Protocorm-like Bodies Research Articles

Exogenous toluene gas removal improvement in recombinant rabbit cytochrome P450 2E1 (CYP2E1)-transgenic Ardisia pusilla DC

The cytochrome P450 2E1 (CYP2E1) enzyme encoded by CYP2E1 plays an important role in the metabolism of organic compounds in mammalian liver cells. In this study, the plasmid pCAMBIA2300 harboring the rabbit CYP2E1 was transferred into Agrobacterium tumefaciens; this was then transferred into internodes of Ardisia pusilla. Shoot primordia, whose appearance resembles that of the in vitro protocorm-like body of orchids, regenerated from 121 of 332 internodes that were cultured in selection medium supplemented with 5 mg·L−1 kanamycin. PCR analysis confirmed that 25 of 89 putative transgenic plants (28.0%) stably harbored the CYP2E1 and NPTII genes, and Southern blot analysis revealed the presence of one to three copies of CYP2E1 within the genome of the 25 transgenic plants. The mRNA expression of the transgene was confirmed in 12 of 25 CYP2E1-transgenic plants by Northern blot analysis. Western blot analysis indicated the presence of a 53-kDa recombinant rabbit CYP2E1 protein in the 12 CYP2E1-transgenic A. pusilla plants. The aniline 4-hydroxylase activity of rabbit CYP2E1 in nine CYP2E1-transgenic A. pusilla plants was higher than that in non-transgenic A. pusilla plants. There was a statistically significant increase (5.1–6.1-fold) in the toluene removal ability of the transgenic line CYP2E1-2–2 compared to that observed in wild-type Ardisia plants at different times after exogenous toluene exposure.

Plantlet Regeneration and Multiple Shoot Induction from Protocorm-Like Bodies (PLBs) of Medicinal Orchid Species, Dendrobium crumenatum Sw.

To investigate the suitable medium for in vitro shoot regeneration and plantlet growth of Dendrobium crumenatum Sw., individual protocorm-like bodies (PLBs) (about 4 - 5 mm in diameter) of Dendrobium crumenatum Sw. derived from MS medium supplemented with 0.5 mg/L TDZ for 60 days of culture were cultured on 6 culture media; Murashige and Skoog (MS) medium, MS medium supplemented with 15 % (v/v) CW, MS medium supplemented with 15 % (v/v) CW and 0.2 % (w/v) AC, Vacin and Went (VW) medium, VW medium supplemented with 15 % (v/v) CW, VW medium supplemented with 15 % (v/v) CW and 0.2 % (w/v) AC. After 4 months of culture, MS medium containing 15 % coconut water (CW) gave the highest percentage of shooting and number of shoots per explant of 96.0 and 9.5, respectively with a significant difference from other media. The addition of 0.2 % (w/v) activated charcoal (AC) significantly increased the number of leaves and roots. PLBs developed into complete plantlets. MS medium supplemented with 15 % (v/v) CW and 0.2 % (w/v) AC and VW medium supplemented with 15 % (v/v) CW and 0.2 % (w/v) AC gave the highest number of roots per plantlet and root length at 5.3 roots and 34.9 mm, respectively. After the transfer of rooted shoots to the greenhouse, 95 % of the regenerated plantlets survived and grew vigorously. Plantlets grown in vitro were successfully acclimatized in the greenhouse and showed normal development.

Cost-Effective in vitro Multiplication and Phenolic Profile of an Important Medicinal Orchid, Satyrium nepalense D. Don

In this study we report a cost-effective and reproducible protocol for large-scale multiplication of an important medicinal orchid, Satyrium nepalense through a four-step protocol, involving asymbiotic seed germination, multiplication of protocorm-like bodies (PLBs), development and multiplication of shoots, and finally in vitro rooting of the developed shoots. Out of various media tested for seed germination, Mitra’s medium supplemented with 0.1 % peptone (BM) supported the highest (79.19 %) seed germination. Eight-weekold protocorms were sub-cultured on BM alone and supplemented with different concentrations (1-8 μM) of BAP, KN, or TDZ. The highest number of shoots was developed on the medium containing 4 μM KN. Here we also observed that the highest number of shoots were produced from protocorms cultured on a 2 % isubgol gelled medium out of the seven different low-cost gelling agents used. Further, optimal elongation of shoots was observed on BM alone and 2 % guar gum, among the tested gelling agents. Elongated shoots were transferred for rooting on BM containing 0-2 μM of IAA, IBA, or NAA. The best rhizogenic response was observed on BM fortified with 0.5 μM IBA and isubgol (3 %). The rooted plantlets showed a 76 % survival rate on acclimatization after transfer to the potting mixture of sand and vermiculite (1:1). HPLC analysis of therapeutically important phenolic acids of leaves and tubers of in vitro regenerated and in vivo plants revealed the presence of higher levels of selected phenolic acids in in vitro tissues than those from their native habitat. This protocol could facilitate the conservation and propagation of this important medicinal orchid.

In vitro regeneration and clonal fidelity assessment on induction and differentiation of protocorm-like body from Pleione bulbocodioides (Franch.)

In vitro regeneration and clonal fidelity assessment on induction and differentiation of protocorm-like body from Pleione bulbocodioides (Franch.)

Protocorm-like bodies (PLBs) of Dendrobium Sabin Blue: a novel source for in vitro production of dendrobine and anthocyanin

Dendrobium hybrids have been cultivated as commercially important ornamental plants in the floriculture industry. However, the potential of Dendrobium hybrids as a source of valuable secondary metabolites has not been fully exploited. In this study, the effect of various elicitors on the proliferation of protocorm-like bodies (PLBs) of Dendrobium Sabin Blue and the corresponding production of dendrobine and anthocyanin was investigated. Yeast extract (YE) and glutamine (both at 200 mg L−1) increased dendrobine content in PLBs by 2.7- and 6.5-fold (54.874 ± 2.553 and 894.481 ± 26.185 ng mg−1dry weight (DW)) compared to controls (20.051 ± 0.182 and 136.956 ± 7.397 ng mg−1 DW). Glutamine (200 mg L−1) also significantly enhanced PLB biomass (0.190 ± 0.009 g) with respect to the control (0.144 ± 0.003 g). Chitosan and salicylic acid (SA) were not effective in enhancing the dendrobine and anthocyanin contents in PLBs. A combination of 4 mg L−1 NAA and 100 mg L−1 arginine increased dendrobine content by 8-fold (123.793 ± 5.446 ng mg−1 DW) compared to the control (15.554 ± 0.406 ng mg−1 DW). Anthocyanin content was increased by 1.7-fold (0.548 ± 0.019 Color Value (CV) g−1 DW) when PLBs treated with 4 mg L−1 TDZ were exposed to 1.5 h of UV irradiation as compared to those without UV treatment (0.331 ± 0.019 CV g−1 DW). Despite the low dendrobine content, PLBs as a source of dendrobine remains appealing due to its short culture duration (6 wk) compared to mother plants (2 yr).

Genome-wide identification and analysis of DNA methyltransferase and demethylase gene families in Dendrobium officinale reveal their potential functions in polysaccharide accumulation

BackgroundDNA methylation is a conserved and important epigenetic modification involved in the regulation of numerous biological processes, including plant development, secondary metabolism, and response to stresses. However, no information is available regarding the identification of cytosine-5 DNA methyltransferase (C5-MTase) and DNA demethylase (dMTase) genes in the orchid Dendrobium officinale.ResultsIn this study, we performed a genome-wide analysis of DoC5-MTase and DodMTase gene families in D. officinale. Integrated analysis of conserved motifs, gene structures and phylogenetic analysis showed that eight DoC5-MTases were divided into four subfamilies (DoCMT, DoDNMT, DoDRM, DoMET) while three DodMTases were divided into two subfamilies (DoDML3, DoROS1). Multiple cis-acting elements, especially stress-responsive and hormone-responsive ones, were found in the promoter region of DoC5-MTase and DodMTase genes. Furthermore, we investigated the expression profiles of DoC5-MTase and DodMTase in 10 different tissues, as well as their transcript abundance under abiotic stresses (cold and drought) and at the seedling stage, in protocorm-like bodies, shoots, and plantlets. Interestingly, most DoC5-MTases were downregulated whereas DodMTases were upregulated by cold stress. At the seedling stage, DoC5-MTase expression decreased as growth proceeded, but DodMTase expression increased.ConclusionsThese results provide a basis for elucidating the role of DoC5-MTase and DodMTase in secondary metabolite production and responses to abiotic stresses in D. officinale.

Exogenous ethylene increases the viability of cryopreserved Dendrobium protocorm-like bodies by regulating the hydrogen peroxide and its mediated oxidative stress

Exogenous ethylene increases the viability of cryopreserved Dendrobium protocorm-like bodies by regulating the hydrogen peroxide and its mediated oxidative stress

Effects of Plant Growth Regulators on In vitro Growth and Development of Orchid Dendrobium sp. from Protocorm Like Bodies (PLBs)

The present study was undertaken to find out the optimum concentration and combination of plant growth regulators (PGRs) and their effects on in vitro growth and development of Dendrobium sp. orchid. Protocorm-like bodies (PLBs) were inoculated as explants on MS medium supplemented with four levels (0, 0.5, 2.5, and 5.0 mg/l) of auxin [Naphthalene acetic acid (NAA)] and cytokinin [6-Benzylaminopurine (BAP)] independently to proliferate and multiply the PLBs. Then, these multiplied PLBs were cultured on different concentrations and combinations of NAA (0, 0.5, 2.5, and 5.0 mg/l) and BAP (0, 0.5, 2.5, and 5.0 mg/l) along with 0.5 mg/l kinetin (Kn) for the growth and development of Dendrobium sp. plantlets. The significantly maximum fresh weight and the highest percentage of PLBs were observed from the MS medium supplemented with 2.5 mg/l NAA at both 30 and 60 days after culture (DAC). Similarly, the significantly maximum fresh weight and the highest percentage of PLBs were achieved when MS medium supplemented with 0.5 mg/l BAP and 2.5 mg/l BAP at both 30 DAC and 60 DAC, respectively. In this study, shoots and roots regeneration from PLBs were observed simultaneously at different concentrations and combinations of NAA and BAP with 0.5 mg/l Kn. The significantly highest numbers of leaves (8.3), the longest length of leaves (4.26 cm), and the highest percentage of shoot regeneration (85%) were found from the medium supplemented with 2.5 mg/l BAP + 0.5 mg/l NAA+ 0.5 mg/l Kn. On the other hand, MS medium with the same concentrations of BAP and Kn but a higher concentration of NAA (5.0 mg/l) produced the maximum number of roots (6.17), the longest length of roots (5.10 cm), and the highest percentage of root regeneration (76%). From the findings of this study, these two concentrations and combinations of PGRs (2.5 mg/l BAP+ 0.5 mg/l NAA+ 0.5 mg/l Kn and 2.5 mg/l BAP + 5.0 mg/l NAA+ 0.5 mg/l Kn) found optimum for in vitro culture of Dendrobium orchid. Therefore, these concentrations and combinations of PGRs can be supplemented with the culture medium for mass propagation and conservation of endangered species like Dendrobium orchid.

Characterization of LEA genes in Dendrobium officinale and one Gene in induction of callus

Late embryogenesis abundant (LEA) proteins are widely involved in plant stress responsive, while their involvement in callus formation is largest unknown. In this study, we identified and conducted expression analysis of the LEA genes from Phalaenopsis equestris and Dendrobium officinale, and characterized a LEA gene from D. officinale. A total 57 and 59 LEA genes were identified in P. equestris and D. officinale, respectively. A phylogenetic analysis showed that AtM, LEA_5 and Dehydrin groups were absent in both orchids. LEA_1 group genes were strongly expressed in seeds, significantly down-regulated in flowers, and absent in vegetative organs (leaves, stems and roots) in both orchids. Moreover, LEA_1 and LEA_4 group genes from D. officinale were abundant in the protocorm-like body stage and were dramatically up-regulated in response to abscisic acid and salinity stress. A LEA_1 gene (DoLEA43) was selected for further functional analysis. DoLEA43 protein was localized in the cytoplasm and nucleus, and its promoter contained a WUN-motif that was modulated by wounding. Overexpression of DoLEA43 in Arabidopsis enhanced callus induction, causing changes to callus formation-related genes such as WIND1. Our results indicate the involvement of LEA genes in the induction of callus, which provide insights into plant regeneration.

Plant Regeneration from Protocorm Like Body (PLB) derived Callus of Dendrobium barbatulum Lindl.

In vitro regenerative potential of protocorm like bodies (PLBs) of Dendrobium barbatulum were assessed. The formation of secondary PLBs from the primary PLBs that formed from the callus, it is derived from a few larger injured protocorm like bodies. The injured larger PLBs were cultured on half strength MS supplemented with different concentrations of BAP (0.32, 1.62, 3.23 and 16.15 μM), Zn (0.46, 2.28, 4.56 and 22.80 μM) and Kn (0.47, 2.33, 4.65 and 23.25 μM). The highest percentage of secondary PLBs (66.33%) were obtained on half strength MS medium supplemented with BAP 3.23 μM, after 5 weeks of culture. Then these clumps of PLBs were subcultured on half strength MS fortified with coconut water (CW), cane juice (CJ), peptone (P) and casein hydrolysate (CH) for regeneration. Among the complex organic additives 20% CW and 2.0 g/l P to half strength MS resulted in the development of plantlet 78.31 and 62.60%, respectively. Well developed plantlets were successfully acclimatized in the community pots having brick pieces, charcoal, decaying litter and coconut husk (1 : 1 : 1 : 1) gave maximum survival rate of 87.03%.
 Plant Tissue Cult. & Biotech. 30(2): 243-252, 2020 (December)

Differentially expressed proteins in the development of protocorm-like bodies of Cattleya tigrina A. Richard

Cattleya tigrina is an endangered Brazilian native orchid with ornamental potential. Biotechnological tools based on tissue culture techniques are effective for its mass propagation and conservation. Protocorm-like bodies (PLBs) were induced from leaves in culture medium supplemented with plant growth regulators, and generated complete in vitro plants. To deepen on the mechanism underlying this morphogenetic route, this study is aimed to identify and characterize differentially expressed proteins during C. tigrina PLBs development. Leaves were inoculated in in culture medium Murashige and Skoog (MS) supplemented with 9 μM Thidiazuron, and PLBs t were collected after 30, 60 and 100 days in culture. Proteomic analysis performed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry detected 122, 132 and 447 protein after 30, 60 and 100 days, respectively. During PLBs development, 36 proteins were differentially expressed. Proteins related to energy and carbohydrate metabolism, cell proliferation, protein processing, and secondary metabolism and phytohormones were assessed showing their relevance in the process of development of PLBs. Many proteins related to metabolic and energy processes were identified after 60 and 100 days in culture, suggesting their role in which is cell division and differentiation. Enolase was only present after 100 days in culture, thus being a candidate as a molecular marker in this developmental phase.

Asymbiotic Germination, Effect of Plant Growth Regulators, and Chitosan on the Mass Propagation of Stanhopea hernandezii (Orchidaceae)

Background: Stanhopea hernandezii was collected from natural habitat in Mexico for its beautiful fragrant flowers. Biotechnological strategies of propagation may satisfy the market demand and are useful for conservation programs.
 Hypothesis: Vigorous seedlings of S. hernandezii can be produced in vitro by asymbiotic seed germination techniques and the addition of chitosan to the culture medium in the temporary immersion system (RITA®) and in semi-solid medium systems.
 Methods: The first step was the in vitro germination of seeds obtained from a mature capsule of wild plants, followed by multiplication via adventitious protocorm induction known as protocorm-like bodies, using plant growth regulators. For this purpose, we utilized Murashige and Skoog (MS) basal medium amended with 0.5 mg/L ?-Naphthaleneacetic acid, combined with different concentrations of 6-Benzylaminepurine (1, 3, and 5 mg/L). The following step comprised the growth and development of protocorms to obtain plantlets in RITA® flasks containing 250 mL of liquid MS medium combined or not with different chitosan concentrations (5, 10, 15, 20, and 25 mg/L).
 Results: The results showed that media supplemented with 5, 10, and 15 mg/L chitosan concentrations enabled the obtaining of a larger biomass with a range of 40-48 seedlings/RITA® and an average height of 13 mm. The last step was the development from seedlings into plantlets, the latter being, vigorous and achieving up to 100 % survival after 12 weeks of ex vitro cultivation.
 Conclusion: This paper describes an efficient process of asymbiotic germination and mass propagation of S. hernandezii , a vulnerable orchid species endemic to Mexico.

Effect of microalgae Messastrum gracile and Chlorella vulgaris on the in vitro propagation of orchid Cattleya labiata

The aim of this study was to evaluate the effect of two green microalgae (Messastrum gracile and Chlorella vulgaris) in comparison with plant growth regulators (6-benzylaminopurine, BAP; thidiazuron, TDZ; zeatin, ZEA) on the in vitro propagation of Cattleya labiata, an endangered orchid, using the thin cell layer (TCL) technique from protocorms. TCL sections were cultivated in MS/2 medium containing M. gracile extract (EM) and biomass (BM); C. vulgaris extract (EC) and biomass (BC); and BAP, TDZ, and ZEA in different concentrations. Subsequently, the explants were grown in MS/2 medium, with 2 g L−1 of activated charcoal, to induce elongation and roots formation. For acclimatization, plants were transplanted in trays using Sphagnum sp. as substrate. TCL explants showed a higher formation of protocorm-like bodies (PLBs) than entire protocorms. Explants cultivated in media supplemented with BM showed a high rate of PLB regeneration (59%) and high mean number of PLBs (4 per explant) and 85% survival after acclimatization of the plants. Supplementation with BAP stimulated similar morphogenic responses to those observed with BM and superior results obtained with ZEA and TDZ. Mass propagation of C. labiata plants was successfully achieved using TCL, and it is recommended to supplement the MS/2 medium with 4 g L−1 of BM or 4 mg L−1 of BAP. Microalgae extracts and biomasses are effective alternatives for in vitro propagation of C. labiata that can replace plant growth regulators, as they favored the formation of PLBs and plants.

In vivo-induced polyploidy in Dendrobium ‘Sonia’ in a bubble bioreactor system using colchicine and oryzalin

Development of polyploidy in Dendrobium ‘Sonia’ using colchicine and oryzalin is described. Five-month-old plantlets derived from protocorm-like bodies produced in vitro were used. These plantlets were immersed into a bubble bioreactor containing different concentrations of colchicine (0.05, 0.10 and 0.15% for 3 days) and oryzalin (14.40 and 28.90 µM for 4 days) together with Nonidet (P-40) as a surfactant. Plantlets were aerated to prevent hypoxia. Tetraploidy was successfully induced by 0.10 and 0.15% colchicine. Polyploidy levels were estimated using flow cytometry and then confirmed by chromosome counting, and through anatomical, cytological and morphological studies. The highest percentage (26.60%) of polyploids was induced with 0.15% colchicine. Oryzalin treatment did not yield tetraploid plantlets. The relative fluorescence intensity emitted by propidium iodide (PI), a fluorescent intercalating agent or a DNA-binding dye, was almost twofold higher in tetraploids and nearly fourfold higher in octaploid as compared to diploids. Plantlets with changed chromosome number were obtained at 0.05–0.15% colchicine concentrations (tetraploid induction) and 14.40 µM oryzalin concentration (mixoploid induction). The chromosome number was 2n = 38 in diploids and 2n = 76 in the tetraploids. The tetraploid and diploid plantlets exhibited significant differences in morphological, anatomical and cytological parameters. Stomata of tetraploid plantlets were longer than diploids. Survival rates from treatments were above 70%. In all treatments with colchicine and oryzalin, 0.10 and 0.15% of colchicine proved the most effective for generation of polyploid plantlets. This is beneficial for further breeding purposes of D. ‘Sonia.’ Oryzalin was found lethal. New ornamental varieties with improved traits can be produced by polyploidy using colchicine.

In vitro plant regeneration through protocorm formation in orchid Calanthe odora

The regeneration of plants through formation of protocorm and protocorm-like bodies was achieved by in vitro asymbiotic immature seed culture of Calanthe odora. This is the first report on in vitro regeneration of plantlets of Calanthe odora Griff., the IUCN red listed threatened species. The immature seeds as explants were most regenerative than mature seeds. The MS media fortified with different concentrations of Kinetin alone or in combination with NAA were tested to sort out as the best regeneration media. From the experimental results the best medium was sorted as MS medium supplemented with 1.0 mg/l Kinetin and 1.0 mg/l NAA for germination and formation of protocorm-like bodies from immature seed explants followed by transfer to MS media fortified with 1.5 mg/l Kinetin and 1.0 mg/l NAA for development of protocorm to plantlets. The plantlets after hardening in in vitro were transferred to polyhouse where 86.7% survival rate was recorded on vermiculite potting media.

Short-term cold storage of encapsulated somatic embryos and retrieval of plantlets in grey orchid (Vanda tessellata (Roxb.) Hook. ex G.Don)

Vanda tessellata (Roxb.) Hook. ex G.Don (grey orchid) is a horticultural and medicinally potent, and conservation prioritized orchid species. The present study investigates asymbiotic seed germination, callus induction, somatic embryogenesis, encapsulation of somatic embryos (SEs), and retrieval of plantlets of V. tessellata from the artificial seeds after short-term cold storage at − 4 °C for 12 months. Seeds cultured on full strength Murashige and Skoog’s (MS) medium containing 2.0 mg L−1 6-benzylaminopurine (BAP) had the highest frequency of asymbiotic seed germination (94%) as compared with half- and one-fourth strength MS media supplemented with various concentrations and combinations of 6-furfurylaminopurine (kinetin) and BAP. The protocorm like bodies (PLBs) gained dedifferentiation and proliferated into embryogenic calli on MS medium containing 2.0 mg L−1 BAP and 0.5 mg L−1 indole-3 acetic acid (IAA). Somatic embryos (SEs) were differentiated from the callus when cultured on MS medium in combination with 1.0 mg L−1 BAP and 0.5 mg L−1 IAA. The morpho-anatomical elucidations authenticated somatic embryogenesis and various developmental stages of SEs from embryogenic calli. Short-term cold storage of SEs was carried out by encapsulating with 2% sodium alginate and 100 mM calcium chloride and stored under dark at − 4 °C for 12 months to conserve this valuable germplasm. The stored artificial seeds were germinated (91%) on MS medium incorporated with additives and 0.5 mg L−1 each of BAP, kinetin, and IAA. Complete retrieval of plantlets was achieved in 8 week with formation of shoots (4.2 cm average length) and roots (2.5 cm). The well developed plantlets were acclimatized in the greenhouse with 90% survival rate. Vanda tessellata (grey orchid) is a horticultural and medicinally potent, and conservation prioritized orchid species. A reproducible protocol has been successfully established for asymbiotic seed germination, callus induction, somatic embryogenesis, encapsulation of somatic embryos, and retrieval of plantlets of V. tessellata from the artificial seeds after short-term cold storage at − 4 °C for 12 months.

Efficient Regeneration of Hedychium coronarium through Protocorm-Like Bodies

Hedychium coronarium J. Koenig is a multipurpose plant with significant economic value, but it has been overexploited and listed as a vulnerable, near threatened or endangered species. In vitro culture methods have been used for propagating disease-free propagules for its conservation and production. However, explant contamination has been a bottleneck in in vitro propagation due to the use of rhizomes as the explant source. Plants in the family Zingiberaceae have pseudostems that support inflorescences, while rhizomes are considered true stems. The present study, for the first time, reported that the pseudostem bears nodes and vegetative buds and could actually be true stems. The evaluation of different sources of explants showed that mature node explants derived from the stem were the most suitable ones for in vitro culture because of the lowest contamination and the highest bud break rates. Culture of mature node explants on MS medium supplemented with 13.32, 17.76, and 22.20 μM 6-benzylaminopurine (BA), each in combination with 9.08 μM thidiazurin (TDZ) and 0.05 μM α-naphthaleneacetic acid (NAA) induced the conversion of buds to micro-rhizomes in six weeks. More than 96% of the micro-rhizomes cultured on MS medium supplemented with 17.76 μM BA, 6.81 μM TDZ, and 2.46 μM indole-3-butyric acid (IBA) were converted to globular-shaped clumps with protocorm-like bodies (PLBs). Further culture of a piece of the clumps induced more than 15 adventitious shoots. Adventitious roots were produced at the base of adventitious shoots, and plantlets were readily transplanted to a substrate for acclimatization in a shaded greenhouse. The survival rate of the plants in the greenhouse was up to 90%. Plants grew vigorously, and there were no off-types from the regenerated 11,100 plants. Our study also, for the first time, shows that H. coronarium can be regenerated via PLBs, which may represent a new way of the in vitro propagation of H. coronarium. The established protocol could be used for the increased propagation of H. coronarium for conservation or commercial production.

Elevation of secondary metabolites production through light-emitting diodes (LEDs) illumination in protocorm-like bodies (PLBs) of Dendrobium hybrid orchid rich in phytochemicals with therapeutic effects

Gas-chromatography-mass-spectrometry revealed the presence of various bioactive compounds with anticancer properties in protocorm-like-body (PLB) cultures of a Dendrobium hybrid orchid (Dendrobium Enopi x Dendrobium Pink Lady). Pre-illumination of red fluorescent light lessened the stimulating effects of light-emitting diodes (LEDs) on secondary metabolites production among in vitro PLB cultures, possibly due to habituation. The highest flavonoid content of 16.79 μmol/ g of fresh weight (FW) was achieved under blue-red (1:1) LED for PLBs pre-treated with white LED for more than 3 subculture cycles. Phenolics content significantly reduced as PLBs pre-cultured under red fluorescent light for 2 subculture cycles were exposed to LED illuminations, where far red LED resulted in the lowest total phenolic content (18.85 μmol/ g FW). High intensity green LED (16.9 μmol/s) enhanced the accumulation of phenolics while amino acids such as L-leucine, glycine and proline exhibited no significant stimulating effect for secondary metabolites production.

Organic Compounds: Contents and Their Role in Improving Seed Germination and Protocorm Development in Orchids

In nature, orchid seed germination is obligatory following infection by mycorrhizal fungi, which supplies the developing embryo with water, carbohydrates, vitamins, and minerals, causing the seeds to germinate relatively slowly and at a low germination rate. The nonsymbiotic germination of orchid seeds found in 1922 is applicable toin vitropropagation. The success of seed germinationin vitrois influenced by supplementation with organic compounds. Here, we review the scientific literature in terms of the contents and role of organic supplements in promoting seed germination, protocorm development, and seedling growth in orchids. We systematically collected information from scientific literature databases including Scopus, Google Scholar, and ProQuest, as well as published books and conference proceedings. Various organic compounds, i.e., coconut water (CW), peptone (P), banana homogenate (BH), potato homogenate (PH), chitosan (CHT), tomato juice (TJ), and yeast extract (YE), can promote seed germination and growth and development of various orchids. They also stimulate seedling development, formation of protocorm-like bodies (PLBs), plantlet growth, and multiple shoot formation. The addition of organic compounds to culture media, individually or in combination, accelerates seed germination and seedling development. Different types and concentrations of organic nutrients are needed for the success ofin vitrocultures, depending on the species and genotype.

Clonal propagation of Trichocentrum stramineum (Orchidaceae), a threatened species endemic to Mexico

Background: Cloning techniques are applied to an endangered orchid species in order to reproduce individual plants and to preserve their genetic characteristics.
 Objectives: To establish a new protocol for clonal propagation of a threatened orchid of horticultural importance.
 Study species: Trichocentrum stramineum , a threatened orchid endemic to Mexico.
 Study site and duration: Totutla, Veracruz, Mexico. All experiments were designed and carried out at the Botanical Garden-UNAM and the IIAF-UMSNH over a course of six years.
 Methods: Seeds were germinated in a modified KC basal medium; protocorms and apical bud explants were obtained from the resulting in vitro plants and cultivated with or without plant growth regulators (PGRs). Both experimental groups were subcultured in order to evaluate the number of protocorm-like bodies (PLBs) and buds per explant.
 Results: On average, protocorms generated 51.2 and 54.1 PLBs in the absence or presence of 1 mg l-1 6-benzyladenine (BA), respectively, while 13.1 and up to 23.7 PLBs and / or shoots were observed on the apical bud explants in the absence or presence of 1 mg l-1 kinetin, respectively. In both cases, responses were direct, without the formation of an intervening callus. Approximately 200 PLBs were subcultured and developed into whole plants within 14 weeks. These were acclimatized to greenhouse conditions with a 90 % survival rate after 12 weeks. After 44 weeks, flowering was observed (3 %) individuals measuring at least 12 cm in height.
 Conclusions: The developed protocol proved to hold great potential for commercial propagation and conservation programs.