Dirigent (DIR) proteins are important regulators of stereoselective lignan biosynthesis and play significant roles in lignification and plant defense against both biotic and abiotic stresses. However, the role of the DIR gene family in banana (Musa spp.) under such stress conditions remains largely unexplored. Therefore, a genome-wide analysis of the DIR gene family was undertaken to characterize these genes and to gain insights into their potential involvement in stress responses, with the aim of supporting the development of improved stress-resistant banana cultivars. A total of 26 DIR genes were identified in Musa acuminata (A genome) and 30 in M. balbisiana (B genome), and classified into nine subfamilies based on phylogenetic relationships. Duplication analysis indicated that purifying selection has played a major role in their evolution. Promoter analysis showed enrichment of hormone- and stress-responsive cis-elements, particularly those related to jasmonic acid, salicylic acid, and abscisic acid signaling. Expression profiling under Pseudocercospora eumusae, Pratylenchus coffeae, and moisture deficit stresses revealed that several dir genes, especially MaDIR-BD-unc4, were strongly induced in resistant cultivars at early stress stages. SNP analysis further identified resistance-associated variants in tolerant genotype. These findings provide a foundation for functional validation and highlight the potential of dir genes for improving stress resistance in banana through molecular breeding.

Pathogenesis-related protein 1 (PR-1) is a key plant defense protein, but its gene family evolution in Musa, Ensete, and Musella is not well understood. In this study, we conducted a comprehensive pangenome wide analysis of pr-1 genes across species, subspecies, and cultivars of Musa, Ensete and Musella using genomic datasets. A total of 104 pr-1 genes were identified and characterized through multiple in-silico comparative analyses. The PR-1 gene family exhibited notable variation in gene number reflecting extensive pangenome level diversity. Motif architecture and gene structure assessments revealed both conserved features and lineage-specific modifications among homoeologous pr-1 genes. Ka/Ks analysis indicated that pr-1 clade orthologs have predominantly undergone positive selection, followed by members of the intermediate clade. Collinearity analysis detected a single tandem duplication event in Musa textilis (abacá), while segmental duplications were predominant across other species. Promoter analysis revealed a wide array of cis-regulatory elements responsive to drought, cold, ethylene, methyl jasmonate (MeJA), abscisic acid (ABA), and gibberellic acid (GA). Transcriptome data mining further showed strong induction of several pr-1 genes under major biotic and abiotic stresses. Collectively, this study provides new insights into the evolutionary patterns, structural diversity, and functional specialization of the pr-1 gene family across Musa, Ensete and Musella. Importantly, it establishes the first pangenome resource for pr-1 genes in these genera, offering a valuable foundation for identifying key candidate genes that can be strategically utilized to enhance stress resilience in banana breeding programs.

Banana (Musa spp.) is a globally important staple and cash crop where an efficient anther culture protocol has been developed to enable the production of doubled haploid (DH) lines for use in breeding programs and genomic research. This study compared the effectiveness of different culture media Murashige and Skoog (MS), N6, and Nitsch in inducing embryogenic callus formation from anthers of five different banana genotypes across three genomic groups (BB, AA, AAA). Among the media tested, MS medium proved to be most effective for embryogenic callus induction. Among the five genotypes tested, Bhimkol (BB) produced embryogenic callus on MS medium supplemented with 4.0 mg L−1 2.4-Dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L−1 each of Indole-3-acetic acid (IAA), and α-Naphthaleneacetic acid (NAA). The callus were sub-cultured three times at regular intervals onto MS medium fortified with 2.0 mg L−1 IAA and 0.5 mg L−1 6-Benzyl amino purine (BAP). The resultant shoots were then transferred to MS medium supplemented with 2.0 mg L−1 BAP and 0.5 mg L−1 IAA to enhance shoot multiplication and elongation. Rooting was successfully achieved by culturing the single shoots on MS medium containing 1.0 mg L−1 each of Indole-3-butyric acid (IBA) and NAA. The rooted plantlets were acclimatized successfully, with 60

Incomplete editing and chimeric phenotypes are major challenges in CRISPR/Cas9-mediated genome editing of polyploid crops. In this study, a single guide RNA (gRNA) was designed to target a conserved dinucleotide-binding motif within exon 3 of the phytoene desaturase (PDS) gene in ‘Grand Naine’ banana. The gRNA was carefully selected for GC content, guanine residues near the PAM, and predicted secondary structure to enhance Cas9 cleavage efficiency. Agrobacterium-mediated transformation of embryonic cell suspensions produced 102 putative transgenic plants, all exhibiting altered phenotypes, with 91% displaying albino and 9% pale green coloration, indicating efficient PDS gene knockout and absence of chimerism. Sequencing confirmed tri-allelic editing, with all edited plants consistently showing two identical and one distinct mutation. Notably, small in-frame deletions of two to six amino acids within the conserved motif were sufficient to abolish PDS function, confirming its critical role in carotenoid biosynthesis. This strategy is adaptable to clonally propagated polyploid crops, providing a practical framework for achieving high-efficiency, uniform genome edits and supporting the development of precise, non-chimeric CRISPR/Cas9 editing approaches.

Enhancing bananas (Musa sp.) through traditional breeding methods is difficult because of the intricate reproductive biology of the plant. This study evaluated the breeding potential of Kothia (ABB)-based progenies, which are typically discarded due to poor fruit filling. Hybridization of Kothia with diverse AA diploid accessions resulted in progenies with low seed set, poor regeneration rates (5.4%), and low frequency of parthenocarpy progenies (0.09%) reflecting inherent cross-compatibility limitations. Among 56 full-sib progenies (FSPs), only one exhibited parthenocarpy, while the rest were non-parthenocarpic with variable female fertility. Two parthenocarpic half-sib progenies (HSPs) were also obtained from these FSPs. Flow cytometry revealed ploidy variation, with most progenies being diploid, and some showing triploid or aneuploid status. Heterosis analysis found that most agronomic traits in FSPs had a negative relative heterosis, but HSPs showed positive heterosis for the number of hands per bunch. Interestingly, six FSPs and both HSPs were resistant to Fusarium wilt (Foc race 1), suggesting they could be useful in breeding programs for disease resistance. One HSP (Pr. 933) displayed a phenotype closely resembling the commercial cultivar Grand Nain (AAA). Another HSP (Pr. 932) showed significantly higher beta-carotene content than commercial cultivars and even surpassed some provitamin A-rich plantains. Volatile compound analysis revealed a unique aroma profile in Pr. 932, contributing to its superior flavor. This study shows that using non-parthenocarpic, poor-fruit-filling progenies can help increase the genetic diversity of bananas, allowing for the use of disease resistance, better nutrition, and unique taste qualities.

The diverse ploidy level of banana derived from Musa acuminata (AA) and Musa balbisiana (BB) are pivotal for crop improvement. Accurate ploidy determination is essential but hindered by costly, expertise intensive methods like root-tip chromosome counting and flow cytometry. Hence, this study aimed to explore morphological, stomatal and palynological differences between Ney Poovan diploid (NP2) and anther/microspore derived autotetraploid (NP4) for practical ploidy differentiation. Twenty-five morphological traits along with stomatal and palynological attributes were evaluated. Additionally, leaf droopiness traits were compared with flow cytometry. Key findings revealed distinctions in 17 morphological traits using Bonferroni correction, with pronounced leaf droopiness in NP4 as a standout indicator. Stomatal traits corroborated this, with NP4 exhibiting larger stomata and lower density. Furthermore, pollen analysis indicated restored male fertility in NP4 with high pollen production, viability and germination. Flow cytometry validated the reliability of leaf droopiness traits in distinguishing NP4 from NP2. In conclusion, this study offered critical insights for advancing banana breeding programs, with each method contributing to a comprehensive strategy for improving the success of ploidy based breeding efforts.

Automation in somatic embryogenesis offers a promising solution to meet the demand for high-quality planting material and to reduce production costs compared to current in vitro shoot multiplication techniques. This study presents a novel temporary immersion bioreactor (TIB) system designed specifically for somatic embryogenesis, utilizing embryogenic cell suspensions (ECS) for plant germination. The system demonstrated high efficiency, with one milliliter of settled cell volume (SCV) of embryogenic cells producing 12.54 ± 0.4 g of somatic embryos. Subsequently, 300 milligrams of these embryos yielded 2392 ± 60 number of plants in banana. Genetic integrity of the plants was evaluated using flow cytometry and simple sequence repeats (SSR) marker analysis, revealing no variations in nuclear DNA peaks or SSR banding patterns, and field planting showed no adverse effects on vegetative or yield characteristics. Based on these insights, we highlight key challenges, knowledge gaps, technical solutions, research opportunities, and the need for a strategic framework to support industry for the adoption of integrated ECS and TIB technology for commercial production of high quality planting material. Addressing these gaps is essential to promoting industry-wide adoption, enabling cost-effective, large-scale propagation of high-quality planting material for sustainable agriculture and enhanced food security. A newly developed temporary immersion bioreactor for somatic embryogenesis enhances efficiency, scalability, and maintain genetic stability, facilitating cost-effective, large-scale propagation of high-quality planting material for sustainable agriculture.

Fusarium wilt, caused by Fusarium oxysporum f.sp. cubense (Foc), poses a significant threat to banana crops. Circular RNAs (circRNAs), a newly discovered group of non-coding RNA, play a role in gene regulation. However, Musa spp. has not been explored for circRNA detection. This study presents the first identification of circRNAs in resistant and susceptible banana cultivars infected with Foc race 1 and TR4. A substantial number of circRNAs were identified from RNA-seq libraries of infected corm and root tissues. Differential expression analysis revealed specific circRNAs associated with Foc-resistant and susceptible cultivars. Functional annotation and pathway analysis highlighted involvement in secondary metabolite biosynthesis, Mitogen-activated protein kinases (MAPK) signalling pathway, and plant-pathogen interaction. Utilizing the differentially expressed circRNAs, we conducted a microRNA miRNA-mediated interaction analysis to identify potential sponge interactions. Subsequently, a comprehensive circRNA–miRNA–messenger RNA (mRNA) network was constructed, incorporating target genes directly or indirectly associated with plant resistance. This network sheds light on the intricate regulatory mechanisms underlying plant resistance to Fusarium wilt and provides valuable insights into the complex interplay between circRNAs, miRNAs, and mRNAs in banana defense responses.

Nudix hydrolases, which catalyze the hydrolysis of nucleoside diphosphate derivatives, play critical roles in plant detoxification and homeostasis. Despite their significance, a comprehensive analysis of the NUDX gene family in Musa spp. remains unexplored. This study identified 30 and 31 putative Nudix hydrolases in the A and B genomes of Musa spp., respectively, categorizing them into eight subfamilies based on substrate specificity. Segmental and tandem duplications have driven the expansion of these genes. Phylogenetic analysis identified that the NUDX genes Macma4_08_g27360, Macma4_04_g28130, and Macma4_10_g25320 are closely related to AtNUDT7, a known negative regulator of basal immunity. Transcriptome and real-time PCR analyses further revealed significant up-regulation of these NUDX genes in banana cultivars susceptible to Fusarium wilt and Eumusae leaf spot. These findings suggest that these genes may act as susceptibility factors, highlighting their potential role in plant immunity against these biotic stresses.

Success of banana (Musa acuminata L.) breeding mainly depends on the male diploid parents used in the hybridization program. Developing intermediate diploid breeding lines with fertile pollen production ability, resistance to biotic and abiotic stresses, and fruit quality through the diploid x diploid strategy is the key step in improving commercial cultivars. Till now, at the global level only a limited number of diploid accessions is being used in banana breeding programs. In the present study, through the diploid x diploid breeding strategy, the genetic diversity of diploids has been broadened by developing nine polleniferous biotic-stress-resistant diploid lines. Among these, except for two progenies (P), all were resistant to the eumusae leaf spot disease. A single progeny (P 134) showed resistance to root-lesion and root-knot nematodes, whereas P 148 showed moderate resistance to root-knot nematodes alone. Three progenies of cv. Rose x Pisang Lilin (P 427, P 428 and P 429) were moderately resistant to banana stem weevil. Irrespective of cross combinations, barring two, all other progenies exhibited resistant reactions to fusarium wilt, Foc race 1 under both hot spot and pot culture screening. Among the nine diploid progenies, P 427, P 428 and P 429 were the best prospects to contribute multiple resistance against pests (banana stem weevil) and diseases (eumusae leaf spot and Foc race 1), whereas P 134 could be used as the resistant source for root-knot nematodes. The pyramiding of resistant/defense-related alleles in the progenies has been confirmed through SSR markers associated with Foc resistance. These polleniferous resistant improved diploid progenies are the potential source for accelerating the banana breeding program for improving biotic resistances in the triploid commercial cultivars through reconstructive breeding and/or for stacking the resistant genes in other genetic backgrounds.

AbstractBananas (Musa spp.) are one of the most highly consumed fruits globally, grown in the tropical and sub‐tropical regions. We evaluated 856 Musa accessions from the breeding programs of the International Institute of Tropical Agriculture of Nigeria, Tanzania, and Uganda; the National Agricultural Research Organization of Uganda; the Brazilian Agricultural Research Corporation (Embrapa); and the National Research Centre for Banana of India. Accessions from the in vitro gene bank at the International Transit Centre in Belgium were included to provide a baseline of available global diversity. A total of 16,903 informative single nucleotide polymorphism markers were used to estimate and characterize the genetic diversity and population structure and identify overlaps and unique material among the breeding programs. Analysis of molecular variance displayed low genetic variation among accessions and diploids and a higher variation among tetraploids (p < 0.001). Structure analysis revealed two major clusters corresponding to genomic composition. The results indicate that there is potential for the banana breeding programs to increase the diversity in their breeding materials and should exploit this potential for parental improvement and to enhance genetic gains in future breeding efforts.

Background Pathogen-related proteins (PR) are pivotal in plant defense, combating diverse biotic and abiotic stresses. While multiple gene families contribute to banana resistance against Fusarium oxysporum f sp. cubense (Foc), Pseudocercospora eumusae , and Pratylenchus coffeae , the significance of PR-1 genes in defense is paramount. Methods Three PR-1 genes, up-regulated under diverse biotic stresses, were cloned from both resistant and susceptible cultivars of Foc, P. eumusae , and P. coffeae . Molecular characterization, phylogenetic analysis, and docking studies with the Foc TR4 CP gene were conducted. Results Through transcriptomic and real-time studies, three PR-1 genes (Ma02_g15050, Ma02_g15060, and Ma04_g34800) from Musa spp. were identified. These genes exhibited significant up-regulation in resistant cultivars when exposed to Foc, P. eumusae , and P. coffeae . Cloning of these genes was successfully performed from both resistant and susceptible cultivars of Foc race 1 and TR4, P. eumusae , and P. coffeae . Distinct characteristics were observed among the PR-1 genes, with groups 1 and 2 being acidic with signal peptides, and group 3 being basic without signal peptides. All cloned PR-1 proteins belonged to the CAP superfamily (PF00188). Phylogenetic analysis revealed clustering patterns for acidic PR-1 proteins, and KEGG orthology showed associations with vital pathways, including MAPK signaling, plant hormone signal transduction, and plant-pathogen interaction. Secondary and tertiary structure analyses confirmed sequence conservation across studied species. Docking studies explored interactions between the cerato-platanin (CP) gene from Foc TR4 and Ma02_g15060 from banana, suggesting the potential hindrance of PR-1 antifungal activity through direct interaction. Conclusions The findings underscore the crucial role of cloned PR-1 genes in banana plant defense mechanisms against a broad spectrum of biotic stresses. These genes, especially those in groups 1 and 2, hold promise as candidates for developing stress-tolerant banana cultivars. The study provides valuable insights into the molecular aspects of banana defense strategies, emphasizing the potential applications of PR-1 genes in enhancing banana resilience.

Bananas are an important staple food crop, but their production is often hindered by pests and diseases. Improvement of banana is possible with the identification and isolation of superior alleles for key genes from the banana gene pool. Allele mining in banana is a promising approach with the freely available genome and transcriptome sequences of banana species, subspecies, and cultivars to dissect naturally occurring allelic variation at candidate genes regulating key traits. Additionally, allele mining can help trace allele evolution, identify new haplotypes, and develop allele-specific markers. This chapter discusses the concepts, approaches, and applications of allele mining in banana, while also highlighting the challenges and the need for more sophisticated 'mining' strategies to speed up allele discovery and its use in molecular banana breeding.

This is the first initiative study to provide quantified levels of four endogenous phytohormones during the induction, regeneration, maturation and germination stages of somatic embryogenesis (SE) in banana, using immature male bud explants, in the popular commercial triploid cultivar Grand Naine (AAA). Phytohormones are known to play a major role in the regulation of SE and most protocols have been developed for various cultivars of banana following empirical approaches. Although, several reports are available with regard to the impact of growth regulators exogenously supplemented to the media, the vital role of endogenous growth hormones in various developmental stages of SE remain unclear in banana. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) was used to analyze endogenous status of indole acetic acid (IAA), zeatin, gibberellic acid (GA) and abscisic acid (ABA). Results from this study revealed that endogenous IAA and GA along with exogenous auxin play a major role in the induction of embryogenic competence of male bud explants. The levels of endogenous IAA and zeatin were found to be higher in embryogenic calli than non-embryogenic calli, although three different types of exogenous auxins were supplemented in callogenesis medium, but not cytokinin. Higher level of ABA and GA was observed in early and late developmental stages of somatic embryo, respectively. GA also played a predominant role in germination of somatic embryos. These findings will be applied for improvement of SE protocol in recalcitrant banana cultivars by suitably altering or supplementing exogenous growth hormones.

Temporary immersion bioreactor (TIB) technology offers a cost-effective means of in vitro micropropagation for large-scale plantlet production, and is increasingly being recognized as a valuable tool in the plant tissue culture industry. However, the limited accessibility of the bioreactor in certain markets is hindering its potential benefits. Here, we report three different TIB systems for large-scale plantlet production in banana. Shoot tip explants of commercially important banana cultivars such as Grand Naine (AAA), Red Banana (AAA), Nendran (AAB), and Ney Poovan (AB) were initiated aseptically in semisolid medium and cultured in the TIB systems using liquid medium supplemented with 4.0 mg/l 6-benzylaminopurine (BAP), 0.2 mg/l indole-3-acetic acid (IAA), and 30 g/l sucrose for shoot multiplication and liquid medium supplemented with 1.0 mg/l indole-3-butyric acid (IBA) and 2.0 mg/l 1-naphthaleneacetic acid (NAA) for rooting of shoots. The liquid media were supplied periodically (2 min for every 6-hr frequency) to the growth vessels and recorded the data on number of shoots per explant, shoot length and number of roots per shoot. The data were com-pared with explants cultured in semisolid medium. The three TIBs showed superior response over semisolid culture system in shoots per explant, shoot length and number of roots per shoot. There was no significant difference within TIBs in photosynthetic efficiency as measured by chlorophyll pigment analysis. Plantlets cultured in TIBs showed better survival under ex vitro conditions. The genetic integrity of the plantlets grown in TIBs were confirmed by flow cytometry and simple-sequence repeat (SSR) markers analysis. The devel-oped TIBs and protocol can be utilized for commercial purpose. (c) 2023 SAAB. Published by Elsevier B.V. All rights reserved.

Summary Banana with different genomic groups has a greater varietal diversity. New varieties are also developed which have unique tastes, and tolerance to biotic or abiotic stresses. Eleven varieties belonging to different genomic groups are evaluated for their nutrients, secondary metabolites, techno‐functional characteristics, antioxidant activities, glycemic indices, and shelf life. Bioactive profiling was also carried out using Gas Chromatography–Mass Spectroscopy (GCMS). Results revealed that plantain and cooking bananas recorded higher fruit weight than dessert bananas. Pulp to peel ratio was higher in the dessert variety Ney Poovan and dual‐purpose variety Popoulu compared to cooking varieties indicating the presence of a thicker skin. Higher resistant starch (39% dry weight) and lower glycemic index (45%–50%) were observed in Kaveri Saba and Monthan. Total phenolics in ripe bananas were significantly higher than in unripe stages. GCMS results showed the predominance of esters and hydrocarbons among banana varieties and the presence of unique volatiles like linoelaidic acid, 9,12‐octadecadienoic acid in Kaveri Sugantham. PCA and cluster analysis indicated that the biochemical parameters also varied with the genomic groups. The study creates new business opportunities for varieties beyond cv. Grand Naine both in the domestic and export markets.

A reliable and reproducible embryogenic cell suspension culture system established successfully for regeneration of plants using pro-embryogenic cell mass derived from secondary somatic embryos as explants. A semi-solid medium was employed, which consisted of Murashige and Skoog (MS) basal salts and vitamins supplemented with 4 mg l− 1 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg l− 1 biotin, 1 mg l− 1 indole acetic acid (IAA), 1 mg l− 1 naphthalene acetic acid (NAA), 25 mg l− 1 ascorbic acid, 3

The prized Red banana, selected for superior qualities, demands strong genetic uniformity for successful clonal propagation and preservation. Ensuring this uniformity early in the growth of in vitro Red banana plants is essential, as gene mutations and chromosome rearrangements during tissue culture can jeopardize both cloning and germplasm conservation. In this situation, molecular markers play a pivotal role in confirming genetic stability. Thus the study aims to discover a marker that identifies tissue-cultured Red bananas from their virescent variants during initial sub-culturing. A marker linked to anthocyanin has been identified which effectively differentiated Red bananas from virescent variants and it was further validated in various banana cultivars, ornamental Musa species and their interspecific hybrids. The PCR-based marker showed remarkable specificity, discerning Red bananas from virescent variants during tissue culture. It also distinguished green and red offspring, cutting time and resource costs, and shortening the banana breeding cycle.