Process for extracting enhanced amounts of plant secondary metabolites with limited loss of plant viability

A process for extracting enhanced amounts of a plant secondary metabolite from plant tissue with limited loss of tissue viability by reversibly permeabilizing the tissue membrane is disclosed.

BACKGROUND OF THE INVENTION 
Plants grown in vitro can provide a major source of specialty chemicals, 
which are plant secondary metabolites. For example, artemisinin, a 
terpenoid found in the herb, Artemisia annua, is a promising therapeutic 
for treatment of malaria. However, this highly effective compound is 
produced by native plants in minute quantities, and by tissue cultured 
plants at levels significantly less than the best native plants. Efforts 
to obtain higher production levels in either native plants or their 
cultured tissues would make therapeutic use of artemisinin on a large 
scale, a reality. 
Taxol is a novel diterpene isolated primarily from the bark of Taxus 
brevifolia and other species of Taxus. Although knowledge about the sites 
of taxol biosynthesis and distribution is limited, it is known that the 
product is next most abundant in the root. Taxol has been shown to be an 
especially effective antitumor agent. However, there is considerable 
difficulty in obtaining sufficient quantities of taxol for clinical 
testing. The yield of taxol from Taxus bark is low (500 gm per 10,000 lbs 
bark), and native plants are rapidly dwindling in number. Like many woody 
plants the genus is recalcitrant to propagation by in vitro culture, 
rooted cuttings, or reseeding for reforestation. Further therapeutic 
development will depend largely on solving problems of biomass supply. 
Because Taxus biomass grows so slowly and taxol is produced at low levels 
in the tissues, the biomass is valuable. 
Except for shikonin and berberine, there has been little success in the 
profitable production of secondary metabolites from plant cultures. The 
lack of success is due in part to the fact that these chemicals are 
present only in small amounts within the plant. In addition, in vitro and 
in vivo cloning of plants, especially woody plants is extremely difficult, 
because of slow growth rates, reduced or lack of rooting ability, frequent 
systemic microbial contamination, phenotypic instability, and phenolics 
build-up. Micropropagation is no different. 
There has been some success in the establishment of undifferentiated callus 
and cell culture lines for the production of secondary metabolites. (See, 
for example, U.S. Pat. No. 5,019,504 entitled, "Production of taxol or 
taxol-like compounds in cell culture," by Christen et al.) However, 
secondary metabolism is frequently linked to differentiation. Therefore, 
most undifferentiated cultures are not useful for producing secondary 
metabolites. 
Most secondary metabolites of plants accumulate within the plant tissue and 
are not readily exported into the growth medium. In addition, most 
secondary metabolites are chemically complex and therefore are difficult 
to synthesize. Thus, there is a need to develop nondestructive methods 
whereby products produced by plant tissues can be easily recovered for 
processing while still retaining the valuable biomass for additional 
product biosynthesis. 
SUMMARY OF THE INVENTION 
The subject invention relates to a process for extracting enhanced amounts 
of a plant secondary metabolite from plant tissue with limited loss of 
tissue viability by reversibly permeabilizing the plant tissue membrane. 
The process includes three steps: 1) destabilizing plant tissue membrane 
to effect partial release of a plant secondary metabolite; 2) removing the 
secondary metabolite to enhance the diffusion gradient and thereby 
increase secondary metabolite efflux; and 3) restabilizing the plant 
tissue membrane to inhibit secondary metabolite release. 
In order to obtain enhanced amounts of a plant secondary metabolite, it is 
preferred that the tissue is differentiated. Plant roots including 
genetically transformed hairy roots provide an especially preferred tissue 
for obtaining many plant secondary metabolites. In addition, the plant 
tissue should be actively synthesizing the metabolite or contain high 
levels of stored metabolite. Generally, plant cultures which are growing 
in late exponential growth phase and stationary phase produce enhanced 
amounts of secondary metabolite. A particularly useful method of obtaining 
plant tissue which contains enhanced amounts of secondary metabolite is by 
culturing in a nutrient mist bioreactor. 
According to the method of the subject invention, plant tissue membrane is 
destabilized to effect partial release of the plant secondary metabolite 
from the tissue. Partial release is important, because if the level of 
secondary metabolite released is too high, the tissue loses viability. 
Destabilization of plant tissue membranes can be accomplished by any of a 
number of techniques, performed alone or in combination. For example, the 
plant tissue membrane can be exposed to elevated temperatures for various 
periods of time. Culturing plant tissue in temperatures in the range of 
25.degree.-45.degree. C. for times ranging generally from 1 minute to 2 
hours is useful for obtaining partial release of most secondary 
metabolites, while maintaining plant tissue viability. 
Alternatively, a plant membrane can be contacted with a permeabilizing 
agent at an appropriate temperature and for an appropriate length of time. 
An example of a permeabilizing agent is a substance which prevents the 
binding of divalent cations to plant cell membranes. Particularly useful 
permeabilizing agents are (NH.sub.4).sub.2 SO.sub.4 and EDTA. 
Permeabilizing agents can be used alone or in combination with other 
permeabilizing agents and/or other methods of destabilizing the plant cell 
membrane. Membrane destabilization can also be accomplished by excluding 
membrane stabilizers (e.g., divalent cations) from the culture medium. 
Subsequent to destabilizing the plant membrane, solvents can optionally be 
added to plant culture medium to effect greater extraction of secondary 
metabolites. For example, some secondary metabolites are nonpolar 
compounds and therefore are not soluble in aqueous solutions, such as the 
culture medium. Therefore, a nonaqueous solvent, which does not decrease 
the plant tissue viability, can be added to the plant tissue surroundings 
(e.g., the culture medium) to enhance extraction of nonpolar secondary 
metabolites. Alternatively, the permeabilizing agent itself can be a 
nonaqueous solvent. 
In the next step of the subject method, secondary metabolite is removed 
from the plant tissue surroundings. For example, removal of the released 
secondary product can be accomplished by exchanging the medium containing 
the released product with fresh medium. As near continuous removal of 
released product is approached, secondary metabolite release is increased. 
In the final step of the method of the invention, the plant tissue membrane 
is restabilized to inhibit secondary metabolite release-and to enhance 
plant tissue viability. Restabilization of plant tissue membranes can be 
accomplished by any of a number of techniques, performed alone or in 
combination. One approach is to remove the condition which promoted 
destabilization. For example, the temperature of the culture medium can be 
cooled (e.g., to room temperature). Alternatively, the destabilizing agent 
can be removed. Further, divalent cations can be introduced into cultures 
which were destabilized by being cultured in medium which lacked divalent 
cations, or other plant cell membrane components which contained a 
permeabilizing agent. Addition of sterols or other components of cell 
membranes can also effectively restabilize plant cell membranes and 
enhance plant tissue viability. 
The disclosed methods for secondary metabolite release can be applied to a 
viable culture without significant loss of biomass. This biomass is 
therefore conserved and available for further permeabilizations to obtain 
product release without the need to accumulate more biomass, which could 
require weeks to accomplish in a large scale bioreactor. Therefore, the 
cost of processing large amounts of biomass in batch production is 
effectively reduced. In addition, the degree of selectivity toward 
specific products offered by the choice of destabilization methods 
provides additional control over process development.

DETAILED DESCRIPTION OF THE INVENTION 
The subject invention relates to processes for extracting enhanced amounts 
of a plant secondary metabolite (also referred to as secondary product) 
from plant tissue with limited loss of tissue viability. Plant secondary 
metabolites are chemical compounds synthesized in plants, that are not 
specifically required by the plant to maintain cellular processes. 
Examples of secondary metabolites include flavors (e.g., spearmint, 
peppermint), fragrances (e.g., jasmine), medicinals (e.g., taxol, 
artemisinin, ginkgolides, vinblastine, vincristine) and cidal agents (e.g. 
thiophenes, thiarubrines). Most secondary metabolites accumulate in plant 
vacuoles and are not readily released from the plant. 
Some secondary metabolites are polar compounds and therefore are soluble in 
aqueous solvents. However, most alkaloids (e.g., serpentine, berberine and 
atropine), phenols (e.g., benzoquinones, coumarins and quinones), 
terpenoids (e.g., taxol, artemisinin) and polyacetylenes are nonpolar and 
therefore are most soluble in nonaqueous solutions. 
In order to obtain enhanced amounts of a plant secondary metabolite, it is 
preferred that the tissue is differentiated (e.g., plant shoots and 
roots). Plant secondary metabolism is frequently linked to 
differentiation. Therefore, most undifferentiated cultures are not useful 
for producing secondary metabolites. 
Many secondary metabolites are produced in plant roots. In addition, roots 
can be transformed into "hairy roots"; hairy roots are genetically stable, 
grow rapidly and produce levels of secondary products equal to or greater 
than the whole plant. Therefore, hairy root tissue is particularly useful 
for obtaining most plant secondary metabolites. 
Hairy roots can be induced by genetic transformation (i.e., by introducing 
the Ri (root inducing) plasmid from Agrobacterium rhizogenes into plant 
seeds or root tissue) Hamill, D. et al., Plant Cell Reports 5:111-114 
(1986). The production of hairy roots is partially a result of auxin 
regulation by this plasmid. A preferred method for transforming root 
tissue is explained in detail in Example 1. Transformation of root tissue 
by the Ri plasmid was found to be easier to accomplish and to result in 
greater rooting than transformation of germinated seedlings. 
Most dicotyledonous plants producing secondary metabolites can be 
transformed. Some monocots and conifers are also transformable. Some 
plants are resistant to transformation by Agrobacteria species, but may be 
made susceptible to transformation through the use of virulence inducers, 
which are compounds, some produced naturally by susceptible plants, which 
have been found to promote transformation. Certain virulence inducers have 
proven especially useful in transforming gymnosperms, which are otherwise 
resistant, with A. tumefaciens, J. W. Morris and R. O. Morris Proc. Natl. 
Acad. Sci. (USA) 87:3614-3618 (1990). 
In addition to producing enhanced levels of secondary metabolite and 
growing quickly, hairy root tissue may be better able to tolerate 
destabilization methods than normal differentiated tissue. For example, 
when tested, beet hairy roots were found to tolerate higher temperatures 
than normal beet roots. For beet roots, viability (measured as CO.sub.2 
evolution) began to decrease at 35.degree.-40.degree. C. However, for 
transformed hairy root tissue, CO.sub.2 evolution did not begin to 
decrease significantly until treatment of 45.degree. C. In addition, the 
rapid growth associated with transformation by Agrobacterium rhizogenes is 
believed to be due in part to endogenous control of auxin synthesis by the 
Ri plasmid. It is also known that plant regulators such as auxins can 
control polyamine biosynthesis, although the exact mechanism is not known. 
Polyamines are directly linked to stress tolerance in plants. Therefore, 
the transformation to hairy roots, besides improving growth, may also 
enable more vigorous destabilization methods to be utilized while still 
maintaining viability. 
In order to obtain enhanced amounts of a plant secondary metabolite, the 
plant tissue should be actively synthesizing the metabolite or contain 
high levels of stored metabolite. Generally, plant cultures which are 
growing in late exponential growth phase and stationary phase produce 
enhanced amounts of a secondary metabolite. A particularly useful method 
of obtaining plant tissue, which contains enhanced amounts of secondary 
metabolite, is by culturing in a nutrient mist bioreactor (NMB), as 
described in U.S. Pat. No. 4,857,464 entitled "Mist Cultivation of Cells" 
by Weathers and Giles, the teachings of which are incorporated herein by 
reference. Cultivation of plant cells in a nutrient mist bioreactor offers 
the advantage of providing cells with a readily available gaseous and 
liquid nutrient supply. In addition, use of the bioreactor permits rapid 
change in culture conditions (e.g., nutrient or extractant addition) to 
allow for precise control of the culture environment. Further, in a 
bioreactor, cells are supported on screens within a sterile chamber which 
allows cell products and media to continuously drain away from the tissue 
into a collection chamber. In order to accommodate the growth of and 
extraction from transformed hairy roots, the Nutrient Mist Bioreactor 
should be modified as described in Example 2. 
Although, cultivation in a nutrient mist bioreactor is a preferred method 
of obtaining tissue which produces high levels of secondary metabolites, 
for the purposes of the subject invention, any method of cultivation known 
in the art can be used. For example, cells can be grown on solidified agar 
gels or in liquid (suspension) cultures. Alternatively, the subject 
methods of extraction can potentially be performed on plants growing in 
the wild. In order to obtain enhanced amounts of a plant secondary 
metabolite, plant growth can be improved by adding plant growth regulators 
and hormones (e.g., cytokinins and auxins) and/or by optimizing 
environmental conditions such as temperature, light intensity, water 
stress, salinity, media composition and exposure to CO.sub.2. 
The following describes the results of experiments conducted to determine a 
method of obtaining enhanced amounts of secondary metabolites from beets 
with limited loss of beet tissue viability. The protocol for beet root 
disks is described in detail in Example 3 and for beet hairy roots in 
Example 4. The removal of secondary metabolites from beets represents a 
worst case scenario, and therefore is an ideal model system, since no 
basal release of secondary product occurs. Secondary metabolites in beets 
are stored intracellularly in vacuoles, requiring the traversal of 2 
membranes before the metabolite can be released. Because the beet model 
represents the most difficult conditions for extracting secondary 
metabolites, the following results demonstrate the success of the present 
method for the extraction of secondary metabolite from virtually any plant 
cell tissue. In addition, the release of secondary metabolites (e.g., 
betacyanin, betanin, betaxanthins and betalamic acid) was easy to assay 
spectrophotometrically. 
As used herein, the phrase "limited loss of tissue viability" refers to 
post-extraction viability in the range of about 80 to 100%, as measured by 
respiratory carbon dioxide evolution (from growing nonphotosynthetic 
tissue) and/or change in biomass (i.e., change in dry weight). Brodelius, 
P., Appl. Microbiol. Biotechnol. 27:561-566 (1988). 
The viability of beet hairy roots is presented in terms of a viability 
index, V defined as: 
##EQU1## 
where X, is the dry weight of the tissue prior to permeabilization (day 
0); Y is the dry weight untreated tissue (day 3, approximately 1-1.5 
doublings from day 0); and Z is the dry weight of treated tissue after 
permeabilization (day 3). The viability index has a variability of 
approximately 4%, which indicated that for V.gtoreq.96%, the tissue is 
considered to have been unaffected by the heat treatment. This value is 
determined by averaging the dry weights of the controls (% variability) in 
the heat treatment experiments. Viability measurements were taken over a 
relatively short period after destabilization (1-3 day). Brodelius found 
that for 3 days after the exposure to DMSO and Triton X-100, no growth 
occurred in suspension cultures of C. roseus, but that growth was 
reestablished relative to untreated controls after 4 days. Brodelius, P., 
Appl. Microbiol. BioTechnol. 27:561-566 (1988). Therefore, measuring 
viability at day 3, provides a worst case value. 
Secondary Product Release From and Viability of Beet Root Disks 
Beet root disks placed in the culture chamber of a nutrient mist 
bioreactor, released secondary product upon heating of the chamber and the 
incoming mist. The tissue treated at 35.degree. C. released more product 
than the control (treated at 25.degree. C.), which released some pigment 
even though the tissues were rinsed for 1 hr in cold running tap water. 
The tissues misted with B5 medium with 20 Mm additional CaCl.sub.2 
released less pigment than the control (treated at 25.degree. C.). 
Secondary product release increased with increasing temperature, while 
viability, (measured as CO.sub.2 evolution, post-heat treatment) decreased 
with increasing temperature. For both parameters, the effects were more 
pronounced at temperatures above 35.degree. C. 
Beet disks rinsed for 20 minutes with 20 mM CaCl.sub.2 (in B5 medium) 
produced more CO.sub.2 than disks not exposed to calcium regardless of 
treatment temperature, except at 55.degree. C. At 25.degree. C. the 
CO.sub.2 production by disks treated with CaCl.sub.2 was greater than that 
produced by disks with no CaCl.sub.2 treatment. During the preparation 
procedure, the disks were cored from a tap root, then sliced into disks. 
This preparation may have stressed the tissue, and the difference noted at 
25.degree. C. was probably due to the improved membrane stability of the 
calcium treated disks. 
The rate of decline of CO.sub.2 production with increasing temperature was 
not as steep as for the tissues not rinsed with CaCl.sub.2 post-heat 
treatment. At 55.degree. C. however no CO.sub.2 evolution was detected 
from any of the disks. The disks appeared bleached, having released all of 
their remaining pigment after 1 day on solid medium. 
After 3 days, the tissues heated to 55.degree. C. were visibly necrotic, 
while the tissues exposed to lower temperatures (40.degree. C. to 
25.degree. C.) retained some membrane integrity evidenced by no further 
pigment leakage. Only the tissues rinsed with CaCl.sub.2 suffered no 
additional pigment leakage 1 day after the 45.degree. C. heat treatment. 
Removal of the released product from beet root disks, during the heat 
treatment, was accomplished by exchanging the medium containing released 
product with fresh medium. The results indicate that as near continuous 
removal of released product is approached, pigment release relative to the 
controls (no rinses of tissue in the same medium volume), increased. For 
an extraction time of 8 minutes in a 20 ml volume, tissue rinsed 4 times 
(5 ml volume rinses) released 40% more product in the same time period. 
For longer extraction times of 60 minutes, the rinsed tissue (four 5 ml 
rinses) yielded the same amount of product released as the tissue that was 
not rinsed (20 ml volume). This equivalence of product release indicated 
that a concentration gradient began to effect the release of product as 
early as 15 minutes into the treatment period. Continuous rinsing occurs 
in the NMB, which can stimulate additional release. 
Secondary Product Release From and Viability of Hairy Root Tissue 
Hairy root tissue of safflower and beet were heat treated as described in 
Example 4. As was observed for beet root disks, secondary product release 
from beet hairy roots increased with increasing temperature, while 
viability decreased. However, hairy roots heated at 35.degree. C. for 1 
hr, unlike the disks, produced more CO.sub.2 after 1 day than the same 
amount of root tissue cultured at 25.degree. C. otherwise similarly 
treated. Based on the evolution of CO.sub.2 from heated beet hairy roots, 
temperatures of 35.degree.-45.degree. C. were tested further for secondary 
product release while preserving viability. 
Post-heat treatment of beet hairy roots with CaCl.sub.2, for short 
exposures of 10, 20 and 60 minutes, improved viability. The improvements 
were determined by comparing the growth, post-heat treatment, of 
CaCl.sub.2 treated tissue with tissue not exposed to CaCl.sub.2 post-heat 
treatment. Although the cultures were at different stages of growth (5 
days; mid exp. phase, 7 days; late exp. phase and 10 days; early 
stationary phase), the ratio of root mass to liquid volume was maintained 
constant. Failure to control this ratio produced inconsistent results 
since the tissues were not exposed to equal amounts of CaCl.sub.2 /gram of 
biomass. For longer exposures to CaCl.sub.2 (&gt;60 minutes), viability 
decreases. Finally, for exposures of 3 days (the entire recovery period), 
viability was less than the control. The control tissue was heat treated, 
but not exposed to CaCl.sub.2 and by definition, is represented by a 0.0 
gram dry weight change. The most effective exposures to CaCl.sub.2, in 
terms of preserving viability, were: the 10 and 20 minute exposures for 
the 5 day (mid-exponential phase) culture, the 10 minutes exposure for the 
8 day (late exponential phase) culture and the 20 minute exposure for the 
10 day (early stationary phase) culture. 
As percent of total betanin, the highest value of secondary product 
released from beet hairy roots (heated at 42.degree. C. for 1 hr) was 
obtained from the 7 and 9 day cultures, at 15 and 14%, respectively. The 
viability indices for these heat treated cultures were 96 and 83%. Very 
little pigment (2% of total betanin; 2 .mu.g) was released from the 5 day 
culture, which was in early exponential phase. The value of V for this 
culture was 84%. 
Although the 11 day culture only released 12% of its pigment, the amount of 
pigment released was 3.6 times the amount released from the 7 day culture 
(44 .mu.g vs 160 .mu.g). The percentages of betanin released were based on 
1.3 and 0.29 mg of total betanin contained in the 11 and 7 day cultures, 
respectively. 
The ratio of tissue biomass to medium must be considered in order to 
isolate the differences, if any, between the stage of growth of the tissue 
and, in this case, pigment release. The betanin concentrations obtained 
after heat treatment of hairy roots in different volumes of media for the 
7 day cultures indicate that a diffusion gradient began to restrict 
pigment release at betanin concentrations approaching 0.9 mg/1. 
With increasing time of exposure to heat at 42.degree., 45.degree. and 
50.degree. C., secondary product release from beet hairy roots increased. 
After heating at 42.degree. C., secondary product release increased to 10% 
of total betanin after 20 minutes with no loss of viability. After heating 
at 45.degree. C., V decreased to 80% after 2 minutes, while at 50.degree. 
C., V was zero after 2 minutes of heating. The maximum temperature to 
which beet hairy roots could be heated while still retaining viability was 
42.degree. C. 
The rates of betanin release from beet hairy roots increased with 
increasing treatment temperature. After just 10 minutes of heating, the 
concentrations of released product (in a 50 ml volume) were 0.25, 0.69 and 
1.37 mg/1 after heat treatments of 42.degree., 45.degree. and 50.degree. 
C., respectively. Similar results were also obtained for the other two 
beet pigments. After 45 minutes heating at 42.degree. C., pigment efflux 
was no longer increasing at a linear rate. The concentration of betanin at 
this time was 0.9 mg/1. Based on the concentration of betanin released 
from 7 day cultures, further heating of the tissue at 42.degree. C. would 
net a diminishing return in terms of product release vs. retention of 
viability. 
Since viability of the heat treated tissue was not 100%, the source of the 
released pigment, whether from viable or non-viable (heat damaged) tissue, 
had to be determined. Using the method described in Example 4, the 
estimated pigment released from non-viable tissue vs. the actual pigment 
released, was calculated. In all cases, except for the 5 day culture, the 
actual pigment released was greater than the amount of pigment which was 
calculated to have been released from only the non-viable tissue. 
Furthermore, even under extreme conditions of heating (50.degree. C. for 
10 minutes; 100% non-viable tissue), beet hairy roots did not release all 
of their pigment, rather, only 25%. The assumption that the non-viable 
tissue released all of its pigment represents a worst-case scenario. 
Therefore, the actual pigment released from non-viable tissue must be less 
than the estimated value. 
Production of secondary metabolite post-heat treatment was measured as 
described in Example 4. Heat treatments were performed on an 8 day culture 
at which time, 18%.of the total product was released (0.097 mg betanin). 
After treatment with CaCl.sub.2 and 3 days in culture, the tissue 
synthesized 0.71 mg of betanin compared to 0.74 mg from the tissue that 
was not heated. The net betanin produced by the heated tissue was 0.81 mg, 
a 9.5% increase in production over the non-heated tissue. This result 
demonstrates that removal of product can stimulate an increase in 
secondary metabolite production. 
Secondary product release from beets was stimulated by treatment with 
(NH.sub.4).sub.2 SO.sub.4 and EDTA. Treatments using B5 medium at pH 
values of 3.5 and 7.0, did not stimulate any product release. Ammonium 
sulfate concentrations up to 20 mM stimulated product release. The maximum 
product released was 12% of total betanin in 20 mM (NH.sub.4).sub.2 
SO.sub.4 after 2 hour extraction at 5.degree. C. The viability index, 
determined 3 days after the heat treatment, decreased to 87%. 
Five percent of total betanin was released after a 2 hour treatment with 1 
mM (NH.sub.4).sub.2 SO.sub.4 with no loss of viability. This effective 
concentration of (NH.sub.4).sub.2 SO.sub.4 was achieved by excluding 
membrane stabilizers normally included in B5 medium (e.g., Ca and Mg). 
Concentrations of (NH.sub.4).sub.2 SO.sub.4 greater than 20 mM decreased 
the amount of product released relative to the treatment using only 1 mM 
(NH.sub.4).sub.2 SO.sub.4. Finally, the use of EDTA (1 mM), a calcium 
chelator, in addition to 20 mM (NH.sub.4).sub.2 SO.sub.4, increased 
product release to 15% of total betanin with no additional loss in 
viability beyond 87%. 
The rate of betanin pigment released from beets using ammonium sulfate 
treatments (20 mM) at 25.degree. C. increased linearly up to 2 hrs. with 
an initial lag in pigment release of 15 minutes. The same result occurred 
with betaxanthin release. However, release of betalamic acid occurred 
immediately. 
Betanin release was initiated in beet hairy roots cultured for 1 week in 
the nutrient mist bioreactor (See Example 4). From 1 gram dry weight of 
tissue, 18% of total betanin (0.49 mg) was released into the surrounding 
culture medium (865 ml) in two hours. Although the percent release of 
total product was higher than that obtained from shake flasks (15%), the 
ratio of tissue dry weight to medium volume must be considered. In shake 
flasks, the ratio was 2.0 g dry wt/1 vs 1.16 g dry wt/1 in the nutrient 
mist bioreactor (865 ml of B5+20 mM (NH.sub.4).sub.2 SO.sub.4 was used in 
the extraction). In previous experiments in shake flasks.sub., using heat 
treatments at 42.degree. C. to permeabilize the tissue, 15% and 23% of 
total pigment were released when the ratios of tissue to medium volume 
were 2.0 and 1.0 g/l, respectively. Therefore, product release in the 
culture chamber of the nutrient mist bioreactor can be directly compared 
to shake flasks containing equivalent tissue biomass to extraction medium 
volume ratios. 
Viability of the tissue after the heat treatment was again comparable to 
the value obtained from shake flasks. The viability index was 84% for the 
tissue treated in the nutrient mist bioreactor vs. 87% for the tissue 
treated in shake flasks. 
Applicability of Extraction Methods to Other Plant Tissue 
The results of experiments using beet root disks and beet hairy roots are 
applicable for extraction of plant secondary metabolites from any other 
plant tissue. The properties of plant cell membranes and in fact all 
eukaryotic cell membranes are universal. All biological membranes, 
including the plasma membrane and the internal membranes of eukaryotic 
cells, have a common overall structure; they are assemblies of lipid and 
protein molecules held together by noncovalent interactions. The lipids 
are arranged as a bilayer, which provides the basic structure of the 
membrane and serves as a relatively impermeable barrier to the flow of 
most water-soluble molecules. The protein molecules are within the lipid 
bilayer and mediate the various functions of the membrane; some service to 
transport specific molecules into or out of the cell; (e.g., channel 
proteins, .such as passive channel proteins, carrier proteins, and 
proteins involved in active transport) others are enzymes that catalize 
membrane-associate reactions; and still others serve as structural links 
between the cell's cytoskeleton and the extracellular matrix, and or as 
receptors for receiving and transducing chemical signals from the cell's 
environment. 
Therefore, methods of destabilizing and restabilizing beet cell membranes 
will produce the same effects when used on other plant cell tissue. The 
beet model results show that plant tissue membranes can be destabilized 
using any of a number of techniques performed alone or in combination. For 
example, the tissue can be exposed to elevated temperatures for various 
periods of time. Culturing plant tissue in temperatures ranging from 
25.degree.-45.degree. for times ranging from about 1 minute-to two hours 
increases the fluidity of plant cell membrane and is therefore useful for 
obtaining partial release of most secondary metabolites, while maintaining 
plant tissue viability. Viability generally decreases with length of 
exposure to the method of permeabilization. The optimal temperature and 
time of exposure can be determined by one of skill in the art without 
requiring undue experimentation. 
Alternatively, destabilization can be accomplished by contacting plant 
membranes with a permeabilizing agent (i.e., a substance which fluidizes a 
channel protein within a plant cell membrane) at an appropriate 
temperature and for an appropriate amount of time. Shorter exposures to 
the permeabilizing agent, (e.g., 1 min to 2 hrs) result in minimal loss of 
viability. 
Examples of permeabilizing agents are substances which prevent the binding 
of divalent cations to plant cell membranes. Divalent cations stabilize 
cell membranes by reducing the electrostatic repulsion between ionic 
centers on the membrane. Siegel, S. M. and O. Daly Plant Phys. 
41:1429-1434 (1966). Therefore, any substance, which prevents the binding 
of divalent cations destabilizes and permeabilizes the membrane. The order 
of effectiveness for destabilizing beet plant cell membranes by preventing 
divalent cation binding to the plant cell membrane is that removal of 
manganese destabilizes better than removal of calcium, which is better 
than removal of strontium, which is better than removal of barium, which 
is better than removal of magnesium (i.e., Mn&gt;Ca&gt;Sr&gt;Ba&gt;Mg). This order of 
effectiveness should prove to hold true for all plant cell membranes. 
Additional examples of permeabilizing agents include compounds which 
contain the ammonium ion-(e.g, (NH.sub.4).sub.2 SO.sub.4, especially in 
concentrations ranging from 1 to 20 mM) and (ethylenediamine) tetraacetic 
acid trisodium salt (EDTA) which is a chelator of divalent cations (4 EDTA 
molecules chelate 1 divalent cation). Since divalent cations stabilize 
membranes, chelating any free divalent cations away from the tissue, 
indirectly permeabilizes the membrane, increasing product release. 
Membrane destabilization can also be accomplished by excluding membrane 
stabilizers, such as methods of divalent cations, from the culture medium. 
For example, culturing plant tissue in medium that lacks membrane 
stabilizers such as Mg.sup.+2 or Ca.sup.+2, or both, can result in release 
of secondary metabolites from the plant tissue. For plant tissue which is 
not growing in an NMB, controlled electroporation and/or sonication can be 
used to destabilize plant cell membranes. 
The results of beet root experiments indicate that different secondary 
metabolites are released at different rates as a result of different 
destabilization methods. For example, the rate of betanin and betaxanthin 
pigments released from beets using ammonium sulfate treatments (20 mM) at 
25.degree. increased linearly up to 2 hrs. with an initial lag in pigment 
release of 15 minutes. However, release of betalamic acid occurred 
immediately. The degree of selectivity toward specific products offered by 
the choice of destabilization methods provides additional control over 
process development. The choice of which destabilization method to employ 
to extract a particular secondary metabolite, as well as optimization of 
the conditions used, such as time of exposure and temperature, can be 
determined by one of skill in the art without undue experimentation. 
Subsequent to destabilizing the plant membrane, a solvent can be added to 
the culture medium in order to effect greater extraction. For example, 
some secondary metabolites (e.g., taxol and artemisinin) are nonpolar 
compounds and therefore are non-soluble in aqueous solutions, such as the 
culture medium. Therefore, a nonaqueous solvent can be added to the 
culture medium to enhance extraction of nonpolar secondary metabolites. 
Preferred nonaqueous solvents include: ethanol, 65% and 75%, polyethylene 
glycol (PEG-400), tomatine, poly-L-Lysine, 50% Cremaphor EL in a short 
chain alcohol (e.g., methanol), DMSO, Triton X-100, Brij Tween-80 and 
cumene peroxide. Ethanol and 50% Cremaphor EL in methanol or in any other 
short chain alcohol are particularly useful for extracting taxol from 
Taxus species. Alternatively, the permeabilizing agent itself can be a 
nonaqueous solvent. 
However, it is important that the particular solvent used does not decrease 
the plant tissue viability. Example 5 presents experiments, which tested 
the effect of nonpolar solvents in combination with heat treatment on 
plant cell viability. The results of the experiment indicate that tissue 
which is treated with lower temperatures (e.g., 25.degree. C.) and shorter 
times of exposure (e.g., 1-35 mins) released more secondary metabolite, 
while maintaining tissue viability. 
In the final step of the method of the invention, plant tissue membrane is 
restabilized to stop secondary metabolite release and to enhance plant 
tissue viability. The results of the beet root experiment show that plant 
tissue membranes can be restabilized using any of a number of techniques 
performed alone or in combination. One approach is to remove the condition 
which promoted destabilization. For example, if destabilization was caused 
by raising the temperature of the culture medium, restabilization can be 
effected by cooling the culture medium (e.g., to room temperature), to 
return the fluidity of the plant cell membrane to its normal level. 
Alternatively, the destabilizing agent can be removed. Further, divalent 
cations can be introduced into cultures which were destabilized by being 
cultured in medium which lacked divalent cations or in medium which 
contained a permeabilizing agent. The order of effectiveness for 
restabilizing plant cell membranes by introducing divalent cations into 
the medium, so as to make them available for binding to the plant cell 
membrane, is the same for the order of effectiveness for destabilizing by 
removal (i.e., Mn&gt;Ca&gt;Sr&gt;Ba&gt;Mg). Addition of substances which intercalate 
into the lipid bilayer, such as sterols (e.g., cholesterol, B-sitosterol, 
and stigmasterol), phospholipids, or glycolipids and/or glycoproteins can 
also effectively restabilize plant cell membranes. 
The present invention will now be illustrated by the following Examples, 
which are not to be seen as limiting in any way. 
EXAMPLE 1 
Method of Obtaining Hairy Roots 
Maintenance of Agrobacterium rhizogenes strains 
Agrobacterium rhizogenes strain ATCC 15834 was maintained on YMB medium (in 
g/l: K.sub.2 HPO.sub.4 -0.5, MgSO.sub.4.7H.sub.2 O-2.0, NaCl-0.1, 
Mannitol-10.0, yeast extract-0.4, and agar-15.0, pH 7.0) at 
25.degree.-28.degree. C. Only fresh (2-3 days old) cultures subcultured 
directly from the original stock were used for purposes of infecting 
tissue since the bacteria lose their virulence after successive 
subculturings. 
Hairy root initiation using beet root disks 
Garden variety beets (Detroit dark red) were harvested, the stems and 
leaves were cut away and the beets were scrubbed under running tap water 
to remove surface dirt. The whole beets were surface sterilized in 10% 
commercial bleach (Clorox) for 30 minutes. The area to be cored was cut 
away and discarded, and the beet was placed in 10% bleach for 20 minutes 
more. The area to be cored was again cut away and discarded. A sterile 
cork borer (1.5 cm diameter) was forced through the center of the beet. It 
was necessary to manually hold the beet (without gloves) at this point 
since forceps and gloves proved ineffective. The ends of the corings were 
cut off and discarded and the remaining core was placed in sterile 
distilled water. The cores were sliced into disks approximately 3 mm 
thick, rinsed three times with sterile distilled water in sterile Petri 
dishes and blotted dry on sterile Whatman #1 filter paper. The disks were 
placed on solid B5 salts with 0.2% Gel-rite, 20 g/1 sucrose and 0.025 % 
(w/v) carbenicillin (Pfizer). The disks were infected with A. rhizogenes 
by swabbing the surface of the disk with a sterile toothpick which had 
been dipped in a 2-3 day old bacterial culture grown in Petri dishes. 
Typically, 3 disks were placed on each plate; 2 of these were infected and 
the third was left as a control. After approximately 2 weeks, roots began 
to grow from the site of the infection. 
Obtaining axenic hairy root cultures of Beta vulgaris 
After excising the roots from the primary explant, the tissue was cultured 
on solid B5 media with 20 g/l sucrose+0.025% carbenicillin. According to 
Flores, et al., roots growing up off the agar surface will be free of 
contamination (Flores H. E., et al., Trends in Biotechnology 64-69 (1987)) 
and can be subcultured to obtain an axenic culture. This method was 
ineffective for beet hairy roots probably due to the excessive root hairs 
which could have trapped and carried contamination off the surface of the 
Gel-rite. The tissue, although contaminated, was cultured in liquid B5 
media (B5 salts+B5 vitamins+30 g/l sucrose, pH 5.5-6.0) containing 0.025% 
carbenicillin for up to 2 weeks. (Liquid B5 media, unless otherwise 
specified contains 30 g/l sucrose, B5 salts and vitamins at a pH of 
5.5-6.0). This procedure increased the amount of tissue enabling a variety 
of harsh tissue sterilization treatments to be performed without danger of 
losing the transformed tissue. Hairy roots were then blotted dry on 
sterile Whatman #1 filter paper and subcultured onto solid media with 
0.25% carbenicillin. After about 1-2 weeks, these tissues were subjected 
to dips in commercial bleach (3-10% Clorox) for periods of up to 10 
minutes. The tissues were then rinsed in sterile deionized water, blotted 
dry as previously described and placed on solid media with 0.25% 
carbenicillin. This procedure was repeated until no contamination was 
visible in subsequent liquid cultures without antibiotics for 2 weeks. 
EXAMPLE 2 
Modifications to the Nutrient Mist Bioreactor to Accommodate the Growth of 
Transformed Hairy Roots 
Modification 1 
Increasing the Mist Volumetric Throughput 
The mist volumetric throughput can be increased by placing the ultrasonic 
transducer inside the culture chamber (i.e., either at the top [which is 
preferred] or the bottom). The theoretical transducer output is 
575.+-.175ml/hr. The growth results were obtained with a mist flow of 12 
ml/hr. The mixing time for the culture changes containing roots calculated 
for these conditions was 63 minutes, which means that 3 hours are required 
for 95% turnover of the retained liquid by the roots in the culture 
chamber. In order to remove released product as well as to treat with a 
restabilizing agent, too much time would be needed and the tissue would 
probably die in the process. For this reason, tissue cultured in the 
growth chamber was destabilized as follows: 
Beet hairy root tissue was cultured for 1 week in the NMB. NMB was operated 
in 2 different configurations: continuous mode (no medium recycle) and 
batch mode (recycle of nutrient mist and coalesced medium from the culture 
chamber into the medium reservoir). Different culture conditions were 
established based on varying the following parameters: mist cycle time 
(range of 5/6 to 5/20, and 2/2 to 2/10), mode of operation (batch or 
continuous), inoculum size and sucrose concentration in B5 medium for 
beets (10-35 g/l). (A mist cycle denoted as 5/6 represent a repeating 
cycle of 5 minutes of misting followed by 6 minutes without misting). 
Carbon dioxide enhancement (1.0%) of the carrier gas (air) was also 
performed. The best conditions for growth were determined by comparing dry 
weight increases after 1 week in culture. Secondary metabolite production 
was also measured, using a small representative sample (approx. 1-2 grams 
wet weight) of the total root mass. 
Beet hairy root tissue growth was monitored as fresh weight increases. The 
entire culture chamber (sterile and loaded with tissue) was placed on a 
Mettler top loading balance (2600 kg maximum load). Growth was measured 
beginning at 3 days after inoculation as an equilibration time was 
necessary for coalesced medium to achieve a steady state within the 
culture chamber. The balance was tared to zero each day, and the 
subsequent weight increases were recorded. Dry to wet weight correlations 
for the tissue in the culture chamber were obtained by comparing the 1 
week values of dry tissue. 
After 1 week, the culture chamber effluent was clamped off and a vent was 
opened at the top of the culture chamber. Fresh B5 liquid medium without 
CaCl.sub.2 +20 mM (NH.sub.4).sub.2 SO.sub.4 was pumped into the culture 
chamber through sterile tubing, until the liquid level was just above the 
tissue in the nylon matrix. After 2 hours of exposure, the culture chamber 
was drained, then filled as before with B5 liquid medium +20 mM CaCl.sub.2 
and immediately drained. The normal misting cycle was resumed, and the 
tissue was cultured with 30 g/l sucrose in B5 medium. After the second 
week of growth, the tissue was analyzed for pigment production and growth. 
The residence time could be reduced to 1-2 minutes with the proposed 
modification. Therefore, product release and recovery could be 
accomplished using nutrient mist. 
The increased availability of mist will also allow smaller air volumes 
(carrier gas) to be used since the mist does not have to be transported 
from the mist generator to the culture chamber. Reduced air volumes 
reduces the effect of medium acidification that was noted after operating 
the bioreactor in a recycle configuration. Mist distribution in the 
modified design could be carried out by rotating the support trellis. 
Another method of mist distribution could be to have the exit from the 
culture chamber just above the top of the support trellis. This would 
force the entire culture volume to be covered with mist, and coalesced 
mist would be effectively recycled since the medium reservoir-is, in 
effect, at the bottom of the culture chamber. If desired, media recycle, 
or fresh media can be added by simultaneously draining and. adding sterile 
nutrient media from the sump at the bottom of the culture chamber. Air 
enriched with CO.sub.2 could be added during the off cycle to minimize 
medium acidification due to dissolved CO.sub.2 in the medium. 
Increased mist volume may enable the culturing of root tips which would 
greatly improve the rate of biomass production in the nutrient mist 
bioreactor. In the past root tips could not be used as inocula for the 
bioreactor because of a naturally produced mucilage layer which prevented 
nutrient uptake. The increased mist flow (liquid contact) could wash away 
this layer (as it is washed away in liquid culture) and allow for the 
culture of rapidly growing root tips. 
Finally, in order to scale-up the culture chamber volume, increased flow, 
which is only possible by the proposed modification, is vital. In the 
previous design, substantial coalescence of the generated mist occurs in 
the mist generator (specifically, at the orifice). For liquid throughput 
determinations, the wider the internal diameter, the better the 
throughput. 
Modification 2 
A collapsible trellis in the culture chamber for use in scale-up. 
Inoculation of the 2 liter culture chamber with hairy root tissue can be 
accomplished by simply removing the lid (in a sterile hood) and placing 
the tissue directly on the 3 levels of nylon mesh. To scale up, this 
method is both inefficient and ineffective, because it requires 
considerable skilled manual labor and the size limits the ability to load 
roots aseptically. In addition, the time required for large numbers of 
roots means early loaded roots will have desiccated. By using a collapsing 
trellis suspension of hairy roots could be pumped into the bottom of the 
culture chamber to effectively load each level. The top level can be 
raised (containing the tissue), and the procedure can be repeated for the 
next level until the entire trellis has been raised. Lowering and raising 
of the trellis can be accomplished without breaching the internal sterile 
environment. A magnetic coupling on the side of the culture chamber could 
be used to raise and lower the trellis or the trellis position could be 
controlled pneumatically. 
EXAMPLE 3 
Secondary Product Release From and Viability of Beet Root Disks 
Growth of beet root disks in the nutrient mist bioreactor. 
A cork borer (1 cm diameter) was used to obtain uniform disks from washed 
and peeled garden variety beets (Detroit Dark Red). The disks were sliced 
approximately 3-5 mm thick, rinsed in running tap water for 1 hour and 
blotted dry. The Nutrient Mist Bioreactor (NMB, Mistifier.TM.) 
(Bio-Rational Technologies, Stow, Mass.) was configured in a continuous 
flow mode and 12 pre-weighed disks were placed in the nylon matrix which 
was then placed into the polycarbonate chamber (pre-heated to 35.degree. 
C. in a Lab-Line incubator). The culture chamber containing the disks was 
returned to the incubator. 
The chamber was misted on a 5/10 cycle using preheated B5 medium 
(35.degree. C.), with an air flow of 1400 ml/min. After 1 hour, the disks 
were removed and placed in 2 ml of B5 medium. After 30 seconds, the disks 
were removed and the liquid was assayed for released pigments based on 
individual absorbance maxima for each of the three major beet pigments. 
The disks were then ground, the pigments were extracted and assayed by 
measuring an absorption spectrum (380-640 nm) of a 1 ml sample containing 
released pigment in a Beckman DU-64 recording spectrophotometer (slit 
dimensions 0.3 mm wide.times.2 mm high). Absorbance values from 380-640 nm 
were measured over 10 nm increments as well as at the absorbance maxima 
for the pigments (426, 478, and 537 nm, for betalamic acid, betaxanthins 
and betanin, respectively). (Saguy I. Mizrahi S. and I. J. Kopelman, 
Journal of Food Science 43:121-123 (1978)). Culture media was used as a 
blank. The error or drift in the reading over the entire spectrum using 
the blank was no more than 0.002 absorbance units. 
Betacyanin was assayed using the methods of Saguy, et al., to resolve the 
individual pigment concentrations within the mixture. (Saguy I., Kopelman 
I. J., and Mizrahi S., Journal of Food Science, 43:124-127 (1974)) The 
procedure was repeated using 12 different disks at a temperature of 
25.degree. C., as a control measure. The experiment was also performed 
using B5 medium with an additional 20 mM CaCl.sub.2 preheated to 
35.degree. C. (12 different disks) in the mist generator. 
Secondary product release and viability of beet root disks after heat 
treatment in B5 medium 
Sterile beet root disks were obtained as described in Example 1. The disks 
were weighed and 6 disks were placed into a series of sterile, 1.times.10 
cm test tubes. The total wet weight of the disks in each of the tubes (2 
g) was kept within 5%. Sterile B5 medium (3 ml)-was added to each test 
tube. The test tubes were placed in heating blocks at temperatures ranging 
from 25.degree. C. (ambient) up to 55.degree. C. for periods of 15, 30 and 
45 minutes. After the specified time had elapsed, the sterile tissues were 
removed and rinsed 3 times with sterile distilled water in Petri dishes. 
The tissues were blotted dry and placed into T25 tissue culture flasks 
containing 20 ml of solid B5 medium. The flasks were sealed with a rubber 
septum and incubated in the dark at 25.degree. C. After 5 days, 200 .mu.l 
of gas was removed from each flask and assayed for CO.sub.2 using gas 
chromatography. Patriquin, D. and Knowles, P. Canadian Journal of 
Microbiology 20:1037-1041 (1974). This experiment was repeated using B5 
medium +20 mM CaCl.sub.2 in the heating medium. 
Application of CaCl.sub.2 to enhance viability after heat treatments 
Beet root disks were heated to release secondary product as described above 
with the following modifications: 1) After coring the beets, all 
subsequent rinses of the tissues were performed using half strength B5 
medium so as not to stress the tissue causing product release. 2) After 
the heat treatments, the tissues to be treated with CaCl.sub.2 were placed 
on solid B5 medium (in T25 flasks) containing an additional 20 mM 
CaCl.sub.2. 3) Five, rather than six disks were used in each flask. 4) 
Carbon dioxide concentrations were measured daily over a 4 day period 
after the heat treatment. 
The experiment described here was repeated a third time with the additional 
modification of rinsing the tissue after the heat treatment. The tissues 
to be treated with CaCl.sub.2 were rinsed in B5 medium+20 CaCl.sub.2. The 
other tissues were rinsed in standard B5 medium. 
Secondary product release from beet root disks with rinsing cycles. 
Beet root disks (1 cm.times.2-3 mm) were rinsed in running tap water for 1 
hr, blotted dry, weighed and placed in sterile 1.times.10 cm test tubes (3 
disks/tube). The total disk weight/tube was kept to within 10%. Preheated 
MS media (Murashige T and F. Skoog Physiologia Plantatum 15:473-482 
(1962)) (40.degree. C.) was added to each tube and the tubes were placed 
in heating blocks at 40.degree. C. for periods of 8, 16 and 60 minutes 
with periodic rinsing cycles according to Table 2. The total volume of 
releasing buffer (MS media pH 6.0) was kept constant at 20 ml. The 
controls for this experiment involved exposing equivalent amounts of 
tissue to the entire treatment volume (20 ml) for 8, 16 or 60 minutes 
(labeled "b" in Table 1). After the tissues were rinsed, the control disks 
were dried and the average dry weight was calculated. 
TABLE 1 
______________________________________ 
Procedural summation for the 
periodic rinsing of beet root 
disks. 
Time Vol. 
Between Used/ Total 
Sample Rinses No. of Rinse Total vol. 
No. (min) Rinses (ml) Time (ml) 
______________________________________ 
1a 2 4 5 8 20 
1b* -- 0 20 8 20 
2a 4 4 5 16 20 
2b* -- 0 20 16 20 
3a 15 4 5 60 20 
3b* -- 0 20 60 20 
______________________________________ 
*The tissues were not passed through multiple rinses, but were exposed to 
the same total volume as the rinsed tissue. 
The disks (5 for each experimental condition) were treated with preheated 
(35.degree. C.) B5 medium containing 30 g/l sucrose. 
EXAMPLE 4 
Secondary Product Release From and Viability of Beet Hairy Root Tissue 
Heat treatment of beet hairy root tissue and the effect of exposure to 
CaCl.sub.2 on viability 
Beet hairy root tissue (0.3 g wet wt.) was cultured in 50 ml B5 liquid 
media for 5, 7, 8, 9, 10, or 11 days. After the initial growth period, 
spent medium was removed aseptically and 50 ml of preheated B5 liquid 
medium (42.degree. C.) was added to each flask (Table 3). For the cultures 
grown 5, 8 and 10 days, the preheated medium added to each flask was 
adjusted to maintain a constant ratio of tissue wet weight to treatment 
medium volume (based on 50 ml added to the 8 day culture). The cultures 
were incubated for 1 hr at 42.degree. C., then the released product was 
assayed spectrophotometrically as explained in Example 3. Tissues were 
treated with 20 mM CaCl.sub.2 for 10', 20', 60' or 3 days. For the tissues 
not treated 3 days, fresh B5 liquid medium was exchanged aseptically and 
the roots were cultured 3 days at 25.degree. C. at which time dry weights 
were measured. Experimental controls were provided. A set of 2 control 
flasks were routinely used for each condition in this and all experiments. 
Heat treatment of beet hairy roots at a range of temperatures and 
exposures. 
Beet hairy root tissue (0.3 g wet wt.) was cultured in 50 ml B5 liquid 
medium for 7 days. Spent media was removed and replaced with 50 ml of 
preheated, B5 liquid media at temperatures of 25.degree., 35.degree., 
40.degree., 42.degree., 50.degree., or 55.degree. C. for 30, 45 or 60 
minutes. Heat treatments at 42.degree. C. were also performed on 1 week 
old cultures using 25 and 100 ml of preheated B5 medium. Treatment medium 
was removed, assayed for betacyanins and replaced with fresh B5 liquid 
media+20 mM CaCl.sub.2 for 10 minutes. Then the CaCl.sub.2 medium was 
replaced with fresh B5 liquid medium and the tissues were cultured an 
additional 3 by days before measuring dry weights. Experimental controls 
(similarly cultured prior to the onset of heat treatment), included: 1) 
root tissue which was not heat treated but was otherwise treated exactly 
the same as the heated tissue, and 2), root tissue which was not heat 
treated, but instead of removing the spent medium, the tissue was cultured 
with no medium exchanges. 
The source of the released pigment, whether from viable or non-viable 
tissue, was estimated by making the following assumptions; 1) The pigments 
were uniformly distributed throughout the tissue biomass. 2) The 
percentage difference between the dry weight of the heated tissue vs. the 
non-heated tissue represented non-viable tissue. 3) The non-viable tissue 
released all of its pigment. This amount of pigment was calculated based 
on assumption 1). Finally, the pigment released by the non-viable tissue 
(Pnv) was compared to the actual pigment released (P) at 42.degree. C., 
after 1 hour. If P&gt;Pnv, then some of the released pigment had to have been 
released from viable tissue. 
Secondary metabolite production and growth after heat treatment 
A series of flasks containing 50 ml B5 liquid medium were inoculated, 
cultured and heat treated at 40.degree. C. as described above, but after 
the heat and CaCl.sub.2 treatments, the spent medium removed prior to 
heating was returned to the flasks aseptically. Another series of flasks 
were also heated, but after the CaCl.sub.2 treatment, fresh B5 liquid 
medium was added. 
At the time of the heat treatment and every day afterward up to 3 days, 2 
flasks of each type (fresh medium and spent medium) were sacrificed. The 
tissue from flask #1 was blotted dry, weighed (fresh weight), then ground 
for total pigment extraction and analysis as described above. The tissue 
from flask #2 was blotted dry, weighed (fresh weight), dried at 60.degree. 
C. overnight and weighed again (dry weight). The fresh weight and dry 
weight of #2 were used, along with the fresh weight of #1, to determine 
the dry weight of #1. 
Treatment of hairy root tissues with (NH.sub.4).sub.2 SO.sub.4, EDTA and 
varying medium pH values. 
The procedure for heat treatment described above was followed with the 
exception that instead of heating, the tissue was treated in one of the 
following variations of B5 media for 2 hrs: 1) B5 liquid medium without 
CaCl.sub.2 or Mg, 2) B5 liquid medium without CaCl.sub.2 +magnesium 5, 10, 
15, 20, 25, 40, 60, 80 or 100 mM (NH.sub.4).sub.2 SO.sub.4, 3) B5 liquid 
medium, pH 4.0, 6.5 or 7.5, and 4), B5 liquid medium+20 mM 
(NH.sub.4).sub.2 SO.sub.4 +1, 3 or 4 mM EDTA. Also, as described above, 
the release of product over time (30', 60', 90' and 120') was measured. 
After treatment for product release, the tissues were treated with 
CaCl.sub.2, etc. as described above. 
EXAMPLE 5 
Effect of Non-Polar Solvents on Plant Cell Viability 
To test the effect that non-polar conditions have on plant cell viability, 
beet root disks were incubated in a solution containing 50% Cremaphor EL 
and 50% methanol and incubated for various periods of time at the 
following temperatures: 25.degree. C., 35.degree. C., and 40.degree. C. 
Since beet disks are mature tissue, which therefore do not increase 
biomass, viability was measured in terms of carbon dioxide respiration. 
Because different beet root disks were used for the various times and 
different temperatures, the left side of the table indicates the dry 
weight of the beet disk used and the right side indicates the respiration 
measured for that amount of beet root disk. 
Disks which were incubated in 50% Cremaphor EL and 50% methanol at 
25.degree. C. had the greatest viability relative to those incubated at 
35.degree. C. and 45.degree. C. The data which were obtained are presented 
in Table 2. Qualitatively, red pigment appeared to leach out of the disks 
much more quickly in disks which were treated with lower temperatures and 
shorter times of exposures. This phenomenon manifested itself as a lack of 
red pigment in incubation media of the T25 flasks as well as in the disks 
which implies that the pigment left the disks upon rinsing. In the medium 
of these disks, there was still a predominance of yellow pigment. The 
plates which were exposed for longer periods of time and/or at higher 
temperatures had medium which was predominantly red in color, although it 
was not possible to determine the presence of yellow pigment because it 
was overshadowed by the red pigment. 
TABLE 2 
______________________________________ 
Heat Treatment of Beet Root Disks 
Incubated in a Solution Containing 
50% Cremaphor EL and 50% Ethanol 
Dry Weight (g) % CO.sub.2 Released 
Minutes 
25.degree. 
35.degree. 
45.degree. 
25.degree. 
35.degree. 
45.degree. 
______________________________________ 
0 .0702.sup.y 
n.d. n.d. 12.79 n.d. n.d. 
5 .0766.sup.y 
.0583.sup.y 
.0593.sup.y 
11.11 7.25 11.18 
15 .0646.sup.y 
.0629.sup.y 
.0569.sup.r 
10.80 6.45 .75 
30 .0597.sup.y 
.0588.sup.r 
.0565.sup.r 
10.58 4.43 .51 
45 .0604.sup.r 
.0546.sup.r 
.0505.sup.r 
.537 
.19 .63 
60 a .0576.sup.r 
.0475.sup.r 
a .23 .44 
______________________________________ 
n.d. = not determined 
a = this sample was contaminated 
y = yellow pigment was predominant in incubation media 
r = red pigment was predominant in incubation media 
Equivalents 
Those skilled in the art will recognize, or be able to ascertain using no 
more than routine experimentation, many equivalents to the specific 
embodiments of the invention described herein. Such equivalents are 
intended to be encompassed by the following claims.