Protocorm-like Bodies Research Articles

Cattleya tigrina (Orchidaceae) in vitro regeneration: Main factors for optimal protocorm-like body induction and multiplication, plantlet regeneration, and cytogenetic stability

The induction and subsequent regeneration of protocorm-like bodies (PLB) is the most common morphogenetic pathway for orchid micropropagation. The regeneration of plants from PLB can be a bottleneck for some species due to asynchronicity. Genetic fidelity is another issue, and the cytogenetic stability of regenerated plantlets deserves special attention due to the common occurrence of endopolyploidy in orchids. In the present work, we defined optimal conditions for PLB induction and plant regeneration for Cattleya tigrina, a threatened orchid from the Brazilian Atlantic Rain Forest. We estimated the optimum thidiazuron (TDZ) concentration for PLB induction from leaf explants, while also comparing whole leaves vs thin cell layer (TCL) as explants. For plantlet regeneration from PLB clusters, we investigated the effects of different concentrations of GA3 and sucrose, as well as the dehydration of PLBs and the use of temporary vs continuous immersion systems. Furthermore, the cytogenetic stability of regenerated plantlets was analyzed using flow cytometry, and the differences in stomatal morphology were studied. Exogenous TDZ (25 µM) was the most effective concentration for PLB induction, with 17.5% induction rate. Leaf basal regions, either in whole leaves or in TCL, were the most responsive for PLB induction. The dehydration of PLBs for 120 min increased the fresh weight increment (FWI) in plantlet regeneration, but at the expense of decreased shoot number and increased mortality. Temporary-immersion bioreactors were found to be the most suitable system for PLB multiplication compared to continuous immersion system. Shoot development was challenging due to previously acquired habituation to TDZ. Sucrose and GA3 showed an interactive effect on shoot growth. GA3 improved shoot formation, and maximum shoot production was estimated to occur at 10.3 and 9.7 µM GA3 on media containing 1.5% and 2% sucrose, respectively, resulting in larger number of developed plantlets (∼220) per culture flask. The flow cytometry analysis showed that 26.5% of plants regenerated from PLBs had doubled ploidy levels. The ploidy alterations were reflected in changes on leaf epidermis, such as increased stomatal density and number.

Low Concentration of Anti-Auxin and Anti-Fungal Agent Accelerates the PLB Regeneration of Dendrobium okinawense under Green LED.

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.

Pemberian air rebusan kentang pada media Murashige-Skoog terhadap pertumbuhan protokorm anggrek sendu (Grammatophyllum stapeliiflorum) secara in vitro

Grammatophyllum stapeliiflorum is one of the most popular orchid species because of its beauty and is characterized by the shape of the flower stalk that sticks down and the brown flowers with white and yellow spots that look like drops of water or sad. This type of orchid is almost extinct and is rarely found in its natural habitat, so it is necessary to perform in vitro propagation of orchids. The culture in vitro method is one of the most effective methods for propagating rare orchids. This study aimed to determine the effect of adding potato water on protocorm growth and determine the best concentration for the growth of G. stapeliiflorum protocorm in vitro. This study used a Completely Randomized Design (CRD) with the addition of potato water (concentrations 0, 50, 100, 150, and 200 ml/L) on Murashige-Skoog (MS) medium. The results showed that the percentage of live explants was 100% in all treatments. The addition of potato water to MS medium had no effect on the percentage of live explants but still affected the growth percentage of Protocorm-like bodies (PLBs) in vitro. The best concentration of potato water for the protocorm growth of G. stapeliiflorum in vitro to MS medium was at a concentration of 150 ml/L, which was indicated by the highest percentage of PLBs growth at 100% and the color of PLBs green (2.5GY 6/8) compared to other potato water treatments and controls.

Co-option of the SHOOT MERISTEMLESS network regulates protocorm-like body development in Phalaenopsis aphrodite.

The protocorm is a structure that is formed upon germination of an orchid seed. It lacks cotyledons and is ovoid in shape. The protocorm-like body (PLB), on the other hand, is a protocorm-like organ induced from somatic tissues. PLBs have been widely used for orchid micropropagation. Because of its unique structure and its application in the orchid industry, PLB development has drawn considerable interest from orchid and developmental biologists. Our previous genome-wide comparative transcriptome study demonstrated that protocorms and PLBs share similar molecular signatures and suggested that SHOOT MERISTEMLESS (STM)-dependent organogenesis is important for PLB development. Here, we show that overexpression of Phalaenopsis aphrodite STM (PaSTM) greatly enhances PLB regeneration from vegetative tissue-based explants of Phalaenopsis orchids, confirming its regulatory role in PLB development. Expression of PaSTM restored shoot meristem function of the Arabidopsis (Arabidopsis thaliana) stm-2 mutant. Moreover, we identified class S11 MYB transcription factors (TFs) as targets downstream of PaSTM. A cis-acting element, TTGACT, identified in the promoters of S11 MYB TFs was found to be important for PaSTM binding and activation. Overexpression of PaSTM or its downstream targets, PaMYB13, PaMYB14, and PaMYB17, enhanced de novo shoot regeneration in Arabidopsis, indicating the active role of the PaSTM-S11 PaMYB module in organogenesis. In summary, our data demonstrate that PaSTM is important for PLB development. The STM-S11 MYB regulatory module is evolutionarily conserved and may regulate shoot or shoot-related organ development in plants.

Shoot Development through Modified Transverse Thin Cell Layer (tTCL) Culture of Phalaenopsis Hybrid Protocorms

This first-attempt study used microtome-based methods to generate a thin cell layer culture for the micropropagation of Phal. Hwafeng Redjewel × Phal. New Cinderella. Protocorms were embedded in various agarose concentrations (8–12%, w/v) and dried from 1 to 8 h before sectioning with a microtome. Optimal conditions for slicing sections of 100 to 300 μm were achieved when the protocorms were embedded at 10% (w/v) agarose and dried for 4 h under laminar flow, and the hardness of the agarose block under these conditions reached 641.8 ± 9.5 g·cm−2. The sectioned protocorms that were cultured on an MS medium supplemented with 1.2 mg·L−1 6-benzylaminopurine and 0.1 mg·L−1 α-naphthaleneacetic acid were capable of growth and differentiated through the neoformation of protocorm-like bodies (PLBs) and/or callus before subsequent regeneration into plantlets and development into healthy plants in a nursery environment. The results of this study demonstrate that microtome-based tTCL is a reliable and promising approach for mass propagation and possible virus-free propagation objectives for Phalaenopsis.

Advances in Breeding, Bioprospecting, and In Vitro Culture of Laelia Orchid Species

Orchids (Orchidaceae) are plants that are highly appreciated by their beautiful flowers worldwide. Moreover, they represent a source of metabolites with applications in medicine and biotechnology. Within the Orchidaceae family, the Laelia genus is a group of orchid species from the Neotropics and is probably one of the most representative genera of America. Laelia orchids are cultivated by their splendid flowers and are widely used in orchid breeding. Here, we revise the use of the Laelia genus in orchid breeding and metabolite bioprospecting. We also analyze the use of plant tissue culture (PTC) as an alternative to conventional propagation and as a strategy for the recovery of those Laelia species threatened with extinction. We summarize and discuss the recent advances in the application of different PTC techniques for mass multiplication based on asymbiotic germination, organogenesis, protocorm-like bodies development, and somatic embryogenesis, and the advances of in vitro conservation by cryoconservation and the use of slow-growth promoting hormones. Finally, we suggest future directions and venues in research for Laelia species.

Ploidy mosaics: does endopolyploidy in explants affect the cytogenetic stability of orchids regenerated from PLBs?

The induction and regeneration of protocorm-like bodies (PLBs) is a morphogenetic pathway widely used for orchid micropropagation. As endopolyploidy, i.e., the coexistence of cells with different ploidy levels, is a common feature in orchid tissues, a natural question arises when using somatic tissues as explants for orchid micropropagation: does endopolyploidy in explants affect the cytogenetic stability of regenerated plantlets? To answer this question, Epidendrum fulgens was used as a model plant, and flow cytometry was used to analyze endopolyploidy in pollinia, petals, labella, leaf bases, leaf tips, root tips, and protocorm bases and apices, which were subsequently used as explants for PLB induction and plant regeneration. Ploidy screenings showed contrasting ploidy patterns in samples, endopolyploidy being detected in all tissues, with C-values ranging from 1 to 16C. Protocorm bases and root tips presented the highest proportion of endopolyploidy, while petals and protocorm apices showed the lowest proportion. Flower parts exhibited high oxidation for PLB induction and pollinia failed to produce PLB or callus. The highest induction rate occurred at 10 µM TDZ, with 92%, 22%, and 0.92% for protocorm bases, leaves, and root tips, respectively. Plantlets were more easily regenerated from PLBs induced from protocorm bases than from leaves and roots. Doubled ploidy levels were registered in a proportion of 11% and 33% for PLB-regenerated plantlets obtained from protocorm bases and leaf bases, respectively, which was not directly associated with the proportion of endopolyploid cells or cycle value of explants. Explants have different endopolyploid patterns and cycle values. Polyploid regenerants were obtained from leaves and protocorms, although in rates not associated with the proportion of endopolyploid cells in the explants.

Impact of culture vessels on micro-morphological features of in vitro Dendrobium Sabin Blue orchid.

Different designs of the plant tissue culture vessel, such as size, material, and shape, may alter its microenvironment atmosphere. The present study was conducted on protocorm-like bodies (PLBs) of Dendrobium Sabin Blue orchid to determine the development of PLBs on plastic and glass culture vessels of different sizes. PLBs were cultured in half-strength Murashige and Skoog (MS) medium with the same initial weight of 0.5 g in 10 replicates. The growth index of the PLBs was calculated after 11 weeks to study their growth in every vessel; additionally, biochemical analysis was performed to determine carbohydrate content, proline concentration, and photosynthesis pigments in the PLBs. Scanning electron microscopy (SEM) was performed to study stomata development on PLBs in each vessel, and histological analyses were conducted to study the cell structure. Overall, the PLBs cultured in a large 470 ml plastic vessel showed successful growth with a high growth index, high carbohydrate content, low-stress condition, and high chlorophyll content. SEM confirmed that the presence of trichome and rhizoid in PLBs cultured in the 470 ml plastic vessel. Histological analysis showed the formation of the shoot on the PLBs and the presence of starch granules. Thus, the use of plastic as a culture vessel provides a good impact for culturing PLBs and has low cost.

Induction Regeneration and Artificial Seed Production of Etiolation Stems of <i>Cymbidium hookerianum</i>

The protocorm-like bodies (PLBs) of Cymbidium hookerianum were used as the explants, the effects of different hormones and hormone concentrations on the induction of etiolation stems, the subsequent regeneration of etiolation stems, and artificial seeds production and conservation using etiolation stems were studied. The results showed that the most efficient etiolation stems production induced by PLBs was obtained by adding 0.1 mg/L NAA and 0.1 mg/L 6-BA. The induced etiolation stems cultivated on the medium containing 2.0/0.1 mg/L NAA/6-BA showed the highest induction rate of PLBs, with an average of 80.40%, and the induction number of PLBs was higher, with an average of 1.36. The Induced etiolation shoot tips could regenerate rootless seedlings on the medium, and these seedlings could root and strengthen on the medium of 1/2MS + 20.0 g/L sucrose + 7.5 g/L agar. The highest germination rate of artificial seeds produced by encapsulating etiolation shoot tips was obtained on the MS medium containing 1.0 mg/L 6-BA, with an average of 86.67%. After storing for 6 weeks, the germination rate of artificial seeds preserved at 4°C was the highest, with an average of 48.00%.

Characterization of phytohormone and transcriptome profiles during protocorm-like bodies development of Paphiopedilum

BackgroundPaphiopedilum, commonly known as slipper orchid, is an important genus of orchid family with prominent horticultural value. Compared with conventional methods such as tillers and in vitro shoots multiplication, induction and regeneration of protocorm-like bodies (PLBs) is an effective micropropagation method in Paphiopedilum. The PLB initiation efficiency varies among species, hybrids and varieties, which leads to only a few Paphiopedilum species can be large-scale propagated through PLBs. So far, little is known about the mechanisms behind the initiation and maintenance of PLB in Paphiopedilum.ResultsA protocol to induce PLB development from seed-derived protocorms of Paphiopedilum SCBG Huihuang90 (P. SCBG Prince × P. SCBG Miracle) was established. The morphological characterization of four key PLB developmental stages showed that significant polarity and cell size gradients were observed within each PLB. The endogenous hormone level was evaluated. The increase in the levels of indoleacetic acid (IAA) and jasmonic acid (JA) accompanying the PLBs differentiation, suggesting auxin and JA levels were correlated with PLB development. Gibberellic acid (GA) decreased to a very low level, indicated that GA inactivation may be necessary for shoot apical meristem (SAM) development.Comparative transcriptomic profiles of four different developmental stages of P. SCBG Huihuang90 PLBs explore key genes involved in PLB development. The numbers of differentially expressed genes (DEGs) in three pairwise comparisons (A vs B, B vs C, C vs D) were 1455, 349, and 3529, respectively. KEGG enrichment analysis revealed that DEGs were implicated in secondary metabolite metabolism and photosynthesis. DEGs related to hormone metabolism and signaling, somatic embryogenesis, shoot development and photosynthesis were discussed in detail.ConclusionThis study is the first report on PLB development in Paphiopedilum using transcriptome sequencing, which provides useful information to understand the mechanisms of PLB development.

SUBSTITUTION OF SYNTHETIC HORMONES WITH ORGANIC MATERIALS ON THE GROWTH OF ORCHID PLANTS (Phalaenopsis amabilis) AS A GROWTH REGULATORY SUBSTANCE IN VITRO

In Indonesia, the agribusiness sector has begun to expand due to the increase in entrepreneurship of orchid plants. Several genera and species of orchid plants are promising to be traded as ornamental plants, one of which is the moon orchid (Phalaenopsis amabilis). However, the limited number of seeds or plants produced by conventional propagation methods takes a long time to get new tillers. Therefore, tissue culture techniques can be used to grow and reproduce moon orchid plants. In the success of in vitro culture, the balance of growth regulators is an essential factor. On the other hand, synthetic hormones such as 1-naphthalenaecetic acid (NAA) and 6-benzylaminopurine (BAP) are relatively expensive. The study in this research is to substitute the synthetic hormones NAA and BAP with organic ingredients that are cheap and easy to find. Such as Purple Sweet Potatoes and Beans and find out the right concentration of organic matter. This study used a completely randomized design with two factorials (concentration of purple sweet potato and green beans) with three repetitions. PLB (Protocorm Like Bodies) moon orchid (Phalaenopsis amabilis) was treated with: positive control (2 ppm), negative control (without the addition of organic or synthetic hormones), and variations in the concentration of purple sweet potato and green beans organic matter. The result was that C4D0 (500g/L purple sweet potato extract and 0 g/L green bean extract) was an excellent formulation, although there was no significant interaction between purple sweet potato and green bean extracts against moon orchid.

Protocorm-like body formation, stem elongation, and enhanced growth of Anthurium andraeanum ‘Tropical’ plantlet on medium containing silver nanoparticles

Protocorm-like body formation, stem elongation, and enhanced growth of Anthurium andraeanum ‘Tropical’ plantlet on medium containing silver nanoparticles

OPTIMIZATION PARAMETER FOR PRODUCTION OF PROTOCORM-LIKE BODY AND GROWTH OF GRAMMATOPHYLLUM SPECIOSUM

Micropropagation of orchids are often through the production of Protocorm Like Bodies (PLBs) and this research conducted a study to discover the optimal condition for the growth of Protocorm Like Bodies (PLBs) and also plantlets regeneration of Grammatophyllum speciosum Several aspects such as different carbon sources (sucrose, maltose) and the presence of charcoal alongside with the variety of concentration of agar were taken into account for the optimization. This study discovered that the 30 g/L of sucrose in 1 mg/L of BAP was superior in producing heavy PLBs. However, the trend differs in the number of plantlets where maltose (5 g/L) is seen to produce a higher number of plantlets compared to sucrose. Charcoal plays an important role in the growth of PLBs where the heaviest PLBs were recorded in the medium supplemented with 0.20 % of charcoal along with 4.5 g of agar. Vice versa relationship was recorded for the number of plantlets where without charcoal in 4.5 g of agar produce the highest number of plantlets.

Putative genes in alkaloid biosynthesis identified in Dendrobium officinale by correlating the contents of major bioactive metabolites with genes expression between Protocorm-like bodies and leaves

BackgroundDendrobium officinale, an endangered Chinese herb, possesses extensive therapeutic effects and contains bioactive ingredients such as major polysaccharides, alkaloids, and minimal flavonoids. We first obtained the protocorm-like bodies (PLBs) of this plant through tissue culture in order to determine the distribution of the main secondary metabolites in each organelle and the PLBs. We then analyzed the correlation between gene expression level from comparative transcriptome sequencing and metabolite content in different organs to identify putative genes encoding enzymes involved in the biosynthesis of polysaccharides, alkaloids, and flavonoids.ResultsWe used seeds as explants for protocorm induction and PLB propagation of D. officinale. The optimal medium formula for PLB propagation was 1/2 MS + α-NAA 0.5 mg·L− 1 + 6-BA 1.0 mg·L− 1 + 2, 4-D 1.5–2.0 mg·L− 1 + potato juice 100 g·L− 1. Stems, PLBs and leaves of D. officinale had the highest content of polysaccharides, alkaloids and flavonoids, respectively. Naringenin was only produced in stem; however, PLBs with high alkaloid content can replace other organs producing alkaloids. The hot water extraction method outperformed the ultrasound-assisted extraction method for extracting polysaccharides from D. officinale. A comparative transcriptome analysis of PLBs and leaves of D. officinale revealed differential expression of genes encoding enzymes involved in polysaccharide, alkaloid and flavonoid biosynthetic pathways. Putative genes encoding enzymes involved in these biosynthetic pathways were identified. Notably, we identified genes encoding the alkaloid biosynthesis enzymes strictosidine β-D-Glucosidase, geissoschizine synthase and vinorine synthase in D. officinale.ConclusionsThe identification of candidate genes encoding enzymes involved in metabolite biosynthesis will help to explore and protect this endangered species and facilitate further analysis of the molecular mechanism of secondary metabolite biosynthesis in D. officinale.

Combined Ultrahigh Pressure Extraction and High-Speed Counter-Current Chromatography for Separation and Purification of Three Glycoside Compounds from Dendrobium officinale Protocorm.

As an alternative to Dendrobium candidum, protocorm-like bodies (PLBs) of Dendrobium candidum are of great value due to their high yield and low cost. In this work, three glycoside compounds, β-D-glucopyranose 1-[(E)-3-(4-hydroxyphenyl)-2-propenoat] (I), β-D-glucopyranose 1-[(E)-3-(3, 4-dihydroxyphenyl)-2-propenoat] (II), and 1-O-sinapoyl glucopyranoside (III), were extracted and isolated by ultrahigh pressure extraction (UPE) coupled with high-speed counter-current chromatography (HSCCC) from PLBs of D. officinale. First, the target compounds were optimized and prepared with 50% ethanol solution at a 1:30 (g/mL) solid/liquid ratio in 2 min under 300 MPa by UPE. Then, the crude extract was chromatographed with a silica gel column, and primary separation products were obtained. In addition, the products (150 mg) were separated by HSCCC under the solvent system of MTBE-n-butyl alcohol-acetonitrile-water (5:1:2:6, v/v/v/v), yielding 31.43 mg of compound I, 10.21 mg of compound II, and 24.75 mg of compound III. Their structures were further identified by ESI-MS, 1H NMR, and 13C NMR. The antioxidant results showed that the three compounds expressed moderate effects on the DPPH· scavenging effect. Compound II had the best antioxidant capacity and its IC50 value was 0.0497 mg/mL.

In vivo polyploidy induction of Phalaenopsis amabilis in a bubble bioreactor system using colchicine

Abstract Phalaenopsis amabilis Blume var. grandiflora Bateman is economically important as cut and pot flower. Polyploidy is considered as a valuable tool in improvement and evolution of ornamental plants. Protocorm-like bodies (PLBs) of P. amabilis were cultured on Murashige and Skoog medium containing 0.20 mg L-1 IBA together with 2.00 mg L-1 KIN and 1.00 g L-1 activated charcoal and grown for a period of five months. Fully-developed plantlets from in vitro grown PLBs were immersed in a bubble reactor filled with half-strength Hoagland solution containing the antimitotic agent colchicine (0.05%, 0.10% and 0.15%, w/v) for 72 h with a few drops (1 mL of 0.1%) of octylphenoxypolyethoxyethanol or Nonidet (P-40) as a surfactant. Plantlets were aerated to prevent hypoxia. Colchicine-treated and untreated plantlets were transferred to pots for a period of 60 days. Tetraploidy was successfully induced by 0.15% colchicine. Polyploidy levels were firstly detected using flow cytometry and then confirmed by cytological and morphological observations. The chromosome number was 2n = 2x = 38 in diploids and 2n = 4x = 76 in tetraploid. Incubation of plantlets in liquid medium containing 0.15% colchicine induced the maximum recovered tetraploids with minimum frequency of survival (50%). The tetraploid plants were more compact and exhibited round and thick leaves with darker green color than diploids. Stomata size in tetraploids were larger with less density than diploids. Chloroplast number in guard cells of tetraploids was about two times more than that of control. These results indicate that induction of tetraploids are a reliable and powerful tool for generation of novel phenotypes with ornamental and horticultural value for genetic improvement and breeding. Produced tetraploids in current study have potential in the ornamental/floriculture trade.

Genome-Wide Identification and Analysis of the APETALA2 (AP2) Transcription Factor in Dendrobium officinale.

The APETALA2 (AP2) transcription factors (TFs) play crucial roles in regulating development in plants. However, a comprehensive analysis of the AP2 family members in a valuable Chinese herbal orchid, Dendrobium officinale, or in other orchids, is limited. In this study, the 14 DoAP2 TFs that were identified from the D. officinale genome and named DoAP2-1 to DoAP2-14 were divided into three clades: euAP2, euANT, and basalANT. The promoters of all DoAP2 genes contained cis-regulatory elements related to plant development and also responsive to plant hormones and stress. qRT-PCR analysis showed the abundant expression of DoAP2-2, DoAP2-5, DoAP2-7, DoAP2-8 and DoAP2-12 genes in protocorm-like bodies (PLBs), while DoAP2-3, DoAP2-4, DoAP2-6, DoAP2-9, DoAP2-10 and DoAP2-11 expression was strong in plantlets. In addition, the expression of some DoAP2 genes was down-regulated during flower development. These results suggest that DoAP2 genes may play roles in plant regeneration and flower development in D. officinale. Four DoAP2 genes (DoAP2-1 from euAP2, DoAP2-2 from euANT, and DoAP2-6 and DoAP2-11 from basal ANT) were selected for further analyses. The transcriptional activation of DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11 proteins, which were localized in the nucleus of Arabidopsis thaliana mesophyll protoplasts, was further analyzed by a dual-luciferase reporter gene system in Nicotiana benthamiana leaves. Our data showed that pBD-DoAP2-1, pBD-DoAP2-2, pBD-DoAP2-6 and pBD-DoAP2-11 significantly repressed the expression of the LUC reporter compared with the negative control (pBD), suggesting that these DoAP2 proteins may act as transcriptional repressors in the nucleus of plant cells. Our findings on AP2 genes in D. officinale shed light on the function of AP2 genes in this orchid and other plant species.

IN VITRO REGENERATION OF ARUNDINA GRAMINIFOLIA (D. DON) HOCHR

We can see Orchids come in a wide variety of shapes, sizes, colours, and textures far beyond the human mind’s imagination. They emerge from seeds in nature, but in the absence of suitable hosts, they do not germinate in sufficient numbers. This problem was solved by using the tissue culture technique for its germination. One of the successful method used for mass propogation of orchid plantlets is in vitro techniques. Therefore, an initial analysis was conducted in order to establish an appropriate procedure for mass multiplication of Arundina graminifolia. MS (Murashige and Skoog) medium was found to be suitable for the asymbiotic seed germination of Arundina graminifolia. Direct protocorm like bodies were induced by using combinations and individual supplement of MS medium with IAA (Indole-3-acetic acid), IBA (Indole-3- butyric acid), BAP (6-Benzylaminopurine) and KIN (Kinetin). Hormone-free MS basal medium was found suitable for the conversion of PLBs (protocorm-like bodies) into complete plantlets

Elicitation of protocorm-like bodies (PLBs) of Dendrobium ‘Sabin Blue’ using methyl jasmonate, salicylic acid and melatonin for in vitro production of anthocyanin

High demands for Dendrobium cut flowers, as well as the secondary metabolite products, have led to the over-exploitation of the orchid from its natural habitat. The use of in vitro hybrid orchid is one of the prominent strategies to meet the demand. Dendrobium Sabin Blue (a hybrid species between Dendrobium Blue Angel and Dendrobium Sanan Blue) orchid was used in this study to produce anthocyanin. This study aimed to improve in vitro production of anthocyanin (Petunidin) from protocorm-like bodies (PLBs) of Dendrobium Sabin Blue by using elicitors such as methyl jasmonate (MeJA), salicylic acid (SA), and melatonin. After four weeks of culture, results showed that all three elicitors had stimulated the production of petunidin. Although the insignificant or low petunidin content was achieved in elicited PLBs as compared to the mother plant, in vitro culture of PLBs can be considered as a more economical strategy than using mother plants.

Modified basal culture medium improves proliferation of Dendrobium Sabin Blue’s protocorm-like bodies (PLBs)

This study was carried out to reformulate half-strength Murashige and Skoog (MS) basal medium, one of the standard media for orchid tissue culture for protocorm-like bodies (PLB) culture of Dendrobium Sabin Blue. Initial PLB fresh mass of 0.5 g was inoculated in various culture treatments for six weeks of culture period. Alteration of half MS medium yielded a modified medium without Fe-EDTA and containing half-strength macronutrients, double-strength micronutrients, full-strength vitamins and 30 g/L sucrose. The modified medium enhanced the proliferation of PLBs by 23% (0.299 ± 0.017 g) as compared half MS medium (0.243 ± 0.012 g) on a dry mass basis. PLB suspension culture was established using liquid medium of the revised formula for a period of 7 weeks. Supplementing the modified medium with quarter-strength of Fe-EDTA is beneficial for long-term culture of PLBs. Improvement of PLB biomass through the use of modified culture medium devoid of plant growth regulators reduces production cost for micropropagation and secondary metabolite production. Besides, it can reduce the likelihood of somaclonal variations related to the use of plant growth regulators in the culture medium.