion

We have generated a barley mutant collection using CRISPR/Cas9 and the advanced materials have increased yield potential, nitrogen use efficiency, or altered plant stature, phenology and coleoptile length. Such materials can be developed for future varieties to increase grain profitability and improve environmental adaptation.

Acknowledgments

We are grateful to the Department of Primary Industries and Regional Development and Murdoch University for supporting this research.

References

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Cheng J, Hill C, Han Y, He T, Ye X, Shabala S, Guo G, Zhou M, Wang K, Li C. New semi-dwarfing alleles with increased coleoptile length by gene editing of gibberellin 3-oxidase 1 using CRISPR-Cas9 in barley (Hordeum vulgare L.). Plant Biotechnol J. 2023 Jan 1. doi: 10.1111/pbi.13998.

Daba SD, Tyagi P, Brown-Guedira G, Mohammadi M (2020) Genome-wide association study in historical and contemporary U.S. winter wheats identifies height-reducing loci. Crop J 8:243–251

Han, Y., Broughton, S., Liu, L., et al 2021. Highly efficient and genotype-independent barley gene editing based on anther culture. Plant Commun, 2(2):100082.

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Teplyakova, S., Lebedeva, M., Ivanova, N. et al. (2017) Impact of the 7-bp deletion in HvGA20ox2 gene on agronomic important traits in barley (Hordeum vulgare L.). BMC Plant Biol 17 (Suppl 1), 181.

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Wendt T, Holme I, Dockter C, Preuß A, Thomas W, Druka A, et al (2016) HvDep1 is a positive regulator of culm elongation and grain size in barley and impacts yield in an environment-dependent manner. PLoS ONE 11(12): e0168924.

Contact details

Yong Han
Department of Primary Industries and Regional Development
3 Baron-Hay Court, Kensington WA 6151
Ph: [08 9360 7590]
Email: yong.han@dpird.wa.gov.au

Chengdao Li
Western Crop Genetics Alliance, Murdoch University
90 South Street, Murdoch, WA 6150
Ph: 08 9360 7519
Email: c.li@murdoch.edu.au