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.

[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.

[Cloning and function analysis of promoter of DcCDPK8 from Dendrobium catenatum].

DcCDPK8 involved in abiotic stress such as low temperature and signal transduction of hormones ABA and MeJA,but the transcriptional regulation is still unclear. In order to study the core promoter region of DcCDPK8 gene in Dendrobium catenatum and explore its transcriptional regulation mechanism,the DcCDPK8 gene promoter sequence was cloned by PCR from D. catenatum. Promoter sequence function was studied by fusion of 5 'terminal deletion and GUS gene. The results showed that the promoter sequence of DcCDPK8 gene has a low-temperature responsive element( LTR) between~(-1) 749 bp and-614 bp,two MeJA responsive elements between~(-1) 749 bp and-230 bp,and one ABA responsive elements between-614 bp and-230 bp. Three 5'-end different deletion fragments were constructed to fuse the eukaryotic expression vectors p BI121 with GUS,which were transformed into tobacco leaves. The GUS activity under cold stress treatment was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. GUS activity under exogenous ABA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3,and GUS activity under exogenous MeJA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. It is speculated that the ABA response element( ARE) in the promoter sequences of DcCDPK8 is positive regulatory role in response to exogenous ABA,the MeJA cis-acting element plays a negative role in response to exogenous MeJA.

Single-stranded DNA modified protonated graphitic carbon nitride nanosheets: A versatile ratiometric fluorescence platform for multiplex detection of various targets.

Facile and cost-effective detection of multiple targets is essential for a variety of applications ranging from life sciences to environmental monitoring. Here, we report a versatile ratiometric fluorescence platform for multiple detection of various targets based on the conjugation of single-stranded DNA (ssDNA) with protonated graphitic carbon nitride nanosheets (Pg-C3N4 NSs). We demonstrate that intrinsic peroxidase-like activity of Pg-C3N4 NSs is enhanced by conjugating with ssDNA, and thus the oxidation of substrate o-phenylenediamine (OPD) is promoted in the presence of H2O2. The oxidation product 2,3-diaminophenazine (DAP) can deliver a new fluorescence signal at 564 nm, and concurrently quench the intrinsic fluorescence of conjugates ssDNA/Pg-C3N4 NSs at 443 nm upon excitation at 370 nm. The transformation of fluorescence provides us a novel strategy for ratiometric fluorescence-based analytical sensing. Taking ssDNA as the target-recognition element of the conjugates ssDNA/Pg-C3N4 NSs, we favorably present ratiometric fluorescence detection of various targets including heavy metal ions (Hg2+) and biomolecules (Aflatoxin B1 (AFB1) and adenosine triphosphate (ATP)) in real samples by varying the ssDNA sequences. The present work provides a new strategy to develop facile methods for quantitative determination of various analytes and uncovers an innovative horizon for Pg-C3N4 NSs-based sensing platform fabrication.

[Effects of germplasm and host tree trunk on endophytic fungal communities in epiphytic Dendrobium catenatum].

In order to investigate the effects of germplasm and host tree trunk on endophytic fungal communities in epiphytic Dendrobium catenatum, a total of 3 835 isolates were recovered from roots, stems and leaves of four D. catenatum germplasms attached to one kind of host tree trunk and one germplasm attached to four kinds of epiphyte-host tree trunks. A total of 152 taxa were identified and classified based on the fungal cultural characteristics and phylogenetic analyses of ITS sequences. The taxa were assigned to 60 genera, 35 families, 21 orders and 5 classes of 2 phyla. The results indicated that D. catenatum cultivated in stereo cultivation harbor variety of fungi. The dominant fungal groups were different between Lin'an and Yiwu. Moreover, several groups showed geographical specificity, such as Arthrinium, Coniochaeta, Fusarium, Neofusicoccum and Zopfiella only dominating in Panshan of Lin'an, while Alternaria, Bjerkandera, Cercophora, Nigrospora and Trichoderma only dominating in Shangxi of Yiwu. There was no significant difference in diversity or species richness of endophytic fungi neither among germplasm nor host tree trunk. However, the richness and diversity indices exhibited a strong dependence on tissue type (P<0.05). The germplasm and host tree trunk impact the distribution patterns of endophytic fungi less than tissue type. Nevertheless, the relative frequencies of the dominant fungal groups were different among germplasms or host tree trunk types. Furthermore, there were some fungal species specific to certain germplasm or host tree trunk. This might be due to the distinctions in growth traits and chemical compositions of D. catenatum owning to the differences in D. catenatumgenetic background and microenvironment of host tree. Most of fungal taxa exhibit tissue specificity or preference. These results provide the basis for the study on the relationship between endophytic fungi and D. catenatum in stereo cultivation mode.

[Effects of sampling time on polysaccharides of Dendrobium catenatum under temperature-controlled condition].

In order to reveal the accumulation trend of polysaccharides in Dendrobium catenatum and determine the effect of sampling time on polysaccharides, D. Catenatum D21 clone was harvested from January to December after culturing for 2 to 5 months in the growth chamber with constant temperature. Polysaccharides were determined by phenol-sulfuric acid method and the monosaccharide compositions were analyzed by pre-column derivative-UPLC. The results showed that the content of polysaccharide and its key component mannose was positively correlated with the culture time, but the contents of polysaccharides in all kinds of culture peaked from 5 to 6 months, which were consistent with the trend of field planting. The results suggested that the trend of polysaccharide accumulation in the plant could be related to the life rhythm of the sensory seasons of D. catenatum, which was significantly affected by the harvesting season, even under the constant condition of the culture chamber.

[Effects of cultivation environments on Dendrobium catenatum].

The study was aimed to clarify the effect of three cultivation environments on the growth and metabolism of Dendrobium catenatum C13 group. There were three different cultivation conditions including rock epiphytic cultivation, pear epiphytic cultivation and pot cultivation. Morphological characteristics and agronomic characters of D. catenatum were observed and measured. Microstructure, contents of polysaccharide and alcohol-soluble extracts were measured by paraffin section method, phenol-sulfuric acid method and hot-dip method, respectively. The result showed that the cultivation environment significantly affected the growth of D. catenatum, the leaves of D. catenatum that cultivated on the rock and pear were sparse and small, the stems were short and purple and the root system was developed. Compare with potted cultivation, D. catenatum from rock epiphytic cultivation and pear epiphytic cultivation showed the following characteristics in the microstructure: the upper epidermis became thicker, the epidermal hair in the epidermis became denser, stomatal showed smaller and denser, the cell wall of exodermis, endoderm and medulla became thicker, the cell of velamen, exodermis, endoderm and medulla were smaller and arranged more closely, but the cultivation environment did not produce specific tissue structure, mainly changed in the structural parameters of size and quantity. The growth environments also influenced contents of polysaccharides and alcohol-soluble extracts. The dontents of polysaccharides and alcohol-soluble extracts in D. catenatum from rock epiphytic were the highest, reached 37.34% and 11.66%, the second was pear epiphytic, both higher than pot cultivation, alcohol-soluble extracts contents in D. catenatum from rock epiphytic are more complex, which shows that rock epiphytic is conducive to the accumulation of secondary metabolites in D. catenatum.

[Dendrobium officinale cliff epiphytic cultivation method].

To solve the issues of costly planting of facility cultivation method and inferior efficacy than wild herbs of Dendrobium officinale, the cliff epiphytic cultivation method was studied. To research the growth, agronomic traits, yield, polysaccharide and alcohol-soluble extract contents were measured on the D. officinale from different water regulation and cliff slope gradients treatments. The results showed that D. officinale epiphytic at 85 degrees-90 degrees cliff and sprayed water 1-2 h x d(-1) at the growing season can get better growth and obtain high yield, and the morphology has no different from wild cliff D. officinale, even in the environments without shade. The contents of polysaccharide and alcohol-soluble extract are closely related to the physiological ages, but significantly higher than the facility cultivation. It is possible that environmental stresses benefit the accumulation of polysaccharides, alcohol-soluble extract and other efficient ingredients.

[Comparison of different harvest ways of Dendrobium officinale].

To standardize the harvest ways of Dendrobium officinale and improve the quality and yield of D. officinale, a field experiment was carried out to study the effect of two kinds of harvest ways, which were keeping some of the axial shoot and harvesting all of the shoot by the end of the year. Then, the agronomic traits and yield were measured and the contents of polysaccharides and extractum were determined. The results showed that the harvest ways significantly affected the growth of D. officinale. Keeping some of the axial shoot could significantly improved the number of sprout, stem length, internode number and the internodal length, which also triggered increase the weight of fresh stems, leaves and the total of them and dry stems in per unit area, but it could not promote the stem diameter and the polysaccharide content in stems. Keeping some of the axial shoot moderately was conducive to the improvement of the production of medicinal materials in the process of harvesting by promoting the germination and growth of new buds, and to ensure the polysaccharide content by regulating the illumination and the density of cultivation.

[Effect of ABA on transcriptionally active Ty1-copia retrotransposons in Dendrobium officinale].

Using universal primer Tyl-copia retrotransposon RT, the conserved reverse transcriptase domain of about 260 bp was amplified by RT-PCR from the Dendrobium officinale which induced by 100 micromol x L(-1) abscisic acid (ABA), indicating these retrotransposons activated by 100 micromol x L(-1) ABA. The amplicons were recovered and cloned,then sequenced and analyzed by related bioinformatics software. Forty-two Ty1-copia like retrotransposon RT transcriptionally activated were obtained with high heterogeneity. The length of these sequences varied from 247 to 266 bp, and was rich in AT and homology ranged from 46.3% to 98.9%. The same to Ty1-copia like retrotransposon RT of genome, different c/s-acting regulatory elements induced by stress conditions and the starting transcription signals, corresponding to CAAT box, TATA box conserved sequences and some other regulatory elements. The c/s-acting regulatory elements induced by stress conditions of reverse transcriptase transcriptionally activated of Tyl-copia retrotransposons were significantly increased than that of Ty1-copia like retrotransposon RT of genome. When being translated into amino acids, fifteen sequences presented stop codon mutation, nineteen sequences presented frameshift mutation, and all sequences presented conserved sequence "SLYGKQ" mutation. Five categories were identified through phylogenic analysis after alignment analyses of their amino acid sequences, and with Ty1-copia like retrotransposon RT of genome having low homology, which indicated that reverse transcriptase transcriptionally activated of Ty1-copia retrotransposons which induced by ABA had Significantly differences with Ty1-copia like retrotransposon RT of genome.

[Cloning and analysis of reverse transcriptase(RT) of Ty1-copia retrotransposons in Dendrobium officinale].

Using universal primer Ty1-copia retrotransposon RT,43 Ty1-copia like retrotransposon RT with high heterogeneity, stop codon mutation and frameshift mutation were amplified by PCR from genomic DNA of Zhejiang Lin'an (C15) and Yunnan Guangnan (A39) of Dendrobium officinale. The length of these sequences varied from 260 to 266 bp, and was rich in AT and consistency ranged from 47.1% to 97.7%. Different c/s-acting regulatory elements induced by low temperature, heat, light, all kinds of plant growth regulating substances and the starting transcription signals, corresponding to CAAT box, TATA box conserved sequences and some other regulatory elements. When being translated into amino acids, ten sequences presented stop codon mutation, five sequences presented frameshift mutation, and thirty-seven sequences presented conserved sequence "SLYGKQ" mutation. Six categories were identified through phylogenic analysis after alignment analyses of their amino acid sequences, and with other plants (eg. Triticum aestivum, Eleocharis quinqueflora) having high homology, which indicated that horizontal transmission of retrotransposon occurred among the plants in the past.

[Dendrobium officinale stereoscopic cultivation method].

The study is aimed to make the most of available space of Dendrobium officinale cultivation facility, reveal the yield and functional components variation of stereoscopic cultivated D. officinale, and improve quality, yield and efficiency. The agronomic traits and yield variation of stereoscopic cultivated D. officinale were studied by operating field experiment. The content of polysaccharide and extractum were determined by using phenol-sulfuric acid method and 2010 edition of "Chinese Pharmacopoeia" Appendix X A. The results showed that the land utilization of stereoscopic cultivated D. officinale increased 2.74 times, the stems, leaves and their total fresh or dry weight in unit area of stereoscopic cultivated D. officinale were all heavier than those of the ground cultivated ones. There was no significant difference in polysaccharide content between stereoscopic cultivation and ground cultivation. But the extractum content and total content of polysaccharide and extractum were significantly higher than those of the ground cultivated ones. In additional, the polysaccharide content and total content of polysaccharide and extractum from the top two levels of stereoscopic culture matrix were significantly higher than that of the ones from the other levels and ground cultivation. Steroscopic cultivation can effectively improves the utilization of space and yield, while the total content of polysaccharides and extractum were significantly higher than that of the ground cultivated ones. The significant difference in Dendrobium polysaccharides among the plants from different height of stereo- scopic culture matrix may be associated with light factor.

[Special culture mediums of breeding clones of Dendrobium officinale].

The explants were obtained from stem segments of hybrid combination of Dendrobium officinale germplasms (Zhejiang Yandang Mountain x Yunnan Guangnan). The screened culture mediums for buds induction and multiplication, which were 1/2MS + IBA 1.0 mg x L(-1) +6-BA 1.0 mg x L(-1) and 1/2MS + IBA 1.5 mg x L(-1) +6-BA 0.5 mg x L(-1) respectively, were applied to 159 germplasms of D. officinale from Zhejiang, Yunnan, Guangxi, Hunan, etc. The medium for axillary buds induction had universality with 94.3% induction rate. During buds multiplication, there were significant differences in proliferation effect among germplasms, and three proliferation forms i.e. single bud, multiple buds and protocorm were differentiated from different germplasms. The results showed that different germplasms of D. officinale had specific requirements for culture medium. Therefore, developing special culture medium for breeding clones of D. officinale is urgent and has important application values.

[Studies on agronomic traits of seedlings from different F1 generations of Dendrobium officinale].

OBJECTIVE: To select superior mating combinations from different F1 generations of Dendrobium officinale. METHOD: Twenty-six superior parents from four provenances were selected to make up 26 mating combinations. The agronomic traits of different F1 generation were measured, counted, and analyzed by DPS software. RESULT: There were significant differences between different mating combinations among F1 generations. The variation among full-sib families was significantly higher than that among half-sib families, while the variation inside full-sib families was significantly lower than that inside half-sib families. Twelve agronomic traits were simplified into four principal components, of which the accumulative contribution rate was 84.127 3%. Twenty-six mating combinations have been divided into six groups, plants from the fifth and sixth group grew much better than the others. CONCLUSION: The selection of parents should be emphasized by using hybrid vigor. There were higher genetic gains and superior uniformity inside families when selection and breeding occurs in full-sib families than in half-sib families. Eight superior full-sib families (83 x 34, 66 x 9, 68 x 2, 91 x 69, 66 x 65, 69 x 91, 17 x 66, 66 x 17) have been selected which showed a good agronomic traits of seedlings.

[Cloning and expression analysis of HSP70 gene from Dendrobium officinale under low temperature stress].

OBJECTIVE: To investigate HSP70 gene expression from Dendrobium officinale under low temperature stress, which will provide the molecular biological foundation for breeding the low temperature resistant strain. METHOD: HSP70 gene full length cDNA was cloned by rapid amplification of cDNA ends (RACE) on the basis of HSP70 gene fragment sequences, and the structure and function of HSP70 gene were deduced. The expression of HSP70 under low temperature stress was detected by RT-PCR. RESULT: The full length of HSP70 gene cDNA was 2 296 bp containing a 1 944 bp open reading frame (ORF) that encoded a protein of 647 amino acids. Its amino acids sequence had typical HSP70 characteristics and high homology with other plant's HSP70. Cold stress expression analysis showed that expression of the HSP70 gene could be induced by low temperature. CONCLUSION: The HSP70 gene of D. officinale was successfully cloned and reported for the first time which proved that the expression could be induced by low temperature. The cloning of HSP70 gene provides a stable foundation for further study of D. officinale cultivation and the breeding of the cold resistance strains.

[Effects of storage time on magnolol and honokiol contents in bark of Magnolia officinalis].

OBJECTIVE: To reveal the relationship between the storage time of the bark of Magnolia officinalis and the content of phenols in it, and lay a theoretical foundation for the harvest, processing, management and storage. METHOD: The contents of magnolol and honokoiol in 15 bark samples, collected from the main producing areas in China, were determined in the time of freshly harvest and 3 and 10 years after respectively by HPLC method. RESULT: It showed that within a certain period of time, bark storage was favorable to conversion and accumulation of phenols, that the content of magnolol tended to increase from year 0 to year 3, then followed by slight decrease with years on account of volatilization of phenols, but was still higher when the bark was stored for 10 years than that that when the bark was freshly harvested, and the content of honokoiol still tended to increase when the bark had been stored for 10 years. CONCLUSION: The phenols in bark of M. officinalis is quite stable and the bark can be stored for 10 years or longer.

[Comparison study of different extraction methods of Sarcandra glabra].

OBJECTIVE: To compare the different extraction methods of Sarcandra glabra (Thunb.) Nakai. METHODS: To compare the yield of extraction and determination content of extraction by 6 kinds of different extraction methods as cold-soaked extraction, percolation extraction, reflux extraction, continuous reflux extraction, ultrasonic extraction and microwave extraction, and the content of furmaric acid was determinated as index. RESULTS: The yields of extraction and contents of availability component were very different through 6 kinds of different extraction methods. Among them, the yields of reflux extraction and continuous reflux extraction were higher, the contents of availability components of ultrasonic extraction and microwave extraction were higher. CONCLUSION: Microwave extraction method is better than other methods. It is fast economical, solvent and energy, and also has higher yield of availability component.

[Study on tissue culture and rapid propagation of Atractylodes macrocephala].

A study was conducted by tissue culture on the explant of Atractylodes macrocephala Koidz.. The result showed that NAA was a leading factor for induction of callus from leaf blades and the petiole as well as bud differentiation. MS + BA 1.0 mg/L + NAA 0.3 mg/L + GA3 0.2 mg/L was an optimal medium for induction of callus from leaf blades and the petiole and for the bud differentation also. MS + BA 3.0 mg/L + IBA 0.5 mg/L was suitable for the proliferation of axillary buds, with an induction rate of 95%. 1/2MS + IBA 0.1 - 0.5 mg/L was optimum for rooting, with a rooting percentage more than 90%. The survival rate of transplanted plantlets was more than 90%.