Proteins Produced during Salt Stress in Tobacco Cell Culture

The protein pattern of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells that have become adapted to a medium containing 10 grams NaCl per liter was compared to that of unadapted cells on one-dimensional sodium dodecyl sulfate gels. Two protein bands (32,000 and 20,000 daltons) were much more abundant in the salt-adapted cells, and one protein (26,000 daltons) was unique to the salt cells. This protein pattern did not change during the growth cycle of the cells. When salt-adapted cells are transferred to control medium, their ability to grow in the salt-containing medium returns to that of control cells after one passage in the control medium (Hasegawa, Bressan, Handa 1980 Plant Cell Physiol 21: 1347). Within this time the levels of the 32,000 and 20,000 dalton proteins also return to that of the control cells, but the 26,000 dalton protein does not disappear until after at least two passages in control medium. Amino acid analyses of these three proteins revealed that they all contain some hydroxyproline.     

Salt Adaptation of K+ Channels in the Plasma Membrane of Tobacco Cells in Suspension Culture

The patch-clamp technique was used to study and compare thecharacteristics of cation channels in the plasma membrane ofcultured lines of tobacco (Nicotiana tabacum L. cv. Bright Yellow-2)cells that were unadapted (NaCl-unadapted cells) and adaptedto 50 and 100 mM NaCl (Na50-adapted and Na100-adapted cells).In these three types of tobacco cell, the outward whole-cellcurrent activated by depolarization was dominated mainly bythe activity of the outward rectifying K⁺ channels with a single-channelconductance of 20 pS. The steady-state amplitude of the outwardwhole-cell currents at all the positive potentials examineddecreased in the following order: NaCl-unadapted cells>Na50-adaptedcells>Na100-adapted cells. There were no significant differencesbetween the NaCl-unadapted and the Na50-adapted cells in termsof the ratio of permeabilities of these channels to K⁺ and Na⁺ions. Furthermore, no significant differences in terms of thesingle-channel conductance of these channels were observed amongthe NaCl-unadapted, the Na50-adapted and the Na100-adapted cells.These observations suggest that adaptation to salinity of tobaccocells in suspension results in reduced permeability of the K⁺channels to both K⁺ and Na⁺ ions, without any change in theK⁺/Na⁺ selectivity and single-channel conductance of these channels. ¹Present address: Research Laboratory of Applied Biochemistry,Tanabe Seiyaku Co., Ltd.16-89 Kashima 3-chome, Yodogawaku, Osaka,532 Japan     

烟草脆散型愈伤组织获取及其细胞悬浮培养(简报)

以烟草叶片为外植体,以2/3MS BA 0.5mg/L NAA 0.1mg/L 为愈伤组织诱导培养基,继代培养3次后,转入添加3332.48mg/L Ca(NO3)2的该培养基上继代,可获得脆散型愈伤组织,并由此建立细胞悬浮培养系统。     

The Effect of Cadmium on Tobacco Cell Culture and the Selection of Potentially Cd-Resistant Cell lines

The effect of different concentrations of cadmium on the viability, cell division, and the total increase in the biomass of the VBI-O cell strain of tobacco (Nicotiana tabacumL., Virginia Bright Italia) was followed. The concentration of 10^-6 mol 1^-1 Cd²⁺ was fully tolerated by this strain, a nearly total inhibition of cell division and high cell mortality rate ocsicurred at the concentration of 10^-4 mol1{si-1} Cd²⁺. Following a long-term exposure of the culture to gradually increasing cadmium concentrations, seven cell lines able to grow on media containing 10^-4 mol 1^-1 Cd²⁺ were derived. Phenotype diversity of the isolated cell lines likely causes of the disappearance of the resistance character are discussed.     

除虫菊细胞的悬浮培养

除虫菊悬浮细胞从培养后第8天开始迅速生长,14 d达到最大生长量,其最适培养基为:MS 4mg·L-12,4-D 0.1mg·L-1NAA 0.3 mg·L-16-BA 30 g·L-1蔗糖,最适接种量为15 g·L-1,摇瓶装液的体积分数为40%,初始培养的最适pH值为5.8,收获时pH下降为4.6±1,最适培养温度为25℃.     

云南红豆杉细胞的悬浮培养

在云南红豆杉细胞悬浮培养中,适宜的培养基为B5,接种量为0．5～0．8g干重细胞／100ml培养基,2,4－D浓度为1．0mg／L;培养细胞的生长周期约30d;培养基中较高浓度的蔗糖（40g／L）可提高紫杉醇含量;添加的椰子汁（CM）、酪蛋白氨基酸（C）和水解乳蛋白（LH）3种有机添加剂均能提高培养细胞中紫杉醇的含量,但只有CM和CA能促进细胞的生长。于B5培养基中添加不同浓度的NH4NO3对培养细胞无明显影响。     

Effect of Altered Sterol Composition on Growth Characteristics of Saccharomyces cerevisiae

The function of sterols in mitochondrial structures of yeast was examined. Sterol mutant strains were employed to examine the effects of altered sterolic content on optimal and permissive growth temperatures in respiring and fermenting cultures. Although fermentative growth was unaffected by sterol composition, a definite decrease in both the optimal and the permissive growth temperatures of respiring cultures was observed when ergosterol was replaced by Delta(8(9), 22)-ergostadiene-3beta-ol. In vitro studies showed a similar decrease in membrane phase transition temperatures of the mitochondrial enzyme S-adenosylmethionine: Delta(24)-sterol methyltransferase in the mutant strains. Increased sterol and methyltransferase levels were detected in strains incapable of synthesizing ergosterol. A possible control function governing sterol synthesis is proposed for ergosterol.     

草麻黄细胞悬浮培养体系的建立

目的:建立草麻黄悬浮培养体系.方法:采用组织培养的方法探讨了水解酪蛋白(CH)、基本培养基、取材时间、和摇床转速对麻黄愈伤组织悬浮培养的影响.结果:MS基本培养基、300mg/l水解酪蛋白、继代25d的愈伤组织、转速110r/min是愈伤组织悬浮培养的适宜条件,愈伤组织细胞增殖量分别为0.76g、0.80g、0.80g、0.80g.结论:初步选择出草麻黄愈伤组织细胞的悬浮培养条件,为麻黄细胞扩大培养及有效成分提取奠定基础.     

Expression of Oat-phyA-cDNA in a Suspension Cell Culture of Transgenic Tobacco: A Single-Cell System for the Study of Phytochrome Function

A culture of callus cells has been developed from a transgenicline of tobacco which contains an introduced phyA-cDNA encodingphytochrome A. Suspension cultures of the cells were shown toaccumulate a significant immunodetectable level of the heterologousphytochrome, but not of the native phyA-gene product. The red-irradiatedform (P_fr) of the heterologous phytochrome was specificallydegraded in vivo, and the red-irradiated (P_fr) and far-red-irradiated(P_r) forms demonstrated different patterns of in vitro proteolyticcleavage. These results strongly suggested that the phytochromeapoprotein was associated with a chromophore moiety which mediatedred/far-red sensitive conformational changes of the molecule.Exogenous application of 4-amino-5-hexynoic acid (AHA) to thetransgenic suspension cultures resulted in the accumulationof a population of phytochrome which was stable under red lightand gave identical patterns of in vitro digestion in the redand far-red irradiated forms, i.e. the spectral activity ofphytochrome was inhibited. Application of exogenous 5-aminolevulinicacid (ALA) or biliverdin overcame the inhibitory effects ofAHA to restore spectral sensitivity of the phytochrome pool.These results are consistent with the proposed pathway of phytochromechromophore biosynthesis in intact plant systems. Thus, thetransgenic suspension cultures provided a single-cell systemin which spectrally-active phytochrome, apparently indistinguishablefrom the native phytochrome synthesized in etiolated seedlings,was accumulated. Photoregulation of expression of the genesencoding the small subunit of ribulose-1,5-bisphosphate carboxylaseand chlorophyll a/b binding proteins demonstrated that the heterologousphytochrome population mediated rapid changes in gene expressionin the de-differentiated cells. It is therefore proposed thatsuch a suspension culture of transgenic cells offers a modelsystem for the study of phytochrome function. Key words: Cell cultures, transgenic tobacco, phytochrome, oat-phy A-cDNA, gene expression     

Salt Tolerance in Cell Suspension Cultures of the Halophyte Kosteletzkya virginica

Tolerance to NaCl was studied in cell suspension cultures ofKosteletzkya virginica (L.) Presl. (Malvaceae), a dicotyledonoushalophyte that grows in tidal marshes of the eastern UnitedStates. Growth of salinized cultures was significantly inhibitedat high (255 mol m^–3 NaCl), but not at lower externalsalinities. Adjustment of cell suspensions to Nacl was rapid,with the duration of the normal growth cycle unaffected by salinity.Maximum biomass was attained when cultures were exposed to NaClduring early log growth. Patterns of inorganic ion accumulationreflected the utilization of both Na⁺ and K⁺ as osmotica, withNa⁺ content substantially increasing when cells were grown atan external salinity sufficient to reduce growth. K⁺ uptakeselectivity was high and Na⁺/K⁺ ratios were low in salt-treatedcultures even though K⁺ content was somewhat lower comparedto unsalinized cultures. Free proline and microsomal lipid contentincreased in salt-treated cell cultures. Key words: Kosteletzkya virginica, halophyte, salt tolerance, cell suspension culture     

沙枣细胞悬浮培养体系的建立

分别以沙枣幼嫩叶片,茎段为外植体诱导沙枣愈伤组织,选择生长旺盛的松散愈伤组织进行细胞悬浮培养,探讨了培养基种类和激素组合对其产生的影响.结果表明,最适的沙枣愈伤组织诱导培养基为NT(包括0.1 mg·L-1BA和0.1 mg·L-1TDZ);沙枣悬浮细胞在MS,NT,WPM,B5和IS培养基中均可生长,其中在NT培养基中生长状态最好,呈现生长均一的绿色疏松状态.激素组合对沙枣细胞生长影响很大,其中附加BA 0.1 mg·L-1和TDZ 0.1 mg·L-1组合的NT液体培养基可获得大量繁殖速度快、生长均匀一致的悬浮细胞,细胞增长系数达5.73.HPLC测定结果证明沙枣细胞中含有一定量的浓缩鞣质达5.2022 mg/gDW,这为利用沙枣悬浮细胞生产次生代谢产物提供了一条有效途径.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Induction of Proteinase Inhibitors in Tobacco Cell Suspension Culture by Elicitors of Phytophthora parasitica var. nicotianae

An elicitor preparation obtained from Phytophthora parasitica var. nicotianae, a pathogen of tobacco, induced an accumulation of proteinase inhibitors and a stimulation of ethylene synthesis in a tobacco (Nicotiana tabacum) cell suspension culture. About 30 micrograms per milliliter of elicitor were necessary for maximal induction of proteinase inhibitor accumulation, and the response was detectable after 12 hours of incubation with elicitor. Accumulation of proteinase inhibitors required de novo protein synthesis, since cycloheximide completely inhibited its elicitation, and actinomycin D inhibited it partially. One of the inhibitors was purified by a procedure that included heating, (NH₄)₂SO₄ precipitation, ion-exchange chromatography, and affinity chromatography. The purified inhibitor was shown to be a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of about 10,500. It inhibited trypsin but not chymotrypsin.     

水翁悬浮细胞系的建立及其悬浮培养的生长特性

建立了水翁悬浮细胞系,并对其悬浮培养的生长特性作了初步探讨。以水翁新生芽尖作为外植体,接种于添加有不同浓度和配比的生长调节物质及各种附加物的MS固体培养基中,诱导培养产生初代愈伤组织;挑选Ⅰ和Ⅱ型的愈伤组织进行继代培养改良,考察愈伤组织的生长状况和统计生长量来决定最佳继代培养基的配方和得到适合悬浮培养的愈伤组织;将以上得到的愈伤组织转接于最佳继代液体培养基中,于24±1℃,120r/min条件下振荡培养,筛选分散度好、较均匀、生长快、色浅透明的细胞作为种子传代,数次传代后得到性能良好的悬浮细胞系;以细胞生长量(鲜重)为指标,绘制了水翁悬浮细胞的生长曲线。研究表明:2.0mg/L的2,4-D的诱导率最高(92%,初代愈伤组织为Ⅰ型),Ⅱ型愈伤组织的最高诱导率为75%;最佳的继代培养基配方为MS 0.5mg/L 2,4-D 0.5mg/L 6-BA 1.0mg/L IAA 0.5mg/L IBA 0.5mg/L NAA 0.1mg/L KT 700mg/L LH,形成Ⅱ型愈伤组织的生长量可达3.28g/瓶(鲜重);液体继代培养3代后,可得到性能良好的悬浮细胞系;水翁悬浮细胞的生长曲线表明,最佳接种期为培养后的16~18d。     

Effect of Modification of Membrane Phospholipid Composition on Phospholipid Methylation in Aggregating Cell Culture

The effect of the presence of nitrogenous bases in the growth medium of fetal rat brain aggregating cell cultures was investigated. The presence of either N-methylethanolamine (MME) or N,N-dimethylethanolamine (DME) in the growth medium resulted in significant increase of the corresponding phospholipid, phosphatidyl-N-monomethylethanolamine (PMME) or phosphatidyl-N,N-dimethylethanolamine (PDME). They represented 28% and 32% of the total phospholipids, respectively. The presence of the new phospholipids was accompanied by a significant decrease of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Cells grown in the presence of ethanolamine or choline had only barely detectable amounts of PMME and PDME. Intact cells previously grown with the bases were incubated with [methyl-3H]methionine. Incubation of cells previously grown in presence of the bases MME and DME resulted in a marked increase of radioactivity in the corresponding phospholipids possessing one additional methyl group, PDME and PC respectively. The incorporation of S-adenosyl[methyl-3H]methionine (AdoMet) was examined in cell homogenates incubated in presence or absence of either PMME or PDME acceptors. The addition of these exogenous phospholipids caused a three-or fourfold stimulation of radioactivity incorporated into the total phospholipids of cells grown in the absence of nitrogen bases. The cells grown in presence of either MME or DME in the culture medium did not show an increased incorporation of methyl groups from AdoMet into the total phospholipids after addition of exogenous acceptors. This work suggests that MME and DME incorporated into the corresponding phospholipids function as effective substrates for phospholipid-N-methylation.     

New Medium Composition for High Betacyanin Production by a Cell Suspension Culture of Table Beet (Beta vulgaris L.)

The effect of a revised Linsmaier-Skoog (LS) medium on betacyanin production was investigated in suspension cultures of table beet (Beta vulgaris L.). The effects of a high iron concentration and low concentration of zinc on betacyanin production were not cumulative. The composition of the new revised medium for high betacyanin production was established by reducing the concentration of inorganic nitrogen (30 mM), modifying the ratio of ammonium to nitrate (1:14), reducing the concentration of zinc (0.0003 mM), and removing copper and cobalt. The revised LS medium enabled the maximum betacyanin yield of 550 mg/l to be obtained from a 14-day culture. This medium promoted the betacyanin production in three types of cell line differing in the betacyanin productivity. The betacyanin productivity (40 mg/l?day) was higher than that quoted in any other previous reports.     

Characteristics of a Galactose-adapted Sugarcane Cell Line Grown in Suspension Culture

Although d-galactose is normally toxic to sugarcane (Saccharum sp.) cells, a cell line that grows on 100 mm galactose has been propagated. Nonadapted cells in a medium containing galactose instead of sucrose accumulate UDP-galactose; these cells also have much lower UDP-galactose 4-epimerase (EC 5.1.3.2) activity than do adapted cells. This enzyme may determine whether or not galactose will cause toxicity symptoms to develop. The growth rate of galactose-adapted cells is similar to most cell lines on several other carbohydrates. The galactose-adapted cells are also similar to sucrose stock cells in cell wall composition and sugar phosphate concentrations, but, like the nonadapted cells, accumulate free galactose.     

Growth Profile of Crown Gall Cells of Tobacco in Suspension Culture

In order to obtain a basic information of plant cell suspension culture as a step toward the development of large scale culture, culture conditions of crown gall cells (auxin non-requiring cells) were investigated. Addition of yeast extract to culture medium was significantly effective for the growth and cell dispersion.

In experiments on the ability of the cultured cells to utilize sugars as the carbon source, it was observed that galactose, added to the culture medium, markedly inhibited the cell growth.

Pasteurization of the medium containing fructose as carbon source made it brownish by Maillard reaction and the medium apparently restrained the cell growth. However, the fructose medium sterilized by filtration was excellent for the cell growth as well as sucrose or glucose medium. In a jar fermentor, even the glucose medium became brownish by heat sterilization and the brown colored medium restrained the cell growth. Under optimum conditions, the doubling time was 1.1 day in exponential phase and 2.0 g of cell (dry weight) per 100 ml culture was obtained as the maximum yield.     

Compensatory Aspects of the Biosynthesis of Spermidine in Tobacco Cells in Suspension Culture

The relationship between the biosynthesis of polyamines andethylene was examined in suspension cultures of Nicotiana tabacumL. cells. Aminooxyacetic acid (AOA), an inhibitor of 1-aminocyclopropane-1-carboxylicacid synthase, inhibited the production of ethylene and raisedlevels of spermidine by increasing the availability of S-adenosylmethionine(SAM) for the synthesis of polyamines. In contrast, methylglyoxalbis (guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethioninedecarboxylase (SAMDC), an enzyme involved in the biosynthesisof polyamines, caused a slight increase in the rate of biosynthesisof ethylene. However MGBG did not decrease the rate of biosynthesisof polyamines in 10-day-old senescing cells. Although MGBG inhibitedthe conversion of L-[U-^l4C]methionine into labeled spermidinevia SAM both in 4-day-and in 10-day-cultured cells, it stimulatedthe conversion of L-[U-^l4C]aspartic acid into labeled spermidinein 10-day-cultured cells. In actively dividing 4-day-culturedcells, L-[U-¹⁴C]homo-serine was also converted into polyamines.In senescing cells, which produce large amounts of ethylene,the biosynthesis of spermidine from aspartic acid coincidedwith that from methionine. In actively growing cells, whichproduce large amounts of polyamines, the biosynthesis of spermidinefrom homoserine coincided with that from methionine. These resultsindicate that homoserine and aspartic acid can be both usedas precursors in the biosynthesis of polyamines and help tomaintain appropriate titers of polyamines, when SAMDC is inhibitedand the level of decarboxylated SAM becomes limiting. (Received May 14, 1990; Accepted March 11, 1991)