[Quality evaluation of clinical practice guidelines for obstetric venous thromboembolism based on AGREE â ¡].

Objective: To evaluate the quality of the current foreign clinical practice guidelines (CPG) on obstetric venous thromboembolism (VTE), providing reference for standardizing the prevention and treatment of domestic VTE clinical practice. Methods: The GPGs for the management of obstetric VTE published abroad from inception to July 2020 were electronically searched. Two researchers independently evaluated the quality of the included guidelines in accordance with the internationally recognized guideline evaluation tool, AGREE Ⅱ. Results: A total of 12 guidelines, 2 recommendations and 1 consensus for obstetric VTE were included, covering 3 continents and 9 countries, and the published time span was 2009-2020. The standardized scores of the six domain (scope & purpose, stakeholder involvement, rigor of development, clarity of presentation, applicability and editorial independence) included in the guidelines were: 99.44%, 62.78%, 70.35%, 95.74%, 68.80% and 76.94% respectively, and the scores in each domain were ≥ 50.00%, indicating that the included CPGs were of good quality. Except for the domain scope & purpose, the ranges of the other five domains were ≥ 50.00%, suggesting that the quality of the CPGs differed significantly. Among the 15 included CPGs, 4 CPGs were level A (recommended), 11 CPGs were level B (recommended after being revised), and there was no CPG in level C (not recommended). The difference in the recommended content of obstetric VTE management mainly focused on the risk assessment, drug type, dosage and period. Conclusions: The overall quality of the CPGs for obstetric VTE management is high, while the quality of the CPGs varies greatly. In addition, there are differences in the recommended content of obstetric VTE management. More high-quality researches are required to provide evidence-based support for the improvement of the CPGs.

Effects of Moringa oleifera silage on milk yield, nutrient digestibility and serum biochemical indexes of lactating dairy cows.

This study investigated the effects of Moringa oleifera (MO) as a partial substitute of alfalfa hay on milk yield, nutrient apparent digestibility and serum biochemical indexes of dairy cows. MO was harvested at 120 days post-seeding. Fresh MO was cut, mixed with chopped oat hay (425:575 on a DM basis), ensiled and stored for 60 days. Sixty healthy Holstein dairy cows were allocated to one of three groups: NM (no MO or control), LM (low MO; 25% alfalfa hay and 50% maize silage were replaced by MO silage) or HM (high MO; 50% alfalfa hay and 100% maize silage were replaced by MO silage). The feeding trial lasted 35 days. The LM and HM diets did not affect dry matter (DM) intake, milk yield or milk composition (lactose, milk fat, milk protein and somatic cell count). The apparent digestibility of DM and NDF was lower for HM group than NM group. Additionally, there were no significant differences in serum biochemical indexes between the LM and NM groups. The HM group had lower serum concentrations of total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol and higher serum concentrations of urea than the NM group. The partial replacement of alfalfa hay (≤50%) and maize silage with MO silage had no negative effects on milk yield, in vivo nutrient apparent digestibility or serum biochemical indexes of lactating cows.

[Application of (125)I seeds combined with biliary stent implantation in the treatment of malignant obstructive jaundice].

OBJECTIVE: To study the feasibility and therapeutic effect of the application of (125)I seeds combined with biliary stent implantation on the treatment of malignant obstructive jaundice. METHODS: Fifty patients with malignant obstructive jaundice treated from September 2010 to February 2013 in Yantai Yuhuangding Hospital were included in this study. Among them, 24 patients received biliary stent implantation combined with (125)I seeds intraluminal brachytherapy as experimental group, and 26 were treated by biliary stent implantation as control group.The total bilirubin, direct bilirubin and tumor markers (CA-199, CA-242, CEA) before and after surgery, the biliary stent patency status was assessed, and the survival time was evaluated. RESULTS: The 24 patients in experimental group were implanted with 30 (125)I seeds successfully in a total of 450 seeds. Jaundice was improved greatly in both groups. The CA-199 and CA-242 after treatment in the experimental group were significantly decreased than that before treatment (P=0.003 and P=0.004). CEA was also decreased, but showed no statistical significance (P>0.05). There were no significant improvement comparing the CA-199, CA-242 and CEA before and 2 months after surgery in the control group (P>0.05). The rate of biliary stent patency was 83.3% (20/24) in the experimental group and 57.7% (15/26) in the control group (P=0.048). The mean biliary stent patency time in the experimental group was 9.84 months (range 1-15.5 months). The mean biliary stent patency time in the control group was 5.57 months (range 0.8-9 months). There was a significant difference between the two groups (P=0.018). The median survival time was 10.2 months in the experimental group and 5.4 months in the control group (P<0.05). CONCLUSION: (125)I seeds combined with biliary stent implantation can inhibit the proliferation of vascular endothelial cells and the growth of tumor effectively, and can prolong the biliary stent patency time and the survival time obviously for patients with malignant obstructive jaundice, therefore, is a safe and effective treatment in this malignancy.

Single nucleotide polymorphism variants within tva and tvb receptor genes in Chinese chickens.

Avian leukosis is an immunosuppressive neoplastic disease caused by avian leukosis viruses (ALV), which causes tremendous economic losses in the worldwide poultry industry. The susceptibility or resistance of chicken cells to subgroup A ALV and subgroup B, D, and E ALV are determined by the receptor genes tumor virus locus A (tva) and tumor virus locus B (tvb), respectively. Four genetic resistant loci (tva(r1), tva(r2), tva(r3), and tva(r4)) in tva receptor gene and a genetic resistant locus tvb(r) in the tvb receptor gene have been identified in inbred lines of White Leghorn. To evaluate the genetic resistance to subgroup A, B, D, and E ALV, genetic variations within resistant loci in tva and tvb genes were screened in Chinese local chicken breeds and commercial broiler lines. Here, the heterozygote tva(s1/r1) and the resistant genotype tva(r2/r2), tva(r3/r3), and tva(r4/r4) were detected in Chinese chickens by direct sequencing. The heterozygote tva(s1/r1) was detected in Huiyang Bearded chicken (HYBC), Rizhaoma chicken, and commercial broiler line 13 to 15 (CB13 to CB15), with the frequencies at 0.08, 0.18, 0.17, 0.25, and 0.15, respectively. The resistant genotype tva(r2/r2) was detected in Jiningbairi chicken (JNBRC), HYBC, and CB15, with the frequencies at 0.03, 0.08, and 0.06, respectively, whereas tva(r3/r3) and tva(r4/r4) were detected in 19 and 17 of the 25 Chinese chickens tested, with the average frequencies at 0.13 and 0.20, respectively. Furthermore, the resistant genotype tvb(r/r) was detected in JNBRC, CB07, CB12, CB14, and CB15 by pyrosequencing assay, with the frequencies at 0.03, 0.03, 0.11, 0.09, and 0.15, respectively. These results demonstrated that the potential for genetic improvement of resistance to subgroup A, B, D, and E ALV were great both in Chinese local chickens and commercial broilers. This study provides valuable insight into the selective breeding for chickens genetically resistant to ALV.

ZEB1 causes the production of hsa-microRNA-99b/let-7e/microRNA-125a cluster and promotes invasion of liver cancer cells.

OBJECTIVE: To explore the role of hsa-microRNA-99b/let-7e/microRNA-125a cluster in the progression of liver cancer and its possible regulatory mechanisms. PATIENTS AND METHODS: Ten liver cancer tissues were randomly selected and matched with normal liver tissue samples. The mRNA expression levels of miR-99b, let-7e and miR-125a were detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). The three miRNA mimics were transfected alone or together into hepatoma cells SMMC-7721; at the same time, the knockdown of the three miRNAs was also performed. Then the cell invasive and migratory abilities were examined through transwell assay. Bioinformatics analysis was used to detect the potential transcription factors that bind to the hsa-microRNA-99b/let-7e/microRNA-125a promoter sequence, and the binding of the two was verified by the Luciferase reporting assay. The level of hsa-microRNA-99b/let-7e/microRNA-125a mature mRNA was detected after ZEB1 was inhibited by ZEB1 siRNA. Meanwhile, after interfering with Drosha and ZEB1, the expression level of hsa-microRNA-99b/let-7e/microRNA-125a of the primary transcript was examined. Rescue experiments were carried out to assess the role of hsa-microRNA-99b/let-7e/microRNA-125a in the ZEB1 regulation of invasive cell capacity. RESULTS: The mRNA levels of microRNA-99b, let-7e, and microRNA-125a in 10 selected hepatocarcinoma tissues were significantly higher than those in the matched paracancerous tissues. After overexpressing the three miRNA mimics either alone or together, cell invasive and migratory abilities were extensively enhanced, and vice versa. It was found that there is a binding site in the upstream sequence of the promoter region of the hsa-microRNA-99b/let-7e/microRNA-125a cluster for ZEB1. The Luciferase reporter gene results showed an increase in the Luciferase activity of the cells transfected with E-box element wild-type sequence, while mutant E-box element group did not change. After knocking out ZEB1, the levels of mature microRNA-99b, let-7e and microRNA-125a were reduced. However, when DROSHA was knocked out, the levels of immature microRNA-99b, let-7e and microRNA-125a were increased, while simultaneous knocking out ZEB1 reversed this effect. Besides, the invasive ability of SMMC-7721 cells decreased after KEB1 was knocked down, while the opposite result was observed after transfection of hsa-microRNA-99b/let-7e/microRNA-125a alone or together. CONCLUSIONS: Hsa-microRNA-99b/let-7e/microRNA-125a cluster is highly-expressed in hepatocarcinoma, and its expression can be regulated by ZEB1. In addition, the overexpression of this cluster can promote the invasion of liver cancer cells and advance liver cancer progression.

MicroRNA-132 reverses cisplatin resistance and metastasis in ovarian cancer by the targeted regulation on Bmi-1.

OBJECTIVE: To explore the role of micro ribonucleic acid-132 (miR-132) in cisplatin (DDP) resistance and metastasis of ovarian cancer and its related mechanisms. MATERIALS AND METHODS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting were applied to detect the expression levels of miR-132 and B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) in maternal SKOV3 cells and cisplatin-resistant SKOV3/DDP cells. SKOV3/DDP cells were transfected with miR-132 mimic and miR-132 mimic negative control (NC). QRT-PCR and Western blotting were used to detect the expression changes in Bmi-1, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was adopted to detect the sensitivity of cells to DDP after transfection with miR-132 mimic. The effect of transfection on the apoptosis was detected via flow cytometry, and that on cell invasion and migration abilities were examined using wound healing assay and transwell assay. Bmi-1 wild-type (wt) and mutant-type (mut) luciferase reporter plasmids were co-transfected with miRNA-132 mimic or miRNA-132 NC, and luciferase activity was analyzed by dual-luciferase reporter system. RESULTS: QRT-PCR and Western blotting results manifested that the miR-132 expression level in SKOV3/DDP cells was significantly lower than that in SKOV3 cells, while the expression level of Bmi-1 in SKOV3/DDP cells was significantly higher than that in SKOV3 cells. The overexpression of miR-132 could reduce the expression level of Bmi-1 in SKOV3/DDP cells, increase the sensitivity of SKOV3/DDP cells to DDP, and inhibit cell invasion and metastasis. Data detected by the luciferase activity revealed that miR-132 could bind to the three prime untranslated region (3'-UTR) of the Bmi-1 gene and negatively regulate the protein expression. CONCLUSIONS: MiR-132 may regulate ovarian cancer's sensitivity to DDP and inhibit its invasion and metastasis by targeted regulation on Bmi-1.

Xanthophyll supplementation reduced inflammatory mediators and apoptosis in hens and chicks.

This study investigated effects of xanthophylls (containing 40% lutein and 60% zeaxanthin) on gene expression of inflammatory mediators ( [] and []) and apoptosis ( [] and ) of breeding hens and chicks. In Exp. 1, 432 hens were divided into 3 groups and fed diets supplemented with 0 (as the control group), 20, or 40 mg/kg xanthophylls. The liver, duodenum, jejunum, and ileum were sampled after 35 d. Results showed that 40 mg/kg of xanthophyll addition decreased in the liver, in the liver and duodenum, and in the liver and jejunum while increasing level in the liver and jejunum. Experiment 2 was a 2 × 2 factorial design. Male chicks hatched from hens fed 0 or 40 mg/kg xanthophyll diets were fed diets containing either 0 or 40 mg/kg xanthophylls. The liver, duodenum, jejunum, and ileum were sampled at 0, 7, 14, and 21 d after hatching. Results showed that in ovo xanthophylls reduced inflammatory mediators and apoptosis in the liver, duodenum, and jejunum of chicks mainly within 1 wk after hatching, whereas dietary xanthophylls only decreased expression in the liver from 2 wk onward. These results underlined important anti-inflammatory and antiapoptotic effects of maternal but not progeny dietary xanthophylls. In conclusion, xanthophylls can suppress inflammatory mediators and apoptosis in different tissues of hens and chicks.

Comparison of the clinical performance, contraceptive efficacy and acceptability of levonorgestrel-releasing IUD and Norplant-2 implants in China.

The levonorgestrel-releasing IUD (LNG-IUD, 20 micrograms/d) and the Norplant-2 implants were studied in a randomized comparative clinical trial with 200 women for one year. No pregnancy or expulsion occurred with the Norplant-2. In the LNG-IUD group, there was one pregnancy at the 12th month of use after an unnoticed expulsion. The net cumulative pregnancy and expulsion rates of LNG-IUD were 1.0 and 3.0. The removal rate due to menstrual disturbances in both LNG-IUD and Norplant-2 groups was 3.0. The use-related discontinuation rates were 9.0 and 3.0, and the continuation rates were 90.0 and 96.0, respectively. The differences were not statistically significant. Menstrual disturbance was the main side effect. It was improved after long time use, especially in the LNG-IUD group. After one year, the percentages of women with amenorrhea were 18.9 for the LNG-IUD and 8.3 for the Norplant-2. The menstrual blood loss was statistically significantly reduced in the LNG-IUD group, and blood haemoglobin concentration increased in both groups. The LNG-IUD and Norplant-2 are highly effective, safe, long-acting, and well accepted by the Chinese women.

Menstrual blood loss, haemoglobin and ferritin concentration of Beijing women wearing steel ring, VCu 200, and TCu 220c IUDs.

Menstrual blood loss (MBL), serum ferritin and whole blood cyanmethemoglobin were measured at pre- and 1, 3, 6, 12, 18 and 24th postinsertion cycles in 60 women wearing the Steel Ring, the Copper V (VCu 200) or the Copper T (TCu 220c). The MBL, incidence of menorrhagia and iron deficiency anemia were lower among users of the Steel Ring than among users of the Copper V and T. Anemia occurred later and the duration of menstruation was shorter with the Steel Ring than with the Copper T. There were no statistically significant differences between the Copper V and T.

PIP: The effects of the Steel Ring, the Copper V (VCu 200), and the Copper T (TCu 220c) were compared in terms of menstrual blood loss, serum ferritin, the incidence of menorrhagia, and anemia before and after insertion to evaluate the safety of these IUDs and to determine if any of them are appropriate for longterm use in Chinese women. 60 healthy women, 20-39 years, with regular menstrual periods, normal pelvic examination, who had used no oral contraceptives (OCs) for more than 1 year were recruited. Those who previously had used an IUD or had an abortion or parturition were admitted after at least 3 normal menses. Each subject was instructed in the method for complete collection of menstrual blood for 1 entire menstrual period. Menstrual blood loss was measured by alkaline hematin photometry and a Stomacher Lab-blender was used for extraction. The insertions of the IUDs were uneventful. After insertion, the mean blood loss for each group at all intervals was significantly higher than that of preinsertion, especially in the 1st postinsertion cycle. Thereafter, blood loss remained high or gradually decreased, but it did not return to the preinsertion levels even at the 24th postinsertion cycle. The differences were still significant, especially for the T group. The mean increase 25.4 ml (55.8%) for the Ring group, 32.3 ml (56.8%) for the V group, and 39.9 ml (82.2%) for the T group. The highest blood loss for the Ring group was 154 ml, for the V group 290.9 ml, and for the T group 211 ml. The incidence of menorrhagia of all IUDs was significantly increased in the 1st postinsertion cycle. Except for the Ring group, the differences were still significant even in the 24th postinsertion cycle. The mean value of serum ferritin for the Ring group was significantly decreased at the 18 and 24th postinsertion cycles. For the V group, the mean value decreased significantly at the 6, 12, and 18th postinsertion cycles and was below the normal level (16 ng/ml). For the T group, the mean value decreased significantly at the 12th and 18th postinsertion cycles, but the difference was not significant at the 24th postinsertion cycle as compared with preinsertion in either the V or T groups. After insertion, the mean hemoglobin (Hb) concentrations were still above 12 gm/dl in each cycle, but the incidence of Hb less than 12 gm/dl, which was zero before insertion in all groups, for the Ring group was 12.5% and 6.2% at the 12 and 24th postinsertion cycles and for the V group, 10% at the 1st postinsertion cycle, then gradually increased to 21.1% at the 24th postinsertion cycle. Menstrual flow was significantly prolonged only in the T group. This study indicated that blood loss, incidence of menorrhagia, and iron deficiency anemia were lower among Steel Ring users than among VCu 200 and TCu 220c users.

Menstrual blood loss and hematologic indices in healthy Chinese women.

Menstrual blood loss, cyanmethemoglobin and serum ferritin were determined in 421 healthy, noncontracepting Chinese women. The range of menstrual blood loss (MBL) was 4.1-273.6 mL, the mean value was 54.2 mL, and the median was 42.9 mL. The range of hemoglobin was 8.3-16.7 g/dL, and the mean value was 13.2 g/dL. The range of ferritin was 1.2-180.0 ng/mL, the mean value was 22.8 ng/mL, and the geometric mean was 17.1 ng/mL. The upper normal limit of MBL in Chinese women was set at 80 mL.

Effects of Ginkgo biloba extract on somatosensory evoked potential, nitric oxide levels in serum and brain tissue in rats with cerebral vasospasm after subarachnoid hemorrhage.

This study aimed to investigate the protective effects of Ginkgo biloba extract on cerebral vasospasm and neural damage following subarachnoid hemorrhage (SAH) in rats. It was found that the regional cerebral blood flow decreased immediately and persistently after SAH in SAH rats. The latency of somatosensory evoked potential delayed progressively. The nitric oxide levels in serum and brain tissue decreased and increased, respectively, after SAH. Ginkgo biloba extract effectively antagonized the changes of above parameters. It was concluded that somatosensory evoked potential is useful for the judgement of cerebral ischemic damage during cerebral vasospasm after SAH. Decrease in serum nitric oxide and increase in brain tissue nitric oxide are important factors leading to cerebral vasospasm and neural damage, respectively, after SAH. Ginkgo biloba extract relieves cerebral vasospasm and cerebral ischemic damage by reversing the pathological alteration of nitric oxide.

Effects of blockade of cerebral lymphatic drainage on cerebral ischemia after middle cerebral artery occlusion in rats.

This experiment aimed to investigate the effects of blockade of cerebral lymphatic drainage on cerebral ischemic damage. Seventy six Wistar rats were divided randomly into middle cerebral artery occlusion (MCAO) group and MCAO plus cerebral lymphatic blockade (MCAO+CLB) group for the experiment. The contents of water and electrolytes, the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the ischemic brain tissue were detected at 24, 48 and 72 hours after the operation. The morphologic examination was also performed. In MCAO group, contents of water, sodium and calcium in the ischemic brain tissue increased significantly at any time after the operation. The SOD activity decreased while the MDA content increased markedly. The morphologic findings showed severe damage of ischemic brain tissue and neurons. In MCAO+CLB group, the above parameters were altered more obviously. The present observation suggests that blockade of cerebral lymphatic drainage may deteriorate ischemic brain damage after MCAO.

[Clinical analysis and percutaneous transhepatic cholangiography drainage treatment of 25 patients with clonorciosis].

The clinical data of 25 patients with clonorchiasis were analyzed. Some features shown by B-mode ultrasonography and percutaneous transhepatic cholangiography (PTC) were discussed. Application of percutaneous transhepatic cholangiography drainage (PTCD) for treatment of complete biliary tract obstruction caused by clonorchiasis was introduced.

[Effects of ginkgo biloba extract on somatosensory evoked potential and nitric oxide after subarachnoid hemorrhage].

OBJECTIVE: To observe the changes of somatosensory evoked potential(SEP) and nitric oxide (NO) after subarachnoid hemorrhage(SAH), and the influence of Ginkgo biloba extract (EGb). METHOD: Rats in sham-operated group, SAH model group and EGb-treated group underwent measurement of dynamic changes of regional cerebral blood flow(rCBF), SEP and NO levels both in serum and in brain tissue within 24 h after operation. RESULT: In SAH group, rCBF decreased immediately after operation, with no tendency to recover within 24 h. The latency of SEP delayed progressively from 1 h to 24 h after SAH. NO levels in serum and in brain tissue decreased and increased respectively from 1 h to 24 h after SAH. EGb effectively antagonized the changes of above parameters. CONCLUSION: SEP is helpful in the judgement on brain ischemic damage after SAH. Decrease of NO in serum and increase of that in brain tissue may lead to cerebral vasospasm and ischemic brain damage respectively after SAH. EGb relieves SAH-induced brain ischemic damage by reversing the pathological alterations of NO.

Ancient and recent patterns of geographic speciation in the oyster mushroom Pleurotus revealed by phylogenetic analysis of ribosomal DNA sequences.

Evidence from molecular systematic studies suggests that many mushroom species may be quite ancient. Gene phylogenies were developed to examine the relationship between reproductive isolation, genetic divergence, and biogeography in oyster mushrooms (Pleurotus). Sequence data were obtained for two regions of DNA from populations belonging to eight intersterility groups (biological species). Phylogenetic analysis of sequences from the 5' portion of the nuclear encoded large subunit rDNA demonstrates an ancient origin for four intersterility groups of broad geographic distribution (world-wide), with a more recent radiation of several intersterility groups that are restricted to the Northern Hemisphere. An expanded analysis using sequence data from the more variable rDNA internal transcribed spacer region also reveals a phylogenetically based pattern of genetic divergence associated with allopatric speciation among populations from different continents in the Northern Hemisphere. The ability of rDNA sequences to resolve phylogenetic relationships among geographically isolated populations within intersterility groups illustrates the importance of biogeography for understanding speciation in Pleurotus. Patterns of geographic distribution among intersterility groups suggest that several species lineages evolved quite early, with recently evolved groups restricted to the Northern Hemisphere and older lineages occurring throughout the world. Based on phylogenetic evidence, analysis of historical biogeography using area cladograms shows that multiple dispersal and vicariance events are responsible for patterns of speciation observed.

[Changes of nitric oxide and endothelin-1 levels in rat brain tissue during cerebral vasospasm following subarachnoid hemorrhage and protective effect of nimodipine].

AIM: To investigate the changes of nitric oxide and endothelin-1 levels in brain tissue in rat with cerebral vasospasm (CVS) following subarachnoid hemorrhage (SAH), and the protective effect of nimodipine. METHODS: Wistar rats were divided randomly into SAH group, nimodipine-treated group and sham-operated group. Diameters of basilar artery before and after operation were measured, and dynamic changes of regional cerebral blood flow (rCBF), brain nitric oxide and endothelin-1 levels were determined within 24 h. Pathological examination of hippocampus was also performed. RESULTS: SAH resulted in significant and persistent decrease of rCBF. Diameter of basilar artery was reduced markedly. Neurons in hippocampus CA1 subfield were damaged severely. Both nitric oxide and endothelin-1 levels in brain tissue were increased significantly from 1 h to 24 h after SAH. However, the pathological changes of above parameters were not so severe in nimodipine-treated rats. CONCLUSION: Increase of nitric oxide and endothelin-1 levels in brain tissue is probably involved in the development of ischemic brain damage caused by CVS after SAH. Nimodipine exerts its protective effect on brain neurons by antagonizing CVS and the pathological changes of nitric oxide and endothelin-1 in brain tissue.

High frequency plant regeneration from protoplasts of wheat.

Calli were initiated from the mature seeds of wheat (Triticum asetivum L.cv. Xuzhou 211), and suspension cultures were established. The protoplasts isolated from suspension cells were cultured in the modified MS medium solidified with 0.8% agarose. Regenerated cells divided and calli formed. Whole plants were regenerated from protoplast-derived calli. Colony formation was promoted when the medium with lower osmotic pressure was added after two weeks culture. The frequency of regenerated plants was increased with lower concentration of sucrose in the differentiation medium. Shoots were induced effectively with high concentration of cytokinins and calliferous shoots were avoided. The frequency of regenerated plants was affected when protoplast-derived calli were transferred onto the differentiation medium in different periods.