Somatic embryogenesis-mediated plant regeneration is essential for the genetic manipulation of agronomically important traits in upland cotton. Genotype specific recalcitrance to regeneration is a primary challenge in deploying genome editing and incorporating useful transgenes into elite cotton germplasm. In this study, transcriptomes of a semi-recalcitrant cotton (Gossypium hirsutum L.) genotype ‘Coker312’ were analyzed at two critical stages of somatic embryogenesis that include non-embryogenic callus (NEC) and embryogenic callus (EC) cells, and the results were compared to a non-recalcitrant genotype ‘Jin668’. We discovered 305 differentially expressed genes in Coker312, whereas, in Jin668, about 6-fold more genes (2155) were differentially expressed. A total of 154 differentially expressed genes were common between the two genotypes. Gene enrichment analysis of the upregulated genes identified functional categories, such as lipid transport, embryo development, regulation of transcription, sugar transport, and vitamin biosynthesis, among others. In Coker312 EC cells, five major transcription factors were highly upregulated: LEAFY COTYLEDON 1 (LEC1), WUS-related homeobox 5 (WOX5), ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and WRKY2. In Jin668, LEC1, BABY BOOM (BBM), FUS3, and AGAMOUS-LIKE15 (AGL15) were highly expressed in EC cells. We also found that gene expression of these embryogenesis genes was typically higher in Jin668 when compared to Coker312. We conclude that significant differences in the expression of the above genes between Coker312 and Jin668 may be a critical factor affecting the regenerative ability of these genotypes.

Fruit tree rootstock breeding is prolonged by extended juvenile phases, high heterozygosity, limited germplasm diversity, and hybrid incompatibilities, often requiring four decades to release new cultivars. Direct somatic embryogenesis (DSE) in established peach rootstocks presents a promising avenue for rapid genetic transformation and breeding. However, peach is highly recalcitrant to in vitro regeneration, posing major challenges for organogenesis and somatic embryogenesis (SE). This study evaluated the effects of 2,4-dichlorophenoxyacetic acid (2,4-D) and Kinetin (KIN) on SE %, SE productivity, and callus % rate in the widely used Guardian^® peach rootstock. A 5 × 3 full factorial completely randomized design was used to test 15 different combinations of 2,4-D and KIN on immature cotyledons, classified as upper or lower based on their position on the preculture medium. Media formulation containing a higher concentration (3.2 µM) of 2,4-D and KIN induced SE in ~50% of lower and ~85% of upper cotyledons. Optimal SE productivity occurred with higher KIN (3.2 µM) and reduced 2,4-D (2.6 µM). Callus formation peaked with 1.8 µM 2,4-D and 3.2 µM KIN. This highly reproducible research establishes a robust whole plant regeneration system via DSE in Guardian^® peach rootstock using immature cotyledons, providing a foundation for expedited trait manipulation through biotechnological approaches.

Upland cotton (Gossypium hirsutum L.) is a major global commodity crop whose production is threatened by the reniform nematode (Rotylenchulus reniformis Linford and Oliveira), a plant-parasitic pest that causes substantial yield losses. Host-plant resistance offers a sustainable management strategy, but currently available resistant cotton cultivars provide only partial protection and often require supplemental control methods. In this study, Clustered Regularly Interspaced Palindromic Repeats (CRISPR)–CRISPR-associated 9 (Cas9) gene editing was used to generate targeted knockouts of Mildew Resistance Locus O (GhiMLO3) in cotton and assess its role in resistance to R. reniformis. Four independent knockout lines (A1, D3, E1, and P3) were developed, confirmed by sequencing, and evaluated for nematode resistance under controlled greenhouse conditions. Nematode reproduction was significantly reduced on lines D3 and E1, with lower egg counts and fewer vermiform life stages compared with the control genotypes, Coker 312 (WT), Delta Pearl, and Jin668. The edited lines also showed characteristic mesophyll cell-death phenotypes, suggesting potential pleiotropic effects associated with MLO-mediated resistance. Sequence analysis confirmed multiple homozygous and heterozygous mutations in MLO3 alleles from both the A and D subgenomes, with D3 and E1 lines displaying the strongest resistance profiles. These findings demonstrate that MLO3 gene editing is a promising approach for improving R. reniformis resistance in cotton.

Background/Objectives: Fusarium wilt of cotton, caused by Fusarium oxysporum f. sp. vasinfectum (FOV), is a destructive vascular disease that severely impacts cotton production. Among its variants, race 4 (FOV4) is especially aggressive, leading to early season stand losses and yield reductions. While resistant cultivars of Gossypium barbadense (pima cotton) have been developed, the molecular basis of this resistance remains unclear. This study aimed to characterize transcriptomic responses associated with FOV4 resistance in pima cotton. Methods: We conducted an in vitro infection assay using two G. barbadense cultivars with contrasting phenotypes: the highly resistant ‘DP348RF’ and the highly susceptible ‘GB1031’. Root tissues were sampled at multiple stages of infection, and RNA sequencing was performed to identify differentially expressed genes and pathways contributing to resistance. Results: Resistant plants ‘DP348RF’ showed strong induction of genes related to reactive oxygen species (ROS) metabolism, chitinase activity, and lignification compared to the susceptible cultivar. Notably, genes involved in the biosynthesis and reinforcement of the Casparian strip, a critical biochemical barrier limiting pathogen penetration into vascular tissues, were uniquely and significantly upregulated in resistant roots. These transcriptional responses suggest that fortification of cell wall barriers and enhanced antimicrobial defenses contribute to effective restriction of FOV4 colonization. Conclusions: Our findings identify a distinct molecular signature of resistance to FOV4 in pima cotton, with Casparian strip reinforcement emerging as a potential mechanism limiting vascular infection. These insights provide a foundation for breeding strategies aimed at improving Fusarium wilt resistance in cotton.

Fiber cell initiation and development affect cotton fiber yield and quality. Cotton fiber develops from the ovular epidermis of a seed, and approximately 25% to 30% of protodermal cells in each cotton ovule develop into fiber. However, the molecular basis for fiber cell development remains elusive. Here, we analyzed single-cell RNA-seq (scRNA-seq) data from over 40,000 cells during early stages of fiber cell development in Upland and Pima cotton and in a naked seed mutant. We found concerted expression changes of 900 to 1,700 genes in fiber-cell clusters involving gene expression, translation, and peptide biosynthesis, which were substantially delayed or absent in the mutant. Expression of ∼500 and ∼300 genes in Upland and Pima cotton, respectively, was distinguishably different and consistent with overrepresentation of the genes in transcriptional and translational regulation, implying their roles in fiber yield and quality traits. Gene coexpression network analysis of scRNA-seq along with scATAC-seq data revealed two modules of fiber gene coexpression networks. One module of the fiber coexpressed genes was associated with elevated chromatin accessibility for transcriptional regulation, whereas the other module of the genes was related to translational regulation and ribosome biogenesis. Indeed, expression of cotton putative translation factor genes was elevated in fiber cell clusters in both Upland and Pima cotton. Finally, cotton transgenic plants expressing promoter::GFP confirmed expression patterns of fiber-expressed GhRDL2_D5 during fiber cell initiation. These single-cell genomic resources provide insights into fiber cell development for breeding and biotechnological improvement of fiber yield and quality in Upland and Pima cotton.