[Correlation between traditional Chinese medicine and reduced risk of readmission in rheumatoid arthritis patients with hypoproteinemia:a retrospective cohort study].

This study aimed to explore the correlation between traditional Chinese medicine(TCM) and reduced risk of readmission in patients having rheumatoid arthritis with hypoproteinemia(RA-H). A retrospective cohort study was conducted on 2 437 rheumatoid arthritis patients in the information system database of the First Affiliated Hospital of Anhui University of Chinese Medicine from 2014 to 2021, and 476 of them were found to have hypoproteinemia. The patients were divided into TCM users and non-TCM users by propensity score matching. Exposure was defined as the use of oral Chinese patent medicine or herbal decoction for ≥1 month. Cox regression analysis was performed to explore the risk factors of clinical indicators of rheumatoid arthritis. Additionally, the use of TCM during hospitalization was analyzed, and analysis of association rules was conducted to investigate the correlation between TCM, improvement of indicators and readmission of patients. Kaplan-Meier survival curve was plotted to compare the readmission rate of TCM users and non-TCM users. It was found the readmission rate of RA-H patients was significantly higher than that of RA patients. By propensity score matching, 232 RA-H patients were divided into TCM group(116 cases) and non-TCM group(116 cases). Compared with the conditions in the non-TCM group, the readmission rate of the TCM group was lowered(P<0.01), and the readmission rate of middle-aged and elderly patients was higher than that of young patients(P<0.01). Old age was a risk factor for readmission of RA-H patients, while TCM, albumin(ALB) and total protein(TP) were the protective factors. During hospitalization, the TCMs used for RA-H patients were mainly divided into types of activating blood and resolving stasis, relaxing sinew and dredging collaterals, clearing heat and detoxifying, and invigorating spleen and resolving dampness. The improvement of rheumatoid factor(RF), immunoglobulin G(IgG), erythrocyte sedimentation rate(ESR), C-reactive protein(CRP) and ALB was closely related to TCM. On the basis of western medicine treatment, the application of TCM could reduce the readmission rate of RA-H patients, and longer use of TCM indicated lower readmission rate.

Polymerase chain reaction-based assays facilitate the breeding and study of mouse models of Klinefelter syndrome.

Klinefelter syndrome (KS) is one of the most frequent genetic abnormalities and the leading genetic cause of nonobstructive azoospermia. The breeding and study of KS mouse models are essential to advancing our knowledge of the underlying pathological mechanism. Karyotyping and fluorescence in situ hybridization are reliable methods for identifying chromosomal contents. However, technical issues associated with these methods can decrease the efficiency of breeding KS mouse models and limit studies that require rapid identification of target mice. To overcome these limitations, we developed three polymerase chain reaction-based assays to measure specific genetic information, including presence or absence of the sex determining region of chromosome Y (Sry), copy number of amelogenin, X-linked (Amelx), and inactive X specific transcripts (Xist) levels. Through a combined analysis of the assay results, we can infer the karyotype of target mice. We confirmed the utility of our assays with the successful generation of KS mouse models. Our assays are rapid, inexpensive, high capacity, easy to perform, and only require small sample amounts. Therefore, they facilitate the breeding and study of KS mouse models and help advance our knowledge of the pathological mechanism underlying KS.

New paraconic acids from the endophytic fungus Xylariaceae sp. J4.

Three new paraconic acids, xylariacinics A-C (1-3), were isolated from the endophyte Xylariaceae sp. J4 harbored in the medicinal plant Blumea balsamifera. Their structures were elucidated on the basis of extensive spectroscopic data including HRMS, and NMR. The antibacterial efficacies of compounds 1-3 were evaluated against a panel of bacteria such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. Their antifungal activities were also tested against Colletotrichum gloeosporioides. Unfortunately, all of them were inactive.

Cerebral palsy in children born after assisted reproductive technology: a meta-analysis.

BACKGROUND: Several studies have assessed the association between cerebral palsy (CP) and assisted reproductive technology (ART), but the results remain controversial. We conducted a meta-analysis to evaluate the risk of CP after ART compared with natural conceptions and to examine CP risk separately in ART singletons, multiples and preterm births. METHODS: Web-based databases (PubMed, Embase, the Cochrane Library, and Web of Science) were searched until November 22, 2020. Studies which compare CP rates after ART with natural conceptions were included. The Newcastle-Ottawa Scale was used to assess the quality of the included studies. Effect estimates were extracted and combined using the fixed-effects or random-effects model depending on the heterogeneity test. RESULTS: There were nine studies included in the meta-analysis. The included studies were of moderate or high quality. A significantly higher risk of CP [odds ratio (OR) = 2.17, 95% confidence interval (CI) 1.72-2.74] was found in ART children (n = 89,214) compared with naturally conceived children (n = 4,160,745). The significantly higher risk decreased when data were restricted to singletons (OR = 1.36, 95% CI 1.16-1.59) and disappeared when data were restricted to multiples (OR = 1.05, 95% CI 0.86-1.29) or preterm births (OR = 1.53, 95% CI 0.66-3.56). Subgroup and sensitivity analyses indicated that the overall results were robust. CONCLUSIONS: The risk of CP is increased more than two-fold after ART. This increased risk is largely due to increased rates of multiple birth and preterm delivery in ART children.

[Traditional medicinal plants for arthropod-borne diseases of five countries in Lancang-Mekong region:a review].

Arthropod-borne diseases, such as malaria and dengue fever, have frequently beset five countries(Cambodia, Vietnam, Laos, Myanmar, and Thailand) in the tropical rainy Lancang-Mekong region, which pose a huge threat to social production and daily life. As a resort to such diseases, chemical drugs risk the resistance in plasmodium, non-availability for dengue virus, and pollution to the environment. Traditional medicinal plants have the multi-component, multi-target, and multi-pathway characteristics, which are of great potential in drug development. Exploring potential medicinals for arthropod-borne diseases from traditional medicinal plants has become a hot spot. This study summarized the epidemiological background of arthropod-borne diseases in the Lancang-Mekong region and screened effective herbs from the 350 medicinal plants recorded in CHINA-ASEAN Traditional Medicine. Based on CNKI, VIP, and PubMed, the plants for malaria and dengue fever and those for killing and repelling mosquitoes were respectively sorted out. Their pharmacological effects and mechanisms were reviewed and the material basis was analyzed. The result is expected to serve as a reference for efficient utilization of medicinal resources, development of effective and safe drugs for malaria and dengue fever, and the further cooperation between China and the other five countries in the Lancang-Mekong region.

Microglia-derived TNF-α mediates endothelial necroptosis aggravating blood brain-barrier disruption after ischemic stroke.

Endothelium (EC) is a key component of blood-brain barrier (BBB), and has an important position in the neurovascular unit. Its dysfunction and death after cerebral ischemic/reperfusion (I/R) injury not only promote evolution of neuroinflammation and brain edema, but also increase the risk of intracerebral hemorrhage of thrombolytic therapies. However, the mechanism and specific interventions of EC death after I/R injury are poorly understood. Here we showed that necroptosis was a mechanism underlying EC death, which promoted BBB breakdown after I/R injury. Treatment of rats with receptor interacting protein kinase 1 (RIPK1)-inhibitor, necrostatin-1 reduced endothelial necroptosis and BBB leakage. We furthermore showed that perivascular M1-like microglia-induced endothelial necroptosis leading to BBB disruption requires tumor necrosis factor-α (TNF-α) secreted by M1 type microglia and its receptor, TNF receptor 1 (TNFR1), on endothelium as the primary mediators of these effects. More importantly, anti-TNFα (infliximab, a potent clinically used drug) treatment significantly ameliorate endothelial necroptosis, BBB destruction and improve stroke outcomes. Our data identify a previously unexplored role for endothelial necroptosis in BBB disruption and suggest infliximab might serve as a potential drug for stroke therapy.

Postoperative Guillain-Barré Syndrome, a Neurologic Complication that Must Not Be Overlooked: A Literature Review.

BACKGROUND: Guillain-Barré syndrome (GBS) is an uncommon, yet life-threatening postoperative neuropathic complication that is easily neglected, and hence, timely treatment is not provided in the clinics. This review aims to summarize the clinical features of postoperative GBS, and thus, improve the understanding of postoperative GBS. METHODS: We reviewed the literature on postoperative GBS and assessed the demographic information, clinical manifestation, operation, time of onset of postoperative GBS, and prognosis. RESULTS: A total of 33 cases of postoperative GBS were included in this study. The average age of patients with postoperative GBS was 46.9 years, and there was a peak in occurrence of GBS between ages 50 and 70 years. Men seemed more likely to have postoperative GBS than did women, with a ratio of 2.67:1. Progressive muscular weakness, present in 31 of the cases, was the most common presentation. Patients with spinal surgery were at further increased risk for GBS, and 84.8% of the patients with GBS had a good prognosis after prompt treatment. CONCLUSIONS: Surgery is probably a potential risk factor for the occurrence of GBS. Early diagnosis and prompt treatment are imperative to reduce mortality.

NIR Rapid Assessments of Blumea balsamifera (Ai-na-xiang) in China.

Blumea balsamifera (Ai-na-xiang) is used as an important plant source of natural borneol, which is widely used in the pharmaceutical industry. The aim of this study was to establish the methods based on near infrared (NIR) spectroscopy for determining the geographical origin of B. balsamifera, as well as developing a method for the quantitative rapid analysis of the active pharmaceutical ingredients (APIs). A total of 109 samples were collected from China in 2013 and arbitrarily divided into calibration and prediction sets using the Kennard-Stone algorithm. The l-borneol and total flavone contents of the samples were measured by gas chromatography and ultraviolet-visible spectroscopy, respectively. The NIR spectra were acquired using an integrating sphere and a partial least squares (PLS) model was built using the optimum wavelength regions, which were selected using a synergy interval partial least-squares (SiPLS) algorithm. The root mean square errors of prediction of the l-borneol and total flavone models were 0.0779 and 2.2694 mg/g, with R² of 0.9069 and 0.8013, respectively. A discriminant model to determine the geographical origin of B. balsamifera (Guizhou and Hainan) was also established using a partial least squares discriminant analysis method with an optimum pretreatment method. The prediction accuracy rate of the model was 100%. NIR spectroscopy can be used as a reliable and environmentally friendly method to determine the API and the origin of different B. balsamifera samples.

Twins methods quantitatively explore the genetic impact on children and adolescents brain gray matter volume.

The gray matter volumes of 58 pairs of twins ranging in age from 12 to 18 were measured by MRI to explore the genetic and environmental impacts on gray matter volume in twin children and adolescents. By means of A/C/E structural equation modeling, it was found that the gray matter volume in children and adolescents was jointly affected by genetic (A: 0.89) and environmental factors while genetic factors play a greater role. The gray matter volume in frontal lobe, parietal lobe, occipital lobe and lateral temporal lobe was mainly affected by genetics (A: 0.7-0.89), where as the gray matter volume in medial temporal lobe and cingulate cortex was affected by both genetics and environment.

Variations in Essential Oil Yield, Composition, and Antioxidant Activity of Different Plant Organs from Blumea balsamifera (L.) DC. at Different Growth Times.

Blumea balsamifera, also named Ainaxiang, is widely used as an ancient medicinal herb in tropical and subtropical Asia. It is rich in essential oils. In this work the essential oils of B. balsamifera from different plant organs and in different months were extracted, and then analyzed by gas chromatography-mass spectrometry. The results showed that essential oil yield of young leaves was the highest (0.65 mL/100 g), followed by mature leaves (0.57 mL/100 g), and the oil yield was higher in October (0.47 mL/100 g) than other months. A total of 44 compounds were identified, representing 92.64%-96.71% of the oil. Eighteen common chemical components were found among the six plant organs, representing >80% of the oil constituents. l-borneol was the main ingredient in leaves, and its content was the highest in senescent leaves and in December. In the essential oils of young shoots and young stems, the main component was dimethoxydurene. Antioxidant activity was also determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ß-carotene bleaching (BCB) assays. The results indicated that the ß-carotene bleaching activity was far stronger than the DPPH radical-scavenging capacity, and the young leaves and young shoots showed stronger antioxidant activity. Dimethoxydurene, ß-caryophyllene, and α-caryophyllene play a positive role in good antioxidant activity, while ß-eudesmol, phytol, and tetradecanal play a negative role. The antioxidant activity revealed in this study might help in developing this promising bioresource for use in the medicinal and cosmetic industries.

MiR-150 Regulates Poststroke Cerebral Angiogenesis via Vascular Endothelial Growth Factor in Rats.

AIMS: Angiogenesis is a harmonized target for poststroke recovery. Therefore, exploring the mechanisms involved in angiogenesis after stroke is vitally significant. In this study, we are reporting a miR-150-based mechanism underlying cerebral poststroke angiogenesis. METHODS: Rat models of middle cerebral artery occlusion (MCAO) and cell models of oxygen-glucose deprivation were conducted. Capillary density, tube formation, cell proliferation, and cell migration were measured by FITC-dextran assay, matrigel assay, Ki-67 staining, and wound healing assay, respectively. The expression of miR-150 and vascular endothelial growth factor (VEGF) was, respectively, measured by RT-PCR and Western blotting. Dual-luciferase assay was conducted to confirm the binding sites between miR-150 and VEGF. RESULTS: We found that miR-150 expression in the brain and serum of rats subjected to cerebral ischemia, and in oxygen-glucose-deprived brain microvascular endothelial cells (BMVECs) and astrocytes. Upregulation of miR-150 expression could decrease vascular density of infarct border zone in rat after MCAO and decrease tube formation, proliferation, and migration of BMVECs. We also found that miR-150 could negatively regulate the expression of VEGF, and VEGF was confirmed to be a direct target of miR-150. Moreover, VEGF mediated the function of miR-150 on tube formation, proliferation, and migration of BMVECs. CONCLUSIONS: Our data suggested that miR-150 could regulate cerebral poststroke angiogenesis in rats through VEGF.

MicroRNA-150 regulates blood-brain barrier permeability via Tie-2 after permanent middle cerebral artery occlusion in rats.

The mechanism of blood-brain barrier (BBB) disruption, involved in poststroke edema and hemorrhagic transformation, is important but elusive. We investigated microRNA-150 (miR-150)-mediated mechanism in the disruption of BBB after stroke in rats. We found that up-regulation of miR-150 increased permeability of BBB as detected by MRI after permanent middle cerebral artery occlusion in vivo as well as increased permeability of brain microvascular endothelial cells after oxygen-glucose deprivation in vitro. The expression of claudin-5, a key tight junction protein, was decreased in the ischemic boundary zone after up-regulation of miR-150. We found in brain microvascular endothelial cells that overexpression of miR-150 decreased not only cell survival rate but also the expression levels of claudin-5 after oxygen-glucose deprivation. With dual-luciferase assay, we confirmed that miR-150 could directly regulate the angiopoietin receptor Tie-2. Moreover, silencing Tie-2 with lentivirus-delivered small interfering RNA reversed the effect of miR-150 on endothelial permeability, cell survival, and claudin-5 expression. Furthermore, poststroke treatment with antagomir-150, a specific miR-150 antagonist, contributed to BBB protection, infarct volume reduction, and amelioration of neurologic deficits. Collectively, our findings suggested that miR-150 could regulate claudin-5 expression and endothelial cell survival by targeting Tie-2, thus affecting the permeability of BBB after permanent middle cerebral artery occlusion in rats, and that miR-150 might be a potential alternative target for the treatment of stroke.-Fang, Z., He, Q.-W., Li, Q., Chen, X.-L., Baral, S., Jin, H.-J., Zhu, Y.-Y., Li, M., Xia, Y.-P., Mao, L., Hu, B. MicroRNA-150 regulates blood-brain barrier permeability via Tie-2 after permanent middle cerebral artery occlusion in rats.

[Comparative Study on Quality of Volatile Oil of Acori Tatarinowii Rhizoma from Two Habitats].

OBJECTIVE: To compare the quality of volatile oil of Acori Tatarinowii Rhizoma from Guangxi and Sichuan. METHODS: The volatile oil was extracted from Acori Tatarinowii Rhizoma by using steam distillation method, and analyzed by GC-MS. Peak area normalization method was used for calculating the relative percentage contents of chemical constituents, and hierarchical cluster analysis was used for classifying the 20 batches of samples by their relative contents of the main components that were methyleugenol, cis-methylisoeugenol, γ-asarone, ß-asarone and α-asarone. RESULTS: The average extraction rate of 10 batches of volatile oil in Acori Tatarinowii Rhizoma from Guangxi was 1. 61%, and 10 batches of samples from Sichuan was 1. 72%. The relative percentage contents of five main components totaled 78. 19% and 88. 84%, respectively. By t-test, there was no statistical difference between samples from Guangxi and Sichuan. In the hierarchical cluster analysis,10 batches of samples from Guangxi and 10 batches of samples from Sichuan could respectively be classified into four clusters subcategories and five clusters subcategories, while the mean of samples of Guangxi and the mean of samples of Sichuan respectively analyzed with 20 batches of the two habitats that all were classified in the same clusters subcategories. The results of similarity showed that the correlation coefficients of 8 batches in 10 batches of samples from Guangxi were over 0. 9, while 1 batch was only 0. 466. The correlation coefficients of 7 batches in 10 batches of samples from Sichuan had were over 0. 9, while 1 batch was only 0. 069. The correlation coefficients between the mean of samples of Guangxi and the mean of samples of Sichuan was 0. 996. CONCLUSION: Quality of the different batches of volatile oil from Acori Tatarinowii Rhizoma have significant differences, but it has no obvious correlation with the habitats.

[Chemical constituents from roots of Rubus parvifolius].

OBJECTIVE: To investigate the chemical constituents from the roots of Rubus parvifolius. METHODS: The chemical constituents were isolated by silica gel column chromatography, Sephadex LH-20, as well as preparative high performance liquid chromatography. Their structures were identified on the basis of physicochemical properties and NMR analysis. RESULTS: Six comounds were isolated and identified as 3-O-Acetyl-11α, 12α-epoxy-oleanan-28,ß-olide( I ) ,3-O-Acetyl-pomolic acid( II ), Ursolic acid( Ill),Ursolic acid acetate (1V ), Euscaphic acid ( V) and ß-Sitosterol ( VI). CONCLUSION: Compounds I , II and IV are isolated from Rubus parvifolius for the first time.

External application of the volatile oil from Blumea balsamifera may be safe for liver--a study on its chemical composition and hepatotoxicity.

Ainaxiang (Blumea balsamifera), also known as Sambong, is an important ancient medicinal herb in Southeast Asia. It is rich in volatile oil, and still widely used nowadays for skin wound healing and treatment of sore throats. We analyzed the volatile oil from Blumea balsamifera (BB oil) by gas chromatography-mass spectrometry (GC-MS). Forty one components, including l-borneol, were identified. Next, the damaging effects of BB oil diluted with olive oil on liver at different concentrations (100%, 50%, 20%), were evaluated, using both normal and wounded skin. Plasma ALT, AST, ALP and TBili were assessed, along with liver histopathology. The results showed that serum levels of liver toxicity markers in the high concentration groups (100% w/v) increased compared with control groups, whereas no significant changes was observed in histopathology of liver samples. In the wound groups, treatment with BB oil resulted in a decrease in serum toxicity index, compared with normal animal groups. This study confirms the safety of short term BB oil consumption, though high BB oil doses may lead to mild liver injury and this response might be weakened in the case of cutaneous wounds. These results are expected to be helpful for guiding appropriate therapeutic use of BB oil.

An uncommon plant growth regulator, diethyl aminoethyl hexanoate, is highly effective in tissue cultures of the important medicinal plant purple coneflower (Echinacea purpurea L.).

We investigated the effects of various concentrations of diethyl aminoethyl hexanoate (DA-6) on the regeneration and growth of adventitious buds in in vitro purple coneflower cultures. Among the 3 types of explants tested, leaf explants required higher concentrations of DA-6 than petiole and root explants in order to obtain high regeneration rates, while root explants required the lowest concentration of DA-6. Additionally, explants with higher ploidy levels were more sensitive to the addition of DA-6, while explants with lower ploidy levels required higher concentrations of DA-6 to achieve its maximal regeneration rate. Interestingly, the application of a concentration that was conducive to the regeneration of explants with lower ploidy levels was inhibitory to the regeneration of explants with higher ploidy levels. Moreover, during the growth of regenerated buds, DA-6 application significantly improved plant height and weight, root weight, root thickness, root number, primary root length, total root length, and root/top ratio. Differences in the responses of explants to supplementation with DA-6 were also observed among explants with different ploidy levels, with buds having lower ploidy levels responding to lower concentrations of DA-6. Taken together, the results of the present experiments showed that proper application of DA-6 could increase in vitro culture efficiency in purple coneflower.

[Prediction of blood tacrolimus concentration in liver transplantation recipients by artificial neural network].

This study is to establish an artificial neural network (ANN) for predicting blood tacrolimus concentration in liver transplantation recipients. Tacrolimus concentration samples (176 samples) from 37 Chinese liver transplantation recipients were collected. ANN established after network parameters were optimized by using momentum method combined with genetic algorithm. Furthermore, the performance of ANN was compared with that of multiple linear regression (MLR). When using accumulated dose of 4 days before therapeutic drug monitoring (TDM) of tacrolimus concentration as input factor, mean prediction error and mean absolute prediction error of ANN were 0.02 +/- 2.40 ng x mL(-1) and 1.93 +/- 1.37 ng x mL(-1), respectively. The absolute prediction error of 84.6% of testing data sets was less than 3.0 ng x mL(-1). Accuracy and precision of ANN are superior to those of MLR. The correlation, accuracy and precision of ANN are good enough to predict blood tacrolimus concentration.

Recurrent bacterial meningitis caused by an occult basilar skull fracture.

BACKGROUND: We present a rare case of recurrent bacterial meningitis caused by an occult basilar skull fracture. METHODS: A 9-year-old boy presented with acute headache, fever and vomiting. He had recurrent meningitis episode one month earlier and a head trauma 6 months ago. Laboratory findings and lumbar puncture suggested an intracranial bacterial infection. Computerized tomography, magnetic resonance imaging and nasal endoscopy failed to find the presence of rhinorrhea. Spiral computed tomography was performed and a three-dimensional reconstruction of the bony cranium was done. RESULTS: A diagnosis of bone defect in the ethomoid was made. An endoscopic operation was performed to repair the defect successfully and the child was completely normal during a 4-month follow-up. CONCLUSIONS: The skull base should be evaluated radiologically to find one or multiple bony defects in case of recurrent meningitis in absence of cerebrospinal fluid rhinorrhea. Compared to other neuroradiological technologies, three-dimensional computed tomography provides a better three-dimensional definition of the basilar skull fracture for both diagnosis and surgical planning. When the fracture is located in the anterior skull base, an endoscopic transnasal approach is considered the best option.

[Physical training improves spatial learning and memory impairments following hypoxic ischemic brain damage in neonatal rats].

OBJECTIVE: To investigate the effect of physical training on cerebral structure and spatial learning and memory in neonatal rats submitted to hypoxic-ischemic brain damage (HIBD). METHODS: Forty-eight 7-day-old Sprague-Dawley rats were randomly divided into three groups: a group that was subjected to left carotid ligation followed by 2 hrs hypoxic stress (HIBD); a group that received physical training 2 weeks after the HIBD event; a control group that was subjected to a sham-operation without ligation and hypoxic stress. Following four weeks physical training, motor function test and water maze tasks were performed. Bilateral brain weight, cerebral morphology and left hippocampal ultrastructrue of the animals were examined. The expression levels of phosphor calmodulin-dependent protein kinase II (CaMKII) and brain derived neurotrophic factor (BDNF) were determined by immunohistochemistry. RESULTS: Compared with the control group, the motor function and the spatial learning and memory ability in the non-trained HIBD group were significantly decreased, whereas there was no significant difference between the trained-HIBD and the control groups. The left hemisphere weight and neurons in the left hippocampal CA1 zone of both HIBD groups decreased and the reduction was more significant in non-trained HIBD group. The ultrastructure of the left hippocampus was remarkably abnormal in the non-trained HIBD group, while no obvious abnormality was observed in the trained HIBD and the control groups. Phosphor-CaMKII and BDNF expression in the left hippocampus in the trained HIBD group increased significantly compared with that in the non-trained HIBD group. CONCLUSIONS: Physical training can restrain brain damage and ameliorate spatial learning and memory impairments in rats with HIBD.

Induction of tetraploids from petiole explants through colchicine treatments in Echinacea purpurea L.

Petiole explants were obtained from in vitro grown diploid (2x = 22) Echinacea purpurea plantlets. Shoots were regenerated by culturing the explants on MS basal medium containing 0.3 mg/L benzyladenine (BA), 0.01 mg/L naphthaleneacetic acid (NAA) and four concentrations (30, 60, 120, and 240 mg/L) of colchicine for 30 days, or 120 mg/L of colchicine for various durations (7, 14, 21, and 28 days). The regenerated shoots were induced to root on MS basal medium with 0.01 mg/L NAA, and then the root-tips of the regenerated shoots were sampled for count of chromosome number. It was found that a treatment duration of >7 days was necessary for induction of tetraploid (4x = 44) shoots, and treatment with 120 mg/L colchicine for 28 days was the most efficient for induction of tetraploids, yielding 23.5% of tetraploids among all the regenerated shoots. Chimeras were observed in almost all the treatments. However, the ratio of tetraploid to diploid cells in a chimeric plant was usually low. In comparison with diploid plants, tetraploid plants in vitro had larger stomata and thicker roots with more root branches, and had prominently shorter inflorescence stalk when mature.