Search Results (111)

Eurasian aspen (Populus tremula L.) is a tree species with recognised ecological and economic importance for both natural and plantation forests. For the fast cloning of selected aspen genotypes, the method of plant propagation through in vitro culture (micropropagation) is often recommended. The efficiency of this method is related to the use of shoot-inducing chemical growth regulators, among which cytokinins, a type of plant hormone, dominate. Although cytokinins can inhibit rooting, this effect is avoided by using cytokinin-free media. This study sought to identify concentrations and combinations of growth regulators that would stimulate one type of P. tremula organogenesis (either shoot or root formation) without inhibiting the other. The investigated growth regulators included cytokinin 6-benzylaminopurine (BAP), auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA), auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), gibberellin biosynthesis inhibitor paclobutrazol (PBZ), and a gibberellin mixture (GA₄/₇). Both BAP and TIBA increased shoot number per P. tremula explant and decreased the number of adventitious roots, but TIBA, in contrast to BAP, did not inhibit lateral root formation. However, for the maintenance of both adventitious shoot and root formation above the control level, the combination of PBZ and GA_4/7 was shown to be especially promising. 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

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

Urban expansion has led to two significant environmental challenges: the reduction in green spaces and the rise in urban temperatures, decreasing city livability. Green roofs have emerged as a sustainable solution to mitigate these issues, offering ecological and economic benefits while improving building energy efficiency. Some species of the genus Sedum, particularly Sedum sediforme and Sedum album, are ideal for such green infrastructure due to their non-aggressive and superficial root system, high drought tolerance, low nutrient needs, pest and disease resistance, and metabolic adaptability during dry periods. This study aims to optimize the large-scale production of two native ecotypes of S. sediforme and S. album from the Valencian Community through an efficient propagation system that enables uniform plant production in limited space. For this purpose, we have developed micropropagation systems that allow a rapid multiplication of these two species. A direct morphogenesis system was established using axenic plant shoots, and a protocol for adventitious organogenesis from leaves was also developed. These methods significantly enhance propagation speed, spatial efficiency, and plant uniformity. Notably, the metabolic plasticity of S. sediforme and S. album reduces abiotic stress during acclimatization, promoting efficient ex vitro establishment and functional integration into extensive green roof ecosystems. Full article

Interspecific and intersectional crosses have introduced valuable genetic traits for blueberry (Vaccinium sect. Cyanococcus) cultivar improvement. Introgression from Vaccinium species at the diploid, tetraploid, and hexaploid levels has been found in cultivated blueberries. Continued efforts to integrate wild blueberry genetic resources into blueberry breeding are essential to broaden the genetic diversity of cultivated blueberries. However, performing heteroploid crosses among Vaccinium species is challenging. Polyploid induction through tissue culture has been useful in bridging ploidy barriers. Mixoploid or chimeric shoots often are produced, along with solid polyploid mutants. These chimeras are mostly discarded because of their genome instability and the difficulty in identifying periclinal mutants carrying germline mutations. Since induced polyploidy in blueberries often results in a low frequency of solid mutant lines, it is important to recover solid polyploids through chimera dissociation. In this study, two vegetative propagation methods, i.e., axillary and adventitious shoot induction, were evaluated for their efficiency in chimera dissociation. Significantly higher rates of chimera dissociation were found in adventitious shoot induction compared to axillary shoot induction. Approximately 89% and 82% of the adventitious shoots induced from mixoploid lines 145.11 and 169.40 were solid polyploids, respectively, whereas only 25% and 53% of solid polyploids were recovered through axillary shoot induction in these lines. Effective chimera dissociation provides useful and stable genetic materials to enhance blueberry breeding. Full article

Triadica sebifera, an economically and medicinally valuable tree species native to China, was investigated for its in vitro regeneration potential using leaf explants from nodal cuttings of young stems and sprouts. This study evaluated the effects of basal media, plant growth regulators (PGRs), explant sources, and incision methods on adventitious shoot induction, supplemented by histological analysis. The highest shoot regeneration frequency (98.89%) and maximum shoot number (72) were achieved via direct organogenesis using sucker-derived nodal cuttings cultured on MS medium with 2 mg/L 6- benzyladenine (6-BA), 0.3 mg/L kinetin (KT), and 0.2 mg/L α-naphthaleneacetic acid (NAA). Under identical conditions, branch-derived explants showed lower regeneration (84.44%, 64 shoots). Transverse midvein incision proved most effective, with sucker-derived leaves exhibiting superior regeneration. Shoots elongated completely (100%) on Murashige and Skoog (MS) medium containing 0.3 mg/L 6-BA, 0.03 mg/L NAA, and activated charcoal. Rooting was optimal on MS medium with 0.3 mg/L indole-3-butyric acid (IBA), yielding a 98% acclimatization survival rate. Histological analysis revealed de novo meristem formation from parenchyma cells, confirming direct organogenesis without callus intermediation, further validating the enhanced regenerative capacity of sprout-derived explants. This efficient in vitro regeneration system provides a foundation for large-scale propagation and germplasm conservation of T. sebifera, while offering insights for woody plant regeneration studies. Full article

Populus species are important resources for ecological conservation and certain industry productions, and are also considered model tree species for scientific research. For tree species, in vitro plant regeneration is an important method of propagation due to the advantage of high multiplication rate. Although many molecular determinants for poplar regeneration have been investigated, the complete regulatory hierarchy network remains unclear. In this study, we tracked the temporal changes of endogenous hormone contents, physiological characteristics and transcriptional profiles during callus induction and adventitious organogenesis in a stem of Populus alba L. to explore the regulatory dynamics of in vitro regeneration in poplars. The results imply that auxin may promote the formation of callus in P. alba by activating the expression of WOX11/12. By up-regulating the expression of CUC1/2, the development of callus begins to initiate apical meristem (SAM) at day 12. The cytokinin-mediated pathway regulates the adventitious shoot formation by ESR1 and WUS. The precursors of active gibberellin GA1, GA53 and GA19 were accumulated in the early stage of callus induction, and then they continued to decrease. JA may function on adventitious shoot regeneration due to its accumulation after 12 days of induction. The dominant hormonal components and regulatory factors during regeneration were identified. Based on the results, a regeneration pathway regulated by auxin and cytokinin for poplars is proposed. The key regulators identified in this study will accelerate the exploration and understanding of the asexual reproduction mechanism of poplar trees. 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

Nandina domestica ‘Firepower’ is one of the most popular colorful foliage species in landscaping. However, it is currently propagated mainly by seeding and cuttings, with a low reproduction coefficient, hindering the cultivation of this species. Therefore, establishing an in vitro regeneration system would be beneficial for the industrialized production of Nandina domestica ‘Firepower’. In this study, an ex vivo regeneration system was established using the direct organogenesis pathway. In early April, the new shoots of Nandina domestica ‘Firepower’ were selected, and the stem segments of 1~2 cm were cut as the disinfection materials for the explants. The optimal formulation for inducing axillary shoots was 1/2 MS + 1.5 mg L⁻¹ 6-benzylaminopurine (BA) + 0.3 mg L⁻¹ indole-3-butric acid (IBA). The optimal formulation for the differentiation and proliferation of axillary shoots was 1/2 MS + 1.5 mg L⁻¹ BA + 0.01 mg L⁻¹ IBA with a multiplicity of proliferation of 9.22. We determined that the rooting of axillary shoots required a combination of IBA, naphthalene acetic acid (NAA), and activated carbon (AC). The optimal formulation for rooting was 1/2 MS + 0.2 mg L⁻¹ NAA + 0.3 mg L⁻¹ IBA + 0.2 mg L⁻¹ AC. After a two-day hardening period for tissue-cultured plantlets, a substrate consisting of peat soil, vermiculite, and perlite at a ratio of 2:2:1 was determined to be the optimal cultivation formulation. This system provides a framework for the industrialized production of Nandina domestica ‘Firepower’. Full article

Cannabis sativa L. cv. ‘Cheungsam’ is an industrial hemp plant of Republic of Korea origin, primarily cultivated for fiber and seed production. In vitro seed germination and tissue culture are valuable tools for developing various biotechnological techniques. In the present study, we aimed to develop a tissue culture process for hemp plants using Cheungsam as a model plant and examine the secondary metabolites produced from its callus. We also developed a method to prepare pathogen-free seedlings from field-derived seeds using hydrogen peroxide (H₂O₂) solution as a liquid germination medium. Treating seedlings with removed seed coat in 3% H₂O₂ significantly reduced the contamination rate. Callus formation and de novo organogenesis of shoots and roots from callus were successfully achieved using cotyledon and leaf tissues prepared from the pathogen-free seedlings. The most effective in vitro regeneration results were obtained using the Murashige and Skoog (MS) medium supplemented with certain targeted growth regulators. An optimal combination of 0.5 mg/L thidiazuron (TDZ) and 1.0 mg/L 1-naphthalene acetic acid proved highly effective for callus induction. The addition of 0.5 mg/L TDZ in the MS medium significantly stimulated shoot proliferation, while robust root development was best supported by MS medium supplemented with 2.5 mg/L indole-3-butyric acid for both cotyledon and leaf explants. Finally, gas chromatography–mass spectrometry (GC–MS) analysis of ethanol extract from Cheungsam leaf callus revealed the presence of different secondary metabolites, including 9-octadecenamide, methyl salicylate, dodecane, tetradecane, and phenol, 2,4-bis-(1,1-dimethylethyl). This study provides a comprehensive de novo regeneration protocol for Cheungsam plants and insight into the secondary metabolite profiles of its callus. Full article

Soybean’s lengthy protocols for transgenic plant production are a bottleneck in the transgenic breeding of this crop. Explants cultured on a medium for an extended duration exhibit unanticipated modifications. Stress-induced somaclonal variations and in vitro contaminations also cause substantial losses of transgenic plants. This effect could potentially be mitigated by direct shoot regeneration without solid media or in-planta transformation. The current study focused primarily on developing a rapid and effective media-free in-planta transformation technique for three soybean genotypes (Wm82) and our newly developed two hybrids, designated as ZX-16 and ZX-3. The whole procedure for a transgenic plant takes the same time as a stable grown seedling. Multiple axillary shoots were regenerated on stable-grown soybean seedlings without the ectopic expression of developmental regulatory genes. An approximate amount of 200 µL medium with a growth regulator was employed for shoot organogenesis and growth. The maximal shoot regeneration percentages in the Wm82 and ZX-3 genotypes were 87.1% and 84.5%, respectively. The stable transformation ranged from 3% to 8.0%, with an average of 5.5%. This approach seems to be the opposite of the hairy root transformation method, which allowed transgenic shoots to be regenerated on normal roots. Further improvement regarding an increase in the transformation efficiency and of this technique for a broad range of soybean genotypes and other dicot species would be extremely beneficial in achieving increased stable transformation. Full article

Gaillardia pulchella is an important plant species with pharmacological and ornamental applications. It contains a wide array of phytocompounds which play roles against diseases. In vitro propagation requires callogenesis and differentiation of plant organs, which offers a sustainable, alternative synthesis of compounds. The morphogenetic processes and the underlying mechanisms are, however, known to be under genetic regulation and are little understood. The present study investigated these events by generating transcriptome data, with de novo assembly of sequences to describe shoot morphogenesis molecularly in G. pulchella. The RNA was extracted from the callus of pre- and post-shoot organogenesis time. The callus induction was optimal using leaf segments cultured onto MS medium containing α-naphthalene acetic acid (NAA; 2.0 mg/L) and 6-benzylaminopurine (BAP; 0.5 mg/L) and further exhibited a high shoot regeneration/caulogenesis ability. A total of 68,366 coding sequences were obtained using Illumina150bpPE sequencing and transcriptome assembly. Differences in gene expression patterns were noted in the studied samples, showing opposite morphogenetic responses. Out of 10,108 genes, 5374 (53%) were downregulated, and there were 4734 upregulated genes, representing 47% of the total genes. Through the heatmap, the top 100 up- and downregulating genes’ names were identified and presented. The up- and downregulated genes were identified using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Important pathways, operative during G. pulchella shoot organogenesis, were signal transduction (13.55%), carbohydrate metabolism (8.68%), amino acid metabolism (5.11%), lipid metabolism (3.75%), and energy metabolism (3.39%). The synthesized proteins displayed phosphorylation, defense response, translation, regulation of DNA-templated transcription, carbohydrate metabolic processes, and methylation activities. The genes’ product also exhibited ATP binding, DNA binding, metal ion binding, protein serine/threonine kinase -, ATP hydrolysis activity, RNA binding, protein kinase, heme and GTP binding, and DNA binding transcription factor activity. The most abundant proteins were located in the membrane, nucleus, cytoplasm, ribosome, ribonucleoprotein complex, chloroplast, endoplasmic reticulum membrane, mitochondrion, nucleosome, Golgi membrane, and other organellar membranes. These findings provide information for the concept of molecular triggers, regulating programming, differentiation and reprogramming of cells, and their uses. Full article

We present data on the ability for organogenesis in 22 genotypes of grapevine and developed a direct organogenesis protocol for the cultivar Podarok Magaracha and the rootstock Kober 5BB. The protocol does not require replacement of culture media and growth regulators, and the duration is 11 weeks. The cultivation of explants occurs on modified MS medium with the addition of 2.0 mg L⁻¹ benzyladenine and indole-3-butyric acid (0.15 mg L⁻¹ for the rootstock Kober 5BB or 0.05 mg L⁻¹ for the cultivar Podarok Magaracha). The direct organogenesis protocol consists of three time periods: (1) culturing explants for 2 weeks in dark conditions for meristematic bulk tissue, (2) followed by 4 weeks of cultivation in light conditions for regeneration, and (3) 5 weeks of cultivation in dark conditions for shoot elongation. Based on this protocol, conditions for the Agrobacterium-mediated transformation of the Podarok Magaracha cultivar were developed with an efficiency of 2.0% transgenic plants per 100 explants. Two stably transformed lines with integration into the genome of the pBin35SGFP plasmid construction, confirmed by Southern blotting, were obtained. Full article

Mango (Mangifera indica L.) is one of the most significant tropical and subtropical fruit species, with high ecological and economic value. However, research on the in vitro culture of mangoes is relatively weak, so establishing an efficient and stable mango plant regeneration system is of great significance. In this study, a preliminary mango regeneration system was established with Mangifera indica L. cv. Keitt from young branches as the starting explants. The results showed that the optimal plant growth regulator (PGR) formula for direct adventitious shoot induction on the branches was 1 mg/L 6-benzylaminopurine (6-BA) + 0.1 mg/L a-naphthaleneacetic acid (NAA), with an adventitious shoot induction rate of 73.63% and an average of 6.76 adventitious shoots. The optimal basal medium for adventitious shoot induction was wood plant medium (WPM), with an adventitious shoot induction rate of 63.87% and an average of 5.21 adventitious shoots. The optimal culture medium for adventitious shoot elongation was WPM + 1 mg/L 6-BA + 0.5 mg/L NAA, with an adventitious shoot elongation rate of 89.33% and an average length of 5.17 cm. The optimal formula for the induction of mango rooting was Douglas fir cotyledon revised medium (DCR) + 3 mg/L indole-3-butyric acid (IBA), with a maximum rooting rate of 66.13% and an average rooting quantity of 6.43. The genetic fidelity of the in vitro-regenerated plants was evaluated using inter-simple sequence repeat (ISSR) molecular markers. There was no difference between the in vitro-regenerated plants and the parent plant. This study provides an efficient and stable propagation system for Mangifera indica L., laying the foundation for its rapid propagation and genetic improvement. Full article

Allium microdictyon Prokh. is a rare, endemic species possessing good taste qualities and listed in the Red Book of Kazakhstan; therefore, it is subject to anthropogenic impact (food gathering, grazing, logging, fires, etc.), which leads to a substantial reduction of its area. The aim of the study was to develop a protocol for microclonal propagation of A. microdictyon. Mature seeds of A. microdictyon collected from natural habitats in the Kazakhstani Altai were used as explants. Optimization of seed sterilization methods, selection of growth regulators for inducing adventitious shoot formation and microclonal propagation, and optimization of conditions for adaptation of regenerants to ex vitro conditions were carried out. Surface sterilization of seeds with 70% EtOH and 0.01% HgCl₂ is optimal for obtaining sterile and viable A. microdictyon seedlings. Sterile seedlings obtained in vitro on ½ Murashige and Skoog medium supplemented with 10 mg L⁻¹ gibberellic acid and 0.1 mg L⁻¹ indole-3-acetic acid (IAA) were used as a source for obtaining micropropagation cultures. Induction of adventitious organogenesis of A. microdictyon was effective on media containing 0.5 mg L⁻¹ 6-benzylaminopurine (BAP) and 1.5–2 mg L⁻¹ zeatin. On these variants, leaf conglomerates consisting of abundantly overgrown thin leaves were formed. The effect of 0.2 mg L⁻¹ indole-3-butyric acid (IBA) on further development of organogenesis and formation of microbulbs in A. microdictyon was shown in comparison with IAA, NAA, and PAC. Regenerated A. microdictyon plants were adapted to ex vitro conditions and resumed growth after 16–20 weeks of relative dormancy. The developed micropropagation protocol can be used to preserve germplasm and propagate for subsequent restoration of A. microdictyon populations in natural habitats. Full article