Functional analysis of WOX family genes in Dendrobium catenatum during growth and development.

The WUSCHEL-Related Homeobox (WOX) family is a group of transcription factors unique to plants that play an important role in regulating key developmental processes such as stem cell maintenance and organ morphogenesis. As a rare and valuable Chinese herb, Dendrobium catenatum has a unique epiphytic lifestyle and growth and developmental characteristics, and a functional investigation of its WOX family genes can help to further understand the conserved and specific development of D. catenatum. In this study, we analyzed the phylogeny, spatio-temporal expression pattern and heterologous expression function of D. catenatum WOX family genes (DcWOX). The results showed that members of the D. catenatum WOX gene family could be divided into three evolutionary branches with significantly different tissue expression profiles. In transgenic Arabidopsis, overexpression of DcWOX4 resulted in significant dwarfism, pinnately leaf margins, and delayed flowering for 2 weeks; overexpression of DcWOX9 resulted in plant dwarfing, serrated leaf margin, delayed flowering for 1 week, and even male and female sterility in strong phenotype plants; overexpression of DcWOX11 caused curl downward leaf. The abnormal morphogenesis of DcWOX4/9/11 overexpression Arabidopsis leaves are related to the down-regulation of TCP family genes, CUC family genes and the up-regulation of KNOX family genes; Postponement of flowering is related to down-regulation of early flowering genes such as FT, SOC1 and CO. Therefore, this study showed that D. catenatum WOX family genes have important functions in regulating plant morphogenesis, leaf development, flowering time and fertility, further expanding the understanding of the WOX gene family function, and providing clues for the conservation and specificity during orchid development and evolution.

[Inhibitory effect of three strains of biocontrol microbes on pathogens causing rhizome rot of Polygonatum cyrtonema].

Rhizome rot is one of the main disease in the cultivation of Polygonatum cyrtonema, and it is also a global disease which seriously occurs on the perennial medicinal plants such as Panax notoginseng and P. ginseng. There is no effective control method at present. To identify the effects of three biocontrol microbes(Penicillium oxalicum QZ8, Trichoderma asperellum QZ2, and Brevibacillus amyloliquefaciens WK1) on the pathogens causing rhizome rot of P. cyrtonema, this study verified six suspected pathogens for their pathogenicity on P. cyrtonema. The result showed that Fusarium sp. HJ4, Colletotrichum sp. HJ4-1, and Phomopsis sp. HJ15 were the pathogens of rhizome rot of P. cyrtonema, and it was found for the first time that Phomopsis sp. could cause rhizome rot P. cyrtonema. Furthermore, the inhibitory effects of biocontrol microbes and their secondary metabolites on three pathogens were determined by confrontation culture. The results showed that the three tested biocontrol microbes significantly inhibited the growth of three pathogens. Moreover, the secondary metabolites of T. asperellum QZ2 and B. amyloliquefaciens WK1 showed significant inhibition against the three pathogens(P<0.05), and the effect of B. amyloliquefaciens WK1 sterile filtrate was significantly higher than that of high tempe-rature sterilized filtrate(P<0.05). B. amyloliquefaciens WK1 produced antibacterial metabolites to inhibit the growth of pathogens, and the growth inhibition rate of its sterile filtrate against three pathogens ranged from 87.84% to 93.14%. T. asperellum QZ2 inhibited the growth of pathogens through competition and antagonism, and P. oxalicum QZ8 exerted the inhibitory effect through competition. The research provides new ideas for the prevention and treatment of rhizome rot of P. cyrtonema and provides a basis for the di-sease control in other crops.

[Relative molecular mass distribution and monosaccharide composition of polysaccharides in Polygonatum kingianum var.grandifolium].

Polygonatum kingianum var.grandifolium, different from most Polygonatum species in biological characteristics, sprouts and blooms in spring and autumn, respectively, and it is evergreen in winter.It is difficult to learn from the patterns of other Polygonatum plants because the chemical composition in P.kingianum var.grandifolium changes with phenology, which consequently restricts the production of high-quality medicinal and eatable substances.Samples of P.kingianum var.grandifolium in different months and ages collected from Xiushan, Chongqing were analyzed for polysaccharide content, polysaccharide relative molecular mass, and mono-saccharide composition by anthrone-sulfuric acid colorimetric assay, high-performance gel-permeation chromatography, and trimethylsilane(TMS) derivatization prior to GC-MS.The results showed that the polysaccharide content and composition in the rhizome of P.kingianum var.grandifolium were closely related to the growth period.New shoot sprouting promoted the accumulation of polysaccharides, and flowering and fruiting consumed polysaccharides.The highest polysaccharide content was found in April, reaching 134.04 mg·g~(-1).Polysaccharides in P.kingianum var.grandifolium were divided into five fractions according to the weight-average M_W, i.e., P1(2.02×10~7), P2(5.09×10~6), P3(1.37×10~6), P4(4.73×10~5), and P5(5.11×10~3), and P5 had the highest content.In terms of monosaccharide composition, polysaccharides were mainly composed of fructose, glucose, galactose, mannose, xylose, and arabinose with the average molar ratio of 1.31∶1.00∶0.90∶0.53∶0.22∶0.21.The results of the study provide a scientific basis for the precise harvesting and resource utilization of P.kingianum var.grandifolium.

[Quality evaluation of Polygonatum cyrtonema based on HPLC fingerprint and multi-component quantitative analysis].

The medicinal and edible Polygonatum cyrtonema is one of the original species of Polygonati Rhizoma. In this study,HPLC fingerprints for 25 batches of P. cyrtonema from 6 provinces were established. A total of 14 common peaks were identified and the similarities of the fingerprints were in the range of 0. 939-0. 999. In additon, the partial least squares-discriminant analysis(PLSDA) demonstrated that the samples had low discriminability except for JX-1 and most components of them had no significant correlation with environmental factors such as longitude, latitude, and altitude. Thus, chemical composition specificity of P. cyrtonema in natural distribution areas had no obvious regularity and their variation might be induced by the local environment. This conclusion explained the lack of records about Dao-di area of Polygonati Rhizoma. However, JX-1 boasted significantly higher content of 5-hydroxymethylfurfural(HMF) and 4',5,7-trihydroxy-6,8-dimethylhomoisoflavone( HIF), thick and long inflorescence and rhizome, and extremely high yield. Therefore, excellent variety of P. cyrtonema might have great potential to improve the quality and yield of Polygonati Rhizoma. Moreover, three components of HMF, polygonalline A(PA), and HIF were identified in the fingerprint. Among them, HMF has the activities of blood rheology improvement, antioxidation, and anti-myocardial ischemia and PA is an indolizine alkaloid with potential anti-inflammatory activity. HIF, the characteristic homoisoflavone in Polygonatum, has the pharmacological activities of regulating blood glucose and anti-tumor. A quantitative analysis method can provide a theoretical basis for the improvement of the quality evaluation of Polygonati Rhizoma.

[Effects of different storage conditions on edible quality and antioxidant activity of Polygonatum cyrtonema flowers].

The flower of Polygonatum cyrtonema has good edible and medicinal values. In this study, four samples of P. cyrtonema flowers from different regions were selected as test materials. The contents, composition and antioxidant activities of lipid-soluble pigments and alcohol-soluble components were determined under different light and temperature conditions, which help to reveal the discoloration reason and the composition variation patterns during storage. The results showed that light and temperature had different effects on the lipid-soluble pigments and alcohol-soluble components in the dried flowers during storage. After storage for 4 weeks, the contents of total chlorophyll, carotenoids, phenols and saponins in the samples exposed to light respectively decreased by 62.62%, 66.4%, 68.7% and 43.4% compared with those in the dark. The decreases in the contents of chlorophyll a, chlorophyll b, lutein, ß-carotene and zeaxanthin were 64.64%, 56.74%, 59.2%, 77.7% and 45.4%, respectively. The contents of pigments and components in the samples stored at-20 ℃ were significantly higher than those at room temperature and 4 ℃, indicating that low temperature was conductive to the stability of lipid-soluble pigments and alcohol-soluble components. The samples stored at low temperature and in the dark had the strongest free radical scavenging activity. The results suggest that P. cyrtonema dried flowers should be stored in low temperature environment without light, which can slow down the degradation of internal components. The study provides a theoretical basis for the production, processing and storage of P. cyrtonema flowers.

Effects of Various Processing Methods on the Metabolic Profile and Antioxidant Activity of Dendrobium catenatum Lindley Leaves.

The metabolite profiles and antioxidant activity of Dendrobium catenatum Lindley leaf, a new functional ingredient for food product development, were evaluated in samples that had been prepared using various methods, including freeze-drying, hot-air drying, rolling before drying, steaming before drying, steaming and rolling before drying, and drying at 100, 80, and 60 °C. The concentrations of polysaccharides and flavonoids, as well as the antioxidant capacity of each sample, were determined. Furthermore, two nucleosides, four amino acids, one monoaromatic compound, and eight flavonoids were identified in dried leaves using high-performance liquid chromatography-diode array detector-electrospray ionization-multistage mass spectrometry (HPLC-DAD-ESI-MSn) and ultraviolet (UV) spectral analyses. The content of polar compounds such as cytidylic acid, arginine, tyrosine, and hydroxybenzoic acid hexose increased dramatically during hot-air-drying and rolling-before-drying treatments, while flavonol C-glycosides remained stable throughout the various treatments and drying temperatures. Rolling before drying at 100 °C was identified as the most suitable process when manufacturing tea products from D. catenatum leaves. This process resulted in a high-antioxidant-activity and visually appealing tea. This report details a potential strategy that should be applied in the manufacturing processes of high-quality products from D. catenatum leaves.

[Investigation on reproductive characteristics of Polygonatum cyrtonema].

The study is aimed to investigate the reproductive biology characteristics of Polygonatum cyrtonema, especially including phenology, flower bud differentiation, flowering timing, floral traits, pollen vigor and stigma receptivity. The results showed that P. cyrtonema forms inflorescence before the leaves spread. In the wild, P. cyrtonema is mainly pollinated by insects such as bumblebees, with a seed setting rate of 65.12%. The seed setting rate of indoor single plant isolation or self-pollination enclosed by parchment paper bag is 0, indicating that it is self-incompatible. In Lin'an city, seedlings begin to emerge from mid-March to early April(the temperature is higher than 7.5 ℃), buds begin to emerge from the end of March to mid-April, and then undergo the full bloom stage from mid-to-late April, and the final flowering stage from the end of April to mid-May. The whole flowering period lasts 36 to 45 days. There are obvious differences in the phenology of different provenances. The flowers come into bloom from the base to the top along the aboveground main axis, which usually contain 4-22 inflorescences with(2-) 4-10(-21) flowers per inflorescence. The flowering pe-riod for a single plant is 26-38 days. The single flower lasts about 20-25 days from budding to opening and withers 2 days after pollination, and then the ovary will gradually expand. If unpollinated, it will continue to bloom for 3-5 days and then wither. Flower development period is significantly related to pollen vigor and stigma remittance. The pollen viability is the highest when the flower is fully opened with anthers gathering on the stigma, and the receptivity is the strongest when the stigma protrudes out of the perianth and secretes mucus. The fruits and seeds ripen in October, and proper shading can ensure the smooth development and maturity of the seeds. This study provides a basis for the hybrid breeding and seed production of P. cyrtonema.

Transcriptomic and Metabolomic Approaches Deepen Our Knowledge of Plant-Endophyte Interactions.

In natural systems, plant-symbiont-pathogen interactions play important roles in mitigating abiotic and biotic stresses in plants. Symbionts have their own special recognition ways, but they may share some similar characteristics with pathogens based on studies of model microbes and plants. Multi-omics technologies could be applied to study plant-microbe interactions, especially plant-endophyte interactions. Endophytes are naturally occurring microbes that inhabit plants, but do not cause apparent symptoms in them, and arise as an advantageous source of novel metabolites, agriculturally important promoters, and stress resisters in their host plants. Although biochemical, physiological, and molecular investigations have demonstrated that endophytes confer benefits to their hosts, especially in terms of promoting plant growth, increasing metabolic capabilities, and enhancing stress resistance, plant-endophyte interactions consist of complex mechanisms between the two symbionts. Further knowledge of these mechanisms may be gained by adopting a multi-omics approach. The involved interaction, which can range from colonization to protection against adverse conditions, has been investigated by transcriptomics and metabolomics. This review aims to provide effective means and ways of applying multi-omics studies to solve the current problems in the characterization of plant-microbe interactions, involving recognition and colonization. The obtained results should be useful for identifying the key determinants in such interactions and would also provide a timely theoretical and material basis for the study of interaction mechanisms and their applications.

Endophytic fungi, host genotype, and their interaction influence the growth and production of key chemical components of Dendrobium catenatum.

To examine how host plant genotype, endophytic fungal species, and their interaction may affect growth and key chemical content and composition in an important orchid species, we assessed four Dendrobium catenatum cultivars co-cultured with three fungi previously isolated from D. catenatum. Fungal endophytes (Tulasnella sp., Leptosphaeria microscopica, and Guignardia sp.) specifically affected the growth and chemical composition of the four cultivars. Fungal infection significantly increased certain growth traits, especially mid-stem thickness, stem biomass, stem polysaccharide and ethanol-soluble extractive content, and leaf flavonoid and phenol content. Presence or abundance of some key chemical components was also altered by fungal treatment. These increases and alterations were highly dependent on the host genotype. The findings of this study contribute to our understanding of Dendrobium and endophytic fungi interactions, and provide vital information for improving the development and use of endophytic fungi in D. catenatum breeding.

[Genome-wide identification and expression analysis of CSLA gene family of Dendrobium catenatum].

Glucomannan is the key active ingredient of Dendrobium catenatum, and CSLA family is responsible for glucomannan biosynthesis. In order to systematically evaluate the CSLA family members of D. catenatum, the bioinformatics methods were performed for genome-wide identification of DcCSLA gene family members through the genomic data of D. catenatum downloaded from the NCBI database, and further analyses of their phylogenetic relationship, gene structure, protein conserved domains and motifs, promoter cis-elements and gene expression profiles in response to stresses. The results showed that D. catenatum contains 13 CSLA members, all of which contain 9-10 exons. In the evolutionary relationship, CSLA genes were clustered into 5 groups, DcCSLA genes were distributed in all branches. Among which the ancestral genes of groupI existed before the monocot-dicot divergence, and groupⅡ-Ⅴ only existed in the monocot plants, indicating that group Ⅰ represents the earliest origin group. CSLA proteins are characteristic of the signature CESA_CaSu_A2 domain. Their promoter regions contain cis elements related to stresses and hormones. Under different stress treatments, low temperature induces the expression of DcCSLA5 and inhibits the expression of DcCSLA3. Infection of Sclerotium delphinii inhibits DcCSLA3/4/6/8/9/10 expression. Under the treatment of jasmonic acid, DcCSLA11 expression was significantly up-regulated, and DcCSLA2/5/7/12/13 were significantly down-regulated. These results laid a foundation for further study on the function of DcCSLA genes in glucomannan biosynthesis and accumulation.

[Changes of pigment components in Dendrobium catenatum flower under different storage conditions].

The flower color of Dendrobium catenatum(D. officinale) tends to fade during storage. In order to clarify the influence of storage conditions on the pigment components in flowers, two conditions were applied:temperature and illumination. The contents of pigments in the D. catenatum flower were determined by UV-Vis spectrophotometry and HPLC, and the changes of them during storage were analyzed. The results showed that illumination and temperature had an effect on the pigments of D. catenatum flower during sto-rage. Illumination significantly promoted the degradation of pigments. The contents of total chlorophyll, carotenoids and anthocyanins in the light samples were significantly lower than those in the dark. The total chlorophyll, carotenoids and anthocyanins in the light samples were decreased by 46.5%, 63.4%, and 69.2% respectively. Illumination had a greater effect on fat-soluble pigments than water-soluble pigments. Among the three temperature treatments, the contents of chlorophyll, carotenoid and anthocyanin were as follows:-20 ℃>4 ℃>room temperature, it is indicated that-20 ℃ was the best temperature to maintain the stability of pigment composition. The contents of chlorophyll a, chlorophyll b, ß-carotene, lutein and zeaxanthin in the light samples decreased by 34.8%, 69.0%, 72.5%, 61.6%, 36.1%, respectively. After storage for 5 months, the contents of chlorophyll, carotenoid and anthocyanin constituent at-20 ℃ was significantly higher than those at 4 ℃ and room temperature. The results show that light avoiding and low-temperature can effectively slow down the degradation of pigment components. Therefore, it is suggested that D. catenatum flower should be stored in light avoiding and low-temperature conditions in actual production and processing, which can prolong the usable time.

[Main nutrients and functional ingredients in shoots of Polygonatum cyrtonema].

In order to reveal the main nutrients and functional ingredients in the shoots of Polygonatum cyrtonema, the polysaccharides, proteins, amino acids, and total phenols were determined. The tested samples cultured in Ma'nijiaonong, Hengtang village, Tianmushan town, Lin'an, Zhejiang, which were collected from three provenances(Pan'an and Longquan in Zhejiang and Qingyang in Anhui). The results showed that the polysaccharide content of the shoots varied from 2.34% to 12.73%, roughly one-third of rhizomes. The protein content varied from 107.75 to 192.49 mg·g~(-1), nearly 5.50 times more than rhizomes. Moreover, the average of total amino acid content was 193.13-248.74 mg·g~(-1), approximately 4.16 times of rhizomes. And the essential amino acids account for 35.57%-39.44% of the total amino acids content, which was close to the standard of the ideal protein proposed by FAO/WHO(the essential amino acid/total amino acid is about 40%). In addition, the taste amino acids(TaAA) changed from 160.12 to 208.29 mg·g~(-1), revealing the material basis of "shoots were extremely delicious" in Chinese ancient herbal medicine. Additionally, the total phenols varied from 51.21-58.76 mg·g~(-1), about 2.96 times of rhizomes. The DPPH free radical scavenging rate of tested shoots was over 95%, which obviously superior to rhizomes. Therefore, the shoots of P. cyrtonema is a very high-quality vegetable and functional food with good development potential. Furthermore, the main nutrients and functional substances in P. cyrtonema shoots are closely related to the provenances and harvesting seasons. It is important to improve the quality and yield of the shoots by strengthening the variety of breeding and cultivation techniques.

[Main nutrients and functional ingredients in flowers of Polygonatum cyrtonema and P. filipes].

To reveal the main nutrients and functional ingredients in the flowers of Polygonatum cyrtonema and P. filipes, the content of the polysaccharides, saponins, amino acids, total phenols, mineral elements, and the DPPH free radical scavenging rates were determined. The flowers and rhizomes of P. cyrtonema were collected from Qingyang in Anhui and Qingyuan in Zhejiang, while the flowers and rhizomes of P. filipes were collected from Longyou in Zhejiang, respectively. The results showed that the polysaccharides content in flowers varied from 60.88 to 97.00 mg·g~(-1), about half of that in rhizomes. The saponins content in flowers varied from 32.55 to 40.93 mg·g~(-1), which was close to the content in rhizomes. The content of total phenols ranged from 40.79 to 50.95 mg·g~(-1), approximately 4.5 times of that in rhizomes. The total amino acids content in flowers was 111.85 to 131.03 mg·g~(-1), about 2.3 times of the content in rhizomes. The essential trace element content was abundant in flowers. The contents of heavy metal elements were all within the limits set by the standards. The DPPH free radical scavenging rate IC_(50) varied from 1.77 to 3.25 mg·mL~(-1), less than one-fifth of that in rhizomes, showing a significant superiority of antioxidant activity compared to rhizomes. The results initially revealed the fundamental of "the flowers exceed the rhizomes in effect", the common saying about the traditional Chinese medicinal herbs over the years, indicating a great developing potential of the flowers. Besides, as polysaccharides, saponins, amino acids, total phenols and other nutritive substances in flowers differ widely among species and provenances, it's important to develop variety breeding to improve the quality and yield of flowers.

Genome-wide identification and expression profiling of SET DOMAIN GROUP family in Dendrobium catenatum.

BACKGROUND: Dendrobium catenatum, as a precious Chinese herbal medicine, is an epiphytic orchid plant, which grows on the trunks and cliffs and often faces up to diverse environmental stresses. SET DOMAIN GROUP (SDG) proteins act as histone lysine methyltransferases, which are involved in pleiotropic developmental events and stress responses through modifying chromatin structure and regulating gene transcription, but their roles in D. catenatum are unknown. RESULTS: In this study, we identified 44 SDG proteins from D. catenatum genome. Subsequently, comprehensive analyses related to gene structure, protein domain organization, and phylogenetic relationship were performed to evaluate these D. catenatum SDG (DcSDG) proteins, along with the well-investigated homologs from the model plants Arabidopsis thaliana and Oryza sativa as well as the newly characterized 42 SDG proteins from a closely related orchid plant Phalaenopsis equestris. We showed DcSDG proteins can be grouped into eight distinct classes (I~VII and M), mostly consistent with the previous description. Based on the catalytic substrates of the reported SDG members mainly in Arabidopsis, Class I (E(z)-Like) is predicted to account for the deposition of H3K27me2/3, Class II (Ash-like) for H3K36me, Class III (Trx/ATX-like) for H3K4me2/3, Class M (ATXR3/7) for H3K4me, Class IV (Su (var)-like) for H3K27me1, Class V (Suv-like) for H3K9me, as well as class VI (S-ET) and class VII (RBCMT) for methylation of both histone and non-histone proteins. RNA-seq derived expression profiling showed that DcSDG proteins usually displayed wide but distinguished expressions in different tissues and organs. Finally, environmental stresses examination showed the expressions of DcASHR3, DcSUVR3, DcATXR4, DcATXR5b, and DcSDG49 are closely associated with drought-recovery treatment, the expression of DcSUVH5a, DcATXR5a and DcSUVR14a are significantly influenced by low temperature, and even 61% DcSDG genes are in response to heat shock. CONCLUSIONS: This study systematically identifies and classifies SDG genes in orchid plant D. catenatum, indicates their functional divergence during the evolution, and discovers their broad roles in the developmental programs and stress responses. These results provide constructive clues for further functional investigation and epigenetic mechanism dissection of SET-containing proteins in orchids.

Screening of endophytic fungi against southern blight disease pathogen-Sclerotium delphinii in Dendrobium catenatum / 中国中药杂志

In order to screen the endophytic fungi that can enhance the host(Dendrobium catenatum) resistance to Sclerotium delphinii, the antagonism between each of the 43 endophytic fungi and the pathogen S. delphinii were tested. The results showed that 6 endophytic fungi(DCR2, DCR5, DCR21, DCR22, DCR42, DCR43) have strong activities against the pathogen, the inhibition rates were 49.2%, 49.2%, 47.2%, 56.2%, 53.2%, 48.0%, respectively. Then D. catenatum plantlets were inoculated with both S. delphinii and each of these six endophytic fungi. As a result, the incidence rates of leaves and stems of the D. catenatum plantlets inoculated with DCR2 and the pathogen were both significantly lower than those with other treatments, and the plantlet death rate was 0. It showed that DCR2 Trichoderma polysporum could effectively inhibit the southern blight disease of D. catenatum. Through the endophytic fungal re-isolation test, it was found that DCR2 can colonize in the roots, stems, and leaves of D. catenatum. The research will provide new ideas for the prevention and treatment of the southern blight disease of D. catenatum. It is also significant for reducing pesticide use, ensuring food safety, and promoting the sustainable development of D. catenatum industry. Furthermore, it will provide a basis for the disease control in other crops.

[Study on quantification method of non-starch polysaccharides in Dendrobium catenatum].

The non-starch polysaccharides,mainly composed of glucomannans,are the major bioactive compounds in Dendrobium catenatum. In order to evaluate the quality of the medicinal materials and guide the production and processing,a quantification method of non-starch polysaccharides was established by stems of D. catenatum C15 strain collected from the pear epiphytic cultivation. The non-starch polysaccharides were obtained by " water extraction,α-amylase pretreatment,and alcohol precipitation once" method. The contents of starches,non-starch polysaccharides and monosaccharides were analyzed. In addition,the system suitability was tested. Compared with method of the Chinese Pharmacopoeia( 2015 edition),the contents of total polysaccharides,glucose,and mannose were decreased by 20. 9%,58. 8% and 1. 6% respectively. The method effectively digested starch and retained non-starch polysaccharides,and the analysis result was accurate and repeatable. Therefore,it is suitable for the content measurement of non-starch polysaccharides of D. catenatum. Furthermore,it could be an alternative method for quality control of D. catenatum and a reference in the determination of non-starch polysaccharides in other starch-containing medicinal materials.

[Study on tissue culture system of Polygonatum cyrtonema].

In order to accelerate the breeding of the excellent seedlings of Polygonatum cyrtonema,tissue culture system of P. cyrtonema was established through the comprehensive regulation of key factors( leaf age,leaf location,basic media and plant growth regulators) and cytological basis of callus formation and differentiation was analyzed through paraffin section. The results showed that the 30-day-old leaf base explanton medium MS+6-BA 1. 50 mg·L~(-1)+2,4-D 0. 20 mg·L~(-1) had the highest induction rate( 80. 00%). The callus was initiated from cells on leaf base epidermis and near cortex,formed by the differentiation of middle vascular bundle cells. The optimal medium for adventitious bud differentiation was MS+ 6-BA 4. 00 mg·L~(-1)+ 2,4-D 0. 20 mg·L~(-1) with the differentiation rate of90. 33%,and the average number of buds was 5. 16. The adventitious buds had two origin types: exogenous and endogenous origin,formed by callus proximal cells and callus internal meristemoid. The adventitious bud proliferation medium was screened by orthogonal design,which determined the optimum combination was MS+ 6-BA 2. 00 mg·L~(-1)+NAA 0. 10 mg·L~(-1) and MS+ 6-BA 2. 00 mg·L~(-1)+NAA 0. 20 mg·L~(-1). The tubers with three leaves were cut and inoculated in the medium 1/2 MS+IBA 2. 00 mg·L~(-1),showing the highest rooting rate of 94. 00%. The rooting seedlings transplanted into the peat-vermiculite( 1 ∶ 1) matrix grew healthy and the survival rate was over 85. 00%. This research provided a novel solution for large-scale cultivation of P. cyrtonema seedling.

[Occurrence regularity of Dendrobium catenatum southern blight disease].

In order to scientifically prevent and control Dendrobium catenatum southern blight disease,the main factors related to this disease occurrence,the pathogen( Sclerotium delphinii),environmental factors( temperature and humidity) and D. catenatum germplasms,were investigated. The results showed that reaching 25-30 ℃ temperature and over 95% humidity simultaneously should be the main conditions for the occurrence and prevalence of D. catenatum southern blight disease. Moreover,the S. delphinii-infected plants and their contaminated substrates were the disease spreading sources. Therefore,removing the infected plants,dealing with the contaminated substrates,keeping air ventilation,and reducing air humidity are the effective ways to prevent and control the occurrence and prevalence of D. catenatum southern blight disease. The research also indicated that D. catenatum has different resistances to the southern blight disease depending on germplasm. The present study lays important foundations for the breeding of D. catenatum diseaseresistant varieties and the further analysis of the infection and resistance mechanisms underlying southern blight disease.

[Composition analysis and antioxidant activity of polysaccharide from Hibiscus syriacus].

The flower of Hibiscus syriacus has good ornamental and edible-medicinal values.In this study,four samples of two varieties,namely white multiple petals flower and pink multiple petals flower,were selected as test materials.And the optimum extraction conditions,relative molecular weight,monosaccharide composition and antioxidant activity of polysaccharides in flower were investigated.Through single factor experiment and response surface,the optimal extract conditions of polysaccharide were designed as follows:extraction temperature at 96.8℃,ratio of material to liquid of 43.5∶1 m L·g~(-1),extraction time of 3.1 h.Polysaccharides of H.syriacus flowers were analyzed by high performance gel chromatography.The average molecular masses of the 4 polysaccharide samples were1.49×10~5,1.25×10~5,1.01×10~5,1.37×10~5,respectively.Polysaccharides of H.syriacus flowers were mainly composed of glucose,mannose,galactose,rhamnose and arabinose by pre-column derivatization HPLC.The ratio of galactose was the highest in five monosaccharide,and the ratio of galactose to glucose was 1.656-4.496.In addition,crude polysaccharides of H.syriacus flowers showed potential antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl radical(DPPH)assay,total reducing capacity assay and ABTS assay in vitro,and its antioxidant effect showed a good dose-effect relationship with the concentration of crude polysaccharides.Among the tested varieties,polysaccharides of pink multiple petals flower and white multiple petals flower had the same molecular masses and monosaccharides composition,but the antioxidant activity of the polysaccharides of pink multiple petals flower was higher than that of the white flowers.The results of this study can provide a theoretical basis for the application of H.syriacus flower in the field of functional foods.

[Temporal and spatial variation of polysaccharides and alcohol-soluble extractives of Polygonatum cyrtonema].

To reveal the variation of polysaccharides and alcohol-soluble extractives of rhizomes Polygonatum cyrtonema from different producing areas,growing years,and harvesting seasons,25 wild samples from the main producing areas( provenances) of China and 6 artificial cultivated samples were collected and detected. The 6-year-old rhizomes of the artificial cultivation were gathered from Qingyuan,Zhejiang every 2 months during 2016 to 2017. Anthrone-sulfuric acid method and hot-dip method were used to determine the contents of polysaccharides and alcohol-soluble extractives respectively. The results showed that provenances,age sections and harvesting seasons affected the quality of Polygonati Rhizoma significantly. The content of polysaccharides from different provenances varied from 6. 96% to 20. 09%,and the content of extractives varied from 32. 08% to 78. 99%. The polysaccharides and extractives' content were the highest at 2-year-age sections,active constituents accumulation peaked in the third year,then decreased significantly with age increasing. The contents of polysaccharides and extractives accumulated were highest when aerial parts,up to 15. 39% and 78. 99% respectively. In summary,the activity and consistency fluctuated with producing areas. while such phenomenon indicated a huge potential of breeding. Additionally,the results showed that collecting Polygonati Rhizoma in February or August as herbal textual recorded were unreasonable,neither in spring or autumn according to the Chinese Pharmacopoeia( 2015 edition). Because these collecting periods didn't have the highest contents of polysaccharides and extractives. Therefore,based on phenological options,the rhizomes of P. cyrtonema should be harvested when the aboveground parts had just withered.