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.

Histone demethylases in neurodevelopment and neurodegenerative diseases.

Neurodevelopment can be precisely regulated by epigenetic mechanisms, including DNA methylations, noncoding RNAs, and histone modifications. Histone methylation was a reversible modification, catalyzed by histone methyltransferases and demethylases. So far, dozens of histone lysine demethylases (KDMs) have been discovered, and they (members from KDM1 to KDM7 family) are important for neurodevelopment by regulating cellular processes, such as chromatin structure and gene transcription. The role of KDM5C and KDM7B in neural development is particularly important, and mutations in both genes are frequently found in human X-linked mental retardation (XLMR). Functional disorders of specific KDMs, such as KDM1A can lead to the development of neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). Several KDMs can serve as potential therapeutic targets in the treatment of neurodegenerative diseases. At present, the function of KDMs in neurodegenerative diseases is not fully understood, so more comprehensive and profound studies are needed. Here, the role and mechanism of histone demethylases were summarized in neurodevelopment, and the potential of them was introduced in the treatment of neurodegenerative diseases.

Exogenous GA3 promotes flowering in Paphiopedilum callosum (Orchidaceae) through bolting and lateral flower development regulation.

Paphiopedilum orchids have a high ornamental value, and their flower abundance and timing are both key horticultural traits regulated by phytohormones. All one-flowered Paphiopedilum have additional lateral buds in the apical bract that fail to develop. In this study, an exogenous gibberellin (GA3) application promoted flowering of Pathiopedilum callosum by inducing its early bolting instead of the floral transition of dominant flowers. Applying GA3 effectively promoted lateral flower differentiation, resulting in a two-flowered inflorescence. GA-promoted lateral flower formation involved GA interacting with indole-3-acetic acid (IAA) and cytokinins (CTKs), given the decreased CTK content and downregulated expression of CTK synthesis genes, the increased IAA content and downregulated expression of IAA degradation, and the upregulated expression of transport genes. Further, GA acted via PcDELLA, PcTCP15, and PcXTH9 expressed in stage 5 to promote bolting, and via expression of PcAP3, PcPI, and PcSEP to promote flowering. This study provides insight into mechanisms regulating flower development of P. callosum.

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.

Shoot organogenesis and somatic embryogenesis from leaf and root explants of Scaevola sericea.

An efficient regeneration system via shoot organogenesis and somatic embryogenesis from in vitro leaf and root explants was established for Scaevola sericea for the first time. The highest axillary shoot proliferation coefficient (4.8) was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 6-benzyladenine (BA) and 0.1 mg/L α-naphthaleneacetic acid (NAA) every 45 days. Young in vitro leaves and roots, which were used as explants, were cultured onto medium supplemented with different plant growth regulators. Our results showed that only cytokinins BA and thidiazuron (TDZ), could induce adventitious shoots and somatic embryos from leaf and root explants. The optimal medium to achieve this was MS medium supplemented with 2.5 mg/L BA and which induced most adventitious shoots (2.7) and somatic embryos (17.3) from leaf explants within 30 days. From root explants, 1.1 adventitious shoots and 7.6 somatic embryos could be induced on MS medium supplemented with 2.5 mg/L TDZ. Histological observation showed that both somatic embryos and adventitious shoots were originated from homogeneous parenchyma and the development of somatic embryos was visible. Maximum rooting percentage (99.0%) was achieved on half-strength MS medium supplemented with 2.5 mg/L NAA. Well-rooted plantlets, which were transplanted into a substrate of pure river sand, displayed a high survival percentage of 91.7% after transplanting for 45 days while the best substrate for plantlet growth was river sand: coral sand (1:1).

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.

Selection and Validation of Novel RT-qPCR Reference Genes under Hormonal Stimuli and in Different Tissues of Santalum album.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used technique to investigate gene expression levels due to its high throughput, specificity, and sensitivity. An appropriate reference gene is essential for RT-qPCR analysis to obtain accurate and reliable results. To date, no reliable reference gene has been validated for the economically tropical tree, sandalwood (Santalum album L.). In this study, 13 candidate reference genes, including 12 novel putative reference genes selected from a large set of S. album transcriptome data, as well as the currently used ß-actin gene (ACT), were validated in different tissues (stem, leaf, root and callus), as well as callus tissue under salicylic acid (SA), jasmonic acid methyl ester (MeJA), and gibberellin (GA) treatments using geNorm, NormFinder, BestKeeper, Delta Ct and comprehensive RefFinder algorithms. Several novel candidate reference genes were much more stable than the currently used traditional gene ACT. ODD paired with Fbp1 for SA treatment, CSA and Fbp3 for MeJA treatment, PP2C and Fbp2 for GA treatment, as well as Fbp1 combined with Fbp2 for the total of three hormone treatments were the most accurate reference genes, respectively. FAB1A, when combined with PP2C, was identified as the most suitable reference gene combination for the four tissues tested, while the combination of HLMt, PPR and FAB1A were the most optimal reference genes for all of the experimental samples. In addition, to verify our results, the relative expression level of the SaSSy gene was evaluated by the validated reference genes and their combinations in the three S. album tissues and under MeJA treatment. The evaluated reference genes in this study will improve the accuracy of RT-qPCR analysis and will benefit S. album functional genomics studies in different tissues and under hormone stimuli in the future.