Adaptation of the Spodoptera exigua Se301 insect cell line to grow in serum-free suspended culture. Comparison of SeMNPV productivity in serum-free and serum-containing media

Spodoptera exigua Se301 cells have been successfully adapted to two different commercial serum-free media (SFM; Ex-Cell 420 and Serum-Free Insect Medium-1) by gradually reducing the 10 %-added serum-containing medium content from 100 % to 0 % (v/v) in suspended cultures. Both direct adaptation to a serum-free medium and cell growth in the absence of protective additives against fluid dynamic stress [polyvinyl pyrrolidone and polyvinyl alcohol] and disaggregation [dextran sulfate] proved impossible. Cells grew reproducibly in both SFMs once the serum had been completely removed, although the use of Ex-Cell 420 resulted in higher growth rates and cell densities. Turbulence was sufficiently high to reduce growth rates and final cell densities at the highest Reynolds number investigated, although no clear influence of agitation was observed on virus productivity. Both attached and suspended Se301 cell cultures were successfully infected with the SeMNPV baculovirus. Cells adapted to different conditions (attached or suspended culture, serum-containing or serum-free medium) showed different occlusion bodies productivities at the two multiplicities of infection assayed (0.1 and 0.5).     

Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines

Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID₅₀) titers of the two cell lines reached 4.51×10⁶ cells/mL, 2.94Log₁₀(HAU/50 μL) and 8.49Log₁₀(virions/mL), and 5.97×10⁶ cells/mL, 3.88Log₁₀(HAU/50 μL), and 10.34Log₁₀(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.     

Evaluation of the new serum-free medium (MDSS2) for the production of different biologicals: Use of various cell lines

The presence of serum in cell culture raises safety problems for the production of biologicals, thus a new serum-free medium (MDSS2) was developed. The evaluation of this medium for the growth of different cell lines (BHK-21 C13, BSR and Vero) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static (as monolayer) as well as in agitated systems (in suspension in spinner and perfusion reactors). BHK-21 and BSR cells grew as aggregate cultures and could proliferate in both static and agitated culture systems. Vero cells stayed attached to a substrate and proliferated equally in static and in agitated microcarrier-culture systems. The cell densities obtained with BHK-21 cells depended only on the culture system used. They ranged from 2–3×10⁶ to 6–12×10⁶ cells per ml for static batch and perfusion reactor cultures respectively. The cell concentration was 3 to 6 times higher than in classical cultures performed in serum-containing medium. The cell densities obtained with Vero cells were indistinguishable from those obtained in serum-containing medium, whatever the cell culture system used. These cell lines have been used for the production of rabies virus. With respect to BHK-21 and BSR, similar production rates of rabies glycoprotein have been found as in the standard roller bottle process. The production of rabies virus and of viral glycoprotein by Vero cells cultivated in serum-free medium was augmented 1.5-fold and 2.5-fold, respectively, when compared to serum-containing medium.A recombinant BHK-21 cell line, producing human IL-2, can also proliferate in MDSS2, after addition of insulin. The specific IL-2 production rate was augmented 3–4 fold in comparison to serum-containing medium.For the cells tested, the MDSS2 serum-free medium is a good growth and production medium. Its use for cultivating other cell lines and/or for the production of other biologicals is discussed.     

Comparative recombinant protein production of eight insect cell lines

Summary A recombinantAutographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived fromSpodoptera frugiperda, Trichoplusia ni, Mamestra brassicae andEstigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, theT. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than theS. frugiperda-derived Sf9 or Sf21 cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.     

Serum suppresses the expression of hormonally induced functions in cultured granulosa cells

Growth and function of primary cultures of granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were compared in serum-containing and serum-free media. In serum-free medium (1:1 mixture of DMEM:F-12) supplemented with insulin, hydrocortisone, transferrin and fibronectin (4F medium), the cells remained healthy and steroidogenically responsive for at least 60 days in culture. The growth profile of the granulosa cells in 4F medium was similar to that obtained in serum-containing medium. In both media cell proliferation did not exceed more than one cell doubling. DMEM:F-12 alone did not support the cell viability. Upon FSH stimulation, the cells produced 25 fold more progestin and estrogen per cell in 4F medium than in medium supplemented with 5% serum. This effect was not directly related to serum proteins which mediate cell adhesion since cells cultured in dishes precoated with serum remained steroidogenically responsive to FSH. Cholera toxin and Bt₂-cAMP readily stimulated progestin production in the presence of serum. The inhibitory effect of serum was not reversed by adding the four factors to serum-containing medium. The factors were essential for the FSH-induced steroidogenesis in serum-free medium. After four days of incubation in 4F medium, the cells showed a transient loss of their ability to produce progestin in response to FSH. In both 4F medium as well as in serum-containing medium, the cells regained their hormonal responsiveness after 35 days in culture. Since the loss of hormonal responsiveness occurred at the same time as growth was initiated in the cultures, it is suggested that the FSH-induced steroidogenesis is negatively controlled by growth-related processes.     

Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells

The necessity to perform serum-free cultures to produce recombinant glycoproteins generally requires an adaptation procedure of the cell line to new environmental conditions, which may therefore induce quantitative and qualitative effects on the product, particularly on its glycosylation. In previous studies, desialylation of EPO produced by CHO cells was shown to be dependent on the presence of serum in the medium. In this paper, to discriminate between the effects of the adaptation procedure to serum-free medium and the effects of the absence of serum on EPO production and glycosylation, adapted and non-adapted CHO cells were grown in serum-free and serum-containing media. The main kinetics of CHO cells were determined over batch processes as well as the glycosylation patterns of produced EPO by HPCE-LIF. A reversible decrease in EPO production was observed when cells were adapted to SFX-CHO^TM medium, as the same cells partially recovered their production capacity when cultivated in serum-containing medium or in the enriched SFM^TM serum-free medium. More interestingly, EPO desialylation that was not observed in both serum-free media was restored if the serum-independent cells were recultured in presence of serum. In the same way, while the serum-independent cells did not release a sialidase activity in both serum-free media, a significant activity was recovered when serum was added. In fact, the cell adaptation process to serum-free conditions did not specifically affect the sialidase release and the cellular mechanism of protein desialylation, which appeared to be mainly related to the presence of serum for both adapted and non-adapted cells.     

Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium

Summary Five different mouse mammary tumor cell lines were propagated in a serum free medium. Evaluation of growth characteristics, including logarithmic growth, cell population increase, protein production and days to confluency, showed serum-free medium comparable to serum-containing medium. Mouse mammary tumor virus expression and production, in C₃H and GR tumor cell lines, as determined by virus particle counting and RNA dependent DNA polymerase assays, subsequent to dexamethasone stimulation revealed equivalent to higher levels of virus in serum-free medium as compared to serum-containing medium.     

Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.     

Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media

The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.     

Continuous production of human erythropoietin by immobilized recombinant L-929 cells

Recombinant L-929 cells transfected with the human erythropoietin (EPO) gene were immobilized in a macroporous cellulosic support and its derivatives in which charged groups or cell attachment factors were introduced. The immobilized cells were cultured in serum-containing and serum-free media. Comparable production of EPO was observed even in the serum-free medium when a support modified by polyethyleneimine was used for immobilization. The cells immobilized on the supports were cultured in fluidized-bed and inner-loop type air-lift bioreactors for continuous production of EPO. A high cell density of more than 2 × 10⁷ cells/cm³-support and high EPO productivity were achieved and maintained for 50 d through the use of the inner-loop type air-lift bioreactor. The productivity was 13.4-fold higher than that of conventional static cultures in petri-dishes.     

Serum-free medium conditions for steroidogenesis of bovine follicular thecal cells cultured on collagen gel matrix

Summary Thecal cells isolated from bovine ovarian follicles were cultured with a serum-free basal medium or a serum-free complete medium in the presence or absence of collagen gel matrix, and their cellular proliferation and steroidogenesis were compared with those of cells cultured with a serum-containing medium. The cells cultured with the serum-free basal medium produced larger amounts of progesterone, androstenedione, and estradiol than the cells cultured with the serum-containing medium, but no appreciable cell proliferation was observed in the serum-free medium. Response of thecal cells to 8 bromo-cAMP, a steroidogenic agent, varied according to the type of steroid production examined and the type of culture medium used. In a cultivation period of 4 d, progesterone production was stimulated about five-fold by 8 bromo-cAMP in the serum-free complete medium on collagen gel matrix and in the serum-free basal medium without collagen matrix, whereas androstenedione production was stimulated about three- to fourfold in the serum-free complete medium on collagen gel matrix and in the serum-free basal medium with or without collagen matrix. Estradiol production, however, was significantly suppressed by 8 bromo-cAMP in the serum-free complete medium on collagen gel matrix and also in the serum-containing medium. Thus, among the conditions examined, the most suitable primary culture media for steroidogenesis of thecal cells were the serum-free media, especially serum-free complete medium on collagen gel matrix.     

类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

近年来,因病毒侵害人类每年都要蒙受巨大的经济损失和社会损失。犬肾细胞MDCK以其具有的培养容易、增殖快、流感病毒感染效率高等特点,成为适用于流感病毒疫苗生产的重要细胞系之一。以MDCK细胞为研究对象,在自制无血清培养基中成功实现了MDCK细胞从有血清培养到无血清培养的驯化;并通过单细胞悬浮培养驯化过程实现了MDCK细胞的无血清单细胞悬浮培养,获得了适于无血清单细胞悬浮生长的ssf-MDCK细胞株,无血清单细胞悬浮批培养最大活细胞密度可达3.81×106 cells/mL,最大比生长速率可达0.056 h?     

犬肾细胞MDCK无血清贴壁及单细胞悬浮培养

旨在挖掘用于鉴定金黄色葡萄球菌的高特异性靶点及其PCR检测引物。采用C++语言编程,以金黄色葡萄球菌Staphylococcus aureus MRSA 252基因组编码序列为对象,对2 656个可编码区进行分析,获得特异性靶点序列,并设计PCR扩增引物。对包括葡萄球菌属11个种及其他细菌属在内的共计137株细菌验证引物特异性,筛选获得9个DNA序列,并设计了4对引物。经验证2对引物的特异性较好,其中引物SA3的基因组DNA检测限为13.7 fg/μL,菌体检测限为9.25×102 CFU/mL。结果验证     

Enhancement of cell growth and viability of CHO cells in serum-free media by 30Kc6 gene expression

Many attempts have been made to develop a serum-free medium on account of the problems caused by serum in mammalian cell culture. However, serum deprivation inhibits cell growth and induces apoptosis. Moreover, adapting host cells to the serum-free medium is difficult and time-consuming. In a previous study, the anti-apoptotic 30K proteins were identified from silkworm hemolymph, which suggests that the 30K genes coding for the anti-apoptotic compound can be used for the anti-apoptosis engineering of mammalian cells. In this study, the 30K genes (30Kc6, 30Kc19, and 30Kc123) were introduced to DG44 CHO cells, which are the mammalian cell line most commonly used by industry for the production of biopharmaceuticals, in order to make them resistant to the apoptosis induced by serum deprivation. Among the 30K genes, the 30Kc6 gene exhibited the highest apoptosis-inhibition activity. When the 30Kc6-expressing cells cultivated in the serum-containing medium were transferred directly to commercially available serum-free media, 30Kc6 expression increased the viable cell density by four-fold through inhibiting serum deprivation-induced apoptosis.     

Optimization of serum free medium for cord blood mesenchymal stem cells

Human umbilical cord blood harbors mesenchymal stem cells (MSCs), which can give rise to several mesenchymal lineages. In order to explore their usages in medical applications, the ex vivo expansion of MSCs to sufficient cell numbers is necessary. Additionally, the development of a serum-free medium becomes indispensable for elimination of possible contaminants from the serum-containing medium during expansion. Using fractional factorial designs combined with the steepest ascent approach, we have developed a serum-free medium that could ex vivo expand MSCs over nine passages, resulting in at least 1000-fold increases in cell number within 1-month. Based on Iscove's modified Dulbecco's medium, this medium formulation includes bFGF (17.91 ng/mL), human albumin (2.80 mg/mL), hydrocortisone (27.65 μM) and SITE (1.18%, v/v). The expanded MSCs in the designed medium preserved differentiation potentials into three mesenchymal lineages in vitro, including chondrocytes, adipocytes and osteoblasts. In conclusion, we optimized a serum-free and defined culture medium for cord blood-derived MSCs, which could be applied to cell-based therapy and biomedical research.     

黄牛表皮细胞分离与体外培养最适条件的探索

为了建立黄牛表皮细胞分离与体外培养的最适条件,比较了组织块法与单细胞悬液法、不同蛋白酶(胰蛋白酶和分离酶)的消化以及有无血清培养基对细胞生长的影响,以克隆形成率来检测细胞生长和存活情况。结果证明：采用分离酶分离表皮细胞进行无血清培养是黄牛表皮细胞体外培养的最适条件。     

Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line

A serum-free medium, WCM5, has been developed for the large scale propagation of CHO (Chinese hamster ovary) cells which express recombinant protein using dihydrofolate reductase as a selectable marker. WCM5 was prepared by supplementing Iscoves medium without lecithin, albumin or transferrin with a number of components which were shown to benefit growth. WCM5 medium contained 5 mg l^–1 human recombinant insulin (Nucellin) but was otherwise protein-free. CHO 3D11^* cells which had been engineered to express a humanised antibody, CAMPATH^*-1H, were routinely grown using serum-containing medium. From a seeding density of 10⁵ cells ml^–1, cells grown in static culture with serum reached a maximal cell density of 6.5×10⁵ cells ml^–1 after 6 days in culture and produced a maximal antibody concentration of 69 mg l^–1 after 11 days in culture. CHO 3D11^* cells grown with serum were washed in serum-free medium then cultured in WCM5 medium. Following a period of adaptation the cell growth and product yield was superior to that achieved with serum-containing medium. CHO cells producing CAMPATH-1H grown in an 8000 l stirred bioreactor seeded with 2×10⁵ cells ml^–1 reached a maximal viable cell density of 2.16×10⁶ cells ml^–1 after 108 h in culture and a maximal antibody concentration of 131.1 mg l^–1 after 122 h in culture.Abbreviations CHO Chinese hamster ovary - dhfr dihydrofolate reductase - dhfr dihydrofolate reductase deficient - MTX methotrexate - H hypoxanthine - T thymidine - T/V trypsin versene - F12 Hams F12 medium - NEAA non essential amino acids     

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran^®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.List of symbols c substrate or product concentration mmol l^–1 - c₀ substrate or product concentration in the feed mmol l^–1 - c_Glc glucose concentration mmol l^–1 - c_Gln glutamine concentration mmol l^–1 - c_Amm ammonia concentration mmol l^–1 - c_Lac lactate concentration mmol l^–1 - c_FAB concentration of Fab# 10 antibody fragment g l^–1 - c_MAb monoclonal antibody concentration mg l^–1 - D dilution rate d^–1 - q cell-specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q_Glc cell-specific glucose uptake rate mmol cell^–1 h^–1 - q_Gln cell-specific glutamine uptake rate mmol cell^–1 h^–1 - q_MAb cell-specific MAb production rate mg cell^–1 h^–1 - q^* volume-specific substrate uptake or metabolite production rate mmol l^–1 h^–1 - q^*FB volume-specific substrate uptake or metabolite production rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Glc volume-specific glucose uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Gln volume-specific glutamine uptake rate related to the fixed volume mmol l_FB ^–1 h^–1 - q^*FB,MAb volume-specific MAb production rate related to the fixed volume mg l_FB ^–1 h^–1 - q^*FB,02 volume-specific oxygen uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - t time h - U superficial flow velocity mm s^–1 - V medium volume in the conditioning vessel of the fixed bed reactor l - V_FB volume of the fixed bed l - x_v viable cell concentration cells ml^–1 - y_Amm,Gln yield of Ammonia from glutamine - y_Lac,Glc yield of lactate from glucose - specific growth rate h^–1 - _d specific death rate h^–1     

Enhancement of the attachment on microcarriers and tPA production by fibroblast cells in a serum-free medium by the addition of the extracts ofCentella asiatica

The addition of ethanol extracts ofCentella asiatica showed a remarkable enhancement of fibroblast cells attachment to Cytodex beads in serum-free (SF) medium. It also improves tPA production in both batch and perfusion cultivations. The optimal concentration for SF medium was determined as 2 ppm of the extracts when using Cytodex III. In batch cultivation a high specific tPA production rate was obtained, compared to that from 5% FBS containing medium. However, a fast specific growth rate was observed in 5% FBS medium. In perfusion cultivation a reasonably good cell density and tPA production was achieved at a perfusion rate of 2.4×10⁶ (viable cell/ml) and 0.65 (g/ml), respectively at 22 ml/min.     

Induction and Proliferation of Callus Derived from Fruits of Different Varieties of Capsicum annuum

Callus was initiated on Murashige and Skoog (MS) medium containing different combinations of growth regulators or different concentrations of vitamins from pericarp of six varieties of Capsicum annuum differing in their capsaicin content. Callus derived from pericarp of low capsaicin containing varieties was snowy white, friable and showed excellent growth. Callus initiation was delayed (10-15 days) in Punjab Lal, which had very high fruit capsaicin content (7.0 mg g^?1 DW). It also showed relatively slow proliferation although callus obtained was white and friable. Several different media were tried to improve the initiation and the proliferation of the callus in this variety. Callus growth improved greatly by doubling the concentration of MS salts. Initiation time was reduced to 4-6 days by adding 10 mg l^?1 NAA and 0.5 mg l^?1 Kin in MS medium. Other combinations of growth regulators or increase in concentration of vitamins or activated charcoal (0.1%) resulted in poor callus growth and callus texture. Of all media tried, MS medium containing 2 mg l^?1 2,4 D and 0.5 mg l^?1 Kin was the best for callus initiation and proliferation.