CRISPR-knockout of ß-kafirin in sorghum does not recapitulate the grain quality of natural mutants.

MAIN CONCLUSION: When gene editing was applied to knockout beta-kafirin, there was a compensatory increase of gamma-kafirin which does not occur in domesticated null varieties, so enhanced grain quality was not achieved. Sorghum bicolor is an important animal feedstock cereal crop throughout Australia and the southern United States, where its use as a food product is limited by issues with low calorific and nutritive value. Qualities such as reduced digestibility and low essential amino acid content are directly attributed to the kafirin grain storage proteins, the major components of protein bodies within the endosperm. Specifically, the ß- and γ-kafirins have few protease cleavage sites and high levels of cysteine residues which lead to a highly cross-linked shell of intra- and inter-molecular disulphide linkages that encapsulate the more digestible α- and δ-kafirins in the core of the protein bodies. Naturally occurring ß-kafirin mutants exist and are known to have improved grain quality, with enhanced protein contents and digestibility, traits which are often attributed to the lack of this cysteine-rich kafirin in the mature grain. However, when CRISPR/Cas9 editing was used to create ß-kafirin knockout lines, there was no improvement to grain quality in the Tx430 background, although they did have unique protein composition and changes to protein body morphology in the vitreous endosperm. One explanation of the divergence in quality traits found the lines lacking ß-kafirin are due to a drastic increase of γ-kafirin which was only found in the gene edited lines. This study highlights that in some germplasm, there is a level of redundancy between the peripheral kafirins, and that improvement of grain protein digestibility cannot be achieved by simply removing the ß-kafirin protein in all genetic backgrounds.

Biotechnological Road Map for Innovative Weed Management.

In most agriculture farmlands, weed management is predominantly reliant on integrated weed management (IWM) strategies, such as herbicide application. However, the overuse and misuse of herbicides, coupled with the lack of novel active ingredients, has resulted in the uptrend of herbicide-resistant weeds globally. Moreover, weedy traits that contribute to weed seed bank persistence further exacerbate the challenges in weed management. Despite ongoing efforts in identifying and improving current weed management processes, the pressing need for novel control techniques in agricultural weed management should not be overlooked. The advent of CRISPR/Cas9 gene-editing systems, coupled with the recent advances in "omics" and cheaper sequencing technologies, has brought into focus the potential of managing weeds in farmlands through direct genetic control approaches, but could be achieved stably or transiently. These approaches encompass a range of technologies that could potentially manipulate expression of key genes in weeds to reduce its fitness and competitiveness, or, by altering the crop to improve its competitiveness or herbicide tolerance. The push for reducing or circumventing the use of chemicals in farmlands has provided an added incentive to develop practical and feasible molecular approaches for weed management, although there are significant technical, practical, and regulatory challenges for utilizing these prospective molecular technologies in weed management.

Endogenous U6 promoters improve CRISPR/Cas9 editing efficiencies in Sorghum bicolor and show potential for applications in other cereals.

KEY MESSAGE: Endogenous U6 promoters increase CRISPR/Cas9 editing efficiency in sorghum and may be useful for gene editing applications in other cereals.

Hotter, drier, CRISPR: the latest edit on climate change.

KEY MESSAGE: Integrating CRISPR/Cas9 genome editing into modern breeding programs for crop improvement in cereals. Global climate trends in many agricultural regions have been rapidly changing over the past decades, and major advances in global food systems are required to ensure food security in the face of these emerging challenges. With increasing climate instability due to warmer temperatures and rising CO2 levels, the productivity of global agriculture will continue to be negatively impacted. To combat these growing concerns, creative approaches will be required, utilising all the tools available to produce more robust and tolerant crops with increased quality and yields under more extreme conditions. The integration of genome editing and transgenics into current breeding strategies is one promising solution to accelerate genetic gains through targeted genetic modifications, producing crops that can overcome the shifting climate realities. This review focuses on how revolutionary genome editing tools can be directly implemented into breeding programs for cereal crop improvement to rapidly counteract many of the issues affecting agriculture production in the years to come.

Biolistic DNA Delivery and Its Applications in Sorghum bicolor.

Biolistic DNA delivery has been considered a universal tool for genetic manipulation to transfer exotic genes to cells or tissues due to its simplicity, versatility, and high efficiency. It has been a preferred method for investigating plant gene function in most monocot crops. The first transgenic sorghum plants were successfully regenerated through biolistic DNA delivery in 1993, with a relatively low transformation efficiency of 0.3%. Since then, tremendous progress has been made in recent years where the highest transformation efficiency was reported at 46.6%. Overall, the successful biolistic DNA delivery system is credited to three fundamental cornerstones: robust tissue culture system, effective gene expression in sorghum, and optimal parameters of DNA delivery. In this chapter, the history, application, and current development of biolistic DNA delivery in sorghum are reviewed, and the prospect of sorghum genetic engineering is discussed.

Correction to: Applications and potential of genome editing in crop improvement.

Following publication of the original article [1], the authors reported the following two errors.

Applications and potential of genome editing in crop improvement.

Genome-editing tools provide advanced biotechnological techniques that enable the precise and efficient targeted modification of an organism's genome. Genome-editing systems have been utilized in a wide variety of plant species to characterize gene functions and improve agricultural traits. We describe the current applications of genome editing in plants, focusing on its potential for crop improvement in terms of adaptation, resilience, and end-use. In addition, we review novel breakthroughs that are extending the potential of genome-edited crops and the possibilities of their commercialization. Future prospects for integrating this revolutionary technology with conventional and new-age crop breeding strategies are also discussed.

Group II introns in wheat mitochondria have degenerate structural features and varied splicing pathways.

Mitochondrial introns in flowering plant genes are virtually all classified as members of the group II ribozyme family although certain structural features have degenerated to varying degrees over evolutionary time. We are interested in the impact that unconventional intron architecture might have on splicing biochemistry in vivo and we have focused in particular on intronic domains V and VI, which for self-splicing introns provide a key component of the catalytic core and the bulged branchpoint adenosine, respectively. Notably, the two transesterification steps in classical group II splicing are the same as for nuclear spliceosomal introns and release the intron as a lariat. Using RT-PCR and circularized RT-PCR, we had previously demonstrated that several wheat mitochondrial introns which lack a branchpoint adenosine have atypical splicing pathways, and we have now extended this analysis to the full set of wheat introns, namely six trans-splicing and sixteen cis-splicing ones. A number of introns are excised using non-lariat pathways and interestingly, we find that several introns which do have a conventional domain VI also use pathways that appear to exploit other internal or external nucleophiles, with the lariat form being relatively minor. Somewhat surprisingly, several introns with weakly-structured domain V/VI helices still exhibit classical lariat splicing, suggesting that accessory factors aid in restoring a splicing-competent conformation. Our observations illustrate that the loss of conventional group II features during evolution is correlated with altered splicing biochemistry in an intron-distinctive manner.

Whole Genome Sequencing Reveals Potential New Targets for Improving Nitrogen Uptake and Utilization in Sorghum bicolor.

Nitrogen (N) fertilizers are a major agricultural input where more than 100 million tons are supplied annually. Cereals are particularly inefficient at soil N uptake, where the unrecovered nitrogen causes serious environmental damage. Sorghum bicolor (sorghum) is an important cereal crop, particularly in resource-poor semi-arid regions, and is known to have a high NUE in comparison to other major cereals under limited N conditions. This study provides the first assessment of genetic diversity and signatures of selection across 230 fully sequenced genes putatively involved in the uptake and utilization of N from a diverse panel of sorghum lines. This comprehensive analysis reveals an overall reduction in diversity as a result of domestication and a total of 128 genes displaying signatures of purifying selection, thereby revealing possible gene targets to improve NUE in sorghum and cereals alike. A number of key genes appear to have been involved in selective sweeps, reducing their sequence diversity. The ammonium transporter (AMT) genes generally had low allelic diversity, whereas a substantial number of nitrate/peptide transporter 1 (NRT1/PTR) genes had higher nucleotide diversity in domesticated germplasm. Interestingly, members of the distinct race Guinea margaritiferum contained a number of unique alleles, and along with the wild sorghum species, represent a rich resource of new variation for plant improvement of NUE in sorghum.

Multiple splicing pathways of group II trans-splicing introns in wheat mitochondria.

Trans-splicing of discontinuous introns in plant mitochondria requires the assembly of independently-transcribed precursor RNAs into splicing-competent structures, and they are expected to be excised as Y-branched molecules ("broken lariats") because these introns belong to the group II ribozyme family. We now demonstrate that this is just one of several trans-splicing pathways for wheat mitochondrial nad1 intron 4 and nad5 intron 2; they also use a hydrolytic pathway and the liberated 5'-half-intron linear molecules are unexpectedly abundant in the RNA population. We also observe a third productive splicing pathway for nad5 intron 2 that yields full-length excised introns in which the termini are joined in vivo and possess non-encoded nucleotides. In the case of trans-splicing nad1 intron 1, which has a weakly-structured and poorly-conserved core sequence, excision appears to be solely through a hydrolytic pathway. When wheat embryos are germinated in the cold rather than at room temperature, an increased complexity in trans-splicing products is seen for nad1 intron 4, suggesting that there can be environmental effects on the RNA folding of bipartite introns. Our observations provide insights into intron evolution and the complexity of RNA processing events in plant mitochondria.