Search Results (34)

This study established an efficient in vitro regeneration system using stem nodes from root collar suckers as explants. Subsequently, regenerated shoots were used to establish an in vitro medicinal production protocol that achieved ginkgolic acid production. The self-developed Ginkgo biloba medium (GBM), first reported in this study, was pivotal to system establishment. The plantlet propagation system showed that the bases of stem nodes dipped in GBM with 2 mg·L⁻¹ 6-benzyladenine (BA) and 0.2 mg·L⁻¹ 1-naphthaleneacetic acid (NAA) achieved near-complete axillary bud induction (99.56%). Adventitious shoot induction reached 82.22% (3.5 shoots/explant) using GBM with 0.2 mg·L⁻¹ BA, 0.02 mg·L⁻¹ kinetin (Kin) and 0.2 g·L⁻¹ proline (Pro). Maximum adventitious shoot elongation (92.22%, average 3.35 cm) was observed on GBM containing 0.1 mg·L⁻¹ zeatin (ZT) and 0.01 mg·L⁻¹ BA. After 3-week preculture with 15 mg·L⁻¹ phloroglucinol (PG), treatment with 0.6 mg·L⁻¹ indole-3-butyric acid (IBA) and 0.2% activated carbon (AC) yielded 96.67% rooting (6.19 roots/explant) and 85% acclimatization survival. For medicinal resource production, bud cluster induction at 94.44% (20.89 buds/explant) on GBM with 1 mg·L⁻¹ BA, 0.03 mg·L⁻¹ Kin, and 0.2 g·L⁻¹ Pro. Leaf organs in GBM with 0.3 mg·L⁻¹ BA, 0.01 mg·L⁻¹ Kin, 0.01 mg·L⁻¹ IBA, 0.3 g·L⁻¹ Pro, and 0.01 mg·L⁻¹ glutamine (Gln) accumulated 20.64 g fresh weight and 41.910 mg·g⁻¹ DW ginkgolic acids, representing a 4.93-fold increase over mother plants. This system enables large-scale Ginkgo biloba L. propagation and provides an in vitro strategy for producing medicinal compounds in endangered plants. Full article

A pluripotent callus is central to genetic transformation in Cinnamomum parthenoxylon; however, the molecular and cellular mechanisms regulating callus formation and subsequent differentiation remain unelucidated, hindering progress in its genetic improvement. This study systematically investigated the dynamic changes during the in vitro regeneration of C. parthenoxylon through morphological observations, physiological assays, and transcriptomic analyses, while comparing differences in callus formation under varying induction conditions to elucidate the mechanism of its high-efficiency regeneration. The results showed that the formation of a pluripotent callus is a critical step in C. parthenoxylon regeneration, characterized by the presence of highly proliferative cell zones. Compared to an ordinary callus (P3C), a pluripotent callus (P3) exhibited higher activities of polyphenol oxidase (PPO) and indole-3-acetic acid oxidase (IAAO), as well as elevated levels of zeatin riboside (ZR) and abscisic acid (ABA). In contrast, P3 showed lower levels of soluble sugars, soluble proteins, malondialdehyde (MDA), indole-3-acetic acid (IAA), and gibberellins (GA), a reduced IAA/ZR ratio, and diminished peroxidase (POD) activity. Weighted gene co-expression network analysis (WGCNA) identified 27 hub transcription factors (TFs) strongly associated with IAA/ZR, primarily from the ERF, bHLH, MYB, WRKY, and C3H families. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed the significant enrichment of differentially expressed genes (DEGs) related to plant hormone signal transduction and cell wall metabolism during pluripotent callus acquisition. Further investigations revealed that five genes encoding a putative indole-3-acetic acid-amido synthetase GH3.1, protein TIFY 10A, a two-component response regulator ARR2-like isoform X2, and xyloglucan endotransglucosylase/hydrolase, likely promoting callus pluripotency by modulating plant hormone signaling and cell wall metabolism, thereby enhancing in vitro regeneration in C. parthenoxylon. In summary, this study provides critical insights into the molecular mechanisms of C. parthenoxylon regeneration and offers valuable germplasm resources for establishing an efficient and stable genetic transformation system via tissue culture. Full article

Datong in Shanxi Province, known as the “Daylily Capital of China,” still primarily relies on traditional propagation by division for daylily seedling production. Although traditional seedling propagation methods are simple and low-cost, they suffer from limitations such as low propagation efficiency, which restricts large-scale production. The application of tissue culture in seedling production not only enables rapid large-scale propagation but also helps maintain desirable genetic traits through virus elimination. This study aimed to establish an efficient in vitro regeneration system for Hemerocallis citrina ‘Datong Huanghua’ through optimization of key culture stages using scape explants. The results demonstrated that during the stages of callus induction, adventitious bud differentiation, and proliferation culture, the best results were achieved using MS medium supplemented with 3 mg/L zeatin (ZT) and 0.3 mg/L α-naphthylacetic acid (NAA), yielding a callus induction rate of 83.33%, an adventitious bud differentiation rate of 83.40%, and a proliferation coefficient of 4.05. For root induction, MS medium containing 0.25 mg/L indole-3-butyric acid (IBA) and 0.25 mg/L NAA resulted in an average of 4.7 roots per plantlet with a 100% rooting rate. In addition, endogenous hormone analysis showed that lower ABA/GA₃ and ABA/ZR ratios in scape explants promoted callus formation during the induction and differentiation stages. Full article

Catalpa bungei C.A.Mey is an economically significant deciduous tree valued for timber production and landscaping applications. An efficient regeneration system is crucial for clonal propagation and serves as a foundation for future molecular breeding in C. bungei. This study established two in vitro regeneration pathways—indirect somatic embryogenesis and shoot organogenesis utilizing mature zygotic embryos as explants. Primary callus was induced from cotyledon, hypocotyl, and plumule explants. A high frequency (45.73%) of yellow-green compact callus was achieved on De-Klerk and Walton (DKW) medium supplemented with 2.0 mg/L 6-BA, 1.0 mg/L zeatin (ZT), and 0.1 mg/L NAA. Subsequent transfer to 1.5× Murashige and Skoog (MS) medium containing 1.5 mg/L 6-BA, 0.2 mg/L ZT, and 0.1 mg/L NAA yielded the highest embryogenic callus induction rate (16.67%). Embryogenic callus demonstrated bipotent potential, generating both adventitious shoots and somatic embryos under specific hormonal conditions. Histological analyses confirmed the typical developmental stages of somatic embryos, from globular to cotyledonary forms, validating the embryogenic origin of regenerated structures. Furthermore, hormone or osmotic additives such as abscisic acid (ABA), Phytagel, and polyethylene glycol 4000 (PEG4000) significantly enhanced somatic embryo induction, with Phytagel at 5.0 g/L achieving the highest rate (76.31%). For shoot organogenesis, the optimal hormonal combination of the 0.6 mg/L 6-BA, 0.4 mg/L KT, and 0.15 mg/L NAA achieved the highest bud induction rate (88.89%) and produced an average of 4.07 adventitious buds per explant. This study presents an efficient regeneration system for C. bungei, providing a practical platform for large-scale propagation and basis for biotechnological applications in woody plants. Full article

Callicarpa peichieniana is an important traditional Chinese medicinal plant with pharmacological benefits for digestive system diseases and wounds, as well as high ornamental value. The goal of this study is to establish an effective in vitro regeneration system in order to satisfy the expanding market demand. Extracts from algae can enhance the proliferation and rooting effect of adventitious buds and can improve the survival rate of transplantation. This study developed an in vitro regeneration system using apical bud explants of C. peichieniana associated with Chlorella sorokiniana (an alga species). Inter simple sequence repeat (ISSR) molecular markers confirmed the genetic fidelity of the regenerated plantlets. The highest number of adventitious buds (5.00 buds) was induced from the apical buds with 0.5 mg/L 6-BA in a Murashige and Skoog (MS) medium, and the highest proliferation coefficient (5.83) was achieved with 2.0 mg/L 6-BA. A rooting rate of 100% was achieved by using 0.1 mg/L NAA, MS with 50% macroelements, and 20 g/L sucrose, averaging 6.36 roots per explant and a root length of 1.32 cm. In all micropropagation stages, C. sorokiniana coexisted and proliferated alongside C. peichieniana materials. ISSR showed that the genetic fidelity of C. peichieniana regenerated plants was 95.45%. Coconut coir/perlite = 1∶1 (v/v) was identified as the optimal transplantation substrate, achieving a 100% survival rate. The “C. peichieniana–C. sorokiniana association” in vitro regeneration system established in this study not only enables the mass production of high-quality regenerated plantlets but provides new ideas and demonstrations for culturing multiple species in the same in vitro system. Full article

This study established an efficient in vitro regeneration system for Tibouchina granulosato (Desr.) Cogn. petiolated leaves to address the low propagation efficiency and propagatable germplasm shortages. The results revealed that the Murashige and Skoog (MS) medium supplemented with 1.1 mg/L of Zeatin (ZT) and 0.2 mg/L of 1-naphthyl acetic acid (NAA) was the optimal formulation for callus induction, yielding callus induction of 89.59%. For adventitious bud induction, the combination of 2.0 mg/L of 6-benzyladenine (BA) and 0.4 mg/L of NAA proved most effective, achieving an induction rate of 83.33%. Additionally, the adventitious shoots exhibited remarkable elongation when cultured in a medium containing 0.2 mg/L of BA and 0.04 mg/L of NAA. All explants rooted when treated with 0.5 mg/l NAA, inducing a mean number of 6.90 roots per plant and a survival percentage of 91.00%. This study provided technical support for the promotion of superior varieties and genetic improvement of Tibouchina granulosa. Full article

Bombax ceiba is an important medicinal and ornamental tree widely distributed in tropical and subtropical areas. However, its seeds lose viability rapidly after harvest, which has created hurdles in large-scale propagation. Here, we describe the development of a rapid and efficient de novo organogenesis system for Bombax ceiba, incorporating both indirect and direct regeneration pathways. The optimal basal medium used throughout the protocol was ½ MS supplemented with 30 g/L glucose, with all cultures maintained at 26–28 °C. For the indirect pathway, callus was induced from both ends of each hypocotyl on basal medium supplemented with 0.2 mg·L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg·L⁻¹ 6-Benzylaminopurine (6-BA) under dark conditions. The induced calluses were subsequently differentiated into adventitious shoots on basal media containing 0.5 mg·L⁻¹ Indole-3-butyric acid (IBA), 0.15 mg·L⁻¹ Kinetin (KIN), and 1 mg·L⁻¹ 6-BA under a 16 h photoperiod, resulting in a callus induction rate of 140% and a differentiation rate of 51%. For the direct regeneration pathway, shoot buds cultured on medium with 0.5 mg·L⁻¹ IBA and 1 mg·L⁻¹ 6-BA achieved a 100% sprouting rate with a regeneration coefficient of approximately 3.2. The regenerated adventitious shoots rooted successfully on medium supplemented with 0.5 mg·L⁻¹ Naphthylacetic acid (NAA) and were acclimatized under greenhouse conditions to produce viable plantlets. This regeneration system efficiently utilizes sterile seedling explants, is not limited by seasonal or environmental factors, and significantly improves the propagation efficiency of Bombax ceiba. These optimized micropropagation methods also provide a robust platform for future genetic transformation studies using hypocotyls and shoot buds as explants. 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

Prunus xueluoensis, a unique Prunus germplasm resource native to China, exhibits significant ornamental value due to its short juvenile phase, early flowering period, abundant flowers, and elegant tree form. However, the lack of an efficient regeneration and genetic transformation system has hindered its genetic improvement and wider application. In this study, we focused on optimizing the tissue culture conditions for P. xueluoensis and establishing an Agrobacterium-mediated transient genetic transformation system. We first determined the optimal medium compositions for different stages of tissue culture, including seed germination, callus induction, adventitious bud differentiation, and rooting. For seed germination, the optimal medium was MS supplemented with 200 mg/L GA3 and 4 mg/L 6-BA. For callus induction, the best medium was MS containing 2.00 mg/L 6-BA, 1.00 mg/L NAA, and 200 mg/L VC. Adventitious bud differentiation was favored on MS medium with 1.00 mg/L 6-BA, 0.10 mg/L NAA, and 200 mg/L VC, while rooting was optimal on 3/4 MS medium supplemented with 0.50 mg/L NAA. Subsequently, we established an Agrobacterium-mediated transient genetic transformation system using stem segments of P. xueluoensis as explants. Through orthogonal experiments, we identified the optimal conditions for genetic transformation as pre-cultivation for 2 days, an Agrobacterium concentration of OD₆₀₀ = 0.6, an infection time of 30 min, and co-cultivation for 3 days. Under these conditions, the transient genetic transformation efficiency reached 10.42%, as confirmed by PCR and GFP fluorescence detection. This study provides a reliable transient genetic transformation system for P. xueluoensis, facilitating further functional gene analysis and genetic improvement of this valuable ornamental species. Full article

Propagation of hinoki cypress (Japanese cypress, Chamaecyparis obtusa, Cupressaceae) through adventitious bud multiplication was performed using leaf-segment explants from cutting plants of selected adult trees. Explants were successfully surface-sterilized (>90% asepsis) by agitating them in 2.5% (w/v available chlorine) sodium hypochlorite solution for 15 min and then rinsed with sterile distilled water. Explants approximately 2 cm long were cultured on plates containing medium supplemented with 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/L sucrose, and 7 g/L agar. The cultures were kept at 25 ± 1 °C under a 16-h photoperiod with a photon flux density of approximately 65 µmol m⁻² s⁻¹. The optimal adventitious bud multiplication (31.5 buds per explant) was obtained on a medium supplemented with 10 µM BAP in combination with 1 µM 2,4-D. Proliferated adventitious buds were elongated better on medium supplemented with 1 µM trans-zeatin. The best rooting result (86%) was achieved on a rooting medium supplemented with 1 µM 3-indolebutyric acid in combination with 0.1 µM 1-naphthaleneacetic acid. However, rooting response varied according to genotypes. Clones related to the cultivar ‘Nangouhi’ (Na18, Na14 x Isa, Na14-14, Isa x Na14, and NaS) were easier to root than those derived from the cultivar ‘ShizuokaKenZairai’ (SKZ5 and SKZ8). Regenerated plantlets did not show morphological abnormalities and showed a high survival rate after acclimatization (>90%). Full article

Actinidia kolomikta (Maxim) Maxim. is a winter-hardy species of the genus Actinidia Lindl., whose fruits are valued for their high content of vitamin C and other bioactive compounds. The use of biotechnological propagation methods significantly accelerates the production of quality planting materials for this crop. This study revealed the regeneration features of promising A. kolomikta cultivars. The main morphometric parameters of explants were determined in regard to the effect of different iron chelates (FeEDTA and FeEDDHA) and cytokinins (6-benzylaminopurine, meta-Topolin, and 2-izopentyladenine) in the Quoirin and Lepoivre medium. FeEDTA-supplemented media were optimal for explant culture. Meta-Topolin was found to promote the formation of adventitious microshoots at the base of explants and bud activation, which increased the multiplication rate by 1.5 and 1.7 times compared to the media with 6-benzylaminopurine and 2-izopentyladenine. The morpho-anatomical studies revealed the structural organization of assimilation tissues and the stomatal apparatus of A. kolomikta under different culture conditions (field, in vitro, and ex vitro). The stomata in vitro were round and had a larger area, lower thickness, and a lower layer number of mesophyll compared to field conditions. The transfer from in vitro to ex vitro caused gradual normalization of the leaf structure: a decrease in the stomatal number and area, changes in shape (from round to elliptical), and an increase in the mesophyll thickness. Full article

Chrysanthemum morifolium ‘11-C-2’ is a variety of chrysanthemums with high ornamental and tea value, experiencing significant market demand. However, as cultivation areas expand, issues such as viral infection, germplasm degradation, low proliferation coefficient, and slow proliferation rate arise, necessitating the establishment of an efficient in vitro regeneration system. This study, based on the principles of orthogonal experimental design, explored the regeneration system of Chrysanthemum cultivar ‘11-C-2’ using sterile seedlings. The research focused on three key stages: adventitious bud differentiation, rooting culture, and acclimatization–transplantation, employing shoot-bearing stem segments and leaves as explants. The findings indicate that the optimal explant for the Chrysanthemum ‘11-C-2’ sterile seedlings is the shoot-bearing stem segment. The best medium for adventitious bud differentiation was determined to be MS supplemented with 1.5 mg/L 6-BA and 0.5 mg/L NAA. Bud differentiation began on day 17 with a 100% differentiation rate, completing around day 48. The maximum differentiation coefficient reached 87, with an average of 26.67. The adventitious buds were then cultured for rooting in the optimal medium of 1/2 MS supplemented with 0.1 mg/L NAA. Rooting was initiated on day 4 and was completed by day 14, achieving a rooting rate of 97.62%. After a 5-day acclimatization under natural light, the rooted seedlings were transplanted into a growth substrate with a peat-to-vermiculite ratio of 1:2. The plants exhibited optimal growth, with a transplantation survival rate of 100%. The findings provide data support for the efficient large-scale propagation of ‘11-C-2’ and lay the foundation for germplasm preservation and genetic transformation research of tea chrysanthemums. Full article

In our research, we utilized six small-fruited pepper germplasms as materials, selected cotyledons with the petiole and hypocotyls as explants, and conducted in vitro regeneration studies. Our outcomes specify that the most suitable explant is cotyledon with the petiole, and the suitable genotype is HNUCA341. The optimal medium for inducing and elongating adventitious buds for this genotype is Murashige and Skoog medium (MS) + 9.12 μM Zeatin (ZT) + 0.57 μM 3-Indoleacetic acid (IAA), with a bud induction rate of 44.4%. The best rooting induction medium is MS + 1.14 μM IAA, with a rooting rate of 86.7%. Research on the addition of exogenous hormones has revealed that the induction speed of buds in small-fruited pepper (HNUCA341) in the combination of ZT and IAA hormones (abbreviated as ZI) is quicker, and the induction effect is better. The histological observations indicate that ZI treatment accelerates the initiation of explant division and differentiation, causing a shorter duration of vascular-bundle tissue production. The plant hormone signaling pathway was significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, including ARR9 (LOC107843874, LOC107843885), ARR4 (LOC107848380, LOC107862455), AHK4 (LOC107870540), AHP1 (LOC107839518), LAX2 (LOC107846008), SAUR36 (LOC107852624), IAA8 (LOC107841020), IAA16 (LOC107839415), PYL4 (LOC107843441), and PYL6 (LOC107871127); these significantly enriched genes may be associated with in vitro regeneration. In addition, the carbon metabolism pathway and plant mitogen-activated protein kinase (MAPK) signaling pathway are also significantly enriched in KEGG. The results of the Gene Ontology (GO) analysis revealed that differentially expressed genes related to carbon metabolism and fixation, photosynthesis and MAPK signaling pathways were upregulated under ZI treatment. It was found that they might be associated with enhanced regeneration in vitro. Furthermore, we also screened out differentially expressed transcription factors, primarily from the MYB, bHLH, AP2/ERF, and NAC families. Overall, our work accumulated important data for the in-depth analysis of the molecular mechanism of in vitro regeneration of pepper, and provides valuable germplasm for establishing an efficient stable pepper genetic-transformation system based on tissue culture. Full article

Microsorum pteropus ‘Windeløv’ is a water fern from Asia commonly used in aquarium landscapes. This study aimed to develop a mass production method through the induction of green globular bodies (GGBs) from leaf explants of M. pteropus. Surface sterilization was performed on adventitious buds on the fronds of M. pteropus ‘Windeløv’ as explants. The results showed that the optimal explant disinfection condition was 1% sodium hypochlorite solution for 20 min, achieving a successful rate of 87.5 ± 9.6%. The most effective GGB induction was achieved with MS medium supplemented with 5.0 mg/L of NAA (1-Naphthaleneaceticacid) and 0.5 mg/L of 6-BA (6-benzylaminopurine), producing the highest number and size of GGBs, with a mean value of 4.0 ± 0.8 GGBs per explant and a diameter of 2.45 ± 0.61 mm after 8 weeks of culture. The regeneration of multiple sporophytes from the GGBs was only observed under hormone-free MS medium, with a mean value of 34.2 ± 7.3 sporophytes per GGB after 6 weeks of culture. Subsequently, rootless and separated sporophytes could be directly transplanted into the aquarium with a 100% survival rate. Full article

The optimization of explant selection and adjustment of plant growth regulators (PGRs) ratio may enhance the efficiency of micro-propagation of Gardenia jasminoides Ellis. The findings of the study suggest that the shoot tip proved to be the optimal explant for regenerating adventitious buds, with an impressive regeneration rate of 77.78% and the average number of adventitious buds being 2.86. The ideal medium consisted of Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (6-BA) at a 2 mg L⁻¹, indoleacetic acid (IAA) at a 0.2 mg L⁻¹, kinetin (KT) at 0.15 mg L⁻¹, resulting in an outstanding regeneration rate of adventitious buds reaching up to 91.11%. For rooting purposes, the best medium was found be half-strength MS supplemented with indoleacetic acid (IAA) 0.5 mg L⁻¹, achieving an rate for adventitious roots amounting to as high as 97.78%. The culture plantlets ultimately thrived, achieving an impressive transplanting survival rate of 93.33%. The application of PGRs was also found to enhance the regeneration of adventitious buds by increasing the ratios of endogenous hormones ZR/IAA and GA₃/IAA. Additionally, it facilitated the differentiation of adventitious roots by elevating the ratios of endogenous hormones IAA/ZR, IAA/GA₃, and ABA/GA₃. Our study would provide a theoretical reference for the establishment of an efficient gardenia tissue culture system and the industrial production of gardenia. Full article