Method for producing azadirachtin by cell culture of Azadirachta indica

A plant cell suspension culture to derive azadirachtin-producing cells is described. The culture overcomes the disadvantages of the present processes of seed extraction. The culture allows for the continuous production of azadirachtin that is free of pathogens and toxins, independent of environmental conditions. It yields a predictable quantity and quality of bioactive product and permits continuous production at a desired, contained location.

FIELD OF THE INVENTION 
The present invention relates to a phytohormone-independent cell culture. 
In particular, the present invention relates to the use of plant cell 
cultures for the production of bioactive compounds. In particular, the 
present invention relates to plant cell cultures that produce the 
insecticidal tetranortriterpeniod azadirachtin, and processes for 
obtaining azadirachtin from such cell cultures. 
BACKGROUND OF THE INVENTION 
Industrial-scale quantities of bioactive compounds may be produced by 
growing cultured cells in bioreactors of up to 100,000 liters (Payne, et 
al. (1992) Plant Cell Culture and Tissue Culture in Liquid Systems, Oxford 
University Press, New York; Scragg A. H. (1990); Fermentation systems for 
plant cells IN: Charlwood, B. V. and M. J. C. Rhodes, Eds Secondary 
Products from Plant Tissue Culture, Oxford University Press, New York). 
Few commerical-scale examples of plant cell culture of secondary 
metabolites exist. This is because plant cell culture is generally a more 
expensive route to desired compounds than is extraction of plant parts 
harvested from nature. The costs of plant cell culture presently exceed 
the costs of bacterial and yeast fermentation. 
U.S. Pat. No. 4,717,664 (Method of Producing Secondary Metabolites of 
Plants issued Jan. 5, 1988) discloses a two-stage process for obtaining 
secondary metabolites from suspension cultures of certain families of 
plants. In order to maximize the production of desired secondary 
metabolites, at least two stages of liquid media are used, a growth medium 
and a production medium. At least one of the components of the production 
medium is substantially changed from the growth medium. 
Agrobacterium tumefaciens is a plant pathogenic bacterium that mediates the 
transfer of genetic material into the chromosomal DNA of suseptible plant 
cells by a process known as transformation. Transformation alters plant 
secondary metabolism by the introduction of genes coding for phytohormones 
into plant chromosomal DNA. As a result, these tumorous cells are able to 
grow in culture in the absence of exogenously added phytohormones. 
Zambryski, et al. ((1989) Cell 56:193-202) describe the sequence of 
molecular genetic events that occur when Agrobacterium infects susceptible 
plant cells. The authors further disclose genetic modifications occurring 
to plant chromosomal DNA, and characteristics of the tumorous growth of 
transformed plant cells. Saito, et al. ((1992) J. Nat Prod (LLoydia) 
55(149-162)) discuss Agrobacterium-mediated gene transfer as a strategy to 
enhance secondary metabolite production in transgenic medicinal plants. 
Saito, et al. disclose that suspension cells derived from crown gall 
tumors have been used for production of some specific secondary 
metabolites, specifically quinoline alkaloids and isoflavonoid glucosides. 
Berlin, et al., (1989; Planta Medica, 55:685) disclose the use of 
Agrobacterium tumefaciens transformation of Lupinus cell cultures to alter 
secondary metabolite pathways and made quantitative comparisons between 
productivity of normal and transformed cultures. Berlin, et al., disclose 
that nontransformed and transformed suspension cultures produced the same 
spectrum of compounds, and that transformed cells produced up to ten times 
more isoflavonoid diglucosides than nontransformed lines. However, the 
authors also disclose elevated yields of isoflavones in nontransformed 
cells when those cells were aged ("very lumpy") when compared to yields 
observed in transformed cells. Berlin, et al., further disclose that 
addition of phytohormones to transformed suspensions did not enhance 
isoflavone production. 
Cosio, et al., (1986; J. Plant Physiol, 124:155) disclose that thiarubrine 
synthesis by cells transformed by Agrobacterium tumefaciens is influenced 
by the degree of cellular organization rather than as a direct result of 
cellular transformation. Norton, et al., (1985; Phytochemistry, 24:719) 
discloses thiophene production in Tagetes cells transformed with 
Agrobacterium tumefaciens and concludes that it is not possible to predict 
the amounts of secondary metabolites produced as a result of transfers of 
genetic material from infected plants to crown galls and then to 
transformed callus tissues. Eilert, et al., (1987; Plant Cell Reports, 
6:271) disclose that transformation of Catharanthus roseus cells with 
Agrobacterium tumefaciens did not enhance the accumulation of the alkaloid 
vindoline as compared to habituated, nontransformed cells. Additionally, 
transformed cells did not respond to elicitation with Pythium homogenate. 
Tremouillaux-Guiller, et al., (1988; Plant Cell Reports, 7:456) examined 
alkaloid production in tissue cultures of Choisya ternata, and disclose 
that transformed cells showed lower production of balfourodinium in 21 
transfrormed cell cultures compared to nontransformed cell cultures, and 
no difference in platydesminium between transformed and nontransformed 
lines. 
It is known that Agrobacterium rhizogenes may be used to generate 
transformed root cultures. Transformed roots grow on hormone-free media 
and generally make the same secondary compounds found in nontransformed, 
differentiated root tissues (M. J. C. Rhodes, et al. (1990) IN: Charlwood, 
B. V. and M. J. C. Rhodes, Ed., Secondary Products from Plant Tissue 
Culture, Oxford University Press, New York). 
Compounds referred to in the art as elicitors may be used to enhance the 
production of certain secondary metabolites. Elicitors may be biotic or 
abiotic compounds that stimulate a rapid increase in de novo synthesis of 
some plant secondary metabolites. Examples of elicitors include crude 
fungal extracts, metal ions, carbohydrates, and proteins (Chapter 11, pg. 
333 IN: Payne, et al., Plant Cell and Tissue Culture in Liquid Systems, 
Oxford University Press, New York (1992). Repeated addition of elicitors 
to cell suspensions using a semi-continuous cultivation method is 
disclosed by Kurz (1987; IN: T. Mabry, Ed., Plant Biotechnology Research 
Bottlenecks for Commercialization and Beyond, IC.sup.2 Institute, Austin, 
Tex.; Questions and strategies for productivity improvements). van der 
Heijden, et al., (1988; Plant Cell Reports, 7:51) disclose the use of 
elicitors to induce de novo synthesis of antimicrobial triterpenes in some 
Tabernaemontana species. The study focuses upon whether these 
antimicrobial triterpenes are phytoalexins. EP-A-0378921 (Crawford, et 
al.) discloses methods for monitoring the physiological state of cultured 
cells so that elicitors can be applied at an optimum time for enhancement 
of secondary metabolite production, that time being the end of the growth 
phase. The addition of cell viability stabilizers and/or nutrients at the 
time of elicitation is also disclosed. 
Members of the family Melieae are known to contain bioactive compounds. In 
particular, compounds obtained from the neem tree, Azadirachta indica A. 
Juss (synonymous with Melia azadirach), are used in many products 
including soaps, toothpaste, cosmetics, pharmaceuticals, disinfectants, 
fertilizers, and insecticides (Jacobson M (1986), Am. Chem. Soc. Symp. 
Ser., 296:220-232; Schmutterer, H. and K. R. S. Ascher (1987), Natural 
Pesticides from the Neem Tree (Azadirachta indica A. Juss) and other 
tropical plants: Proceedings of the 3rd International Neem Conference, 
Nairobi, Kenya 10-15 Jul., 1986, Deutsche Gesellschaft fur Technische 
Zusammenarbeit (GTZ) Eschborn Germany). 
Azadirachtin, also referred to as neem, is an insecticidal 
tetranortriterpenoid present in Azadirachta indica A. Juss. Azadirachtin 
has insecticidal and antifeedant activities against a broad spectrum of 
insects (Schmutterer, H. and K. R. S. Ascher (1987) Natural Pesticides 
from the Neem Tree (Azadirachta indica A. Juss) and other tropical plants 
: Proceedings of the 3rd International Neem Conference, Nairobi, Kenya, 
10-15 Jul., 1986, Deutsche Gesellschaft fur Technische Zusammenarbeit 
(GTZ) Eschborn, Germany; Jacobson, M., (1986) Am. Chem. Soc. Symp. Ser., 
296:220-232). Seeds are the primary source of azadirachtin for industrial 
scale production because they are the most concentrated source of 
azadirachtin, and they can most easily be harvested, transported, and 
extracted in large quantities. 
Azadirachta indica has been introduced into cell culture in order to 
regenerate whole plants. Gautam, et al., (1991; In Vitro, 27(3): Pt 
2:146A) disclose callus derived from anthers of Azadirachta indica and 
shoot organogenesis. Kokate, et al., (1989; In Vitro, 25(3) Pt 2 60A) 
disclose generation of callus from Azadirachta indica leaves for the study 
of electrokinetic potentials. Sanyal and Datta (1988; Current Science, 
India, 57 (1): 40-41) disclose generation of callus from tissues of 
Azadirachta indica to produce the triterpenoid nimbin. Sanyal and Datta 
disclose that nimbin gradually disappeared as cells dedifferentiated. 
Nimbin reappeared only after organogenesis. Naina, et al. (Current Science 
(1989) 58(4):184-187) disclose the regeneration of transformed whole 
plantlets from regions of Azadirachta indica seedlings infected with 
Agrobacterium tumefaciens strains K12.times.562E and K12.times.167. Naina, 
et al. disclose that Azadirachta indica offers great potential for 
agricultural, industrial and commercial exploitation as it is an excellent 
source of a variety of secondary metabolites. Naina, et al. point out that 
utilization of neem is limited by lack of knowledge about the tree and its 
specific climatic requirements. Schulz (Tissue Culture of Azadirachta 
indica IN: Schmutterer, H. and K. R. S. Ascher (1984) Natural Pesticides 
from the Neem Tree (Azadirachta indica, A. Juss) and other tropical 
plants: Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ) 
Eschborn, Germany) discloses tissue culture of Azadirachta indica. Callus 
formation and shoot organogenesis are described. Schulz reports that 
callus stopped growing after about six weeks in the presence of the 
phytohormones indole acetic acid and 2,4-dichlorophenoxyacetic acid 
(2,4-D). Although Shulz discloses the desirability of using tissue culture 
as a means for "biotechnical production of the insecticidal plant 
constituents" of Azadirachta indica. Schulz reports no specific disclosure 
of enhanced production of the insecticidally active substances. 
It is important to realize that Azadirachta trees only grow in limited 
regions of the world. Seed production is therefore seasonal. However, an 
obstacle that has prevented the industrial-scale production of 
azadirachtin is establishing a reliable source of seeds containing 
adequate amounts of azadirachtin. Plant productivity and secondary 
metabolite content are influenced by the plant's environment, and 
season-to season variation in the azadirachtin content of seeds from the 
same geographical region may occur. Prior to collecting a large quantity 
of seed for extraction, it is important to measure the azadirachtin 
content of seeds to assure that the azadirachtin content is adequate for 
processing. In the subtropical regions where Azadirachta trees grow, the 
azadirachtin content of seeds may be substantially reduced if seeds are 
improperly stored or invaded by pathogens. Additionally, pathogens may 
produce toxic metabolites such as aflatoxin which may accumulate on seed 
surfaces and be extracted with azadirachtin during processing. Because 
Azadirachta seeds are available only seasonally, industrial processing is 
restricted to periods when seeds are available. The failure to identify 
sources of seeds of acceptable quality in any season can lead to 
disruptions in the manufacturing process. 
In the case of azadirachtin and many other small molecules that are 
products of secondary metabolism, synthesis is achieved by complex, 
multienzyme pathways whose regulation is often not well understood. 
Because of the large number of enzymes involved, cloning of genes into 
microorganisms is not technically feasible. Up until now, the only routes 
to obtaining the desired product azadrachtin have been chemical synthesis 
and seed extraction. Chemical synthesis is too costly to be an economical 
source of azadirachtin. The disadvantages of seed extraction have already 
been described. 
The present invention seeks to overcome the problems associated with the 
known processes. 
BRIEF SUMMARY OF THE INVENTION 
According to a first aspect of the present invention, there is provided a 
phytohormone-independent cell culture that is capable of producing 
bioactive compounds. 
Preferably, the cell culture comprises cells from a member of the family 
Melieae, preferably Azadirachta indica. 
According to a second aspect of the present invention, there is provided a 
phytohormone-independent cell culture of Azadirachta indica that is 
capable of producing bioactive compounds. 
According to a third aspect of the present invention, there is provided 
azadirachtin free of contaminants from Azadirachta endocarps. 
According to a fourth aspect of the present invention, there is provided a 
method for producing azadirachtin using cultured plant cells. 
According to a fifth aspect of the present invention, there is provided a 
method for producing commercial-scale amounts of Azadirachta using 
suspension cultures from callus. 
According to a sixth aspect of the present invention, there is provided a 
method for producing azadirachtin in aqueous solution, comprising 
inoculating sterile Azadirachta plant tissues with an oncogenic strain of 
Agrobacterium tumefaciens, removing the bacteria from the plant cells, 
generating callus cultures that grow on plant cell culture media in the 
absence of exogenously applied plant hormones, inoculating the callus 
cultures into liquid plant cell culture media (preferably 
phytohormone-free) to generate suspension cultures and harvesting 
azadirachtin secreted by the suspension cells from the culture media, 
optionally without killing the cells. 
Preferably, the callus cultures are inoculated directly into the liquid 
plant cell culture media and not via an intermediate scale-up process. 
Preferably, the Agrobacterium is the strain of Agrobacterium tumefaciens 
A281. 
Preferably, one or more supplements selected from vitamins, carbohydrates, 
ions or phytohormones are added to the media. 
Preferably, the cells are immobilized. Preferably, the cells are in beads; 
preferably beads are of calcium alginate. 
Preferably, elicitors are added to the media at levels to increase 
azadirachtin production. 
Preferably, the Azadirachtin is harvested using adsorbents. 
Preferably, the azadirachtin is separated from other components and cell 
extracts by chromatographic techniques wherein a gradient of increasing 
concentration of acetonitrile in water, from about 20 percent to about 90 
percent, is used. 
The present invention therefore relates to the use of plant cell suspension 
culture of cells that yield bioactive compounds, such as 
azadirachtin-producing cells. The present invention overcomes the 
disadvantages of the known processes for the production of bioactive 
compounds, such as the isolation of azadirachtin by seed extraction. The 
aseptic culture of the present invention allows for the continuous 
production of azadirachtin that is free of pathogens and toxins, 
independent of environmental conditions, yields a predictable quantity and 
quality of bioactive product and permits continuous production at a 
desired, contained location. We recognized that plant cell culture 
presented a possible alternative to seed extraction for production of 
azadirachtin and other bioactive products. Known plant cell culture 
techniques could be used, such as those discussed in the literature, for 
example in Vasil, I, Ed. (1984) Cell Culture and Somatic Cell Genetics of 
Plants, Volume 1, Academic Press, New York; Charlwood, B. V. and M. J. C. 
Rhodes, Eds Secondary Products from Plant Tissue Culture, Oxford 
University Press, New York. One of the main advantages of plant cell 
culture is that it provides a process for predictable, consistent, 
year-round production of bioactive compounds that is independent of 
climate and free of pathogens and toxins. 
The other advantages associated with the present invention are that it 
provides cells that produce useful bioactive compounds, it eliminates the 
known obstacles for the industrial production of useful bioactive products 
of Azadirachta indica, it provides a method for producing azadirachtin 
using cultured plant cells, it produces azadirachtin directly in aqueous 
solution, it provides a method for rapid production of commercial-scale 
Azadirachta suspension cultures from callus and it provides azadirachtin 
free of metabolites, including toxins, produced by pathogens that infest 
Azadirachta seeds. 
DETAILED DESCRIPTION OF THE INVENTION 
In one embodiment, the present invention involves the generation of 
Azadirachta indica cells in cell culture that produce the 
tetranortriterpenoid azadirachtin. It has been found that cells of 
Azadirachta indica, for example, leaf, hypocotyl or cotyledon cells, can 
be infected with oncogenic strains of Agrobacterium tumefaciens to produce 
callus cultures that grow on phytohormone-free media. Suspension-cultured 
cells of Azadirachta indica derived from those callus cultures secrete 
azadirachtin into the culture media. Azadirachtin secretion is preferred 
because it is not necessary to kill the cells to obtain azadirachtin. In 
brief, the process involves the steps of inoculating sterile plant tissues 
with an oncogenic strain of Agrobacterium tumefaciens, removing the 
bacteria from the plant cells, and generating callus cultures that grow on 
plant cell culture media in the absence of exogenously applied plant 
hormones. The callus cultures are inoculated into phytohormone-free liquid 
plant cell culture media to generate suspension cultures. Azadirachtin 
secreted by the suspension cells is harvested from the culture media 
without killing the cells. Cell viability may be maintained in culture 
over long periods of time, and the media from the same cell population may 
be repeatedly harvested to extract insecticidally-active azadirachtin. 
In another embodiment of the present invention, and in order to obtain 
bioactive compounds from plant tissue culture, viable aseptic cell 
cultures are established. In order to achieve commercial levels of 
azadirachtin production, it is desired to produce cell suspension cultures 
that can be grown in bioreactors of appropriate scale to meet commercial 
production requirements, usually 10,000 to 20,000 liters. The first step 
in plant cell culture is generally to establish callus cultures. Various 
plant tissues may be used to establish cell cultures, for example, seeds 
(cotyledons, hypocotyls, embryos), leaves, meristems, stems, or roots. In 
a first step, tissue is surface-sterilized to kill microorganisms on 
tissue surfaces. Hypochlorite, chlorine dioxide or other 
surface-sterilizing agents known in the art may be used. Sterile leaf 
tissue may be obtained by surface-sterilizing seeds, germinating the seeds 
under aseptic conditions, then harvesting sterile tissues from the 
seedlings. Alternatively, leaf tissue from trees grown in a greenhouse or 
in the open environment may also be used. Leaf tissue grown under aseptic 
conditions is preferred because it is not necessary to supplement media 
with antibiotics to suppress the growth of diverse populations of 
microorganisms present on leaves grown in nonsterile environments. 
It is preferred that phytohormone-independent callus cultures are generated 
by transformation of plant tissues with a competent oncogenic strain of 
Agrobacterium tumefaciens. Leaf tissue transformation is preferred, 
however, other tissues, such as seeds, roots, anthers, and hypocotyls may 
also be used. The hypervirulent strain of Agrobacterium tumefaciens A281 
is the strain that is preferred for transformation, however, other 
oncogenic strains of Agrobacterium tumefaciens that are competent to 
infect Melieae may also be used. Sterile leaf sections are cut into two or 
more sections and inoculated by soaking in a dilute suspension of 
Agrobacterium tumefaciens (i.e., for one minute). The Agrobacterium 
tumefaciens is prepared by diluting an aliquot of a suspension culture of 
bacteria into sterile Murashige Skoog media lacking phytohormones. Leaf 
sections are removed from the Agrobacterium tumefaciens suspension, and 
any excess liquid is blotted off and placed on plant tissue culture media 
containing a gelling agent. Inoculation continues by incubating leaf 
sections for two to five days at 24 to 26 degrees centigrade. At the end 
of the inoculation time period, the tissues are transferred to a second 
media which contains one or more antibiotics which are toxic to 
Agrobacterium, but not to plant tissues, to kill viable Agrobacterium 
remaining in the culture. Suitable antibiotics for use in such a medium 
include cefotaxime and carbenicillin. After approximately three to five 
weeks, callus tissues may be observed at one or more sites on the infected 
leaf surfaces. As soon as discrete callus masses are large enough to 
excise with a scalpel, they may be cut away from the leaf surface and 
transferred to fresh hormone-free media. 
The leaf and callus tissues are maintained on phytohormone-free media 
containing sufficient antibiotic to suppress bacterial growth until all 
bacterial cells are killed. Following eradication of the Agrobacterium, 
plant tissues may be maintained on phytohormone-free, antibiotic-free 
media. It is preferred that a number of independent callus lines be 
generated so that selection of lines with different levels of azadirachtin 
or (other bioactive compound) production may be identified. 
In addition to callus tissues, transformed root cultures may be established 
by transformation of plant tissues using Agrobacterium rhizogenes 
(American Type Culture Collection (ATCC), Rockville, Md., strain 15834). 
Substantially the same transformation procedure is used for A. rhizogenes 
transformation as is used for A. tumefaciens transformation. Within two to 
four weeks after infection, hormone-independent roots may be excised from 
the leaf surface and transferred to fresh, hormone-free media. Optionally, 
callus and suspension cultures may also be derived from transformed roots 
by addition of appropriate phytohormones to induce dedifferentiation of 
transformed root tissues. 
Although callus lines resulting from A. tumefaciens infection are preferred 
for azadirachtin production, clonal lines of nontransformed callus tissues 
may also be generated by placing surface-sterilized tissues, for example, 
leaves, seeds or stems, on a tissue culture media that induces callus cell 
growth. A suitable tissue culture media for induction and maintenance of 
callus is Murashige Skoog media supplemented with the phytohormones 
napthaleneacetic acid and benzyladenine each at between 0.1 and 2.0 
milligrams per liter, and solidified with a gelling agent. Other 
formulations of salts and phytohormones may also be used to establish 
callus cultures. Additionally, antibacterial and antifungal agents may be 
added to the medium to maintain sterility. The callus generated may be 
unorganized and undifferentiated, or some degree of cellular organization 
or differentiation may occur. 
The next step of establishing suspension cultures is accomplished by 
inoculating liquid media with callus cells. Suspension media may be 
identical in composition to the callus media except that in suspension 
media, no gelling agent is present. 
The cellular material that is used to inoculate suspension cultures for 
scale-up of production processes is referred to as inoculum. It was 
surprisingly found that the transformed Azadirachta callus can be used 
directly as inoculum to initiate rapidly growing suspension cultures. 
Transformed callus is the preferred inoculum for beginning scale-up 
processes. Alternatively, the inoculum for large-scale production can be 
prepared by initiating a small scale suspension culture from callus or 
suspension cells, maintaining the culture as suspension for at least one 
serial transfer to fresh liquid culture media, and then using this 
suspension culture to inoculate the first of a series of increasingly 
larger culture vessels. 
An advantage of initiating suspension cultures directly from callus is that 
such direct inoculation would substantially reduce the time required to 
achieve desired production cell volumes, thereby eliminating the time 
required to gradually increase cell numbers required for serial transfer 
using suspension cultures. 
To enhance suspension cell growth, other media supplements, such as 
vitamins, carbohydrates, or phytohormones may optionally be added. 
Additionally, cells may be immobilized in beads of calcium alginate or 
other gelling agents, a technique known to those skilled in the art. 
The productivity of suspension cell lines generated from transformed and 
nontransformed calli may be assessed by measuring extracellular 
azadirachtin in the suspension cell media that is secreted by suspension 
cells. Alternatively, azadirachtin may be measured by harvesting callus or 
suspension cells and extracting intracellular azadirachtin. Intracellular 
azadirachtin may be extracted by homogenizing cells in methanol or other 
solvent, such as acetonitrile or ethyl acetate. Methanol is preferred 
because of high extraction efficiency. At intervals of time after the 
callus or suspension culture is initiated, for example, every three days 
for thirty days, an aliquot of one or more cell cultures may be analyzed 
for azadirachtin content. Azadirachtin contents may be compared among 
different cell lines. The peak time of productivity may also be determined 
for each individual line. 
High performance reverse-phase liquid chromatography may be used to analyze 
the concentration of azadirachtin in media or in cell extracts. To 
separate azadirachtin from other components, a gradient of increasing 
concentration of acetonitrile in water, from about 20 percent to about 90 
percent, is preferred to achieve separation of azadirachtin from other 
components in media and in cell extracts. After the sample is applied to 
the chromatography column, the eluate is monitored at about 210 nanometers 
in the ultraviolet absorption spectrum. The elution pattern of an unknown 
mixture is compared to an elution pattern of authentic azadirachtin at a 
known concentration. A standard curve may be developed by chromatographic 
resolution and quantitation of several known concentrations of standard. 
Using the standard curve the concentration of azadirachtin in the unknown 
sample may be determined by linear regression. While reverse phase column 
chromatography is preferred, other methods of chromatographic separation 
may also be suitable for azadirachtin determination. Alternatively, 
azadirachtin may be detected by bioassay using insects susceptible to 
azadirachtin, for example, Mexican Bean Beetles (Epilachna varivestis). 
The mortality rate caused by exposure of the insects to the sample extract 
is compared to the mortality rate caused by authentic azadirachtin 
standard at a known concentration. Analytical methods such as mass 
spectrometry may be used to further confirm the identity of azadirachtin 
in the sample extracts. 
Optionally, the azadirachtin in media or from cell extracts may be 
concentrated prior to analysis using chromatographic media such as resins 
in batch or column. It is preferred that the styrenic resin CG161 
(TosoHaas Inc., Montgomeryville, Pa.) be used for azadirachtin 
concentration, however, other chromatographic media such as C.sub.18 
reverse phase resin may also be suitable. Azadirachtin in aqueous 
suspension binds to CG161, and contaminants are washed away using water, 
buffer or culture media. Azadirachtin is eluted from CG161 using solvent. 
The preferred solvent for azadirachtin elution is methanol, because 
azadirachtin is very soluble in methanol and may be quantitatively eluted 
from CG161 by methanol. The azadirachtin eluted from the resin may be 
directly analyzed by HPLC or by insect bioassay. 
Production of azadirachtin in the suspension culture media may be enhanced 
by supplementing the suspension media with one or more phytohormones, for 
example, 2,4-dichlorophenoxyacetic acid (2,4-D), napthaleneacetic acid 
(NAA) and benzyladenine (BA) each at between one tenth and ten milligrams 
per liter. The effects of hormone addition may be additive. Other hormones 
alone or in combination may also be used. 
Additionally, one or more elicitors may be added to the media to increase 
azadirachtin production compared with cells in media to which no elicitor 
is added. To enhance azadirachtin production, it is preferred that an 
autoclaved preparation of yeast extract at 1 to 40 milligrams per 
milliliter of media be added at least one time to the media. Extracts of 
killed Agrobacterium or killed fungi derived from Azadirachta seed coats 
are also effective elicitors. Additionally, other media amendments, such 
as phosphate starvation, phytohormones; ions, or other compounds may also 
be applied to enhance azadirachtin production.

The present invention will now be described by way of the following 
non-limiting procedures and examples. 
PROCEDURE 1 
Preparation of Standard Murashige Skoog Media and One-Half Strength 
Murashige Skoog Media 
Unless indicated otherwise, Murashige Skoog media was prepared by 
dissolving into one liter of glass distilled water: 1 package of Murashige 
Skoog basal salts (Gibco-BRL, Bethesda, Md.); 30 grams sucrose; 100 
milligrams myo-inositol; 1 milliliter of 1000.times.B5 vitamin stock 
(nicotinic acid, 1 milligram per liter; thiamine-HCL, 10 milligrams per 
liter; pyridoxine-HCL, 1 milligram per liter). The media solution was 
adjusted to between pH 5.7 and 5.8. Nine grams of Difco Bacto Agar (Fisher 
Scientific, Pittsburg, Pa.) was added, and the suspension autoclaved at 
250 degrees Farenheit, 15 PSI for 25 minutes. The media was poured into 
100.times.20 millimeter plastic Petri Dishes (Fisher Scientific, 
Pittsburgh, Pa.) and allowed to solidify. When appropriate, 
filter-sterilized phytohormone solutions, prepared at 1 milligram per 
milliliter were added to molten media before pouring into plates. 
One-half strength Murashige Skoog media was prepared by dissolving one 
package of Murashige Skoog basal salts and 15 grams of sucrose in two 
liters of glass distilled water. The solution was adjusted to between pH 
5.7 and 5.8, and autoclaved using the conditions described above. 
PROCEDURE 2. 
Recovery of Azadirachtin from Cultured Cells and from Cell Culture Media 
Azadirachtin was extracted from one to five grams of callus cells which 
were harvested from petri plates. The fresh weight of the harvested cells 
was recorded. Suspension cells were separated from media by centrifugation 
at 2,500.times.G for 5 minutes. The culture media was poured off, and 
cells were collected with a spatula. Excess liquid was blotted away using 
absorbent towels. The cell fresh weight was recorded. Callus or suspension 
cells were homogenized in methanol using a mortar and pestle. 
Approximately 1 to 5 milligrams of acid-washed sea sand (Fisher 
Scientific, Pittsburgh, Pa.) was added to the mortar to facilitate 
homogenization. Callus or suspension cells were extracted with methanol at 
the proportion of one milliliter of methanol per gram fresh weight of 
cells. The homogenates were poured into two milliliter microcentrifuge 
tubes and centrifuged for 15 minutes at 12,000 rpm (revolutions per 
minute) in a Jouan Model MR14.11 refrigerated microcentrifuge (Jouan 
Winchester, Va.). The supernatants were collected and their volumes were 
recorded. When media was to be analyzed, a three to five milliliter 
aliquot of media was drawn from the supernatant after centrifugation at 
2,500.times.G for 5 minutes. 
Azadirachtin in media or in cell extracts was concentrated by incubating 
two or three milliliters of media or extract with 500 microliters of a 1:1 
(v:v resin:water) slurry of Amberchrom CG161 (TosoHaas, Montgomeryville, 
Pa.) for 30 minutes. The resin mixture was poured into a 12 milliliter 
PrepTorr.TM. column (Fisher Scientific, Pittsburg Pa.) and placed on a 
PrepTorr.TM. vacuum chamber. Excess aqueous media was removed from the 
resin by applying gentle vacuum. Azadirachtin was eluted from the resin by 
adding 500 microliters of methanol adjusted to pH 5.8, applying a vacuum 
to the column and collecting the eluate in a two milliliter 
microcentrifuge tube. 
PROCEDURE 3 
Quantitation of Azadirachtin by High Performance Liquid Chromatography 
(HPLC) 
Azadirachtin in cultured plant tissues was measured by reverse phase high 
performance liquid chromatography. Extracts of callus, suspension cells, 
or cell culture media prepared as described above were applied to a Tosoh 
TSK- ODS120T C.sub.18 reverse phase HPLC column (15 cm.times.4.6. mm; 
Thomson Instrument Company, Wilmington, Del.), equilibrated in 30 percent 
HPLC-grade acetonitrile in filtered, glass-distilled water. The high 
performance liquid chromatograph used was a TosoHaas Model TSK-6010 pump 
with a TosoHaas Model TSK-6041 ultraviolet detector (Thomson Instrument 
Co., Wilmington, Del.). Upon injection of the sample onto the column, a 
gradient of 30% to 60% acetonitrile was commenced and continued for twenty 
minutes at a flow rate of one milliliter per minute. The area under the 
peak eluting at the same retention time as authentic azadirachtin was 
recorded using a Hewlett Packard Model HP3396A integrator attached to the 
TSK-6010 pump. Authentic azadirachtin standards were purchased from Sigma 
Chemical Company (St. Louis, Mo.). Azadirachtin in the unknown samples was 
determined by linear regression, using a standard curve generated by 
running authentic azadirachtin in methanol of at least three 
concentrations under the same gradient conditions as the unknowns. The 
column was washed with 100% acetonitrile for five minutes following each 
sample run and then reequilibrated in 30% acetonitrile before beginning 
another sample run. The azadirachtin peak, under these running conditions, 
eluted at approximately 14.5 minutes after starting the gradient. 
PROCEDURE 4 
Insect Bioassay 
Methanol extracts of callus and suspension cells were diluted to a 
concentration of 1 microgram per 62.5 microliters. To set up leaf disc 
assays, 47 millimeter circular adsorbent cellulose filter pads (Gelman 
Sciences, Ann Arbor, Mich.) were placed one per dish in 50.times.9 
millimeter petri dishes. The filter paper discs were each moistened with 1 
milliliter of sterile glass distilled water. Circular leaf discs (3 
centimeters in diameter) were cut from fresh lima bean (Phaseolus lunatus) 
leaves, avoiding major veins. The leaf discs were placed adaxial surfaces 
down on the filter paper. One leaf disc was placed in each petri dish. 
Control and unknown azadirachtin samples were spread onto the entire area 
of the leaf surfaces, without damaging the leaf surfaces in the process. 
The extracts were allowed to evaporate to dryness. One Mexican Bean Beetle 
(second larval instar) was placed on each treated leaf disc and exposed to 
the sample extracts for three days. At three days, the filter paper and 
leaf disc in each plate was replaced with fresh, moistened filter paper 
and an untreated leaf disc. For each experiment, two control samples were 
used, one being no treatment to the leaf disc, and a second being 50 
percent acetonitrile on the leaf disc. For each sample, ten replicate leaf 
discs were tested. The insects were observed at 1, 3, 6, 7 and 8 days and 
scored for mortality. 
EXAMPLE 1 
Establishing sterile plant cultures 
Sterile trees were obtained by surface-sterilizing viable seeds within 
their endocarps for 30 minutes in a mixture of 20 milliliters of 
commercial bleach (Clorox (Procter and Gamble, Cincinatti, Ohio.) diluted 
to 100 milliliters with water. Then 0.01 milliliters Triton.times.100 
(Sigma Chemical Company, St. Louis, Mo.) was added and the solution 
agitated to mix the components thoroughly. Fifty seeds were surface 
sterilized by agitating rapidly (200 rpm) for thirty minutes. The seeds 
were drained, then rinsed three times with sterile water for ten minutes 
per rinse. The last rinse was drained off of the seeds, and the wet seeds 
were allowed to sit overnight in aseptic conditions. The next day, the 
seeds were resterilized with Alcide LD disinfectant (Alcide Corporation, 
Norwalk, Conn.) for one and one half hours, with agitation at 200 rpm on a 
rotary platform shaker. The seeds were drained and used without further 
rinsing. 
The endocarps (hardshells) were removed, using a scalpel and forceps, in a 
laminar flow hood under aseptic conditions. The seeds were placed in petri 
dishes containing one-half strength of Murashige Skoog media prepared as 
described in Procedure 1. The media was supplemented with 20 parts per 
million Dithane fungicide (Rohm and Haas Company, Philadelphia, Pa.), 50 
parts per million Captan 50WP (ICI Americas, Inc., Wilmington, DE), and 
250 milligrams per liter cefotaxime (Calbiochem, La Jolla, Calif.). The 
seeds were placed in an incubator set at 27 degrees centigrade, on a 16 
hour light/8 hour dark cycle. During the approximately two weeks required 
for germination, uncontaminated seeds were transferred to fresh plates 
when any seeds on a plate became contaminated. At the end of two to four 
weeks, sterile germinated seedlings were transferred to Magenta boxes 
(Sigma, St. Louis, Mo.) containing one-half Murashige Skoog salts without 
fungicides or antibiotics, and returned to the incubator. 
EXAMPLE 2 
Generation of Hormone-Independent Callus Cultures 
Sterile leaves were cut under aseptic conditions from plants described in 
Example 1 and incubated for one minute in a suspension of Agrobacterium 
tumefaciens A281. A. tumefaciens was prepared by inoculating 50 
milliliters of Luria broth (10 grams per liter bacto tryprone, 5 grams per 
liter sodium chloride, 5 grams per liter yeast extract, pH 7.0) and 
incubating overnight in a rotary shaker at 250 rpm, 30 degrees C. A dilute 
suspension (OD.sub.600 =0.05 ) of Agrobacterium was prepared in sterile 
Murashige Skoog salts, pH 5.7. Leaf pieces were cut into sections 
approximately 1.times.2 cm, wounded slightly on their surfaces by pinching 
with forceps, to increase the number of potential infection sites. The 
leaf pieces were incubated for one minute in the Agrobacterium suspension, 
touched on one edge to sterile filter paper to remove excess liquid, and 
placed on hormone-free Murashige Skoog media. After an incubation at 28 
degrees C. in the dark for 48 hours, leaf pieces were transferred to 
hormone-free Murashige Skoog media supplemented with 250 milligrams per 
milliliter cefotaxime, and incubated at 28 degrees C. with a 16 hour 
light/8 hour dark cycle. After approximately two weeks, callus appeared 
around wound sites. When the callus was of sufficient size to remove from 
the leaf surface, individual callus masses were subcultured on fresh 
plates containing the same media. Cefotaxime was maintained in the media 
for at least six subcultures, then omitted from the media. If bacterial 
growth was observed after removal of cefotaxime, callus was transferred to 
fresh plates of hormone-free media supplemented with 250 milligrams per 
liter of cefotaxime and maintained on the antibiotic until no further 
bacterial growth was observed. Callus was transferred to fresh media every 
four to six weeks and maintained under the same culture conditions. 
Azadirachtin was extracted from cells and from culture media using 
Procedure 2. Azadirachtin was analyzed by HPLC using Procedure 3. 
EXAMPLE 3 
Generation of Hormone-Independent Suspension Cell Cultures 
Rapidly growing callus maintained on hormone-free Murashige Skoog media was 
inoculated into 125 milliliter Erlenmeyer flasks containing 30 milliliters 
of standard Murashige Skoog media (Procedure 1) lacking agar. Callus was 
inoculated at one to one and one half grams per 30 milliliters of media. 
Cultures were incubated at room temperature, which fluctuated between 25 
and 30 degrees C., shaken at 110 rpm on a New Brunswick incubator shaker 
(Model G10 gyrotory shaker; New Brunswick, Edison, N.J.). for three to 
four weeks, at which time a thick suspension was obtained (approximately 
two to three grams per 30 milliliters, depending upon the cell line being 
tested). Data are presented in Table 1. 
TABLE 1 
______________________________________ 
Azadirachtin (for suspension cell culture media, 
micrograms per milliliter of media; for suspension cell 
culture cells, micrograms per milliliter of extract) 
Culture time in Days 
Cell Line 
Sample 10 15 17 21 24 27 
______________________________________ 
A281/118 
cells nd -- 0.03 -- nd -- 
A281/118 
media nd -- 0.06 -- 0.10 -- 
A281/125 
cells 0.14 -- nd -- nd -- 
A281/125 
media 0.31 -- 0.06 -- 0.10 -- 
A281/130 
cells 0.19 -- 0.60 -- 0.32 -- 
A281/130 
media 0.32 -- 0.11 -- 0.16 -- 
A281/135 
cells 0.12 -- 0.08 -- 0.34 -- 
A281/135 
media 0.09 -- 0.l2 -- 0.04 -- 
A281/167 
cells -- nd -- 0.65 -- 0.23 
A281/167 
media -- 0.04 -- 0.63 -- 0.49 
______________________________________ 
nd -- not detected; 
-- not analyzed 
EXAMPLE 4 
Generation of Hormone-Dependent Callus 
Sterile leaves, cotyledons and hypocotyls of Example 1 were placed on 
Murashige Skoog media supplemented with 1 milligram per liter 
napthaleneacetic acid and 1 milligram per liter benzyladenine. Tissues 
were incubated for three to six weeks at 28 degrees C., 16 hours light/8 
hours dark. When individual callus masses could be excised, they were 
subcultured onto fresh media of the same composition and maintained under 
the same culture conditions. Calli were subcultured every four to six 
weeks. 
EXAMPLE 5 
Generation of Transformed Root Cultures 
Sterile leaves of Example 1 were infected with Agrobacterium rhizogenes 
(-ATCC 15834). A. rhizogenes was prepared by inoculating 50 milliliters of 
Luria broth (10 grams per liter bacto tryptone, 5 grams per liter sodium 
chloride, 5 grams per liter yeast extract, pH 7.0) and incubating 
overnight in a rotary shaker at 250 rpm, 30 degrees C. A dilute suspension 
(OD.sub.600 =0.05) of Agrobacterium was prepared in sterile Murashige 
Skoog salts, pH 5.7. Leaf pieces were cut into sections approximately 
1.times.2 cm, wounded slightly on their surfaces by pinching with forceps, 
to increase the number of potential infection sites. The leaf pieces were 
incubated for one minute in the Agrobacterium suspension, touched on one 
edge to sterile filter paper to remove excess liquid, and placed on 
hormone-free Murashige Skoog media. After an incubation at 28 degrees C. 
in the dark for 48 hours, leaf pieces were transferred to standard 
Murashige Skoog media supplemented with 250 milligrams per milliliter 
cefotaxime, and incubated at 28 degrees C. with a 16 hour light/8 hour 
dark cycle. After approximately two weeks, roots appeared at original 
wound sites. When the roots were of sufficient size to remove from the 
leaf surface, individual roots were excised, then subcultured on fresh 
plates containing the same media. Cefotaxime was maintained in the media 
for at least six subcultures, then omitted from the media. If bacterial 
growth was observed after removal of cefotaxime, roots were transferred to 
fresh plates of hormone-free media supplemented with 250 milligrams per 
liter of cefotaxime and maintained on the antibiotic until no further 
bacterial growth was observed. Roots were transferred to fresh media every 
four to six weeks and maintained under the same culture conditions. 
Representative concentrations of azadirachtin extracted from root cultures 
are presented in Table 2. 
TABLE 2 
______________________________________ 
Root Azadirachtin 
Line Micrograms per gram fresh weight 
______________________________________ 
RKA-1 0.60 
RKA-2 1.68 
RKA-3 0.29 
R201 0.42 
______________________________________ 
EXAMPLE 6 
Enhancing Azadirachtin Production Using Biotic Elicitors 
Suspension cells of Example 3 were grown 10 days in Murashige Skoog media. 
At 10 days, an elicitor was added to the culture media at the final 
concentration indicated in Table 3. Agrobacterium elicitor was prepared by 
growing Agrobacterium tumefaciens A281 in (media) overnight at 28 degrees 
C. at 250 rpm on a rotary shaker. The stationary phase culture was 
autoclaved for 15 minutes at 120 degrees C., 43.9.times.10.sup.-4 
kgm.sup.2 (15 psi) to kill bacterial cells. The autoclaved extract was 
centrifuged at 10,000 rpm for 10 minutes to remove cell debris, and the 
supernatant was used for elicitor studies. An unidentified fungus isolated 
from the coats of azadirachtin seeds was cultured for 5 days in potato 
dextrose broth, then autoclaved and centrifuged using the same conditions 
as used for preparation of the Agrobacterium elicitor. At the time of 
elicitor addition, CaCl.sub.2 (2.5 mm final concentration) and glycerol 
(1% (v/v) final concentration) were also added to the culture media. The 
samples were incubated for 24 hours under standard conditions of 25 to 27 
degrees C., shaken at 110 rpm on a rotary platform shaker. A 5 milliliter 
sample of suspension media was collected at 24 hour intervals for 96 
hours. After each sampling, the cultures were returned to standard 
conditions. The media was sampled by HPLC using Procedure 3. Results are 
presented in Table 3. 
TABLE 3 
______________________________________ 
(Azadirachtin in media) 
(micrograms per milliliter) 
Elicitor 
Hours after addition 
Concen- 
of elicitor to media 
Cell Line 
Elicitor tration 0 24 48 72 144 
______________________________________ 
A281/135 
yeast 10 mg/ml 
nd nd 0.78 0.06 nd 
A281/135 
Agrobacterium 
.16 ml/ml 
nd nd nd nd 0.07 
A281/135 
seed fungus 
.16 ml/ml 
nd nd nd 1.10 nd 
A281/146 
yeast 10 mg/ml 
nd nd 0.41 0.80 0.49 
A281/146 
Agrobacterium 
.16 ml/ml 
nd 0.14 0.10 0.85 nd 
A281/146 
seed fungus 
.16 ml/ml 
nd nd 0.10 nd nd 
A281/167 
yeast 10 mg/ml 
nd 1.37 0.71 0.18 0.17 
A281/167 
Agrobacterium 
.16 ml/ml 
nd 0.12 0.45 0.92 0.09 
A281/167 
seed fungus 
.16 ml/ml 
nd 0.28 0.28 0.43 0.25 
______________________________________ 
nd -- not detected 
EXAMPLE 7 
Repeated Elicitation of Suspension Cells to Enhance Azadirachtin Production 
A 2.sup.5-1 fractional factorial experiment was designed to compare the 
effects on two different cell lines of repeated addition of 5 or 10 
milligrams per milliliter of yeast elicitor, presence of phosphate in the 
medium, and presence of 2.4-D (1-milligram per liter) in the media. For 
this experiment, media were made according to the following recipe and 
adjusted to a final pH of 5.7 to 5.8: 
TABLE 4 
______________________________________ 
Murashige Skoog Media with and without phosphate salt 
Constituent milligrams per liter 
______________________________________ 
ammonium nitrate 1650 
potassium nitrate 1900 
magnesium sulfate heptahydrate 
370 
potassium phosphate, monobasic 
170 
ferric EDTA 43 
sucrose 30,000 
calcium chloride dihydrate 
440 
potassium iodide 0.83 
boric acid 620 
manganese sulfate tetrahydrate 
2230 
zinc sulfate heptahydrate 
860 
sodium molybdate dihydrate 
25 
copper sulfate pentahydrate 
2.5 
cobalt chloride hexahydrate 
2.5 
nicotinic acid 1.0 
thiamine HCl 10.0 
pyridoxine HCl 1.0 
myo-inositol 100 
______________________________________ 
For this example, two versions of the media were prepared; one as described 
and the other as a phosphate-free media (omitting the 170 milligrams of 
monobasic potassium phosphate). Suspension cultures from two independent 
cell lines (A281/124 and A281/135) were initiated by inoculating 
transformed callus from Example 2 (30 g of A281/135 and 40 grams of 
A281/124) into 300 milliliters of Murashige Skoog media with phosphate as 
described in Table 4. Cells were inoculated into one liter erlenmeyer 
baffle flasks (Bellco Glass, Vineland, N.J.) and incubated under standard 
conditions of 28 degrees C. for 14 days on a rotary shaker platform set at 
110 rpm. The following processes were carried out separately for each cell 
line. The suspension culture was sieved through a two millimeter stainless 
steel mesh (Fisher Scientific, Pittsburgh, Pa.). Cells and aggregates of 
less than 2 millimeters were collected and combined into a single flask. 
The cells were centrifuged at 2,500.times.G for 5 minutes, the media was 
poured off. Twenty grams of A281/135 and 50 grams of A281/124 cells were 
placed into a clean sterile flask containing 240 milliliters of fresh 
Murashige Skoog media (Table 4, with phosphate). Differences in amounts of 
cell aggregation account for the differences in fresh cell weights. Line 
A281/124 did not aggregate into clumps of more than 2 mm, whereas the 
proportion of clumps exceeding 2 mm in A281/135 made up a larger 
percentage of the culture. The samples were aged for 4 days. In a next 
step, a sequential halving procedure was used to prepare eight flasks of 
the same cell density. The flask containing 240 milliliters of sieved 
cells was swirled and the contents split equally between two new sterile 
flasks. The cells in these two flasks were each swirled and each split 
into two equal portions to generate four flasks with equal cell densities 
and media volumes. This sequential halving process was continued until 
eight flasks containing 30 milliliters of media in 125 milliliter flasks 
were generated for each of the two cell lines. 
The contents of individual flasks were poured into a 50 milliliter sterile 
polypropylene centrifuge tube, capped and centrifuged for 5 minutes at 
2,500.times.G to pellet the cells. The media was poured off, and the fresh 
weight of each sample was measured using aseptic conditions. The cells 
were returned to clean 125 ml flasks and the media was replaced with 30 
milliliters of Murashige Skoog media either with or without phosphate, 
according to Table 5. The suspension cultures were incubated for three 
days under standard conditions to deplete appropriate cultures of 
phosphate. On the fourth day, designated 0 (zero) hours of the experiment, 
the cultures were poured into sterile 50 milliliter centrifuge tubes, 
centrifuged for five minutes at 2,500.times.G, and the fresh weight of 
each sample was recorded. A 3 milliliter sample of the media was saved and 
prepared according to Procedure 2 for HPLC analysis using Procedure 3. The 
cells were returned to the flasks, and the media was replaced with the 
appropriate amendments of elicitor, phosphate and hormone as listed in 
Table 5. 
The suspensions were incubated for twenty four hours under standard 
conditions. At 24 hours, the cells were harvested by centrifugation at 
2,500.times.G for 5 minutes, and a three milliliter sample of the media 
was collected for HPLC analysis. The remainder of the media was discarded. 
The cells were returned to the flasks, and the media was replaced with 
media containing the appropriate amendments as indicated in Table 5. The 
procedure was repeated at 48, 72 and 96 hours. Samples which were not 
repeatedly elicited did not receive any elicitor after the original 
addition at zero hours. For samples that were repeatedly elicited, 
elicitor at the appropriate level was added a zero, 24, 48, and 72 hours. 
All samples were stored at minus 20 degrees C. until the end of the 
experiment and all were processed according to Procedure 3 at the same 
time in an order designated by the experimental design to reduce sampling 
bias. The results are presented in Table 5. 
TABLE 5 
__________________________________________________________________________ 
Azadirachtin in media 
(mg/ml) 
Sample 
Cell Repeat 
Phosphate 
Mg/ml 
2,4-D 
Hours after addition of elicitor 
Number 
Line Elicitation 
Present 
Elicitor 
present 
24 48 72 96 
__________________________________________________________________________ 
1 A281/135 
No No 5 Yes 0.09 
8.89 
nd 0.08 
2 A281/124 
No No 5 No 0.08 
10.32 
.19 
nd 
3 A281/135 
Yes No 5 No 0.09 
0.11 
0.12 
0.15 
4 A281/124 
Yes No 5 Yes 0.12 
0.12 
0.13 
0.13 
5 A281/135 
No Yes 5 No nd 10.65 
0.12 
0.13 
6 A281/124 
No Yes 5 Yes 0.13 
0.10 
nd nd 
7 A281/135 
Yes Yes 5 Yes 0.09 
0.08 
0.14 
0.14 
8 A281/124 
Yes Yes 5 No nd 0.20 
0.18 
nd 
9 A281/135 
No No 10 No 0.01 
0.16 
nd nd 
10 A281/124 
No No 10 Yes nd 8.16 
nd nd 
11 A281/135 
Yes No 10 Yes nd 1.04 
0.13 
nd 
12 A281/124 
Yes No 10 No 0.06 
1.72 
0.23 
0.43 
13 A281/135 
No Yes 10 Yes nd 2.23 
0.12 
0.13 
14 A281/124 
No Yes 10 No nd 0.34 
nd 0.34 
15 A281/135 
Yes Yes 10 No nd 9.91 
0.17 
nd 
16 A281/124 
Yes Yes 10 Yes nd nd 0.13 
nd 
__________________________________________________________________________ 
nd -- not detected 
EXAMPLE 8 
Repeated Elicitation of Suspension Cell Cultures to Enhance Azadirachtin 
Production 
Thirty grams of callus cells of cell line A281/135 were inoculated in 400 
milliliters of standard Murashige Skoog media in a one liter baffle flask, 
and incubated under standard conditions for three weeks. At three weeks, 
the cell suspension was filtered through a sterile two millimeter mesh 
into a 1 liter baffle flask containing 240 milliliters of Murashige Skoog 
media. The suspension was aged for 24 hours, then the culture was 
centrifuged at 2,500.times.G for 5 minutes. Eight grams of cells were 
placed in 120 milliliters of minimal Murashige Skoog media (Table 4) media 
(media lacking phosphate). A sequential splitting procedure was used to 
divide these cells into four equal 30 milliliter aliquots in 125 
milliliter flasks. Similarly, eight grams of cells were placed into 120 
milliliters of complete Murashige Skoog media (with phosphate, Table 4). A 
sequential splitting procedure was used to divide these cells into four 
equal 30 milliliter aliquots in 125 milliliter flasks. The samples were 
aged for 5 days under standard conditions to deplete cells in media 
lacking phosphate of intracellular phosphate. On the fifth day, designated 
hour 0 (zero). of the experiment, 2,4-D at 1 milligram per liter, or yeast 
elicitor at either 5 or 10 milligrams per milliliter were added according 
to Table 6 and returned to the standard incubation conditions. 
At 24 hours, 3 milliliter media samples were removed from each flask. The 
samples that did not receive repeated elicitation were not further 
treated. Samples 1 and 6, which received repeated elicitation, were 
treated as follows: the suspension were poured into centrifuge tubes, 
centrifuged at 2,500.times.G for 5 minutes, and the media was discarded. 
New media containing elicitor at the same level was added to the cells, 
which were returned to the 125 milliliter flask, then incubated under 
standard conditions. Media samples (3 milliliters) were removed from each 
sample at 48 and 72 hours with no further media changes to any samples. 
Media samples were prepared for HPLC as in Procedure 3, after all samples 
had been collected. Prior to sample preparation, all samples were stored 
at minus 20.degree. C. Azadirachtin production in the samples are 
presented in Table 6. 
TABLE 6 
______________________________________ 
Azadirachtin in media 
(micrograms/milliliter) 
Phos- Hours after 
Sample 
Elicitation 
phate Elicitor 
2,4-d elicitor added 
No. repeated present Level Present 
24 48 72 
______________________________________ 
1 yes no 5 no 0.17 11.11 10.91 
2 no no 10 no 0.24 13.33 14.70 
3 no no 5 no 0.11 0.15 0.14 
4 no no 10 yes 3.75 14.70 17.81 
5 no yes 5 no 0.14 9.78 12.34 
6 yes yes 5 yes 0.44 6.57 6.42 
7 no yes 10 yes 0.99 5.15 15.54 
8 no yes 10 no 021 12.93 16.27 
______________________________________ 
EXAMPLE 9 
Insect Bioassay of Media Samples from Elicitor-Treated Suspension Cell 
Cultures 
Media samples collected from selected samples of Example 8 were bioassayed 
to test for biological activity against Mexican Bean Beetles according to 
Procedure 4. Extracts of culture media were applied to lima bean leaves. 
By eight days, all insects fed extracts of cell suspension media were 
either dead or moribund, while 11 out of 12 insects on control leaf discs 
were alive at 8 days. Results of the assay are presented in Table 7. 
TABLE 7 
__________________________________________________________________________ 
Sample Elicitation 
Phosphate 
Elicitor 
2,4-D 
Insect Condition at 8 Days 
Number. repeated 
present 
level 
present 
Live 
Dead 
Moribund 
__________________________________________________________________________ 
no treatment control 
no no none 
no 6 0 0 
acetonitrile control 
no no none 
no 5 1 0 
1 yes no 5 no 0 7 3 
2 no no 10 yes 0 8 2 
3 no no 5 no 0 8 2 
4 no no 10 yes 0 8 2 
5 no yes 10 no 0 5 5 
6 no yes 10 yes 0 6 4 
__________________________________________________________________________ 
EXAMPLE 10 
Mass Spectroscopic Detection of Azadirachtin in Cultured Azadirachta Cells 
Extracts obtained from three independent suspension cultures generated 
according to Example 3 were analyzed by mass spectroscopy for the presence 
of authentic azadirachtin. The samples analyzed were single peak fractions 
obtained by chromatographic separation of whole cell extracts using the 
high performance liquid chromatography method described in Procedure 3. 
For each sample, the peak eluting at a retention time of approximately 
14.5 minutes, and at an acetonitrile: water mixture of approximately 45:55 
was collected and evaporated to dryness in an air stream of room 
temperature air. The dry extract was redissolved in 10 microliters of 
methanol, and one microliter of this solution was placed on the mass 
spectrometer desorption chemical probe filament. 
The analysis was performed on a Jeol HX-110 (Tokyo, Japan) mass 
spectrometer using the following conditions. The source was operated in 
the desorption chemical ionization (DCI) mode at approximately 40 degrees 
centigrade with isobutane as reagent gas at a source housing pressure of 
2.2.times.10.sup.-6 torr. The filament was heated at 1 ampere/minute, and 
the ionizing electron current was set at 50 microamperes at an energy of 
200 electron volts. At a mass resolving power of 1000 the magnetic field 
was scanned from a mass to charge ratio (m/z) 650 to 750 with a one second 
cycle time. The one microliter of a 300 nanogram per microliter 
azadirachtin standard was used to check the performance of the instrument. 
The diagnostic peaks of the authentic standard were the protonated 
molecular ion at m/z 721 and the peak at m/z 703 due to the loss of water. 
The relative abundance of the peak at m/z 703 was generally two to three 
times that of the peak at m/z 721. Analysis of cell lines A281/130, 
A281/131, and A281/135, each having a peak eluting at approximately 14.5 
minutes and 45 percent acetonitle using HPLC, were all found to contain 
the peaks diagnostic for azadirachtin. Note: The use of freeze-drying 
should be avoided in sample preparation; it was observed to result in loss 
of azadirachtin from commercial standard and samples!. 
EXAMPLE 11 
Use of Resinous,Adsorbents to Enhance Azadirachtin Production 
The removal of accumulated product with resinous adsorbents was used to 
enhance the production of azadirachtin by suspension cell cultures. 
A281/124 and A281/135 suspension cultures were incubated with sterilized 
non-functionalized, macroporous acrylate based polymeric absorbent (sold 
under the trademark Ambersorb.RTM. XAD-7 absorbent, hereinafter ("XAD-7 
absorbent") or non-functionalized, macroporous styrenic-based polymeric 
absorbent (sold under the trademark Ambersorb.RTM. XAD-16 absorbent, 
hereinafter ("XAD-16 absorbent") added directly to the suspension media. 
The resins were washed extensively with distilled water prior to use. 
Interstitial water was removed from the resin immediately before weighing 
by plating resin on several layers of clean filter paper. 1.48 g to 1.52 g 
of the appropriate resin was weighed directly into a tared 250 ml 
erlenmeyer flask. Five milliliters of basal media was added to each flask 
prior to autoclaving at 250 degrees Farenheit, 15 PSI (pounds per square 
inch) for 20 minutes. Control flasks contained 5 ml of basal MSA media. 
These flasks were capped and sterilized by autoclaving. 
Suspension cultures were initiated by inoculating one gram of callus of 
cell lines A281/124 or A281/135 into 30 ml of basal MS media in 125 ml 
erlenmeyer flasks. The cultures were incubated for 14 days at 27.degree. 
C., 100 RPM in a Queue controlled-temperature incubator-shaker at which 
time the suspension cells and media were poured into preweighed sterile 50 
ml certrifuge tubes and centrifuged at 2,500.times.G for 5 minutes to 
gently pellet suspension cells to separate them from the conditioned 
media. The conditioned media was poured out of the tubes and back into the 
original culture flask. Cell fresh weight for each sample was determined 
by weighing the cells in the preweighed 50 ml centrifuge tubes and 
subtracting the weights of the tubes. The cells and conditioned media from 
which they were originally separated were recombined and added to flasks 
containing 5 ml of basal media containing XAD-7 absorbent or XAD-16 
absorbent, or no resin, and returned to the incubator-shaker to incubate 
at 27.degree. C., 100 RPM for 4 to 10 days. On each harvest day, 
triplicate samples of the cells and resin, or cells without resin 
(controls) were transferred to preweighed 50 ml centrifuge tubes, the 
cells and resin were pelleted. Ten to 15 ml of media was collected for 
analysis and the rest of the media was discarded. Cells and resin were 
weighed to determine fresh weight and then were frozen at -20.degree. C. 
until analysis. Analysis was begun by thawing cells plus resin, placing 
them in plastic 250 ml bottles, and adding methanol at a ratio of 
approximately 1:1 cell weight:methanol volume. The bottles were tightly 
capped and placed on a gyratory shaker for 7 days to extract the 
azadirachtin. The cells and resin beads were homogenized together using a 
mortar and pestle. The homogenates were poured into two milliliter 
microcentrifuge tubes and centrifuged for 15 minutes at 12,000 rpm 
(revolutions per minute) in a Jouan Model MR14.11 refrigerated 
microcentrifuge (Jouan Winchester, Va.). The supernatants were collected 
and their volumes recorded. When media was to be analyzed, a three to five 
milliliter aliquot of media was drawn from the supernatant after 
centrifugation at 2,500.times.G for 5 minutes. The samples were either 
measured directly without concentration by HPLC analysis according to 
Procedure 3, or azadirachtin in media or cell extracts. Azadirachtin in 
media or in cell extracts was concentrated by incubating two or three 
milliliters of media or extract with 500 microliter of 1:1 (v:v 
resin;water) slurry of Amberchrom CG161 (TosoHaas, Montgomeryville, Pa.) 
for 30 minutes. The resin mixture was poured into a 12 milliliter Prep 
Torr.TM. column (Fisher Scientific, Pittsburgh, Pa.) and placed on a Prep 
Torr.TM. vacuum chamber. Excess aqueous media was removed from the resin 
by applying gentle vacuum. Azadirachtin was eluted from the resin by 
adding 500 microliters of methanol adjusted to pH5.8, applying a vacuum to 
the column and collecting the eluate in a two milliliter microcentrifuge 
tube. 
The cell and resin samples were homogenized using a mortar and pestle and 
extracted according to the method of Procedure 2 before HPLC analysis 
according to the method of Procedure 3. Several media samples from 
cultures incubated in the presence of resin were selected at random and 
analyzed by HPLC according to the method or Procedure 3. No azadirachtin 
was detected in any media samples. Azadirachtin production was enhanced in 
the presence of XAD-7 absorbent and XAD-16 absorbent as shown in Table 8. 
TABLE 8 
______________________________________ 
Experiment to test effect of addition of XAD-7 and XAD-16 
adsorbents on azadirachtin production in cultured Azadirachta 
suspension cells. Sterile resin was added directly to cultures 
and the analyses reflect the sum of azadirachtin in the cells 
and adsorbed onto the resin. 
Azadirachtin 
Day of .mu./g fresh wt 
Line # Harvest Resin (cels and resin) 
______________________________________ 
A281/135 0 none n.d..sup.(1) 
A281/135 4 none 0.50 (0.50).sup.(2) 
A281/135 4 XAD-7 17.07 (4.63) 
A281/135 4 XAD-16 5.48 (1.31) 
A281/135 10 none n.d. 
A281/135 10 XAD-7 1.48 (.265) 
A281/135 10 XAD-16 22.5 (4.63) 
A281/124 0 none 2.42 (.35) 
A281/124 4 none 0.57 (0.81) 
A281/124 4 XAD-7 57.83 (1.90) 
A281/124 4 XAD-16 27.55 (8.9) 
A281/124 10 none 0.62 (.88) 
A281/124 10 XAD-7 40.58 (4.22) 
A281/124 10 XAD-16 32.19 (16.0) 
______________________________________ 
.sup.(1) n.d. not detected 
.sup.(2) expressed as mean (standard deviation)