Search Results (100)

Rosa rugosa (R. rugosa) is a commercially important ornamental species within the genus Rosa, highly valued in the horticultural market. With the increasing availability and improved annotation of Rosa genomes, establishing an efficient genetic transformation system has become essential for validating candidate gene functions. As a common intermediate tissue in plant regeneration, callus has been successfully used to establish genetic transformation systems in numerous species. In this study, we characterized the morphological and physiological differences between embryogenic and non-embryogenic calli in R. rugosa. The embryogenic callus exhibited significantly higher catalase (CAT) activity and proline (PRO) content than the non-embryogenic callus. However, its growth rate was markedly slower. Antibiotic sensitivity assays identified the optimal selection concentrations for non-embryogenic callus as 35 mg/L for kanamycin and 13 mg/L for hygromycin. We subsequently introduced the phytoene synthase (RrPSY1) gene into non-embryogenic callus, with positive transformants identified using GFP fluorescence detection and PCR analysis. The overexpression of RrPSY1 significantly increased the yellow pigment substances in the callus, confirming the establishment of an effective genetic transformation system for non-embryogenic calli in R. rugosa. This system provides a useful technical platform for the manipulation of metabolic products and the verification of related gene functions in rose. Full article

Plant transformation efficiency is highly dependent on species, individual genotypes, and tissue types. In maize, immature embryos are regularly used for transformation. The process relies heavily on callus development, as it is intricately associated with somatic embryogenesis and subsequent plant regeneration, both of which directly affect transformation efficiency. Immature embryos of the segregation progeny derived from the two inbred parents, a transformation-amenable line A188 and a recalcitrant line B73, can be cultured to form two primary callus types: Type I and Type II. The Type II callus grows faster and is a favorable type for regeneration. Here, Type I and II calli from the B73xA188 F2 population were genotyped by Genotyping-By-Sequencing (GBS). Quantitative trait locus (QTL) analysis of the callus type identified QTLs at chromosomes 2, 5, 6, 8, and 9. The result was largely supported by the bulk segregant RNA-seq (BSR-seq) genetic analysis using RNA from separately pooled Type I and II calli. Both analyses revealed that an allele of A188 on chromosome 6 and B73 alleles on chromosomes 2, 5, 8, and 9 promoted the formation of the Type II callus. Differentially expressed genes (DEGs) between the Type II and I F2 calli were also identified. In addition, the A188 calli developed from the same immature embryos often exhibit heterogeneous morphology, including the fast- and slow-growing callus sectors. The transcriptional comparison between the two sectors was performed to identify DEGs. Both sets of DEGs were enriched in genes involved in cell-wall organization and wax biosynthesis pathways. Full article

Napier grass (Cenchrus purpureus Schumach) is an important forage crop and livestock feed. However, its yield and quality in Kenya are often limited by Napier grass headsmut and stunt disease. Napier grass genetic improvements through mutation breeding and selection could avail cultivars with increased forage. This study investigated the response of embryogenic calli to different levels of colchicine in inducing polyploidy in the two germplasms of Napier grass; South africa and Bana grass. The experiments were carried out as a factorial experiment in a completely randomized design (CRD). The colchicine concentrations used were 0, 0.05, 0.1, and 0.2%, and the exposure durations were 24, 48, and 72 h. During the shoot regeneration stage, culturing explants on an MS medium (Murashige and Skoog) supplemented with 0.2 mg L⁻¹ Benzyl Adenine (BAP), 0.1 mg L⁻¹ dichlorophenoxyacetic acid (2, 4-D), and 0.1 mg L⁻¹ indole-3-butyric acid (IBA) was more suitable for shoot regeneration. Chromosome doubling was confirmed by genomic DNA and the stomata size and number. Culturing explants on an MS medium supplemented with 1 mg L⁻¹ IBA, 1 mg L⁻¹ 2, 4-D, and 0.5 mg L⁻¹ BAP was more suitable in inducing embryogenic calli in both genotypes. Polyploidy results revealed that a 0.1% concentration of colchicine with two days of treatment established the maximum number of octoploid plantlets induced in vitro, while a 0.2% concentration was very toxic. The stomata size and number of derived octoploid plantlets were bigger with a lower density, a shorter plant height, and a smaller stem diameter, and despite being the first to produce tillers, they were significantly higher than their progenitors. Induced mutants also had a significantly higher number of chromosomes and showed different band patterns and distances during gel electrophoresis. However, we recommend the use of flow cytometry to confirm the ploidy level. The superior mutant plantlets can be selected and recommended for characterization across representative agro-ecologies for large-scale production and used in Cenchrus purpureus breeding programs in Kenya and its environments. Full article

Ilex paraguariensis (yerba mate), a culturally and economically important South American species, faces significant challenges in vitro, including contamination, phenolic oxidation, and low regeneration rates. Nanoparticles have recently emerged as promising tools to overcome such limitations. This study evaluated silver (AgNPs) and chitosan nanoparticles (ChNPs) in eight experiments using nodal, leaf, and internodal explants. Surface disinfection with 1% colloidal silver solution 20 ppm significantly reduced contamination (17.2% and 15%) while maintaining viability (62.1%). However, supplementation of culture media with AgNPs (4–75 mg·L⁻¹) or ChNPs (5–120 mg·L⁻¹) did not improve nodal segment responses. In leaf explants, 4 mg·L⁻¹ AgNPs proved most effective, reducing contamination and markedly decreasing callus oxidation from 63.3% to 10.0%. Callogenesis was enhanced when AgNPs were combined with growth regulators, with the highest induction at 6 mg·L⁻¹ AgNPs + zeatin (38.1%) and 4 mg·L⁻¹ AgNPs + BAP (42.9%). Conversely, in internodal segments, AgNPs combined with BAP completely inhibiting callus formation. The resulting calli exhibited compact and friable morphologies but no signs of somatic embryogenesis. Overall, the effectiveness of AgNPs depends on their formulation, explant type, and interaction with cytokinins. Optimization of nanoparticle formulation and hormonal balance remains essential to maximize efficacy while minimizing toxicity. Full article

Iris laevigata is an ornamental plant and so its wild genetic resources need to be protected. However, traditional inefficient propagation limits its landscape applications. In this study, we assessed the effects of phytohormones on growth of I. laevigata at various culture stages using roots and hypocotyls as explants and established an efficient micropropagation system. The highest callus induction of hypocotyl (75.0%) was obtained using Murashige and Skoog medium containing 6-benzylaminopurine (6-BA), 0.5 mg L⁻¹ + 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg L⁻¹ + 1-naphthylacetic acid (NAA), and 0.4 mg L⁻¹. Similarly, the highest callus induction (73.3%) of roots was achieved with 6-BA 0.5 mg L⁻¹ + 2,4-D 0.5 mg L⁻¹ + NAA 0.4 mg L⁻¹. The calli induced from hypocotyl and root tissues achieved 39.7% and 49.5% adventitious shoot induction on a medium containing indole-3-butyric acid (IBA) 0.5 mg L⁻¹ + 6-BA 1.5 mg L⁻¹ + NAA 1.0 mg L⁻¹ and 6-BA 2.0 mg L⁻¹ + NAA 0.4 mg L⁻¹ + kinetin (KT) 1.0 mg L⁻¹, respectively. The rooting of adventitious shoots reached 93.3% in the medium supplemented with NAA 0.2 mg L⁻¹. The survival of regenerated plants reached 90.0% after being transplanted into soil. This study provides an efficient and reliable propagation method for I. laevigata for landscape applications and the preservation of wild genetic material. Full article

Light influence on shoot regeneration in Prunus salicina is a complex interaction that has been studied for the first time. Japanese plum plants were regenerated from calli and seeds of the scion cultivar ‘Victoria’. The effect of four different light spectra (white, blue, red, and mixed), along with three 6-benzyladenine (BA) concentrations (1, 1.5, and 2 mg L⁻¹), was studied in these two sources of explants. Organogenic calli were derived from the base of stem explants of the scion cultivar ‘Victoria’, whereas cotyledons and embryogenic axis slices were used as seed explants. Calli cultured with 2 mg L⁻¹ of BA and mixed light or 2.5 mg L⁻¹ of BA and control light showed the highest regeneration rates, with no significant differences compared to other treatments. Seed explants exposed to 2.5 mg L⁻¹ of BA and red light exhibited significantly higher organogenesis. In comparison, those in 1.5 mg L⁻¹ of BA with blue light or 2.5 mg L⁻¹ of BA with mixed/control light showed no regeneration. BA concentration did not have a significant effect in the induction of somatic shoots from any explant source. In contrast, a strong interaction between light and BA was noticed. This work presents a protocol that can be applied in transformation and editing research as light spectrum studies continue to advance. Full article

In the severe environment of Hunshandake Sandy Land, the uncommon and indigenous Chinese tree species Picea mongolica is an important biological component. Conventional seed propagation in P. mongolica is constrained by low germination rates, prolonged breeding cycles, and hybrid offspring genetic instability, limiting efficient varietal improvement. In contrast, somatic embryogenesis (SE) offers superior propagation efficiency, exceptional germination synchrony, and strict genetic fidelity, enabling rapid mass production of elite regenerants. However, SE in P. mongolica is hampered by severe genotype dependence, poor mature embryo induction rates, and loss of embryogenic potential during long-term cultures, restricting the production of high-quality seedlings. In this study, we aimed to analyze the transcriptome and metabolome of three crucial phases of SE: mature somatic embryos (MSEs), globular somatic embryos (GSEs), and embryogenic calli (EC). Numerous differentially expressed genes (DEGs) were found, especially in pathways linked to ribosomal functions, flavonoid biosynthesis, and the metabolism of starch and sucrose. Additionally, 141 differentially accumulated metabolites (DAMs) belonging to flavonoids, organic acids, carbohydrates, lipids, amino acids, and other metabolites were identified. An integrated study of metabolomic and transcriptome data indicated considerable enrichment of DEGs and DAMs in starch and sucrose metabolism, as well as phenylpropanoid biosynthesis pathways, all of which are required for somatic embryo start and development. This study revealed a number of metabolites and genes linked with SE, offering important insights into the molecular mechanisms driving SE in P. mongolica and laying the groundwork for the development of an efficient SE system. Full article

The rhizome of Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine used extensively owing to its antimicrobial properties. It is utilized to treat nyctalopia and problems related to the gastrointestinal tract. However, its yield is limited because of its endangered status, long growth period, and restricted reproductive ability. Ancillary approaches have not been established to ensure sustainable resource utilization by applying efficient plant regeneration technologies and producing bioactive metabolites via genome editing. This study reports the effects of explants, hormones, and culture conditions on embryogenic callus induction, plant regeneration, adventitious and hairy root cultivation, and essential oil production. Embryogenic calli were successfully induced in MS and 2.0 mg/L 2,4-D and 1.0 mg/L NAA and 1/2MS medium supplemented with 4.0 mg/L 6-BA and 0.4 mg/L NAA, which were optimal for callus differentiation. Maximum proliferation (12-fold) of cluster buds was observed with a select combination of hormones [NAA (0.2 mg/L) and 6-BA (2.0 mg/L)]. “Efficient plant regeneration and bioactive metabolite production” can provide technical support for the protection and sustainable utilization of A. lancea germplasm resources in terms of resource preservation and new variety breeding, natural product production, and industrial breeding of medicinal plants. Full article

Callus browning is a significant problem that hinders plant tissue regeneration in Paeonia ostii “Fengdan” by causing cell death and inhibiting growth. However, the molecular mechanism underlying callus browning in P. ostii remains unclear. In this study, we investigated the metabolites and potential regulatory genes involved in callus browning of P. ostii using metabolomic and transcriptomic analyses. We found a significant accumulation of phenolic compounds in the browned callus, represented by flavonoid compounds. Notably, the accumulations of luteotin and disomentin were higher in browning calli compared to non-browning calli. Transcriptomic analysis identified that candidate genes associated with flavonoid biosynthesis, including flavonoid 3-hydroxylase (PoF3H) and flavone synthase II (PoFNSII), were highly expressed in the browned callus of P. ostii “Fengdan”. Weighted gene co-expression network analysis (WGCNA) further highlighted that polyphenol oxidase (PoPPO) which encoded polyphenol oxidase, together with flavonoid biosynthesis-related genes such as flavanone 3-hydroxylase (PoF3H) and flavonone Synthase II (PoFNSII), as well as cellular totipotency-related genes wuschel-related homeobox 4 (PoWOX4), were involved in callus browning. Based on these findings, we proposed the molecular mechanism by which flavonoid accumulation, polyphenol oxidation, and cellular totipotency pathways contribute to callus browning in P. ostii. Our study provides new insights into the molecular mechanism underlying callus browning and offers the foundations to facilitate the establishment of an efficient plant tissue regeneration system in P. ostii. Full article

This study aimed to develop an aromatic thermosensitive genic male sterile (TGMS) line in indica rice using CRISPR/Cas9 technology. The TMS5 and FGR in the high-quality conventional rice variety Huahang 48 were targeted for editing using CRISPR/Cas9 technology. CRISPR/Cas9 vectors designed for TMS5 and FGR were constructed and introduced into rice calli through Agrobacterium-mediated transformation. Transgenic seedlings were subsequently regenerated, and the target sites of the edited plants were analyzed via sequencing. A total of fifteen T₀ double mutants were successfully obtained. Three mutants without T-DNA insertion were screened in the T₁ generation by the PCR detection of hygromycin gene fragments, and homozygous mutants without T-DNA insertion were screened in the T₂ generation by the sequencing analysis of the mutation sites, named Huahang 48s. Huahang 48s exhibited complete sterility at 24 °C and pollen transfer at 23 °C. The 2-acetyl-1-pyrroline (2-AP) content was detected in the young panicles, leaves, and stems of Huahang 48s. The leaves of Huahang 48s had the highest 2-AP content, contrasting with the absence of 2-AP in HuaHang 48. F₁ hybrids that crossed Huahang 48s with two high-quality restorer lines were superior to the two parents in terms of yield per plant and 1000-grain weight. Huahang 48s has a certain combining ability and application potential in two-line cross breeding. The successful application of CRISPR/Cas9 technology in Huahang 48 established a foundation for developing aromatic TGMS lines, providing both theoretical insights and practical materials for breeding efforts. Full article

Roses are one of the most important flowers applied to landscape, cut flowers, fragrance and food industries widely. As an effective method for plant reproduction, the regeneration via somatic embryos is the most promising method for breed improvement and genetic transformation of woody plants. However, lower somatic embryogenesis (SE) induction rates and genotypic constraints impede progress in genetic transformation in rose. This study describes a plant regeneration system for the famous red cut flower cultivar Rosa hybrida ‘Carola’. The stems without petioles cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg·L⁻¹ 6-benzylaminopurine (6-BA), 0.05 mg·L⁻¹ a-naphthalene acetic acid (NAA) and 30 g·L⁻¹ sucrose showed the maximum proliferation coefficient of shoots with 3.41 for the micropropagation system. We evaluated the effects of different plant growth regulators (PGRs) on the induction, proliferation and conversion of somatic embryos. The induction rate of calli reached 100% on MS medium supplemented with 2.0 g·L⁻¹ NAA and 30 g·L⁻¹ glucose. The highest induction rate of somatic embryos achieved a frequency of 13.33% on MS medium supplemented with 2.0 mg·L⁻¹ zeatin (ZT), 0.1 mg·L⁻¹ NAA and 30 g·L⁻¹ glucose. The most suitable carbohydrate with 60 g·L⁻¹ glucose resulted in a proliferation rate of somatic embryos (4.02) on MS medium containing 1.5 mg·L⁻¹ ZT, 0.2 mg·L⁻¹ NAA and 0.1 mg·L⁻¹ gibberellic acid (GA₃). The highest somatic embryos germination rate (43.33%) was obtained from the MS medium supplemented with 1.0 mg·L⁻¹ 6-BA, 0.01 mg·L⁻¹ IBA and 30 g·L⁻¹ glucose. Finally, the germinated somatic embryos successfully rooted on 1/2 MS medium containing 1.0 mg·L⁻¹ NAA, 30 g·L⁻¹ sucrose, and the vigorous plantlets were obtained after hardening-off culture. This study provided a stable and efficient protocol for plant regeneration via somatic embryos in R. hybrida ‘Carola’, which will be beneficial to the further theoretical study and genetic improvement in roses. Full article

Camellia hainanica is one of the camellia plants distributed in tropical regions, and its regeneration system and genetic transformation are affected by callus browning. However, the underlying mechanism of Camellia hainanica callus browning formation remains largely unknown. To investigate the metabolic basis and molecular mechanism of the callus browning of Camellia hainanica, histological staining, high-throughput metabolomics, and transcriptomic assays were performed on calli with different browning degrees (T1, T2, and T3). The results of histological staining revealed that the brown callus cells had obvious lignification and accumulation of polyphenols. Widely targeted metabolomics revealed 1190 differentially accumulated metabolites (DAMs), with 53 DAMs annotated as phenylpropanoids and flavonoids. Comparative transcriptomics revealed differentially expressed genes (DEGs) of the T2 vs. T1 associated with the biosynthesis and regulation of flavonoids and transcription factors in Camellia hainanica. Among them, forty-four enzyme genes associated with flavonoid biosynthesis were identified, including phenylalaninase (PAL), 4-coumaroyl CoA ligase (4CL), naringenin via flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), Chalcone synthase (CHS), Chalcone isomerase (CHI), hydroxycinnamoyl-CoA shikimate transferase (HCT), Dihydroflavonol reductase (DFR), anthocyanin reductase (LAR), anthocyanin synthetase (ANS), and anthocyanin reductase (ANR). Related transcription factors R2R3-MYB, basic helix-loop-helix (bHLH), and WRKY genes also presented different expression patterns in T2 vs. T1. These results indicate that the browning of calli in Camellia hainanica is regulated at both the transcriptional and metabolic levels. The oxidation of flavonoids and the regulation of related structural genes and transcription factors are crucial decisive factors. This study preliminarily revealed the molecular mechanism of the browning of the callus of Camellia hainanensis, and the results can provide a reference for the anti-browning culture of Camellia hainanica callus. Full article

Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species. Full article

Purslane (Portulaca oleracea L.) is highly valued for its nutritional, medicinal, and ecological significance. Genetic transformation in plants provides a powerful tool for gene manipulation, allowing for the investigation of important phenotypes and agronomic traits at the genetic level. To develop an effective genetic transformation method for purslane, various organ tissues were used as explants for callus induction and shoot regeneration. Leaf tissue exhibited the highest dedifferentiation and regeneration ability, making it the optimal explant for tissue culture. By culturing on Murashige and Skoog (MS) medium supplemented with varying concentrations of 6-benzyleaminopurine (6-BA) and 1-naphthaleneacetic acid (NAA), somatic cells from leaf explants could be developed into calli, shoots, and roots. The shoot induction results of 27 different purslane accessions elucidated the impact of genotype on somatic-cell regeneration capacity and further confirmed the effectiveness of the culture medium in promoting shoot regeneration. On this basis, a total of 17 transgenic plants were obtained utilizing the genetic transformation method mediated by Agrobacterium. The assessment of GUS staining, hygromycin selection, and polymerase chain reaction (PCR) amplification of the transgenic plants as well as their progeny lines indicated that the method established could effectively introduce foreign DNA into the purslane nucleus genome, and that integration was found to be stably inherited by offspring plants. Overall, the present study demonstrates the feasibility and reliability of the Agrobacterium-mediated genetic transformation method for introducing and integrating foreign DNA into the purslane genome, paving the way for further research and applications in purslane genetic modification. Full article

Somatic embryogenesis (SE) is a biotechnological tool used to generate new individuals and is the preferred method for rapid plant regeneration. However, the molecular basis underlying somatic cell regeneration through SE is not yet fully understood, particularly regarding interactions between the proteome and post-translational modifications. Here, we performed association analysis of high-throughput proteomics and phosphoproteomics in three representative samples (non-embryogenic calli, NEC; primary embryogenic calli, PEC; globular embryos, GE) during the initiation of plant regeneration in cotton, a pioneer crop for genetic biotechnology applications. Our results showed that protein accumulation is positively regulated by phosphorylation during SE, as revealed by correlation analyses. Of the 1418 proteins that were differentially accumulated in the proteome and the 1106 phosphoproteins that were differentially regulated in the phosphoproteome, 115 proteins with 229 phosphorylation sites overlapped (co-differential). Furthermore, seven dynamic trajectory patterns of differentially accumulated proteins (DAPs) and the correlated differentially regulated phosphoproteins (DRPPs) pairs with enrichment features were observed. During the initiation of plant regeneration, functional enrichment analysis revealed that the overlapping proteins (DAPs-DRPPs) were considerably enriched in cellular nitrogen metabolism, spliceosome formation, and reproductive structure development. Moreover, 198 DRPPs (387 phosphorylation sites) were specifically regulated at the phosphorylation level and showed four patterns of stage-enriched phosphorylation susceptibility. Furthermore, enrichment annotation analysis revealed that these phosphoproteins were significantly enriched in endosomal transport and nucleus organization processes. During embryogenic differentiation, we identified five DAPs-DRPPs with significantly enriched characteristic patterns. These proteins may play essential roles in transcriptional regulation and signaling events that initiate plant regeneration through protein accumulation and/or phosphorylation modification. This study enriched the understanding of key proteins and their correlated phosphorylation patterns during plant regeneration, and also provided a reference for improving plant regeneration efficiency. Full article