Search

Surface immobilization of plant cells avoids the problem of hydrodynamic or shear stress, which tends to be characteristic of suspended cells cultured in typical, mechanically agitated bioreactor systems. Surface immobilization also promotes the natural tendency for plant cells to aggregate, which may improve the synthesis and accumulation of secondary metabolites. In addition, exchange of medium is made simple in surface-immobilized systems, and extracellular secondary products are easily recovered on a continuous basis. However, problems related to regulation of the thickness of the immobilized cell layer, maintenance of the biomass in a productive condition, and vacuolar retention of secondary products have yet to be resolved satisfactorily. This review focusses on two surface-immobilization technologies, differing primarily in the nature and the configuration of the inert support. Prototypes of these designs have been applied to a variety of plant cell systems at bioreactor volumes up to 20 litres. Results obtained with several alternative technologies are also summarized.

A full-length complementary DNA clone encoding tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus (De Luca V, Marineau C, Brisson N [1989] Proc Natl Acad Sci USA 86: 2582-2586) driven by the CaMV 35S promoter was introduced into tobacco (Nicotiana tabacum) to direct the synthesis of the protoalkaloid tryptamine from endogenous tryptophan. Young, fully expanded leaves of CaMV 35S-TDC transformed plants had from four to 45 times greater TDC activity than did controls. Tryptamine accumulated in transgenic plants to levels that were directly proportional to their TDC specific activity. Despite their increased tryptamine content, the growth and development of the CaMV 35S-TDC plants appeared normal with no significant differences in indole-3-acetic acid levels between high tryptamine and control plants. Plants with the highest TDC activity contained more than 1 milligram of tryptamine per gram fresh weight, a 260-fold increase over controls.

The enzyme acetylcoenzyme A: deacetylvindoline 4- O -acetyltransferase (EC 2.3.1.-) (DAT), which catalyzes the final step in vindoline biosynthesis in Catharanthus roseus , was purified 3300-fold using ammonium sulfate precipitation followed by gel filtration, anion exchange, hydroxyapatite, and affinity chromatographies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified DAT showed the presence of two major proteins having M r values of 33,000 and 21,000, whereas native PAGE showed three protein bands, and isoelectric focusing-PAGE one diffuse protein band (p I = 4.7−5.3) plus two minor protein bands (p I = 5.7 and 6.1). Purified DAT possessed K m values of 6.5 μ m and 1.3μ m for acetylcoenzyme A and deacetylvindoline, respectively, and V max values of 12.6 pkat/μg protein (acetylcoenzyme A) and 10.1 pkat/μg protein (deacetylvindoline). Inhibition of DAT by tabersonine, coenzyme A, and cations (K + , Mg 2+ , and Mn 2+ ) was observed, while the pH optimum of this enzyme was determined to be 7.5 to 9.

The medicinal value of monoterpene indole alkaloids continues to attract interest in their phytochemistry, chemical structure, biosynthesis, and chemothera-peutic activity. Structural complexity and non-profitability of total synthesis, perceived or real shortages in the supply of botanicals, and recent concerns for the environmental impact of wild cropping, made production of these alkaloids in vitro, i.e. by plant cells cultured in bioreactors, a viable prospect. The synthesis and accumulation of monoterpene indole alkaloids in cell cultures received great attention when commercial production of compounds like quinine (1), aj-malicine (2) and catharanthine (6), vinblastine (8) and vincristine (9) appeared attainable. Technologies aimed at increasing alkaloid accumulation by employment of production media, by precursor feeding, elicitation, semi-continuous culture, and by application of enhanced bioreactors were designed to achieve this goal. At present, however, efforts to further promote cell cultures as medici-nals have softened somewhat due to regulatory barriers, but also due to biological barriers. Vinblastine and vincristine, the most desirable Catharanthus alkaloids, did not occur in concentrations which would warrant commercial exploitation of an in vitro process. Research will be required to overcome these barriers. Research into the biosynthesis of monoterpene indole alkaloids, when supplemented with molecular biological approaches, appears to be the most sensitive approach.

Upon exposure to a variety of elicitors, suspension cultures of the Papaver somniferum L. cell line PBI #2009, initiated more than ten years ago (2), produce the benzophenanthridine alkaloids, sanguinarine and dihydrosanguinarine (1). Cells of this line, which when unelicited accumulate, at best, only trace quantities of these alkaloids, release a portion of the product into the culture medium, suggesting a possible long-term, continuous use of the cell material. Because of the potential use of sanguinarine as an antibiotic in oral hygiene products (3), an effort was made to develop efficient production methodology by eliciting and re-eliciting surface immobilized cells. The results of these investigations are presented here.

A method for the analysis of ginkgolides by tlc was developed. It allows for the detection of ginkgolides A, B, and C at the 4-μg level, an 86% increase in sensitivity over previously published methods

Papaver somniferum L. (opium poppy) cells were elicited with a Botrytis sp. homogenate and cultured by a semi-continuous process. Elicitation induced synthesis of sanguinarine and dihydrosanguinarine. Significant release of both alkaloids into the culture medium occurred. Medium exchange at 2-day intervals enabled product recovery from spent medium and maintained culture viability. Culture growth was not inhibited by elicitor treatment necessitating sub-culture prior to re-elicitation. Re-elicited cultures displayed an increasing sensitivity (reduced growth rate, higher alkaloid yield) to the elicitor with each successive treatment and did not survive a fourth elicitation.

Cell division in soybean cells (Glycine max (L.) Merr.) grown as 100 ml batch cultures with a defined medium under continuous light has been partially synchronized by daily exchange of 2 5 of the cell suspension for an equal amount of fresh medium. Oscillations of the mitotic index showed 30 h periods, MI(max) = 25%. In cell suspensions maintained as a continuous culture partial synchrony was induced by flushing the cultures with N2-pulses of 1 10 sec every 3–4 sec for 90 min per 24 and 30 h, MI(max) = 12–15%. When the suspensions were grown as a batch culture and N2-pulses administered 8 × 30 min 24 h over a period of 4 days, cell division during the following period of constant aeration was synchronized for 1–2 cycles, MI(max) = 26%

Two cell lines of Vinca major L. cv. variegata produced strictosidine lactam as the main alkaloid. Quantities varied from 500–1000 μg/g DW in the high-yielding line and 1–100 μg/g DW in the low-yielding line. Transfer of cells to alkaloid production medium resulted in a 6–8 fold increase in alkaloid production with the high-yielding line, some increase in the low-yielding line and in both lines induced intense greening (up to 200 μg chlorophyll/g DW) indicating chloroplast differentiation. Though in the source plant leaves are the main storage site, no correlation between alkaloid accumulation and chloroplast differentiation could be found.

Several hundred serially cultured cell suspensions derived from three cultivars of periwinkle ( Catharanthus roseus ) were established in Gamborg's B 5 medium and then transferred to Zenk's alkaloid production medium. Total alkaloid concentration ranged from 0.1 to 1.5% of dry weight. Alkaloids present were of the corynanthe, strychnos and aspidosperma types, with the greatest diversity arising during the third to the fifth week of subculturing. The alkaloid content appeared both specific for, and reproducible in, individual cell lines.

A procedure for cryogenic storage of alkaloid producing cell lines of periwinkle, Catharanthus roseus (L.) G. Don., has been developed. The procedure differs from established cryopreservation protocols in several aspects. Specifically, 4-day-old suspension subcultures of three cell lines were precultured in nutrient media supplemented with 1 molar sorbitol for 6 to 20 hours. The cells were then incubated in nutrient media with 1 molar sorbitol plus 5% DMSO in an ice bath for 1 hour and, thereafter, were frozen in this solution at a cooling rate of 0.5 degrees C per minute to -40 degrees C prior to immersion in liquid nitrogen (LN). After rapid thawing in a 40 degrees C water bath, the regrowth of LN stored cells was achieved by transferring them without washing onto filter paper discs over nutrient media solidified with agar for a period of 4 to 5 hours. The filter paper discs with the cells were then transferred to fresh media of the same composition for regrowth. The viability immediately after thawing as evaluated by the 2,3,5-triphenyl tetrazolium chloride method was about 60% of controls. Suspension cultures established from LN stored cells retained the capability for alkaloid synthesis and accumulation.

Habituated and tumorous Catharanthus roseus cells grown in the absence of hormones accumulated indole alkaloids. Total alkaloids and alkaloid pattern were the same when cells were cultured in medium without hormones or in alkaloid production medium with and without indole acetic acid. Treatment of cells with Pythium homogenate as elicitor did not increase total alkaloids or change the pattern of alkaloids produced. When either habituated or tumorous cells were grown in 1B5 medium after Gamborg et al (1968) containing 2,4-dichlorophenoxyacetic acid (2,4-D), their capacity to accumulate alkaloids decreased with time. The levels of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) specific activities were constant throughout growth except when cells were exposed to 2,4-D in 1B5 medium, where enzyme activities declined in step with the decrease in alkaloid accumulation. Neither habituated nor tumorous cell suspension cultures accumulated vindoline, nor could they be induced to produce this alkaloid by any of the given treatments.

The 953 cell line, one of the cell lines from Catharanthus roseus, has been investigated in terms of its ability to produce indole alkaloids. Various parameters (mitotic index, pH, cell dry weight, growth period) were evaluated in the fermentation process and their relative importance for alkaloid production was determined. The alkaloids isolated and identified are ajmalicine, yohimbine, isositsirikine, vallesiachotamine, strictosidine lactam, lochnericine, horhammericine, horhammerinine, vindolinine, 19-epivindolinine, 19-acetoxy-11-methoxytabersonine, 19-hydroxy-11-methoxytabersonine and dimethyltryptamine.

Summary Cells of established callus and suspension cultures derived from anther walls of periwinkle, Catharanthus roseus (L.) G. Don, responded to 0.2 x 10-3 M 3′,4′-dehydrovinblastine (dVBL) and vincristine with C-mitosis. Cells derived from protoplasts of periwinkle leaves and cultured for 3 weeks tolerated treatment with dVBL. The frequency of C-mitosis increased significantly as, over several weeks, protoplast derivatives continued to multiply and became callus cells, and in the presence of 5 x 10-3 M dVBL. The effect of vincristine was similar to that of dVBL. Vindoline and catharanthine, monomeric moieties of dVBL, did not exert mitotoxicity.

Biotransformation studies of 3′,4′-anhydrovinblastine (1) in Calharanthus roseus cell suspension cultures are reported. A detailed study of one cell line, coded at „916”, has revealed that 1 is transformed to the natural bisindole alkaloids leurosine (2) and Catharine (3) in approximately 25–30 and 9–16% yield, respectively. In 24–48 h, about 40% of 1 is utilized by the cells. Conditions for biotransformation of 1 have not been optimized so the above yields quoted are not final.

Upon treatment of 5 cell lines of Catharanthus roseus with homogenates of various fungi, as well as with chemically defined phytoalexin elicitors, all except one (non-alkaloid producing #916) responded with browing and accumulation of tryptamine within 6 – 24 h. Cells of line #615 responded with not only accumulating tryptamine, but also N-acetyl tryptamine, strictosidine lactam, ajmalicine, tabersonine, lochnericine, and catharanthine. Based on amounts of alkaloids accumulated, cells of line #615 performed best when treated with homogenates of Alternaria zinnae, Pythium apbanidermatum, Verticillium dabliae, and Rhodotorula rubs. A Pythium homogenate concentration of 5 % and a Rhodotorula homogenate concentration of 0.5 % effected maximum alkaloid yields, and, thus, were used in subsequent studies. These revealed a temporary increase of the level of alkaloids in cells and in their medium after 12 – 24 h of treatment. Ten-day-old subcultures responded better than younger and older ones. The elicitor stimulated accumulation of alkaloids and alkaloid composition did not depend on the use of 1-B5 or alkaloid production medium. A 5 l cell suspension of #615 grown in a 7.5 l bioreactor and treated with 5 % Pythium homogenate for 18 h was found to contain strictosidine lactam, ajmalicine, and catharanthine in concentrations of 27, 10, and 13 μg/g DW respectively, the medium contained 42 % of total ajmalicine.

Developing seedlings of Catharanthus roseus were analyzed for appearance of tryptophan decarboxylase (TDC), strictosidine synthase (SS), N-methyltransferase (NMT) and O-acetyltransferase (DAT) enzyme activities. SS enzyme activity appeared early after germination and was present throughout most of the developmental study. TDC activity was highly regulated and peaked over a 48 hour period achieving a maximum by day of 5 of seedling development. Both TDC and SS were present in all tissues of the seedling. NMT and DAT enzyme activities were induced after TDC and SS had peaked and these activities could only be found in hypocotyls and cotyledons. TDC, SS, and NMT did not require light for induction whereas DAT enzyme activity was increased approximately 10-fold after light treatment of dark grown seedlings.

Chlorophyllous, heterotrophic periwinkle (Catharanthus roseus (L.) G. Don) cells were capable of sustained photoautotrophic growth in sugar-free B5 medium containing naphthaleneacetic acid and kinetin when provided with a CO2-enriched atmosphere. An increase in cell fresh weight, first observed approximately 2 weeks after transfer from heterotrophic to photoautotrophic conditions, coincided with the development of maximum chlorophyll content and photosynthetic activity. Electron micrographs revealed that chloroplasts of cells cultured photoautotrophically in continuous light contained large starch granules and exhibited a less extensive thylakoid system than did periwinkle mesophyll chloroplasts. Photoautotrophic cells did not accumulate vindoline or dimeric alkaloids.

Studies involving plant tissue cultures of Catharanthus roseus are described. Investigations concerning the propagation of cell lines of this plant for the purposes of producing indole alkaloids within the Corynanthe, Aspidosperma and Iboga families are presented. The utilization of such tissue culture systems for studies in biosyntheses and isolation of enzymes are also discussed.

Vanadyl sulphate (10-500 mg/l), when added to cell suspension cultures of Catharanthus roseus stimulated increased intracellular accumulation of catharanthine and ajmalicine. This response was demonstrated in both flask and fermenter (30 litre) systems. The response varied, and depended upon cell line, concentration of vanadyl sulphate and the stage of the growth phase at which the cells were treated. This process has the potential to increase the yield and reduce the production time for commercially useful secondary plant metabolites.

Summary Seeds of Catharanthus roseus (L.) G. Don cv. Little Delicata were germinated in the presence of different dark-light regimes. Alkaloid profiles as well as tryptophan decarboxylase and acetyl-CoA: deacetylvindole 0-acetyltransferase were monitored. Growth of seedlings in the dark resulted in an early ubiquitous accumulation of tabersonine as a major alkaloid and in the subsequent day 5–10 cotyledon restricted accumulation of vindoline and its immediate precursors. Transfer of 5-day old seedlings to the light resulted in rapid loss of vindoline precursors followed by a more gradual disappearance of tabersonine and the subsequent enhanced accumulation of vindoline. Although light enhanced vindoline biosynthesis, it was not essential. This sequence of biogenetic events suggested the following biosynthetic pathway: tabersonine → 16-hydroxytabersonine → 16-methoxytabersonine → 16-methoxy-2,3-dihydro-3-hydroxytabersonine → N(1)-methyl-l6-methoxy-2,3-dihydro-3-hydroxytabersonine (i.e. desacetoxyvindoline) → deacetylvindoline → vindoline. The time course of induction indicated that increase of tryptophan decarboxylase activity coincided with tabersonine accumulation, whereas increase of acetyl-CoA: deacetylvindoline 0-acetyltransferase activity coincided with vindoline accumulation. Results suggested that tabersonine biosynthetic pathway enzymes occur in all plant parts whereas the last 5 steps in vindoline biosynthesis are restricted to aerial parts of the plant and that the whole pathway to vindoline biosynthesis is developmentally regulated.

Periwinkle (Catharanthus roseus) cells cultured in 1-B 5 medium display the ultrastructure of parenchyma cells. The parenchyma character remained unchanged when cells were exposed to any one of three different conditions effecting alkaloid accumulation. Transfer of cells to “alkaloid production medium” for 2 weeks (condition 1) accorded two special features,i.e., unusually big lipid droplets in the cytoplasm and, upon fixation, one or several electron-dense droplets of spongy precipitate in vacuoles. Among hormone-autotrophic cultures (condition 2) some cells showed a fine electron-dense vacuolar precipitate. Addition ofPhythium homogenate (fungal elicitor) to cells cultured in 1-B 5-medium for 10 days (condition 3), cells showed a frequent appearance of singular big lipid droplets in the cytoplasm, whereas vacuoles remained devoid of precipitate. The appearance of big lipid droplets and of vacuolar precipitate is interpreted as progressing cytodifferentiation, but is coincidental with alkaloid accumulation.

Summary The uptake of liposomal contents into Catharanthus roseus protoplasts has been quantitated. Hi.phest uptake was obtained from positively charged vesicles (1.09% of vesicle contents per 10 7 protoplasts) in the presence of 10% w/v polyethylene glycol 4000. Significant uptake was also observed from negatively charged vesicles (0.1% of vesicle contents per 10 7 protoplasts) in the presence of 25% polyethylene glycol 1540. The uptake of vesicle contents from negatively charged liposomes was confirmed by light microscopy after treatment of the protoplasts with liposomes loaded with the fluorescent dye 6-carboxyfluorescein. About 10% of the treated protoplasts exhibited intracellular fluorescence. No uptake from neutral liposomes was detected.

Hairy root cultures of Catharanthus roseus were established by infection with six different Agrobacterium rhizogenes strains. Two plant varieties were used and found to exhibit significantly different responses to infection. Forty-seven hairy root clones derived from normal plants and two derived from the flowerless variety were screened for their growth and indole alkaloid production. The growth rate and morphological appearance showed wide variations between the clones. The alkaloid spectra observed were qualitatively but not quantitatively very similar to that of the corresponding normal plant roots. No vindoline or deacetyltransferase activity could be detected in any of the cultures studied. O-acetylval-lesamine, an alkaloid which has not been previously observed in C. roseus was identified from extracts of hairy root clone No. 8. Two root clones were examined for their growth and alkaloid accumulation during a 26-day culture period. Alkaloid accumulation parallelled growth in both clones with ca. 2 mg ajmalicine and catharanthine per g dry weight being observed.

l-Tryptophan decarboxylase (TDC) (EC 4.2.1.27) enzyme activity was induced in cell suspension cultures of Catharanthus roseus after treatment with a Pythium aphanidermatum elicitor preparation. The enzyme was extracted from lyophilized cells containing high levels of TDC and the protein was purified to homogeneity. The pure protein was used to produce highly specific polyclonal antibodies, and an enzyme-linked immunosorbent assay (ELISA) was developed to quantitate the level of TDC antigen during seedling development and in leaves of the mature plant. Western immunoblotting of proteins after SDS-PAGE with anti-TDC antibodies detected several immunoreactive proteins (40, 44, 54.8, 55, and 67 kilodaltons) which appeared at different stages during seedling development and in leaves of the mature plant. The major 54.8 and 55 kilodalton antigenic proteins in immunoblots appeared transiently between days 1 to 5 and 5 to 8 of seedling development, respectively. The 54.8 kilodalton protein was devoid of TDC enzyme activity, whereas the appearance of the 55 kilodalton protein coincided with the appearance of this decarboxylase activity. The minor immunoreactive proteins (40, 44, and 67 kilodaltons) appeared after day 5 of seedling development and in older leaves of the mature plant, and their relationship, if any, to TDC is presently unknown. Results suggest that the synthesis and degradation of TDC protein is highly regulated in Catharanthus roseus and that this regulation follows a preset developmental program.

Cells of a seven year old strain of Papaver somniferum L. when cultured for 2 weeks and incubated with substances known to elicit the formation of phytoalexins, responded by turning reddish brown within 6 h and accumulating sanguinarine. Morphinan alkaloids were not detected. Media (100 ml) containing 1 ml of Botrytis spec. preparations raised the level of sanguinarine in the cells 26 times over the maximum level found in controls. Over a culture period of 79 h the cells achieved a sanguinarine concentration of 2.9% of dry weight. Media (100 ml) with 1 ml of Rhodotorula rubra preparation, 15 mg arachidonic acid, 1 mg actinomycin, 0.5 ml of Helminthosporium gramineum, Sclerotinia sclerotiorum , or 5 ml Colletotrichum gloeosporoides preparation elicited a considerable, but relatively weaker response. Sanguinarine accumulation was also found to occur in the medium and reached a concentration of 43% of total sanguinarine per culture when cells were cultured in 100 ml medium with 5 ml Colletotrichum preparation for 24 h. Young poppy cell cultures initiated 9 months ago responded to the presence of Botrytis material as did 7-year-old cultures.

Partial synchrony of cell division in continuous cultures of soybean cell suspensions was obtained by flushing the cultures with ethylene at intervals of 36 h. The most pronounced synchrony resulted from flushing the suspensions with 3% ethylene for 3 h, followed immediately by 3% CO 2 for 3 h and 30 h aeration prior to the next ethylene treatment. Soybean cells responded to this regime of gassing also with a significant enhancement of growth.

An apparatus for the continuous cultivation of plant cells in suspension culture is described. A type of agitation and aeration is employed which prevents the cells from forming cell aggregates and produces a homogeneous cell culture.

Analysis of the leaves of Vinca major L. cv. Variegata led to the isolation of seven alkaloids: reserpinine, majdine, akuammicine, strictosidine lactam, pseudoakuammigine, akuammine, and a new alkaloid tentatively assigned as 10-hydroxycathofoline. The yellowish and green laminar sections of the leaves did not differ in their alkaloid profile.

Thebaine is biotransformed to neopine by cell suspension cultures of Papaver somniferum cv. Marianne grown in O-B5 medium. Results of precursor stu

A cell suspension culture of Catharanthus roseus was evaluated with regard to its potential usage in studies of early stage indole alkaloid biosynthesis. Employing this material, 1,1,7-trideutero-10-hydroxygeraniol and 1,1,7-trideutero-10-hydroxynerol were incorporated into the alkaloids ajmalicine and strictosidine lactam in approximate proportions of 50% and 80% respectively.

Callus derived from hypocotyls of periwinkle, Catharanthus roseus, responded to culture on nutrient media supplementedwith IAA, BA, and zeatin with shoot formation at low frequencies. However, shoot regenerating callus could be very successfully propagated and subcultured. Alkaloid profiles of callus derived from the original explants (hypocotyls) as well as callus derived from regenerated shoots were almost identical. Subcultures of old callus (initiated in 1978) failed completely to grow shoots. In programs for long-term preservation of alkaloid producing cell lines by regeneration and storage of shoots, selection for ability to form shoots would have to precede selection for alkaloid production.

At the end of the initial meeting on Primary and Secondary Metabolism of Plant Cell Cultures at Schloss Rauischholzhausen, it was decided to convene similar events on a regular basis midway between the International Congress for Plant Tissue and Cell Cul ture. We felt it was necessary to bring representatives of the research teams working in this field together to assess progress as well as to discuss future directions. The Plant Biotechnology Institute of the National Research Council of Canada in Saskatoon, Saskatchewan was chosen to be the site for the second symposium held in 1988. The theme of this meeting was "The Regulation of Primary and Secondary Metabol ism in Plant Cells" and judging by the contributions published in this volume it was a timely and actual one indeed. I would like to thank my organizing committee and my research group for their efforts, the National Research Council of Canada for the financial assistance and the participants for their contributions which all contributed to make this symposium a success. A special thank you to Mrs. L. -Blashill and R. Gallays for their assistance in hosting this event. Last but not least I would like to thank our publisher, Springer-Verlag, Heidelberg for their deci sion to publish the proceedings of this and future symposia."

A study on the effect of various bioregulators on the biosynthesis of ajmalicine (8) and catharanthine (9) in plant tissue cultures of Catharanthus roseus is described. It is shown that 1,1-dimethylpiperidine bromide (3) and 2-diethylaminoethyl-3,4-dimethylphenylether (7) are effective in increasing these alkaloid levels in the cell line PRL #200. Such studies may prove beneficial in larger scale experiments designed for the production of these alkaloids.

Until very recently (Eilert et al 1986), transfer of cells to alkaloid production medium was the only method available to induce indole alkaloid accumulation and enzymes involved in their biosynthesis. However, the slow rates of enzyme induction observed presented difficulties in performing detailed biochemical investigations. Eilert et al (1986) reported that elicitor treatment of Catharanthus cell suspension cultures stimulated production of indole alkaloids within 12–24 h after treatment and the alkaloid profile obtained was qualitatively and quantitatively identical to that obtained by cultivation of cells in alkaloid production medium.

Publisher Summary This chapter provides an overview on the production of indole alkaloids by Catharanthus plant cell cultures, and gives special attention to the enzymatic regulation of synthesis. The preservation of genetic information on callus formation is an important prerequisite for the possible production of the alkaloids in tissue culture. Biosynthesis of the alkaloids takes place in the leaf and biosynthetic regulation of alkaloid formation is performed on organized, rather than callus, tissue. There are three novel culture systems that eliminate the need to cultivate cells in two successive media for the production of indole alkaloids. An important factor for the commercial production of secondary metabolites by tissue culture methods is the retention of high levels of product formation capability in selected cell lines. Various strategies have been proposed for the maintenance of high yields, such as repeated selection for high-yielding clones, preservation of high-yielding clones by cultivation as slower-growing callus, or cryopreservation of productive cells.

Benzophenanthridine alkaloids occur widely in members of the Papaveraceae (poppy family) and have been detected in cell cultures of several species (Furuya et al. 1972, Ikuta et al. 1979, Berlin et al. 1983, Krane et al. 1984). Aspects of production in elicited cultures of Eschscholtzia californica (Schumacher et al. 1987) and Papaver bracteatum (Cline and Coscia 1988) have recently been described. In this regard, our laboratory has demonstrated that a cell line (PBI# 2009) of Papaver somniferum L. initiated approximately 10 years ago (Tarn et al. 1980) synthesizes and accumulates sanguinarine, a quaternary benzophenanthridine alkaloid, when treated with any of a number of elicitor preparations (Eilert et al. 1985). A portion of the sanguinarine is released into the culture medium. This cell line normally accumulates trace quantities, at best, of benzophenanthridines in unelicited suspension culture.

Young leaves from Catharanthus roseus plants contain a novel N-methyltransferase which transfers the methyl group from S-adenosyl-L-methionine specifically to position 1 of (2R, 3R)-2,3-dihydro-3-hydroxytabersonine, producing the N-methylated product. The enzyme shows a high degree of specificity toward substrates containing a reduced double bond at position 2,3 of tabersonine derivatives but the more substituted N-desmethyldeacetylvindoline did not act as a substrate. The enzyme catalyses the third last step in vindorosine and vindoline biosynthesis, and is associated with chlorophyll-containing fractions in partially purified enzyme preparations. The lack of vindoline accumulation in cell suspension cultures is correlated with the lack of expression of this enzyme activity as well as that of an acetyltransferase which catalyses the last step in vindoline biosynthesis. Neither fungal elicitor treatment of cell line #615 nor transfer to alkaloid production medium resulted in expression of these two enzyme activities, nor was either enzyme activity detected in photoautotrophic or hormone autotrophic cultures. Cell lines #200, 615–767 and 916 could not be induced to produce DAT or NMT enzyme activities.

Treatment of one cell line (No. 615) of Catharanthus roseus c.v. Little Delicata with an elicitor preparation of autoclaved and homogenized Pythium aphanidermatum culture resulted in rapid accumulation of indole alkaloids. Alkaloid formation was preceded by rapid transient increases in the extractable activities of the enzymes tryptophan decarboxylase and strictosidine synthase. The induction of these two enzyme activities occurred when cells were transferred to alkaloid production medium or treatment with fungal elicitors. Treatment of this cell line with translational or transcriptional inhibitors prevented the Pythium-induced increases of enzyme activity as well as alkaloid accumulation. When cells were transferred to alkaloid production medium the induction of strictosidine synthase activity preceded that of tryptophan decarboxylase by many hours even when cells were also treated with Pythium elicitor. Results suggested that tryptophan decarboxylase induction proceeds only when endogenous tryptamine levels were decreased by two-third. The internal cellular level of tryptamine, therefore, could regulate expression of tryptophan decarboxylase, whereas induction of strictosidine synthase or of another enzyme in the biosynthetic pathway could control channeling of tryptamine into alkaloids. The results demonstrate that fungal elicitors can be used to facilitate studies of the factors which regulate expression of indole alkaloid pathway enzymes and their ultimate pathway products.

Callus derived from hypocotyls of periwinkle, Catharanthus roseus, responded to culture on nutrient media supplementedwith IAA, BA, and zeatin with shoot formation at low frequencies. However, shoot regenerating callus could be very successfully propagated and subcultured. Alkaloid profiles of callus derived from the original explants (hypocotyls) as well as callus derived from regenerated shoots were almost identical. Subcultures of old callus (initiated in 1978) failed completely to grow shoots. In programs for long-term preservation of alkaloid producing cell lines by regeneration and storage of shoots, selection for ability to form shoots would have to precede selection for alkaloid production.

When added to suspension cultures of CATHARANTHUS ROSEUS, abscisic acid (ABA) stimulated intracellular accumulation of the indole alkaloids catharanthine and ajmalicine in both flask and 30 litre fermenter-scale systems. The response varied, and depended upon the cell line, the concentration and source of the ABA, and the growth phase at which the cells were treated. Precise timing of ABA addition to cells in a 301 fermenter resulted in a catharanthine yield of 85.25 mg/l after 10 days of cultivation. We propose that ABA may be useful for increasing the yield and reducing the production time for commercially useful secondary plant metabolites.

Catharanthus roseus cells suspended in production medium showed the presence of four Aspidosperma-type alkaloids, however, no vindoline. Cells grown in media with the pH adjusted to 7.0 produced 3.3 mg total alkaloids/g dry weight. At pH 5.0, 1.7 mg of alkaloid/g dry weight was produced. (S)-Adenosyl-methionine did not stimulate the production of vindoline. When added to cell suspensions for 21 days, vindoline and catharanthine were degraded to non-alkaloidal substances, not dimerized to bisindole alkaloids.

A large body of research has concerned itself with the production of commercially important phytochemicals by using plant cells in culture. These studies have identified several key areas where basic knowledge is generally limiting exploitation [1,2]. One fundamental problem occurs when non-differentiated tissue cultures are unable to produce and to accumulate complex chemicals originally found in specialized tissues or organs in the intact plant [3,4]. It is this need for specialized organs or tissues for metabolite production that has led to the development of rapidly growing and phytochemical producing organized hairy root cultures following genetic transformation of plants with Agrobacterium rhizogenes [5, and references cited therein]. Such cultures offer the possibility of producing root-derived phytochemicals but not those occurring in other plant tissues [5]. These technical innovations, however, fail to address the fundamental regulatory controls that maintain the specific developmental states which induce the production and accumulation of a given phytochemical.

Azotobacter vinelandii OP was synchronized by the continuous phased culture technique. The nitrogenase (nitrogen:(acceptor) oxidoreductase) (EC 1.7.99.2) activity of the culture was determined continuously within the fermentor by acetylene reduction. Addition of NH4+ in excess of 5 × 10−3 M to the culture lowered nitrogenase activity immediately. Other sources of fixed nitrogen had no immediate effect on nitrogenase activity, but nitrogenase synthesis decreased in the cell cycle following the one in which the fixed nitrogen was added.