Protocorm-like Bodies Proliferation Research Articles

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

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.

Gamma Radiation Induced In-Vitro Mutagenesis and Isolation of Mutants for Early Flowering and Phytomorphological Variations in Dendrobium 'Emma White'.

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

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.

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).

An Overview of Orchid Protocorm-Like Bodies: Mass Propagation, Biotechnology, Molecular Aspects, and Breeding

The process through induction, proliferation and regeneration of protocorm-like bodies (PLBs) is one of the most advantageous methods for mass propagation of orchids which applied to the world floricultural market. In addition, this method has been used as a tool to identify genes of interest associated with the production of PLBs, and also in breeding techniques that use biotechnology to produce new cultivars, such as to obtain transgenic plants. Most of the molecular studies developed have used model plants as species of Phalaenopsis, and interestingly, despite similarities to somatic embryogenesis, some molecular differences do not yet allow to characterize that PLB induction is in fact a type of somatic embryogenesis. Despite the importance of species for conservation and collection purposes, the flower market is supported by hybrid cultivars, usually polyploid, which makes more detailed molecular evaluations difficult. Studies on the effect of plant growth regulators on induction, proliferation, and regeneration of PLBs are the most numerous. However, studies of other factors and new technologies affecting PLB production such as the use of temporary immersion bioreactors and the use of lighting-emitting diodes have emerged as new tools for advancing the technique with increasing PLB production efficiency. In addition, recent studies on Phalaenopsis equestris genome sequencing have enabled more detailed molecular studies and the molecular characterization of plantlets obtained from this technique currently allow the technique to be evaluated in a more comprehensive way regarding its real applications and main limitations aiming at mass propagation, such as somaclonal variation.

Changes in Morphological Characteristics, Regeneration Ability, and Polysaccharide Content in Tetraploid Dendrobium officinale

Dendrobium officinale Kimura et Migo is a famous traditional Chinese medicinal plant. It produces various phytochemicals, particularly polysaccharides, which have nutraceutical and pharmaceutical values. To increase its biomass production and polysaccharide content, our breeding program has generated a series of polyploid cultivars through colchicine treatment of protocorm-like bodies (PLBs). The present study compared two tetraploid cultivars, 201-1-T1 and 201-1-T2, with their diploid parental cultivar, 201-1, in an established in vitro culture system. Tetraploid ‘201-1-T1’ and ‘201-1-T2’ had shorter leaves and shorter and thicker stems and roots, and they produced higher biomass compared with the diploid cultivar. The length and width of stomata significantly increased, but stomatal density decreased in tetraploid cultivars. The PLB induction rates from the stem node explants of the tetraploid cultivars were significantly higher than those of diploid. However, the PLB proliferation of tetraploids was lower than that of the diploid. The mean number of plantlets regenerated from tetraploid PLBs was also lower than that of the diploid after 4 months of culture. Polysaccharide contents in stems, leaves, and roots of 6-month-old tetraploid plantlets were significantly higher than those of diploids. The polysaccharide content in the stem of ‘201-1-T1’ was 12.70%, which was a 2-fold increase compared with the diploid cultivar. Our results showed that chromosome doubling could be a viable way of improving D. officinale in biomass and polysaccharide production.

Effect of plant growth regulators and carbon sources on proliferation and shoot formation of PLBs in Dendrobium candidum

Dendrobium candidum Wallich ex Lindley is a traditional Chinese medicine plant and has been widely used for medicinal and ornamental purposes. In this study, several different factors affecting micro propagation of protocormlike bodies (PLBs) such as basal media, plant growth regulators, and carbon sources. The proliferation PLB derived from seeds was the best in H3P4 basal medium containing 0.1 mg · L-1 NAA and 0.1 mg · L-1 Kinetin. PLB growth was the best when 10 g · L-1 sucrose was added to the carbon atoms in the medium. The rate of shoot formation from the propagated PLB was the highest in 1/4 MS or H1P2 medium containing 10 g · L -1 sucrose, and the shoot length was longer than the others.

Clonal Fidelity of Micro-propagated Phalaenopsis Plantlets Based on Assessment Using Eighteen Ph-Pto SNAP Marker Loci

Phalaenopsis amabilis is an Indonesia native orchid species having large, white flowers with yellow labellum coloration. This studies aimed to develop Phal. amabilis micropropagation methods and evaluate the regenerated plantlet fidelity. Media supplemented with Thidiazuron (TDZ) and Polyvinylpyrrolidone (PVP) and medium pH adjustment effects to induce protocorm-like bodies (PLBs) from leaf explants and proliferate secondary PLBs were investigated. Clonal fidelity among regenerated plantlets was evaluated using eighteen SNAP marker loci. The results showed that the ½ MS medium supplemented with 3 mg L-1 TDZ and 0.5 g L-1 PVP was the best for PLB induction while the ½ MS medium supplemented with 0.5 mg L-1 TDZ was the best for PLB proliferation. For PLB induction, the media pH was adjusted into pH=7 for efficient PLB regeneration. Based on the assessment using 18 SNAP marker loci, four variant alleles in three loci (11.8%) out of a total 34 plantlets were detected. The mutation frequency at the evaluated SNAP marker loci was 2.5 x 103 (0.25%). Changes in SNP alleles may not always result in phenotype changes and allele variant occurrences may not affect phenotype fidelity of micro-propagated Phal. amabilis plantlets. Therefore, further studies about the phenotype fidelity among plantlets are necessary.

Highly Efficient Proliferation and Regeneration of Protocorm-like Bodies (PLBS) of the Threatened Orchid, Phalaenopsis bellina

Phalaenopsis bellina is an important indigenous fragrant orchid threatened with extinction. In this study, we evaluated the effect of medium strength, sucrose, nitrogen (NH4NO3) and potato extract on proliferation of P. bellina protocorm-like bodies (PLBs) to improve micropropagation in this species. Optimal treatment for PLBs proliferation rate with an average fresh weight (FW) of 0.97±0.16 g was obtained through culturing on half strength (½) MS medium containing 20 g/L sucrose, 15 mM NH4NO3 and 20% w/v potato extract supplemented with 0.8 μM 2,4 dichlorophenoxyacetic acid (2,4-D). The optimal treatment produced large, healthy and greenish PLBs with reduction in the occurrence of culture browning. In contrast, treatments with high potato extract (>20% w/v) or NH4NO3 (>30 mM) concentrations tend to have inhibitory effect and resulted in low PLBs proliferation rate, with an average FW of 0.77±0.15 g and 0.69±0.15 g, respectively. Plant regeneration of PLBs was achieved on plant growth regulator (PGR)-free ½ MS medium. In total, 60 healthy PLBs from the optimal treatment were successfully regenerated, acclimatized with 100% survival percentage and grew well in a mixture of soil, sand and vermicompost (8:4:2 (w/w/w)). With the optimal treatment, PLBs proliferation rate was enhanced by 27.63%. Our findings offer an improved micropropagation protocol of the endangered P. bellina for conservation and commercial production.

Proliferation of Protocorm-Like Bodies of Dendrobium mannii and Dendrobium mirbelianum in Chitosan-containing Media In Vitro

Dendrobium mannii and Dendrobium mirbelianum were rare and endangered orchid species. In vitro micro propagation could be used to conserve and proliferate these species. The aims of this study were to determine the effects of chitosan on proliferation of the protocorm-like-bodies (PLBs) of in vitro grown Dendrobium mannii and D. mirbelianum. Chitosan has been reported as an important component to promote orchid growth in vitro. In this study chitosan was included in the in-vitro media at the concentrations of 0, 1, 2, 3, and 4 mg.L-1. The highest PLBs formation, clump diameter, and fresh weight on D. mannii were obtained from explants cultured on medium supplemented with 1 mg.L-1 chitosan. After 14 weeks of culture, fresh weight, clump diameter and average number of PLBs of D.mannii were 29.52 %, 33.00 % and 35.88% greater at chitosan (1 mg.L-1) compared to control media. D. mirbelianum treated with low chitosan concentrations (1 and 2 mg.L-1) had 65.71% more leaves than those without chitosan treatment. Chitosan, however, had an adverse effects on growth of both species at 4 mg.L-1. Medium contain 4 mg.L-1 chitosan induced PLBs proliferation of D.mannii, but inhibited PLBs proliferation of D.mirbelianum. These results suggested that chitosan had important roles in PLB proliferation and could be used for the propagation of D. mannii PLBs in vitro. Keywords: rare orchid species, in-vitro

Effects of Different Culture Media on Growth and Proliferation of Dendrobium ‘Earsakul’ Protocorm-like Bodies

The effects of culture media on growth and proliferation of ‘Earsakul’ dendrobium (Dendrobium) protocorm-like bodies (PLBs) were evaluated in a two-step culture. After culturing on each of the four first step media for 4 months and on each of the four second step media for 4 months, the greatest total PLB fresh weight, increase in number of PLBs and growth rates were obtained when using Vacin and Went medium 1 (VW1) in both culture steps compared with those in 15 other medium combinations. Starting from 0.5 g of PLBs, culturing on VW1 for 8 months achieved a total of 415.25 g of PLBs, a multiplication rate of 830-fold. The supplementation of tomato (Solanum lycopersicum) and substitution of ‘Hom Thong’ banana [Musa acuminata (AAA group)] with ‘Khai’ banana [M. acuminata (AA group)] in this new medium promoted growth and proliferation of dendrobium PLBs 2.4-fold over the control medium, suggesting its usefulness in commercial micropropagation.

Effect of light emutting diode (LED) lamps and N-acetylglucosamine (NAG) on organogenesis in protocorm-like bodies (PLBs) of a Cymbidium hybrid cultured in vitro

Abstract not available
 Plant Tissue Cult. & Biotech. 24(2): 273-277, 2014 (December)

Use of coconut water and fertilizer for in vitro proliferation and plantlet production of Dendrobium ‘Gradita 31’

To improve the micropropagation of Dendrobium ‘Gradita 31’, coconut water (CW) and fertilizer media were used to enhance growth, proliferation, and germination of protocorm-like bodies (PLBs). PLBs formed when shoots approximately 0.4 cm long were cultured on semi-solid half-strength Murashige and Skoog (MS) basal medium containing 1 mg L−1 thidiazuron (TDZ) and 0.5 mg L−1 N6-benzyladenine (BA). After four rounds of 15-d subcultures, PLBs were initially proliferated in liquid half-strength MS medium supplemented with 0.3 mg L−1 TDZ and 0.1 mg L−1 α-naphthalene acetic acid (NAA) and subcultured monthly into fresh medium. When 15% (v/v) CW was added to half-strength MS medium, optimal growth and proliferation of PLBs resulted with the following principal findings: a maximum of 2.86 g total fresh weight (FW) of PLBs formed from 1 g of PLBs, an average of 0.46 g FW of PLBs added per subculture period, a total of 157.1 PLBs formed, an average of 25.3 PLBs added per subculture period, and a low percentage of PLB browning (20.7%). Half-strength MS medium containing 15% (v/v) CW with Rosasol® medium (liquid fertilizer; 1.5 g L−1 18 N:18P:18 K + 1.5 g L−1 25 N:10P:10 K + TE) resulted in a similar organogenic outcome with 25.5% PLB browning. PLBs germinated easily and rooted on Rosasol-supplemented plant growth regulator-free medium. In total, 180 Dendrobium ‘Gradita 31’ plantlets from all treatments were successfully acclimatized with 100% survival on a Cycas rumphii bulk substrate and grew well after repotting in a mixture of wood charcoal and C. rumphii bulk (1:1, v/v).

다양한 배지종류, sucrose 농도 및 갈변억제물질 처리에 의한 팔레놉시스 PLB 증식 및 재분화 체계확립

Abstract To establish an efficient proliferation and rege-neration of PLBs (protocorm-like bodies) of Phalaenopsis plants, a variety of propagation medium types, various concentraions of sucrose as well as liquid and solid type were tested in this study. Further, activated charcoal, citric acid and ascorbic acid were compared whether these agents are suppose to reduce the browning in culture process using PLBs of Phalanopsis plants. With regard to the proper propagation medium, VW medium showed 1.3 ~ 2 times highr than those of other medium in an index of increasing for fresh weight and 50% higher than those of other medium in the frequency of shoot regeneration. However, regarding liquid and solid types of culture, there were no significant differences in the proliferation of PLBs and regeneration of shoots from PLBs. In the experiment for a variety of sucrose concentrations (0 ~ 50 g/l), 10 g of sucrose showed 30 ~ 50% higher than other concentrations in increasing index and 10 ~ 50% higher in the regeneration of shoots from PLBs. Regarding the reduction of browning in tissue culture via PLBs of

In vitro propagation and reintroduction of the endangered Renanthera imschootiana Rolfe.

Renanthera imschootiana Rolfe is an endangered tropical epiphytic orchid that is threatened with extinction due to over-collection and the loss of suitable habitats. In vitro propagation is a useful way to mass produce plants for re-establishment in the wild and for commercial propagation. Seeds collected 150 days after pollination (DAP) were the optimum stage for in vitro culture. Seed germination reached 93.1% on quarter-strength MS (i.e., MS containing a quarter of macro- and micronutrients) medium containing 0.5 mg l−1 α-naphthaleneacetic acid (NAA), 20% coconut water (CW), 1.0 g l−1 peptone, 10 g l−1 sucrose and 1.0 g l−1 activated charcoal (AC). Quarter-strength MS medium supplemented with 1.0 mg l−1 BA, 0.5 mg l−1 NAA, 1.0 g l−1 peptone, 10 g l−1 sucrose and 20% CW was suitable for the sub-culture of protocorm-like bodies (PLBs) in which the PLB proliferation ratio was 2.88. Quarter-strength MS medium containing 1.0 mg l−1 NAA, 1.0 g l−1 peptone, 100 g l−1 banana homogenate (BH), and 1.0 g l−1 AC was suitable for plantlet formation and 95.67% of plantlets developed from PLBs within 60 days of culture. Hyponex N016 medium supplemented with 0.5 mg l−1 NAA, 1.0 g l−1 peptone, 20 g l−1 sucrose, 150 g l−1 BH, and 1.0 g l−1 AC was suitable for the in vitro growth of plantlets about 2-cm in height. Plantlets 3-cm in height or taller were transplanted to Chilean sphagnum moss, and 95% of plantlets survived after 60 days in a greenhouse. Three hundred transplanted of seedlings 360-days old were reintroduced into three natural habitats. Highest percentage survival (79.67%) was observed in Yuanjiang Nature Reserve two years after reintroduction, followed by Huolu Mountain forest park (71.33%). This protocol is an efficient means for the large-scale propagation and in vitro and in vivo germplasm conservation of R. imschootiana.

멸종위기 수종 석곡(Dendrobium moniliforme)의 Protocorm-like Body 형성 및 기내증식

This experiment was conducted to establish the in vitro propagation of Dendrobium moniliforme through protocorm like body (PLB) induction from the culture of leaf, stem and root explants. Frequency of PLB formation from root explants was higher than those of leaf and stem explants. PLB's proliferation in MS medium including 1.0 mg/L NAA was better than 1.0 mg/L IBA. Frequency of PLB's proliferation on medium with various concentrations of BA, Kinetin, Tidiazuron (0. 1.0, 3.0 mg/L) and NAA (0, 1.0 mg/L) was tested. The maximun proliferation of PLB's was obtained on MS medium supplemented with 3.0 mg/L BA and 1.0 mg/L NAA after 6 weeks of culture. Addition of 200 mg/L activated charcoal (AC) and 30 g/L sucrose was effective on PLB proliferation for D. moniliforme.

Efficient micropropagation of Dendrobium sonia-28 for rapid PLBs proliferation

Effective propagation of protocorm-like bodies (PLBs) of Dendrobium sonia-28 was achieved in this study. The highest PLBs proliferation within 21 days was observed in semi-solid media over liquid culture. Half strength of Murashige and Skoog (MS) media produced the highest PLBs growth rate percentage compared to full and double strength MS media in semi-solid culture. In sucrose treatment, the highest increased of PLBs growth rate percentage was in 10g.L-1. PLBs growth rate percentage was the highest (14%) in media devoid of exogenous 6-benzylaminopurine (BAP) or naphthalene acetic acid (NAA). However, the addition of BAP and NAA in combinations of BAP (4.44 or 8.88 µM) and NAA (8.88 µM) resulted in increased PLBs growth rate to 14% compared to when added separately. The optimized micropropagation media and condition of PLBs proliferation are important for the production of large number of plant material required for the development of genetic modification and cryopreservation studies.