PACLOBUTRAZOL-RESISTANCE4 positively regulates cell expansion to promote tendril elongation in cucumber.

As a climbing organ, the tendril undergoes rapid elongation to increase its length to locate support within a short growth time. However, the molecular mechanism underlying this observation is poorly understood. Here, tendril development was divided into 4 stages in cucumber (Cucumis sativus L.) along with its growth. Phenotypic observations and section analyses showed that the rapid elongation of tendril primarily happened during stage 3 and was mainly due to cell expansion. RNA-seq analysis showed that PACLOBUTRAZOL-RESISTANCE4 (CsPRE4) was highly expressed in the tendril. Our RNAi studies in cucumber and transgenic overexpression in Arabidopsis (Arabidopsis thaliana) suggested that CsPRE4 functions as a conserved activator of cell expansion to promote cell expansion and tendril elongation. Through a triantagonistic HLH (helix-loop-helix)-HLH-bHLH (basic helix-loop-helix) cascade, CsPRE4-CsPAR1 (PHYTOCHROME RAPIDLY REGULATED1)-CsBEE1 (BR-ENHANCED EXPRESSION 1), CsPRE4 released the transcription factor CsBEE1, which activated expansin A12 (CsEXPA12) to loosen the cell wall structure in tendrils. Gibberellin (GA) promoted tendril elongation by modulating cell expansion, and CsPRE4 expression was induced by exogenous GA treatment, suggesting that CsPRE4 acts downstream of GA in regulating tendril elongation. In summary, our work suggested a CsPRE4-CsPAR1-CsBEE1-CsEXPA12 pathway in regulating cell expansion in cucumber tendrils, which might enable rapid tendril elongation to quickly locate support.

Cucumber NUCLEAR FACTOR-YC2/-YC9 target translocon component CsTIC21 in chloroplast photomorphogenesis.

Light signals promote photomorphogenesis and photosynthesis, allowing plants to establish photoautotrophic growth. Chloroplasts are organelles responsible for photosynthesis in which light energy is converted into chemical energy and stored as organic matter. However, how light regulates chloroplast photomorphogenesis remains unclear. Here, we isolated a cucumber (Cucumis sativus L.) mutant albino seedling (as) from an ethyl methane sulfonate mutagenesis library with an albino phenotype. Map-based cloning revealed that the mutation occurred in a component of cucumber translocon at the inner membrane of chloroplasts (CsTIC21). Subsequently, virus-induced gene silencing and CRISPR/Cas9 analyses confirmed the association between the mutant gene and the as phenotype. Loss-of-function of CsTIC21 induces malformation of chloroplast formation, leading to albinism and death in cucumber. Notably, CsTIC21 transcription was very low in etiolated seedlings grown in the dark and was upregulated by light, with expression patterns similar to those of Nuclear factor-YC (NF-YC) genes. Here, 7 cucumber NF-YC family genes (CsNF-YC) were identified, among which the expression of 4 genes (CsNF-YC1, -YC2, -YC9, and -YC13) responded to light. Gene silencing of all CsNF-YC genes in cucumber indicated that CsNF-YC2, -YC9, -YC11-1, and -YC11-2 induced distinct etiolated growth and decreased chlorophyll content. Interaction studies verified that CsNF-YC2 and CsNF-YC9 target the CsTIC21 promoter directly and promote gene transcription. These findings provide mechanistic insights on the role of the NF-YCs-TIC21 module in chloroplast photomorphogenesis promoted by light in cucumber.

Modified photoperiod response of CsFT promotes day neutrality and early flowering in cultivated cucumber.

KEY MESSAGE: Map-based cloning and photoperiod response detection suggested that CsFT is the critical gene for cucumber photoperiod domestication. Photoperiod sensitivity is important for sensing seasonal changes and local adaptation. However, day-length sensitivity limits crop geographical adaptation and it should be modified during domestication. Cucumber was domesticated in southern Asia and is currently cultivated worldwide across a wide range of latitudes, but its photoperiod sensitivity and its change during cucumber domestication are unknown. Here, we confirmed wild cucumber (Hardwickii) was a short-day plant, and its flowering depends on short-day (SD) conditions, while the cultivated cucumber (9930) is a day-neutral plant that flowers independently of day length. A photoperiod sensitivity locus (ps-1) was identified by the 9930 × Hardwickii F2 segregating populations, which span a ~ 970 kb region and contain 60 predicted genes. RNA-seq analysis showed that the critical photoperiod pathway gene FLOWERING LOCUS T (CsFT) within the ps-1 locus exhibits differential expression between 9930 and Hardwickii, which was confirmed by qRT-PCR detection. CsFT in Hardwickii was sensitive to day length and could be significantly induced by SD conditions, whereas CsFT was highly expressed in 9930 and was insensitive to day length. Moreover, the role of CsFT in promoting flowering was verified by overexpression of CsFT in Arabidopsis. We also identified the genetic variations existing in the promoter of CsFT among the different geographic cucumbers and suggest they have possible roles in photoperiod domestication. The results of this study suggest that a variation in photoperiod sensitivity of CsFT is associated with day neutrality and early flowering in cultivated cucumber and could contribute to cucumber cultivation in diverse regions throughout the world.

Characterization of lncRNAs and mRNAs Involved in Powdery Mildew Resistance in Cucumber.

Powdery mildew (PM) is a severe fungal disease of cucumber worldwide. Identification of genetic factors resistant to PM is of great importance for marker-assisted breeding to ensure cucumber production. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) have been shown to play important roles in plant development and immunity; however, whether they have a role in PM response in cucurbit crops remains unknown. We performed strand-specific RNA sequencing and miRNA sequencing using RNA from cucumber leaves of two near-isogenic lines (NILs), S1003 and NIL (Pm5.1) infected with PM, and systematically characterized the profiles of cucumber lncRNAs and messenger RNA (mRNAs) responsive to PM. In total, we identified 12,903 lncRNAs and 25,598 mRNAs responsive to PM. Differential expression (DE) analysis showed that 119 lncRNAs and 136 mRNAs correlated with PM resistance. Functional analysis of these DE lncRNAs and DE mRNAs revealed that they are significantly associated with phenylpropanoid biosynthesis, phenylalanine metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and endocytosis. Particularly, two lncRNAs, LNC_006805 and LNC_012667, might play important roles in PM resistance. In addition, we also predicted mature miRNAs and competing endogenous RNA (ceRNA) networks of lncRNA-miRNA-mRNA involved in PM resistance. A total of 49 DE lncRNAs could potentially act as target mimics for 106 miRNAs. Taken together, our results provide an abundant resource for further exploration of cucumber lncRNAs, mRNAs, miRNAs, and ceRNAs in PM resistance, and will facilitate the molecular breeding for PM-resistant varieties to control this severe disease in cucumber.

Repression of miR156 by miR159 Regulates the Timing of the Juvenile-to-Adult Transition in Arabidopsis.

Temporally regulated microRNAs have been identified as master regulators of developmental timing in both animals and plants. In plants, vegetative development is regulated by a temporal decrease in miR156 level, but how this decreased expression is initiated and then maintained during shoot development remains elusive. Here, we show that miR159 is required for the correct timing of vegetative development in Arabidopsis thaliana Loss of miR159 increases miR156 level throughout shoot development and delays vegetative development, whereas overexpression of miR159 slightly accelerated vegetative development. The repression of miR156 by miR159 is predominantly mediated by MYB33, an R2R3 MYB domain transcription factor targeted by miR159. Loss of MYB33 led to subtle precocious vegetative phase change phenotypes in spite of the significant downregulation of miR156. MYB33 simultaneously promotes the transcription of MIR156A and MIR156C, as well as their target, SPL9, by directly binding to the promoters of these three genes. Rather than acting as major players in vegetative phase change in Arabidopsis, our results suggest that miR159 and MYB33 function as modifiers of vegetative phase change; i.e., miR159 facilitates vegetative phase change by repressing MYB33 expression, thus preventing MYB33 from hyperactivating miR156 expression throughout shoot development to ensure correct timing of the juvenile-to-adult transition in Arabidopsis.

Recent progress on the molecular breeding of Cucumis sativus L. in China.

KEY MESSAGE: Molecular breeding of Cucumis sativus L. is based on traditional breeding techniques and modern biological breeding in China. There are opportunities for further breeding improvement by molecular design breeding and the automation of phenotyping technology using untapped sources of genetic diversity. Cucumber (Cucumis sativus L.) is an important vegetable cultivated worldwide. It bears fruits of light fragrance, and crisp texture with high nutrition. China is the largest producer and consumer of cucumber, accounting for 70% of the world's total production. With increasing consumption demand, the production of Cucurbitaceae crops has been increasing yearly. Thus, new cultivars that can produce high-quality cucumber with high yield and easy cultivation are in need. Conventional genetic breeding has played an essential role in cucumber cultivar innovation over the past decades. However, its progress is slow due to the long breeding period, and difficulty in selecting stable genetic characters or genotypes, prompting researchers to apply molecular biotechnologies in cucumber breeding. Here, we first summarize the achievements of conventional cucumber breeding such as crossing and mutagenesis, and then focus on the current status of molecular breeding of cucumber in China, including the progress and achievements on cucumber genomics, molecular mechanism underlying important agronomic traits, and also on the creation of high-quality multi-resistant germplasm resources, new variety breeding and ecological breeding. Future development trends and prospects of cucumber molecular breeding in China are also discussed.

Molecular imaging of biological systems with a clickable dye in the broad 800- to 1,700-nm near-infrared window.

Fluorescence imaging multiplicity of biological systems is an area of intense focus, currently limited to fluorescence channels in the visible and first near-infrared (NIR-I; ∼700-900 nm) spectral regions. The development of conjugatable fluorophores with longer wavelength emission is highly desired to afford more targeting channels, reduce background autofluorescence, and achieve deeper tissue imaging depths. We have developed NIR-II (1,000-1,700 nm) molecular imaging agents with a bright NIR-II fluorophore through high-efficiency click chemistry to specific molecular antibodies. Relying on buoyant density differences during density gradient ultracentrifugation separations, highly pure NIR-II fluorophore-antibody conjugates emitting ∼1,100 nm were obtained for use as molecular-specific NIR-II probes. This facilitated 3D staining of ∼170-µm histological brain tissues sections on a home-built confocal microscope, demonstrating multicolor molecular imaging across both the NIR-I and NIR-II windows (800-1,700 nm).

Genome-Wide Identification and Analysis of Polygalacturonase Genes in Solanum lycopersicum.

Polygalacturonase (PG), a large hydrolase family in plants, is involved in pectin disassembly of the cell wall in plants. The present study aims to characterize PG genes and investigate their expression patterns in Solanum lycopersicum. We identified 54 PG genes in the tomato genome and compared their amino acid sequences with their Arabidopsis counterpart. Subsequently, we renamed these PG genes according to their Arabidopsis homologs. Phylogenetic and evolutionary analysis revealed that these tomato PG genes could be classified into seven clades, and within each clade the exon/intron structures were conserved. Expression profiles analysis through quantitive real-time polymerase chain reaction (qRT-PCR) revealed that most SlPGs had specific or high expression patterns in at least one organ, and particularly five PG genes (SlPG14, SlPG15, SlPG49, SlPG70, and SlPG71) associated with fruit development. Promoter analysis showed that more than three cis-elements associated with plant hormone response, environmental stress response or specific organ/tissue development exhibited in each SlPG promoter regions. In conclusion, our results may provide new insights for the further study of PG gene function during plant development.

Regulation of Vegetative Phase Change by SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA.

Plants progress from a juvenile vegetative phase of development to an adult vegetative phase of development before they enter the reproductive phase. miR156 has been shown to be the master regulator of the juvenile-to-adult transition in plants. However, the mechanism of how miR156 is transcriptionally regulated still remains elusive. In a forward genetic screen, we identified that a mutation in the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) exhibited an accelerated vegetative phase change phenotype by reducing the expression of miR156, which in turn caused a corresponding increase in the levels of SQUAMOSA PROMOTER BINDING PROTEIN LIKE genes. BRM regulates miR156 expression by directly binding to the MIR156A promoter. Mutations in BRM not only increased occupancy of the -2 and +1 nucleosomes proximal to the transcription start site at the MIR156A locus but also the levels of trimethylated histone H3 at Lys 27. The precocious phenotype of brm mutant was partially suppressed by a second mutation in SWINGER (SWN), but not by a mutation in CURLEY LEAF, both of which are key components of the Polycomb Group Repressive Complex 2 in plants. Our results indicate that BRM and SWN act antagonistically at the nucleosome level to fine-tune the temporal expression of miR156 to regulate vegetative phase change in Arabidopsis.

Modulation of miR156 to identify traits associated with vegetative phase change in tobacco (Nicotiana tabacum).

After germination, plants progress through juvenile and adult phases of vegetative development before entering the reproductive phase. The character and timing of these phases vary significantly between different plant species, which makes it difficult to know whether temporal variations in various vegetative traits represent the same, or different, developmental processes. miR156 has been shown to be the master regulator of vegetative development in plants. Overexpression of miR156 prolongs the juvenile phase of development, whereas knocking-down the level of miR156 promotes the adult phase of development. Therefore, artificial modulation of miR156 expression is expected to cause corresponding changes in vegetative-specific traits in different plant species, particularly in those showing no substantial difference in morphology during vegetative development. To identify specific traits associated with the juvenile-to-adult transition in tobacco, we examined the phenotype of transgenic tobacco plants with elevated or reduced levels of miR156. We found that leaf shape, the density of abaxial trichomes, the number of leaf veins, the number of stomata, the size and density of epidermal cells, patterns of epidermal cell staining, the content of chlorophyll and the rate of photosynthesis, are all affected by miR156. These newly identified miR156-regulated traits therefore can be used to distinguish between juvenile and adult phases of development in tobacco, and provide a starting point for future studies of vegetative phase change in the family Solanaceae.

A tomato endoplasmic reticulum (ER)-type omega-3 fatty acid desaturase (LeFAD3) functions in early seedling tolerance to salinity stress.

KEY MESSAGE: Transgenic tomato plants overexpressing LeFAD3 sense and antisense sequences were generated. Salt stress suppressed the growth of WT and antisense plants to a higher extent than that in sense plants. In this study, we investigated the role of the LeFAD3-encoding ER-type omega-3 fatty acid desaturase in salt tolerance in tomato plants. We created transgenic tomato plants by overexpressing its sense and antisense sequences under the control of the cauliflower mosaic virus 35S promoter. Based on the results of northern and western blotting as well as quantitative reverse transcription-polymerase chain reaction, sense plants expressed more desaturase than wild-type (WT) plants, whereas antisense plants expressed less desaturase than WT. Salt stress suppressed the growth of both WT and antisense plants to a higher extent than that in sense plants, which can be attributed to the fact that sense plants performed better in maintaining the integrity of the membrane system, as revealed by electron microscopy. The concomitant increase in superoxide dismutase (EC 1.15.1.1) and ascorbate peroxidase (EC 1.11.1.7) may have alleviated the photoinhibition caused by the increased level of ROS in sense plants. Our results suggest that LeFAD3 overexpression can enhance the tolerance of early seedlings to salinity stress.

Research progress on the mechanisms of fruit glossiness in cucumber.

Cucumber (Cucumis sativus L.) is an important horticultural crop in China. Consumer requirements for aesthetically pleasing appearances of horticultural crops are gradually increasing, and cucumbers having a good visual appearance, as well as flavor, are important for breeding and industry development. The gloss of cucumber fruit epidermis is an important component of its appeal, and the wax layer on the fruit surface plays important roles in plant growth and forms a powerful barrier against external biotic and abiotic stresses. The wax of the cucumber epidermis is mainly composed of alkanes, and the luster of cucumber fruit is mainly determined by the alkane and silicon contents of the epidermis. Several genes, transcription factors, and transporters affect the synthesis of ultra-long-chain fatty acids and change the silicon content, further altering the gloss of the epidermis. However, the specific regulatory mechanisms are not clear. Here, progress in research on the luster of cucumber fruit epidermis from physiological, biochemical, and molecular regulatory perspectives are reviewed. Additionally, future research avenues in the field are discussed.

Advance in sex differentiation in cucumber.

Cucumber belongs to the family Cucurbitaceae (melon genus) and is an annual herbaceous vegetable crop. Cucumber is an important cash crop that is grown all over the world. From morphology to cytology, from canonical genetics to molecular biology, researchers have performed much research on sex differentiation and its regulatory mechanism in cucumber, mainly in terms of cucumber sex determination genes, environmental conditions, and the effects of plant hormones, revealing its genetic basis to improve the number of female flowers in cucumber, thus greatly improving the yield of cucumber. This paper reviews the research progress of sex differentiation in cucumber in recent years, mainly focusing on sex-determining genes, environmental conditions, and the influence of phytohormones in cucumber, and provides a theoretical basis and technical support for the realization of high and stable yield cultivation and molecular breeding of cucumber crop traits.

Genome-Wide Identification and Expression Analysis of ESPs and NSPs Involved in Glucosinolate Hydrolysis and Insect Attack Defense in Chinese Cabbage (Brassica rapa subsp. pekinensis).

Glucosinolates are secondary plant metabolites that are part of the plant's defense system against pathogens and pests and are activated via enzymatic degradation by thioglucoside glucohydrolases (myrosinases). Epithiospecifier proteins (ESPs) and nitrile-specifier proteins (NSPs) divert the myrosinase-catalyzed hydrolysis of a given glucosinolate to form epithionitrile and nitrile rather than isothiocyanate. However, the associated gene families have not been explored in Chinese cabbage. We identified three ESP and fifteen NSP genes randomly distributed on six chromosomes in Chinese cabbage. Based on a phylogenetic tree, the ESP and NSP gene family members were divided into four clades and had similar gene structure and motif composition of Brassica rapa epithiospecifier proteins (BrESPs) and B. rapa nitrile-specifier proteins (BrNSPs) in the same clade. We identified seven tandem duplicated events and eight pairs of segmentally duplicated genes. Synteny analysis showed that Chinese cabbage and Arabidopsis thaliana are closely related. We detected the proportion of various glucosinolate hydrolysates in Chinese cabbage and verified the function of BrESPs and BrNSPs in glucosinolate hydrolysis. Furthermore, we used quantitative RT-PCR to analyze the expression of BrESPs and BrNSPs and demonstrated that these genes responded to insect attack. Our findings provide novel insights into BrESPs and BrNSPs that can help further promote the regulation of glucosinolate hydrolysates by ESP and NSP to resist insect attack in Chinese cabbage.

Antisense-mediated depletion of GMPase gene expression in tobacco decreases plant tolerance to temperature stresses and alters plant development.

In our previous work [1] we investigated the role of tomato GDP-mannose pyrophosphorylase (EC 2.7.7.22) in plants by overexpressing its gene in tobacco leaves and showed its function in AsA metabolism and detoxification of reactive oxygen species under temperature stresses. In this study, we use the antisense technique to block the endogenous GMPase gene expression in tobacco in order to further investigate its function. Northern and western blot analysis confirmed that the expression of endogenous tobacco GMPase mRNA and protein was inhibited by this antisense expression. Consequently, the activity of GMPase and the content of AsA in the leaves of antisense transgenic plants were markedly decreased. This was also the case for the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1), APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves of the transgenic plants. On the contrary, the contents of H(2)O(2) and O(2) (-•) were increased. Meanwhile, the net photosynthetic rate (Pn) and the maximal photochemical efficiency of PSII (Fv/Fm) also declined in the leaves of antisense plants. Under high or low temperature stresses, the seed germination rate of the antisense transgenic plants was significantly decreased in comparison with that of the wild-type tobacco. Interestingly, the antisense plants had smaller leaves and an earlier onset of flowering. In conclusion, the depletion of GMPase decreased the content of AsA, resulting in the plants susceptible to the oxidative damage caused by temperature stresses and subjected to developmental alternations.

Isolation and expression of glucosinolate synthesis genes CYP83A1 and CYP83B1 in Pak Choi (Brassica rapa L. ssp. chinensis var. communis (N. Tsen & S.H. Lee) Hanelt).

CYP83A1 and CYP83B1 are two key synthesis genes in the glucosinolate biosynthesis pathway. CYP83A1 mainly metabolizes the aliphatic oximes to form aliphatic glucosinolate and CYP83B1 mostly catalyzes aromatic oximes to synthesis corresponding substrates for aromatic and indolic glucosinolates. In this study, two CYP83A1 genes named BcCYP83A1-1 (JQ289997), BcCYP83A1-2 (JQ289996) respectively and one CYP83B1 (BcCYP83B1, HM347235) gene were cloned from the leaves of pak choi (Brassica rapa L. ssp. chinensis var. communis (N. Tsen & S.H. Lee) Hanelt) "Hangzhou You Dong Er" cultivar. Their ORFs were 1506, 1509 and 1500 bp in length, encoding 501, 502 and 499 amino acids, respectively. The predicted amino acid sequences of CYP83A1-1, CYP83A1-2 and CYP83B1 shared high sequence identity of 87.65, 86.48 and 95.59% to the corresponding ones in Arabidopsis, and 98.80, 98.61 and 98.80% to the corresponding ones in Brassica pekinensis (Chinese cabbage), respectively. Quantitative real-time PCR analysis indicated that both CYP83A1 and CYP83B1 expressed in roots, leaves and petioles of pak choi, while the transcript abundances of CYP83A1 were higher in leaves than in petioles and roots, whereas CYP83B1 showed higher abundances in roots. The expression levels of glucosinolate biosynthetic genes were consistent with the glucosinolate profile accumulation in shoots of seven cultivars and three organs. The isolation and characterization of the glucosinolate synthesis genes in pak choi would promote the way for further development of agronomic traits via genetic engineering.

Research progress on the relationship between leaf senescence and quality, yield and stress resistance in horticultural plants.

Leaf senescence, the final stage of leaf development, is one of the adaptive mechanisms formed by plants over a long period of evolution. Leaf senescence is accompanied by various changes in cell structure, physiological metabolism, and gene expressions. This process is controlled by a variety of internal and external factors. Meanwhile, the genes and plant hormones involved in leaf aging affect the quality, yield and stress resistance in horticultural plants. Leaf senescence mediated by plant hormones affected plant quality at both pre-harvest and post-harvest stages. Exogenous plant growth regulators or plant hormone inhibitors has been applied to delay leaf senescence. Modification of related gene expression by over-expression or antisense inhibition could delay or accelerate leaf senescence, and thus influence quality. Environmental factors such as light, temperature and water status also trigger or delay leaf senescence. Delaying leaf senescence could increase chloroplast lifespan and photosynthesis and thus improve source strength, leading to enhanced yield. Accelerating leaf senescence promotes nutrient redistribution from old leaves into young leaves, and may raise yield under certain circumstances. Many genes and transcriptional factors involved in leaf senescence are associated with responses to abiotic and biotic stresses. WRKY transcriptional factors play a vital role in this process and they could interact with JA signalling. This review summarized how genes, plant hormones and environmental factors affect the quality, yield. Besides, the regulation of leaf senescence holds great promise to improving the resistance to plant biotic and abiotic stresses.

Research Advances in Genetic Mechanisms of Major Cucumber Diseases Resistance.

Cucumber (Cucumis sativus L.) is an important economic vegetable crop worldwide that is susceptible to various common pathogens, including powdery mildew (PM), downy mildew (DM), and Fusarium wilt (FM). In cucumber breeding programs, identifying disease resistance and related molecular markers is generally a top priority. PM, DM, and FW are the major diseases of cucumber in China that cause severe yield losses and the genetic-based cucumber resistance against these diseases has been developed over the last decade. Still, the molecular mechanisms of cucumber disease resistance remain unclear. In this review, we summarize recent findings on the inheritance, molecular markers, and quantitative trait locus mapping of cucumber PM, DM, and FM resistance. In addition, several candidate genes, such as PM, DM, and FM resistance genes, with or without functional verification are reviewed. The data help to reveal the molecular mechanisms of cucumber disease resistance and provide exciting new opportunities for further resistance breeding.

Induction, Proliferation, Regeneration and Kinsenoside and Flavonoid Content Analysis of the Anoectochilus roxburghii (Wall.) Lindl Protocorm-like Body.

Anoectochilus roxburghii (Wall.) Lindl has been used in Chinese herbal medicine for treating various ailments. However, its wild resources are endangered, and artificial cultivation of the plant is limited by the low regeneration rate of conventional propagation methods. The lack of A. roxburghii resources is detrimental to the commercial production of the plant and kinsenoside, which is unique to Anoectochilus species. To develop highly efficient methods for A. roxburghii micropropagation and find alternative resources for kinsenoside production, we created an induction, proliferation, and regeneration of PLBs (IPR-PLB) protocol for A. roxburghii. We also analyzed the kinsenoside and flavonoid contents during the induction and proliferation of PLBs. The best media of IPR-PLB for PLB induction and proliferation (secondary PLB induction and proliferation), shoot formation, and rooting medium were Murashige and Skoog (MS) + 3 mg/L 6-benzylaminopurine (6-BA) + 0.5 mg/L naphthaleneacetic acid (NAA) + 0.8 mg/L zeatin (ZT) + 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2, 4-D), MS + 3 mg/L 6-BA + 0.5 mg/L NAA, and MS + 0.5 mg/L NAA, respectively. On these optimized media, the PLB induction rate was 89 ± 2.08%, secondary PLB induction rate was 120 ± 5%, secondary PLB proliferation rate was 400 ± 10% and 350 ± 10 % in terms of the quantity and biomass at approximately 1 month, shoot induction rate was 10.5 shoots/PLB mass, and root induction rate was 98%. All plantlets survived after acclimation. Darkness or weak light were essential for PLB proliferation, and light was crucial for PLB differentiation on these optimized media. The kinsenoside contents of PLBs and secondary PLBs were 10.38 ± 0.08 and 12.30 ± 0.08 mg/g fresh weight (FW), respectively. Moreover, the peak kinsenoside content during the proliferation of secondary PLBs was 34.27 ± 0.79 mg/g FW, which was slightly lower than that of the whole plant (38.68 ± 3.12 mg/g FW). Two flavonoids exhibited tissue- or temporal-specific accumulation patterns, and astragalin accumulated exclusively during the first 2 weeks of cultivation. The IPR-PLB protocol for A. roxburghii may facilitate the efficient micropropagation of A. roxburghii plants. Furthermore, the PLBs are a good alternative resource for kinsenoside production.

Intrinsically Negative Photosensitive Polyimides with Enhanced High-Temperature Dimensional Stability and Optical Transparency for Advanced Optical Applications via Simultaneous Incorporation of Trifluoromethyl and Benzanilide Units: Preparation and Properties.

Negative photosensitive polyimides (PSPIs) with the photo-patterned ability via the photocrosslinking reactions induced by the i-line (365 nm) and h-line (426 nm) emitting wavelengths in high-pressure mercury lamps have been paid increasing attention in semiconductor fabrication, optical fiber communications, and other advanced optoelectronic areas. In the current work, in view of the optical and thermo-mechanical disadvantages of the currently used negative PSPIs, such as the intrinsically photosensitive or auto-photosensitive systems derived from 3,3',4,4'-benzophenonetetracarboxylic dianhydride (BTDA) and the ortho-alkyl- substituted aromatic diamines, a series of modified negative PSPIs with the enhanced optical transparency in the wavelength of 365~436 nm and apparently reduced coefficients of linear thermal expansion (CTE) were developed. For this purpose, a specific aromatic diamine with both of trifluoromethyl and benzanilide units in the molecular structures, 2,2'-bis(trifluoromethyl)-4,4'-bis[4-(4-amino-3-methyl)benzamide]biphenyl (MABTFMB) was copolymerized with BTDA and the standard 3,3',5,5'-tetramethyl-4,4'-diaminodiphenylmethane (TMMDA) diamine via a two-step chemical imidization procedure. As compared with the pristine PI-1 (BTDA-TMMDA) system, the new-developed fluoro-containing PSPI systems (FPI-2~FPI-7) exhibited the same-level solubility in polar aprotic solvents, including N-methyl-2-pyrrolidone (NMP) and N,N- dimethylacetamide (DMAc). The FPI films cast from the corresponding FPI solutions in NMP showed the optical transmittances of 78.3-81.3% at the wavelength of 436 nm (T436, h-line), which were much higher than that of the PI-1 (T436 = 60.9%). The FPI films showed the CTE values in the range of 40.7 × 10-6/K to 54.0 × 10-6/K in the temperature range of 50 to 250 °C, which were obviously lower than that of PI-1 (CTE = 56.5 × 10-6/K). At last, the photosensitivity of the FPI systems was maintained and the micro-pattern with the line width of 10 µm could be clearly obtained via the standard photolithography process of FPI-7 with the molar ratio of 50% for MABTFMB in the diamine moiety.