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Synergistic Response of Plant Hairy-Root Cultures to Phosphate Limitation and Fungal Elicitation David S. Dunlop and Wayne R. Curtis' Department of Chemical Engineering and Biotechnology Institute, The Pennsylvania State University, University Park, Pennsylvania 16802
T h e combined effects of phosphate limitation and fungal elicitation on sesquiterpene production were examined in Agrobacterium-transformed"hairy-root" cultures of Hyoscyamus muticus. Limiting the initial supply of inorganic phosphate t o elicited cultures resulted in a 4.2-fold increase in solavetivone production as compared t o elicitation a t control media phosphate levels (1.1mM). Because growth was attenuated at low initial phosphate levels, production per unit cell mass increased 46 times as compared to the control. Both elicited and nonelicited cultures showed enhanced solavetivone production a t lower initial phosphate levels. In fact, the specific productivity of nonelicited roots grown in phosphate-free media was comparable to the specific productivity of fungally elicited roots that are not phosphate limited. Addition of fungal elicitors enhanced specific productivity of solavetivone about 200-fold over nonelicited cultures at nearly all phosphate levels. Phosphate limitation and fungal elicitation are therefore synergistic, and optimal total production is achieved by simultaneous application of these two production strategies.
Introduction Roots synthesize many useful biologically active chemicals (Signs and Flores, 1990). In many cases, this biosynthetic ability is part of the response to external stress, in particular, the challenge by microorganisms and herbivores. These defense compounds may have use as pharmaceuticals and pesticides. The "hairy-root" syndrome caused by Agrobacterium rhizogenes infection provides a convenient method for the growth of isolated root cultures (Flores and Filner, 1985). The advantage of hairy-root cultures is that they retain differentiation while exhibiting growth rates comparable to those of plant cell suspensions. Unlike plant suspensions, which often produce very small amounts of secondary metabolites,hairy-root cultures can displayhigh biosyntheticcapabilitiesthat are often comparable to those of normal roots (Signs and Flores, 1990). Previous research has identified parameters that influencethe production of secondary metabolitesfrom plant cell culture. Phosphate limitation has been used extensively to increase the accumulation of secondary metabolites (Knobloch and Berlin, 1980,1983;Sasse et al., 1982; Balague and Wilson, 1982; Schiel et al., 1984). The rationale for phosphate limitation is that a reduced rate of growth results in increased resource allocation for secondary metabolism. This is typically referred to as the inverse relationship between growth and secondary metabolite production. Elicitation has also been used to enhance secondary metabolite production in plant cell cultures (Eilert et al., 1987;DiCosmo et al., 1987; Lamb et al., 1989). Exposure of root cultures of fungal cell wall fragments results in the induction of antifungal compound synthesis by the roots as a part of the defense mechanism (Darvill and Albersheim, 1984). Fungal elicitation provides a rather specific technique of stimulating secondary metabolism that is
* Address correepondence to this author at the Department of Chemical Engineering, 111 Fenske Laboratory, The Pennsylvania State University, University Park, PA 16802. 8756-7938/9 1/3007-0434$02.50/0
not dependent upon reduction of culture growth rate. Phosphate limitation and fungal elicitation enhance secondary metabolism by two fundamentally different mechanisms;therefore, it may be advantageousto use both techniques simultaneously. We have previously shown that the combination of phosphate limitation and fungal elicitation can enhance the production of plant metabolites in cell suspension cultures (Newell, 1990). In this report, we show that synergistic interaction can also be obtained in secondary metabolism of hairy-root cultures.
Materials and Methods Root Maintenance. Hyoscyamus muticus hairy-root clone A4C17 obtained from Dr. Hector Flores (Flores et al., 1987) was used in all experiments. These cultures have been maintained for over 7 years on a B5 medium (Gamborg et al., 1968). Root tips are subcultured every 2 weeks into 50 mL of fresh medium in 125-mLflasks. The cultures are maintained on a gyratory shaker with a 2-in. stroke at 80 rpm and 25 "C. Phosphate Limitation. Experiments were carried out in nine levels of inorganic phosphate: 0,0.05,0.1,0.2,0.3, 0.5, 0.75, 1.0, and 1.5 times the control level of 1.1 mM phosphate. Because NaHzPOr is the primary source of both phosphate and sodium in B5 medium, the media formulation was altered to minimize counterion fluctuations. Levels of phosphate were manipulated with KH2PO4 plus the addition of 1.1mM NaC1. In this manner, the sodium levels were kept constant, and potassium levels varied less than 6.3% from the standard B5 potassium level of 24.7 mM. This medium composition results in a 3.2 mM chloride ion concentration, which is 50% higher than the standard B5 level of 2.1 mM. To account for the effects of elevated chloride ion, an additional control equivalent to B5 medium (NaHzPO4) was included. Inoculation/Harvest. Flasks containing 50 mL of media were each inoculated with 0.2 g (fresh weight) of roots. Inoculation was accomplished by cutting roots into segments approximately 1cm in length and blotting gently
@ 199 1 American Chemical Society and Amerlcan Instltute of Chemical Englneers
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with sterile Whatman no. 4 filter paper to remove excess medium. Blotted roots were then weighed in a sterile petri dish. This quantitative inoculation procedure provided reproducible growth kinetics. One day after elicitation, fresh root mass was measured after blotting excess media between paper towels, and dry mass was determined after lyophilization and desiccation. Elicitation. After 14 days of growth, three replicate root cultures a t each phosphate level were elicited with 1 %volume (0.5 mL) of fungal elicitor and replaced on the shaker (Signs and Flores, 1989). The elicitor was prepared by growing the soil fungus Rhizoctonia solani in a modified S & H medium (Schenk and Hilderbrandt, 1972) supplemented with 1.00 g/L nyo-inositol, 2.36 g/L asparagine, 15.0 g/L dextrose, 10 pg/L pyridoxine hydrochloride, and 10 pg/L thiamine hydrochloride for 18 days on a gyratory shaker at 80 rpm and 25 "C. The fungal mycelium was resuspended in distilled water (10% of original culture volume) and homogenized for 15 min in a blender on high speed. The homogenate was then autoclaved for 3 h to facilitate release of cell wall fragments, followed by centrifugation at 21000g for 30 min. The final crude elicitor consisted of the filter-sterilized supernatant. Phosphorus Analysis. Total phosphorus content of the root tissue was determined from acid digests. Dry powdered tissue (10-20 mg) was weighed into a test tube and digested at 300 "C for 30 min with 0.25 mL of 70% perchloric acid and 2 drops of 30% peroxide. To prevent interference with the phosphate assay, the digested sample was neutralized with NaOH by using 2 drops of thymol blue (57.4 mg/L) as indicator. After adjustment of the sample volume to 10 mL with deionized, distilled water, the neutralized sample was assayed for inorganic phosphate by the method of Murphy and Riley (1962),adapted as described previously (Curtis, 1990). Sesquiterpene Extraction and Analysis. Sesquiterpenes were recovered from the medium by extraction twice with 25 mL of HPLC-grade chloroform. The organic layer was combined and boiled to dryness on a rotary evaporator. The compounds were rinsed from the boiling flasks three times with 1 mL of chloroform through a 0.2pm nylon syringe filter. The filtrate was then dried under nitrogen atmosphere and resuspended in 2 mL of HPLCgrade methanol for analysis. After separation through a Waters p-Bondapak C18 HPLC column, sesquiterpenes were detected by using a Waters 990 photodiode array detector at 200 nm (2 mL/min, 60:40 Hz0:acetonitrile). The HPLC procedure was calibrated against a standardized GC procedure for sesquiterpenes (Chappell and Nable, 1987).
Rssults Phosphate Effects on Growth. Figure 1 shows the growth response of roots to different initial media phosphate levels. The accumulated cell mass after 2 weeks of culture is shown to increase with phosphate level up to about 0.5 mM. By examination of the phosphorus content of the root tissue, an assessment can be made on the degree of phosphate limitation. Below an initial media phosphate concentration of 0.18 mM, the phosphorus content of the tissue is essentially constant at 25 pmol of P/g of dry weight. This indicates that the roots are yield limited for phosphate and have ceased growing due to phosphate deprivation. The minimum cellular phosphorus content of the root tissue is an important kinetic parameter in the modeling of phosphate-limited growth (Curtis, 1988). The value reported here for H. muticus is in close agreement with the minimum phosphate content of 33 pmol of P / g of dry
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initial Media Phosphate (mM) Figure 1. Growthresponse of H. muticus hairy-rootcultures to initial media phosphate. Dry weight accumulatedafter 14 days at each phosphate concentration. Phosphorus content of cells delimits ranges of yield-limited, rate-limited, and rate-saturated growth on phosphate. Open symbol represents control for chloride ion effects. weight recently obtained for cell-suspension cultures of potato, another solanaceous species (Newell, 1990). Initial phosphate levels between 0.19 and 0.5 mM result in attenuated growth; however,the root phosphate content has not reached the minimum level to stop growth. This indicates that these roots are phosphate rate limited at the time of elicitation and harvest. Within this range, the roots are growing at a reduced rate due to lowered intracellular phosphate reserves. Above an initial medium concentration of 0.5 mM, growth is saturated with respect to phosphate. Since growth is now limited by factor(s) other than phosphate, phosphorus is shown to accumulate to high levels within the root tissue (Figure 1). A growth model developed previously to describe phosphate-limited suspension cultures can be applied here to model the growth response of H. muticus root cultures. Details of the model development are described elsewhere (Curtis, 1988). Application of the model involves integration of the batch growth equation
where the specific growth rate (p) is a function of the intracellular kinetic pool of phosphorus (CF):
The maximum yield on phosphate (Yp)of 0.04 g of cell/ pmol of P is a constant, equal to the inverse of the minimum phosphorus content taken from Figure 1. Initial tissue density (XO= 0.204 g/L) and initial phosphorus content (Cm = 112 pmol of P/g of dry weight) are experimentally determined initial conditions, and initial media phosphate concentration (PO)is known. Equations 1 and 2 are solved numerically both for linear kinetics p =
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and for saturation kinetics (4)
Fit of the model to experimental growth data is shown in Figure 2. Analysis of variance of the residuals shows that saturation kinetics provide an adequate fit (1% F test) to the data over the entire range of initial phosphate levels. The resulting kinetic parameters are pmax= 0.284 day'
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Initial Phosphate Level (mM) Figure 2. Modeling of growth data based on intracellular kinetics. Saturation kinetics provide a statisticallyadequate fit (1% F test) over the entire range of initial phosphate levels. Error bars indicate fl standard deviation based on six replicate flasks at each phosphate level.
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initial Media Phosphate (mM) Figure 4. Total solavetivoneproduction in elicited (m) and nonelicited ( 0 )hairy-root cultures of H.muticus after growth for 14 days on different initial media phosphate levels. Open Symbols represent the control for chloride ion effects.
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Table I. Summary of Solavetivone Production from E. mu tic us
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Initial Media Phosphate (mM) Figure 3. Specific solavetivone production in elicited (m) and nonelicited ( 0 )hairy-root cultures of H. muticus after growth for 14 days on different initial media phosphate levels. Open symbols represent the control for chloride ion effects. and k, = 18.6 pmol of P/g of dry weight. Linear kinetics can only adequately describe growth at lower initial phosphate conditions. On the basis of a 1% F test, linear kinetics cannot provide an adequate fit for initial phosphate levels greater than 0.55 mM. Of particular interest is the linear asymptotic kinetics a t low phosphate levels. Least-squares fitting of linear kinetics between 0.0 and 0.33 mM gives a good visual as well as statistically significant fit [k' = 2.63 mg of dry weight/(pmol of P-day)]. This rate of growth is considerably faster than linear phosphate-limited growth rates observed in plant cell suspension cultures: 1.87,0.76,and 0.73 mg of dry weight/ (pmol of Paday) for carrot (Dougall and Weyrauch, 1980), poppy (Curtis, 19881, and potato (Newell, 1990) cell suspensions, respectively. This enhancement in growth may result from localization of the phosphate within the meristems under phosphate-starvedconditions. The model described here was developed for undifferentiated tissue with uniform cellular distribution of the rate-limiting substrate. Phosphate Effects on Production. Reduced medium phosphate results in increased nonelicited solavetivone production per gram of dry weight (Figure 3B). Specific productivity, a measure of product production per unit mass, is an indicator of cellular metabolic activity. Clearly, phosphate-deprived cells are more metabolically active
specific solavetivone, media concn, initial Pg P g / g of dry w t mg/L phosphate nonnonnonconcn, mM elicited elicited elicited elicited elicited elicited 151.7 2.92 3579.2 67.89 0.OOO 3.32 0.065 0.71 999.1 5.12 0.055 135.9 3.26 0.017 119.2 0.49 648.7 2.42 0.110 2.97 0.013 0.41 308.8 0.220 80.8 1.45 2.44 0.013 93.4 0.17 356.8 0.43 0.330 2.73 0.009 35.9 0.50 87.9 0.550 1.06 1.68 0.028 41.0 0.58 97.5 1.20 0.825 2.41 0.037 1.100 36.2 0.14 77.2 0.28 2.04 0.008 0.29 84.8 0.65 1.650 39.1 1.87 0.016
for the production of solavetivone. These results support the widely observed "inverse relationship" between primary and secondary metabolism. The cells only have a limited amount of energy available for primary metabolism (growth) and secondary metabolism (compound production). When phosphate is available, cells expend energy on primary metabolism to increase cell mass. Alternatively, when phosphate limits growth, resources are more readily allocated for sesquiterpene production. The effects of phosphate on nonelicited cultures are quite significant. The specific productivity of solavetivone in phosphate-deprived cells is 240 times greater than production at initial phosphate levels of 1.1mM. Total nonelicited production is enhanced at low phosphate levels as well (Figure 4B). For example, at an initial phosphate level of 0.0 mM, the total production of sesquiterpenes is 21 times greater than production at the control level of 1.1 mM, despite a 9-fold reduction in root mass. Production in nonelicited cultures declines rapidly with phosphate addition in the medium. Such an observation might suggestcell death and release of metabolites for phosphatestarved tissue; however, the root tissue appeared healthy, and no appreciable solavetivone was measured intracellularly. Despite this large increase in production under phosphate-starved conditions,the levels of production are small in comparison to fungally elicited cultures (Figure 4A, Table I). Fungal Elicitation. The effect of elicitation on specific productivity of solavetivoneis found by comparing elicited and nonelicited cultures at correspondingphosphate levels.
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Initial Media Phosphate (mM) Figure 5. Media solavetivoneconcentrations in elicited (w) and nonelicited ( 0 )hairy-root cultures of H.muticus after growth for 14 days on different initial media phosphate levels. Error bars indicate f l standard deviation. Our results indicate that fungal elicitation increased specific and total productivity by approximately 200 times for initial phosphate levels between 0.055 and 0.8 mM (Figures 3 and 4, Table I). This tremendous enhancement in production was achieved without any observed stress such as browning of the root tissue. Assessment of the relative effectiveness of phosphate limitation and fungal elicitation as production enhancement strategies can be made by comparing the elicited specific productivity of solavetivone at the control phosphate level of 1.1 mM (77 pg/g of dry weight) to the maximum productivity achieved by varying initial phosphate for nonelicited cultures (68 pg/g of dry weight). This comparison reveals that, per unit cell mass, fungal elicitation and phosphate limitation are equally effective at enhancing solavetivone production. However, when total production is considered, elicitation is shown to outperform phosphate limitation by 12 times. The advantage of fungal elicitation is that it can be applied under conditions of rapid growth. Synergistic Effects. When phosphate limitation is coupled with fungal elicitation, large gains in productivity are realized over either method individually. The maximum specific productivity for phosphate-limited cultures challenged with fungal elicitor is 46-fold higher than elicitation at the control initial phosphate level of 1.1mM. Thus, by reducing the rate of growth of the roots with phosphate limitation, over 40 times more solavetivonewas produced per unit mass in response to the fungal elicitor. The combined effects of phosphate limitation and fungal elicitation result in a total solavetivone productivity that is 4.6 times greater than elicitation at control phosphate levels (Figure 4A). Maximum total production was achieved after inoculation into phosphate-free medium, which corresponds to the maximum extracellular solavetivone concentration (Figure 5). It is worth noting that maxima in specific and total productivity for both elicited and nonelicited cultures take place under conditions that are phosphate yield limited (Figure 1). Roots that are yield limited will have ceased growth; therefore, the observed synergism between fungal elicitation and phosphate limitation may require nongrowing tissue. Optimization of production would therefore require a carefully controlled growth phase followed by a nongrowth production phase. Achieving such a phosphateyield-limited condition at a rapid rate is difficult due the utilization of phosphate from intracellular reserves. Modeling of phosphate-limited growth provides a means of predicting and optimizing growth conditions for subsequent gene induction by fungal elicitors. Counterion Effects. In manipulating the initial levels of medium phosphate, the levels of counterions will
necessarily change due to charge neutrality. To minimize variations in the physiologically important sodium and potassium concentrations, the medium was changed from a sodium phosphate based medium to a potassium phosphate based medium as described under Materials and Methods. This effectively traded off large fluctuations in sodium for an increase in the chloride ion concentration. To determine the effect of the elevated “background“ chloride ion, a control was conducted a t the standard phosphate level by using sodium phosphate and eliminating the addition of sodium chloride. The reduced chloride ion control displayed essentially the same rate of growth and phosphate accumulation (Figure 1)and slightly higher levels of solavetivone production (Figures 3 and 4). This suggests that elevated chloride ion may inhibit sesquiterpene production; however, the effect is small in comparison to the effects of phosphate and fungal elicitation that are observed in this study.
Discussion The synergism between nutrient deprivation and fungal elicitation demonstrated here for solavetivone production from hairy-root cultures of H. muticus raises some interesting questions about secondary metabolism in plant roots. Enhanced production a t reduced rates of growth could result from increased enzyme levels, increased activity of enzymes, and/or increased availability of substrate for production. An increase in substrate availability at reduced growth rates can be anticipated as resources are “freed up” from primary metabolism. While this logic is rarely disputed, metabolic availability of substrate is difficult to quantitate. The effect of reduced growth rates on the enzymatic component of secondary metabolism is less clear. This is especially true in the case of fungal elicitation, where the process of induction takes place a t the level of DNA transcription (Darvill and Albersheim, 1984; Templeton and Lamb, 1988). A reduction in DNA content is expected during phosphate deprivation since phosphate is a component of DNA structure. Such a reduction in nucleic acid content with reduced rates of growth has been observed experimentally in microbial and plant cell culture systems (Schaechter et al., 1958; Dougall and Weyrauch, 1980). Reduced levels of cellular nucleic acids might be expected to decrease the capacity for induction of RNA transcripts and subsequent translation of the enzymes required for phytoalexin synthesis. In contrast to this logic, our results demonstrate an increased response to fungal elicitation at reduced growth rates. Sesquiterpene biosynthcsis is an ideal model system to examine the enzymatic component of phosphate effects on induced secondary metabolism because the metabolic pathway has been intensely studied (Stoessl et al., 1976; Whitehead et al., 1990), and a key enzyme in the pathway has recently been isolated (Vogeli and Chappell, 1990). The role of differentiation in this synergistic response also remains to be answered. Since growth in root cultures is localized within the meristem, a large proportion of the root mass is not undergoing division. In this sense, much of the tissue is in the “stationary phase”. Since meristematic regions represent a small fraction of the root mass, manipulation of the rate of growth of roots might not be expected to significantly alter the metabolic state of a large proportion of the roots. Despite this localization of the effects of growth rate reduction, a 46-fold enhancement in cellular productivity is observed under phosphate limitation. In elucidating the basis for the large difference in productivity observed in this work, it must be kept in
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mind that combination of fungal elicitation with other production strategies is a complicated divergence from "typical" fungal elicitation studies, which are carried out at carefully standardized conditions. Both the physiological state and the physical environment of the tissues are altered. In addition to alterations ingrowth rate, other parameters of the system are altered, such as the mass of roots, the extracellular medium volume and composition, the degree of culture mixing, and the rate of oxygen consumption, just to name a few. Phosphate limitation may play only an indirect role in the observed synergistic interaction with fungal elicitation. The large differences in metabolic activity observed with this experimental system will facilitate identification of the contributing factor (9). Sorting out the physical and physiological parameters that alter the metabolism of cultured plant tissue presents a challenging interdisciplinary research problem. By furthering our understanding of the factors that alter root metabolism, we can better develop strategies to take advantage of the biosynthetic potential of roots for the production of useful chemicals.
Notation "bound" intracellular phosphorus content, pmol of P/g of dry wt "free" kinetic intracellular phosphorus content, CF pmol of P/g of dry wt total intracellular phosphorus content,pmol of P/g CT of dry wt k' linear specific growth rate constant, g of dry wt/ pmol of P saturation constant pmol of P/g of dry w t specific growth rate, days-' maximum specific growth rate, days-l media phosphate concentration, rmol of P/L culture tissue density, g of dry wt/L maximum yield on phosphorus, g of dry wt/pmol of P Subscripts 0 initial condition
DiCosmo,F.; Towers,G. H. N. Stress and SecondaryMetabolism in Cultured Plant Cells. In Recent Advances in Phytochemistry, Vol. 18: Phytochemical Adaptations to Stress; Timmermann,B., Ed; Plenum Press: New York, 1984;pp 97-175. Dougall, D. K.; Weyrauch, K. W. Growth and anthocyanin production by carrot suspension cultures grown under chemostat conditions with phosphate as limiting nutrient. Biotechnol. Bioeng. 1980,22, 337-352.
Eilert, U. Elicitation: Methodology and aspects of application. In Cell Culture and Somatic Cell Genetics of Plants; Vasil, I. K., Schell, J., Eds.; Academic Press: San Diego, CA, 1987; Vol. 4, pp 153-188. Flores,H.; Filner, P. Metabolicrelationships of putrescine, GABA and alkaloids in cell and root cultures of Solanaceae. In Primary and Secondary Metabolism of Plant Cell Cultures; Neumann, K., Barz, W., Reinhard, E., Eds.; Springer-Verlag: New York, 1985;pp 174-186. Flores, H.; Hoy,M.; Pickard, J. Secondarymetabolites from root culture. Trends Biotechnol. 1987,5, 64-69. Gamborg, 0.L.; Miller, R. A.; Ojima, K. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 1968,50,151-158. Knobloch, K. H.; Berlin, J. Influence of medium composition in
the formation of secondary compounds in cell suspension cultures of Catharanthus roseus. 2.Naturforsch. 1980, 35, 551-556.
Knobloch,K.; Berlin, J. Influence of phosphate on the formation of the indolealkaloidsand phenoliccompounds in cell cultures of Catharanthus roseus I. Comparison of enzyme activities and product accumulation. Plant Cell, Tissue Organ Cult. 1983,2,333-340.
Lamb, C. J.; Lawton, M. A.; Dron, M.; Dixon, R. A. Signals and transduction mechanisms for activation of plant defences against microbial attack. Cell 1989,56, 215-224. Murphy, J.; Riley, J. P. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 1962,27,31-36.
Newell, J. A. Optimization of secondary metabolite production in plant tissue culture. M. S. Thesis, The Pennsylvania State University, University Park, PA, June 1990. Sasse, F.; Heckenberg, U.; Berlin, L. Accumulation of 8-carboline alkaloids and serotonin by cell cultures of Peganum harmala L. Plant Physiol. 1982, 69, 400-404. Schaechter, M.; Maaloe, 0.; Kjeldgaard, N. 0. Dependency on medium and temperature of cell size and chemicalcomposition during balanced growth of Salmonella typhimurium. J. Gen. Microbiol. 1958,19,592-606.
Acknowledgment We thank Dr. Hector Flores and Mark Signs (Penn State University) for technical expertise and advice in the preparation of elicitors and sesquiterpene analysis. Calibration of the HPLC procedure was conducted by Dr. Joseph Chappell (University of Kentucky). The fitting of our growth model was performed with FORTRAN-callable IMSL subroutines for numerical integration. Literature Cited Balague, C.; Wilson, G. Growth and alkaloid biosynthesis by cell suspensions of Catharanthus roseus in a chemostat under sucroseand phosphate limiting conditions. Physiol. Veg. 1982, 20 (3),515-522.
Chappell, J.; Nable, R. Induction of sesquiterpeneoid biosynthesis in tobacco cell suspension cultures by fungal elicitor. Plant Physiol. 1987,85,469-473.
Curtis, W. R. Kinetics of phosphate limited growth of poppy plant suspension cultures. Ph.D. Thesis, Purdue University, West Lafayette, IN, December 1988. Curtis, W. R. Interference of intracellular phosphate analysis by phosphatase in Papaver somniferum cell suspensions. Phytochem. Anal. 1990,I, 70-73. Darvill, A.; Albersheim, P. Phytoalexins and their elicitors-A defenseagainst microbial infectionin plants. Annu. Rev. Plant Physiol. 1984,35, 243-275.
Schiel, 0.; Jarchow-Redecker,K.; Piehl, G.; Lehmann, J.; Berlin, J. Increased formation of cinnamoylputrescines by feed-batch fermentation of cellsuspensioncultures of Nicotiana tabacum. Plant Cell Rep. 1984,3, 18-20.
Shenk, R. U.; Hilderbrandt, A. C. Medium and techniques for induction and growth of monocotyledonousand dicotyledonous plant cell cultures. Can. J. Bot. 1972,50, 199-204. Signs, M.; Flores, H. Elicitation of sesquiterpene phytoalexin biosynthesis in transformed root cultures of Hyoscyamus muticus L. Plant Physiol. Suppl. 1989,4 , 135. Signs, M.; Flores, H. The Biosynthetic potential of plant roots; In BioEssays 1990, 12 (l),7-13. Stoessl, A.; Stothers, J. B.; Ward, W. B. Sesquiterpenoid stress compounds of the Solanaceae. Phytochemistry 1976,15,855872.
Templeton, M. D.; Lamb, C. J. Elicitors and defense gene activation. Plant, Cell Environ. 1988,11, 345-401. Vogeli, U.; Freeman, J. W.; Chappell, J. Purification and characterization of an inducible sesquiterpene cyclase from elicitor-treatedtobacco cell suspension cultures. Plant Physiol. 1990,93, 182-187.
Whitehead, I. M.; Atkinson, A. L.; Threlfall, D. R. Studies on the biosynthesis and metabolism of the phytoalexin lubimin and related compounds in Datura stramonium L. Planta 1990, 182.81-88.
Accepted May 30, 1991. Registry No. PO4, 14265-44-2;solavetivone, 54878-25-0.
