High frequency regeneration of plants via callus-mediated organogenesis from cotyledon and hypocotyl cultures in a multipurpose tropical tree (Neolamarkia Cadamba).

In this works, a simple, efficient and repeatable protocol was developed for in vitro regeneration via callus-mediated organogenesis of Neolamarkia Cadamba using cotyledonary petioles and hypocotyls. Effects of basal medium, plant growth regulators, the types and age of explant on the formation of adventitious buds/shoots were studied. Meanwhile, histological analysis for early ontogenic stages and genetic stability assessment by flow cytometry were investigated. Our investigation demonstrated that, compared with 6-benzyladenine (BA), N6-(2-isopentenyl) adenine (2-ip), Thidiazuron (TDZ) was the optimal cytokinin for buds/shoots induction on cotyledon and hypocotyl explants. Douglas-fir and sugar pine medium (DCR) supplemented with 22.7 µM TDZ and 0.27 µM α-naphthalene acetic acid (NAA) was most effective on bud induction, with the highest bud-induction rate and numbers of buds on cotyledon and hypocotyl explants. The available shoot per explant hit 35.2 when the induced callus sub-cultured to a medium without TDZ. It was found that TDZ could promote induction of the callus and the buds, however, continuous exposure beyond 4 weeks of supplemented high concentration (exceed 11.35 µM), TDZ was harmful to the proliferation and growth of buds/shoots. DCR appeared more efficiency than Murashige and Skoog medium (MS), Woody Plant medium (WPM), anther culture of cereal crops medium (N6) on bud induction. Age of cotyledon and hypocotyl explants in 20-day to 25-day was most beneficial to adventitious buds/shoots formation. Histological investigation confirmed that the buds originated from the wounded incisions of cotyledonary petiole and hypocotyl fragments, with callus formation. The regeneration plantlets were successfully acclimatized in greenhouse, yielded above 95% survival rate in field, exhibited normal morphology and growth characteristics. The analysis of flow cytometry on N. cadamba indicated no variation in the ploidy levels between the regenerated plantlets and the donor trees. The developed procedure can be used for mass production, germplasm exchange and transgenic studies to improve the resistance of the species via Agrobacterium-mediated.

Cloning and characterization of squalene synthase and cycloartenol synthase from Siraitia grosvenorii.

Mogrosides and steroid saponins are tetracyclic triterpenoids found in Siraitia grosvenorii. Squalene synthase (SQS) and cycloartenol synthase (CAS) are key enzymes in triterpenoid and steroid biosynthesis. In this study, full-length cDNAs of SgSQS and SgCAS were cloned by a rapid amplification of cDNA-ends with polymerase chain reaction (RACE-PCR) approach. The SgSQS cDNA has a 1254 bp open reading frame (ORF) encoding 417 amino acids, and the SgCAS cDNA contains a 2298 bp ORF encoding 765 amino acids. Bioinformatic analysis showed that the deduced SgSQS protein has two transmembrane regions in the C-terminal. Both SgSQS and SgCAS have significantly higher levels in fruits than in other tissues, suggesting that steroids and mogrosides are competitors for the same precursors in fruits. Combined in silico prediction and subcellular localization, experiments in tobacco indicated that SgSQS was probably in the cytoplasm or on the cytoskeleton, and SgCAS was likely located in the nucleus or cytosol. These results will provide a foundation for further study of SgSQS and SgCAS gene functions in S. grosvenorii, and may facilitate improvements in mogroside content in fruit by regulating gene expression.

Transcriptome Analysis of Taxillusi chinensis (DC.) Danser Seeds in Response to Water Loss.

BACKGROUND: Taxillus chinensis (DC.) Danser, the official species of parasitic loranthus that grows by parasitizing other plants, is used in various traditional Chinese medicine prescriptions. ABA-dependent and ABA-independent pathways are two major pathways in response to drought stress for plants and some genes have been reported to play a key role during the dehydration including dehydration-responsive protein RD22, late embryogenesis abundant (LEA) proteins, and various transcription factors (TFs) like MYB and WRKY. However, genes responding to dehydration are still unknown in loranthus. METHODS AND RESULTS: Initially, loranthus seeds were characterized as recalcitrant seeds. Then, biological replicates of fresh loranthus seeds (CK), and seeds after being dehydrated for 16 hours (Tac-16) and 36 hours (Tac-36) were sequenced by RNA-Seq, generating 386,542,846 high quality reads. A total of 164,546 transcripts corresponding to 114,971 genes were assembled by Trinity and annotated by mapping them to NCBI non-redundant (NR), UniProt, GO, KEGG pathway and COG databases. Transcriptome profiling identified 60,695, 56,027 and 66,389 transcripts (>1 FPKM) in CK, Tac-16 and Tac-36, respectively. Compared to CK, we obtained 2,102 up-regulated and 1,344 down-regulated transcripts in Tac-16 and 1,649 up-regulated and 2,135 down-regulated transcripts in Tac-36 by using edgeR. Among them some have been reported to function in dehydration process, such as RD22, heat shock proteins (HSP) and various TFs (MYB, WRKY and ethylene-responsive transcription factors). Interestingly, transcripts encoding ribosomal proteins peaked in Tac-16. It is indicated that HSPs and ribosomal proteins may function in early response to drought stress. Raw sequencing data can be accessed in NCBI SRA platform under the accession number SRA309567. CONCLUSIONS: This is the first time to profile transcriptome globally in loranthus seeds. Our findings provide insights into the gene regulations of loranthus seeds in response to water loss and expand our current understanding of drought tolerance and germination of seeds.

Identification of novel and conserved microRNAs in Panax notoginseng roots by high-throughput sequencing.

BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNAs that are important regulators of gene expression, and play major roles in plant development and their response to the environment. Root extracts from Panax notoginseng contain triterpene saponins as their principal bioactive constituent, and demonstrate medicinal properties. To investigate the novel and conserved miRNAs in P. notoginseng, three small RNA libraries constructed from 1-, 2-, and 3-year-old roots in which root saponin levels vary underwent high-throughput sequencing. METHODS: P. notoginseng roots, purified from 1-, 2-, and 3-year-old roots, were extracted for RNA, respectively. Three small libraries were constructed and subjected to next generation sequencing. RESULTS: Sequencing of the three libraries generated 67,217,124 clean reads from P. notoginseng roots. A total of 316 conserved miRNAs (belonging to 67 miRNA families and one unclassified family) and 52 novel miRNAs were identified. MIR156 and MIR166 were the largest miRNA families, while miR156i and miR156g showed the highest abundance of miRNA species. Potential miRNA target genes were predicted and annotated using Cluster of Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes. Comparing these miRNAs between root samples revealed 33 that were differentially expressed between 2- and 1-year-old roots (8 increased, 25 decreased), 27 differentially expressed between 3- and 1-year-old roots (7 increased, 20 decreased), and 29 differentially expressed between 3- and 2-year-old roots (8 increased, 21 decreased). Two significantly differentially expressed miRNAs and four miRNAs predicted to target genes involved in the terpenoid backbone biosynthesis pathway were selected and validated by quantitative reverse transcription PCR. Furthermore, the expression patterns of these six miRNAs were analyzed in P. notoginseng roots, stems, and leaves at different developmental stages. CONCLUSIONS: This study identified a large number of P. notoginseng miRNAs and their target genes, functional annotations, and gene expression patterns. It provides the first known miRNA profiles of the P. notoginseng root development cycle.

[Study on exogenous hormones inducing parthenocarpy fruit growth and development and quality of Siraitia grosvenorii].

To explore the growth and development and analyze the quality of the parthenocarpy fruit induced by exogenous hormones of Siraitia grosvenorii. the horizontal and vertical diameter, volume of the fruit were respectively measured by morphological and the content of endogenous hormones were determined by ELISA. The size and seed and content of mogrosides of mature fruit were determined. The results showed that the fruit of parthenocarpy was seedless and its growth and development is similar to the diploid fruit by hand pollination and triploid fruit by hand pollination or hormones. But the absolute value of horizontal and vertical diameter, volume of parthenocarpy fruit was less than those of fruit by hand pollination, while triploid was opposite. The content of IAA, ABA and ratio of ABA/GA was obviously wavy. At 0-30 d the content of IAA and ABA of parthenocarpy fruit first reduced then increased, content of IAA and GA parthenocarpy fruit was higher than that of fruit by hand pollination. Mogrosides of parthenocarpy fruit was close to pollination fruit. Hormones can induce S. grosvenorii parthenocarpy to get seedless fruit and the fruit shape and size and quality is close to normal diploid fruit by hand pollination and better than triploid fruit by hormone or hand pollination.

[Rapid propagation system for tissure culture of Smilax glabra].

OBJECTIVE: Establishing the rapid propagation for plant tissue culture of Smilax glabra in order to rationally protect and utilize the resource. METHODS: The seed, shoot, leaf and rhizome of Similax glabra were cultured as explants. Different media and plant growth regulators at different concerntration were used. RESULTS: The best medium for bud induction was MS + 6-BA 2.0 mg/L + NAA 0.05 mg/L; The optimal medium for proliferation culture was MS +6-BA 2.0 mg/L + NAA 0.5 mg/L; The optimal medium for rooting was H + IBA 0.5 mg/L + NAA 0.5 mg/L; The optimal stroma for seedlings was 50% peat + 50% river sand. CONCLUSION: Different explants except leaf can be induced for tissue culture and rhizome has the optimal bud induction rate. An effective approach for rapid propagation has been provided,which can promote the artificial cultivation of Smilax glabra.

[Extraction of total RNA and cloning of sgDHAR gene from Siraitia grosvenorii].

Total RNA was isolated from Siraitia grosvenorii fruit by the method of modified Trizol, according to S. grosvenorii fruit characteristics of rich phenols, polysaccharide, oil and proteins. The OD260/280, OD260/230, RNA integrity (RIN) and yield of the total RNA with this method were 2.01, 2.02, 9.50 and 260 mirog.g-1, respectively. The open reading frame (ORF) of dehydroascorbate reductase (DHAR), named as SgDHAR, was cloned by rapid amplification of cDNA ends (RACE) and RT-PCR method from S. grosvenorii. The GenBank accession number for this gene is KC907731. The SgDHAR gene contains a full-length cDNA of 1,252 bp including ORF of 819 bp and encodes a predicted protein of 272 amino acids. The molecular mass is 30.217 7 kD and the isoelectric point is 8.76. Homology comparison showed that it shared 87% nucleotide sequence homology with Cucumis sativus. Expression patterns using qRT-PCR analysis showed that SgDHAR was mainly expressed in fruit and stem, followed by flower, and was lowest in root, while the expression level was 6.83 times in triploid. T than that in diploid. Therefore, SgDHAR gene may be involved in abortion of triploid seedless S. grosvenorii.

[Effect of Nutrient Solution in Soilless Cultivation on Growth, Antioxidase Activities and Amino Acid Contents of Nervilia fordii].

OBJECTIVE: To select the best formula for soilless cultivation of Nervilia fordii. METHODS: Four different formulas of nutrient solution on growth,antioxidase activities and amino acid contents were investigated. RESULTS: The concentration of 1/2 lettuce nutrient solution formula was the most propitious for the growth of Nervilia fordii, the antioxidase activities and amino acid contents were increased in the treatment of nutrient solution,and the highest amino acid content was observed in 1/2 dose of green leafy vegetables nutrient solution formula. CONCLUSION: The concentration of 1/2 lettuce nutrient solution formula is the optimal solution for growth of Nervilia fordii, the results of this study provides academic and technological basis for soilless cultivation of Nervilia fordii.

[Study on quantitative assessment of Siraitia grosvenorii germplasms by general index].

OBJECTIVE: To establish a weight-marking method and evaluate Siraitia grosvenorii germplasms. METHOD: The characters of 21 kinds of S. grosvenorii germplasms in the field and the lab were analyzed, seven important characters were selected to weight the marks. A general evaluation index was made and used to evaluate S. grosvenorii germplasms. RESULT: The evaluation result of 21 kinds of S. grosvenorii germplasms by the weight-marking method was consistent with production practice. Meanwhile, the new variety Yongqing No. 1 and major cultivars were the superior germplasms. CONCLUSION: A rational technique system of evaluating S. grosvenorii germplasms was established, and the superior germplasms were selected.

[Progress on research of tissue culture of Siraitia grosvenorii].

In this paper we reviewed the development of tissue culture, current situations of virus-free plantlets industrialization and the way to deal with the situations, application prospects of Siraitia grosvenorii so as to give some advice for its further study and application.