Targeted knockout of early nodulin-like 3 (MusaENODL3) gene in banana reveals its function in resistance to Xanthomonas wilt disease.

Nodulins and nodulin-like proteins play an essential role in the symbiotic associations between legumes and Rhizobium bacteria. Their role extends beyond the leguminous species, as numerous nodulin-like proteins, including early nodulin-like proteins (ENODL), have been identified in various non-leguminous plants, implying their involvement in functions beyond nodulation, such as nutrient transport and growth modulation. Some ENODL proteins have been associated with plant defense against pathogens, as evident in banana infected with Xanthomonas campestris pv. musacearum (Xcm) causing banana Xanthomonas wilt (BXW) disease. Nonetheless, the specific role of ENODL in plant defense remains to be fully elucidated. The MusaENODL3 gene was found to be repressed in BXW-resistant banana progenitor 'Musa balbisiana' and 20-fold upregulated in BXW-susceptible cultivar 'Gonja Manjaya' upon early infection with Xcm. To further unravel the role of the ENODL gene in disease resistance, the CRISPR/Cas9 system was employed to disrupt the MusaENODL3 gene in 'Gonja Manjaya' precisely. Analysis of the enodl3 edited events confirmed the accurate manipulation of the MusaENODL3 gene. Disease resistance and gene expression analysis demonstrated that editing the MusaENODL3 gene resulted in resistance to BXW disease, with 50% of the edited plants remaining asymptomatic. The identification and manipulation of the MusaENODL3 gene highlight its potential as a critical player in plant-pathogen interactions, offering new opportunities for enhancing disease resistance in crops like banana, an important staple food crop and source of income for resource-poor farmers in the tropics. This study provides the first evidence of the direct role of the ENODL3 gene in developing disease-resistant plants.

Advances in Somatic Embryogenesis of Banana.

The cultivation of bananas and plantains (Musa spp.) holds significant global economic importance, but faces numerous challenges, which may include diverse abiotic and biotic factors such as drought and various diseases caused by fungi, viruses, and bacteria. The genetic and asexual nature of cultivated banana cultivars makes them unattractive for improvement via traditional breeding. To overcome these constraints, modern biotechnological approaches like genetic modification and genome editing have become essential for banana improvement. However, these techniques rely on somatic embryogenesis, which has only been successfully achieved in a limited number of banana cultivars. Therefore, developing new strategies for improving somatic embryogenesis in banana is crucial. This review article focuses on advancements in banana somatic embryogenesis, highlighting the progress, the various stages of regeneration, cryopreservation techniques, and the molecular mechanisms underlying the process. Furthermore, this article discusses the factors that could influence somatic embryogenesis and explores the prospects for improving the process, especially in recalcitrant banana cultivars. By addressing these challenges and exploring potential solutions, researchers aim to unlock the full potential of somatic embryogenesis as a tool for banana improvement, ultimately benefiting the global banana industry.

Control of Bacterial Diseases of Banana Using CRISPR/Cas-Based Gene Editing.

Banana is an important staple food crop and a source of income for smallholder farmers in about 150 tropical and sub-tropical countries. Several bacterial diseases, such as banana Xanthomonas wilt (BXW), blood, and moko disease, cause substantial impacts on banana production. There is a vast yield gap in the production of bananas in regions where bacterial pathogens and several other pathogens and pests are present together in the same field. BXW disease caused by Xanthomonas campestris pv. musacearum is reported to be the most destructive banana disease in East Africa. The disease affects all the banana varieties grown in the region. Only the wild-type diploid banana, Musa balbisiana, is resistant to BXW disease. Developing disease-resistant varieties of bananas is one of the most effective strategies to manage diseases. Recent advances in CRISPR/Cas-based gene editing techniques can accelerate banana improvement. Some progress has been made to create resistance against bacterial pathogens using CRISPR/Cas9-mediated gene editing by knocking out the disease-causing susceptibility (S) genes or activating the expression of the plant defense genes. A synopsis of recent advancements and perspectives on the application of gene editing for the control of bacterial wilt diseases are presented in this article.

Transgenic expression of the rice Xa21 pattern-recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum.

Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, biocontrol agents or resistant cultivars available to control BXW. Here, we take advantage of the robust resistance conferred by the rice pattern-recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21-mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar 'Gonja manjaya' (AAB) using a rapid bioassay and 12 transgenic lines in the glasshouse for resistance against Xcm. About 50% of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the nontransgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore, this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa.

Cassava molecular genetics and genomics for enhanced resistance to diseases and pests.

Cassava (Manihot esculenta) is one of the most important sources of dietary calories in the tropics, playing a central role in food and economic security for smallholder farmers. Cassava production is highly constrained by several pests and diseases, mostly cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). These diseases cause significant yield losses, affecting food security and the livelihoods of smallholder farmers. Developing resistant varieties is a good way of increasing cassava productivity. Although some levels of resistance have been developed for some of these diseases, there is observed breakdown in resistance for some diseases, such as CMD. A frequent re-evaluation of existing disease resistance traits is required to make sure they are still able to withstand the pressure associated with pest and pathogen evolution. Modern breeding approaches such as genomic-assisted selection in addition to biotechnology techniques like classical genetic engineering or genome editing can accelerate the development of pest- and disease-resistant cassava varieties. This article summarizes current developments and discusses the potential of using molecular genetics and genomics to produce cassava varieties resistant to diseases and pests.

Q&A: Methods for estimating genetic gain in sub-Saharan Africa and achieving improved gains.

Regular measurement of realized genetic gain allows plant breeders to assess and review the effectiveness of their strategies, allocate resources efficiently, and make informed decisions throughout the breeding process. Realized genetic gain estimation requires separating genetic trends from nongenetic trends using the linear mixed model (LMM) on historical multi-environment trial data. The LMM, accounting for the year effect, experimental designs, and heterogeneous residual variances, estimates best linear unbiased estimators of genotypes and regresses them on their years of origin. An illustrative example of estimating realized genetic gain was provided by analyzing historical data on fresh cassava (Manihot esculenta Crantz) yield in West Africa (https://github.com/Biometrics-IITA/Estimating-Realized-Genetic-Gain). This approach can serve as a model applicable to other crops and regions. Modernization of breeding programs is necessary to maximize the rate of genetic gain. This can be achieved by adopting genomics to enable faster breeding, accurate selection, and improved traits through genomic selection and gene editing. Tracking operational costs, establishing robust, digitalized data management and analytics systems, and developing effective varietal selection processes based on customer insights are also crucial for success. Capacity building and collaboration of breeding programs and institutions also play a significant role in accelerating genetic gains.

High Potassium, Calcium, and Nitrogen Application Reduce Susceptibility to Banana Xanthomonas Wilt Caused by Xanthomonas campestris pv. musacearum.

The effect of exogenous applications of potassium (K), calcium (Ca), and nitrogen (N) on the susceptibility of four banana cultivars to Banana Xanthomonas wilt (BXW) was studied. Murashige and Skoog (MS) medium with normal concentrations of K at 783 mg/liter, Ca at 121 mg/liter, and N at 841 mg/liter was modified to contain various concentrations of K, Ca, and N. Each nutrient was varied singly, each with three replicate experiments. The concentrations were K at 78, 157, 391, 783, 1,565, and 3,913 mg/liter; Ca at 12, 24, 60, 121, 241, and 603 mg/liter; and N at 84, 168, 420, 841, and 1,682 mg/liter. Plantlets were generated in vitro on normal MS medium and later exposed to the nutrient concentrations for a total of 8 weeks. Thereafter, they were artificially inoculated with Xanthomonas campestris pv. musacearum using an insulin syringe. In each nutrient, plantlets exposed to higher nutrient concentrations significantly (P < 0.0001) accumulated more nutrient in their tissues compared with those exposed to lesser nutrient concentrations. Wilt incidences were significantly reduced, and incubation periods (time from inoculation to appearance of first disease symptoms) increased, with increasing nutrient application. The study lays a background for in vivo studies aimed at management of BXW using nutrients, such as fertilizer application.

Precision genetics tools for genetic improvement of banana.

Banana is an important food security crop for millions of people in the tropics but it faces challenges from diseases and pests. Traditional breeding methods have limitations, prompting the exploration of precision genetic tools like genetic modification and genome editing. Extensive efforts using transgenic approaches have been made to develop improved banana varieties with resistance to banana Xanthomonas wilt, Fusarium wilt, and nematodes. However, these efforts should be extended for other pests, diseases, and abiotic stresses. The commercialization of transgenic crops still faces continuous challenges with regulatory and public acceptance. Genome editing, particularly CRISPR/Cas, offers precise modifications to the banana genome and has been successfully applied in the improvement of banana. Targeting specific genes can contribute to the development of improved banana varieties with enhanced resistance to various biotic and abiotic constraints. This review discusses recent advances in banana improvement achieved through genetic modification and genome editing.

Transgenic expression of Arabidopsis ELONGATION FACTOR-TU RECEPTOR (AtEFR) gene in banana enhances resistance against Xanthomonas campestris pv. musacearum.

Banana Xanthomonas wilt (BXW) caused by Xanthomonas campestris pv. musacearum (Xcm) is a severe bacterial disease affecting banana production in East and Central Africa, where banana is cultivated as a staple crop. Classical breeding of banana is challenging because the crop is clonally propagated and has limited genetic diversity. Thus, genetic engineering serves as a viable alternative for banana improvement. Studies have shown that transfer of the elongation factor Tu receptor gene (AtEFR) from Arabidopsis thaliana to other plant species can enhance resistance against bacterial diseases. However, AtEFR activity in banana and its efficacy against Xcm has not been demonstrated. In this study, transgenic events of banana (Musa acuminata) cultivar dwarf Cavendish expressing the AtEFR gene were generated and evaluated for resistance against Xcm under greenhouse conditions. The transgenic banana events were responsive to the EF-Tu-derived elf18 peptide and exhibited enhanced resistance to BXW disease compared to non-transgenic control plants. This study suggests that the functionality of AtEFR is retained in banana with the potential of enhancing resistance to BXW under field conditions.

Genome Editing for Sustainable Agriculture in Africa.

Sustainable intensification of agriculture in Africa is essential for accomplishing food and nutritional security and addressing the rising concerns of climate change. There is an urgent need to close the yield gap in staple crops and enhance food production to feed the growing population. In order to meet the increasing demand for food, more efficient approaches to produce food are needed. All the tools available in the toolbox, including modern biotechnology and traditional, need to be applied for crop improvement. The full potential of new breeding tools such as genome editing needs to be exploited in addition to conventional technologies. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing has rapidly become the most prevalent genetic engineering approach for developing improved crop varieties because of its simplicity, efficiency, specificity, and easy to use. Genome editing improves crop variety by modifying its endogenous genome free of any foreign gene. Hence, genome-edited crops with no foreign gene integration are not regulated as genetically modified organisms (GMOs) in several countries. Researchers are using CRISPR/Cas-based genome editing for improving African staple crops for biotic and abiotic stress resistance and improved nutritional quality. Many products, such as disease-resistant banana, maize resistant to lethal necrosis, and sorghum resistant to the parasitic plant Striga and enhanced quality, are under development for African farmers. There is a need for creating an enabling environment in Africa with science-based regulatory guidelines for the release and adoption of the products developed using CRISPR/Cas9-mediated genome editing. Some progress has been made in this regard. Nigeria and Kenya have recently published the national biosafety guidelines for the regulation of gene editing. This article summarizes recent advances in developments of tools, potential applications of genome editing for improving staple crops, and regulatory policies in Africa.

Transgenic Expression of dsRNA Targeting the Pentalonia nigronervosa acetylcholinesterase Gene in Banana and Plantain Reduces Aphid Populations.

The banana aphid, Pentalonia nigronervosa, is the sole insect vector of banana bunchy top virus (BBTV), the causal agent of banana bunchy top disease. The aphid acquires and transmits BBTV while feeding on infected banana plants. RNA interference (RNAi) enables the generation of pest and disease-resistant crops; however, its effectiveness relies on the identification of pivotal gene sequences to target and silence. Acetylcholinesterase (AChE) is an essential enzyme responsible for the hydrolytic metabolism of the neurotransmitter acetylcholine in animals. In this study, the AChE gene of the banana aphid was targeted for silencing by RNAi through transgenic expression of AChE dsRNA in banana and plantain plants. The efficacy of dsRNA was first assessed using an artificial feeding assay. In vitro aphid feeding on a diet containing 7.5% sucrose, and sulfate complexes of trace metals supported aphid growth and reproduction. When AChE dsRNA was included in the diet, a dose of 500 ng/µL was lethal to the aphids. Transgenic banana cv. Cavendish Williams and plantain cvs. Gonja Manjaya and Orishele expressing AChE dsRNA were regenerated and assessed for transgene integration and copy number. When aphids were maintained on elite transgenic events, there was a 67.8%, 46.7%, and 75.6% reduction in aphid populations growing on Cavendish Williams, Gonja Manjaya, and Orishele cultivars, respectively, compared to those raised on nontransgenic control plants. These results suggest that RNAi targeting an essential aphid gene could be a useful means of reducing both aphid infestation and potentially the spread of the disease they transmit.

Strategies to combat the problem of yam anthracnose disease: Status and prospects.

Yam (Dioscorea spp.) anthracnose, caused by Colletotrichum alatae, is the most devastating fungal disease of yam in West Africa, leading to 50%-90% of tuber yield losses in severe cases. In some instances, plants die without producing any tubers or each shoot may produce several small tubers before it dies if the disease strikes early. C. alatae affects all parts of the yam plant at all stages of development, including leaves, stems, tubers, and seeds of yams, and it is highly prevalent in the yam belt region and other yam-producing countries in the world. Traditional methods adopted by farmers to control the disease have not been very successful. Fungicides have also failed to provide long-lasting control. Although conventional breeding and genomics-assisted breeding have been used to develop some level of resistance to anthracnose in Dioscorea alata, the appearance of new and more virulent strains makes the development of improved varieties with broad-spectrum and durable resistance critical. These shortcomings, coupled with interspecific incompatibility, dioecy, polyploidy, poor flowering, and the long breeding cycle of the crop, have prompted researchers to explore biotechnological techniques to complement conventional breeding to speed up crop improvement. Modern biotechnological tools have the potential of producing fungus-resistant cultivars, thereby bypassing the natural bottlenecks of traditional breeding. This article reviews the existing biotechnological strategies and proposes several approaches that could be adopted to develop anthracnose-resistant yam varieties for improved food security in West Africa.

CRISPR/Cas9-based genome editing of banana for disease resistance.

Banana production is severely constrained by many pathogens and pests, particularly where a number of them are co-existing. The use of disease-resistant banana varieties is one of the most effective ways to mitigate the negative impacts of pathogens on banana production. Recent advances in new breeding techniques have the potential to accelerate breeding of banana for disease resistance. The CRISPR/Cas9 based genome editing has emerged as the most powerful tool for crop improvement due to its capability of creating precise alterations in plant genome and trait stacking through multiplexing. Recently, the robust CRISPR/Cas9-based genome editing of banana has been established, which can be applied for developing disease-resistant varieties. This article presents a synopsis of recent advancements and perspectives on the application of genome editing for generating disease-resistant banana varieties. It also summarizes the current status of regulatory requirements for the release of genome-edited crop varieties among different countries.

Genome Sequence of the Banana Aphid, Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae) and Its Symbionts.

The banana aphid, Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae), is a major pest of cultivated bananas (Musa spp., order Zingiberales), primarily due to its role as a vector of Banana bunchy top virus (BBTV), the most severe viral disease of banana worldwide. Here, we generated a highly complete genome assembly of P. nigronervosa using a single PCR-free Illumina sequencing library. Using the same sequence data, we also generated complete genome assemblies of the P. nigronervosa symbiotic bacteria Buchnera aphidicola and Wolbachia To improve our initial assembly of P. nigronervosa we developed a k-mer based deduplication pipeline to remove genomic scaffolds derived from the assembly of haplotigs (allelic variants assembled as separate scaffolds). To demonstrate the usefulness of this pipeline, we applied it to the recently generated assembly of the aphid Myzus cerasi, reducing the duplication of conserved BUSCO genes by 25%. Phylogenomic analysis of P. nigronervosa, our improved M. cerasi assembly, and seven previously published aphid genomes, spanning three aphid tribes and two subfamilies, reveals that P. nigronervosa falls within the tribe Macrosiphini, but is an outgroup to other Macrosiphini sequenced so far. As such, the genomic resources reported here will be useful for understanding both the evolution of Macrosphini and for the study of P. nigronervosa. Furthermore, our approach using low cost, high-quality, Illumina short-reads to generate complete genome assemblies of understudied aphid species will help to fill in genomic black spots in the diverse aphid tree of life.

Introgression Breeding in Cowpea [Vigna unguiculata (L.) Walp.].

The narrow base of genetic diversity characteristic of cowpea can be attributed to it being self-pollinating, evolving from narrow wild germplasm and exhibiting very limited gene flow between wild and cultivated types. Backcrossing to introduce simply inherited desirable traits and utilization of improved breeding lines and varieties as parents in crossing programs further narrowed the genetic base of cowpea varieties. In most cowpea breeding programs, genes for resistance and market traits were pyramided into lines characterized by high levels of acceptance to farmers and consumers. Besides predisposing widely distributed improved varieties to genetic vulnerability, a narrow base of genetic variation may be contributing to the plateauing in cowpea grain yield, which compromises genetic gains. Cross compatible wild relatives have not been used in variety development because breeders shy away from them due to their tiny seed size, unattractive seed coat color and texture, pod shattering, and susceptibility to viruses. A number of wild cowpea relatives, both within and outside section Catiang of Vigna species, have been evaluated for their reaction to cowpea insect pests and diseases. Vigna vexillata lines were resistant to the legume pod borer (Maruca vitrata), the cowpea weevil (Callosobruchus maculatus), and Striga gesnerioides but are cross incompatible with cultivated cowpea. Some lines among the cross compatible wild relative V. unguiculata ssp. dekindtiana were found to be resistant to aphid in the seedling stage, while others showed good levels of drought and heat tolerance. Molecular markers are being generated to identify quantitative trait loci (QTL) with effects on some desirable attributes in cowpea. Modern breeding tools, including transgenics, can be applied for the improvement of cowpea, bypassing the natural barriers of traditional breeding. Transgenic cowpea with Bt gene cry1Ab showing resistance to M. vitrata has been released in Nigeria. Genome editing, a powerful emerging tool, can also be used for developing improved cowpea varieties with durable resistance to pests and diseases.

Application of CRISPR/Cas for Diagnosis and Management of Viral Diseases of Banana.

Viral diseases are significant biotic constraints for banana (Musa spp.) production as they affect the yield and limit the international movement of germplasm. Among all the viruses known to infect banana, the banana bunchy top virus and banana streak viruses are widespread and economically damaging. The use of virus-resistant bananas is the most cost-effective option to minimize the negative impacts of viral-diseases on banana production. CRISPR/Cas-based genome editing is emerging as the most powerful tool for developing virus-resistant crop varieties in several crops, including the banana. The availability of a vigorous genetic transformation and regeneration system and a well-annotated whole-genome sequence of banana makes it a compelling candidate for genome editing. A robust CRISPR/Cas9-based genome editing of the banana has recently been established, which can be applied in developing disease-resistant varieties. Recently, the CRISPR system was exploited to detect target gene sequences using Cas9, Cas12, Cas13, and Cas14 enzymes, thereby unveiling the use of this technology for virus diagnosis. This article presents a synopsis of recent advancements and perspectives on the application of CRISPR/Cas-based genome editing for diagnosing and developing resistance against banana viruses and challenges in genome-editing of banana.