Search Results (32)

An efficient in vitro propagation protocol for Eulophia bicallosa was developed using asymbiotic seed germination and protocorm proliferation. The effect of light on seed germination and development was evaluated on Vacin and Went (VW) medium under five conditions: darkness, white, green, red, and blue light for 24 weeks. Blue and red light significantly accelerated seed development, allowing progression to stage 5 within 24 weeks. For protocorm proliferation, six semi-solid culture media were tested. Half-strength Murashige and Skoog (½MS) medium yielded the best results after 8 weeks, producing the highest numbers of shoots (1.0), leaves (1.1), and roots (4.2) per protocorm, with 100% survival. The effects of organic additives were also evaluated using coconut water and potato extract. A combination of 200 mL L⁻¹ coconut water and 50 g L⁻¹ potato extract enhanced shoot formation (1.7 shoots), while 150 mL L⁻¹ coconut water with 50 g L⁻¹ potato extract increased both leaf (1.9) and root (8.8) numbers. The effects of cytokinins (benzyladenine (BA), kinetin (6-furfurylaminopurine), and thidiazuron (TDZ)) and auxins (indole-3-acetic acid (IAA), α-naphthalene acetic acid (NAA), indole-3-butyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D)) were investigated using ½MS medium supplemented with each plant growth regulator individually at concentrations of 0, 0.1, 0.5, 1.0, and 2.0 mg L⁻¹. Among the cytokinins, 0.1 mg L⁻¹ BA produced the highest survival rate (96%), while 1.0 mg L⁻¹ BA induced the greatest shoot formation (93%, 2.3 shoots). Among the auxins, 0.1 mg L⁻¹ IAA resulted in the highest survival (96%), and 1.0 mg L⁻¹ IAA significantly enhanced root induction (4.2 roots per protocorm). Acclimatization in pots containing a 1:1:1 (v/v) mixture of pumice, sand, and soil resulted in 100% survival. This protocol provides a reliable and effective approach for the mass propagation and ex situ conservation of E. bicallosa. Full article

Aerides rosea (Orchidaceae) boasts high ornamental value due to its pleasant aroma, foxtail spike, and elegant floral morphology. Inducing A. rosea to become tetraploid enhances horticultural traits and facilitates fertile intergeneric hybrids through crosses with other market-available tetraploid species. The experimental design involved the application of colchicine at varying concentrations—0.05%, 0.1%, and 0.2%—to a solid medium. Exposure durations were 5, 10, and 15 days, with treatments conducted under sterile conditions on 6-week-old protocorms post-germination. Results indicated that the protocorms were sensitive to colchicine concentrations exceeding 0.05%, with high concentrations leading to a mortality rate exceeding 50%. Flow cytometry (FCM) with 4′,6-diamidino-2-phenylindole (DAPI) staining confirmed a doubling of chromosome numbers in tetraploid plants (2n = 4x = 76) compared to diploid controls (2n = 2x = 38). Induction efficiency was significantly influenced by colchicine concentration and treatment duration. A 10-day treatment with 0.2% colchicine yielded a 70.00% tetraploid induction rate; however, considering protocorm survival, a 5-day treatment with 0.05% colchicine was preferable, achieving a 63.55% survival rate and a 56.67% tetraploid induction rate. Tetraploid plants exhibited distinct morphological traits, such as a more compact growth habit, thicker leaves, and increased stem and root thickness. Leaf morphology changes included larger stomata with reduced density, denser spongy mesophyll, and more pronounced venation. Tetraploids also demonstrated a 1.94-fold increase in genome size compared to diploids. The tetraploid genotypes developed in this study hold significant potential for future Aerides breeding programs. Full article

Liparis loeselii (L.) Rich, an endangered member of the Orchidaceae family, is found in alkaline fens. With the declining populations of L. loeselii, there is a pressing need to reintroduce this species in Central Europe. As in vitro germination is a crucial tool for obtaining plants for introduction into the environment, we looked at the morphological changes occurring during the early stages of L. loeselii development in vitro. As the early stages of orchid development, especially the protocorm stage, are thought to be responsible for SAM formation and the initiation of symbiotic association, we focused on cell wall elements whose epitopes have been found in similar processes in other species: the extensin and pectin rhamnogalacturonan I (RG-I) side chain epitopes. We addressed the following questions: Does the cell wall of L. loeselii change its composition during the early stages of development, as noted in other species? Are there noticeable similarities in the cell wall to organs of different species whose function is to contact microorganisms? Are there regularities that allow the recognition of individual structures on this basis? Immunolocalization revealed changes in the distribution of certain extensins (JIM11 and JIM20) and RG-I (LM5 and LM6) side chain epitopes. Extensins, a type of cell wall protein, were observed during the initial stages of the formation of PLB and the shoot apical meristem of protocorms and PLBs. RG-I, on the other hand, was found to play a significant role in the development of the protocorm and PLB. In pseudobulbs, which appeared on the protocorms, extensins occurred in their storage part. However, RG-I side chains (1→4)-β-galactans (LM5), and (1→5)-α-L-arabinans (LM6) were not found in pseudobulbs. We revealed that a common feature of protocorms and PLBs was an increased amount of extensins, which were detected with the JIM11 antibody, and pectins, which were detected with the LM5 antibody, that were present together, which may prove helpful in determining the identity of the induced structures and distinguishing them from pseudobulbs. Thus, our study unveiled the role of extensins and RG-I during the growth of protocorms and PLBs. We suggest that PLBs may mimic the wall remodelling that occurs in protocorms, which indicates that using cell wall components is an invitation to be colonised by a fungal partner. However, this needs to be tested in future research. The findings of this research can help interpret future studies on the propagation, acclimatisation, and introduction of L. loeselii into the natural environment. Full article

Cymbidium faberi Rolfe is one of the traditional Chinese orchids with important ornamental value, and the cultivation of Cymbidium faberi Rolfe mutant strains with different appearances is essential to increase its economic value. However, at present, their acquisition largely relies on natural mutation. The objectives of this research were to mutagenize Cymbidium faberi Rolfe protocorm-like bodies (PLBs) and shoots in vitro using sodium azide (NaN₃) and to screen and evaluate mutants in the mutagenized seedlings using morphological characteristics. Cymbidium faberi Rolfe PLBs and shoots were used as mutagenic materials. Mutations were induced by the addition of 0.0 (control), 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg·L⁻¹ and 0.0 (control), 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 mg·L⁻¹ NaN₃ to a shoot-growth-inducing medium. The mortality rates of the PLBs and shoots increased with an increase in the NaN₃ concentration. At 14 d of co-cultivation, the PLBs and shoots were most efficiently mutagenized with 1.5 mg·L⁻¹ and 4.0 mg·L⁻¹ NaN₃, respectively. After the explants were cultured for 3 months, changes in leaf and flower morphology were observed in some mutants: shorter and thicker leaves, shorter node length, reduced height, and mid-translucent leaves compared with controls. Some Cymbidium faberi Rolfe bloomed prematurely, with single flowers with large, thick petal sepals and small inflorescences. Colors included light green throughout, with some exhibiting purple stamens. This suggests that NaN₃ can effectively mutagenize Cymbidium faberi Rolfe PLBs and shoots to satisfy people’s demand for this plant’s ornamental properties while increasing its economic value. Full article

Dendrobium officinale Kimura & Migo is a perennial herbaceous plant of the genus Dendrobium in the family of Orchidaceae with high medicinal value. Melatonin (MT) is an indole-like tryptamine with functions such as regulating plant growth and development. This experiment investigated the effects of different concentrations of MT on the growth and development of protocorms of D. officinale protocorm-like bodies (PLBs). The results showed that the changes in morphological indicators such as color, cluster size, and surface changes were more significant under 75 µM MT than those of 0 µM (CK), and the appearance of white on the PLB surface was expedited, which was more conducive to the proliferation of PLBs. MT treatment of 100 µM inhibited the differentiation of adventitious buds, and the contents of photosynthetic pigments, polysaccharides, and flavonoids were significantly increased. Moreover, as compared with CK, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in PLBs increased significantly, while the content of malondialdehyde (MDA) decreased gradually with 75 µM or less. In conclusion, a concentration of 75 µM melatonin can enhance the rapid propagation rate of D. officinale in vitro, providing insights into the effects of melatonin on the growth of tissue-cultured D. officinale seedlings. Full article

Epiphytic orchids comprise 68% of vascular epiphytes globally; nevertheless, many are endangered. One such epiphytic orchid is Phalaenopsis japonica, which is widely used in the floricultural industry. This study aimed to identify the mycorrhizal fungi of adult P. japonica and their roles in seed germination and seedling development. Root samples were collected from 32 adults across 4 sites in southern Japan, and mycorrhizal fungi were identified using Sanger and high-throughput sequencing (HTS). The results show phylogenetically diverse mycobionts, mainly Ceratobasidiaceae (CE) and Tulasnellaceae (TU), with dominant OTUs designated CE6 and CE22. Sanger sequencing found 9 OTUs, 4 CE, and 5 TU; HTS detected 22 OTUs, 4 CE, 16 TU, and 2 Serendipitaceae. Seeds inoculated with CE6 improved germination and protocorm development compared with other strains. In addition, asymbiotic seedlings inoculated with CE6, CE22, and TU18 displayed varying effects in growth, with CE6 being the most notable. While TU18 did not promote seed germination, it effectively promoted leaf development in seedlings. Overall, Ceratobasidiaceae was predominantly associated with seed germination, seedling growth, and the adult stages, with CE6 and CE22 becoming the primary partners throughout the life history of P. japonica. Our findings illuminate mycorrhizal symbiosis in epiphytic habitats, offering conservation and commercial production insights. Full article

Dendrobium officinale Kimura et Migo is a kind of herb with high medicinal, ornamental, and commercial value, and is rich in polysaccharides. Polyploid breeding is an important breeding method for the genome doubling of medicinal species to increase biomass and polysaccharide production. Previous studies have revealed comparative transcriptome analysis and polysaccharide biosynthesis across the growth stages and plant parts, but there have been no studies dissecting such genes and pathways in tetraploid D. officinale. Therefore, this study aimed to unravel the molecular mechanisms of the increase in polysaccharide content in tetraploid D. officinale via the generation of four transcriptomic libraries for protocorm-like bodies and six-month-old seedlings of both diploid and tetraploid D. officinale plants. In this study, a total of 230,786,618 clean reads remained with a total of 34.62 Gb nucleotides generated; 274,403 unigenes were assembled, of which 73.99% were annotated to at least one of the protein databases; and of 17,451 unigenes, 6.35% were annotated to all seven protein databases (NR, NT, KO, Swiss-Prot, FAM, GO, and KOG). Putative genes encoding enzymes related to polysaccharide biosynthetic pathways were determined. RT-qPCR for 11 randomly selected genes involved in polysaccharides indicated consistency with RNA-Seq data and polysaccharide content. The expressions of nine genes were higher in tetraploid than in diploid plants, while the expressions of the other two genes encoding bifunctional enzymes were the opposite. This study has provided a foundation for subsequent works regarding the biosynthetic pathways of metabolites involved in the autoploidy of Dendrobium species in general, and D. officinale in particular. Full article

Wild orchids, especially the terrestrial temperate ones are endangered species due to challenges in their natural habitats. Therefore, there is an urgent need to introduce efficient propagation methods to overcome the natural reproduction problems of these orchids. In this study, the effects of different light spectrums, explant types, wounding, and combinations of different plant growth regulators (PGRs) on direct somatic embryogenesis (DSE) of two species of these endangered orchids listed in the conservation category, were studied. The highest percentages of DSE formation and embryo germination were observed in Dactylorhiza umberosa protocorm explants exposed to white light (400–730 nm) and in Epipactis veratifolia protocorm explants exposed to a combination of red and far-red spectra (R: FR = 70:30). This occurred while red (610–700) alone and in combination with far-red (710–730 nm) spectrum induced embryogenesis more than the blue spectrum and dark condition in E. veratifolia. Thidiazuron (TDZ, 3 mg L⁻¹), produced the highest percentage of protocorm-like bodies (PLBs) on protocorm explants in both orchids. Kinetin (Kin, 2 mg L⁻¹) and Benzyladenine (BA 3 mg L⁻¹) had the most effect on the survival and growth of PLBs, respectively, in D. umberosa and E. veratifolia. Species did not show similar embryogenesis responses under light spectrums. In a medium containing 3 mg L⁻¹ TDZ, white light and R-FR spectra produced the most PLBs on wounded protocorm explants of D. umberosa and E. veratifolia respectively. The developmental stage of apical meristem of PLBs in both species was more advanced under R-B spectra, compared to others. Full article

This study describes the in vitro propagation and ex vitro acclimatization of Philodendron erubescens pink princess, one of the most popular ornamental variegated foliage plants. For shoot proliferation, the protocorm-like bodies of the Philodendron pink princess were cultured on solid Murashige and Skoog (MS) media supplemented with 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) at different concentrations. The results revealed that supplementation with BAP alone at a concentration of 1.0 mg/L yielded the maximum number of shoots and leaves. Furthermore, the application of BAP at 1.0 mg/L significantly enhanced the shoot proliferation of Philodendron pink princess when grown in liquid MS medium, yielding 11.2 shoots/explant and 4.7 leaves/explant. When the established microshoots were subjected to root induction using solid MS media supplemented with different kinds and concentrations of auxins, indole-3-butyric acid (IBA) at 3 mg/L resulted in the highest number of roots (3.2 roots/explant) and longest root length (1.9 cm). Three supporting materials, i.e., peat moss, vermiculite, and perlite, were used as planting media for the ex vitro acclimatization of the Philodendron pink princess plantlets. The results demonstrated that the in vitro plantlets acclimatized and exhibited a relatively high survival frequency in all planting media without morphological abnormalities. Peat moss outperformed all other types of planting media in terms of sustaining the vegetative growth of the plantlets. In the future, the approach established in this study could be employed for the extensive production of Philodendron pink princess. Full article

Paraphalaenopsis labukensis Shim, A. Lamb & C.L. Chan (P. labukensis) is an endangered monopodial epiphytic orchid threatened due to habitat fragmentation and overharvesting. Consequently, this research aimed to achieve in vitro propagation of P. labukensis through asymbiotic seed germination and seedling development. This study focused on identification and optimizing capsule maturity, basal media and nutrient requirements. In addition, after hand pollinating the plant, their capsules’ flowers, length, girth, and colour variations were recorded weekly to ascertain morphological maturity characters of this species. Murashige and Skoog (MS), Knudson C (KC), and Vaccin and Went (VW) basal media were used to determine seed vegetation at 60, 90, and 120 days. Subsequently, for the proliferation and development of protocorms, the best basal media were those supplemented with different organic additives (coconut water (CW) and banana homogenate (BH)) and plant growth regulators (PGRs). As a result, 120 days after pollination (DAP) was chosen as the best capsule age for germination since the capsules had reached maturity. The highest germination rate was attained after 90 days of culture in Knudson C (KC) medium, with 98.78 ± 0.89%, followed by Murashige and Skoog (MS) medium, with 92.80 ± 3.26%. The KC supplemented with 0.5 mg/L NAA media documented a maximum percentage of 17.25 ± 0.96%. The 5 g/L (w/v) BH recorded a higher protocorm proliferation percentage than CW. Additionally, after 150 days of culture (DOC), this medium combination produced the most leaves, averaging four or five. The result of this present study has successfully established an effective in vitro propagation protocol for this species. Full article

The compatibility of mycorrhizal fungi with the early growth stage of orchids is essential for their growth. In this study, the compatibility and promotion effects of 14 Tulasnella strains from different hosts were studied by co-culturing them with the protocorms of Dendrobium crepidatum, which has high ornamental and economic value in China. The ITS–LSU–SSU–TEF combined sequence analysis divided the 14 strains into three clades belonging to Tulasnella calospora (clades A and B) and Tulasnella asymmetrica (clade C). All the strains were compatible with D. crepidatum protocorms within 90 d of the co-culture. Strain T12 in Clade A had a significantly higher (p < 0.05) effect on the biomass and morphology of D. crepidatum, and strain T13 in Clade C had a significantly lower (p < 0.05) effect than the other strains. Through morphological principal component analysis, we constructed a hierarchical cluster analysis tree, which was consistent with the phylogenetic tree of these 14 strains at the clade level. Orthogonal partial least squares-discriminant analysis showed that these strains have an important effect on the plant height, root number, and length of D. crepidatum. The findings of this study will contribute to the identification of Tulasnella strains, conservation of D. crepidatum resources, and commercial utilization of mycorrhizal technology. Full article

Mycorrhizal specificity, i.e., the range of fungi allowing mycorrhizal partnerships, differs among orchid species, but that at early developmental stages is unclear. We investigated whether mycorrhizal specificity during seed germination and seedling development differs among three Dendrobium species, D. officinale, D. okinawense and D. moniliforme, in vitro. Nine mycorrhizal fungal strains were obtained from the roots of these species and cultured with a seed of each Dendrobium species. Five to eight fungal strains stimulated seed germination, whereas one to four fungal isolates significantly promoted protocorm development in the three species. To evaluate effects on leafy seedling growth, seedlings obtained from asymbiotic culture were cultured with nine fungal isolates. D. officinale and D. okinawense showed specificity for a single Serendipitaceae or Tulasnellaceae isolate, whereas D. moniliforme exhibited specificity for three isolates of Serendipitaceae and Tulasnellaceae. Therefore, the three Dendrobium species had a growth bottleneck from seed germination to the protocorm stage, and mycorrhizal specificity of protocorm growth and seedling development in vitro varied among the species. Our findings imply divergent mycorrhizal specificity in Dendrobium species at early developmental stages. This study provides insights into the diversity of orchid mycorrhizal specificity, as well as valuable information for conservation of endangered orchids. Full article

Syngonium podophyllum Schott. cv. ‘White Butterfly’ is recognized as a valuable ornamental plant, and today it is also an important plant species of medicinal interest due to its high contents of phenolic compounds. The purpose of this article is to review the main scientific publications from our laboratory with regard to new scientific achievements dealing with Syngonium species or topics of interest, such as callus formation and further de novo shoot regeneration. The principles and stages necessary to start an industrial-level micropropagation protocol are discussed based on our experience. Different media compositions induced different morphogenetic responses inside the callus—particularly those related to the development of xylematic elements in the organogenetic areas, such as those for rooting, protocorms, and de novo shoot formation. The re-evaluation of old histological images revealed for the first time that xylematic elements are constantly closely positioned to all organogenetic centers, and that their development is closely dependent on the composition of the culture medium. Separate protocorms can be identified only when xylematic tracheary elements are well developed and closely connected to them. The formation of protocorms is strongly dependent on the mineral composition of the culture medium and the balance of plant growth regulators. Full article

Ornamentals come in a variety of shapes, sizes, and colors to suit a wide range of climates, landscapes, and gardening needs. Compared to demand, a shortage of plant materials and diversity force the search for solutions for their constant acquisition and improvement to increase their commercial value, respectively. In vitro cultures are a suitable solution to meet expectations using callus culture, somatic embryogenesis, protoplast culture, and the organogenesis of protocorm-like bodies; many of these techniques are commercially practiced. Factors such as culture media, explants, carbohydrates, plant growth regulators, and light are associated with the success of in vitro propagation. Techniques, especially embryo rescue and somatic hybridization, are widely used to improve ornamentals. The development of synthetic seed allows season-independent seed production and preservation in the long term. Despite the advantages of propagation and the improvement of ornamentals, many barriers still need to be resolved. In contrast to propagation and crop developmental studies, there is also a high scope for molecular studies, especially epigenetic changes caused by plant tissue culture of ornamentals. In this review, we have accumulated and discussed an overall update on cultivation factors, propagation techniques in ornamental plant tissue culture, in vitro plant improvement techniques, and future perspectives. Full article

In vitro mutagenesis offers a feasible approach for developing new orchid cultivars through genetic manipulation. In the present study, protocorm-like bodies (PLBs) were exposed to gamma rays (10, 20, 40, 60, 80 Gy) to study in vitro growth responses and induction of mutants in Dendrobium ‘Emma White’. Both proliferation and regeneration of PLBs decreased progressively with increasing doses, except for a significantly enhanced growth response at 10 Gy. The optimal dose of gamma radiation for mutagenesis was found in the range 10 to 25 Gy based on the growth reduction curve. Analysis using a high-throughput cell analyzer revealed a significant reduction in nuclear DNA content at > 40 Gy doses. At 10 Gy treatment, the growth attributes, such as root length, plant height and leaf number, were significantly increased by 36%, 26% and 20%, respectively, compared to the control. This increase was significant over other tested doses as well. Testing of random amplified polymorphic DNA markers revealed the presence of detectable polymorphism among gamma mutant plantlets with a polymorphism information content value at 0.41. The gamma-ray-induced earliness in flower development was observed within 294 days post ex vitro growth of 10 Gy mutant compared to the control plants flowered after 959 days. Our results highlight the significance of gamma radiation in inducing enhanced growth, morphological variations and early floral initiation in Dendrobium, providing a basic framework for mutation breeding and improvement of orchids. Full article