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.

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.

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.

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.

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.

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.

RNAi technology for management of banana bunchy top disease.

Banana bunchy top disease (BBTD) is one of the world's most destructive viral diseases of banana and plantain, causing up to 100% yield loss in severe cases. The disease is vectored by banana aphids (Pentalonia nigronervosa) and carried long distances through the movement of infected plant materials. The banana aphids harboring banana bunchy top virus (BBTV) present in banana producing regions are the sole vector and the most efficient method of transmitting the virus to the healthy plants. Controlling the spread of BBTD has been very challenging since no known banana germplasm is immune to BBTV. The disease can be managed with the use of virus-free planting material and roguing. However, once BBTD is established in the field, it is very difficult to eradicate or manage it. Therefore, a more sustainable way of controlling the disease is developing host plant resistance against the virus and the vector. Biotechnological strategies via RNA interference (RNAi) could be used to target the banana aphid as well as BBTV to reduce virus-associated yield losses of banana and plantain, which feed over 500 million people around the world. This review discusses the status of BBTD and perspectives on effective RNAi technologies for controlling BBTV and the vector, banana aphid, transmitting the virus as sustainable management of the disease.

Phytochemical Analysis and Establishment of Embryogenic Cell Suspension and Agrobacterium-mediated Transformation for Farmer Preferred Cultivars of West African Plantain (Musa spp.).

Banana and plantain are among the foremost staple food crops providing food and livelihood to over 500 million people in tropical countries. Despite the importance, their production is hampered due to several biotic and abiotic stresses. Plant tissue culture techniques such as somatic embryogenesis and genetic transformation offer a valuable tool for genetic improvement. Identification and quantification of phytochemicals found in banana and plantain are essential in optimizing in vitro activities for crop improvement. Total antioxidants, phenolics, flavonoids, and tannins were quantified in various explants obtained from the field, as well as in vitro plants of banana and plantain cultivars. The result showed genotypic variation in the phytochemicals of selected cultivars. The embryogenic cell suspensions were developed for three farmer-preferred plantain cultivars, Agbagba, Obino l'Ewai, and Orishele, using different MS and B5-based culture media. Both culture media supported the development of friable embryogenic calli (FEC), while MS culture media supported the proliferation of fine cell suspension in liquid culture media. The percentage of FEC generated for Agbagba, Obino l'Ewai, and Orishele were 22 ± 24%, 13 ± 28%, and 9 ± 16%, respectively. Cell suspensions produced from FECs were successfully transformed by Agrobacterium-mediated transformation with reporter gene constructs and regenerated into whole plants.

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.

A simple and rapid protocol for the genetic transformation of Ensete ventricosum.

Enset (Ensete ventricosum), also known as Ethiopian banana, is a food security crop for more than 20 million people in Ethiopia. As conventional breeding of enset is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced disease resistance and nutritional value. Genetic transformation and subsequent regeneration of transgenic enset has never been reported mainly due to challenges in developing transformation protocols for this tropical species. Agrobacterium-mediated transformation could be a practical tool for the genetic improvement of enset. However, the efficiency of the transformation system depends on several parameters such as plant regeneration, genotype, explant, selection agent and Agrobacterium strains. As a first step towards the development of transgenic enset, a simple and rapid plant regeneration system was developed using multiple buds as explants. Induction and proliferation of multiple buds from shoot tip explants was achieved on Murashige and Skoog (MS) medium supplemented with 5 and 10 mg/l of 6-benzylaminopurine (BAP), respectively. Shoots were regenerated from multiple buds on MS media containing 2 mg/l BAP and 0.2% activated charcoal. Based on the optimized regeneration protocol, an Agrobacterium-mediated transformation method was developed using multiple buds as explants and the binary plasmid pCAMBIA2300-GFP containing the green florescent protein (gfp) reporter gene and neomycin phosphotransferase II (nptII) selection marker gene. Transgenic plantlets were obtained within 4 months at a frequency of about 1.25%. The transgenic lines were validated by PCR analysis using primers specific to the nptII gene. To obtain uniformly transformed plantlets, chimerism was diluted by subculturing and regenerating the transgenic shoots on a selective medium containing kanamycin (150 mg/l) for five cycles. The uniformity of the transgenic plants was confirmed by Southern blot hybridization and RT-PCR analyses on different tissues such as leaf, pseudostem and root of same transgenic plant. In the present study, we report a simple Agrobacterium-mediated transformation system for generating transgenic events of enset. To the best of our knowledge, this is the first report on the stable transformation and regeneration of transgenic events of enset. The transformation system established in this study can be used for the generation of transgenic enset with important traits such as disease resistance.

Molecular Basis of Disease Resistance in Banana Progenitor Musa balbisiana against Xanthomonas campestris pv. musacearum.

Banana Xanthomonas wilt disease, caused by Xanthomonas campestris pv. musacearum (Xcm), is a major threat to banana production in east Africa. All cultivated varieties of banana are susceptible to Xcm and only the progenitor species Musa balbisiana was found to be resistant. The molecular basis of susceptibility and resistance of banana genotypes to Xcm is currently unknown. Transcriptome analysis of disease resistant genotype Musa balbisiana and highly susceptible banana cultivar Pisang Awak challenged with Xcm was performed to understand the disease response. The number of differentially expressed genes (DEGs) was higher in Musa balbisiana in comparison to Pisang Awak. Genes associated with response to biotic stress were up-regulated in Musa balbisiana. The DEGs were further mapped to the biotic stress pathways. Our results suggested activation of both PAMP-triggered basal defense and disease resistance (R) protein-mediated defense in Musa balbisiana as early response to Xcm infection. This study reports the first comparative transcriptome profile of the susceptible and resistant genotype of banana during early infection with Xcm and provide insights on the defense mechanism in Musa balbisiana, which can be used for genetic improvement of commonly cultivated banana varieties.

Expression of sweet pepper Hrap gene in banana enhances resistance to Xanthomonas campestris pv. musacearum.

Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum, is the most devastating disease of banana in the Great Lakes region of Africa. The pathogen's rapid spread has threatened the livelihood of millions of Africans who rely on banana fruit for food security and income. The disease is very destructive, infecting all banana varieties, including both East African Highland bananas and exotic types of banana. In the absence of natural host plant resistance among banana cultivars, the constitutive expression of the hypersensitivity response-assisting protein (Hrap) gene from sweet pepper (Capsicum annuum) was evaluated for its ability to confer resistance to BXW. Transgenic lines expressing the Hrap gene under the regulation of the constitutive CaMV35S promoter were generated using embryogenic cell suspensions of two banana cultivars: 'Sukali Ndiizi' and 'Mpologoma'. These lines were characterized by molecular analysis, and were challenged with Xanthomonas campestris pv. musacearum to analyse the efficacy of the Hrap gene against BXW. The majority of transgenic lines (six of eight) expressing Hrap did not show any symptoms of infection after artificial inoculation of potted plants in the screenhouse, whereas control nontransgenic plants showed severe symptoms resulting in complete wilting. This study demonstrates that the constitutive expression of the sweet pepper Hrap gene in banana results in enhanced resistance to BXW. We describe the development of transgenic banana varieties resistant to BXW, which will boost the arsenal available to fight this epidemic disease and save livelihoods in the Great Lakes region of East and Central Africa.