CRISPR/Cas-mediated plant genome editing: outstanding challenges a decade after implementation.

The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.

Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.).

Genetic transformation of perennial ryegrass (Lolium perenne L.) is critical for fundamental and translational research in this important grass species. It often relies on Agrobacterium-mediated transformation of callus tissue. However, callus induction is restricted to a few genotypes that respond well to tissue culture. Here, we report callus induction from different perennial ryegrass genotypes and explants, such as shoot tips, seeds, and anthers, which were transformed with several plasmids for functional genomics. ß-glucuronidase (GUS) histochemical staining showed the LmdsRNAbp promoter sequence was active in stigmas, spikelets, anthers, and leaves. We also transformed calli with plasmids allowing gene silencing and gene knock-out using RNA interference and CRISPR/Cas9, respectively, for which genotypic and phenotypic investigations are ongoing. Using 19 different constructs, 262 transgenic events were regenerated. Moreover, the protocol regenerated a doubled haploid transgenic event from anther-derived calli. This work provides a proof-of-concept method for expanding the range of genotypes amenable to transformation, thus, serving research and breeding initiatives to improve this important grass crop for forage and recreation.

Overexpressing the H-protein of the glycine cleavage system increases biomass yield in glasshouse and field-grown transgenic tobacco plants.

Photorespiration is essential for C3 plants, enabling oxygenic photosynthesis through the scavenging of 2-phosphoglycolate. Previous studies have demonstrated that overexpression of the L- and H-proteins of the photorespiratory glycine cleavage system results in an increase in photosynthesis and growth in Arabidopsis thaliana. Here, we present evidence that under controlled environment conditions an increase in biomass is evident in tobacco plants overexpressing the H-protein. Importantly, the work in this paper provides a clear demonstration of the potential of this manipulation in tobacco grown in field conditions, in two separate seasons. We also demonstrate the importance of targeted overexpression of the H-protein using the leaf-specific promoter ST-LS1. Although increases in the H-protein driven by this promoter have a positive impact on biomass, higher levels of overexpression of this protein driven by the constitutive CaMV 35S promoter result in a reduction in the growth of the plants. Furthermore in these constitutive overexpressor plants, carbon allocation between soluble carbohydrates and starch is altered, as is the protein lipoylation of the enzymes pyruvate dehydrogenase and alpha-ketoglutarate complexes. Our data provide a clear demonstration of the positive effects of overexpression of the H-protein to improve yield under field conditions.

Accelerated ex situ breeding of GBSS- and PTST1-edited cassava for modified starch.

Crop diversification required to meet demands for food security and industrial use is often challenged by breeding time and amenability of varieties to genome modification. Cassava is one such crop. Grown for its large starch-rich storage roots, it serves as a staple food and a commodity in the multibillion-dollar starch industry. Starch is composed of the glucose polymers amylopectin and amylose, with the latter strongly influencing the physicochemical properties of starch during cooking and processing. We demonstrate that CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated targeted mutagenesis of two genes involved in amylose biosynthesis, PROTEIN TARGETING TO STARCH (PTST1) or GRANULE BOUND STARCH SYNTHASE (GBSS), can reduce or eliminate amylose content in root starch. Integration of the Arabidopsis FLOWERING LOCUS T gene in the genome-editing cassette allowed us to accelerate flowering-an event seldom seen under glasshouse conditions. Germinated seeds yielded S1, a transgene-free progeny that inherited edited genes. This attractive new plant breeding technique for modified cassava could be extended to other crops to provide a suite of novel varieties with useful traits for food and industrial applications.

FLOWERING LOCUS T Triggers Early and Fertile Flowering in Glasshouse Cassava (Manihot esculenta Crantz).

Accelerated breeding of plant species has the potential to help challenge environmental and biochemical cues to support global crop security. We demonstrate the over-expression of ArabidopsisFLOWERING LOCUS T in Agrobacterium-mediated transformed cassava (Manihot esculenta Crantz; cultivar 60444) to trigger early flowering in glasshouse-grown plants. An event seldom seen in a glasshouse environment, precocious flowering and mature inflorescence were obtained within 4-5 months from planting of stem cuttings. Manual pollination using pistillate and staminate flowers from clonal propagants gave rise to viable seeds that germinated into morphologically typical progeny. This strategy comes at a time when accelerated crop breeding is of increasing importance to complement progressive genome editing techniques.

Cassava (Manihot esculenta Crantz).

Genetic transformation of plants is an indispensable technique used for fundamental research and crop improvement. Recent advances in cassava (Manihot esculenta Crantz) transformation have facilitated the effective generation of stably transformed cassava plants with favorable traits. Agrobacterium-mediated transformation of friable, embryogenic callus has evolved to become the most widely used approach and has been adopted by research laboratories in Africa. This procedure utilizes axillary meristem tissue (buds) to produce primary and secondary somatic embryos and subsequently friable, embryogenic callus. Agrobacterium harboring a binary expression cassette is used to transform this tissue, which is regenerated via cotyledons and shoot organogenesis to produce rooted in vitro plantlets. This chapter details each step of the procedure using the model cultivar 60444 and provides supplementary notes to successfully produce transgenic cassava.

Cassava: constraints to production and the transfer of biotechnology to African laboratories.

Knowledge and technology transfer to African institutes is an important objective to help achieve the United Nations Millennium Development Goals. Plant biotechnology in particular enables innovative advances in agriculture and industry, offering new prospects to promote the integration and dissemination of improved crops and their derivatives from developing countries into local markets and the global economy. There is also the need to broaden our knowledge and understanding of cassava as a staple food crop. Cassava (Manihot esculenta Crantz) is a vital source of calories for approximately 500 million people living in developing countries. Unfortunately, it is subject to numerous biotic and abiotic stresses that impact on production, consumption, marketability and also local and country economics. To date, improvements to cassava have been led via conventional plant breeding programmes, but with advances in molecular-assisted breeding and plant biotechnology new tools are being developed to hasten the generation of improved farmer-preferred cultivars. In this review, we report on the current constraints to cassava production and knowledge acquisition in Africa, including a case study discussing the opportunities and challenges of a technology transfer programme established between the Mikocheni Agricultural Research Institute in Tanzania and Europe-based researchers. The establishment of cassava biotechnology platform(s) should promote research capabilities in African institutions and allow scientists autonomy to adapt cassava to suit local agro-ecosystems, ultimately serving to develop a sustainable biotechnology infrastructure in African countries.

Infectivity, pseudorecombination and mutagenesis of Kenyan cassava mosaic begomoviruses.

Cloned DNA-A and DNA-B components of Kenyan isolates of East African cassava mosaic virus (EACMV, EACMV-UG and EACMV-KE2), East African cassava mosaic Kenya virus (EACMKV) and East African cassava mosaic Zanzibar virus (EACMZV) are shown to be infectious in cassava. EACMV and EACMKV genomic components have the same iteron sequence (GGGGG) and can form viable pseudorecombinants, while EACMZV components have a different sequence (GGAGA) and are incompatible with EACMV and EACMKV. Mutagenesis of EACMZV has demonstrated that open reading frames (ORFs) AV1 (encoding the coat protein), AV2 and AC4 are not essential for a symptomatic infection of cassava, although mutants of both ORF AV1 and AV2 produce attenuated symptoms in this host. Furthermore, ORF AV1 and AV2 mutants were compromised for coat protein production, suggesting a close structural and/or functional relationship between these coding regions or their protein products.

Deletion and recombination events between the DNA-A and DNA-B components of Indian cassava-infecting geminiviruses generate defective molecules in Nicotiana benthamiana.

Cloned DNA-B components, belonging to the bipartite begomoviruses Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV), family Geminiviridae, when co-inoculated along with previously cloned DNA-A components of the respective viruses onto the experimental host Nicotiana benthamiana, generated defective DNAs (def-DNA) ranging in size from 549 to 1555 nucleotides. All the cloned def-DNAs contained the common region (CR) as well as portions of either DNA-A or DNA-B and, in a few cases, both DNA-A and DNA-B, representing recombinant products, the junction points of which correspond to repeats of 2-11 bases found in the parental molecules. The DNA-B-derived def-DNAs were, in some cases, associated with a decrease in levels of DNA-B, with a concomitant change in the symptoms from downward leaf curling in the older leaves to upward leaf-rolling in newly emerging leaves, more typical of monopartite begomoviruses.

Genetic diversity and phylogeography of cassava mosaic viruses in Kenya.

Cassava is a major factor in food security across sub-Saharan Africa. However, the crop is susceptible to losses due to biotic stresses, in particular to viruses of the genus Begomovirus (family Geminiviridae) that cause cassava mosaic disease (CMD). During the 1990s, an epidemic of CMD severely hindered cassava production across eastern and central Africa. A significant influence on the appearance of virus epidemics is virus diversity. Here, a survey of the genetic diversity of CMD-associated begomoviruses across the major cassava-growing areas of Kenya is described. Because an initial PCR-restriction fragment-length polymorphism analysis identified a much greater diversity of viruses than assumed previously, representative members of the population were characterized by sequence analysis. The full-length sequences of 109 components (68 DNA-A and 41 DNA-B) were determined, representing isolates of East African cassava mosaic virus and East African cassava mosaic Zanzibar virus, as well as a novel begomovirus species for which the name East African cassava mosaic Kenya virus is proposed. The DNA-B components were much less diverse than their corresponding DNA-A components, but nonetheless segregated into western and eastern (coastal) groups. All virus species and strains encountered showed distinct geographical distributions, highlighting the importance of preventing both the movement of viruses between these regions and the importation of the disease from adjacent countries and islands in the Indian Ocean that would undoubtedly encourage further diversification.

Cotton leaf curl Gezira virus-satellite DNAs represent a divergent, geographically isolated Nile Basin lineage: predictive identification of a satDNA REP-binding motif.

Cotton leaf curl Gezira virus (CLCuGV), a species of the genus Begomovirus (family Geminiviridae), was recently cloned from cotton, okra, and Sida alba plants exhibiting leaf-curling and vein-thickening symptoms in Sudan. Here, we describe a previously unknown lineage of single-stranded DNA satellite (satDNA) molecules, which are associated with CLCuGV, and are required for development of characteristic disease symptoms. Co-inoculation of cotton and Nicotiana benthamiana plants with satDNAs cloned from cotton, okra, and S. alba, together with CLCuGV as the 'helper virus' resulted in the development of characteristic leaf-curling and vein-thickening symptoms in both hosts. An anatomical study of symptomatic, virus-infected cotton leaves revealed that spongy parenchyma cells had developed instead of collenchyma cells at the sites of vein thickening. Phylogenetically, the CLCuGV-associated satDNAs from Sudan, together with their closest relatives from Egypt, form a new satDNA lineage comprising only satDNAs from the Upper and Lower Nile Basins. Analysis of satellites and their helper virus sequences identified a predicted REP-binding site consisting of the directly repeated sequence, 'CGGTACTCA', and an inverted repeated sequence, 'TGAGTACCG', which occur in the context of a 17-nucleotide motif. The conserved REP-binding motif identified herein, together with strict geographic isolation, and apparent host-restriction, may be the collective hallmark of these new satDNA-begomovirus lineages, extant in the Nile Basin.

Diversity of DNA 1: a satellite-like molecule associated with monopartite begomovirus-DNA beta complexes.

DNA 1 components are satellite-like, single-stranded DNA molecules associated with begomoviruses (family Geminiviridae) that require the satellite molecule DNA beta to induce authentic disease symptoms in some hosts. They have been shown to be present in the begomovirus-DNA beta complexes causing cotton leaf curl disease (CLCuD) and okra leaf curl disease (OLCD) in Pakistan as well as Ageratum yellow vein disease (AYVD) in Singapore. We have cloned and sequenced a further 17 DNA 1 molecules from a diverse range of plant species and geographical origins. The analysis shows that DNA 1 components are associated with the majority of begomovirus-DNA beta complexes, being absent from only two of the complexes examined, both of which have their origins in Far East Asia. The sequences showed a high level of conservation as well as a common organization consisting of a single open reading frame (ORF) in the virion sense, a region of sequence rich in adenine and a predicted hairpin structure. In phylogenetic analyses, there was some evidence of grouping of DNA 1 molecules according to geographic origin, but less evidence for grouping according to host plant origin. The possible origin and function of DNA 1 components are discussed in light of these findings.

Diversity of DNA beta, a satellite molecule associated with some monopartite begomoviruses.

DNA beta molecules are symptom-modulating, single-stranded DNA satellites associated with monopartite begomoviruses (family Geminiviridae). Such molecules have thus far been shown to be associated with Ageratum yellow vein virus from Singapore and Cotton leaf curl Multan virus from Pakistan. Here, 26 additional DNA beta molecules, associated with diverse plant species obtained from different geographical locations, were cloned and sequenced. These molecules were shown to be widespread in the Old World, where monopartite begomoviruses are known to occur. Analysis of the sequences revealed a highly conserved organization for DNA beta molecules consisting of a single conserved open reading frame, an adenine-rich region, and a region of high sequence conservation [the satellite conserved region (SCR)]. The SCR contains a potential hairpin structure with the loop sequence TAA/GTATTAC; similar to the origins of replication of geminiviruses and nanoviruses. Two major groups of DNA beta satellites were resolved by phylogenetic analyses. One group originated from hosts within the Malvaceae and the second from a more diverse group of plants within the Solanaceae and Compositae. Within the two clusters, DNA beta molecules showed relatedness based both on host and geographic origin. These findings strongly support coadaptation of DNA beta molecules with their respective helper begomoviruses.