Protocorm-like Bodies Research Articles

Production of genetically stable and Odontoglossum ringspot virus-free Cymbidium orchid ‘New True’ plants via meristem-derived protocorm-like body (PLB) subcultures

BackgroundThis study aimed to produce Odontoglossum ringspot virus (ORSV)-free Cymbidium orchid ‘New True’ plants from ORSV-infected mother plants by culturing their meristems and successively repeating subcultures of protocorm-like bodies (PLBs) derived from the meristems.ResultsInitially, ORSV was confirmed as the causative agent of viral symptoms in orchid leaves via reverse transcription-polymerase chain reaction (RT-PCR) analysis. Meristems from infected plants were cultured to generate PLBs, which in sequence were repeatedly subcultured up to four times. RT-PCR and quantitative RT-PCR analyses revealed that while ORSV was undetectable in shoots derived from the first subculture, complete elimination of the virus required at least a second subculture. Genetic analysis using inter-simple sequence repeat markers indicated no somaclonal variation between regenerated plants and the mother plant, suggesting that genetic consistency was maintained.ConclusionOverall, our findings demonstrate that subculturing PLBs for a second time is ideal for producing genetically stable, ORSV-free Cymbidium orchids, thus offering a practical means of generating genetically stable, virus-free plants and enhancing plant health and quality in the orchid industry.

Genome-Wide Identification of DREB Transcription Factor Family and Functional Analysis of PaDREB1D Associated with Low-Temperature Stress in Phalaenopsis aphrodite

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.

DoSPX1 and DoMYB37 regulate the expression of DoCSLA6 in Dendrobium officinale during phosphorus starvation

BackgroundDendrobium officinale Kimura et Migo (D. officinale) is parasitic on rocks or plants with very few mineral elements that can be absorbed directly, so its growth and development are affected by nutritional deficiencies. Previous studies found that phosphorus deficiency promotes polysaccharides accumulation in D. officinale, the expression of DoCSLA6 (glucomannan synthase gene) was positively correlated with polysaccharide synthesis. However, the molecular mechanism by which the low phosphorus environment affects polysaccharide accumulation remains unclear.ResultsWe found that DoSPX1 can reduce phosphate accumulation in plants and promote the expression of PSIs genes, thereby enhancing plant tolerance to low phosphorus environments.Y1H and EMSA experimental show that DoMYB37 can bind the promoter of DoCSLA6. DoSPX1 interact with DoMYB37 transiently overexpressed DoSPX1 and DoMYB37 in D. officinale protocorm-like bodies, decreased the Pi content, while increased the expression of DoCSLA6.ConclusionsThe signaling pathway of DoSPX1-DoMYB37-DoCSLA6 was revealed. This provides a theoretical basis for the accumulation of polysaccharide content in D. officinale under phosphorus starvation.

Mutagenesis and Flowering Promotion through Sodium Azide In Vitro Culture of Cymbidium faberi Rolfe

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.

An acidic polysaccharide promoting GLP-1 secretion from Dendrobium huoshanense protocorm-like bodies: Structure validation and activity exploration

Glucagon-like peptide-1 (GLP-1) as a multifunctional hormone is secreted mainly from enteroendocrine L-cells, and enhancing its endogenous secretion has potential benefits of regulating glucose homeostasis and controlling body weight gain. In the present study, a novel polysaccharide (h-DHP) with high ability to enhance plasma GLP-1 level in mice was isolated from Dendrobium huoshanense protocorm-like bodies under the guidance of activity evaluation. Structural identification showed that h-DHP was an acidic polysaccharide with the molecular weight of 1.38 × 105 Da, and was composed of galactose, glucose, arabinose and glucuronic acid at a molar ratio of 15.7: 11.2: 4.5: 1.0 with a backbone consisting of →5)-α-L-Araf-(1→, →3)-α-D-Galp-(1→, →6)-α-D-Galp-(1→, →3,6)-α-D-Galp-(1→, →6)-β-D-Glcp-(1→ and →4,6)-β-D-Glcp-(1→ along with branches consisting of α-L-Araf-(1→, α-D-Galp-(1→, α-D-GlcAp-(1→, β-D-Glcp-(1→ and →4)-β-D-Glcp-(1→. Animal experiments with different administration routes demonstrated that h-DHP-enhanced plasma GLP-1 level was attributed to h-DHP-promoted GLP-1 secretion in the enteroendocrine L-cells, which was supported by h-DHP-enhanced extracellular GLP-1 level in STC-1 cells. Inhibition of adenylate cyclase and phospholipase C indicated that cAMP and cAMP-triggered intracellular Ca2+ increase participated in h-DHP-promoted GLP-1 secretion. These results suggested that h-DHP has the potential of enhancing endogenous GLP-1 level through h-DHP-promoted and cAMP-mediated GLP-1 secretion from enteroendocrine cells.

In Vitro Seed Germination and Seedling Growth of Staurochilus Ramosum (Lindl.) Seidenf

A comparative study on in vitro seed germination and seedling development of Staurochilus ramosum (Lindl.) Seidenf. was carried out in vitro. Four basal media namely KC, MS, PM and VW media were used as half strength, full strength and full strength supplemented with 0.5mg/l BAP and 0.5mg/l NAA and solidified with agar. Response like protocorm like bodies (PLBs) initiation, seed germination and seedlings development on different basal media were varied distinctly. Full strength PM medium supplemented with 0.5mg/l BAP and 0.5mg/l NAA showed better response within 6 weeks of culture. The overall result indicates that PM medium was more effective than KC, MS and VW media for initiation of germination, PLBs formation and plantlets development. The Chittagong Univ. J. B. Sci., Vol. 10(1 &2):181-190, 2020

Effect of Melatonin on the Growth of Dendrobium officinale Protocorm-Like Bodies

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.

Exogenous Cytokinin Induces Callus and Protocorm-Like-Bodies Formation in In Vitro Root Tips of Vanilla planifolia Andrews.

Vanilla is a popular flavouring essence derived from the pods of vanilla orchid plants. Due to the high demand for vanilla flavour, high yielding vanilla plantlets are necessary for establishing vanilla plantations. Clonal micropropagation is a viable technique for the mass production of high yielding vanilla plantlets. This study reports an efficient regeneration protocol by using cytokinin as the sole plant growth regulator to regenerate plantlets from the root tips of a commercial vanilla orchid species, Vanilla planifolia. Most studies to date have reported using seeds and nodes as starting explants for in vitro micropropagation of vanilla orchids. So far, regeneration from roots has not been very successful. Previous studies favoured the use of auxins only or high auxin to cytokinin ratios to induce callus, and sole cytokinins were used for direct shoot regeneration. However, it was sporadically observed in plantlets regeneration of V. planifolia that multiple shoots were regenerated from the tips of intact aerial roots submerged in media. This study therefore investigated the regeneration of excised vanilla root tips through the application of most commonly used auxins (1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid) and cytokinins (6-benzylaminopurine and thidiazuron). High auxin presence is known to promote callusing in in vitro plants. However, in this study, auxin treatment inhibits callusing in root tips. While cytokinin treatments, even at low levels, has promoted high rate of callusing. These callus cells regenerate into protocorm-like-body (PLB) shoots when cytokinin levels are increased to 0.5 mg/mL 6-benzylaminopurine (BAP) under light conditions. The findings of the study have the potential of providing large quantity of high yielding vanilla plantlets through clonal micropropagation.

Transcriptomic Analysis for Diurnal Temperature Differences Reveals Gene-Regulation-Network Response to Accumulation of Bioactive Ingredients of Protocorm-like Bodies in Dendrobium officinale.

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.

Invitro germination and optimization of basal media for protocorm-like bodies proliferation in Dieniaophrydis(J. Koenig) Seidenf

Dienia ophrydis is an endangered terrestrial orchid used for its ornamental and therapeutic purposes. This orchid possesses an anti-malarial alkaloid known as Malaxin. There is ongoing exploitation in the wild due to illegitimate herbal collection and climate change. To control the potential risk of extinction, there are no conservational measures or in vitro protocols established in this genus. The study focuses on addressing a germination protocol and enhancement of protocorm-like bodies (PLB) proliferation by optimizing the basal media and vitamins. It also aims to improve the misinterpretation of tetrazolium viability results in orchid testa along with a permeability test. Further, the seeds were subjected to 1% TTC staining to perform embryo viability test and 0.4% trypan blue (TB) dye for testa permeability. Ten basal media such as Gamborg's medium, Mitra medium, Thomale GD medium, Vacin and Went medium, Knudson C medium, Knop's medium, MS medium and ½ MS medium, ¼ MS medium, and modified MS medium (MMS) were tested for seed germination. The Half-strength MS medium responded with 92% of the highest germination and T-GD medium is not suitable for seed germination. The seed viability and permeability result showed 93% of permeability, 72% viability, and 9% dead seeds. Protocorms were transferred to the modified mMS medium containing half-strength macronutrients, full micronutrients, and 10 ppm of thiamine in vitamin formulation. PLB proliferation was observed and 12 shoots emerged from a single protocorm explant. Subsequently, the seedlings were acclimatized with 8-0-24 ratios of nitrogen, phosphorus, and potassium (NPK) as determined by soil testing. The study contributes in experimenting with seed germination of terrestrial orchid seeds asymbiotically and to establishing a protocol for conservation programs, especially in this genus.

Tissue Culture via Protocorm-like Bodies in an Orchids Hybrids Paphiopedilum SCBG Huihuang90.

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.

Plant growth regulators and organic additives on the proliferation of protocorm-like bodies and plantlet regeneration of Cattleya gaskelliana (N.E.Br.) B.S.Williams

Plant growth regulators and organic additives on the proliferation of protocorm-like bodies and plantlet regeneration of Cattleya gaskelliana (N.E.Br.) B.S.Williams

Comparative Transcriptome Analysis and Expression of Genes Associated with Polysaccharide Biosynthesis in Dendrobium officinale Diploid and Tetraploid Plants

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.

In vitro propagation of Liparis nervosa (Thunb.) Lindl., an endangered medicinal orchid

In vitro regeneration was studied to protect the rare Chinese medicinal orchid Liparis nervosa (Thunb.) Lindl. The mixtures of protocorm and seeding and the stem tip were used as explants. The results revealed that the best essential medium for L. nervosa growth was 1/3 MS medium with 25 g · L–1 sucrose, 50 g · L–1 banana puree, 40 g · L–1 mashed potato, and 1.0 g · L–1 AC (MS1); MS1 medium with 0.5 mg · L–1 BA, 0.05 mg · L–1 2,4-D, and 1.5 mg · L–1 NAA was optimal for proliferation. When stem tips were cultured in a proliferation medium, four types of proliferation occurred: basal stem cluster bud (occurring at the basal node), tiller bud (occurring at the root), protocorm-like body (occurring at the plant’s base incision), and high-position bud (occurring on plant stem nodes other than the basal nodes). Four methods produced 10.12 proliferation coefficients. In the MS1 medium with 0.5 mg · L−1 NAA, the plantlets rooted 100%, and the rooted plantlets survived 100% after domestication and transplantation.

Invitro and Mass Multiplication of Cymbidium mastersii an Ornamental Orchid through Protocorm-like Body Formation

The epiphytic orchid species known as Cymbidium mastersii can only be found in the region of India known as Northeast India. The preservation of wild populations faces a significant obstacle in the form of a significant threat when it comes to the necessity of obtaining an additional source of the material required to fulfil commercial demands. The objective of this research was to build an in vitro system for the bulk multiplication of C. mastersii, and it was successful in that endeavour. This required the capacity to control the growth of the seedlings through the development of the primary protocorm and germination from a limited number of fruit capsules. Also necessary was the ability to limit the amount of fruit capsules that could be used. Each protocorm resulted in the production of a number of robust plantlings that, once they had been separated from their mother plant, flourished in the conditions of the greenhouse. The child plants, in terms of both their size and the development they underwent, were genetically identical to their parent plant. The streamlined process will make it possible to cultivate Cymbidium mastersii for use in the commercial sector, which will reduce the negative influence on remaining viable wild populations. We offer here for the very first time a method of producing C. mastersii on a commercial scale that makes use of bioreactor technology.

Use of Light Spectra for Efficient Production of PLBs in Temperate Terrestrial Orchids

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.

The Application of Plant Growth Regulators in Propagation of Dendrobium Orchid with Thin Cell Layer (TCL) Technique: A Review

Plant growth regulators is one of the success factors for in vitro propagation. Plant growth regulators are organic compounds that are not nutrients and, in small amounts, promote, inhibit, or regulate physiological processes in plants. The function of growth regulators is to stimulate the growth of morphogenesis in cell, tissue and organ cultures. The most used plant growth regulators in tissue culture are auxins, cytokinins and gibberellins. One of technique for tissue culture is the thin cell layer technique. Thin Cell Layer (TCL) is a technique using small thin sections produced from organ pieces. TCL explants more effective than large conventional explants. Thin explants facilitate contact and diffusion of media into the tissue, better than thick explant slices. This literature review aims to gather information related to the application of plant growth regulators in propagation of Dendrobium orchid with thin cell layer (tcl) technique. A systematic Literature Review (SLR) uses in this paper. The growth regulators commonly used in Dendrobium orchid propagation using the TCL technique are auxins and cytokinins and the type of media that is often used in Dendrobium orchid propagation is Murashige and Skoog (MS). Protocorm Like Bodies (PLB) is the most potential explant in Dendrobium orchid propagation. The TCL technique both transverse and longitudinal has its own specificity with each type of explant used. TCL explants proved to be more effective than large conventional explants.

Effect of Bioreactor Cultures on the Proliferation and Biological Activity of Protocorm-like Bodies of Dendrobium loddigesii

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.

ERF5 enhances protocorm-like body regeneration via enhancement of STM expression in Dendrobium orchid.

Orchid plants develop protocorms upon germination and produce protocorm-like structures called protocorm-like bodies (PLBs) from protocorms and somatic cells via tissue culture. Protocorm-like bodies have broad technical application potential in the orchid industry and their regeneration is a distinct developmental process in the plant kingdom. However, little is known about this unparalleled developmental program. In this study, we identified a PLB-abundant gene, ethylene response factor (ERF), and a transcription factor named DoERF5, and determined its important role in PLB regeneration in Dendrobium orchid. Overexpression of DoERF5 in Dendrobium greatly enhanced the PLB regeneration from PLB and stem explants, and upregulated the expression of WOUND-INDUCED DEDIFFERENTIATION (DoWIND) homologs and SHOOT MERISTEMLESS (DoSTM), as well as the genes involved in cytokinin biosynthesis (DoIPT) and the cytokinin response factors (DoARRs). However, silencing DoERF5 reduced the regeneration rate of PLBs, and downregulated the expression of DoWIND homologs, DoSTM and DoARRs. We demonstrated that DoERF5 is directly bound to the DoSTM promoter and regulates its expression. In addition, overexpression of DoSTM in Dendrobium orchid resulted in favorable regeneration of PLBs. Our results clarify that DoERF5 regulates the regeneration of PLB by enhancing DoSTM expression. Our findings provide new insights into how DoERF5 mediates PLB regeneration and offers technical potential in improving clonal propagation, preservation, and the bioengineering of orchids.

\(\textit{In vitro}\) regeneration of \(\textit{Renanthera imschootiana}\) Rolfe from protocorm-like body

Renanthera imschootiana Rolfe is an extremely rare and endangered tropical epiphytic orchid. Studies on in vitro culture of R. imschootiana were conducted in order to conserve and increase the genetic pool of this wild orchid species. In this study, the effect of plant growth regulators (BA and NAA), ripe bananas in combination with potato, humic acid, and silver nanoparticles (AgNPs) were investigated to find a suitable condition for in vitro plant regeneration from R. imschootiana protocorm-like body (PLB). The results showed that after 45 days of culture, MS medium supplemented with 100 g ripe banana/liter and 100 g potato/liter was suitable for regenerating shoots from the protocorm-like body (PLB) (17.56 shoots/PLBs and 100.00% of shoot-regenerating PLBs); MS medium supplemented with 2.0 mg/L BA, 0.3 mg/L NAA and 4 ppm AgNPs was the most suitable for the growth of shoots after 60 days of culture (10.00 leaves/shoot and shoot length of 4.22 cm). The in vitro shoots were transferred to half-strength MS supplemented with humic acid and AgNPs to investigate the root formation of R. imschootiana. After 60 days of culture, the best rooting was obtained at 2.0 mg/L humic acid and 6 ppm AgNPs (8.33 roots/shoot; root length of 4.00 cm and 100.00% root formation). The findings suggest that the in vitro micropropagation from R. imschootiana protocorm-like body provides a useful alternative tool for the conservation of this endangered species.