Photoautotrophic Culture of Undifferentiated Cells and Shoot-Forming Cultures of Digitalis purpurea L.

Undifferentiated cells and shoot-forming cultures of Digitalispurpurea L. were grown photoautotrophically under 1% CO₂. During3 weeks of culture, the undifferentiated cells multiplied 3-foldand the shoot-forming cultures 2-fold on a fresh weight basis.The chlorophyll content, ribulose 1,5-bisphosphate carboxylaseactivity, Hill reaction activity of the isolated chloroplastsand photosynthetic O₂ evolution of the photoautotrophicallygrown cultures were somewhat higher than the values of the correspondingphotomixotrophic cultures. The digitoxin contents, however,were not improved by photoautotrophic culture. (Received November 9, 1983; Accepted June 11, 1984)     

Production of phytosterols by mature Digitalis purpurea L. plants

Summary The relative rates of production by mature Digitalis purpurea plants of cholesterol, campesterol, stigmasterol and sitosterol isolated from the glycoside and lipid fractions of the plant extract, were estimated. Plants were exposed to an atmosphere of ¹⁴CO₂ in a growth chamber and the radioactivity of the individual sterols assessed at intervals over 25 days on a gas-liquid radio chemical chromatography (GLRC). Incorporation of ¹⁴CO₂ occurred within 12 hours into both fractions of the extract. The 5-ene sterols were produced at a similar rate over a period of 25 days but the lipid fraction was about 100% more radioactive than the glycoside fraction.     

Studies on the Production of Digitalis Cardenolides by Plant Tissue Culture: II. EFFECT OF LIGHT AND PLANT GROWTH SUBSTANCES ON DIGITOXIN FORMATION BY UNDIFFERENTIATED CELLS AND SHOOT-FORMING CULTURES OF DIGITALIS PURPUREA L. GROWN IN LIQUID MEDIA

Undifferentiated, highly chlorophyllous cell cultures; undifferentiated white cell cultures; green, shoot-forming cultures; and white, shoot-forming cultures of Digitalis purpurea L. were established and subcultured every 3 weeks in liquid media in the light or in the dark. The digitoxin content, the chlorophyll content, and the ribulose bisphosphate carboxylase activity of these cultures were assayed. The light-grown, green, shoot-forming cultures accumulated considerable amounts of digitoxin (about 20 to 40 micrograms per gram dry weight), and the white, shoot-forming cultures without chloroplasts accumulated about one-third that amount of digitoxin. The chlorophyll content and the ribulose bisphosphate carboxylase activity of the undifferentiated green cells were about the same as they were in the green, shoot-forming cultures, but the digitoxin content of the former was extremely low (about 0.05 to 0.2 microgram per gram dry weight), which is about the same as that in undifferentiated white cells without chloroplasts. Thus, it was concluded that the chloroplasts are not essential for the synthesis of digitoxin in Digitalis cells. The optimum concentrations of the tested compounds for accumulation of digitoxin were: benzyladenine, 0.01 to 1 milligram per liter; indoleacetic acid, 0.1 to 1 milligram per liter; α-naphthaleneacetic acid; 0.1 milligram per liter; and 2,4-dichlorophenoxyacetic acid, 0.01 milligram per liter.     

Effects of Auxin and Phenobarbital on Morphogenesis and Production of Digitoxin in Digitalis Callus

Pieces of callus obtained from seedlings of Digitalis purpureawere grown on solid Murashige-Skoog's medium supplemented with1 mg liter^–1 BA and 0.1 mg liter^–1 IAA or NAA, withor without phenobarbital (40 mg liter^–1). The replacementof the natural auxin IAA by the synthetic auxin NAA increasedcallus growth and inhibited organogenesis, whereas the additionof phenobarbital had the opposite effect. Morphometric measurementsrevealed a high ratio of vacuole to cytoplasm (v/v) in calluscells. This ratio was affected by the different treatments inthe same way as the fresh weight. The activity of mitochondrialcytochrome P450scc (the enzyme that provides the precursor,pregnenolone, for the biosynthesis of cardenolide in foxgloveplants) was detected in the relevant fraction of callus grownunder all experimental conditions, and its activity was increasedby the addition of phenobarbital. The different treatments testedincreased the cardenolide content and quantifiable amounts ofdigitoxin were detected in all callus tissues. It is of specialinterest that phenobarbital added to the culture medium increasedthe accumulation of digitoxin. The mechanism affecting the developmentand production of cardenolide in callus tissues of D. purpureaby phenobarbital and the replacement of IAA by NAA is discussed. (Received July 18, 1994; Accepted December 14, 1994)     

Biosynthesis of Protein Amino Acids in Plant Tissue Culture. III. Studies on the Biosynthesis of Arginine

Evidence from isotope competition studies and enzymic studies indicates that n-acetyl glutamic semialdehyde, alpha-n-acetyl-l-ornithine, l-ornithine and l-citrulline are intermediates between glucose and arginine in cells of Paul's Scarlet Rose. Evidence for the presence of alpha-n-acetyl-ornithine aminotransferase (E. C. 2.6.1.11) in cell-free extracts was obtained.     

Annual Variation in the Sterol Content of Digitalis purpurea L. Seedlings

Seedings from a single lot of Digitalis purpurea L. seeds were germinated in batches over a period of 13 months. A total lipid extract was made which was resolved into esterified and unconjugated plus glycosylated sterol fractions. The amounts of sterol in each fraction and in the total were compared for seedlings germinated at different times of the year. The amount of esterified sterols reached a maximum value from March until June, and a low value from July until January. In January, a sharp increase began which lasted until March. Amounts of unconjugated and glycosylated sterols were elevated from March until June, low from July until October, and on the rise from November until March. These data correlate with an annual cycle in seed germination. The phase of maximum sterol content of seedlings is followed by a period of null germination.     

Plastid Ontogeny in the Corolla Cells of Digitalis purpurea L. cv. Foxy

Initially the corolla plastids of Digitalis purpurea containsmall grana with negatively stained thylakoids. Degenerationof the grana, loss of chlorophyll and the transient accumulationof starch accompany corolla expansion Starch disappears by thetime the anthers dehisce and granular and amorphous phytoferrtindeposits become prominent in the stroma Concomitant with theseparation of the stigmatic lobes the thylakoid system is reducedto a central membranous network enveloping the phytofemtm aggregatesJust prior to corolla abscission the stroma becomes packed withplastoglobuh Although this developmental sequence closely resemblesthat for chromoplasts in yellow and red flowers, fruits andautumn leaves there is no synthesis of carotenoid pigments inthe corollas of Digitalis purpurea At maturity the plastidsare therefore best described as elaioplasts. Digitalis purpurea L., foxglove, corolla, plastids, elaioplasts, phytofemtin, ultrastructure, X-ray microanalysis     

野生树莓组织培养技术研究

以未萌发的腋芽和单芽茎段为外植体,对野生种刺萼粉枝莓进行了组织培养技术研究。.结果表明,未萌发的腋芽是最佳的外植体;MS 6-BA 1.0 mg/L NAA 0.01 mg/L为较佳的启动培养基;MS 6-BA 1.0 mg/L NAA 0.2 mg/L为最佳的增殖培养基;生根培养基为1/2MS NAA 0.05 mg/L,试管苗生根率高,炼苗移栽成活率达90%以上。     

Effects of Malignant Effusions on the Mitotic Index of L Strain Mouse Cells Grown in Tissue Culture

By employing a clone of L strain mouse fibroblasts (L_E) which does not exhibit cell clumping and lysis (cytolytic antibody reaction), it was possible to screen for the presence of growth-regulating factors in human sera and effusions, exclusive of an antigen-antibody reaction. Under conditions of the test a mitotic index greater than 20% indicated the presence of a growth-promoting factor.A total of 11 pleural effusions was tested. Four of the eight malignant effusions possessed a growth-promoting factor, while none of the three non-malignant effusions or the one sample of human umbilical cord serum possessed such a factor. Overnight storage of the unfiltered effusions at 5° C. resulted in complete loss of the growthpromoting activity.     

Production of a Fungistat and the Role of Fungi during Germination of Digitalis purpurea L.cv. Gloxiniaflora Seeds

The seed coats of Digitalis purpurea L. cv. Gloxiniaflora werecultured for indigenous fungi. Alternaria alternata (Fries)was identified as the sole fungus on seeds freshly harvestedfrom unopened capsules, whereas A. alternata, Rhizopus arrhizus(Fischer), Penicillium sp. and other fungi appeared on storedseeds. The appearance of fungi in seed cultures was seasonal,being more frequent in winter and early spring than in summerand autumn. Alternaria grew well on autoclaved seeds, on dehiscentseed coats, or on seed coats separated from the embryos of ungerminatedseeds. Rhizopus did not grow on these but grew weakly on theculture medium from viable seeds. A. alternata appears to functionas a degradation agent for the seed coat subsequent to germination.Neither fungus was found to be essential to germination of Digitalisseeds. Bioassay of the culture medium from germinating seedsshowed that a fungistat effective against both Alternaria andRhizopus is produced coincident with germination. Based on chromatographicanalysis, the fungistat appears to be a cardenolide. Alternaria alternata (Fries), cardenolides, Digitalis purpurea L. cv, Gloxiniaflora, fungistats, germination, Rhizopus arrhizus (Fischer)     

植物组织培养方法生产药用次生代谢产物研究进展

许多药物来源于植物次生代谢途径,目前植物组织培养方法已成为生产药用成分的重要手段.在植物组织培养中,选择高产的外植体,寻找合适的培养条件,运用两相培养法和毛状根培养技术以及控制组织培养过程中的污染、褐化及玻璃化等问题,则是提高植物细胞生长速度和次生代谢产物产量并实现工业化生产的先决条件.本文主要从以上几个方面介绍其近来的研究进展,并提出了存在的问题及解决对策.     

Studies of Laryngotracheitis Virus in Avian Tissue Cultures: III. Enhancement of Infectivity by Diethylaminoethyl-Dextran

Diethylaminoethyl-dextran (DEAE-D) enhanced the infectivity of laryngotracheitis virus (LTV) for chicken kidney (CK) cells when cultures were treated before inoculation with virus and when DEAE-D was present in the inoculum. Infectivity was not increased when cultures were treated after virus had adsorbed to cells; since infection was not synchronized, most of the virus had probably already penetrated the plasma membrane by the time DEAE-D was added. Maximal enhancement occurred when DEAE-D was present in the inoculum. Enhancement of a lesser degree occurred when virus and DEAE-D were mixed, diluted, and inoculated onto cultures. Adsorption of LTV at 37 C as compared to that at 5 C usually yields about a threefold greater number of plaques after a 2-hr adsorption period. However, when DEAE-D was incorporated in the inoculum, greater enhancement occurred at 5 C than at 37 C, and the number of plaques produced at both adsorption temperatures was about equal. Results are compatible with the hypothesis that increased adsorption is a factor in enhancement of infectivity of LTV by DEAE-D.     

培养基及其组成对水母雪莲悬浮培养细胞生长及黄酮形成的影响

8种不同的基本培养基对水母雪莲(Saussurea medusa)细胞生长和黄酮形成的影响不同。实验结果表明,MS培养基较有利于细胞生长和黄酮形成。从MS培养基修饰得到的MG和MP培养基比前者细胞生长量与黄酮产量分别提高32%和70%。碳源、氮源、植物激素对细胞生长及黄酮形成的影响较为明显。用HPLC对细胞培养物中两种有效黄酮(Jaceosidin和Hispidulin)作了定性定量分析。     

Effects of Culture Conditions on the Growth of Usneaceae Lichen Tissue Cultures

Conditions influencing the in vitro growth of tissues from Usneaceae(sens, lat.) species were investigated. Thallus segments ofall species except the alpine lichen grew at 20?C on malt-yeastextract agar medium in the dark. Tissues of the subtropicallichen grew at 29?C, but those of other species did not. CulturedU. longissima tissues grew rapidly to about 13 times the initialweight in 12 weeks of culture on Lilly-Barnett medium containing2% mannitol. D-Asparagine promoted the growth of cultured lichentissues, as did the corresponding L-amino acid. Cultured tissuesderived from different lichen species used different sugarsand amino acids. Addition of vitamins, phytohormones, and nucleicacid derivatives did not accelerate growth (Received February 9, 1987; Accepted August 26, 1987)     

Trypsin Action on the Growth of Sendai Virus in Tissue Culture Cells III. Structural Difference of Sendai Viruses Grown in Eggs and Tissue Culture Cells

Polypeptides of egg-borne Sendai virus (egg Sendai), which is biologically active on the basis of criteria of the infectivity for L cells and of hemolytic and cell fusion activities, were compared by polyacrylamide gel electrophoresis with those of L cell-borne (L Sendai) and HeLa cell-borne Sendai (HeLa Sendai) viruses, which are judged biologically inactive by the above criteria. Densitometer profiles on the stained gels of egg Sendai resolved six polypeptides (virion protein [VP] 1 to VP6), in which VP2 and VP4 were identified as glycoproteins by PAS stain. Comparative electropherograms of both L Sendai and HeLa Sendai revealed that there were significantly larger amounts in the VP2 region of these viruses but VP4 was present only in greatly reduced amounts as compared to egg Sendai. It was also found that VP2 of L Sendai and HeLa Sendai consisted of two components, VP2a and VP2b, but the one of egg Sendai consisted of only VP2a. A mild trypsin treatment which converts both L Sendai and HeLa Sendai to a biologically active form selectively removed VP2b from these viruses and increased concomitantly the amounts of materials in the VP4 region. The same treatment of egg Sendai affected neither its biological activities nor its electropherogram. Consequently, gross polypeptide profiles on the stained gels of L Sendai and HeLa Sendai after trypsin treatment became favorably comparable to that of egg Sendai. Electrophoresis of labeled L Sendai and HeLa Sendai with a (3)H-amino acids mixture and (14)C-glucosamine resolved at least three glycoproteins, GP1, GP2, and GP3, each corresponding to VP2a, VP2b, and VP4, respectively. The trypsin treatment of these viruses removed almost all the radioactivity of GP2 and simultaneously increased the radioactive counts of GP3 and raised small amounts of rapidly moving heterogeneous glycoprotein, GP4. A possible relationship between the biological modification and the above characteristic polypeptide patterns of Sendai virus was discussed.     

Annual Variation in the Effect of Red Light on Sterol Biosynthesis in Digitalis purpurea L

The effect of varying sequences of red and far red light on sterol biosynthesis in etiolated seedlings of Digitalis purpurea L. was examined. Red light caused a marked increase in the amounts of free and glycosidic sterols and a small decrease in esterified sterols during the first 4 hours after illumination. Far red light elicited the same response but to a lesser degree. Exposure to red followed by far red light or the reverse caused little or no increase in the amounts of free and glycosidic sterols. The magnitude of the increase in the amounts of sterols varied, depending upon the season in which the experiments were performed. The largest increments were obtained during the summer and fall, whereas the smallest were observed during the winter and spring. Correlation of these data with previous observations of an annual cycle in the sterol content of Digitalis seedlings showed that the maximum stimulation in sterol biosynthesis occurs when the endogenous level of sterols is minimal.

Sterol monoglycosides, acylmonoglycosides, and an unidentified sterol conjugate from the lipid extracts were quantitated. Changes in conjugated sterol content were related to the particular light conditions of each experiment. The results are discussed in terms of physiological cycles and the possible influence of hormones upon the control of sterol biosynthesis in Digitalis.

     

Growth and Differentiation of Plant Tissue Cultures III. The Initiation and Pattern of Cell Division in Developing Callus Cultures

The factors affecting the initiation and subsequent patternof cell division in the early stages of callus development havebeen investigated using cultured isolated explants from theJerusalem Artichoke (Helianthus tuberosus). The results obtainedhave been interpreted to explain the possible role played byproducts of autolysis in restricting almost all of the celldivisions to the periphery of the tissue. An attempt has alsobeen made to elucidate the factors which may determine the exponentialincrease in cell number which succeeds the initial lag phase.     

Clonal Variation in Growth and Flavour Production in Tissue Cultures of Allium cepa L.

Callus cultures of onion were initiated from seedling root tissueof three varieties, Rijnsburger, White Lisbon and Red Italian.Twenty separate clones of each variety were sub-cultured fornine passages and estimates made of growth, friability, sliminessand pigmentation at each sub-culture; measurement of alliinaseactivity and precursor level was made at the ninth sub-culture.A number of clones showing a uniformity in one or more charactersappeared but clonal characteristics could not be correlatedwith alliinase activity or flavour precursor levels. The alliinaseactivity was comparable to the normal plant but the precursorlevel was 2–10 per cent of that in the plant. The workshows that despite the large number of separate cultures establishedand the development of stable clonal lines, it was not possibleto select out a clone able to produce a high level of the onionflavour compounds.