Objective To provide a new approach of preservation for germplasm resources of Citrus medica var.sarcodactylis(CMS).Methods A micrografting test was made on CMS shoot-tips after vitrified cryopreservation,resulting in living shoot-tip.Results First the CMS shoot-tip is inoculated in medium consisting of MS,5%DMSO,and 5%sucrose for 48 h preincubation.Then the shoot-tips were cut off 3—4 mm long and fore-treated by 60%PVS_2 at room temperature(25℃) for 30 min.After that,they were treated by PVS_2 at 0℃for another 80 min and conserved in liquid nitrogen for 24 h.Next the shoot-tips were defrosted by aqueous bath at 40℃and cleaned twice at room temperature(25℃) by MT minimal medium with additive 1.2 mol/L sucrose,10 min once.Finally data collected recorded a higher survival rate of 81.25%by TTC inspectation and a better regeneration rate of 52.94%if the CMS shoot-tips had been grafted on darkly-cultured parental stock,growing,and differentiating normally afterwards.The plantlets detected by polymerase chain reaction(PCR) showed that the pathogens of Huanglong disease had been removed.Conclusion Therefore a conclusion is made that the approach of vitrified cryopreservation may be applied to the preservation of CMS germplasm resources.

Objective To lay a foundation for the cultivation of disease-free plantlet of Citrus medica L.var.sarcodactylis Swingle(CMS) by discussion of the factors affecting survival rate for micro-grafted shoot-tip of CMS.Methods With citrus limon seedlings cultured in the dark as the rootstock,shoot-tip of CMS was grafted to the inverse T-shaped cut in the cotyledon of rootstocks.Then an investigation was carried out on the factors which affected the survival rate for micrografted shoot-tip of CMS.The factors included age of rootstocks,scion size,culture medium with different concentrations of phytohormones,and rootstocks with or without cotyledon.Results The survival rate for micro-grafted shoot-tip of CMS was closely related to the factors mentioned above.A higher survival rate was obtained when CMS shoot-tip with 4 pieces of leaf primordia was grafted to the inverse "T" shaped cut in the cotyledon of parental stock(citrus limon seedlings) which was cultured in the dark for 14 days,and when the citrus limon seedlings were cultivated in MT-based medium with ?-naphthylacetic acid 0.1 mg?L-1 added.Conclusion The optimized micro-grafting conditions for CMS shoot-tips improve the propagation of micro-grafted plantlet and the survival rate of micro-grafted shoot-tip of CMS.

Objective To study the effects of the recipe for activating blood circulation and nourishing qi on expression of mitochondrial ribosomal protein L51 (MRPL51) in CVB3 infection model in rat cardiac myocytes, to reveal the pathogenesis of CVB3 myocarditis in the genetic level, to explore the therapeutic mechanism of the recipe for activating blood circulation and nourishing qi on CVB3 myocarditis, and to confirm the validity of the recipe for activating blood circulation and nourishing qi on CVB3 myocarditis. Methods After establishing CVB3 infection model and treatment model with recipe for activating blood circulation and nourishing qi by culturing neonatal rat myocardial cells, a modified suppression subtractive hybridization (SSH) was used to isolate differentially expressed genes between two model groups. These results were further verified by fluorescence RT-PCR. Results The results of SSH showed that gene expression of the treatment group was lower than that of the CVB3 infection group. The results of fluorescent RT-PCR which agreed with that of SSH displayed the threshold cycle number (Ct) in the treatment group was higher than the virus group. Conclusion Up-regulation of MRPL51 might be one of the pathogenesis of CVB3 myocarditis. The recipe for activating blood circulation and nourishing qi could treat viral myocarditis by regulating the expression of MRPL51.

To investigate the quality of different parts of Radix Fici Hirtae. The content and components of volatile oils and ethanol-extracts from different parts of Radix Fici Hirtae were analyzed by the combination of gas chromatography and mass spectrometry (GC-MS). The content of volatile oils in the cortex was higher than that in the xylem and the chemical components either in the cortex or in the xylem are similar. The main components in volatile oils are hexadecanoic acid, oleic acid+linoleic acid, linoleic acid amide, palmitic acid amide,stearic acid amide and dibutyl phthalate. The main components in ethanol-extracts are psoralen, hexadecanoic acid, hexadecanoic acid ethyl ester, bergapten, oleic acid and linoleic acid. The content of psoralen in the cortex was higher than that in the xylem.[Conclusion]The chemical components in different parts of Radix Fici Hirtae are similar but the contents of volatile components and psoralen are higher in the cortex than those in the xylem.

The sterile stem tips, nodular and non-nodular stem of Morinda officinalis How were cultured in vitro. The results showed that the most suitable procedure for sterilization of explants was soaking into 0.1% HgCl 2 solution for 10 to 15 minutes after pretreating with 70% ethanol for 30 seconds. MT culture medium with BA was effective to induce direct shooting of stem tips and nodular stem, the shooting rate of nodular stem being 97.5% . The optimal BA concentration was 1 mg?L -1 , the shooting rate would decrease when the concentration of BA increased. As for the induction of rooting, MT culture medium adding with 0.2 to 0.5 mg?L -1 NAA was optimal, the rooting rate being over 80.0% .

[ Objective ] To supply a technological evidence for the rapid reproduction of Andrographis paniculata (Burm. f.) Nees (APN). [Methods] Different explants from sterile seedling of Andrographis paniculata (Burm. f.) Nees were cultured in vitro to induce the production of fascicular buds. [ Results ] Cotyledon with nodes and half cotyledon with nodes were the most suitable explants for culture, and the budding rate was 100% and over 90% respectively. 6-Benzylaminopurine (BA) in the concentrations of 0.5 ~ 1.0mg/L were beneficial for the reproduction and growth of buds. The age of explants had no correlation with budding. The optimal rooting medium was MT (Murashige and Tucher) medium added with Img/L NAA (naphthaleneacetic acid) or IBA (indolebutyric acid). A highest rooting rate as much as 61.9% was achieved after 10 days of culture. [Conclusion] An effective plant regeneration system has been established for explants of Andrographis paniculata (Burm. f.) Nees.

Objective To establish a molecular identification method for three cultivars of Amomum villosum Lour.(AVL),thus to provide scientific evidence for the identification,selection and breeding of AVL.Methods The fragments of 26S rDNA D1-D3 region and matK gene of three cultivars of AVL and Amomum longiligulare T.L.Wu were amplified by polymerase chain reaction(PCR) and with corresponding primers,and then their sequences were analyzed,and phylogenetic tree was constructed based on the sequences.Results We obtained 739 bp in 26S rDNA D1-D3 sequence.Differences in 4 basic sites of 739 bp were shown between AVL and Amomum longiligulare T.L.Wu.The two cultivars of AVL,Changguo and Yuanguo,had the same sequence,but there was a difference in one basic site of Changguo and Yuanguo from Chunxuan.The phylogenetic tree based on 26S rDNA D1-D3 sequence revealed the difference between Chunxuan and the other two cultivars of AVL.We also obtained 824 bp in matK gene sequence.The three cultivars of AVL showed the consistent sequence,but there was a difference in one basic site of three cultivars of AVL from Amomum longiligulare T.L.Wu.Conclusion We can identify the three cultivars of AVL through the sequence differences at the molecular level,and Chunxuan has a closer genetic relationship with Amomum longiligulare T.L.Wu.

Objective There are few studies on the application of low molecular weight heparin (LMWH) in the therapy of women with recurrent spontaneous abortion (RSA) pregnant by assisted reproductive technology (ART).The article aimed to explore the clinical research of LMWH in therapy of women with RSA pregnant by ART.Methods 126 women with RSA pregnant by ART were enrolled and they were diagnosed and treated in Reproductive Medicine Center in the Affiliated Hospital of Guangdong Medical University from January 2010 to February 2016.According to the patient′s agreement on LMWH treatment, 60 patients in agreement with LMWH treatment were divided into 2 groups: LMWH+IUI group(n=30) and LMWH+ IVF-ET group(n=30).66 patients in disagreement with LMWH treatment were also divided into 2 groups: IUI group(n=32) and IVF-ET group(n=34).Comparison was made in patients from 4 groups concerning pregnancy success rate, live birth rate, pregnancy time involving RSA and incidence of pregnancy complications.At the same time, the occurrence of adverse reactions during the use of LMWH was also observed.Results The pregnancy time involving RSA in LMWH+IUI group significantly increased compared with IUI group([82.67±9.10]d vs [48.17±8.68]d)(P<0.05).The pregnancy success rate and live birth rate in LMWH+ IVF-ET group were both higher than those of IVF-ET group (66.7% vs 35.29%, 85.00% vs 50.00%)(P<0.05) and significant difference was also found in the incidence of RSA, the pregnancy time involving RSA, and the morbidity of gestational hypertension between groups(P<0.05).The results of D2D at 4 weeks of pregnancy in LMWH+IUI group([0.65±0.07]mg/L) and LMWH+ IVF-ET group([0.625±0.06]mg/L) were lower than those of LMWH group ([0.76±0.12]mg/L) and LMWH group([0.77±0.06]mg/L).The result of D2D at 6 weeks of pregnancy in LMWH+IUI group was lower than those of IUI group and IVF-ET group, and D2D in LMWH+ IVF-ET group was lower compared with IVF-ET group(P<0.05).The results of D2D in all the four groups increased with the pregnancy weeks(P<0.05).The prothrombin time(PT) at 4 weeks of pregnancy in LMWH+IUI group, IUI group and LMWH+ IVF-ET group ([12.53±0.38]s, [12.38±0.65]s, [12.47±0.58]s) was significantly higher at pre-pregnancy([12.33±0.52]s, [12.30±0.68]s, [12.22±0.64]s) and 6 weeks of pregnancy([12.13±0.62]s, [12.05±0.60]s, [12.03±0.54]s) (P<0.05).Among 60 cases treated with LMWH, small area ecchymoma were found in 11 cases and the incidence was 18.33%(11/60), only two cases reported with uncomfortable light pain in the location of subcutaneous injection.Conclusion Low-dose LMWH is safe and effective in the therapy of pregnant women with RSA through ART.

Objective To investigate the chemical constituents of Citrus medica var. sareodactylis from Sichuan province, and to provide evidence for the development and utilization and quality evaluation of the medicinal material.Methods Various chromatographic techniques were used to purify the components of this herb. Compounds were identi-fied by their physical characteristics and spectral feature. Results Seven compounds were isolated from Citrus medica var. sarcodactylis, and they were identified as sibiricol (Ⅵ), 7-methylesculetin (Ⅶ), bergapten (Ⅷ), sigmasteryl acetate(Ⅸ), 5-methoxyfurfural(Ⅹ), limonin(Ⅺ), daucosterol (Ⅻ). Conclusion Compound Ⅵ, Ⅶ and Ⅸ are isolated from plants of Rutaceae for the first time.

Objective To investigate the efficacy of computer-assisted postural balance training system combined with Bobath balance training on balance disorders after cerebellar stroke.Methods Forty patients with balance disorders after cerebellar stroke were randomly divided into either a combined training group or a control group (n =20 in each group).The control group was trained with Bobath balance training and routine rehabilitation treatment.On the basis of this,the combined training group used the computerassisted postural balance training system for rehabilitation treatment.The balance function,activities of daily living and motor function were evaluated with the Berg Balance Scale (BBS),modified Barthel Index,mBI)and Fugl-Meyer Assessment (FMA) before and after treatment,and the computer-assisted postural balance training system was used to conduct the postural balance ability according to the center of gravity track length,rectangle area of track,and peripheral area of track.Results There were no significant differences in various indexes before treatment between the patients of both groups.The scores of BBS,mBI and FMA after treatment were increased obviously (all P ＜ 0.001).The the center of gravity track length,rectangle area of track,and peripheral area of track were decreased compared before treatment (P ＜0.001 or 0.05).In addition to the FMA score,all the indexes of the combined training group after treatment were significantly better than those of the control group (all P＜0.05).Conclusions On the basis of Bobath balance training,using computer-assisted postural balance training system can obviously improve the balance disorder and extremities motion ability after cerebellar stroke,and thus improving the activities of daily living in cerebellar stroke patients.