Production of 8-epi-tomentosin by plant cell culture ofXanthium strumarium

This study was conducted to establish a plant cell culture system for the production of medically important secondary metabolites fromXanthium strumarium. The effects of plant growth regulators including NAA, 2,4-D, kinetin, and ABA were examined in terms of callus induction, maintenance of callus and suspension cultures. It was shown that callus was induced upon treatment with NAA while embryo was induced after treatment with 2,4-D. Callus formation was further improved by treatment with ABA and NAA. The level of callusing increased by 17–29% for the seed case, cotyledon, leaf, and hypocotyl and by 96% in the case of the root. Suspension cell lines were established using calli produced from cotyledon, hypocotyl and root and cultured at 25°C under light conditions. The cells grew up to 15 g/L with NAA 2 ppm, BA 2 ppm, and ABA 1 ppm treatment. Supernatants of suspension cultures of cell lines derived from coyledon and hypocotyl produced some distinctive secondary metabolites, one of which was identified as 8-epi-tomentosin, which belongs to the xanthanolides. The amounts of 8-epi-tomentosin produced by the cotyledon-and hypocotylderived cell lines were 13.4 mg/L and 11.0 mg/L, respectively.     

Production of human serum albumin by sugar starvation induced promoter and rice cell culture

Human serum albumin (HSA) is the most widely used clinical serum protein. Currently, commercial HSA can only be obtained from human plasma, due to lack of commercially feasible recombinant protein expression systems. In this study, inducible expression and secretion of HSA by transformed rice suspension cell culture was established. Mature form of HSA was expressed under the control of the sucrose starvation-inducible rice α Amy3 promoter, and secretion of HSA into the culture medium was achieved by using the α Amy3 signal sequence. High concentrations of HSA were secreted into culture medium in a short time (2–4 days) by sucrose depletion after cell concentrations had reached a peak density in culture medium containing sucrose. The recombinant HSA had the same electrophoretic mobility as commercial HSA and was stable and free from apparent proteolysis in the culture medium. In a flask scale culture with repeated sucrose provision-depletion cycles, HSA was stably produced with yields up to 11.5% of total medium proteins or 15 mg/L per cycle after each sucrose provision-depletion cycle. A bubble column type bioreactor was designed for production of HSA. In the bioreactor scale culture, HSA was produced with yields up to 76.4 mg/L 4 days after sucrose depletion. HSA was purified from the culture medium to high purity by a simple purification scheme. Enrichment of HSA in culture medium simplifies downstream purification, minimizes protease degradation, and may reduce production cost. The combination of a DNA construct containing the α Amy3 promoter and signal sequence, and the use of a rice suspension cell culture can provide an effective system for the production of recombinant pharmaceutical proteins.     

紫茉莉悬浮细胞培养体系的建立

为建立紫茉莉(Mirabilis jalapa L.)悬浮细胞培养体系,以紫茉莉无菌苗叶片诱导的愈伤组织为材料,筛选紫茉莉悬浮细胞的适宜培养体系。结果表明,紫茉莉愈伤组织在MS+2,4-D 1 mg L-1+KT 0.5 mg L-1的液体培养基中悬浮继代培养3~4次,能得到稳定的悬浮细胞系。培养基的pH值为5.5~5.9,蔗糖浓度为30 g L-1更适合悬浮细胞的生长。紫茉莉悬浮细胞的生长曲线大致呈S型。最佳继代培养时间是10 d,培养液的体积为40 mL时,接种量为7.5 mL,可以较好地保持悬浮细胞系。1 L培养液中可提取分泌蛋白(0.42±0.15) g。这些有助于对悬浮细胞提取分泌蛋白的研究。     

Influence of processing parameters on disintegration of Chlorella cells in various types of homogenizers

The following bead mills used for disruption of the microalga Chlorella cells were tested: (1) Dyno-Mill ECM-Pilot, grinding chamber volume 1.5 L; KDL-Pilot A, chamber volume 1.4 L; KD 20 S, chamber volume 18.3 L; KD 25 S, chamber volume 26 L of Willy A. Bachofen, Basel, Switzerland, (2) LabStar LS 1, chamber volume 0.6 L of Netzsch, Selb, Germany, (3) MS 18, chamber volume 1.1 L of FrymaKoruma, Neuenburg, Germany. Amount of disrupted cells decreased with increasing Chlorella suspension feed rate and increased up to about 85% of the beads volume in the grinding chamber of the homogenizers. It also increased with agitator speed and number of passes of the algae suspension through the chamber. The optimum beads diameter was 0.3–0.5 mm in the homogenizers Dyno-Mill and LabStar LS 1 and 0.5–0.7 mm in the homogenizer MS 18. While the degree of the cell disruption decreased with increasing cell density in Dyno-Mill and LabStar, the cell disruption in the MS 18 increased. Depending on processing parameters, more than 90% of algae cells were disrupted by passing through the bead mills and bacteria count in algae suspension was reduced to about two orders.     

Cryopreservation of photosynthetic plant cell suspension cultures

Attempts were made to cryopreserve in liquid nitrogen six different photomixotrophic suspension cultured lines of five different species:Amaranthus powellii Wats.,Datura innoxia Mill.,Glycine max (L.) Merr.,Gossypium hirsutum L. andNicotiana tabacum xNicotiana glutinosa L. fusion hybrid. Only theD. innoxia line, DAT, and theG. max line, SB1, could be successfully recovered as viable, growing, dark green cultures. The successful method utilized a preculture treatment of from 2 to 8 days in a medium containing 3% starch and 3% sorbitol for DAT and 3% sucrose and 3% sorbitol for SB1 cells. The cells survived if frozen with 10% dimethylsulfoxide (DMSO) and 9.1% sorbitol or with 10% DMSO and 8% sucrose. Following a programmed slow-cooling, the cells were thawed in a 40° C bath and could be recovered directly when added to fresh liquid medium. Cryostorage of these lines will save labor and prevent further genetic changes from occurring in these unique suspension cultures.     

Plant regeneration from embryogenic cell suspension cultures of <Emphasis Type="Italic">Lolium temulentum</Emphasis>

Summary Lolium temulentum L. (Darnel ryegrass) is a self-fertile and diploid grass species with a relatively short life cycle. We propose to use L. temulentum as a model system for genetic manipulation studies in forage and turf grasses, since most of the important grasses are outcrossing, require vernalization to flower, and in some cases are polyploid. As the first step to develop an efficient regeneration and transformation system, we performed a large-scale genotype screening for tissue culture responses using 46 L. temulentum accessions. Embryogenic callus formation frequency ranged from <1% to 11% across all accessions tested. Embryogenic calluses of a few responsive accessions were used to establish cell suspension cultures. The regeneration frequency of green plantlets from the established cell suspension ranged from 15% to 39%. After transferring the regenerants to the greenhouse, fertile plants were readily obtained without any vernalization treatment. This efficient plant regeneration system is being used for genetic transformation studies. With the development of genomics approaches for the improvement of forage and turf grasses, L. temulentum could serve as a model system for testing gene functions.     

Abscisic acid (ABA) treatment increases artemisinin content in <Emphasis Type="Italic">Artemisia annua</Emphasis> by enhancing the expression of genes in artemisinin biosynthetic pathway

Artemisinin, a sesquiterpene lactone endoperoxide derived from Artemisia annua L., is the most effective antimalarial drug. In an effort to increase the artemisinin production, abscisic acid (ABA) with different concentrations (1, 10 and 100 μM) was tested by treating A. annua plants. As a result, the artemisinin content in ABA-treated plants was significantly increased. Especially, artemisinin content in plants treated by 10 μM ABA was 65% higher than that in the control plants, up to an average of 1.84% dry weight. Gene expression analysis showed that in both the ABA-treated plants and cell suspension cultures, HMGR, FPS, CYP71AV1 and CPR, the important genes in the artemisinin biosynthetic pathway, were significantly induced. While only a slight increase of ADS expression was observed in ABA-treated plants, no expression of ADS was detected in cell suspension cultures. This study suggests that there is probably a crosstalk between the ABA signaling pathway and artemisinin biosynthetic pathway and that CYP71AV1, which was induced most significantly, may play a key regulatory role in the artemisinin biosynthetic pathway.     

High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures

Stable transformation of cotton (Gossypium hirsutum L.) at a high frequency has been obtained by particle bombardment of embryogenic cell suspension cultures. Transient and stable expression of the β-glucuronidase (GUS) gene was monitored in cell suspension cultures. Transient expression, measured 48 h after bombardment, was abundant, and stable expression was observed in over 4% of the transiently expressing cells. The high efficiency of stable expression is due to the multiple bombardment of rapidly dividing cell suspension cultures and the selection for transformed cells by gradually increasing the concentrations of the antibiotic Geneticin (G418). Southern analysis indicated a minimum transgene copy number of one to four in randomly selected plants. Fertile plants were obtained from transformed cell cultures less than 3 months old. However, transgenic and control plants from cell cultures older than 6 months produced plants with abnormal morphology and a high degree of sterility. Received: 20 January 1999 / Revision received: 1 October 1999 / Accepted: 11 October 1999     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of embryogenic cell suspension cultures of <Emphasis Type="Italic">Santalum album</Emphasis> L.

An efficient method for Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L. is described. Embryogenic cell suspension cultures derived from stem internode callus were transformed with Agrobacterium tumefaciens harbouring pCAMBIA 1301 plant expression vector. Transformed colonies were selected on medium supplemented with hygromycin (5 mg/l). Continuously growing transformed cell suspension cultures were initiated from these colonies. Expression of β-glucuronidase in the suspension cultures was analysed by RT-PCR and GUS histochemical staining. GUS specific activity in the transformed suspension cultures was quantified using a MUG-based fluorometric assay. Expression levels of up to 105,870 pmol 4-MU/min/mg of total protein were noted in the transformed suspension cultures and 67,248 pmol 4-MU/min/mg of total protein in the spent media. Stability of GUS expression over a period of 7 months was studied. Plantlets were regenerated from the transformed embryogenic cells. Stable insertion of T-DNA into the host genome was confirmed by Southern blot analysis. This is the first report showing stable high-level expression of a foreign protein using embryogenic cell suspension cultures in S. album. U. K. S. Shekhawat and T. R. Ganapathi contributed equally to this work.     

High-level production of recombinant trypsin in transgenic rice cell culture through utilization of an alternative carbon source and recycling system

Productivity of recombinant bovine trypsin using a rice amylase 3D promoter has been studied in transgenic rice suspension culture. Alternative carbon sources were added to rice cell suspension cultures in order to improve the production of recombinant bovine trypsin. It was demonstrated that addition of alternative carbon sources such as succinic acid, fumaric acid and malic acid in the culture medium could increase the productivity of recombinant bovine trypsin 3.8–4.3-fold compared to those in the control medium without carbon sources. The highest accumulated trypsin reached 68.2 mg/L on day 5 in the culture medium with 40 mM fumaric acid.The feasibility of repeated use of the cells for recombinant trypsin production was tested in transgenic rice cell suspension culture with the culture medium containing the combination of variable sucrose concentration and 40 mM fumaric acid. Among the used combinations, the combination of 1% sucrose and 40 mM fumaric acid resulted in a yield of up to 53 mg/L five days after incubation. It also increased 31% (W/W) of dry cell weight and improved 43% of cell viability compared to that in control medium without sucrose. Based on these data, recycling of the trypsin production process with repeated 1% sucrose and 40 mM fumaric acid supplying-harvesting cycles was developed in flask scale culture. Recombinant bovine trypsin could be stably produced with a yield of up to 53–39 mg/L per cycle during five recycling cycles.     

青扦胚性细胞悬浮培养中影响体细胞胚发生因素的研究

试验以青扦（Ｐｉｃｅａｗｉｌｓｏｎｉ）的胚性愈伤组织为材料,以改良５９基本成分附加２４－Ｄ１ｍｇ／Ｌ及ＫＴ１ｍｇ／Ｌ为培养介质,比较了液体悬浮与半固体二种培养方式对胚性愈伤组织增殖和体细胞发生的影响,研究了液体悬浮培养过程中影响体细胞胚发生的因素。结果表明：液体悬浮培养好于半固体培养,它的胚性愈伤组织的生长率为２６８％,是半固体培养的１２４倍;体细胞胚的分化率为９３％,是半固体培养的２２倍;悬浮培养较佳的培养条件为：初始细胞密度为２％（鲜重）,蔗糖浓度为２０ｇ／Ｌ,摇床转速为１００ｒ／ｍｉｎ,ｐＨ为５８。经过两个月悬浮培养,将培养物转至１／２改良５９附加ＡＢＡ１ｍｇ／Ｌ的分化培养基上,３个月后每ｇ培养物上可获得２８５个正常的子叶期体细胞胚。     

“红颜”草莓原生质体制备及瞬时转化体系的建立

为探索“红颜”草莓悬浮细胞系原生质体提取的最优条件,并建立“红颜”草莓原生质体瞬时转化体系,以“红颜”草莓悬浮细胞为材料,对酶液组成、酶解温度、酶解方式进行研究。用PEG介导的瞬时转化法将标记基因GFP转化到“红颜”草莓原生质体中。结果显示：以“红颜”草莓悬浮细胞系作为分离材料,酶液组合为CPW中含有0.5%PVP+0.1%MES+1%纤维素酶+0.5%离析酶+0.01%半纤维素酶+0.9 mol/L甘露醇,在低速（50 r/min）恒温（31 ℃）震摇下进行酶解反应,酶解10 h时,达到“红颜”草莓原生质体最佳分离效果,每克鲜重产量可得原生质体6×10⁸ 个,活力值可达93.0%。PEG介导法成功将含有绿色荧光蛋白（green fluorescent protein, GFP）的植物表达载体转化“红颜”草莓悬浮细胞原生质体,转化效率达44%。通过实验筛选得到“红颜”草莓悬浮细胞原生质体的最佳制备条件,建立“红颜”草莓悬浮细胞原生质体的瞬时转化体系,为进一步开展“红颜”草莓功能基因及合成生物学研究奠定基础。     

Bacoside production by suspension cultures of Bacopa monnieri (L.) Pennell

Cell suspension cultures of Bacopa monnieri (L.) Pennell, grown in modified MS medium, grew some 5–6 fold over 40 days. Selected cell lines produced the important saponin, bacoside A, up to 1 g/100 g dry wt after this time.     

Cell Cultures of Ecdysteroid-Containing Ajuga reptansand Serratula coronata Plants

Cell suspension cultures of Ajuga reptans L. and Serratula coronata L. were derived from long-cultured calluses. Their growth patterns and morphophysiological characteristics indicated that these cells adapted well to grow in suspensions. During the growth cycle of cell suspension, the main ecdysteroid, among others present in plants, was 20-hydroxyecdyson (20E) for both cell lines. The content of 20E in cell suspension of A. reptans was 4–8 times higher than in the intact plant. After long culturing, the ecdysteroid profile in cell suspensions of S. coronata (20E, inokosterone, makisterone A, ecdyson, and a nonidentified metabolite) became similar to that of the intact plant. The ecdysteroids accumulated with a periodicity during subculturing cycle.     

Effect of sugar concentration on camptothecin production in cell suspension cultures ofCamptotheca acuminata

Studies for the effects of sugar concentration on camptothecin production in suspension cultures ofCamptotheca acuminata were made with different concentrations of sucrose, glucose, and fructose. Sucrose among tested carbon sources increased the camptothecin production. The highest camptothecin, 29×10⁴ mg/L, was obtained at 6% of sucrose that was 11 times higher than that at 2% of sucrose. Kinetics of camptothecin production with 6% of sucrose showed the camptothecin production was increased up to 3 days and then decreased after 6 days from inoculation. The highest camptothecin was obtained on the third day from inoculation.     

Characterisation of extracellular polysaccharides from suspension cultures of members of the Poaceae

Microscopic examination of suspension- cultured cells of Phleum pratense L., Panicum miliaceum L., Phalarisaquatica L. and Oryza sativa L. showed that they were comprised of numerous root primordia. Polysaccharides secreted by these suspension cultures contained glycosyl linkages consistent with the presence of high proportions of root mucilage-like polysaccharides. In contrast, suspension-cultured cells of Hordeum vulgare L. contained mostly undifferentiated cells more typical of plant cells in suspension culture. The polysaccharides secreted by H. vulgare cultures contained mostly linkages consistent with the presence of glucuronoarabinoxylan. The soluble polymers secreted by cell-suspension cultures of Phleum pratense contained 70% carbohydrate, 14% protein and 6% inorganic material. The extracellular polysaccharides were separated into four fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7.0. From glycosyl-linkage analyses, five polysaccharides were identified: an arabinosylated xyloglucan (comprising 20% of the total polysaccharide), a glucomannan (6%), a type-II arabinogalactan (an arabinogalactan-protein; 7%), an acidic xylan (3%), and a root-slime-like polysaccharide, which contained features of type-II arabinogalactans and glucuronomannans (65%). Received: 27 April 1999 / Accepted: 5 June 1999     

Oxidative stress induces alkaloid production in Uncaria tomentosa root and cell cultures in bioreactors

The effect of oxidative stress on indole alkaloids accumulation by cell suspensions and root cultures of Uncaria tomentosa in bioreactors was investigated. Hydrogen peroxide (H₂O₂, 200 μM) added to U. tomentosa cell suspension cultures in shaken flasks induced the production of monoterpenoid oxindole alkaloids (MOA) up to 40.0 μg/L. In a stirred tank bioreactor, MOA were enhanced by exogenous H₂O₂ (200 μM) from no detection up to 59.3 μg/L. Root cultures grew linearly in shaken flasks with a μ=0.045 days^?1 and maximum biomass of 12.08±1.24 g DW/L (at day 30). Roots accumulated 3α‐dihydrocadambine (DHC) 2354.3±244.8 μg/g DW (at day 40) and MOA 348.2±32.1 μg/g DW (at day 18). Exogenous addition of H₂O₂ had a differential effect on DHC and MOA production in shaken flasks. At 200 μM H₂O₂, MOA were enhanced by 56% and DHC by 30%; while addition of 800 and 1000 μM H₂O₂, reduced by 30–40% DHC accumulation without change in MOA. Root cultures in the airlift reactor produced extracellular H₂O₂ with a characteristic biphasic profile after changing aeration. Maximum MOA was 9.06 mg/L at day 60 while at this time roots reached ca. 1 mg/L of DHC. Intracellular H₂O₂ in root cultures growing in the bioreactor was 0.87 μmol/g DW compared to 0.26 μmol/g DW of shaken flasks cultures. These results were in agreement with a higher activity of the antioxidant enzymes superoxide dismutase and peroxidase by 6‐ and 2‐times, respectively. U. tomentosa roots growing in the airlift bioreactor were exposed to an oxidative stress and their antioxidant system was active allowing them to produce oxindole alkaloids.     

l-Phenylalanine ammonia-lyase in suspension culture cells of spruce (Picea abies)

The activity of l-phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) was determined in seedlings, callus cells, cell suspension cultures and in young needles of spruce (Picea abies) (L.) (Karst). PAL activity increased up to 10 fold in response to transferring suspension cultured cells into new cultivation medium. PAL was also induced about 10 fold when callus cells were transferrd into liquid medium. The increase was transient and it required the presence of a carbohydrate.In cell suspension cultures, grown in the dark (white cells), but not in light-grown cultures (green cells), PAL activity was induced up to 30 fold by UV-light.With a cell wall preparation of Rhizosphaera kalkhoffii, a forest pathogenic fungus, used as elicitor, the activity of PAL could be induced more than 10 fold. The degree of induction depended on the elicitor concentration. Induction was prevented by cycloheximide but not by actinomycin D.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

Production of betalains by suspension cultures of Chenopodium rubrum L.

Red-violet cell suspension cultures of Chenopodium rubrum were found to accumulate the betacyanins amaranthin, celosianin and betanin and the betaxanthins vulgaxanthin I and vulgaxanthin II. Under a 16-h daylight regime the cells accumulated 0.3–0.4% betacyanins on a dry mass basis after 2–3 weeks of cultivation on the growth medium. Experiments to define a production medium for betacyanins failed with this habituated line. The accumulation could however be increased up to 1% or 100 mg betacyanins/1 by feeding tyrosine and by adaptation of the inoculum size to the nutrient concentration.