Plants

BackgroundUrtica dioica, also known as stinging nettle, is a widespread plant that can indicate high nitrogen availability in the soil. It is probably best known for the pain caused by touching it. U. dioica is also recognized as a medicinal plant with reports claiming applicability against numerous diseases. ResultsA highly continuous genome sequence was constructed based on nanopore long read sequencing data. The total assembly size is 1.1 Gbp with an N50 of 40.7 Mbp. RNA-seq data and hints from other species were integrated to produce a high quality annotation of the protein encoding genes. This genomic resource enabled the identification of genes involved in the flavonoid biosynthesis. A particular focus was on anthocyanin biosynthesis genes as these are crucial for high light and nitrogen deprivation stress response, which is revealed by redding of the leaves. ConclusionThis genomic resource provides the basis for future studies unraveling the biosynthesis pathways underlying various medically important compounds produced by stinging nettles.

Belemia belongs to Nyctaginaceae and comprises two species of delicate vines. Both species are endemic to Brazil. Belemia fucsioides, the type species, described in 1981, occurs in a restricted area of the Atlantic Forest in southeastern Brazil. Belemia cordata, described in 2020, is known from only two records from the same area in the Cerrado of northern Brazil. Here, we describe the taxonomic history of Belemia and provide the first synopsis for the genus. We include species description, distribution map, identification key, and anatomical data. We used field observations over the past decade and modeled anthropogenic changes in the species range to conduct a conservation assessment in accordance with the IUCN Red List criteria. Conservation assessments indicate significant concerns for Belemia, classified as either endangered (B. fucsioides) or critically endangered (B. cordata). The species are threatened primarily by habitat loss to land used for agriculture, forestry, and livestock production. This study contributes to ongoing initiatives exploring plant diversity in the Neotropics and supports efforts to identify threats to biodiversity.

Heat tolerance determines the vitality of tree species under climate change independently of drought tolerance, but has been much less studied than tree water relations. We studied species-specific differences and the capacity for seasonal heat acclimation in Central Europes naturally most important tree species, Fagus sylvatica, in comparison with two exotic tree species (Fagus orientalis, Pseudotsuga menziesii) that are considered for silvicultural climate change adaptation in managed forests. Foliage of mature trees was incubated at temperatures from 35-50 {degrees}C for up to 4 h to simulate daily heat maxima during heat waves. The maximum quantum yield (Fv/Fm) of photosystem II (PS II) of dark-adapted leaves was measured, because the PS II is particularly sensitive to heat and its functionality can decide on plant survival under heat. Fagus sylvatica was much more tolerant to heat than Pseudotsuga menziesii, but weakly (albeit significantly) less tolerant than Fagus orientalis. Within its limits, Pseudotsuga menziesii showed high seasonal heat acclimation with constantly increasing tolerance during the growing season. Fagus orientalis, but practically not Fagus sylvatica, also acclimated to heat. This makes Fagus orientalis slightly superior over Fagus sylvatica in terms of heat tolerance, whereas the suitability of Pseudotsuga menziesii for silvicultural climate change adaptation is questionable. Strong heat acclimation, but also overall low heat tolerance, in Pseudotsuga menziesii might be the result of evergreenness, which requires the generation of both cold and heat tolerance during the year.

The production of reactive oxygen species (ROS) in response to cadmium (Cd) has been extensively studied, demonstrating that they play a key role in the plants response to this heavy metal. While the role of enzymes like RBOHs has been thoroughly studied, the function of other ROS-producing enzymes, such as peroxisomal glycolate oxidase (GOX), remains largely overlooked. Peroxisomal GOX is a core metabolic enzyme of the photorespiratory pathway occurring in chloroplasts, mitochondria and peroxisomes. Using Arabidopsis (Arabidopsis thaliana) mutants lacking the main peroxisomal GOX genes, GOX1 (gox1-1) and GOX2 (gox2-1) we explored their function in plant response to Cd. Although photosynthetic capacity appears to be affected to the same extent in both mutants under control and Cd stress conditions, GOX2 seems to play a greater role in ROS production in response to the metal. Transcriptomic analyses on WT and gox2-1 pointed to the mitochondrial electron transport chain (mETC) as a target of Cd stress. We further investigated the individual GOX1 and GOX2 functions in mETC regulation and redox state. Although oxidative ratio of mitochondria was higher in both mutants, it was more pronounced in the absence of GOX1. Furthermore, the mETC is affected in both mutants but the regulation of its components differs in each mutant. These results point out the different functions of the two photorespiratory GOX isoforms in Arabidopsis, leading to a better understanding of the photorespiratory pathway.

This paper presents the results of a structural analysis of chlorogenic acid isolated from a 70% ethanol extract of red clover (Trifolium pratense) callus culture. X-ray phase analysis showed that the sample was crystalline and single-phase and crystallized in an orthorhombic unit cell with the following parameters: a = 36.7548(5) [A], b = 11.0770(3) [A], c = 7.7947(2) [A], V = 3173.46(11) [A]3, R-Bragg = 0.347 %, Rexp = 4.75 %, Rwp = 5.83 %, Rp = 4.39 %, GOF = 1.23 %. NMR spectroscopy data (1H, 13C{1H}, 2D 1H1H-COSY, 1H13C-HSQC, 1H13C-HMBC) confirmed that the chemical structure and purity of the sample fully corresponded to chlorogenic acid, with no chemical impurities detected. Complete proton and carbon atom assignments are provided.

Prosopis juliflora is an invasive alien plant species and a problematic weed that poses significant ecological and socio-economic challenges in Ethiopia, particularly in the Afar rangelands. The study explored the diversity and effects of insect herbivores communities feeding on the flowers and pods of P. juliflora to determine their role in limiting reproductive success across three selected ecological sites: Amibara, Gewanne, and Aysayita. A total of 118 adult insect specimens were collected between January and November 2021 using a sweep net and hand collection methods. Community structure, analysis via the Shannon Wiener diversity index, strongly influenced damage pattern. Amibara exhibited the highest insect diversity resulting in significant reproductive damage, including 5.98% of flower loss and 10.39% pods tunneling, primarily caused by Chrysomelidae and Pyralidae. Conversely, Gewanne was showed lower diversity, but higher sap-sucking (13.39 % shriveled pods; 5.11 % flower curling) were caused by Aphididae. Overall, 18.41 % of the pods, and 11.59 % of the flowers were exhibited insect related injury. These finding confirm that more internal seed predation and nutrient depletion were revealed significantly reduce viable seed production. The result was suggested that natural insect communities currently function as partial biological control agents. This indicates strong potential for developing integrated biological control strategies to manage P. juliflora invasion in Ethiopia rangelands.

Two threatened new species of Podostemaceae belonging to the genus Inversodicraea, I. joulei and I. lebbiei, both from the Republic of Sierra Leone, are described and illustrated. A first record in Sierra Leone of the genus Lestestuella is also reported. Inversodicraea is the most species-rich genus of Podostemaceae in Africa and now comprises 38 species. Inversodicraea joulei is easily recognised because it has a persistent spine distally on the median rib of each fruit valve, and scattered, membranous scale-leaves with broadly rounded apices, while Inversodicraea lebbiei is distinct in having narrowly triangular robust scale-leaves which are inrolled, spreading distally, and completely covering the stem, arranged in five ranks. Inversodicraea joulei is known from a single location with three sites while I. lebbiei is known from two locations each with one site. Using the latest IUCN Red List guidance, Inversodicraea joulei is assessed as Critically Endangered and I. lebbiei is assessed as Endangered, due to threats from dam construction projects, agricultural practices and mining activities, resulting in high levels of siltation on rocks in the fast-flowing rivers where these species grow.

{gamma}-Glutamyl cyclotransferases (GGCTs) belongs to class of cytosolic enzymes that are responsible for glutathione (GSH) degradation under stress conditions. They regulate GSH homeostasis through the {gamma}-glutamyl cycle which is responsible for maintaining the synthesis of GSH as well as its breakdown, enabling recycling of its constituent amino acids. Although GGCTs have been implicated in enhancing heavy metal (HMs) tolerance in plants, their role in biotic stress remains largely unexplored. Previously, OsGGCT1 was identified as a gene strongly upregulated in Fusarium stress. In this study, the GGCT1 homolog from Oryza sativa japonica was characterized for its role in conferring tolerance to Fusarium oxysporum (F.O.). Similar to abiotic factors, biotic stresses significantly impact crop yield and productivity. The rhizosphere harbors diverse microbial communities, including harmful pathogens such as F. oxysporum. Fusarium causes wilt disease in a variety of plant species, such as: tomato, legumes, rice, and Arabidopsis thaliana. Our results demonstrate that overexpression of OsGGCT1 enhanced tolerance to F. oxysporum in A. thaliana, primarily by reducing fungal spore accumulation. Transgenic plants showed elevated expression of OsGGCT1 along with AtGSH1 and AtGSH2, reduced levels of reactive oxygen species (ROS), improved growth and photosynthetic performance and enhanced activities of the antioxidant enzymes. OsGGCT1 serves as a key component in maintaining GSH homeostasis by supporting glutamate (Glu) regeneration necessary for sustained GSH biosynthesis. Overall, these findings identify OsGGCT1 as an important constituent of the GSH-mediated detoxification pathway against Fusarium oxysporum and provide valuable molecular insights for developing Fusarium-tolerant rice varieties with reduced fungal accumulation.

The fully mycoheterotrophic, non-photosynthetic Afrothismia fonensis Cheek & G.Delhaye sp. nov. (Afrothismiaceae), is described and illustrated from two sites in submontane forest in or adjacent to the Pic de Fon Foret Classee, Simandou Range, Republic of Guinea. This is the first record of the genus and family in West Africa west of Nigeria. The new species is remarkable for its small size, and for being unique in the genus in the entirely connate intertepaline lobes (in other species of the genus they are free or only partly united) and the longitudinal ridges on the outer perianth tube (unknown in other species). The provisional extinction risk assessment for Afrothismia fonensis is Critically Endangered (CR B1ab (iii)+2ab(iii)+D1) using the IUCN 2012 categories and criteria, due to less than 50 individuals being recorded, and due to the both the very small range and the immediate threats from foraging by red river hogs, trampling by cattle and from de-watering of the adjacent Oueleba iron-ore body where mining began in 2025. It should be noted that mitigation actions are expected to adequately address the risks associated with mining activities, and direct impacts to both areas of Afrothismia fonensis habitat have been fully avoided through relocation of planned infrastructure. We review the importance of the Boyboyba forest, Simandou range, as the West African centre of diversity for non-photosynthetic heteromycotrophs. This new discovery is examined in the context of other recently discovered range extensions to Guinea of Central African genera and families.

This study presents the structural verification of baicalin isolated from a hydroethanolic extract of an in vitro Scutellaria baicalensis root culture using X-ray diffraction analysis and a set of NMR spectroscopy techniques. The crystalline molecular structure of the sample was found to correspond to baicalin. The 1H, 13C{1H}, 2D 1H1H-COSY, 1H13C-HSQC, 1H13C-HMBC spectra confirmed that the chemical shifts, signal multiplicities, integral intensities, and spin-spin coupling constants were fully consistent with the structure of the target compound. Minor impurity signals were detected in the aliphatic region of the spectra, with a total content not exceeding 5 mol%. These results confirm the high purity and structural individuality of baicalin, a biologically active flavonoid glycoside of considerable interest.

The transformation of the free nuclear syncytium into cellular endosperm tissue with starch and protein accumulation is a well-established phenomenon, at least in the fruits of cereals of the Triticeae tribe. The present article demonstrates that there is considerable diversity inherent in this type of caryopsis morphogenesis. By examining various taxa (species, varieties, and cultivars) of wheat, oats, and some wild grasses, this research reveals significant deviations in endosperm morphogenesis from the typical state. A new developmental pattern of endosperm was identified, characterized by several distinctive features such as incomplete cellularization of the syncytium and starch accumulation within the acellular endosperm domains and the endosperm cavity. A large number of plastids were observed in the syncytium stage, which served as the basis for the later amyloplast stage. The acellular endosperm domains and the cavity domain exhibited connections at specific discontinuities in the modified aleurone layer surrounding the cavity. The peripheral parts of the caryopsis received fewer assimilates necessary for starch synthesis, which was attributed to their increased distance from the transfer system and a likely reduction in the efficiency of assimilate transport through the apoplast in these areas. The starch cavity volume constituted a few percent of the overall caryopsis volume, which could serve as a foundation for potential breeding improvements to enhance starch yields across different varieties.

Attracting specific pollinators can be favoured by natural selection to avoid reproductive interference between sympatric plant species. However, the ways in which fine differences in floral traits lead to the attraction of specific pollinators are diverse and unknown in many pollination interactions. We surveyed pollinators on three sympatric Aristolochia species (A. clematitis, A. pistolochia and A. rotunda) pollinated by Diptera to investigate if specific pollination occurs. To decipher if specific pollination may be mediated by different floral odours, we characterized the volatile organic compounds (VOCs) emitted by flowers and highlighted those VOCs electrophysiologically detected by pollinators in A. rotunda and A. pistolochia. Among the most abundant pollinators, Forcipomyia monilicornis was a specific pollinator of A. pistolochia while two Dasyhelea species were specific pollinators of A. clematitis. Forcipomyia aristolochiae and T. ruficeps were non-specific pollinators of A. rotunda, although they were more frequently found in A. rotunda flowers. The floral odours of A. rotunda and A. pistolochia differed significantly from each other and elicited specific electrophysiological responses in their respective pollinators. Although several pollinator species visit more than one Aristolochia species, those pollinators are preferentially found in one Aristolochia species. Selective attraction is likely mediated by specific VOCs.

Indica rice, being a tropical crop, is highly sensitive to cold temperature. Cold stress affects vegetative growth, photosynthetic efficiency, along with reproductive features. Genetic resource screening in diverse landraces is an approach for identifying cold-tolerant traits. Here, we have characterised a boro germplasm, CB1, with an efficient germination rate and growth vigour when treated at chilling temperatures. CB1 seedlings show a higher survival rate compared to IR36 when subjected to prolonged chilling stress. Biochemical analyses indicated efficient ROS modulation, higher chlorophyll content, enhanced photosystem II efficiency and unique stomatal traits, leading to higher relative water content in CB1 plants during stress and recovery. Transcriptome analysis supported upregulation of chlorophyll biosynthesis, photosystem, & light harvesting complex and ROS scavenger genes in CB1 seedlings. Interestingly, high D1 protein turnover in CB1 promotes damage-repair of PSII for efficient photosynthesis. Furthermore, key transcription factors for stomatal development and expression of photosynthetic genes were upregulated in CB1 during stress recovery. Notably, higher expression of OsGLK1 and enrichment of GLK1 targets were observed in CB1 plants during chilling stress and recovery. Taken together, our results suggested that CB1 plants exhibit cold tolerance by modulating photosynthesis efficiency and stomatal behavior for better adaptability and survival against chilling temperature. HIGHLIGHTSThe efficient photosynthetic recovery, active ROS scavenging system and maintenance of water content through regulating stomatal traits, enhance the survival of indica germplasm CB1 against chilling stress.

Bread wheat (Triticum aestivum) is a staple food crucial for global caloric intake and food security. The current climate emergency demands the development of sustainable agricultural practices, particularly in the context of drought-induced yield reductions in bread wheat. Microalgae-based biostimulants have emerged as promising tools to enhance crop tolerance to drought stress while concurrently mitigating atmospheric CO2 accumulation. This study characterizes the transcriptomic responses to the foliar application of the microalgae-based biostimulant LRMTM in drought-stressed and fully irrigated wheat plants unveiling its mode of action. Drought stress at the tillering stage significantly altered gene expression activating key pathways related to phosphate starvation response (PSR), inositol phosphate signaling, and tocopherol biosynthesis. The application of the microalgae-based biostimulant LRMTM in drought-stressed plants further enhanced the expression of drought-responsive genes, particularly those involved in PSR and carbon fixation. Specific responses to LRMTM treatment in drought-stressed plants were also found related to abscisic acid (ABA) signaling activating genes involved in stomata closure, which plays a critical role in drought tolerance. In fully irrigated plants, LRMTM treatment was also beneficial modulating circadian rhythms, shade avoidance and attenuating stress responses. Phenotypic analysis showed that LRMTM-treated plants exhibited enhanced drought tolerance, increased height and spike length even under fully irrigated conditions. These results indicate that the microalgae-based biostimulant LRMTM not only enhances wheat response to drought but also promotes growth and productivity in both stressed and non-stressed conditions which could contribute to the development of sustainable agriculture in the face of the current climate challenges.

This investigation aimed at compiling all phylogenetic lineages within and around genus Cyanoboletus. The evolutionary inference obtained from the nuclear ribosomal genes internal transcribed spacer region (ITS) suggests that part of the species currently classified in Cyanoboletus belong in lineages separate from the genus, thus suggesting a narrower boundary that includes only the species that develop a strong staining reaction to touch and to air exposure of the context. The separate lineages are the monotypic Cupreoboletus genus and a few species that do not develop such reaction, which are part of a clade together with genera Cacaoporus and Acyanoboletus, thus broadening the concept of Cacaoporus to encompass all of them. The emerging 3C perspective of Cupreoboletus, Cacaoporus and Cyanoboletus offers a remarkably consistent morphological diagnosis, overcoming the problems of a too broad concept for Cyanoboletus. This work reveals that Boletus neotropicus, B. novae-zelandiae and B. sensibilis belong respectively in Cyanoboletus, Cacaoporus and Lanmaoa, and by studying multigene alignment concatenates it identifies lineages that probably represent undescribed species: at least four in Cacaoporus and at least five in Cyanoboletus. Diagnostic tables and dichotomic keys are presented by geographic region. The present work also includes a study of the phylogenetic position of Neoboletus flavosanguineus, a species once classified in Cyanoboletus. The complexity of assigning species epithets in some lineages is addressed, namely for the boundaries between Cacaoporus instabilis and Ca. fagaceophilus as well as the diversity under the names Cyanoboletus sinopulverulentus and Cy. pulverulentus. The overall picture of evolutionary lineages sets a framework for the choice of reference data that can provide, in future phylogenetic studies that involve the 3C, a balanced and efficient coverage. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/724631v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@7f618corg.highwire.dtl.DTLVardef@dd6a14org.highwire.dtl.DTLVardef@5f7399org.highwire.dtl.DTLVardef@9e7443_HPS_FORMAT_FIGEXP M_FIG C_FIG

Functional validation of genetic components in plants often requires cloning them separately into both plant and bacterial expression vectors, a process that is both time-consuming and laborious. This study aimed to simplify this workflow by developing plant-bacteria dual-host promoter systems that drive high-level constitutive expression in both environments. To achieve this, two variants of the chloramphenicol acetyltransferase promoter (PCAT), a bacterial {sigma} factor-dependent promoter, were integrated into the cauliflower mosaic virus 35S promoter (P35S), and their performance was evaluated using a hygromycin phosphotransferase (HPT)-GFP fusion reporter. One of these variants, PCAT1, conferred hygromycin resistance to Escherichia coli (DH5 and BL21 (DE3)) and maintained high-level expression comparable to the original P35S in onion epidermal cells. A hybrid P35S enhancer-PNOS system also conferred hygromycin resistance to E. coli, but its activity in inducing GFP signals in onion cells remained lower than that of P35S. Due to its compact size (89 bp) and efficiency, PCAT1 can serve as a module for converting standard plant vectors into dual-host systems, accelerating gene characterization and the development of new gene-based tools.

(1) BackgroundSaffron crocus (Crocus sativus) is the source of saffron, the most expensive spice in the world. It evolved about 3000 years ago as a sterile triploid clone in Greece. Since then, saffron has spread across the globe, where regionally distinct practices of saffron cultivation have developed. Despite differences in morpho-physiological traits, genetic variability is low, if present at all. Here, we aim to resolve chromosomal and sequence-associated variability across saffron crocus cultivars from the crops main cultivation areas in Africa, Asia and Europe. (2) MethodsWe used genome-wide DNA polymorphisms obtained through genotyping-by-sequencing (GBS) of 33 saffron and 14 closely related Crocus accessions, which we place into a phylogenetic context. For karyotyping, we compare nine saffron accessions by multi-color fluorescent in situ hybridisation (FISH) with repetitive DNA probes. (3) Key resultsPhylogenetic analyses confirmed the single origin and clonal nature of all saffron accessions. We detected slight DNA differences among saffron crocus genotypes, which were minor compared with those in wild C. cartwrightianus populations. Still, the Iranian saffron accessions form a genetically very narrow group that differs from the other proveniences in population genetic analyses. However, chromosomes of some saffron accessions display variable FISH signals, likely resulting from gains and losses of tandemly repeated DNA. (4) Main conclusionsBased on the high genetic identity and small karyotypic differences, we confirm the clonal origin of the saffron accessions. Nevertheless, as we detected small and regional chromosomal variability, we conclude that at least four somaclonal saffron lineages emerged after saffrons origin. Societal Impact StatementFor millennia, many cultures developed cultivation practices and regional crop varieties. A notable case is saffron, the worlds most expensive spice that is harvested from stigmas of saffron crocus. This flower crop arose 3000 years ago in a singular genome triplication event and since then spread clonally across the globe. By identifying genetic and chromosomal variability in clonal saffron accessions, we highlight regional diversity, support the preservation of traditional knowledge, and underscore the risk of relying on only one clonal lineage. This informs strategies for saffron cultivation, linking cultural heritage with modern genomics to address biodiversity, evolution, and food security.

Soybean (Glycine max [L.] Merr.) productivity is frequently compromised by soil-borne pathogens. Macrophomina phaseolina (Mp), the causal agent of charcoal rot, can produce important soybean yield losses especially when hot and dry weather prevails. Integrating biological control agents with chemical seed treatments represents a promising strategy for improving disease management. This study aimed to (i) assess the in vitro compatibility of Trichoderma koningiopsis with commercial fungicide seed treatments, and (ii) evaluate the field performance of T. koningiopsis, alone or combined with compatible fungicides, across three soybean growing seasons. Compatibility assays revealed fungicide-specific effects, with Acronis(R) classified as non-fungitoxic and Topseed Extra as moderately fungitoxic. Across field seasons, Mp inoculation reduced seedling emergence, while several seed treatments improved emergence compared to the inoculated control, however, treatment effects varied markedly among years. Disease severity did not differ significantly among treatments in any season, and yield responses were strongly modified by environmental conditions rather than treatment effects. Temperature-response assays showed that T. koningiopsis exhibited optimal growth between 28 to 30{degrees}C and complete inhibition above 40{degrees}C, indicating high thermal sensitivity. The results demonstrate that T. koningiopsis can be integrated with compatible fungicides and may enhance early stand establishment under favorable conditions, but its field performance is strongly limited by high temperatures. These findings highlight the importance of environmental conditions when biological seed treatments are used.

RAP2.6, an AP2/ERF transcription factor (TF), regulates plant stress responses; however, its role in floral transition remains unexplored. Here, we evaluated RAP2.6s role in flowering and the associated transcriptional changes in Arabidopsis thaliana under long-day conditions. RAP2.6-overexpressing line showed early flowering with fewer rosette leaves, whereas rap2.6-1 mutant flowered later, had more rosette leaves, and higher expression of the floral repressor FLOWERING LOCUS C (FLC). Early flowering in the overexpressing line was accompanied by transcriptional activation of the floral integrators GIGANTEA (GI), FLOWERING LOCUS T (FT), and COSTANS (CO), potentially through RAP2.6 interaction with GCC/DRE cis-regulatory elements. RAP2.6-mediated floral transition depended on nitric oxide (NO), with flowering time largely varying based on NO bioactivity. RAP2.6 was found to be a downstream regulator of Arabidopsis S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1) in controlling S-nitrosothiol (SNO) levels, flowering time, and silique formation. The NITRIC OXIDE-ASSOCIATED 1 (NOA1)-dependent reduction in NO levels abolished early flowering in 35S::RAP2.6 plants without affecting silique formation. Furthermore, enhanced cytokinin sensitivity and upregulation of cytokinin biosynthetic genes suggest cytokinin involvement in RAP2.6-mediated flowering. Together, these findings highlight the crucial role of RAP2.6 in regulating flowering time by integrating redox and hormonal signaling to coordinate reproductive development in A. thaliana.

Banana (Musa spp.) is a vital staple food and cash crop cultivated in over 140 countries, providing nourishment and livelihoods to more than 400 million people worldwide. In this context, Bhimkol (Musa balbisiana, BB genome), a diploid banana variety native to Northeast India holds significant nutritional and commercial value. Its high iron and nutrient content have already been commercially validated through products like Bhimvita and Bhimshakti, which utilize fresh fruit pulp as nutrient-rich food for infants. However, Bhimkol fruits typically contain 100-150 seeds, an undesirable trait for product development. The manual removal of these seeds significantly increases production time and labour costs. Furthermore, because bananas are recalcitrant to traditional breeding, there is a constant need for rapid in vitro transformation protocols. To address these challenges, as a proof of concept, our research aims to knockout the INNER NO OUTER (INO) gene, which is responsible for ovule development. Using CRISPR/Cas12a technology, we established an efficient and reproducible in vitro regeneration and transformation system using Embryogenic Cell Suspensions (ECS). The resulting CRISPR-edited plantlets exhibited various mutations, including insertions and deletions (INDELs) within the targeted INO gene. These INDELs resulted in frameshift mutations that triggered premature stop codons. While these genetic changes are expected to render the banana seedless, phenotypic verification is currently underway to confirm the absence of seeds in mature fruit. Significance StatementDespite its superior nutritional profile, the commercial viability of the Bhimkol banana (Musa balbisiana) is restricted due to abundance of seeds (100-150 per fruit). This study employs CRISPR/Cas12a-mediated knockout the INNER NO OUTER (INO) gene in Bhimkol and expected to develop seedless fruits. The resulting plantlets exhibit targeted indels that trigger frameshift mutations, effectively disrupting ovule developmental INO gene.