The biggest challenge in agriculture is to feed the growing human population, which is projected to reach 9.7 billion in 2050 compared to 7.7 billion in 2019. There is a critical need to close the yield gap in staple crops and enhance food production to feed the world. In Africa, the emphasis should be on root, tubers, and banana rather than on cereals, unlike other parts of the world, as they are the main crops used for staple food and income generation.
To fulfill the increasing demand for food with limited resources, better and efficient ways to produce food are required. Modern biotechnological tools such as genome-editing (GE) offer cost-effective strategies for developing improved varieties. GE is a type of genetic engineering in which DNA is inserted, knocked out, modified, or replaced in the genome (set of genes or genetic materials) of a living organism. Unlike genetic engineering that randomly inserts genetic material into the host genome, GE targets the gene modification to site-specific locations.
In recent years, GE using sequence-specific nucleases (enzymes that split nucleic acids) has emerged as a powerful technique to generate targeted mutation in the genome organisms and applied plant gene function studies and crop improvement. Of these nucleases, clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) was widely adopted as the most robust GE tool in plants because of its simplicity, design flexibility, high efficiency and its ability to edit multiple genes simultaneously (Tripathi et al., 2020).
GE technology uses “molecular scissors” that create precise double-strand breaks at the desired target site in the genome. These double-stranded breaks are then repaired by the cell’s natural repair mechanism and produce a user-desired mutation or genetic outcome.
A recent development in GE can accelerate breeding by making efficient and precise changes in the plant genome to develop new traits such as resistance to various biotic and abiotic stresses. The advantage of targeted gene editing is that it can be used for ‘trait stacking,’ whereby several desired traits are physically linked to ensure their co-segregation during the breeding processes. This precise GE has the potential to revolutionize crop improvement in sub-Saharan Africa. Researchers at IITA have established the GE system for banana and yam.
Banana production is severely constrained by many pathogens and pests, particularly where a number of them are co-existing. The use of disease and pest-resistant banana varieties is one of the most effective options to improve banana, which is of great importance in enhancing food security. Recent advances in GE have the potential to accelerate breeding of banana for disease and pest resistance. The availability of a well-annotated, whole-genome sequence of banana and well-established genetic transformation protocols makes the banana a strong candidate for GE.
Recently, a robust CRISPR/Cas9-based GE system was developed for banana and plantain using phytoene desaturase (PDS) gene as a marker as mutations disrupting PDS causing albinism and dwarﬁng of plants (Ntui et al., 2020). This system could open up avenues for efficient and targeted genome manipulations for disease resistance in banana (Tripathi et al., 2019a; 2020). At IITA, we are using GE as a tool for improving banana for disease resistance.
Once the efficient protocol for GE was established at IITA, this technology was used to inactivate the endogenous banana streak virus (eBSV) integrated into the B genome of plantain, overcoming a significant challenge in breeding and the dissemination of hybrids (Tripathi et al. 2019b). BSV is a prevalent virus pathogen showing symptoms as chlorotic streaks on leaves, and advancement of the disease leads to the killing of the plant. When the banana plants are stressed, the eBSV recombines to produce a functional episomal viral genome and infectious viral particles that result in disease symptoms development.
The GE plantain was generated using a multiplex CRISPR/Cas9 construct targeting the editing of integrated eBSV sequences in the host genome. Sequencing and phenotyping of the edited events showed targeted mutations and confirmed the inactivation of eBSV for its ability to be converted into infectious viral particles. This strategy can be applied to improve breeding lines, which can then be used to develop plantain hybrids with no risk of activation of the functional virus.
We are currently trying to develop the genome-edited banana varieties by disrupting the function of disease-causing susceptibility (‘S’) genes, the negative regulators of plant defense, and sugar transporters as a strategy to develop resistance against bacterial and fungal pathogens.
The target genes in a banana for resistance to bacterial disease have been identified through the comparative transcriptomics of the resistant wild type banana Musa balbisiana and susceptible banana Pisang Awak (Tripathi et al. 2019c).
Yam production is severely limited by pests and diseases. The genetic improvement of yam by conventional breeding is very challenging and can be complemented using the GE tool. Therefore, an efficient CRISPR-Cas9 based GE system has been developed for Dioscorea rotundata using PDS gene in Amola, a farmer-preferred variety (Fig. 2). The establishment of CRISPR/Cas9 system will facilitate the improvement of yam for economically important traits.
GE has shown immense potential for crop improvement, but its regulation is still in the early stages. There are differences among the countries regarding the regulation of genome-edited crop varieties (Fig. 3). GE technologies are capable of creating genotypic and phenotypic variations in plants that are indistinguishable from those produced through natural means or conventional mutagenesis methods. The genome-edited varieties with no foreign gene integration are not regulated in several countries like Argentina, Australia, Brazil, Chile, Canada, Japan, and the USA. Several other countries like Kenya, Nigeria, and India are in the process of developing the regulatory guidelines for the application of genome editing. Science-based regulatory guidelines will enhance the adoption of disease-resistant GE varieties, hence contributing to food security.
Authors: Leena Tripathi, Valentine Otang Ntui, and Jaindra Nath Tripathi