Comparative chloroplast genomics of 24 species shed light on the genome evolution and phylogeny of subtribe Coelogyninae (Orchidaceae).

BACKGROUND: The orchids of the subtribe Coelogyninae are among the most morphologically diverse and economically important groups within the subfamily Epidendroideae. Previous molecular studies have revealed that Coelogyninae is an unambiguously monophyletic group. However, intergeneric and infrageneric relationships within Coelogyninae are largely unresolved. There has been long controversy over the classification among the genera within the subtribe. RESULTS: The complete chloroplast (cp.) genomes of 15 species in the subtribe Coelogyninae were newly sequenced and assembled. Together with nine available cp. genomes in GenBank from representative clades of the subtribe, we compared and elucidated the characteristics of 24 Coelogyninae cp. genomes. The results showed that all cp. genomes shared highly conserved structure and contained 135 genes arranged in the same order, including 89 protein-coding genes, 38 tRNAs, and eight rRNAs. Nevertheless, structural variations in relation to particular genes at the IR/SC boundary regions were identified. The diversification pattern of the cp. genomes showed high consistency with the phylogenetic placement of Coelogyninae. The number of different types of SSRs and long repeats exhibited significant differences in the 24 Coelogyninae cp. genomes, wherein mononucleotide repeats (A/T), and palindromic repeats were the most abundant. Four mutation hotspot regions (ycf1a, ndhF-rp132, psaC-ndhE, and rp132-trnL) were determined, which could serve as effective molecular markers. Selection pressure analysis revealed that three genes (ycf1a, rpoC2 and ycf2 genes) might have experienced apparent positive selection during the evolution. Using the alignments of whole cp. genomes and protein-coding sequences, this study presents a well-resolved phylogenetic framework of Coelogyninae. CONCLUSION: The inclusion of 55 plastid genome data from a nearly complete generic-level sampling provide a comprehensive view of the phylogenetic relationships among genera and species in subtribe Coelogyninae and illustrate the diverse genetic variation patterns of plastid genomes in this species-rich plant group. The inferred relationships and informally recognized major clades within the subtribe are presented. The genetic markers identified here will facilitate future studies on the genetics and phylogeny of subtribe Coelogyninae.

Rational Design of Activatable Lanthanide NIR-IIb Emissive Nanoprobe for In Situ Specific Imaging of HOCl In Vivo.

Hypochlorous acid (HOCl), as an indispensable signaling molecule in organisms, is one of the key members of reactive oxygen species (ROS). However, in vivo, real-time dynamic near-infrared fluorescence imaging of HOCl levels in the 1400-1700 nm sub-window (NIR-IIb) remains a major challenge due to the lack of suitable detection methods. Herein, a general design of HOCl-responsive NIR-IIb fluorescence nanoprobe is proposed by integrating NaLuF4Yb/Er@NaLuF4 downshift nanoparticles (DSNPs) and HOCl recognition/NIR-IIb emissive modulation unit of M2-xS (M = Cu, Co, Pb) nanodots for real-time monitoring of HOCl levels. The fluorescence modulation unit of M2-xS nanodots presents remarkably enhanced absorption than Yb sensitizer at 980 nm and greatly inhibits the NIR-IIb fluorescence emission via competitive absorption mechanism. While, the M2-xS nanodots are easily degraded after triggering by HOCl, resulting in HOCl responsive turn-on (≈ten folds) NIR-IIb emission at 1532 nm. More importantly, in vivo highly precise and specific monitoring of inflammatory with abnormal HOCl expression is successfully achieved. Thus, the explored competitive absorption mediated quenching-activation mechanism provides a new general strategy of designing HOCl-responsive NIR-IIb fluorescence nanoprobe for highly specific and sensitive HOCl detection.

An Activated Structure Transformable Ratiometric Photoacoustic Nanoprobe for Real-Time Dynamic Monitoring of H2S In Vivo.

H2S has emerged as a promising biomarker for many diseases such as colon cancer and metformin-induced hepatotoxicity. Real-time monitoring of H2S levels in vivo is significant for early accurate diagnosis of these diseases. Herein, a new accurate and reliable nanoprobe (Au NRs@Ag) was designed for real-time dynamic ratiometric photoacoustic (PA) imaging of H2S in vivo based on the endogenous H2S-triggered local surface plasmon resonance (LSPR) red-shift. The Au NRs@Ag nanoprobe can be readily converted into Au NRs@Ag2S via the endogenous H2S-activated in situ sulfurative reaction, subsequently leading to a significant red-shift of the LSPR wavelength from 808 to 980 nm and enabling accurate ratiometric PA (PA980/PA808) imaging of H2S. Moreover, dynamic ratiometric PA imaging of metformin-induced hepatotoxicity was also successfully achieved by the designed PA imaging strategy. These findings provide the possibility of designing a new ratiometric PA imaging strategy for dynamic in situ monitoring of H2S-related diseases.

Transcriptomic analysis reveals biosynthesis genes and transcription factors related to leaf anthocyanin biosynthesis in Aglaonema commutatum.

BACKGROUND: Aglaonema commutatum 'Red Valentine', as a foliage ornamental plant, is widely used for interior and exterior decoration because of its easy cultivation and management. However, reduced proportion of red foliage during large-scale production of A. commutatum seedlings is a frequent occurrence, which has considerable implications on the plant's ornamental and market value. However, the molecular mechanisms underlying this phenomenon remain unclear. RESULTS: To explore the molecular basis of the variation in leaf color of A. commutatum Red Valentine, we performed transcriptome sequencing with the Illumina platform using two different varieties of A. commutatum, namely Red Valentine and a green mutant, at three different stages of leaf development. We annotated 63,621 unigenes and 14,186 differentially expressed genes by pairwise comparison. Furthermore, we identified 26 anthocyanin biosynthesis structural genes. The transcript per million (TPM) values were significantly higher for Red Valentine than for the green mutant in all three developmental stages, consistent with the high anthocyanin content of Red Valentine leaves. We detected positive transcription factors that may be involved in the regulation of anthocyanin biosynthesis using BLAST and through correlation analysis. Downregulation of these transcription factors may downregulate the expression of anthocyanin genes. We obtained full-length cDNA of the anthocyanin biosynthesis and regulatory genes and constructed phylogenetic trees to ensure accuracy of the analysis. CONCLUSIONS: Our study provides insights into the molecular mechanisms underlying leaf variation in A. commutatum Red Valentine and may be used to facilitate the breeding of ornamental cultivars with high anthocyanin levels.

Identification of HpMYB1 inducing anthocyanin accumulation in Hippeastrum Hybridum tepals by RNA-seq.

BACKGROUND: Cultivated Hippeastrum × hybridum is a popular ornamental plant with large and colorful flowers, long flowering duration, and high commercial value. As its main ornamental feature, its flower color is related to the anthocyanin content in the tepals. However, the molecular regulatory mechanisms of anthocyanin biosynthesis in H. × hybridum have not yet been elucidated. RESULTS: In the present study, 12 cDNA libraries of four stages of H.× hybridum 'Royal Velvet' tepal development were used for RNA-seq, obtaining 79.83 gigabases (GB) of clean data. The data were assembled into 148,453 unigenes, and 11,262 differentially expressed genes were identified. Forty key enzymes participating in anthocyanin biosynthesis were investigated, and the results showed that most of the anthocyanin structural genes were expressed at low levels in S1 and were markedly upregulated in S2 and S3. The expression profiles of 12 selected genes were verified by qRT-PCR. Furthermore, the R2R3-MYB transcription factor (TF), HpMYB1, involved in the regulation of anthocyanin biosynthesis was identified by sequence, expression pattern, and subcellular localization analyses. Its overexpression in tobacco significantly increased the anthocyanin levels in various tissues and activated anthocyanin-related genes. CONCLUSIONS: Using RNA-seq technology, we successfully identified a potential R2R3-MYB gene, HpMYB1, that regulates anthocyanin biosynthesis in H.× hybridum 'Royal Velvet'. Our findings provide basic transcript information and valuable transcriptome data for further identification of key genes involved in anthocyanin biosynthesis and can be applied in the artificial breeding of new H. × hybridum cultivars with enhanced ornamental value.

Comparative analyses and phylogenetic relationships of thirteen Pholidota species (Orchidaceae) inferred from complete chloroplast genomes.

BACKGROUND: The orchid genus Pholidota Lindl. ex Hook. is economically important as some species has long been used in traditional medicine. However, the systematic status of the genus and intergeneric relationships inferred from previous molecular studies are unclear due to insufficient sampling and lack of informative sites. So far, only limited genomic information has been available. The taxonomy of Pholidota remains unresolved and somewhat controversial. In this study, the complete chloroplast (cp.) genomes of thirteen Pholidota species were sequenced and analyzed to gain insight into the phylogeny of Pholidota and mutation patterns in their cp. genomes. RESULTS: All examined thirteen Pholidota cp. genomes exhibited typical quadripartite circular structures, with the size ranging from 158,786 to 159,781 bp. The annotation contained a total of 135 genes in each cp. genome, i.e., 89 protein-coding genes, 38 tRNA genes, and eight rRNA genes. The codon usage analysis indicated the preference of A/U-ending codons. Repeat sequence analysis identified 444 tandem repeats, 322 palindromic repeats and 189 dispersed repeats. A total of 525 SSRs, 13,834 SNPs and 8,630 InDels were detected. Six mutational hotspots were identified as potential molecular markers. These molecular markers and highly variable regions are expected to facilitate future genetic and genomic studies. Our phylogenetic analyses confirmed the polyphyletic status of the genus Pholidota, with species grouped into four main clades: Pholidota s.s. was resolved as the sister to a clade containing species of Coelogyne; the other two clades clustered together with species of Bulleyia and Panisea, respectively; species P. ventricosa was placed at the basal position, deviated from all other species. CONCLUSION: This is the first study to comprehensively examine the genetic variations and systematically analyze the phylogeny and evolution of Pholidota based on plastid genomic data. These findings contribute to a better understanding of plastid genome evolution of Pholidota and provide new insights into the phylogeny of Pholidota and its closely related genera within the subtribe Coelogyninae. Our research has laid the foundation for future studies on the evolutionary mechanisms and classification of this economically and medicinally important genus.

In Vivo High-Resolution Bioimaging of Bone Marrow and Fracture Diagnosis Using Lanthanide Nanoprobes with 1525 nm Emission.

Bones play vital roles in human health. Noninvasive visualization of the full extent of bones is highly demanded to evaluate many bone-related diseases. Herein, we report poly (acrylic acid) (PAA)-modified NaLuF4:Yb/Er/Gd/Ce@NaYF4 nanoparticles (PAA-Er) with second near-infrared emission beyond 1500 nm (also referred as NIR-IIb) for high-resolution bone/bone marrow imaging and bone fracture diagnosis. The NIR-IIb optical-guided bone marrow imaging presents a high signal to noise ratio, which is superior to that for imaging in the NIR-II window (1000-1400 nm, NIR-IIa). Importantly, we also investigated the size-dependent accumulation of the nanoparticles and the possible accumulation mechanism of the designed PAA-Er nanoprobes in bone marrow. Due to the high affinity capability of the PAA-Er nanoprobes, a highly sensitive NIR-IIb optical-guided bone fracture diagnosis was successfully achieved. This novel technology paves the way to design lanthanide nanoprobes for NIR-IIb optical-guided high-resolution bone marrow imaging and bone-related disease diagnosis.

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

BACKGROUND: Paphiopedilum, 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. RESULTS: A 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. CONCLUSION: This study is the first report on PLB development in Paphiopedilum using transcriptome sequencing, which provides useful information to understand the mechanisms of PLB development.

A H2S-Triggered Dual-Modal Second Near-Infrared/Photoacoustic Intelligent Nanoprobe for Highly Specific Imaging of Colorectal Cancer.

An endogenous H2S-triggered intelligent optical nanoprobe combining second near-infrared (NIR-II) fluorescence with photoacoustic (PA) imaging can provide more comprehensive information to further improve the sensitivity and reliability of diagnosis for colorectal tumor, which is rarely explored. Herein, an endogenous H2S-triggered SiO2@Ag nanoprobe was designed for in situ dual-modal NIR-II/PA imaging of colorectal cancer. The designed dual-modal nanoprobe can be converted to SiO2@Ag2S after in situ biosynthesis via a sulfuration reaction with the over-expressed endogenous H2S in the colorectal tumor. More importantly, the designed SiO2@Ag nanoprobe exhibits high sensitivity and specificity for diagnosing colorectal cancer in vivo via dual-modal NIR-II/PA imaging. These results provide a new NIR-II/PA dual-modal imaging strategy for noninvasive intelligent detection of colorectal cancer.

Transcriptome analysis provides insights into the non-methylated lignin synthesis in Paphiopedilum armeniacum seed.

BACKGROUNDS: Paphiopedilum is an important genus of the orchid family Orchidaceae and has high horticultural value. The wild populations are under threat of extinction because of overcollection and habitat destruction. Mature seeds of most Paphiopedilum species are difficult to germinate, which severely restricts their germplasm conservation and commercial production. The factors inhibiting germination are largely unknown. RESULTS: In this study, large amounts of non-methylated lignin accumulated during seed maturation of Paphiopedilum armeniacum (P. armeniacum), which negatively correlates with the germination rate. The transcriptome profiles of P. armeniacum seed at different development stages were compared to explore the molecular clues for non-methylated lignin synthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that a large number of genes associated with phenylpropanoid biosynthesis and phenylalanine metabolism during seed maturation were differentially expressed. Several key genes in the lignin biosynthetic pathway displayed different expression patterns during the lignification process. PAL, 4CL, HCT, and CSE upregulation was associated with C and H lignin accumulation. The expression of CCoAOMT, F5H, and COMT were maintained at a low level or down-regulated to inhibit the conversion to the typical G and S lignin. Quantitative real-time RT-PCR analysis confirmed the altered expression levels of these genes in seeds and vegetative tissues. CONCLUSIONS: This work demonstrated the plasticity of natural lignin polymer assembly in seed and provided a better understanding of the molecular mechanism of seed-specific lignification process.

NaCeF4:Gd,Tb Scintillator as an X-ray Responsive Photosensitizer for Multimodal Imaging-Guided Synchronous Radio/Radiodynamic Therapy.

Photosensitizers (PSs) that are directly responsive to X-ray for radiodynamic therapy (RDT) with desirable imaging abilities have great potential applications in cancer therapy. Herein, the cerium (Ce)-doped NaCeF4:Gd,Tb scintillating nanoparticle (ScNP or scintillator) is first reported. Due to the sensitization effect of the Ce ions, Tb ions can emit fluorescence under X-ray irradiation to trigger X-ray excited fluorescence (XEF). Moreover, Ce and Tb ions can absorb the energy of secondary electrons generated by X-ray to produce reactive oxide species (ROS) for RDT. With the intrinsic absorption of X-ray by lanthanide elements, the NaCeF4:Gd,Tb ScNPs also act as a computed tomography (CT) imaging contrast agent and radiosensitizers for radiotherapy (RT) sensitization synchronously. Most importantly, the transverse relaxation time of Gd3+ ions is shortened due to the doping of Ce and Tb ions, leading to the excellent performance of our ScNPs in T2-weighted MR imaging for the first time. Both in vitro and in vivo studies verify that our synthesized ScNPs have good performance in XEF, CT, and T2-weighted MR imaging, and a synchronous RT/RDT is achieved with significant suppression on tumor progression under X-ray irradiation. Importantly, no systemic toxicity is observed after intravenous injection of ScNPs. Our work highlights that ScNPs have potential in multimodal imaging-guided RT/RDT of deep tumors.

Abscisic Acid Inhibits Asymbiotic Germination of Immature Seeds of Paphiopedilum armeniacum.

Paphiopedilum armeniacum is a rare orchid native to China with high ornamental value. The germination of P. armeniacum seeds is difficult, especially for the mature seeds, which is the major limitation for their large-scale reproduction. This study explored the reasons for seed germination inhibition from the aspects of the important plant endogenous hormone-abscisic acid (ABA). The major endogenous hormone contents of seeds were determined at different developmental stages. The ABA content was 5.8 ng/g in 73 days after pollination (DAP) for the immature seeds, peaked at 14.6 ng/g in 129 DAP seeds, and dropped to 2.6 ng/g in the late mature stage of the 150 DAP seeds. The reduction of ABA content in the mature seed suggests a possible contribution to the increased expression of CYP707A, an ABA catabolism gene. The germination rate of the immature seeds was reduced to 9% from 69% when 5 µg/mL ABA was added to the Hyponex N026 germination medium. The result showed that ABA can inhibit the germination of P. armeniacum immature seeds. However, for the heavily lignified mature seeds, reduction in endogenous ABA level does not result in an increase in the germination rate. Lignin accumulation in the seed coat imposes the physical dormancy for P. armeniacum. In summary, the germination of P. armeniacum is regulated by both ABA and lignin accumulation.

Ovule Development and in Planta Transformation of Paphiopedilum Maudiae by Agrobacterium-Mediated Ovary-Injection.

In this paper, the development of the Paphiopedilum Maudiae embryo sac at different developmental stages after pollination was assessed by confocal laser scanning microscopy. The mature seeds of P. Maudiae consisted of an exopleura and a spherical embryo, but without an endosperm, while the inner integument cells were absorbed by the developing embryo. The P. Maudiae embryo sac exhibited an Allium type of development. The time taken for the embryo to develop to a mature sac was 45-50 days after pollination (DAP) and most mature embryo sacs had completed fertilization and formed zygotes by about 50-54 DAP. In planta transformation was achieved by injection of the ovaries by Agrobacterium, resulting in 38 protocorms or seedlings after several rounds of hygromycin selection, corresponding to 2, 7, 5, 1, 3, 4, 9, and 7 plantlets from Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, respectively. Transformation efficiency was highest at 50 DAP (2.54%), followed by 2.48% at 53 DAP and 2.45% at 48 DAP. Four randomly selected hygromycin-resistant plants were GUS-positive after PCR analysis. Semi-quantitative PCR and quantitative real-time PCR analysis revealed the expression of the hpt gene in the leaves of eight hygromycin-resistant seedlings following Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, while hpt expression was not detected in the control. The best time to inject P. Maudiae ovaries in planta with Agrobacterium is 48-53 DAP, which corresponds to the period of fertilization. This protocol represents the first genetic transformation protocol for any Paphiopedilum species and will allow for expanded molecular breeding programs to introduce useful and interesting genes that can expand its ornamental and horticulturally important characteristics.

RNA-Seq analysis of Clerodendrum inerme (L.) roots in response to salt stress.

BACKGROUND: Clerodendrum inerme (L.) Gaertn, a halophyte, usually grows on coastal beaches as an important mangrove plant. The salt-tolerant mechanisms and related genes of this species that respond to short-term salinity stress are unknown for us. The de novo transcriptome of C. inerme roots was analyzed using next-generation sequencing technology to identify genes involved in salt tolerance and to better understand the response mechanisms of C. inerme to salt stress. RESULTS: Illumina RNA-sequencing was performed on root samples treated with 400 mM NaCl for 0 h, 6 h, 24 h, and 72 h to investigate changes in C. inerme in response to salt stress. The de novo assembly identified 98,968 unigenes. Among these unigenes, 46,085 unigenes were annotated in the NCBI non-redundant protein sequences (NR) database, 34,756 sequences in the Swiss-Prot database and 43,113 unigenes in the evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) database. 52 Gene Ontology (GO) terms and 31 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were matched to those unigenes. Most differentially expressed genes (DEGs) related to the GO terms "single-organism process", "membrane" and "catalytic activity" were significantly enriched while numerous DEGs related to the plant hormone signal transduction pathway were also significantly enriched. The detection of relative expression levels of 9 candidate DEGs by qRT-PCR were basically consistent with fold changes in RNA sequencing analysis, demonstrating that transcriptome data can accurately reflect the response of C. inerme roots to salt stress. CONCLUSIONS: This work revealed that the response of C. inerme roots to saline condition included significant alteration in response of the genes related to plant hormone signaling. Besides, our findings provide numerous salt-tolerant genes for further research to improve the salt tolerance of functional plants and will enhance research on salt-tolerant mechanisms of halophytes.

Identification and functional characterization of three new terpene synthase genes involved in chemical defense and abiotic stresses in Santalum album.

BACKGROUND: It is well known that aromatic essential oils extracted from the heartwood of Santalum album L. have wide economic value. However, little is known about the role of terpenoids in response to various adverse environmental stresses as other plants do in the form of signals during plant-environment interactions. RESULTS: In this study, trace amounts of volatiles consisting of α-santalene, epi-ß-santalene, ß-santalene, α-santalol, ß-santalol, (E)-α-bergamotene, (E)-ß-farnesene and ß-bisabolene were found in the leaves of mature S. album trees. We identified more than 40 candidate terpene synthase (TPS) unigenes by mining publicly-available RNA-seq data and characterized the enzymes encoded by three cDNAs: one mono-TPS catalyzes the formation of mostly α-terpineol, and two multifunctional sesqui-TPSs, one of which produces (E)-α-bergamotene and sesquisabinene as major products and another which catalyzes the formation of (E)-ß-farnesene, (E)-nerolidol and (E,E)-farnesol as main products. Metabolite signatures and gene expression studies confirmed that santalol content is closely related with santalene synthase (SaSSY) transcripts in heartwood, which is key enzyme responsible for santalol biosynthesis. However, the expression of three new SaTPS genes differed significantly from SaSSY in the essential oil-producing heartwood. Increased activities of antioxidant enzymes, superoxide dismutase, catalase, peroxidase and ascorbate peroxidase, were detected in different tissues of S. album plants after applying 1 mM methyl jasmonate (MeJA) and 1 mM salicylic acid (SA), or exposure to 4°C, 38°C and high light intensity. MeJA and SA dramatically induced the expression of SaTPS1 and SaTPS2 in leaves. SaTPS1 to 3 transcripts were differentially activated among different tissues under adverse temperature and light stresses. In contrast, almost all SaSSY transcripts decreased in response to these environmental stresses, unlike SaTPS1 to 3. CONCLUSIONS: Multifunctional enzymes were biochemically characterized, including one chloroplastic mono-TPS and two cytosolic sesqui-TPSs in sandalwood. Our results suggest the ecological importance of these three new SaTPS genes in defensive response to biotic attack and abiotic stresses in S. album.

Advances in Dendrobium molecular research: Applications in genetic variation, identification and breeding.

Orchids of the genus Dendrobium are of great economic importance in global horticultural trade and in Asian traditional medicine. For both areas, research yielding solid information on taxonomy, phylogeny, and breeding of this genus are essential. Traditional morphological and cytological characterization are used in combination with molecular results in classification and identification. Markers may be useful when used alone but are not always reliable in identification. The number of species studied and identified by molecular markers is small at present. Conventional breeding methods are time-consuming and laborious. In the past two decades, promising advances have been made in taxonomy, phylogeny and breeding of Dendrobium species due to the intensive use of molecular markers. In this review, we focus on the main molecular techniques used in 121 published studies and discuss their importance and possibilities in speeding up the breeding of new cultivars and hybrids.

In vitro propagation of Paphiopedilum orchids.

Paphiopedilum is one of the most popular and rare orchid genera. Members of the genus are sold and exhibited as pot plants and cut flowers. Wild populations of Paphiopedilum are under the threat of extinction due to over-collection and loss of suitable habitats. A reduction in their commercial value through large-scale propagation in vitro is an option to reduce pressure from illegal collection, to attempt to meet commercial needs and to re-establish threatened species back into the wild. Although they are commercially propagated via asymbiotic seed germination, Paphiopedilum are considered to be difficult to propagate in vitro, especially by plant regeneration from tissue culture. This review aims to cover the most important aspects and to provide an up-to-date research progress on in vitro propagation of Paphiopedilum and to emphasize the importance of further improving tissue culture protocols for ex vitro-derived explants.

Methods for genetic transformation in Dendrobium.

KEY MESSAGE: The genetic transformation of Dendrobium orchids will allow for the introduction of novel colours, altered architecture and valuable traits such as abiotic and biotic stress tolerance. The orchid genus Dendrobium contains species that have both ornamental value and medicinal importance. There is thus interest in producing cultivars that have increased resistance to pests, novel horticultural characteristics such as novel flower colours, improved productivity, longer flower spikes, or longer post-harvest shelf-life. Tissue culture is used to establish clonal plants while in vitro flowering allows for the production of flowers or floral parts within a sterile environment, expanding the selection of explants that can be used for tissue culture or genetic transformation. The latter is potentially the most effective, rapid and practical way to introduce new agronomic traits into Dendrobium. Most (69.4 %) Dendrobium genetic transformation studies have used particle bombardment (biolistics) while 64 % have employed some form of Agrobacterium-mediated transformation. A singe study has explored ovary injection, but no studies exist on floral dip transformation. While most of these studies have involved the use of selector or reporter genes, there are now a handful of studies that have introduced genes for horticulturally important traits.

Identification of genes involved in biosynthesis of mannan polysaccharides in Dendrobium officinale by RNA-seq analysis.

Dendrobium officinale is a traditional Chinese medicinal plant. The stems of D. officinale contain mannan polysaccharides, which are promising bioactive polysaccharides for use as drugs. However, the genes involved in the biosynthesis of mannan polysaccharides in D. officinale have not yet been identified. In this study, four digital gene expression profiling analyses were performed on developing stems of greenhouse-grown D. officinale to identify such genes. Based on the accumulation of mannose and on gene expression levels, eight CELLULOSE SYNTHASE-LIKE A genes (CSLA), which are highly likely to be related to the biosynthesis of bioactive mannan polysaccharides, were identified from the differentially expressed genes database. In order to further analyze these DoCSLA genes, a full-length cDNA of each was obtained by RACE. The eight genes, belonging to the CSLA family of the CesA superfamily, contain conserved domains of the CesA superfamily. Most of the genes, which were highly expressed in the stems of D. officinale, were related to abiotic stress. Our results suggest that the CSLA family genes from D. officinale are involved in the biosynthesis of bioactive mannan polysaccharides.

Symbiotic in vitro seed propagation of Dendrobium: fungal and bacterial partners and their influence on plant growth and development.

The genus Dendrobium is one of the largest genera of the Orchidaceae Juss. family, although some of its members are the most threatened today. The reason why many species face a vulnerable or endangered status is primarily because of anthropogenic interference in natural habitats and commercial overexploitation. The development and application of modern techniques and strategies directed towards in vitro propagation of orchids not only increases their number but also provides a viable means to conserve plants in an artificial environment, both in vitro and ex vitro, thus providing material for reintroduction. Dendrobium seed germination and propagation are challenging processes in vivo and in vitro, especially when the extreme specialization of these plants is considered: (1) their biotic relationships with pollinators and mycorrhizae; (2) adaptation to epiphytic or lithophytic life-styles; (3) fine-scale requirements for an optimal combination of nutrients, light, temperature, and pH. This review also aims to summarize the available data on symbiotic in vitro Dendrobium seed germination. The influence of abiotic factors as well as composition and amounts of different exogenous nutrient substances is examined. With a view to better understanding how to optimize and control in vitro symbiotic associations, a part of the review describes the strong biotic relations of Dendrobium with different associative microorganisms that form microbial communities with adult plants, and also influence symbiotic seed germination. The beneficial role of plant growth-promoting bacteria is also discussed.