Protocorm-like Bodies Regeneration Research Articles

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.

Protocorm-like-body extract of Phalaenopsis aphrodite combats watermelon fruit blotch disease.

Bacterial fruit blotch, caused by the seedborne gram-negative bacterium Acidovorax citrulli, is one of the most destructive bacterial diseases of cucurbits (gourds) worldwide. Despite its prevalence, effective and reliable means to control bacterial fruit blotch remain limited. Transcriptomic analyses of tissue culture-based regeneration processes have revealed that organogenesis-associated cellular reprogramming is often associated with upregulation of stress- and defense-responsive genes. Yet, there is limited evidence supporting the notion that the reprogrammed cellular metabolism of the regenerated tissued confers bona fide antimicrobial activity. Here, we explored the anti-bacterial activity of protocorm-like-bodies (PLBs) of Phalaenopsis aphrodite. Encouragingly, we found that the PLB extract was potent in slowing growth of A. citrulli, reducing the number of bacteria attached to watermelon seeds, and alleviating disease symptoms of watermelon seedlings caused by A. citrulli. Because the anti-bacterial activity can be fractionated chemically, we predict that reprogrammed cellular activity during the PLB regeneration process produces metabolites with antibacterial activity. In conclusion, our data demonstrated the antibacterial activity in developing PLBs and revealed the potential of using orchid PLBs to discover chemicals to control bacterial fruit blotch disease.

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.

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.

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.

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.

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.

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.

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.

Molecular spectrum of somaclonal variation in PLB-regenerated Oncidium revealed by SLAF-seq

Protocorm-like body (PLB) formation is an efficient method that has been widely used in the propagation of orchids. There is little information regarding the genome-wide molecular spectrum of somaclonal variation in PLB-regenerated orchids. We used specific-locus amplified fragment sequencing (SLAF-seq) to investigate the variations of single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) in somaclones of Oncidium ‘Milliongolds’, which was regenerated via PLB formation after in vitro culturing for 10 years. A high average variation rate (1.00 × 10− 3) was detected in 11 somaclones, and the most abundant variations were line specific. The average Ti/Tv ratio was 2.17, and G:C > C:G was the rarest type of SNP. The SNPs adjacent to adenine and thymine were more frequent than those adjacent to guanine and cytosine. The most prominent InDels were mononucleotide InDels. More than half of the InDels occurred within or adjacent to homopolymeric or polynucleotide repeats, and a majority of the repeats were thymine or adenine homopolymers. This study expands the current understanding of the somaclonal variation that occurs in PLB regeneration systems and long-term subcultures, which will be useful for improving the use of PLB formation in the propagation and breeding of orchids. We also confirmed that SLAF-seq is an efficient technology for genome-wide analysis of somaclonal variations for most species without reference genome sequences. We expanded the understanding of somaclonal variation in PLB regeneration and long-term subcultures of orchids, and confirmed SLAF-seq is efficient for genome-wide analysis of somaclonal variations without reference genome sequences.

Micropropagation of two near threatened orchid. Part 1: Catasetum pileatum cv. Alba

Many orchid species are threatened. In this study, a reliable and efficient protocol was outlined for in vitro propagation of Catasetum pileatum cv. Alba, a near threatened orchid species with the proper usage of plant growth regulators (PGRs). Protocorms as explants were cultured on Murashige and Skoog (MS) medium containing different concentrations of kinetin (Kn; 0.00, 0.20, 0.50, 1.00, 2.00, 3.00 and 5.00 mg l-1) and indole-3-butyric acid (IBA; 0.00, 0.10, 0.20, 0.50 and 1.00 mg l-1), either individually or in combination. The frequency of protocorm-like bodies (PLBs) regeneration significantly relied on concentrations of PGRs used. A combination of 1.00 mg l-1 Kn and 1.00 mg l-1 IBA was found to be suitable for maximum PLB regeneration (8.63 per explant) and the largest number of leaf (12.70 per explant). The highest rooting frequency with 7.40 roots per explant was achieved on protocorms grown in medium enriched with 1.00 mg l-1 Kn and 0.50 mg l-1 IBA. Plantlets were transplanted to pots filled with a mixture of peat moss, leca and perlite (1:1:1) and transferred to the greenhouse. The plantlets were successfully acclimatized in the greenhouse with a survival rate of 80% exhibiting normal developmental patterns.

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.

Protocorms and Protocorm-Like Bodies Are Molecularly Distinct from Zygotic Embryonic Tissues in Phalaenopsis aphrodite.

The distinct reproductive program of orchids provides a unique evolutionary model with pollination-triggered ovule development and megasporogenesis, a modified embryogenesis program resulting in seeds with immature embryos, and mycorrhiza-induced seed germination. However, the molecular mechanisms that have evolved to establish these unparalleled developmental programs are largely unclear. Here, we conducted comparative studies of genome-wide gene expression of various reproductive tissues and captured the molecular events associated with distinct reproductive programs in Phalaenopsis aphrodite Importantly, our data provide evidence to demonstrate that protocorm-like body (PLB) regeneration (the clonal regeneration practice used in the orchid industry) does not follow the embryogenesis program. Instead, we propose that SHOOT MERISTEMLESS, a class I KNOTTED-LIKE HOMEOBOX gene, is likely to play a role in PLB regeneration. Our studies challenge the current understanding of the embryonic identity of PLBs. Taken together, the data obtained establish a fundamental framework for orchid reproductive development and provide a valuable new resource to enable the prediction of gene regulatory networks that is required for specialized developmental programs of orchid species.

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

Optimization of genetic transformation of orchid (Dendrobium Sonia) mediated by Agrobacterium tumefaciens

Dendrobium Sonia is a popular orchid and used for many purposes in Vietnam. In this study, the transformation is set on protocorm like bodies (PLBs) mediated by Agrobacterium tumefaciens containing vector with GUS gene and kanamycin resistance gene. The main factors influencing the transformation including medium for regeneration and growth of PLBs, acetosyringone (AS) concentration, the co-cultivation time, Agrobacterium strain, bactericidal antibiotic concentrations and screening of transformed PLBs were surveyed. Results showed that MS medium supplemented with 1.0 mg/l NAA in combination with 0.5 mg/l or 0.3 mg/l BA was suitable for PLBs regeneration from stem tissue slices. The highest transformation efficiency (percentage of PLBs with GUS gene expression) was obtained when PLBs were infected and co-cultivated with Agrobacterium on the medium containing 100 µM AS for 3 days (with strain EHA105 and C58) or 4 days (with strain LBA4404). Medium containing 300 mg/l cefotaxime and 7-15 mg/l hygromycin was suitable for the disinfection and screening of transformed PLBs.

Callus-mediated and direct protocorm-like body formation of Bletilla striata and assessment of clonal fidelity using ISSR markers

Two efficient morphogenetic pathways for micropropagation of Bletilla striata (Thunb.) Reichb. f. have been established through the callus-mediated and direct formation of protocorm-like bodies (PLBs) from protocorms and shoot tips. Green calli were induced from the basal surface of protocorms and the cut-end of shoot tips on Vacin and Went (VW) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalene acetic acid (NAA) after 3–5 weeks, with the highest frequency of explants forming callus (48.0 %) from protocorms at 1.0 mg l−1 2,4-D. The calli obtained from all plant growth regulator (PGR) treatments could proliferate and differentiate PLBs on the PGR-free medium. NAA and 2,4-D significantly enhanced the growth of callus. The fastest growth rate of callus was achieved at the combination of 1.0 mg l−1 2,4-D and 1.0 mg l−1 TDZ with 46.2-fold within 3 months. The regeneration of PLBs from callus was significantly improved by 6-benzyladenine (BA), and a mean number of 48.4 PLBs was produced from 100 mg calli at 1.0 mg l−1 BA within 3 months. BA and thidiazuron (TDZ) promoted the direct formation of PLBs from explants. The highest frequency of direct PLBs formation (76.0 %) and the highest mean number of PLBs per explant (30.2) were observed in protocorms cultured with 0.5 mg l−1 BA. Assessment of clonal fidelity by inter-simple sequence repeat (ISSR) markers revealed similarity ranges of 99.8–100.0 % between the regenerants and their mother plants and 99.5–100.0 % among the regenerants, which suggested the micropropagation protocols were genetically stable.

Multiple regeneration pathways in Spathoglottis plicata Blume — A study in vitro

Asymbiotic germination of immature seeds (embryos), and mature seeds and micropropagation of Spathoglottis plicata were described. Effects of three nutrition media namely, Murashige & Skoog (MS); Phytamax (PM); and Phyto-Technology orchid seed sowing medium (P723), two carbon sources such as glucose and sucrose at 2–3% (w/v), two plant growth regulators such as 6-benzylaminopurine (BAP; 0.5–3.0mgl−1) and α-naphthalene acetic acid (NAA; 0.5–2.0mgl−1) and peptone (2.0gl−1) were examined on seed germination, early protocorm development and micropropagation. The maximum germination of mature seeds (95%) was recorded in PM medium supplemented with 2% (w/v) sucrose+2.0gl−1 peptone. For germination of embryos P723 medium supplemented with 1.0mgl−1 BAP proved best. Multiple shoot buds or protocorm-like bodies (PLBs) were produced from stem segments of in vitro raised seedlings. Both direct organogenesis and embryogenesis were observed and the morphogenetic response was initiated by different concentrations and combinations of PGRs. The optimum PGR combination for maximal PLB regeneration was 1.0mgl−1 NAA+2.5mgl−1 BAP, while 1.0mgl−1 NAA+1.0mgl−1 BAP for shoot bud development. Strong and stout root system was induced in half strength PM medium supplemented with 0.5mgl−1 IAA. The well-rooted plantlets were transferred to pots containing a potting mixture composed of saw dust, coconut coir, humus, and coal pieces at 1:1:1:2 (w/w) with 80% survival in outside environment and flowered after two years of transfer.

Asymbiotic seed germination, mass propagation and seedling development of Vanda coerulea Griff ex.Lindl. (Blue Vanda): An in vitro protocol for an endangered orchid

The application of modern biotechnology for conservation of any endangered species requires an efficient in vitro regeneration protocol. In this study a reliable protocol was developed for in vitro seed germination, protocorm multiplication and subsequent plantlet regeneration of Vanda coerulea, an endangered orchid species. Among the four basal media evaluated for asymbiotic seed germination, Phytamax was found to be the best followed by Murashige and Skoog (MS). Phytamax was also found good for protocorm development. For protocorm like body (PLB) regeneration, protocorms were then further cultured on Phytamax media fortified with different phytohormones either individually or in combinations. The frequency of protocorm like body (PLB) regeneration significantly relied on kinds and concentrations of plant growth regulators used. A combination of 1-naphthaleneacetic acid (NAA) (5.36 μM) and 6-benzyle amino purine (BAP) (3.80 μM) was found to be suitable for maximum PLB regeneration. Healthy plantlets were induced from PLBs when cultured on same basal medium supplemented with activated charcoal (AC – 3.0 g/l). Plantlets with well developed leaves and roots were transplanted to pots filled with a mixture of charcoal, brick pieces and sphagnum moss and transferred to the greenhouse. This protocol will enable mass propagation and conservation of this exquisite orchid.

Direct regeneration of protocorm-like bodies (PLBs) from leaf apices of Oncidium flexuosum Sims (Orchidaceae)

The present study describes the direct regeneration of protocorm-like bodies (PLBs) in leaf explants of the tropical species Oncidium flexuosum. The explants were inoculated in a solid, modified Murashige and Skoog (MS) medium with different concentrations of the growth regulator thidiazuron (TDZ) and with or without 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA), and kept away from light or in a 16-h photoperiod. The presence of auxins, 2,4-D, and NAA inhibited the formation of PLBs. The highest frequency of explants that regenerated PLBs (80%) was obtained when they were maintained in a culture medium containing 1.5 μM TDZ under dark conditions. In the same culture medium but under a 16-h photoperiod, 95% of the leaf explants presented necrosis. Therefore, darkness was crucial for the regeneration of PLBs in O. flexuosum leaf explants, which is in disagreement with the literature. PLBs developed from the division of epidermal and subepidermal cells mainly on the adaxial side of the apex region of the explant. Plants with well-developed leaves and roots grew after the PLBs were transferred to growth regulator-free medium under a 16-h photoperiod.