Salvia miltiorrhiza ameliorates endometritis in dairy cows by relieving inflammation, energy deficiency and blood stasis.

Introduction: According to traditional Chinese veterinary medicine, endometritis is caused by a combination of Qi deficiency, blood stasis, and external evil invasion. Salvia miltiorrhiza is a traditional Chinese medicine that counteracts blood stasis and has additional demonstrated effects in boosting energy and restraining inflammation. Salvia miltiorrhiza has been employed in many traditional Chinese prescriptions that have proven effective in healing clinical dairy cow endometritis. Methods: the in vivo effect of Salvia miltiorrhiza in treating endometritis was evaluated in dairy cows. In addition, bovine endometrial epithelium cell inflammation and rat blood stasis models were employed to demonstrate the crosstalk between energy, blood circulation and inflammation. Network analysis, western blotting, qRT-PCR and ELISA were performed to investigate the molecular mechanism of Salvia miltiorrhiza in endometritis treatment. Results: The results demonstrate that treatment with Salvia miltiorrhiza relieves uterine inflammation, increases blood ATP concentrations, and prolongs blood clotting times. Four of the six Salvia miltiorrhiza main components (SMMCs) (tanshinone IIA, cryptotanshinone, salvianolic acid A and salvianolic acid B) were effective in reversing decreased ATP and increased IL-1ß, IL-6, and IL-8 levels in an in vitro endometritis model, indicating their abilities to ameliorate the negative energy balance and external evil invasion effects of endometritis. Furthermore, in a blood stasis rat model, inflammatory responses were induced in the absence of external infection; and all six SMMCs inhibited thrombin-induced platelet aggregation. Network analysis of SMMC targets predicted that Salvia miltiorrhiza may mediate anti-inflammation via the Toll-like receptor signaling pathway; anti-aggregation via the Platelet activation pathway; and energy balance via the Thermogenesis and AMPK signaling pathways. Multiple molecular targets within these pathways were verified to be inhibited by SMMCs, including P38/ERK-AP1, a key molecular signal that may mediate the crosstalk between inflammation, energy deficiency and blood stasis. Conclusion: These results provide mechanistic understanding of the therapeutic effect of Salvia miltiorrhiza for endometritis achieved through Qi deficiency, blood stasis, and external evil invasion.

Identification of BBX gene family and its function in the regulation of microtuber formation in yam.

BBX proteins play important roles in all of the major light-regulated developmental processes. However, no systematic analysis of BBX gene family regarding the regulation of photoperiodic microtuber formation has been previously performed in yam. In this study, a systematic analysis on the BBX gene family was conducted in three yam species, with the results, indicating that this gene plays a role in regulating photoperiodic microtuber formation. These analyses included identification the BBX gene family in three yam species, their evolutionary relationships, conserved domains, motifs, gene structure, cis-acting elements, and expressional patterns. Based on these analyses, DoBBX2/DoCOL5 and DoBBX8/DoCOL8 showing the most opposite pattern of expression during microtuber formation were selected as candidate genes for further investigation. Gene expression analysis showed DoBBX2/DoCOL5 and DoBBX8/DoCOL8 were highest expressed in leaves and exhibited photoperiod responsive expression patterns. Besides, the overexpression of DoBBX2/DoCOL5 and DoBBX8/DoCOL8 in potato accelerated tuber formation under short-day (SD) conditions, whereas only the overexpression of DoBBX8/DoCOL8 enhanced the accelerating effect of dark conditions on tuber induction. Tuber number was increased in DoBBX8/DoCOL8 overexpressing plants under dark, as well as in DoBBX2/DoCOL5 overexpressing plants under SD. Overall, the data generated in this study may form the basis of future functional characterizations of BBX genes in yam, especially regarding their regulation of microtuber formation via the photoperiodic response pathway.

Construction of SNP fingerprint and population genetic analysis of honeysuckle germplasm resources in China.

Introduction: The flower buds of Lonicera japonica Thunb. are widely used in Chinese medicine for their anti-inflammatory properties, and they have played an important role in the fight against SARS COVID-19 and other major epidemics. However, due to the lack of scientific and accurate variety identification methods and national unified standards, scattered and non-standardized management in flower bud production has led to mixed varieties that have caused significant difficulties in the cataloging and preservation of germplasm resources and the identification, promotion, and application of new L. japonica varieties. Methods: In this study, we evaluated the population structure, genetic relationships, and genetic fingerprints of 39 germplasm resources of Lonicera in China using simplified genome sequencing technology. Results: A total of 13,143,268 single nucleotide polymorphisms (SNPs) were identified. Thirty-nine samples of Lonicera were divided into four subgroups, and the population structure and genetic relationships among existing Lonicera germplasm resources were determined using principal component analysis, population structure analysis, and phylogenetic tree analysis. Through several stringent selection criteria, 15 additional streamlined, high-quality DNA fingerprints were filtered out of the validated 50 SNP loci and verified as being able to effectively identify the 39 Lonicera varieties. Discussion: To our knowledge, this is the first comprehensive study measuring the diversity and population structure of a large collection of Lonicera varieties in China. These results have greatly broadened our understanding of the diversity, phylogeny, and population structure of Lonicera. The results may enhance the future analysis of genetic diversity, species identification, property rights disputes, and molecular breeding by providing a scientific basis and reference data for these efforts.

Identification of CmbHLH Transcription Factor Family and Excavation of CmbHLHs Resistant to Necrotrophic Fungus Alternaria in Chrysanthemum.

Chrysanthemum morifolium Ramat. 'Huaihuang' is a traditional Chinese medicinal plant. However, a black spot disease caused by Alternaria sp., a typical necrotrophic fungus, has a serious damaging influence on the field growth, yield, and quality of the plant. 'Huaiju 2#' being bred from 'Huaihuang', shows resistance to Alternaria sp. bHLH transcription factor has been widely studied because of their functions in growth development, signal transduction, and abiotic stress. However, the function of bHLH in biotic stress has rarely been studied. To characterize the resistance genes, the CmbHLH family was surveyed in 'Huaiju 2#'. On the basis of the transcriptome database of 'Huaiju 2#' after Alternaria sp. inoculation, with the aid of the Chrysanthemum genome database, 71 CmbHLH genes were identified and divided into 17 subfamilies. Most (64.8%) of the CmbHLH proteins were rich in negatively charged amino acids. CmbHLH proteins are generally hydrophilic proteins with a high aliphatic amino acid content. Among the 71 CmbHLH proteins, five CmbHLHs were significantly upregulated by Alternaria sp. infection, and the expression of CmbHLH18 was the most significant. Furthermore, heterologous overexpression of CmbHLH18 could improve the resistance of Arabidopsis thaliana to necrotrophic fungus Alternaria brassicicola by enhancing callose deposition, preventing spores from entering leaves, reducing ROS accumulation, increasing the activities of antioxidant enzymes and defense enzymes, and promoting their gene expression levels. These results indicate that the five CmbHLHs, especially CmbHLH18, may be considered candidate genes for resistance to necrotrophic fungus. These findings not only increase our understanding of the role CmbHLHs play in biotic stress but also provide a basis by using CmbHLHs to breed a new variety of Chrysanthemum with high resistance to necrotrophic fungus.

Integrated volatile metabolomic and transcriptomic analysis provides insights into the regulation of floral scents between two contrasting varieties of Lonicera japonica.

Lonicera japonica Thunb., belonging to the Caprifoliaceae family, is an important traditional Chinese medicinal plant. The L. japonica flower (LJF) is widely used in medicine, cosmetics, drinks, and food due to its medicinal and sweet-smelling properties. Considerable efforts have been devoted to investigating the pharmacological activities of LJF; however, the regulatory mechanism of the floral scents remains unknown. We previously selected and bred an elite variety of L. japonica var. chinensis Thunb. called 'Yujin2', which has a strong aroma and is used in functional drinks and cosmetics. In order to reveal the regulatory mechanism of the floral scents of LJF, volatile metabolomic and transcriptomic analyses of the LJF at the silver flowering stage of 'Yujin2' (strong aroma) and 'Fengjin1' (bland odor) were performed. Our results revealed that a total of 153 metabolites and 9,523 genes were differentially regulated in LJF between 'Yujin2' and 'Fengjin1'. The integrated analysis of omics data indicated that the biosynthetic pathways of terpenoids (i.e., monoterpenoids, including geraniol and alpha-terpineol; sesquiterpenoids, including farnesol, farnesal, and alpha-farnesene; triterpenoid squalene), tryptophan and its derivatives (methyl anthranilate), and fatty acid derivatives, were major contributors to the stronger aroma of 'Yujin2' compared to 'Fengjin1'. Moreover, several genes involved in the terpenoid biosynthetic pathway were characterized using quantitative real-time PCR. These results provide insights into the metabolic mechanisms and molecular basis of floral scents in LJF, enabling future screening of genes related to the floral scent regulation, such as alpha-terpineol synthase, geranylgeranyl diphosphate synthase, farnesyl pyrophosphate synthase, anthranilate synthase, as well as transcription factors such as MYB, WRKY, and LFY. The knowledge from this study will facilitate the breeding of quality-improved and more fragrant variety of L. japonica for ornamental purpose and functional beverages and cosmetics.

The integration of transcriptomic and transgenic analyses reveals the involvement of the SA response pathway in the defense of chrysanthemum against the necrotrophic fungus Alternaria sp.

Chrysanthemum morifolium cv. 'Huaihuang' has ornamental, edible, medicinal, and tea product uses. However, its field growth, yield, and quality are negatively affected by black spot disease caused by Alternaria sp. (Strain: HQJH10092301; GenBank accession number: KF688111). In this study, we transcriptionally and transgenically characterized a new cultivar, 'Huaiju 2#' (Henan Traditional Chinese Medicine Plant Cultivar identification number: 2016002), which was bred from 'Huaihuang' and shows resistance to Alternaria sp. Numerous 'Huaiju 2#' plants were inoculated with Alternaria sp. for three or five days. Metabolic analysis showed increases in both salicylic acid (SA) and jasmonic acid (JA) in infected plants compared to the control. Protein activity analysis also revealed a significant increase in defense enzyme activities in infected plants. RNA-Seq of plants infected for 3 or 5 days produced a total of 58.6 GB of clean reads. Among these reads, 16,550 and 13,559 differentially expressed genes (DEGs) were identified in Cm_3 dpi (sample from 3 days post-inoculation labeled as Cm_3 dpi) and Cm_5 dpi (sample from 5 days post-inoculation labeled as Cm_5 dpi), respectively, compared with their controls (Cm_0 d: a mixture samples from 0 d (before inoculation) and those treated with sterile distilled water at 3 dpi and 5 dpi). Gene annotation and cluster analysis of the DEGs revealed a variety of defense responses to Alternaria sp. infection, which were characterized by increases in resistance (R) proteins and the reactive oxygen species (ROS), Ca2+, mitogen-activated protein kinase (MAPK), and JA signaling pathways. In particular, SA signaling was highly responsive to Alternaria sp. infection. The qPCR analysis of 12 DEG candidates supported their differential expression characterized by using the RNA-Seq data. One candidate was CmNPR1 (nonexpressor of pathogenesis-related gene 1), an important positive regulator of SA in systemic acquired resistance (SAR). Overexpression of CmNPR1 in 'Huaiju 2#' increased the resistance of transgenic plants to black spot. These findings indicate that the SA response pathway is likely involved in the defense of 'Huaiju 2#' against Alternaria sp. pathogens.

Transcriptome Analysis Reveals Key Pathways and Hormone Activities Involved in Early Microtuber Formation of Dioscorea opposita.

Chinese yam (Dioscorea opposita) is an important tuberous crop used for both food and medicine. Despite a long history of cultivation, the understanding of D. opposita genetics and molecular biology remains scant, which has limited its genetic improvement. This work presents a de novo transcriptome sequencing analysis of microtuber formation in D. opposita. We assembled cDNA libraries from different stages during the process of microtuber formation, designated as initial explants (EXP), axillary bud proliferation after three weeks (BUD), and microtuber visible after four weeks (MTV). More differentially expressed genes (DEGs) and pathways were identified between BUD vs. EXP than in MTV vs. BUD, indicating that proliferation of the axillary bud is the key stage of microtuber induction. Gene classification and pathway enrichment analysis showed that microtuber formation is tightly coordinated with primary metabolism, such as amino acid biosynthesis, ribosomal component biosynthesis, and starch and sucrose metabolism. The formation of the microtuber is regulated by a variety of plant hormones, including ABA. Combined with analysis of physiological data, we suggest that ABA positively regulates tuberization in D. opposita. This study will serve as an empirical foundation for future molecular studies and for the propagation of D. opposita germplasm in field crops.

Artemisinin Biosynthesis in Non-glandular Trichome Cells of Artemisia annua.

Artemisinin-based combination therapy (ACT) forms the first line of malaria treatment. However, the yield fluctuation of artemisinin has remained an unsolved problem in meeting the global demand for ACT. This problem is mainly caused by the glandular trichome (GT)-specific biosynthesis of artemisinin in all currently used Artemisia annua cultivars. Here, we report that non-GT cells of self-pollinated inbred A. annua plants can express the artemisinin biosynthetic pathway. Gene expression analysis demonstrated the transcription of six known pathway genes in GT-free leaves and calli of inbred A. annua plants. LC-qTOF-MS/MS analysis showed that these two types of GT-free materials produce artemisinin, artemisinic acid, and arteannuin B. Detailed IR-MALDESI image profiling revealed that these three metabolites and dihydroartemisinin are localized in non-GT cells of leaves of inbred A. annua plants. Moreover, we employed all the above approaches to examine artemisinin biosynthesis in the reported A. annua glandless (gl) mutant. The resulting data demonstrated that leaves of regenerated gl plantlets biosynthesize artemisinin. Collectively, these findings not only add new knowledge leading to a revision of the current dogma of artemisinin biosynthesis in A. annua but also may expedite innovation of novel metabolic engineering approaches for high and stable production of artemisinin in the future.

Identification and validation of reference genes for qRT-PCR analysis in mulberry (Morus alba L.).

Mulberry (Morus alba L.) is an important economic tree species in many countries. Quantitative real time PCR (qRT-PCR) has become a widely used method for gene expression studies in plants. A suitable reference gene is essential to ensure accurate and reliable results for qRT-PCR analyses. However, no reports describing the selection of reference genes have been published for mulberry. In this work, we evaluated the stability of twenty candidate reference genes in different plant tissues and under different stress conditions by qRT-PCR in mulberry using algorithms in two programs-geNorm and NormFinder. The results revealed that TUB2, UBI4, ACTIN3 and RPL4 were ranked as the most stable reference genes in the samples subsets, whereas EF1α4 and TUB3showed the least stability with both algorithms. To further validate the stability of the reference genes, the expression patterns of six genes of mulberry were analyzed by normalization with the selected reference genes. Our study will benefit future analyses of gene expression in mulberry.

Validation of Suitable Reference Genes for RT-qPCR Data in Achyranthes bidentata Blume under Different Experimental Conditions.

Real-time quantitative polymerase chain reaction (RT-qPCR) is a sensitive technique for gene expression studies. However, choosing the appropriate reference gene is essential to obtain reliable results for RT-qPCR assays. In the present work, the expression of eight candidate reference genes, EF1-α (elongation factor 1-α), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), UBC (ubiquitin-conjugating enzyme), UBQ (polyubiquitin), ACT (actin), ß-TUB (ß-tubulin), APT1 (adenine phosphoribosyltransferase 1), and 18S rRNA (18S ribosomal RNA), was evaluated in Achyranthes bidentata samples using two algorithms, geNorm and NormFinder. The samples were classified into groups according to developmental stages, various tissues, stresses (cold, heat, drought, NaCl), and hormone treatments (MeJA, IBA, SA). Suitable combination of reference genes for RT-qPCR normalization should be applied according to different experimental conditions. In this study, EF1-α, UBC, and ACT genes were verified as the suitable reference genes across all tested samples. To validate the suitability of the reference genes, we evaluated the relative expression of CAS, which is a gene that may be involved in phytosterol synthesis. Our results provide the foundation for gene expression analysis in A. bidentata and other species of Amaranthaceae.

Transcriptome Analysis to Identify the Putative Biosynthesis and Transport Genes Associated with the Medicinal Components of Achyranthes bidentata Bl.

Achyranthes bidentata is a popular perennial medicine herb used for 1000s of years in China to treat various diseases. Although this herb has multiple pharmaceutical purposes in China, no transcriptomic information has been reported for this species. In addition, the understanding of several key pathways and enzymes involved in the biosynthesis of oleanolic acid and ecdysterone, two pharmacologically active classes of metabolites and major chemical constituents of A. bidentata root extracts, is limited. The aim of the present study was to characterize the transcriptome profile of the roots and leaves of A. bidentata to uncover the biosynthetic and transport mechanisms of the active components. In this study, we identified 100,987 transcripts, with an average length of 1146.8 base pairs. A total of 31,634 (31.33%) unigenes were annotated, and 12,762 unigenes were mapped to 303 pathways according to the Kyoto Encyclopedia of Genes and Genomes pathway database. Moreover, we identified a total of 260 oleanolic acid and ecdysterone genes encoding biosynthetic enzymes. Furthermore, the key enzymes involved in the oleanolic acid and ecdysterone synthesis pathways were analyzed using quantitative real-time polymerase chain reaction, revealing that the roots expressed these enzymes to a greater extent than the leaves. In addition, we identified 85 ATP-binding cassette transporters, some of which might be involved in the translocation of secondary metabolites.

Identification and Validation of Reference Genes for qRT-PCR Studies of Gene Expression in Dioscorea opposita.

Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most common methods for gene expression studies. Data normalization based on reference genes is essential for obtaining reliable results for qRT-PCR assays. This study evaluated potential reference genes of Chinese yam (Dioscorea opposita Thunb.), which is an important tuber crop and medicinal plant in East Asia. The expression of ten candidate reference genes across 20 samples from different organs and development stages was assessed. We identified the most stable genes for qRT-PCR studies using combined samples from different organs. Our results also suggest that different suitable reference genes or combinations of reference genes for normalization should be applied according to different organs and developmental stages. To validate the suitability of the reference genes, we evaluated the relative expression of PE2.1 and PE53, which are two genes that may be associated with microtuber formation. Our results provide the foundation for reference gene(s) selection in D. opposita and will contribute toward more accurate gene analysis studies of the genus Dioscorea.

A multiplex RT-PCR for simultaneous detection and identification of five viruses and two viroids infecting chrysanthemum.

Pathogens causing significant economic losses in chrysanthemum include tomato aspermy virus (TAV), chrysanthemum virus B (CVB), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), potato virus Y (PVY), chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd). A multiplex reverse transcription polymerase chain reaction (RT-PCR) method, using specific primer sets for each virus or viroid, was developed for simultaneous detection and differentiation of TAV, CVB, CMV, TMV, PVY, CChMVd, and CSVd. The RT-PCR method was validated by testing chrysanthemum samples collected from different regions of China. In this study, CVB, TAV, TMV, PVY, CSVd, CMV, and CChMVd were detected, respectively, in 24.7 %, 17.5 %, 4.4 %, 4.4 %, 2.9 %, 2.5 %, and 1.5 % of the samples tested. These results indicate that CVB and TAV (24.7 % and 17.5 %) are common, whereas CMV, TMV, CChMVd, CSVd, and PVY (all below 5 %) are less frequently encountered. This new multiplex RT-PCR method has potential to be used routinely in large-scale virus and viroid surveys.

Multiplex reverse transcription loop-mediated isothermal amplification for the simultaneous detection of CVB and CSVd in chrysanthemum.

A multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) assay was developed for the simultaneous detection of Chrysanthemum Virus B (CVB) and Chrysanthemum stunt viroid (CSVd), which are the major viral pathogens of chrysanthemum worldwide. Two sets of mRT-LAMP primers were designed for the coat protein gene of CVB and the complete nucleotide sequence of CSVd, and a restriction enzyme cleavage site was inserted into two pairs of species-specific primers. The mRT-LAMP assay was designed by combining these two sets for a total of eight primers. The mRT-LAMP method distinguished between CVB and CSVd due to the subsequent restriction enzyme analysis. The sensitivity of the mRT-LAMP method was 10(3) times higher than classical PCR regarding the detection limits for CVB and CSVd. No positive results were observed when RNA from other chrysanthemum pathogens were used as mRT-LAMP templates. The method was verified by testing chrysanthemum samples collected from Beijing and Henan Province and showed high reliability and sensitivity. The developed mRT-LAMP assay also offers an efficient, convenient, and rapid tool for screening chrysanthemum virus and viroid, especially CVB and CSVd, and can be diagnosed in a single reaction. These results suggest that the new mRT-LAMP method may be used routinely for virus and viroid surveys.

A simple and rapid method for visualization of male meiotic chromosomes in Arabidopsis thaliana.

Meiotic chromosomes are of basic interest to the geneticist and cell biologist who study their behavior. A rapid and highly repeatable method for visualization of meiotic chromosomes is useful. Here we describe a fast staining protocol for Arabidopsis male meiotic chromosomes. Meiocytes were squashed into a labeling buffer, the chromosome morphology could be analyzed using fluorescence without any additional treatment.

Identifying local co-regulation relationships in gene expression data.

Identifying interesting relationships between pairs of genes, presented over some of experimental conditions in gene expression data set, is useful for discovering novel functional gene interactions. In this paper, we introduce a new method for id entifying L ocal C o-regulation R elationships (IdLCR). These local relationships describe the behaviors of pairwise genes, which are either up- or down-regulated throughout the identified condition subset. IdLCR firstly detects the pairwise gene-gene relationships taking functional forms and the condition subsets by using a regression spline model. Then it measures the relationships using a penalized Pearson correlation and ranks the responding gene pairs by their scores. By this way, those relationships without clearly biological interpretations can be filtered out and the local co-regulation relationships can be obtained. In the simulation data sets, ten different functional relationships are embedded. Applying IdLCR to these data sets, the results show its ability to identify functional relationships and the condition subsets. For micro-array and RNA-seq gene expression data, IdLCR can identify novel biological relationships which are different from those uncovered by IFGR and MINE.

Cryopreservation of plantlet nodes of Dioscorea opposita Thunb. using a vitrification method.

A cryopreservation method by vitrification was developed for long-term storage of Dioscorea opposita Thunb., a valuable native medicinal plant species in Henan Province of China. The cryopreservation protocol was established with cultivar B and evaluated with another four cultivars, Tiegun, 47, Taigu and Huaiqing 1. The results showed that nodes with a bud excised from 60 d plantlets were desirable for the cryopreservation. The optimum procedure was established as: 1) the plantlets were cultured on the Murashige and Skoog (MS) medium supplemented with 2 mg L(-1) KT and 0.02 mg L(-1) NAA at 4 degree C for 7 d before nodes with length of 1-1.5 cm were excised; 2) the nodes were precultured at 4 degree C for 7 d on the MS supplemented with 10 percent dimethyl sulfoxide (DMSO) followed by loading with 60 percent Dioscorea vitrification solution 1 (DVS1): 22 percent (w/v) glycerol + 13 percent (w/v) ethylene glycol + 13 percent (w/v) polyethylene glycol + 10 percent (w/v) DMSO for 60 min at 0 degree C and dehydrated with 100 percent DVS1 for 60 min at 0 degree C; 3) the nodes were then immersed into liquid nitrogen (LN) directly and conserved for 180 d; 4) after rapid thawing in a water-bath at 37 degree C, the nodes were rinsed four times with MS medium supplemented with 5 percent sucrose, then transferred to the MS medium supplemented with 2 mg L(-1) kinetin (KT) and 0.02 mg L(-1) NAA for regeneration. In the present research the regeneration rate of cv. B was about 77.1 percent, those of cvs. Tiegun and Huaiqing 1 were 67.2 percent and 54.0 percent respectively, while cvs. Taigu and 47 were about 40 percent. There were no visual changes observed between the plantlets regenerated from nodes with and without cryopreservation in terms of the morphology indices, indicating that the method established could be applicable to D. opposita with optimized protocol.

[Effect of medium components on callus induction and contents of polysaccharides in calli from Achyranthes bidentata].

OBJECTIVE: To study the effect of medium components on the callus induction and the contents of polysaccharides in calli from Achyranthes bidentata. METHOD: Leaves and stems were selected as explants. The effects of six kinds of factors including basal culture medium, carbon source, 2,4-D, 6-BA, TDZ, CH on the callus induction and the contents of ABPS in calli on the high growth point were studied by orthogonal design method. The data were analyzed with range analysis and variance analysis. RESULT: To leaf, the optimal medium of callus induction was B5 with 2 mg x L(-1) 2,4-D, 0.5 mg x L(-1) 6-BA, 30 g x L(-1) glucose and 1 g x L(-1) CH; to stem, the optimal one was B5 with2 mg x L(-1) 2,4-D, 1 mg x L(-1) 6-BA, 30 g x L(-1) glucose and 0.5 g x L(-1) CH. In order to obtain higher contents ABPS, to leaf calli, the optimal medium was LS with 1 mg x L(-1) 6-BA, 1 mg x L(-1) TDZ and 30 g x L(-1) sucrose; to stem calli, the optimal one was LS with 1 mg L(-1) 2,4-D, 0.5 mg x L(-1) 6-BA, 1 mg x L(-1) TDZ and 30 g x L(-1) glucose. CONCLUSION: The optimal media of callus induction were established with stems and leaves of A. bidentata as explants and with a view to an industrial production of polysaccharides by tissue and cell culture.