Search Results (233)

Since the status of C. jambhiri is “rare” and C. aurantifolia is endowed with folk medicinal properties, our study aimed at producing true-to-type seedlings for further conservation by using PGRs, ferrous sulfate, and casein hydrolysate in MS medium to induce in vitro germination and “callus” formation from seeds of C. jambhiri Lush. It also focused on evaluation of suitable species and the best type of explant for organogenesis. The present study was undertaken to develop an efficient micropropagation protocol for C. jambhiri Lush. and C. aurantifolia. The frequency of callus induction increased to its maximum when 2.0 mg/L 2,4-D and 1.0 mg/L picloram were added individually. The calli derived from 2,4-D exhibited maximum regeneration potentiality. In addition, sucrose (30 g/L), dextrose (60 g/L), and coconut water (10 mL/L and 15 mL/L) also enhanced callus induction. Regarding heavy metals, 100 ppm of Fe₂SO₄ exhibited maximum germination percentage (84.33%) from seeds of C. jambhiri Lush. However, maximum callus induction (50.00%) was induced from the seeds of C. aurantifolia incubated in Fe₂SO₄ (400 ppm). The maximum number of shoots per callus was produced (5.13) with the addition of 200 mg/L casein hydrolysate in the callus-induction medium. Pearson correlation analysis revealed a positive and significant association of the number of plantlets/calluses with shoot length and regeneration percentage, respectively. It was phenotypically observed that the tissue culture traits performed better from explants derived from C. jambhiri Lush. than C. aurantifolia when subjected to varying concentrations of PGRs, carbon sources, organic adjuvants, and heavy metals, respectively. Full article

Globba bicolor Gagnep., an ornamental ginger of cultural importance in Thailand’s “Tak Bat Dok Mai” festival, faces conservation challenges due to climate change and slow natural propagation. Limited understanding of its cultivation and chemical composition further constrains sustainable utilization. This study provides the first integrated investigation of micropropagation using rhizome-derived explants under various combinations of exogenous hormones, acclimatization strategies, and comparative phytochemical profiling between wild and in vitro-propagated plants. An optimized clonal regeneration system was established from plantlets, with Murashige and Skoog (MS) medium containing 2.0 mg/L 6-benzylaminopurine (BA) and 0.5 mg/L 1-naphthaleneacetic acid (NAA), yielding the highest multiplication (9.10 shoots/explant and 12.40 roots/explant) after eight weeks of cultivation. During acclimatization, sand substrate proved superior, facilitating a 90% survival rate and enhanced physiological vigor. Comparative analysis revealed that while wild plants possessed significantly higher total phenolic (TPC) and total flavonoid (TFC) contents and antioxidant activities (DPPH, ABTS, and FRAP) than their in vitro counterparts, both sources maintained a rich diversity of chemical constituents. HPLC analysis identified cinnamic acid, rutin, and quercetin as major metabolites, while GC–MS detected 90 volatile compounds, with β-caryophyllene and β-pinene as predominant constituents. Notably, rhizomes of wild plants exhibited particularly high-value detections. To provide a rapid and non-destructive approach for linking chemical composition with antioxidant activity, FTIR-based chemometric models were applied, demonstrating high predictive accuracy (R²–cv = 0.9712–0.9862). These results provide a scientific foundation for the conservation and sustainable commercial utilization of G. bicolor as a potential source of bioactive natural products. Full article

Lilium, a traditional plant with dual medicinal and ornamental values, is restricted in its industrial development by the low natural propagation rate of bulbs and dependence on imported high-quality germplasm. To address this bottleneck, this study used new lines of LA hybrid lilies and Asiatic hybrid lilies (three cultivars) as experimental materials to establish an efficient and stable tissue culture and rapid propagation system. Key procedures including disinfection of different explants (bulb scales and capsules), adventitious bud induction and proliferation, rooting culture, as well as acclimatization and transplantation were systematically evaluated. The results showed that bulb scales were superior to capsule seeds as explants in tissue culture (contamination rate 9.44%, regeneration rate 11.92%). After disinfection with 75% ethanol combined with 10% sodium hypochlorite, the contamination rate could be controlled at 14.29–21.43%, and the regeneration rate reached 100%. Supplementation with 50 g·L⁻¹ sucrose + 1 mg·L⁻¹ 6-BA + 0.1 mg·L⁻¹ NAA (Treatment TA1), 50 g·L⁻¹ sucrose + 2 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ NAA (Treatment TA4) in MS medium, combined with dark culture, could effectively promote adventitious bud induction, proliferation and bulblet enlargement. For the rooting stage, the optimal media were 1/2 MS + 0.5 g·L⁻¹ activated charcoal + 2 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ NAA (Treatment TB4) or MS + 0.3 g·L⁻¹ activated charcoal + 2 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ NAA (Treatment TB1), and the highest rooting rate of ‘Pink Renault’ reached 100%. When plantlets from all three cultivars were combined and acclimatized and transplanted into sterilized peat soil, the overall survival rate was 89.33%. The TOPSIS method was also adopted for comprehensive evaluation to screen out the optimal culture conditions for different varieties. Based on phenotypic observation and physiological index data, ‘Pink Renault’ showed great potential as an excellent propagation germplasm. The integrated and optimized technical system provides a feasible solution for large-scale and industrialized seedling production of medicinal and ornamental lilies, and is of great practical significance for the efficient utilization of germplasm resources and sustainable development of the lily industry. Full article

As globally important ornamental and medicinal plants, orchids exhibit significant differences in the difficulty and pathways of in vitro regeneration. Most orchid species can directly form protocorm-like bodies (PLBs) through the differentiation of shoot tips or other explants, which then regenerate into new plantlets, while some species form callus through explant dedifferentiation followed by PLB differentiation from the callus. At present, the regenerative mechanisms underlying PLB and callus in orchids, as well as the key factors influencing their differentiation, remain poorly elucidated. In this study, seedlings of Dendrobium officinale obtained from aseptic seed germination were used to investigate the effects of explant type, 2,4-D concentration, temperature, light intensity and photoperiod on the induction of PLBs and callus. The results showed that there were no significant differences in callus induction among the tested explants in D. officinale, whereas stem nodal segments were more suitable for PLB induction. For both internodal and nodal segments, the incidence rate of callus formation was higher than that of PLBs. The concentration of 2,4-D influenced the induction direction of the explants; higher concentration promoted PLB induction, while lower concentration was sufficient for callus formation. Low temperature and low light intensity inhibited PLB induction while promoting callus formation in D. officinale. High temperature and intense light partially caused desiccation of explants. A temperature of 25/22 °C (day/night) and a photosynthetic photon flux density of 50 µmol m⁻² s⁻¹ were more suitable for callus or PLB induction in D. officinale. A shorter photoperiod favored callus induction, while a longer photoperiod was beneficial for PLB induction. This study reveals the differences in influencing factors for PLB and callus induction in D. officinale, providing important insights for the propagation of orchid seedlings and laying a significant foundation for elucidating the mechanisms of PLB and callus induction. Full article

The seedlings of the young Atlas cedar (Cedrus atlantica Manetti) are very important for the regeneration and restoration of forest populations of this endemic species, which inhabits a very fragmented area in the highest mountains of North Africa (Algeria and Morocco). There is very minimal information on the diseases that are afflicting these young plants. In this work, Alternaria strains CHP2, S4.2, and SP1.1 were isolated from different plants and subjected to identification and pathogenicity testing. The infected plants developed clear symptoms of light brown disease spots on the leaves with a yellowish or chlorotic halo around them, which gradually developed to a yellowing of the plantlets and their complete drying. Some spots merged to form large areas of necrosis which covered an average of 80% of the plantlets. The impact of the infection on plant physiology was determined using measurements of photosynthetic pigments, which showed reductions of 46.28% in chlorophyll and 59.90% in carotenoids in strains SP1.1 and CHP2, respectively. Molecular characterization of the ITS region of the isolates revealed that strains CHP2 and S4.2 showed high sequence similarity to reference sequences of Alternaria spp., including taxa related to A. destruens and A. murispora, although species-level identification remains tentative. These findings highlight the growing relevance of fungal pathogens in forest regeneration under global climate change. By revealing the pathogenic role of Alternaria species, this study contributes to sustainable forest management and conservation strategies in changing environments. Full article

To overcome the seasonal constraints of explant availability and facilitate genetic improvement in Catalpa ovata, this study established a dual-pathway in vitro regeneration system (encompassing adventitious shoot organogenesis and somatic embryogenesis) using mature zygotic embryos. We systematically evaluated the synergistic effects of maternal genotypes, plant growth regulators (PGRs), basal media, and the histone deacetylase inhibitor Trichostatin A (TSA). Genotype screening revealed significant divergence in regenerative potential, with the half-sib family 32F17 exhibiting superior responsiveness (84.7% callus induction). A high cytokinin-to-auxin ratio (ZA3 medium) optimally drove direct shoot organogenesis. For adventitious shoot proliferation, the addition of TDZ significantly improved the multiplication coefficient (up to 2.99 on ZB4 medium), although a physiological trade-off with shoot elongation was observed. In parallel, the application of 10 µM TSA significantly enhanced somatic embryogenesis from embryogenic calli, effectively alleviating the inhibitory constraints of exogenous PGRs. For rhizogenesis, the DKW basal medium proved superior to half-strength MS, with the ZE3 treatment (0.1 mg·L⁻¹ NAA + 0.1 mg·L⁻¹ IBA) yielding the highest rooting frequency (69.6%) and robust root architecture. Notably, while somatic embryo conversion remained recalcitrant, plantlets derived exclusively from the adventitious shoot organogenesis pathway were successfully acclimatized ex vitro. These transplanted plantlets exhibited consistently high survival rates (83.1–84.4%) across all tested genotypes, effectively overcoming the initial genotype-dependent recalcitrance. Collectively, this optimized protocol provides a reliable technical platform for the large-scale clonal propagation and biotechnological breeding of C. ovata. Full article

A simple method for the efficient high-throughput transformation of hybrid poplar (Populus tremula x alba clone 717 1B) is described. Factors considered in developing the method included the ease and efficiency of preparing large numbers of explants for transformation, and selection of culture media that enhanced cell and tissue growth while promoting shoot regeneration competence. We found that petiole explants from in vitro-cultured plantlets can be easily collected and prepared for transformation and regenerate shoots comparable to stem or leaf explants. Culturing petiole explants on Driver Kuniyuki Walnut (DKW) medium resulted in significantly greater tissue growth compared to Murashige Skoog (MS) medium. Moreover, the inclusion of low concentrations of thidiazuron (TDZ) in callus-inducing media (CIM) significantly enhanced shoot regeneration competence of cultured petiole explants. As a consequence, the combination of petiole explants cultured on DKW medium along with 2.2 ug/L TDZ during the callus induction phase resulted in rapid and efficient transformation of this hybrid poplar genotype. When applied to genomic approaches such as activation tagging or CRISPR-Cas9 and Cas12a gene editing, we obtained transformation efficiencies ranging between 70% and 90%. The described protocol provides a simple and efficient method that is easily scalable for high-throughput approaches, which could facilitate genome-wide methods for the rapid and efficient production of transformed hybrid poplars. Full article

Efficient in vitro propagation of Sida cordifolia L. from nodal explants is often limited by poor shoot and root elongation. This study aimed to improve elongation of shoots and roots from two-node explants through simple modifications of the Murashige and Skoog (MS) culture medium. Explants were cultured on full- and half-strength MS media. Half-strength MS (1/2MS) significantly increased shoot (3.9-fold) and root (4.2-fold) elongation compared to full-strength MS. Therefore, subsequent experiments were conducted using 1/2MS. Further modifications included supplementation of 1/2MS medium with KH₂PO₄, plant growth regulators (TDZ, BAP, 2iP, GA₃), and pretreatment of nodal segments with Norit buffer before cultivation. KH₂PO₄ did not further affect shoot elongation but significantly enhanced root elongation, with the greatest effect at 170 mg L⁻¹. Pretreatment of nodal segments in Norit buffer had almost the same effect as KH₂PO₄, both on shoots and roots. In contrast, cytokinins and GA₃ inhibited both shoot and root elongation compared to hormone-free 1/2MS medium. The results indicate that 1/2MS medium alone is most suitable for improving shoot and root elongation in vitro in S. cordifolia. Supplementation of the 1/2MS medium with 170 mg L⁻¹ KH₂PO₄ further enhances root elongation. The use of cytokinins and GA₃ should be avoided. Full article

Astragalus glycyphyllos (Fabaceae) is known to be a source of flavones, flavonols, and isoflavones, and its in vitro culture may promote the efficiency and sustainability of obtaining pharmacologically valuable fractions. The aim of this study was to develop an effective plantlet regeneration protocol for A. glycyphyllos, providing the accumulation of phenolic compounds and antioxidants in cultured tissues. The results show a maximum seed germination rate (67.8%) after scarification (mechanical with sandpaper followed by treatment with 50% sulfuric acid) and subsequent sterilization with 1.1% sodium hypochlorite solution. The maximum regeneration rate (95%) was achieved on Murashige and Skoog medium supplemented with 0.5 mg·L⁻¹ thidiazuron. A thidiazuron concentration of 0.05 mg·L⁻¹, combined with a twofold increase in iron chelate content, induced the maximum yield of total flavonoids (8.74 mg·g⁻¹ DW), and significant levels of total phenolics (4.15 mg·g⁻¹) and antioxidants (1.83 mg AAE·g⁻¹) in the microshoot tissues. HPLC analysis showed kaempferol glycosides (1.51 mg·g⁻¹) and acylated kaempferol glycosides (2.76 mg·g⁻¹) as major components. Formononetin in a modest amount (0.09 mg·g⁻¹) was detected in hydrolyzed extracts. The phenolic profiles of the microshoots and native plants coincided in hydroxycinnamic acid composition; meanwhile, quercetin glycosides were present only in in situ plants, and formononetin was found only in the plantlets. The results confirm the prospects of biotechnological methods for the industrial production of standardized medicinal raw materials. Full article

Ulluco (Ullucus tuberosus Caldas) is an Andean tuber crop of high nutritional and genetic importance. However, its vegetative propagation promotes the accumulation of pathogens and limits the availability of uniform, high-quality planting material. In this study, an efficient and reproducible in vitro micropropagation protocol was established for an ulluco genotype from the Amazonas region of Peru. Nodal segments were cultured on MS (Murashige and Skoog) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (KIN) at increasing concentrations (0.0–2.0 mg L⁻¹). For rooting, in vitro-derived shoots were transferred to MS medium supplemented with indole-3-butyric acid (IBA) or 1-naphthaleneacetic acid (NAA) at the same concentration range (0.0–2.0 mg L⁻¹). The explants exhibited a high basal morphogenetic capacity; however, the addition of cytokinins significantly enhanced the response. KIN at 2.0 mg L⁻¹ achieved 100% regeneration, whereas BAP at 0.2 mg L⁻¹ maximized shoot proliferation, producing 2.07 shoots per explant. Shoot elongation was greater with KIN at 1.0 mg L⁻¹, reaching 39.15 mm. In the rooting phase, the response varied depending on the type and concentration of auxin. NAA at 0.1 mg L⁻¹ resulted in 100% rooting and produced the greatest root length (41.93 mm), whereas IBA at 0.1 mg L⁻¹ maximized the number of roots (4.67), although roots were shorter. Rooted plantlets exhibited 100% survival after eight weeks of acclimatization. This protocol provides an effective system for the rapid production of vigorous and uniform clonal plants and represents a useful tool for the propagation, conservation, and future biotechnological improvement of ulluco. Full article

Papaya (Carica papaya L.) is a highly cross-pollinated crop that exhibits considerable genetic variability when propagated through seeds, resulting in non-true-to-type progeny. Therefore, the development of an efficient in vitro regeneration system is essential for large-scale clonal propagation of elite cultivars. In the present study, a highly efficient and reproducible somatic embryogenesis protocol was developed for C. papaya var. TNAU Papaya CO 8 using immature zygotic embryos as explants. This study provides the first comprehensive comparative evaluation of three basal media, viz., Murashige and Skoog Medium, N6 Medium, and Woody Plant Medium, for somatic embryogenesis and plant regeneration in this variety, along with the optimization of polyamine-enriched media for enhanced plantlet recovery. The embryogenic potential of explants was assessed across different stages, including callus induction, somatic embryo development, plant regeneration, shoot elongation, rooting, and acclimatization. Maximum callus induction (81.96%) was observed on half-strength MS medium supplemented with 2,4-Dichlorophenoxyacetic acid under dark conditions, followed by ½ N6 (63.00%) and ½ WPM (58.02%). Somatic embryo initiation was highest on ½ MS medium containing 2.0 mgL⁻¹ 2,4-D (77.82%). Somatic embryos developed through distinct globular, heart, torpedo, and cotyledonary stages. Embryo maturation was significantly enhanced on MS medium supplemented with abscisic acid, polyethylene glycol, benzylaminopurine, and proline. The highest plantlet regeneration (85.02%) was achieved on MS medium enriched with putrescine, whereas comparatively lower regeneration was recorded on N6 (75.99%) and WPM (57.97%). Shoot elongation was significantly improved by supplementation with gibberellic acid (1.0 mgL⁻¹). Root induction was optimal on half-strength MS medium containing Indole-3-butyric acid, 1-Naphthaleneacetic acid, phloroglucinol, and activated charcoal, resulting in well-developed roots. Regenerated plantlets were successfully acclimatized in a cocopeat–vermicompost substrate with a survival rate of 74.01%. The optimized protocol provides a reliable and efficient system for large-scale clonal propagation and offers promising applications in genetic transformation and commercial production of papaya var. TNAU papaya CO 8. Full article

Eucalyptus is a highly diverse genus of the Myrtaceae family that is planted worldwide. Many changes occur during callus development, an important process during in vitro plant regeneration. In this study, we conducted methylome and transcriptome analyses to reveal such changes. The results showed that differentially expressed genes between E. camaldulensis (voucher ID: c0009; high embryogenic potential) and E. grandis × urophylla (voucher ID: j0017; low embryogenic potential) during callus development were enriched in plant hormone signal transduction and MAPK (Mitogen-activated protein kinase) signaling pathways. qRT-PCR analysis showed AHP, BAK1, BSK, CRE1, GID1, MKS1, PR-1, PYL, RbohD, and TCH4 could be involved in the callus development and plantlet regeneration capacity. The differences observed in regenerative potential during callus maturation between the two species under study provide a reliable molecular basis for the study of Eucalyptus regeneration mechanisms. Full article

CRISPR-Cas9 technology has opened new perspectives in genome editing of clonally, asexually propagated and polyploid plants by enabling multiple allelic gene edits. Traditional Agrobacterium- and particle bombardment-mediated transformations, which rely on integration of gene-editing transgene cassettes, have been efficiently applied to several plants; however, concerns about the acceptability of resultant edited transgenic genotypes make these methods less attractive for vegetatively propagated crops. We leveraged and optimized the CRISPR-Cas9/sgRNA-RNPs system for delivery into protoplasts of the hexaploid sweetpotato cultivar PI-318846, targeting eukaryotic translation initiation factor isoform 4E genes to enhance resistance to SPFMV potyviruses. To evaluate the efficiency of pre-assembled Cas9/sgRNA-RNP in sweetpotato transfection, single guide RNAs were designed to target putative host susceptibility genes: IbeIF4E, IbeIF(iso)4E, and IbCBP. Freshly isolated leaf protoplasts were subjected to CRISPR-CAS9-RNP PEG-mediated transfection under different parameters. Sweetpotato regenerants screened using PCR-RE-T7 assay, sequencing, and Inference CRISPR Edit analyses of target-site amplicons revealed the most efficient editing conditions utilizing 25% PEG with a 3:1 (15 µg:45 µg) ratio of Cas9/sgRNA-RNP for 25 min and 48 h incubation period. Different allelic InDels were obtained with editing efficiencies of 10–20% in regenerated plantlets, demonstrating that PEG-mediated CRISPR-RNP transfection system is key for advancing DNA-free editing tools in polyploid and vegetatively propagated crops. Full article

Vanilla planifolia is an endangered orchid of significant commercial relevance, primarily valued for the natural vanillin derived from its cured fruits. However, its global production faces critical threats due to its limited genetic variability and high susceptibility to phytopathogens, particularly vanilla wilt caused by Fusarium oxysporum f. sp. vanillae. This review synthesizes the recent advances in plant biotechnology, evaluating the efficacy of in vitro culture systems, plant growth regulators, and the implementation of semi-automated temporary immersion systems, as compared to traditional semisolid methods. Emphasis is placed on the pivotal role of physical factors, such as LED lighting, and the symbiotic associations with orchid mycorrhizal fungi to enhance plant growth and vigor. By synthesizing advanced in vitro regeneration protocols, this study establishes a strategic guide for the mass production of high-quality disease-free plantlets. Finally, the impact of these biotechnological tools on ex situ conservation at institutions such as the Clavijero Botanical Garden is discussed, aiming to support the sustainability of the vanilla industry and preserve Mexico’s biological heritage. Full article

In the present study, we developed an efficient and reproducible protocol for in vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Broussonetia papyrifera (L.) L’Hér. ex Vent. (paper mulberry) using leaf explants from a hybrid genotype. First, we optimized surface sterilization of leaf explants. Treatment with 0.6% (w/v) sodium hypochlorite for 8 min, followed by three rinses with sterile water and blotting on sterile filter paper, yielded a 33.60% explant survival rate and reduced contamination to 35.84%. Second, we refined the co-cultivation step for transformation using A. tumefaciens strain EHA105 carrying pCAMBIA1300-35S-eGFP. Leaf discs were infected for 20 min and co-cultured for 2 days on co-cultivation medium overlaid with sterile filter paper, which limited the overgrowth of A. tumefaciens. After co-cultivation, explants were transferred sequentially to callus induction, shoot induction, shoot multiplication, and rooting media supplemented with 250 mg·L⁻¹ cefotaxime and 200 mg·L⁻¹ Timentin, as well as 5.0 mg·L⁻¹ hygromycin at a concentration that completely suppressed regeneration of non-transformed explants. Meanwhile, after transfer to the callus induction medium, eGFP fluorescence was detected in resistant calli as an initial screening for transformants. The integration and expression of the transgene were further confirmed by PCR and quantitative reverse transcription PCR (qRT-PCR) after the resistant calli developed into plantlets. Collectively, this streamlined protocol provides a practical platform for functional genomics and genetic improvement of B. papyrifera. Full article