Characterization of Clonal Cultures of Anchusa officinalis Derived from Single Cells of Known Productivity

Characterization of Clonal Cultures of Anchusa officina/is Derived from Single Cells of Known Productivity B. E. ELLIS Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, N1G 2W1, Canada Received November 7, 1984 · Accepted December 14, 1984

Summary Suspension cultures of Anchusa officina/is established by clonal propagation of microspectrophotometrically-analyzed single cells were found to produce rosmarinic acid at different levels. The production level was not related to the productivity of the original single cell, but was a stable characteristic of the clonal suspension. Further cloning appeared to generate a new range of production levels in the new suspension culture lines.

Key words: Anchusa officina/is L., cell cultures, clonal lines, microspectrophotometry, rosmarinic acid, secondary metabolism, single cells.

Introduction

The ability of plant cells cultured in vitro to synthesize and accumulate substantial levels of some of the secondary metabolites which typify the plant kingdom has opened the possibility of large-scale cultures serving as a source of pharmaceuticals or chemical feed-stocks (Nickell, 1980). Considerable effort has therefore been made to maximize the secondary metabolite productivity of various species in suspension culture through screening and selection programs (Zenk, 1978), optimization of media components (Zenk et al., 1975) and manipulation of culture conditions (Mizukami et al., 1978). Selection for high-producing variant lines by visual or analytical procedures has allowed greatly increased yields of various alkaloids (Ogino et al., 1978; Zenk et al., 1977), pigments (Dougall et al., 1980; Sugano et al., 1971), ubiquinone (Matsumoto et al., 1980) and vitamin B6 (Yamada and Watanabe, 1980), but the highproduction trait is sometimes unstable during extended culture (Zenk, 1978; Dougall et al., 1980). Whether this problem arises from genetic instability in culture, from genotypic heterogeneity within the original isolates or from other causes remains uncertain. Microspectrophotometric analysis of the intracellular concentration of rosmarinic acid (a-0-caffeoyl-3,4-dihydroxyphenylactic acid) (RA) in single cultured cells of RA-producing species has revealed that individual cells within suspension culture populations produce RA at very different levels (Chaprin and Ellis, 1984). This cell-to-cell variability could result from the presence of a range of genotypes capable of different levels of RA synthesis, or it might have a physiological (non-genetic) basis. If the former were true, it might be expected that single-cell cloning of high-

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producing cells would yield genetically uniform cell lines whose productivity would reflect that of the original mother cell. By employing a combination of microspectrophotometry and single-cell cloning, cells of known metabolic productivity have been used in the present study to derive true clonal cell suspensions. Nine primary and five secondary clonal lines have been characterized for several traits, including the relationship, if any, between the RA level displayed by the mother cell for each line and the RA production in the single-cell clonal suspension culture.

Materials and Methods Cultures: Suspension cultures of Anchusa officinalis L. (De-Eknamkul and Ellis, 1984) were established and maintained in B5 medium (Gamborg and Eveleigh, 1968) containing 1.0 mg ·1- 1 2,4-D, 0.1 mg ·l- 1 kinetin and 3% sucrose. Subculturing of stock cultures and clonal lines was carried out by a 1 :7 dilution into 50 ml fresh medium (250 ml Erlenmeyer flasks) at seven day intervals. Flasks were aerated by 130rpm gyratory shaking at 25° in continuous light. Single-cell cloning: A suspension culture of the desired age (usually seven days) was filtered aseptically through sequential nylon meshes with porosities of 500 p., 300 p. and 100 p.. The fmal filtrate contained predominantly single cells and 2- or 3-cell clusters. 1-2 ml of this filtrate was spread over the surface of 12 ml Kao and Michayluk medium no. 8 (Kao and Michayluk, 1975) solidified with 1.0% agar in a 100 mm petri dish. After standing for a few minutes, excess liquid was carefully pipetted out, leaving the cells distributed over the surface of the medium. Single cells well separated from any neighbouring cells were located on the inverted plate by lowpower microscopy and marked on the plate bottom. Each chosen cell was then recovered from the plate by excision of an appropriate 3 mm x 3 mm agar block. The block was either set out on a nurse system, or, if microspectrophotometric analysis was required, it was placed on a sterile quartz slide and surrounded by a sterile UV-transparent chamber. This chamber was mounted on the stage of the Zeiss scanning microscope photometer and the concentration of the rosmarinic acid in the cell was measured as described previously (Chaprin and Ellis, 1984), using an illuminating wavelength of 358 nm. The measuring beam was zeroed through the agar block adjacent to the cell. After analysis, the cell on its block was transferred from the chamber to a nurse system consisting of a layer of Anchusa stock suspension culture on solidified B5 medium overlaid with an array of 6 mm discs of Whatman # 1 filter paper. Each disc carried one block, and nurse layer replacement consisted of transferring disc and block together to a fresh nurse layer at 10-day intervals. The condition of the cloned cells was readily monitored in situ by low power microscopy. Once a callus 2-3 mm in diameter had developed on top of a block, it was transferred directly to solidified Kao and Michayluk No. 8 medium and allowed to increase before passing it into B5 liquid medium to establish suspension cultures. Rosmarinic acid extraction and analysis. 1-2 g filtered and rinsed suspension culture was lyophilized in a 15 ml centrifuge tube and the dry weight recorded. To the dry tissue was added 5 ml hot 70% ethanol and the tube was then held at approx. 70° in a sonicator water bath for 20 min. After vortexing briefly and centrifugation at 1200g, the supernatant was diluted 40x with 70% ethanol and the absorbance spectrum recorded from 250 to 400 nm. The rosmarinic acid concentration was calculated from the absorbance at 330 nm using E' = 18,000. The ethanol-soluble aromatic compounds were examined by 2 dim. TLC on microcrystalline cellulose plates using 10% HOAc and nBuOH:EtOH:H20 (4: 1: 1). Phenolic compounds were detected with 254 nm UV light and diazotized p-nitroaniline. The extracts were also subjected to reversed-phase HPLC on a 200 x 4.5 mm Whatman ODS-II column using isocratic elution at 1 mllmin with MeOH: H20: H3P04 (65: 80: 0.02) and detection by UV absorbance at 280nm. ]. Plant Physiol. Vol. 119. pp. 149-158 (1985}

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Growth rate determination. Seven-day old suspension cultures were harvested aseptically by vacuum filtration on Miracloth (Calbiochem., La Jolla, CA) discs and 2 g samples used to inoculate fresh cultures. Seven days later, these cultures were likewise used to inoculate triplicate fresh cultures with 2 g tissue per flask. After nine days growth (the point of the maximum rate of fresh weight increase in the stock cultures), the flasks were harvested, the tissue vacuum filtered to remove free liquid and the fresh weight recorded. Aggregate seiving. Eleven-day old suspension cultures were seived through 300 p. nylon mesh, using extensive rinsing and gentle agitation to ensure efficient passage of the aggregates. The fresh weight of the tissue retained on the mesh was recorded, while the tissue passing through the 300 p. mesh was recovered by filtration on Miracloth and also weighed. Phenylalanine ammonia-lyase (PAL) assay. Suspension cultures were harvested by vacuum filtration after 18 hr growth in fresh medium. The rinsed tissue (1.5-1.7 g) was frozen in liquid Nz and pulverized in a mortar and pestle. The powder was thawed in 5 ml 50 mM Tris-acetate, pH 7.5, containing 5mM mercaptoethanol and 1.5g hydrated Polyclar AT. After 10 min extraction at 4°, the slurry was pressed through Miracloth and centrifuged in a Beckman microcentrifuge for 1 min. The supernatant was desalted on PD-10 columns (Pharmacia, Uppsala) by elution with 50 mM K-borate, pH 8.5, containing 5 mM mercaptoethanol. The protein fraction (300 p.l) was assayed for PAL activity in triplicate incubation mixtures containing 2 mM L-2,63 H-phenylalanine (2 x 105 dpm) (New England Nuclear, Boston) and borate buffer to give a final volume of 1.5 ml. After 40 min incubation at 30°, the reaction was stopped by addition of 100p.l5M HCl, 20p.l O.SM cinnamic acid in ethanol and 2.5ml toluene:ethyl acetate (1:1). The mixture was vortexed for 30 sec to extract cinnamic acid and centrifuged briefly at 1000g to separate the phases. The tritium content of the organic phase was measured by liquid scintillation counting. Enzyme held at 100° for 10 min served as control. Protein in the enzyme extracts was measured using the dye-binding assay (Bradford, 1976) with bovine serum albumin as the standard. Enzyme activity is expressed as pkatals/mg protein.

Results and Discussion Suspensions of Anchusa officina/is were chosen for this study because of their finely divided state and well-characterized growth and metabolism (De-Eknamkul and Ellis, 1984). The ability to measure rosmarinic acid concentrations in individual Anchusa cells by microspectrophotometry had been established previously (Chaprin and Ellis, 1984). While complete optimization of the single-cell cloning procedure was not attempted, preliminary experiments showed that cell survival was improved if the agar block carrying each cell contained a conditioned or highly supplemented medium such as that developed by Kao and Michayluk for low-density protoplast culture (Kao and Michayluk, 1975). It was also observed that Anchusa nurse layers which were completely covered by a large filter paper disc rapidly became senescent. To avoid this, the agar blocks were held on small individual discs which separated the block from the nurse cells without unduly stressing the latter. Finally, the preliminary studies indicated that the overall success rate of the cloning procedure (20-30%) was not substantially reduced when the cells were briefly probed by microspectrophotometry before being set out on the nurse layer. The nine clonal lines described here as primary clones were derived from the stock suspension culture line, which itself was originally derived from a single leaf section explant. The stock culture had previously been shown to contain both cells with high ]. Plant Physiol. Vol. 119. pp. 149-158 (1985}

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Table 1: Rosmarinic acid concentration in single cells of Anchusa officinalis stock culture and in the clonal suspension culture derived from each cell. Suspension cultures were analyzed on day 12 of the growth cycle. Data reported are for six measurements made in duplicate over a period of seven months (mean± 1 S.E.). Suspension culture line

Rosmarinic acid content (% dry wt.)

Stock 81-C-2 JA82D2 FE82A5 FE82B1 FE82B7 MR82A8 MR82A9 MR82A21 MR82B3

6.7±1.0 3.7±0.5 5.5±0.9 2.4±0.4 6.7±1.4 4.3±0.5 8.0±1.0 4.5±0.5 2.4±±0.4 7.4± 1.2

n.d.

=

Mother cell RA concentration (arbitrary units) n.d. 3.9 4.0 4.8 7.4 12.8 3.2 4.9 11.5

not determined.

0,~--------~------~--------~

1-10·83

1-1·84

1·4·84

Fig.1: Variation in rosmarinic acid content of clonal lines FE82A5 (A) and MR82B3 (B) over a period of seven months. Twelve-day old suspension cultures were analyzed in duplicate as described in Materials and Methods.

and with low intracellular rosmarinic acid levels. The random selection of cells from this culture for single cell cloning recovered cells with intracellular RA concentrations ranging from 3.2 to 12.8 arbitrary units. The mother cells for the nine primary clones had the concentrations shown in Table 1; both high and low producing cells are represented. Also shown in Table 1 is the rosmarinic acid production level of the suspension cultures derived from each of these mother cells. While the values vary somewhat between individual analyses (Fig. 1}, it is clear that after more than 100 passages in suspension, each clonal line has a typical level of rosmarinic acid production. It is also clear, however, that the production level of the suspension culture is not strongly correlated with the intracellular content of rosmarinic acid in the mother cell. Although microspectrophotometric analysis of multicellular clusters had earlier shown that physically associated, and presumably progeny-related, cells tended to have similar intracellular rosmarinic acid concentrations (Chaprin and Ellis, 1984} it ]. Plant Physiol. VoL 119. pp. 149-158 (1985)

Clonal cultures of Anchusa officina/is

01_ ...

153

A

0

N

>-

u

z

w ;:)

aw

0

. ...

0:::

LJ... 0

Fig. 2: Frequency distribution of microspectrophotometrically measured intracellvlar rosmarinic acid (RA) concentrations for 200 individual cells in suspension cultures of (A) clonal line FE82A5 and (B) clonal line MR82B3. Seven- day old cultures were analyzed as described in Chaprin and Ellis, 1984. Concentrations in arbitrary units.

B

0

IL

0

N

0

0

~

2 4 6 8 10 12 14 RACONC.

Table 2: Rosmarinic acid concentration in single cells of clonal line MR82B3 and in secondary clonal suspension culture derived from each cell. Data reported are for seven measurements made in duplicate over a period of eight months (mean± S.E.). Suspension culture line

Rosmarinic acid (content% dry wt.)

Mother cell RA concentration (arbitrary units)

AP83A3 AP83A5 AP83A6 AP83A7 MR83A14

5.9±0.7 8.8±0.6 4.2±0.5 7.0±0.5 7.5± 1.2

1.8 18.8 8.0 16.2 4.1

would appear that this metabolic relatedness is not retained through the many cell divisions required to establish a suspension culture. At this point, the cell populations within the clonal suspension cultures display the same type of frequency distribution of cellular RA concentrations observed in the stock culture (Fig. 2), the only difference being the mean value for the population. Since each clonal line presently displays a reasonably stable level of total RA production, however, it seems that the quantitative expression of this trait has been fixed, genetically or epigenetically, in ]. Plant PhysioL VoL 119. pp. 149-158 (1985}

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B. E. Ews 20

16

8

4

Fig. 3: Fresh weight of cells produced after nine days of suspension culture growth from a 2 g inoculum of different A. officina/is lines. Bars indicate actual range of replicate values. Table3: Aggregation level of day old clonal suspension cultures of A. officina/is, expressed as percent of total fresh wt. of tissue which passed through a 300 p. porosity mesh. Suspension culture line

Aggregate size distribution <300p. >300p.

Stock 81-C-2 JA82D2 FE82A5 FE82B1 FE82B7 MR82A8 MR82A9 MR82A21 MR82B3

52% 49% 12% 25% 9% 5% 4% 31% 45% 41%

48% 51% 88% 75% 91% 95% 96% 69% 55% 59%

each line. One possible source of this phenomenon could have been the recovery of individual chemical production genotypes from the original stock culture. To examine this possibility, five secondary clonal lines were established with single cells from primary clone MR82B3. These have now been held in suspension for more than 50 passages and have the RA production characteristics shown in Table2. It can be seen that, once more, the present RA production levels in each clonal line are not strongly correlated with the mother cell RA concentrations and that the quantitative ]. Plant Physiol. Vol. 119. pp. 149-158 {1985)

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RA

Fig. 4: High pressure liquid chromatography profile of the ethanol-soluble aromatic metabolites from twelveday old A. officinalis cultures. Chromatographic conditions as described in Materials and Methods. (RA = rosmarinic acid).

0

10

20

30min

expression of the production trait, while stable and presumably uniform within the MR83B3 line, has apparently been altered in at least some lines during the second cloning cycle. This result is not consistent with the idea that the primary clonal lines differed simply because they were isolated from a genetically heterogeneous population, but rather suggests that the isolation and cloning process itself has a destabilizing effect on the phenotype, and perhaps the genotype, of the cells in culture. This effect is also observed in other traits such as growth rate (Figure 3) and culture morphology (Table 3). These traits, while notoriously prone to spontaneous change during long-term culturing because of the inherent selection pressure for low aggregation state and high growth rate, are expressed at distinctive levels in the primary clonal lines after more than 100 passages. The secondary clones also display differences in these traits (data not shown). An earlier study of the stability of nicotine production in single-cell clonal lines of Nicotiana produced similar evidence of changes in production level associated with successive cycles of cloning, although in that case only a semi-quantitative analytical procedure was employed (Ogino et al., 1978). ]. Plant PhysioL Vol. 119. pp. 149-158 (1985}

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16 14

6

'I

4

•

2 0o~--~~2~~~~4~~--~6~~--~8~~--~10~

RA<%

DRY WT.l

Fig. 5: Relationship between extractable phenylalanine ammonialyase activity (PAL) and production of rosmarinic acid (RA) in nine primary clonal culture lines and the stock culture of Anchusa officina/is. Bars indicate the 95% confidence interval for each data point (RA, n = 6; PAL, n=5).

In contrast to the differences among clones with regard to the level of rosmarinic acid synthesis, no differences could be observed in the qualitative pattern of aromatic metabolites produced as surveyed by two dimensional TLC or by reversed-phase HPLC. The HPLC profile shown in Fig. 4 for the ethanol-soluble metabolites of the stock culture, was identical for each of the clones as well. It is possible, however, that study of larger numbers of clones might reveal some qualitative variants as well, such as have been observed in cloned Catharanthus roseus cells (Constabel et al., 1981). It has been frequently suggested that the critical location of the enzyme phenylalanine ammonia-lyase (PAL) in the pathway leading from phenylalanine to phenylpropanoid products makes it a likely control point for establishing the rate of synthesis of such products Gones, 1984; Hahlbrock and Grisebach, 1979}. The availability of culture lines whose production of a single phenylalanine-derived secondary metabolite, a caffeic acid ester, varied markedly between lines led us to compare the caffeoyl ester accumulation in each line with the phenylalanine ammonialyase activity in freshly subcultured tissue. The results did not show a strong correlation between these two parameters (Fig. 5}, but this type of comparison is fraught with difficulties. The different lines may well have different rates of enzyme indue]. Plant PhysioL VoL 119. pp. 149-158 {1985)

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tion upon subculturing, and/or different requirements for optimum enzyme extraction. While replicates within an experiment were in excellent agreement, the wide variation in enzyme specific activity between experiments for some lines does suggest that one or more unknown factors was not being controlled. Determination of the biochemical basis for the different levels of rosmarinic acid production in these clonal lines will require first developing a clearer understanding of the molecular and structural processes which control expression of phenylpropanoid metabolism in plant cells. Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Can-

ada and was carried out with the excellent technical assistance of Ms. Sylvia Flack. References

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ZENK, M. H., H. EL-SHAGI, H. ARENs, J. STOCKIGT, E. W. WEILER, and B. DEus: Formation of the indole alkaloids serpentine and ajmalicine in cell suspension cultures of Catharanthus roseus. In: BARZ, W., E. REINHAID, and M. H. ZENK {eds.): Plant Tissue Culture and its Biotechnological Application, pp. 27-43. Springer Verlag, Berlin, 1977. ZENK, M. H.: The impact of plant cell culture on industry. In: THORPE, T. A. (ed.): Frontiers of Plant Tissue Culture 1978, pp. 1-14. lnternat. Assoc. Plant Tissue Culture, Calgary, 1978.

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