Roles of circ_0000135/miR-140-3p/PDZK1 network in cervical cancer

PurposeTo explore the effect of circ_0000135/miR-140-3p/PDZ domain containing 1 (PDZK1) on the occurrence and development of cervical cancer.MethodsClinical data were collected to verify circ_0000135/miR-140-3p/PDZK1 expression in cervical cancer. mRNA expressions of circ_0000135 and miR-140-3p were detected by real-time quantitative PCR. Correlation between circ_0000135 and miR-140-3p/miR-140-3p and PDZK1 was analyzed in vitro. Protein expression detection in cells was conducted by Western blot; while cell proliferation, invasion and cycle distribution by CCK8 assay, Transwell chamber assay and flow cytometry, respectively. Rescue and animal experiment were performed to verify the effect of circ_0000135/miR-140-3p/PDZK1 on cervical cancer.Resultscirc_0000135 and PDZK1 expressions were increased, while those of miR-140-3p were decreased in cervical cancer tissues and cells (both P < 0.05). sh-circ_0000135 group had decreased cell viability, arrested cells in G0/G1 phase, decreased CyclinD1 expression, inhibited cell migration and invasion; sh-circ_0000135 group showed reduced tumor volume, weight, and lower Ki67 expression (all P < 0.05). circ_0000135 had conserved target of miR-140-3p. There was a direct interaction between circ_0000135 and miR-140-3p. miR-140-3p might have direct interaction with PDZK1. sh-circ_0000135 and/or miR-140-3p treatment showed obviously decreased PDZK1 expression, decreased cell activity, arrested cells in G0/G1 phase, downregulated cell migration and invasion; sh-circ_0000135 and/or miR-140-3p mimic treatment showed obviously decreased tumor volume, tumor weight, and Ki67 expression (all P < 0.05).Conclusioncirc_0000135 may play an anti-tumor role on the progression of cervical cancer by sponging miR-140-3p to suppress the expression of PDZK1, providing a promising therapeutic target.

Roles of circ_0000135/miR-140-3p/PDZK1 network in cervical cancer.

PURPOSE: To explore the effect of circ_0000135/miR-140-3p/PDZ domain containing 1 (PDZK1) on the occurrence and development of cervical cancer. METHODS: Clinical data were collected to verify circ_0000135/miR-140-3p/PDZK1 expression in cervical cancer. mRNA expressions of circ_0000135 and miR-140-3p were detected by real-time quantitative PCR. Correlation between circ_0000135 and miR-140-3p/miR-140-3p and PDZK1 was analyzed in vitro. Protein expression detection in cells was conducted by Western blot; while cell proliferation, invasion and cycle distribution by CCK8 assay, Transwell chamber assay and flow cytometry, respectively. Rescue and animal experiment were performed to verify the effect of circ_0000135/miR-140-3p/PDZK1 on cervical cancer. RESULTS: circ_0000135 and PDZK1 expressions were increased, while those of miR-140-3p were decreased in cervical cancer tissues and cells (both P < 0.05). sh-circ_0000135 group had decreased cell viability, arrested cells in G0/G1 phase, decreased CyclinD1 expression, inhibited cell migration and invasion; sh-circ_0000135 group showed reduced tumor volume, weight, and lower Ki67 expression (all P < 0.05). circ_0000135 had conserved target of miR-140-3p. There was a direct interaction between circ_0000135 and miR-140-3p. miR-140-3p might have direct interaction with PDZK1. sh-circ_0000135 and/or miR-140-3p treatment showed obviously decreased PDZK1 expression, decreased cell activity, arrested cells in G0/G1 phase, downregulated cell migration and invasion; sh-circ_0000135 and/or miR-140-3p mimic treatment showed obviously decreased tumor volume, tumor weight, and Ki67 expression (all P < 0.05). CONCLUSION: circ_0000135 may play an anti-tumor role on the progression of cervical cancer by sponging miR-140-3p to suppress the expression of PDZK1, providing a promising therapeutic target.

[Research progress on building of disease control and prevention system of the international experience].

Through literature search in regular database and official websites of relevant countries, this paper combs and summarizes the main characteristics of disease prevention and control systems in five countries, the United States, Germany, South Korea, Australia and Japan, and the European Union at key levels including legal construction, organizational structure, financing, personnel construction and international cooperation, in order to provide decision support for the construction of disease prevention and control system in China in the future.

[Correlation analysis of neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio and central cervical lymph node metastasis of papillary thyroid microcarcinoma].

Objective: To investigate the correlation between neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and central lymph node metastasis (CLNM) in patients with cN0 papillary thyroid microcarcinoma (PTMC). Methods: The clinicopathological data of PTMC patients confirmed by surgery and pathology in the 81st Military Hospital of People's Liberation Army from 2016 to 2019 were collected, and the relationship between preoperative NLR, PLR levels and postoperative PTMC CLNM were analyzed. Logistic regression analysis was used for multivariate analysis. Receiver operating characteristic (ROC) curve was used to determine the cutoff value of NLR and PLR. The interaction relative excess risk was used to analyze the relationship between NLR, PLR and CLNM. Results: Among 220 patients with cN0 stage PTMC, 92 were CLNM. The ROC curve showed that when the cutoff value of NLR was 2.5 and the cutoff value of PLR was 175, the highest Youden index was 0.318 and 0.264, respectively. NLR and PLR were both related to CLNM (P<0.05). The tumor long diameter, multifocality, NLR≥2.5 and PLR≥175 were independent impact factors of CLNM (P<0.05). The results of the interaction showed that the relative excess risk of the interaction was 5.531 (95%CI: 0.160, 10.901, P=0.016), the attribution ratio was 0.512 (95%CI: 0.230, 0.794, P=0.009), and the synergy index was 2.294 (95%CI: 1.492, 4.579, P=0.022), suggested that NLR and PLR had an interactive effect, and these two synergistically promoted CLNM. Conclusions: NLR and PLR are independent risk factors for cN0 stage PTMC CLNM. When NLR≥2.5 and PLR≥175, preventive central lymph node dissection should be routinely performed.

[Research Yi Jia on the literature of "Fangji Zhuan" in Songshi].

"Fangji Zhuan"(, Biographies of Technicians), a chapter from Songshi(, History of the Song) uses time as a sequence and adopts a narrative method. It contains 42 talents, including ephemeris, five elements of law, medical practitioners, and Taoists. Among them, there are 25 good medical practitioners, which are rich in content and detailed in history. Based on the biographies of medical doctors in "Fangji Zhuan", this article summarizes and records the life history of 25 different types of medical doctors in terms of divisions, contributions, rewards and punishments, etc., which reflects the career status of medical doctors in the Song Dynasty to a certain extent. The role and contribution in medical development are of great significance.

Decreased long noncoding RNA ADIPOQ promoted cell proliferation and metastasis via miR-219c-3p/TP53 pathway in colorectal carcinoma.

OBJECTIVE: To investigate the expression of Long non-coding RNA ADIPOQ and its facilitating effects on proliferation and invasion of colorectal cancer by modulating the expression of TP53 via sponging with miR-219c-3p. PATIENTS AND METHODS: qRT-PCR was performed to detect the expressions of ADIPOQ and TP53 in human colorectal cancer tissues and cells. CCK-8 assay was performed to evaluate the Caco-2 cells proliferation and transwell assay was performed to evaluate the Caco-2 cells migration. The relationship between ADIPOQ and miR-219c-3p was detected by statistical analysis. Target prediction and Luciferase activity assay were conducted to investigate the binding site and interaction between ADIPOQ and miR-219c-3p. Further, we cloned the mice TP53 3'-UTR into the Luciferase reporter vector and constructed miR-219c-3p binding mutants to verify the inhibited regulation of miR-219c-3p to the TP53 expression. RESULTS: The results suggested that the expression of ADIPOQ and TP53 was downregulated in human colorectal cancer tissues and Caco-2 cells. qRT-PCR and CCK-8 assay showed that ADIPOQ expression is correlated with the proliferation of colorectal cancer cells. Transwell assay showed that ADIPOQ regulated the migration ability of colorectal cancer cells. The bioinformatics prediction and Luciferase assay demonstrated that ADIPOQ serves as ceRNA for miR-219c-3p to further regulate the expression of TP53. CONCLUSIONS: For the first time, we found that lncRNA-ADIPOQ was downregulated in human colorectal cancer cells, which could facilitate tumor proliferation, migration and invasion as a ceRNA by sponging with miR-219c-3p.

[Study on literatures of health preservation and practitioners in Taiping Yulan: Fangshu].

Taiping Yulan(, Imperial overview from the Taiping reign or Readings of the Taiping Era) was a large encyclopedia which compiled by Li Fang in the early Northern Song Dynasty. Its content was broad and the resources of quotation were varied. It contained a large number of medical knowledges. Among this book, the "Fangshu" volume consisted of 18 parts, the first five volumes of health and medical recorded 4 methods of health preservation (nurturing vitality), 88 practitioners, cited 59 kinds of literatures and 160 historical materials. It has important academic value to the study of ancient health preservation and inheritance development of medical school.

[The influence of chronic intermittent hypoxia on hypothalamic-somatotropic axis in rats].

Objective: To investigate the effects of chronic intermittent hypoxia on somatotropic axis hormone levels in rats. Methods: Mature male Wistar rats were exposed to air or intermittent hypoxia randomly.The serum levels of growth hormone-releasing hormone (GHRH), growth hormone (GH) and somatostatin (SS) were measured before exposure, at the 4th, 8th, and 12th week after exposure. Different hormone levels in two groups were compared and analyzed. Results: Compared with the control group, GHRH levels in chronic intermittent hypoxic group showed a significant decline at the 4th week [(732.77±46.99)pg/ml vs. (893.59±40.00) pg/ml, P<0.05], while SS levels at the 8th week [(30.71±2.27) pg/ml vs. (44.69±3.36) pg/ml, P<0.05] and GH levels at the 12th week [(1.20±0.29) ng/ml vs. (2.06±0.13) ng/ml, P<0.05] were similarly reduced. As the duration of intermittent hypoxia was prolonged, the GHRH levels did not decrease further [4th week (732.77±46.99) pg/ml vs. 8th week (607.54±131.61) pg/ml vs. 12th week (730.05±40.63) pg/ml, P>0.05].However, the serum SS levels decreased further from the 8th week to the 12th week [(30.71±2.27) pg/ml vs. (24.41±4.06) pg/ml, P<0.05]. Conclusion: Chronic intermittent hypoxia might inhibit the function of somatotropic axis. Hypothalamic hormones are the earlyonesto be influenced, thereafter the entire axis.

[Thrombosis after establishing internal jugular vein channel in one burn patient].

On September 7th, 2017, one female patient, aged 48 years with deep partial-thickness flame burn on face, upper limbs, trunk, and lower limbs of 40% total body surface area was admitted to the First People's Hospital of Foshan City. After admission, active fluid replacement, anti-infection, nutritional support, and other treatments were performed. After debridement and skin grafting for 3 times and blood transfusion for 2 times, the patient recovered well. On the 20th day post admission, sudden heartbeat and respiratory arrest happened, and the patient died after ineffective rescue. Autopsy showed that thrombus formed in right internal jugular vein and deep veins of lower extremities, and vascular lumina of the bilateral pulmonary artery. The direct cause of death was acute pulmonary thromboembolism, but whether the embolus originated from deep vein of lower extremity or right internal jugular vein was not clear. This case suggests that clinician should not only pay attention to the prevention of deep venous thrombosis of lower extremities of burn patients, but also the possibility of internal jugular vein thrombosis, especially for patients with internal jugular vein access.

Investigating seed dormancy in cotton (Gossypium hirsutum L.): understanding the physiological changes in embryo during after-ripening and germination.

The dormancy of seeds of upland cotton can be broken during dry after-ripening, but the mechanism of its dormancy release remains unclear. Freshly harvested cotton seeds were subjected to after-ripening for 180 days. Cotton seeds from different days of after-ripening (DAR) were sampled for dynamic physiological determination and germination tests. The intact seeds and isolated embryos were germinated to assess effects of the seed coat on embryo germination. Content of H2 O2 and phytohormones and activities of antioxidant enzymes and glucose-6-phosphate dehydrogenase were measured during after-ripening and germination. Germination of intact seeds increased from 7% upon harvest to 96% at 30 DAR, while embryo germination improved from an initial rate of 82% to 100% after 14 DAR. Based on T50 (time when 50% of seeds germinate) and germination index, the intact seed and isolated embryo needed 30 and 21 DAR, respectively, to acquire relatively stable germination. The content of H2 O2 increased during after-ripening and continued to increase within the first few hours of imbibition, along with a decrease in abscisic acid (ABA) content. A noticeable increase was observed in gibberellic acid content during germination when ABA content decreased to a lower level. Coat removal treatment accelerated embryo absorption of water, which further improved the accumulation of H2 O2 and changed peroxidase content during germination. For cotton seed, the alleviation of coat-imposed dormancy required 30 days of after-ripening, accompanied by rapid dormancy release (within 21 DAR) in naked embryos. H2 O2 acted as a core link between the response to environmental changes and induction of other physiological changes for breaking seed dormancy.

Upregulation of miR-132 attenuates osteoblast differentiation of UC-MSCs.

OBJECTIVE: Wnt/ß-catenin signaling pathway plays a role in upregulating expression of osteoblast (OB) specific transcriptional factor Osterix and promoting OB differentiation. It was shown that the elevation of the miR-132 level was associated with sclerotizing inhibition. Bioinformatics analysis revealed the complementary binding site between miR-132 and 3'-UTR of ß-catenin. This study investigated the influence of miR-214 in regulating ß-catenin expression and differentiation of umbilical cord mesenchymal stem cells (UC-MSCs) into OB. MATERIALS AND METHODS: UC-MSCs were induced to differentiate to OB. The expressions of miR-132, ß-catenin, Osterix, and ALP, together with ALP activity were detected on day 0, 5, 10, and 15. The regulatory relationship between miR-132 and ß-catenin was confirmed by dual luciferase reporter gene assay. UC-MSCs were divided into five groups, including agomir-control, miR-132 agomir, pGPH1-NC, pGPH1-ß-catenin, and miR-132 agomir + pGPH1-ß-catenin groups. ß-catenin, Osterix, and ALP expressions, together with ALP activity were tested after induction for 15 days. RESULTS: MiR-132 was downregulated, while ß-catenin Osterix and ALP expressions, together with ALP activity were enhanced in the process of UC-MSCs differentiating into OBs. MiR-132 agomir and/or pGPH1-ß-catenin transfection significantly reduced ß-catenin expression, downregulated Wnt/ß-catenin signaling pathway activity, declined Osterix level, weakened ALP expression and activity, and attenuated OB differentiation of UC-MSCs. CONCLUSIONS: The level of ß-catenin was enhanced, while the miR-132 level was decreased in the process of UC-MSCs differentiating into OBs. Upregulation of miR-132 inhibited the differentiation of UC-MSCs through suppressing ß-catenin expression, attenuating Wnt/ß-catenin signaling pathway activity, and downregulating Osterix level.

Effects of different sources and levels of dietary gossypol on gossypol residues in plasma and milk of lactating cows.

Free gossypol residues in tissues or milk from feeding whole cottonseed and cottonseed meal were measured for their effect on health of dairy cows and humans. Forty lactating cows were randomly assigned to 5 treatments in a 60-d experiment to investigate the effects of sources and dietary level of gossypol on plasma and milk gossypol concentrations in lactating cows. Five experimental diets had identical net energy for lactation and crude protein content on a dry matter (DM) basis. Soybean meal was the main protein ingredient used in the control diet. Cottonseed meal (CSM) or whole cottonseed (WCS) substituted for part of the soybean meal in the other 4 diets. Gossypol levels in the 5 diets were 0 (control), 91.15 mg/kg of DM in CSM1, 117.31mg/kg of DM in CSM2, 385.43 mg/kg of DM in WCS1, and 611.13 mg/kg in WCS2. Yields of 3.5% fat-corrected milk were significantly higher for cows in the WCS2 group; cows in the CSM1 and WCS1 groups showed no differences but both were numerically higher than the control and CSM2 groups. Milk protein concentration was lower for cows consuming WCS1 compared with the control group. Lactose concentration was lower for cows in the CSM2 group compared with the WCS2 group, but no differences were observed among other diets. Aspartate aminotransferase in serum was significantly higher for the WCS2 group compared with the control and WCS1 groups, but no difference was observed with the CSM1 and CSM2 groups. Concentrations of gossypol in plasma and milk of cows in the WCS1 and WCS2 groups were both higher than those of the other groups. No adverse effects were observed on cows fed diets containing 12.0% CSM, and no gossypol was found in plasma and milk. When WCS comprised 15% of the diet DM, yields of 3.5% fat-corrected milk were increased in cows and gossypol was detected in plasma and milk but not at harmful levels.

An hepatitis B virus surface antigen specific single chain of variable fragment derived from a natural immune antigen binding fragment phage display library is specifically internalized by HepG2.2.15 cells.

Hepatitis B virus surface antigen (HBsAg), a specific antigen on the membrane of hepatitis B virus (HBV)-infected cells, provides a perfect target for therapeutic drugs. In order to mediate successful targeted delivery of these therapies, it is essential to have antibodies that recognize HBsAg with high specificity and affinity. In this report, we constructed a natural immune antigen binding fragments (Fab) antibody phage display library against HBsAg and after three rounds of panning, five Fab fragments with significant HBsAg binding ability were selected and analysed. DNA sequencing revealed that all the light chains had the same sequence, while all the Fd genes exhibited different sequences. For further application, all of the Fab antibodies were reconstructed into single chain antibodies (scFvs) and expressed in Escherichia coli BL21 cells. Indirect enzyme-linked immunosorbent assay analysis demonstrated that all five scFvs maintained a high affinity for HBsAg and could bind HBsAg on the membrane of HBV-infected cells. Indirect fluorescent staining analysis revealed that one of the scFvs (scFv15) could be internalized into HBsAg-positive HepG2.2.15 cells through clathrin-mediated endocytosis pathway. The internalizing scFv15 antibody would have great potential for the targeted delivery of therapeutics to HBV-infected cells.

Selective proapoptotic activity of a secreted recombinant antibody/AIF fusion protein in carcinomas overexpressing HER2.

Apoptosis-inducing factor (AIF) represents a caspase-independent apoptotic pathway in the cell, and a mitochondrial localization sequence-truncated AIF (AIFDelta1-120) can be relocated from the cytoplasm to the nucleus and exhibit a constitutive proapoptotic activity. Here, we generated a chimeric immuno-AIF protein, which comprised an HER2 antibody, a Pseudomonas exotoxin translocation domain and AIFDelta1-120. Human Jurkat cells transfected with the immuno-AIF gene could express and secrete the chimeric protein, which selectively recognized HER2-overexpressing tumor cells and was endocytosed. Subsequent cleavage of truncated AIF from immuno-AIF and its release from the internalized vesicles resulted in apoptosis of tumor cells. Intramuscular injection of the immuno-AIF gene caused significant suppression of tumors and substantially prolonged mice survival in an HER2-overexpressing xenograft tumor model. Our study demonstrates the feasibility of the immuno-AIF gene as a novel approach to treating cancers that overexpress HER2.

Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions.

Plants synthesize the osmoprotectant glycine betaine (GB) via choline-->betaine aldehyde-->glycine betaine[1]. Two enzymes are involved in the pathway, choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). A full length CMO cDNA (1,643bp) was cloned from Amaranthus tricolor. The open reading frame encoded a 442-amino acid polypeptide, which showed 69% identity with CMOs in Spinacia oleracea L. and Beta vulgaris L. DNA gel blot analysis indicated the presence of one copy of CMO gene in the A. tricolor genome. The expressions of CMO and BADH proteins in A.tricolor leaves significantly increased under salinization, drought and heat stress (42 degrees C), as determined by immunoblot analysis, but did not respond to cold stress (4 degrees C), or exogenous ABA application. The increase of GB content in leaves was parallel to CMO and BADH contents.

Molecular cloning and functional characterization of two kinds of betaine-aldehyde dehydrogenase in betaine-accumulating mangrove Avicennia marina (Forsk.) Vierh.

Glycinebetaine is an important osmoprotectant in bacteria, plants, and animals, but only little information is available on the synthesis of glycinebetaine in tree plants. Among four mangrove species, glycinebetaine could be detected only in Avicennia marina. Pinitol was the main osmoprotectant in the other three species. The level of glycinebetaine in A. marina increased under high salinity. Betaine-aldehyde dehydrogenase (BADH) was detected in all four species, but choline monooxygenase could not be detected. A cDNA library was constructed from the leaves of A. marina. Two kinds of BADH cDNAs were isolated, one homologous to the spinach chloroplast BADH, and the other with unique residues SKL at the end of C-terminus. The BADH transcription levels of the former were higher than those of the latter. The levels of the former BADH increased at high salinity whereas those of the latter were independent of salinity. BADHs were expressed in Escherichia coli and purified. Two kinds of A. marina BADHs exhibited similar kinetic and stability properties, but were significantly different from those of spinach BADH. A. marina BADHs efficiently catalyzed the oxidation of betainealdehyde, but not the oxidation of omega-aminoaldehydes and were more stable at high temperature than the spinach BADH.

[The development of an automatic dialyser reuse device by microcomputer].

This article introduces the principles, composition and main functions of an automatic dialyser reuse device by microcomputer. It Ras more practical uses, better reliability, easier operations and greater effect than previous ones, and it's an ideal dialyser ruse device.

Overproduction of spinach betaine aldehyde dehydrogenase in Escherichia coli. Structural and functional properties of wild-type, mutants and E. coli enzymes.

Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the synthesis of the osmoprotectant glycine betaine from choline. Although betaine aldehyde has been thought to be a specific substrate for BADH, recent studies have shown that human and sugar beet BADHs also catalyze the oxidation of omega-aminoaldehydes. To characterize the kinetic and stability properties of spinach BADH, five kinds of expression vectors encoding full length, mature, E103Q, E103K, and chimera BADHs were constructed. These enzymes together with Escherichia coli BADH were expressed in E. coli and purified. The affinities for betaine aldehyde were similar in the spinach and E. coli BADHs, whereas those for omega-aminoaldehydes were higher in spinach BADH than in E. coli BADH. A chimera BADH in which part of the Rossmann type fold in the spinach BADH was replaced with that of E. coli BADH, showed properties which resembled spinach BADH more than E. coli BADH. The spinach E103K mutant was almost inactive, whereas the E103Q mutant showed a similar activity for the oxidation of betaine aldehyde to that of wild type BADH, but a lower affinity for omega-aminoaldehydes. All spinach BADHs were dimers whereas E. coli BADH was a tetramer. E. coli BADH was more stable at high temperature than spinach BADHs. The E103Q mutant was most labile to high temperature. These properties are discussed in relation to the structure of spinach BADH.

Coordinated accumulation of (+)-delta-cadinene synthase mRNAs and gossypol in developing seeds of Gossypium hirsutum and a new member of the cad1 family from G. arboreum.

A new member of the (+)-delta-cadinene synthase (CAD1) family was isolated from a Gossypium arboreum cDNA library. This cDNA encodes a protein that showed 97.3%, 96.9%, and 79.2% sequence identities with the proteins encoded by previously isolated cDNAs of cad1-C1, cad1-C14, and cad1-A, respectively. It may be grouped into the cad1-C subfamily as cad1-C2. Seeds of a glanded cotton cultivar, G. hirsutum cv. Sumian-6, were collected at different intervals during maturation, and the cad1 mRNA levels were analyzed by quantitative RT-PCR. The transcripts could be detected in seeds of 27 DPA (days postanthesis) and increased dramatically along with the seed maturation, which coordinated with an increase in sesquiterpene cyclase activities and subsequently the accumulation of gossypol. The transcription level detected with primers specific to cad1-C (including at least C1, C14, and cdn1) was higher than that detected with primers specific to cad1-A, and mRNA was detected also with cad1-C2-specific primers. This investigation indicates that, in developing seeds of the glanded cotton cultivar, genes of both the CAD1-C and CAD1-A subfamilies are expressed and there is an active biosynthesis of cadinene-type sesquiterpenes.

Plant regeneration from protoplasts isolated from callus ofGentiana crassicaulis.

Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1-2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4-5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA3, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.