Search Results (25)

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

Low temperatures are the most significant abiotic stressor for the conservation and production of Phalaenopsis in non-tropical areas. CBF/DREB1 transcription factors play an important role in the plant abiotic stress response. In this study, 31 DREB family members were identified in the Phalaenopsis genome. Expression pattern analysis showed that the expression of different PaDREB members varied among tissue sites. PaDREB1D was isolated from Phalaenopsis aphrodite, and multiple sequence alignment showed that PaDREB1D belonged to the A1 subgroup of the DREB family and was localized in the nucleus. PaDREB1D overexpression in protocorm-like bodies of Phalaenopsis reduced cell damage during low-temperature stress, increased antioxidant enzyme activity, and enhanced the low-temperature tolerance of protocorm-like bodies. The results of this study provide a theoretical basis for breeding for cold resistance and investigating the molecular mechanisms related to low-temperature responses in Phalaenopsis. 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

Dendrobium officinale Kimura et Migo (D. officinale) is one of the most important traditional Chinese medicinal herbs, celebrated for its abundant bioactive ingredients. This study demonstrated that the diurnal temperature difference (DIF) (T1: 13/13 °C, T2: 25/13 °C, and T3: 25/25 °C) was more favorable for high chlorophyll, increased polysaccharide, and total flavonoid contents compared to constant temperature treatments in D. officinale PLBs. The transcriptome analysis revealed 4251, 4404, and 4536 differentially expressed genes (DEGs) in three different comparisons (A: 25/13 °C vs. 13/13 °C, B: 13/13 °C vs. 25/25 °C, and C: 25/13 °C vs. 25/25 °C, respectively). The corresponding up-/down-regulated DEGs were 1562/2689, 2825/1579, and 2310/2226, respectively. GO and KEGG enrichment analyses of DEGs showed that the pathways of biosynthesis of secondary metabolites, carotenoid biosynthesis, and flavonoid biosynthesis were enriched in the top 20; further analysis of the sugar- and flavonol-metabolism pathways in D. officinale PLBs revealed that the DIF led to a differential gene expression in the enzymes linked to sugar metabolism, as well as to flavonol metabolism. Certain key metabolic genes related to ingredient accumulation were identified, including those involved in polysaccharide metabolism (SUS, SUT, HKL1, HGL, AMY1, and SS3) and flavonol (UGT73C and UGT73D) metabolism. Therefore, these findings indicated that these genes may play an important role in the regulatory network of the DIF in the functional metabolites of D. officinale PLBs. In a MapMan annotation of abiotic stress pathways, the DEGs with significant changes in their expression levels were mainly concentrated in the heat-stress pathways, including heat-shock proteins (HSPs) and heat-shock transcription factors (HSFs). In particular, the expression levels of HSP18.2, HSP70, and HSF1 were significantly increased under DIF treatment, which suggested that HSF1, HSP70 and HSP18.2 may respond to the DIF. In addition, they can be used as candidate genes to study the effect of the DIF on the PLBs of D. officinale. The results of our qPCR analysis are consistent with those of the transcriptome-expression analysis, indicating the reliability of the sequencing. The results of this study revealed the transcriptome mechanism of the DIF on the accumulation of the functional metabolic components of D. officinale. Furthermore, they also provide an important theoretical basis for improving the quality of D. officinale via the DIF in production. Full article

This study successfully established an efficient in vitro propagation system for Paphiopedilum SCBG Huihuang90 via protocorm-like body (PLB) formation from seed-derived calluses, PLB proliferation and differentiation, root induction and greenhouse acclimatization. Furthermore, 1/2 Murashige and Skoog (MS) + 0.025 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) was suitable for the proliferation of PLBs, and 1/2MS + 10% coconut water (CW, v/v) + 0.5 g/L activated carbon (AC) was suitable for PLB differentiation. PLBs at different developmental stages required different kinds of sugars. This study provided a reference for research on the propagation techniques of other Paphiopedilum. 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

Dendrobium loddigesii has long been used in traditional folk medicine. The purpose of this study was to optimize the culture conditions for its protocorm-like bodies (PLBs) and explore their biological activities. The use of an air-lift bioreactor demonstrated superior PLB proliferation compared to agitated and solid culture methods. The optimal inoculum quantity of 30 g/vessel, cultured for 28 days in the bioreactor, yielded the highest PLB biomass production. Analysis of PLB extracts revealed that the ethyl acetate (EtOAc) extract exhibited the highest levels of flavonoids and alkaloids, as well as potent antioxidant activity demonstrated by DPPH free radical scavenging assay and reducing power. Furthermore, the antiproliferative effects of the PLB extracts were assessed using MTT assays, and the EtOAc extract showed significant efficacy by reducing cell viability by over 60% in the human colon carcinoma cell line SW620 at the highest tested concentration (200 μg/mL). Mechanistic analysis revealed the downregulation of key regulatory apoptosis genes, including survivin, p53, caspase-3, and caspase-9. These results demonstrate the potential of the bioreactor culture method for the efficient production of D. loddigesii PLBs and the biological activities of the EtOAc extract, suggesting its therapeutic potential. 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

Himantoglossum affine is a threatened terrestrial orchid. We aimed to optimize asymbiotic seed germination and direct embryogenesis and to analyze the phytochemical profile and physico-biochemical analysis of leaf and tuber. The individual use of organic nitrogen compounds resulted in higher germination efficiencies, while the shortest times to germination were observed using coconut water plus casein hydrolysate. Plantlets grown on media supplemented with pineapple juice and peptone had the highest plantlet length and weight. For embryogenesis, the highest regeneration rate (44%) and embryo number/explant (10.12 ± 2.08) were observed in young protocorm-like body (PLB) explants with 0.5 mg/L naphthalene acetic acid (NAA) and 1 mg/L thidiazuron (TDZ). During the acclimatization process, the scattered vascular tubes converted to fully developed vascular tissues, ensuring maximum sap flux. Gas chromatography–mass spectrometry analysis identified 1,2,3-propanetriol, monoacetate, 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, and 2-butenedioic acid, 2-methyl-, (E)- as the most prevalent compounds. We reported higher contents of total phenolics and flavonoids and antioxidant activity compared to other terrestrial orchids. The glucomannan content (36.96%) was also higher than starch content (31.31%), comparable to those reported in other tuberous orchids. Based on the fragmentation of H. affine populations in the Middle East and Euro-Mediterranean countries due to over-harvesting, climate change, and/or human impact, our procedure offers a tool for the re-introduction of in vitro-raised plants to threatened areas. 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

Anoectochilus roxburghii (Wall.) Lindl has been used in Chinese herbal medicine for treating various ailments. However, its wild resources are endangered, and artificial cultivation of the plant is limited by the low regeneration rate of conventional propagation methods. The lack of A. roxburghii resources is detrimental to the commercial production of the plant and kinsenoside, which is unique to Anoectochilus species. To develop highly efficient methods for A. roxburghii micropropagation and find alternative resources for kinsenoside production, we created an induction, proliferation, and regeneration of PLBs (IPR-PLB) protocol for A. roxburghii. We also analyzed the kinsenoside and flavonoid contents during the induction and proliferation of PLBs. The best media of IPR-PLB for PLB induction and proliferation (secondary PLB induction and proliferation), shoot formation, and rooting medium were Murashige and Skoog (MS) + 3 mg/L 6-benzylaminopurine (6-BA) + 0.5 mg/L naphthaleneacetic acid (NAA) + 0.8 mg/L zeatin (ZT) + 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2, 4-D), MS + 3 mg/L 6-BA + 0.5 mg/L NAA, and MS + 0.5 mg/L NAA, respectively. On these optimized media, the PLB induction rate was 89 ± 2.08%, secondary PLB induction rate was 120 ± 5%, secondary PLB proliferation rate was 400 ± 10% and 350 ± 10 % in terms of the quantity and biomass at approximately 1 month, shoot induction rate was 10.5 shoots/PLB mass, and root induction rate was 98%. All plantlets survived after acclimation. Darkness or weak light were essential for PLB proliferation, and light was crucial for PLB differentiation on these optimized media. The kinsenoside contents of PLBs and secondary PLBs were 10.38 ± 0.08 and 12.30 ± 0.08 mg/g fresh weight (FW), respectively. Moreover, the peak kinsenoside content during the proliferation of secondary PLBs was 34.27 ± 0.79 mg/g FW, which was slightly lower than that of the whole plant (38.68 ± 3.12 mg/g FW). Two flavonoids exhibited tissue- or temporal-specific accumulation patterns, and astragalin accumulated exclusively during the first 2 weeks of cultivation. The IPR-PLB protocol for A. roxburghii may facilitate the efficient micropropagation of A. roxburghii plants. Furthermore, the PLBs are a good alternative resource for kinsenoside production. Full article

Dendrobium okinawense is an endangered epiphytic orchid, and there has been no scientific report so far on its propagation. Protocorm is a mass of cells, and protocorm-like bodies (PLBs) are lookalike protocorms produced by vegetative explants in vitro. Regeneration of PLBs is a widely used technique for orchid micropropagation. We used different light-emitting diodes (LEDs) for the PLB regeneration of D. okinawense. The number of PLBs and fresh weight were increased by 81.1% and 80.8%, respectively, under green LED over the white fluorescent (WF) light. We added different concentrations of PCIB (p-Chlorophenoxyisobutyric acid, an anti-auxin) and HMI (3-Hydroxy-5-methyl isoxazole, an anti-fungal agent) in culture media. The number of PLBs was increased in media having 0.01 mg/L of PCIB (35.9%) compared to control (no PCIB), whereas 19.3% increased in media having 0.01 mL/L of HMI compared to control (no HMI). Green LED in combination with 0.01 mg/L of PCIB significantly increased the number of PLBs (69.0%) compared to the WF–without PCIB combination. In LEDs-PCIB and LED-HMI combinations, HMI did not show better PLBs regeneration compared with PCIB. The results suggested that a combination of low concentrations of PCIB and green LED have the potential to accelerate PLB regeneration of D. okinawense. Full article