AU2003244612A1 - Use of plant tissue - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT

ORIGINAL

TO BE COMPLETED BY APPLICANT Name of Applicant: Actual Inventors: Address for Service: Invention Title: RESEARCH DEVELOPMENT FOUNDATION ARNTZEN, Charles J. of 1005 Highland Road, Ithaca, NY 14850- 1447, United States of America; BLAKE, Mary of 6651 N. Campbell Avenue #237, Tucson, AZ 85718-1364, United States of America; GUTTERMAN, Jordan, U. of 1701 Hermann #30 G, Houston, TX 77004, United States of America; HOFFMAN, Joseph, J. of 2718 East Hendrick, Tucson, AZ 85716, United States of America; BAILEY, David T. of 328 Overlook Lane, Boulder, CO 80302, United States of America; JAY-ATILAKE, Gamini, S. of 12347 Wolff Drive, Broomfield, CO 80020, United States of America.

CALLINAN LAWRIE, 711 High Street, Kew, Victoria 3101, Australia USE OF PLANT TISSUE The following statement is a full description of this invention, including the best method of performing it known to us:- 09109/03,swl3567spa,1 -2-

DESCRIPTION

USE OF PLANT TISSUE BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to the field of providing raw materials for medicine. More specifically, the invention relates to methods of obtaining plant tissue, the products of which have therapeutic uses in mammals.

Description of Related Art Plants are valuable sources of biologically active molecules. One diverse class of molecules which has been found in plants is the class of saponins. Saponins are high molecular weight compounds comprising glycosides with a sugar moiety linked to a triterpene or steroid aglycone. Triterpene saponins particularly have been the subject of much interest because of their biological properties.

Pharmacological and biological properties of triterpene saponins from different plant species have been studied, including fungicidal, anti-viral, anti-mutagenic, spermicidal or contraceptive, cardiovascular, and anti-inflammatory activities (Hostettmann et al., 1995). Saponins are known to form complexes with cholesterol by binding plasma lipids, thereby altering cholesterol metabolism (Oakenfull et al.. 1983).

Triterpene glycosides given in feed also have been shown to decrease the amount of cholesterol in the blood and tissues of experimental animals (Cheeke, 1971). Saponins have been found to be constituents of many folk medicine remedies and some of the more recently developed plant drugs.

The triterpene glycyrrhetinic acid, and certain derivatives thereof, are known to have anti-ulcer, anti-inflammatory, anti-allergic, anti-hepatitis and antiviral actions. For instance, certain glycyrrhetinic acid derivatives can prevent or heal gastric ulcers (Doll et al., 1962). Among such compounds known in the art are carbenoxolone S. Patent No.

3,070,623), glycyrrhetinic acid ester derivatives having substituents at the 3' position S.

Patent No. 3,070,624), amino acid salts of glycyrrhetinic acid (Japanese Patent Publication JP-A-44-32798), amide derivatives of glycyrrhetinic acid (Belgian Patent No. 753773), and amide derivatives of 11-deoxoglycyrrhetinic acid (British Patent No. 1346871).

Glycyrrhetinic acid has been shown to inhibit enzymes involved in leukotriene 09/09/03sw I 3567spa.2 -3biosynthesis, including 5-lipoxygenase activity, and this is thought to be responsible for the reported anti-inflammatory activity (Inoue et al., 1986).

Betulinic acid, a pentacyclic triterpene, is reported to be a selective inhibitor of human melanoma tumor growth in nude mouse xenograft models and was shown to cause cytotoxicity by inducing apoptosis (Pisha et al., 1995). A triterpene saponin from a Chinese medicinal plant in the Cucurbitaceae family has demonstrated anti-tumor activity (Kong et al., 1993). Monoglycosides of triterpenes have been shown to exhibit potent and selective cytotoxicity against MOLT-4 human leukemia cells (Kasiwada et al., 1992) and certain triterpene glycosides of the Iridaceae family inhibited the growth of tumors and increased the life span of mice implanted with Ehrlich ascites carcinoma (Nagamoto et al., 1988). A saponin preparation from the plant Dolichos falcatus, which belongs to the Leguminosae family, has been reported to be effective against sarcoma-37 cells in vitro and in vivo (Huang et al., 1982). Soya saponin, also from the Leguminosae family, has been shown to be effective against a number of tumors (Tomas-Barbaren et al., 1988).

Oleanolic acid and gypsogenin glycosides exhibiting haemolytic and molluscicidal activity have been isolated from the ground fruit pods of Swartzia madagascariensis (Leguminosae) (Borel and Hostettmann, 1987).

Genistein, a naturally occurring isoflavonoid isolated from soy products, is a tyrosine kinase inhibitor that has been shown to inhibit the proliferation of estrogenpositive and estrogen-negative breast cancer cell lines (Akiyama et al., 1987). Inositol hexaphosphate (phytic acid), which is abundant in the plant kingdom and is a natural dietary ingredient of cereals and legumes, has been shown to cause terminal differentiation of a colon carcinoma cell line. Phytic acid also exhibits anti-tumor activity against experimental colon and mammary carcinogenesis in vivo (Yang et al., 1995). Some triterpene aglycones also have been demonstrated to have cytotoxic or cytostatic properties, i. stem bark from the plant Crossopteryxfebrifuga (Rubiaceae) was shown to be cytostatic against Co- 115 human colon carcinoma cell line in the ng/ml range (Tomas- Barbaren et al., 1988).

SUMMARY OF THE INVENTION The present application is a divisional application of Australian Patent Application No. 40871/99, the ("parent") specification of which is herein incorporated by reference.

The present invention relates to a hairy root tissue culture comprising cells of an Acacia victoriae plant which have been infected with Agrobacterium rhizogens R-1000 in 09/D09/03.sw13567spa,3 a tissue culture medium. Preferably the tissue culture medium contains 3-4% sucrose by weight.

The present invention also relates to a method of continually harvesting an Acacia victoriae plant tissue comprising: a) cultivating an Acacia victoriae plant in a hydroponic growth system; and b) harvesting said tissue from said plant about 1 to about 4 times per year, wherein said harvesting does not kill said plant.

Preferably the growth system in an aeroponic system. Further preferably the tissue is root tissue.

The present invention provides tissue for the isolation of novel biologically useful compounds. Acacia victoriae seeds have been used as a source of food material by the indigenous people of Australia for generations (Lister et al., 1996). However, the pods and roots were discarded as waste material. Therefore, the inventors of the parent invention have demonstrated the presence of novel anti-cancer and other biologically useful compounds from the parts of the plant that were not used before. For example, the novel biologically active saponin compounds disclosed herein are often specifically cytotoxic to malignant cells.

An important aspect of the invention is that it provides tissue for the production of a nutraceutical composition comprising a triterpene glycoside composition in a pharmacologically acceptable medium such as a buffer, a solvent, a diluent, an inert carrier, an oil, a creme, or an edible material. The nutraceutical composition may comprise dried and ground Acacia victoriae root, pod or combination thereof produced in accordance with the parent invention in a pharmacologically acceptable medium. The nutraceutical compositions disclosed herein may typically be in the form of a tablet, a capsule, or an ointment.

DETAILED DESCRIPTION OF THE INVENTION Acacia victoriae was selected based on factors including native environment and limited prior study of the species. Acacia victoriae originates from Australia, but has been introduced as a horticultural variety throughout the world and is commonly known as prickly wattle or elegant wattle. The tree grows at a rate of 60 to 120 cm per year, is tardily drought deciduous and is hardy to at least-15 0 C. Mature plants grow to 10-15 feet and have bluish-green bipinnate leaves. In the southwest United States, the plant typically flowers from April to May, with pods ripening in June. Acacia victoriae has a number of 09/09/03,sw 13567spa,4 agricultural uses, including wind breaks, shelter belts, food, critical area stabilization, and as a low water-use ornamental. Different Acacia species seeds have been used as a source of food material by the indigenous people of Australia for generations (Lister et al., 1996).

Among the Acacia's, Acacia victoriae is the most common and widespread species, present all over Australia, are therefore, the most widely consumed species. Acacia seeds, commonly called wattleseed, are in high demand for use as a ground product in pastries and breads and also as a flavoring in desserts, especially ice-cream. They are also used to produce a high quality coffee-like beverage and among the Acacia species, Acacia victoriae (Benth.) is generally regarded as having a superior flavor (Lister et al., 1996).

However, there is no record of the use of pods and roots of this plant.

The parent invention relates to the novel use of Acacia victoriae pods and roots for the isolation of biologically useful compounds. The inventors of the parent invention have demonstrated the presence of novel anti-cancer and other biologically useful compounds from parts of the plant that were not used before.

There is no general requirement that the triterpene compositions of the parent invention always be isolated and provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. For example, the inventors envision the use of dried Acacia victoriae root and pod and extracts thereof as nutraceuticals. Nutraceuticals by definition contain a mixture of different bioactive compounds that synergistically have beneficial effects on health. The nutraceuticals of the present invention may be in the form of tablets or capsules and can be taken orally or alternately may contain extracts of the plant in an ointment which can be applied topically. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater"fold" purification than the same technique utilizing a low pressure chromatography system.

Methods exhibiting a lower degree of relative purification may have advantages in total recovery of product, or in maintaining the biological activity of the triterpene compounds.

Isolation and purification methods are fully described in the parent specification.

Growth and Tissue Cultures of Acacia victoriae An important aspect in the preparation of the compounds of the parent invention is the availability of tissue of Acacia victoriae. As the inventors have shown that the 09/09/03,swI3567spa.5 -6compounds of the invention are concentrated in roots and pods of Acacia victoriae, the availability of these tissues is particularly important. The inventors have also shown that young seedlings, are another source for isolating the compounds of this invention. Acacia victoriae grows in the southwest United States and in Australia, and therefore, plant tissue is available to the public. Additionally, a deposit of 2500 seeds of Acacia victoriae has been made by the inventors with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209 on (May 7,1998). Those deposited seeds have been assigned ATCC Accession No. 209835. The deposit was made in accordance with the terms and provisions of the Budapest Treaty relating to deposit of microorganisms and is made for a term of at least thirty (30) years and at least five (05) years after the most recent request for the furnishing of a sample of the deposit was received by the depository, or for the effective term of the patent, whichever is longer, and will be replaced if it becomes non-viable during that period.

Therefore, in light of the instant disclosure, one of skill in the art could plant those deposited seeds, grow plants therefrom, and isolate tissue from the plants for the preparation of the triterpene compounds and nutraceuticals of the parent invention. Also, one could isolate tissue from naturally occurring Acacia victoriae populations. However, the preparation of tissue for isolation of the compounds of the invention would be more readily achieved if a suitable cultivation technique were designed for the propagation of Acacia victoriae tissue. One option for the preparation of tissue would be the large-scale cultivation of the species. More preferable options, however, include tissue cultures of Acacia victoriae and implementation of an aeroponic growth system.

Aeroponic Growth Techniques A number of advantages may be realized by utilization of an aeroponic system of cultivation for Acacia victoriae. First, the growth rate of the plants is approximately twice that achieved with conventional growing techniques. Second, the roots can be easily harvested as needed without harming the plants. The cutting of roots further leads to extensive lateral growth of fibrous roots. Therefore, the roots could be harvested several time a year. In wild populations of Acacia victoriae, collection of pods is limited to several weeks a year, and collection of roots is difficult without harming or killing the plant.

An aeroponic growth system is a closed system in which plant roots are suspended in air and misted with a complete nutrient solution. The roots are enclosed in a watertight box misted at intervals with the nutrient solution. The nutrient solution contains all of the essential elements the plants needs to complete its life cycle. Despite the fact that different 09/09/03,sw 13567spa.6 plants require different levels and formulations for optimum growth, an over-all, singlebalanced solution gives satisfactory results.

(ii) Tissue Cultures of Acacia victoriae Tissue cultures represent another option for cultivation of Acacia victoriae. For the development of tissue cultures, Acacia victoriae seeds are washed thoroughly in tap water with an anti-microbial soap and treated with a 20% solution of commercial bleach for minutes. After repeated washing in deionized water, the seeds are treated with boiling water to induce germination and incubated overnight. The next morning, seeds are once again disinfected with commercial bleach and rinsed 2-3 times in sterile deionized water.

The decontaminated seeds are then cultured on MS medium (Murashige et al., 1962) supplemented with MS vitamins and 2% sucrose (for the explant cultures, 3% sucrose was used) and the medium gelled with either 0.7% agar or 0.2% gelrite.

Explants used for culturing may comprise potentially any tissue type including shoot tips, nodal segments, hypocotyls and root segments. The explants are generally cultured on MS alone or MS supplemented with growth regulators, such as IAA, NAA, IBA, 2,4-D and BAP (either individually or in combination). The cultures are typically maintained at 252 C under a 16 hour light photoperiod at 1000 lux produced by cool white fluorescent tubes. Resulting plantlets are kept under mist in the green house one month for hardening before transferring them to a greenhouse, field or aeroponic growth system.

Hairy root cultures of Acacia victoriae have been developed in the present invention. Infection of the plant with Agrobacterium rhizogenes strain R-1000, leads to the integration and expression of T-DNA in the plant genome, which causes development of a hairy roots. Hairy root cultures grow rapidly, show plagiotropic root growth and are highly branched and hormone-free medium and also exhibit a high degree of genetic stability (Aird et al., 1988). The genetic transformation and induction of hairy root in Acacia victoriae and the optimum conditions for growth are described in detail in the section on Examples. Hairy root cultures allow the rapid growth of tissue on a large scale which can be used for the isolation of the triterpene compounds of the parent invention.

An advantage of tissue culturing is that clonal cultures may potentially be prepared which express the compounds of the invention. These cultures could be grown on a large scale and potentially be expanded to an industrial capacity growth system for the preparation of plant tissue for the isolation of triterpene compounds. Additionally, plants regenerated from tissue cultures frequently display significant variation. Therefore, using tissue cultures, clonal cell lines or plants regenerated from such cultures may be produced 09/09/03.sw I3567spa,7 which are "elite" with regard to their production of the triterpene compounds of the invention. Plants produced could be selfed over generation and selected at each breeding generation to produce true-breeding elite lines.

Elite varieties need not necessarily arise from tissue cultures, however, as significant genetic variation exists within wild populations of Acacia victoriae. It is, therefore, contemplated by the inventors that the genetic variation found in wild populations of Acacia victoriae includes variations in genes controlling the endogenous levels of triterpene production. As such, it should be possible to identify those members of Acacia victoriae populations which produce enhanced levels of triterpenes relative to other members of wild populations, and to select these varieties for use in growth systems directed to producing tissue for the isolation of the triterpene compounds of the invention.

The growth system may constitute, for example, convention farming, aeroponic growth techniques, tissue culturing, or any other suitable technique for the propagation of Acacia victoriae tissue. Still further, these plants may be selected for use in breeding protocols to produce varieties which are more elite and which are also true-breeding.

Definitions means one or more. "Thus, a moiety may refer to one, two, three, or more moieties.

Active constituents refers to the most pure extract that retains activity.

Pods are defined as seedpods of Acacia victoriae.

Cytotoxic is defined as cell death while the term "cytostatic" is defined as an inhibition of growth and/or proliferation of cells.

Apoptosis is defined as a normal physiologic process of programmed cell death which occurs during embryonic development and during maintenance of tissue homeostasis.

Abnormal Proliferation is defined as a series of genetically determined changes that occur in mammalian cells in the pathological state known as cancer.

Malignant cells are defined as cancer cells that escape normal growth control mechanisms through a series of metabolic changes during the initiation and promotion stages of the onset of malignancy.

Extract or fraction refers to consecutive samples collected from tissues by various means.

09/09/03,swl3567spa,8 -9- Triterpene or Triterpene Glycoside refers to the novel and/or biologically active saponin compounds identified in the parent application from Acacia victoriae.

EXAMPLE 1 Localization of Triterpene Compounds Within Acacia victoriae Plants In initial studies, above-ground dry parts of the plants were collected in early summer for extractions. Subsequent re-collection in the fall was without activity. A systematic study was thereafter conducted to determine for the relative absence or presence of the active triterpene compounds in various parts of Acacia victoriae plants. After monitoring the chemistry of the plant, it was determined that essentially all of the active components in the above-ground part of the plant were concentrated in the pods, roots and seedlings while largely or completely absent in the branches, bark, leaves and seeds. Therefore, the active collecting period only lasts about three weeks from the start of pod formation until dehiscence. It was also determined that the roots of the plant produce the same active material with fluctuating ratios of sugars to active components. The latter characteristic indicates that aeroponics, which allows for vigorous root growth while maintaining normal plant development, may be well-suited for Acacia victoriae.

EXAMPLE 2 Aeroponic Growth System In light of the finding that the triterpene compounds of the parent invention were concentrated in the roots and pods of Acacia victoriae plants, it was desired to create a method for propagating suitable tissue from which the compounds may be isolated. In order to achieve this goal, an aeroponic growth system was invented for the cultivation of Acacia victoriae roots. The aeroponic system is a closed system in which plant roots are suspended in air and misted with a complete nutrient solution. An 8 x 4 x 3. 5 ft. box was made out of 3/4 inch plywood sheets held together with screws and lined with fiberglass sheets to produce a watertight box. The top of the box was covered with two (2 x 8 ft) styrofoam sheets, with 12 circular holes drilled all the way through, although a new design incorporating PVC-coated poultry wire covered with opaque co-extruded white-on-black polyethylene is being considered as a chamber cover for future work. A program-repeating timer was used to mist the roots for a period of 12 seconds every 4.5 minutes.

The plumbing system design for the aeroponic chamber is a closed system constructed of 3/4 inch PVC with six whirl-jet hollow cone polypropylene spray nozzles. A 09/09/03,sw13567spa.9 reservoir of 720 liters of nutrient solution is maintained in the bottom of the chamber, and sprayed on the roots of the plants from below using an external pump. The pump used was a Little Giant 4-MD 3250 RPM, 1/12 hp pump.

The pump is controlled by a Tork repeating timer set for intervals of 30 seconds of spray every 4.5 minutes. Temperatures were monitored with a Taylor electronic indoor/outdoor minimum/maximum thermometer and recorded by hand. Two Visi-therm 300W submersible aquarium heaters were used to heat the nutrient solution during the winter months, which was sufficient to keep the plants actively growing without heating the surrounding air in an unheated and uncooled outdoor shade-house in Tucson, Arizona.

The nutrient solution contained all of the essential elements the plants needs to complete its life cycle. Despite the fact that different plants require different levels and formulations for optimum growth, an over-all, single-balanced solution gives satisfactory results. The composition of the solution is given below, in Table 1.

Table 1: Aeroponic Nutrient Solution Compound Element Concentration (ppm) Calcium Nitrate N 150 Potassium nitrate Ca 146 Potassium Nitrate K 200 Mono-potassium phosphate P Magnesium Sulphate Mg S 134 Fe Copper Sulfate Cu 0.07 Manganese Chloride Mn 0.8 Sodium Molybdate Mo 0.03 Boric Acid B 0.3 Zinc Sulfate Zn 0. 1 Seeds of Acacia victoriae were then scarified and sown in a soil-less mix composed of 50% peat moss and 5 0 vermiculite. The seedlings were watered twice a day and fertilized with a single dose of osmacote. Once the seedlings were between 15-20 cm long, which was usually achieved after 3-4 months of growth, the root balls were washed 09109/03.sw 13567spa. 11 thoroughly to remove all traces of peat moss and vermiculite. Next the roots were slipped through holes in the Styrofoam boards, and the top of the seedlings was supported from above by twine coming down from the greenhouse structure. A 7.0 cm tubular piece of foam was wrapped around the crown of the seedlings to prevent misting of the leaves and the surrounding area. The box was then filled with approximately 30 cm of nutrient solution, and the pump turned on.

Once the seedlings were in position and being misted, maintenance was limited to training the growing seedlings up the twine using plastic clips and replenishing the nutrient solution as the level dropped below 10 cm. While the seedlings were growing inside a greenhouse, temperature control of the nutrient solution was not necessary. However, if the aeroponic box is subjected to ambient environmental conditions, it is recommended to increase the nutrient solution temperature to 70 F so that the plants will not become dormant during winter months.

For harvesting of roots, the root mass of a single plant is rinsed with water directly in the aeroponic box and the root mass is cut with scissors a few inches above the sprayer.

The excess water is removed by patting dry with paper towels, followed by weighing of the sample. The root mass is then cut in 3-4 inch sections with scissors and subject to chemical extraction, as described above. Alternatively, for continual harvest of roots, the pump is turned off and roots are clipped from the growing root mass. These roots are then cut into 3-4 inch sections and extracted as described. Care is taken not to damage the non-harvested roots.

A number of advantages were realized by growing plants in the aeroponic system.

First, the growth of the plants was approximately twice that achieved with conventional growing techniques. Second, the roots can be easily harvested as needed without harming the plants. This cutting of roots further leads to extensive lateral growth of fibrous roots.

Therefore, the roots could be harvested several times a year. Further, the aeroponically grown plants flowered in their first year of growth, compared to 3-5 years for plants grown outdoors.

EXAMPLE 3 Tissue Culturing and Germination of Acacia victoriae Seeds/Substrate: Seeds were harvested from plants growing at the Campus Agricultural Center, University of Arizona, Tucson, Arizona. Seeds were washed thoroughly in tap water with an anti-microbial soap (Vionex, Viro Research International Inc., USA Durango, Colorado), then treated with commercial bleach 09/09/03,swl3567spa.I I 12for 15 min. After repeated washing in deionized water, they were treated with boiling water (ca 200 ml for 100 seeds) and left to cool overnight. Then they were treated with commercial bleach for 20 min, rinsed 2-3 times in sterile deionized water, and cultured on MS (Murashige and Skoog, 1962) and 1/2 strength MS medium. The medium was supplemented with MS vitamins, 2% sucrose and gelled with either 0.7% agar or 0.2% Gelrite. In one study, the seeds were scarified with concentrated sulfuric acid, rinsed in sterile water, and cultured on medium. All media was autoclaved at 121 C for 15 min.

Cultures were maintained at 25+2 C under 16-h light photoperiod at 1000 lux produced from cool white fluorescent tubes. Each study contained 18 replications.

Propagation: Shoot tips and nodal segments excised from three-week-old seedlings were cultured on MS medium alone and also MS supplemented with 0.1 mg/L of auxins (IAA, NAA or IBA) and BAP (0.1,0.3,0.5,1.0 and 1.3 mg/L) either separately or in combinations. For rooting of shoots IAA (0.1 mg/L), IBA (0.1 and 0.6 mg/L) and NAA (0.1 and 0.2 mg/L) were tested. For transfer to soil, plantlets were removed from culture tubes, the roots were washed with tap water to remove the nutrients adhering to roots and the transferred to pots filled with desert-type soil. The plants were covered with Magenta boxes to maintain humidity and kept under mist and low light for 3 wk. After 3 wk, the Magenta boxes were removed and the plants were transferred from the mist to higher light in the greenhouse.

Induction of callus: Callus tissue was induced from hypocotyl and root segments excised from 3-week-old in vitro germinated seedlings. The explants were cultured on MS medium supplemented with 2,4-D (1 mg/L), NAA (0.5 1 mg/L), IAA (0.2 and 1 mg/L), Thidiazuron (0.2 mg/L), Dicamba (0.2 2 mg/L), BAP (0.3 mg/L) and KN (0.5 and 3 mg/L) either individually or in combinations.

Seed Germination: Seeds treated with hot water germinated with the emergence of the radicle in 3-4 days and the complete plantlets were obtained within 1 wk. Seeds cultured without hot water treatment did not germinate. A high percentage of seeds germinated on medium solidified with Gelrite as compared to agar The maximum germination percentage of 88.7% was noted on half strength MS medium solidified with Gelrite. The germination responses on different media are summarized in Table 2.

09/09/03,swl3567spa.12 13 Table 2: Seed Germination of Acacia victoriae Media No. of Seeds Cultured No. of Seedsa Germinated MS (agar solidified) 42 36 (85.7) MS (agar solidified) 41 24 (58) (decoated with sulfuric acid) 1/2 strength MS (agar 60 48 solidified) 1/2 strength MS (Gelrite 133 118 (88.7) solidified) a Numbers in parentheses are percent germination.

Transplantable seedlings were obtained in 3-4 wk time. The seeds of A. victoriae have low germination rates in vivo due to high levels of seed dormancy (Kaul and Ganguly, 1965; Grice and Westoby, 1987). To overcome dormancy, seed coats must be either nicked with a sharp instrument, subjected to acid scarification, or covered with boiling water and left to cool in the water overnight. The inventors found that the germination percentage can be increased up to 88.7% by using the boiling water treatment and subsequently culturing the seeds on 1/2 MS medium gelled with 0.2% Gelrite.

According to Larsen (1964), A. victoriae seeds under in vivo conditions treated with boiling water can increase germination by 36%. Without pretreatment, the germination percentage was 19.4% (Kaul and Ganguly, 1965). In addition, it took 12 days for the radicle to emerge and complete seedlings were recovered after 79 days. In our protocol, the percent germination is increased and transplantable seedlings could be obtained in 3-4 wk time.

Shoot tip cultures: To investigate shoot multiplication, the shoot tips (about cm in length) were cultured on either MS alone or MS supplemented with BA, and BA in combination with IAA. On MS alone the shoots had poor vigor, and a poor root growth (1-3 roots/culture). On medium containing BA (1.3 mg/L), the shoot tips produced multiple shoots (average of 3.94 shoots/culture). Among the multiple shoots, one or two shoots elongated and attained a height of 8.6 cm in 4 wk. The combinations of BA and IAA also favored multiple shoot induction. The responses are summarized in Table 3.

09/09/03,swl3567spa,13 14- Table 3: Effect of Different Levels of BA And IAA (0.2 Mg/L) on Multiple Shoot Induction in Acacia victoriae.

Media* Average No. of shoots Shoot Length BA (mg/L IAA (mg/L per shoot tip (cm) 1.3 0 3.94+1.846 8.6+3.0258 0.1 0.2 1.6+0.599 6.8+3.002 0.3 0.2 1.9+0.7071 5.8+2.794 0.2 2.8+1.1659 5.1+2.501 0.2 4.9+2.075 3.2+1.468 *MS. Data represents an average of 18 replicates SE.

At higher BA concentrations (1.0 1.3 mg/L), the number of shoots increased. The combination of BA (1 mg/L) +IAA (0.2mg/L) was found to be better for shoot multiplication. Callus was observed at the cut ends in all the BA-IAA combinations. Kaur, et al. (1998), reported the synergistic effect of BA-NAA on shoot bud induction in Acacia catechu and higher levels of NAA (1-2 mg/L) were not beneficial. They also stated that IAA was not effective in enhancing shoot bud formation; but instead callus was produced from the base of the explants.

To investigate rooting, in vitro-developed shoots were excised and transferred to medium containing IAA, NAA or IBA. The responses are summarized in Table 4. Among the treatments tested, 1/2 MS+NAA (0.2mg/L) was found better for rooting. Almost 100% of the shoots rooted. The shoots attained a height of 9-11 cm in four wk. In Acacia catechu (Kaur, et al., 1998) reported that intermittent callus formation at the junction of root and shoot and they employed reduced sucrose level from 3% to 1.5% to control the callus.

Similar results were also reported in Feronia limonia (Purohit and Tak, 1992) and Acacia auriculiformis (Das, et al., 1993). In the present investigation, slight callusing was also noted at 3% sucrose and it was minimized at 2% sucrose. The rooted shoots were transferred to the greenhouse. The survival after transferring was 100%. The plantlets were acclimatized under mist for 3 wk and later the plantlets were grown in the regular greenhouse.

09/09/03.sw 3567spa.14 Table 4: Effect of IAA, NA and IBA on Rooting of Shoots of Acacia victoriae Media No. of shoots No. of shootsa Mean No. of cultured rooted roots/culture MS 14 6 (42.8) 2+0.816 MS+IAA 12 8(66.6) 13.6+ 1.316 MS+IBA 10 6 (60) 3+0.816 MS+IBA 14 8(57) 1.6+1.111 MS+NAA 10 6(60) 2.16+1.067 1/2MS+NAA 14 14 (100) 3.07+ 1.032 a Numbers in parentheses are percent rooting.

Nodal segment cultures: Nodal segments (cotyledonary node) excised from in vitro germinated seedlings were cultured on MS medium supplemented with 0.1 mg/L IAA, NAA or IBA. Only one or two axillary shoots developed per explant. However, the growth of these shoots was slow. Hence, nodal explants were not used for further studies.

Induction of callus from hypocotyl and root segments: Callus was induced from hypocotyl segments excised from 3-wk-old in vitro germinated seedlings. The callus developed on 2,4-D (1mg/L), Thidiazuron (0.2 mg/L), Dicamba (0.2 mg/L) was greenish, compact and hard. The quantity of callus developed was moderate in most of the concentrations tried (Table 51). Profuse callus development was noted on MS medium supplemented with 2,4-D (4 mg/L) +IAA (I mg/L) +NAA (1 mg/L).

Root segments excised from three-week-old in vitro germinated seedlings were cultured on MS medium supplemented with 2,4-D (Img/L) alone and 2,4-D in combination with KN (0.5 mg/L) showed the development of light yellowish soft callus with a few roots developing from the callus. The callusing was noted in 100% of the cultures. Whitish, soft, friable and profuse callusing was noted from root segments on medium added with BA (0.3 mg/L) +IAA (0.2 mg/L). Light yellowish profuse callusing was noted on the root segments cultured on medium added with 2,4-D (4 mg/L) in combination with 1 mg/L each of IAA and NAA. A similar type of callusing was noted in Thidiazuron (0.2 mg/L) +Dicamba (2 mg/L) and IAA (0.1 mg/L). Root segments cultured on medium with Dicamba (2mg/L) IAA (0.1 mg/L) formed light green compact hard callus. Attempts to regenerate the plantlets from the callus were not successful. Variation among explant types with respect to callus induction has been reported in several woody 09/09/03,sw I 3567spa, 16species such as Albiizzia lebbeck (Lakshmana Rao and De, 1987) and Lonicerajaponica (Georges, et al., 1993). In the inventors' studies, they also found that there is a difference between hypocotyl-and root-derived callus developed on the identical medium. Calli developed from hypocotyl on BA-IAA combinations were light greenish, hard and compact, whereas from the root segments it was whitish, soft, friable and also showed root differentiation from the callus in some of the combinations. In Dalbergia latifolia the callus on regenerating media became compact, hard and dark green and shoot buds were differentiated (Pradhan, et al., 1998). In the inventors' studies, a similar type of callus development was noted, but such callus failed to regenerate. In this investigation the inventors showed that A. victoriae can be propagated in vitro from shoot tips. The technique standardized is useful for the micropropagation of elite individuals detected among the heterogeneous seedling populations and maintenance of elite lines for future studies.

Table 5: Development of Callus from Hypocotyl and Root Segments of Acacia victoriae Media* Nature of callus Hypocotyl 1. MS+2,4-D Moderate, green 2. MS+TD Scanty 3. MS+Dicamba Moderate, compact green 4. MS+2,4-D +KN Scanty, green MS+KN +NAA Moderate, white 6. MS+TD Moderate, light green 7. MS+Dicamba Scanty, compact yellow IAA(0.2) 8. MS+2,4-D Profuse, green, compact, IAA +NAA hard 9. MS+BA +IAA Moderate, compact Numbers in parentheses are mg/L.

Root Moderate, yellow Scanty Moderate, soft yellow Scanty, light green Scanty, light green Moderate, soft yellow Scanty, light green Moderate, yellow soft Profuse, white EXAMPLE 4 Induction of Hairy Roots from Acacia victoriae for the Production of Anti- Cancer Compounds 09/09/03,swl3567spa,16 17- Infection of Acacia victoriae plant material with Agrobacterium rhizogenes leads to the integration and expression of T-DNA in the plant genome, which causes development of a hairy root phenotype (Grant et al., 1991). Hairy root cultures grow rapidly, show plagiotropic root growth and are highly branched on hormone-free medium. Hairy roots also exhibit a high degree of genetic stability (Aird et al., 1988). Many dicotyledonous plants are susceptible to A. rhizogenes, and plants have been regenerated from hairy root cultures in many species (Christey, 1997).

Genetic transformation and the induction of hairy roots were performed by the inventors as a method for the production of the active triterpenes from A. victoriae. The natural ability of the soil bacterium Agrobacterium rhizogenes to transform genes into a host plant genome results in roots being formed at the site of infection is used to produce hairy root cultures. Hairy roots are characterized by numerous fast growing, highly branched adventitious roots, which continues to grow in vitro on hormone-free medium.

The inventors demonstrated induction of hairy roots in Acacia victoriae using Agrobacterium rhizogenes strain R 1000 (an engineered strain of Agrobacterium tumefaciens strain to which Agrobacterium rhizogenes plasmid pRiA4 has been inserted, ATCC Number 43056). The production of the compound of interest in hairy roots was confirmed by HPLC. Induction of hairy roots was carried out as follows. First, Acacia victoriae seeds were collected from field-grown plants in Tucson, Arizona. Boiling water was poured over the seeds, which were soaked overnight as the water cooled and surface sterilized in 15% commercial bleach for 30 minutes. After repeated washing in sterile water, seeds were cultured on liquid MS medium (Murashige and Skoog, 1962) supplemented with MS vitamins and 2% sucrose in 250 ml conical flasks with 50 ml medium. The cultures were maintained in a gyratory shaker in a growth room at 25 2 C in the dark. After four days of culture, embryo-axis were excised from the germinating seedlings and used for agroinfection.

Prior to agroinfection, Agrobacterium rhizogenes strain R1000 was grown overnight on YENB medium. YENB medium was prepared by adding 7.5 g/L Yeast Extract and 8 g/L Nutrient Broth (Difco Laboratories, Detroit, MI). The embryo-axis of the explants was infected with a fine stainless steel needle that had been dipped in bacterial solution. After infection, a drop of bacterial suspension 20 with MS medium) was put on the surface of the explants. Then the explants were transferred to MS medium and MS medium with acetosyringone (100pM) (3,5 dimethoxy-4 hydroxy-acetophenone, Aldrich Chem. Co, Milwaukee, WI) for co-cultivation. Co-cultivation was carried out for three 09/09/03,swl3567spa,17 18days in the dark. After three days of co-cultivation, the explants were transferred to MS+ Cefotaxime (500 mg/1, Agri-Bio, North Miami, Fl) to control the bacterial overgrowth.

Root initiation was noted at the site of infection mostly from the young developing leaves from the embryo-axis in 3-4 weeks time. After 4 weeks, the explants along with the roots were transferred to MS medium alone and the dark incubation was continued for the development of hairy roots. Hairy root development was noted after a further 8 weeks. The hairy roots thus developed were multiplied routinely on MS medium by subculturing. The transgenic nature of the hairy roots was confirmed by PCRTM using a set of primers to amplify a portion of the rol B gene. The primers used were as follows: 1) 5'GAGGGGATCCGATTTGCTTTTG 3' [SEQ ID NO. 7] 2) 5'CTGATCAGGCCCCGAGAGTC [SEQ ID NO. 8] A 50 tl PCRTM reaction mix contained the primers [tM final concentration each), Taq polymerase OU), 125 itM each dNTP, IX PCRTM reaction buffer, 1.5 mM Mg Cl 2 ,300ng of isolated DNA. PCR M conditions employed were 92 0 C initial denaturation for five min followed by 35 cycles of 92 0 C 50 seconds, 55 0 C 1 min for annealing, 72 0 C 1 and 1/2 min for extension and 72°C 7 min final extension. A 645 bp fragment was amplified.

Hairy root cultures in liquid medium: To optimize the conditions for the growth, hairy roots growing on MS semi-solid medium were excised and cultured in MS liquid medium in different capacity flasks (125,250,500 and 1,000 mL) with 20, 50,100 and 400 mL medium respectively. The initial hairy root inoculum was 6 gm/L. The growth of hairy roots was also tested in the following basal media: MS, Nitsch and Nitsch (N and N) (1969), Schenk and Hilderbrandt (SH) (1972) and Woody Plant Medium (WPM) (Lloyd and McCown, 1981). To test the effect of different carbon sources on hairy root growth, 2% of each of the following was added to MS medium: sucrose, glucose, fructose and mannose. The effect of gibberellic acid (0.1,0.5 and 1 mg/L) on hairy root growth was tested by adding the filter-sterilized solution to MS medium after autoclaving.

Initiation of roots at the site of infection was noted in 3-4 weeks. Four independently transformed hairy root clones were established from embryo axes infected with R1000 strain in the presence of acetosyringone (100[M). The embryo axes cocultivated with A. rhizogenes without acetosyringone did not develop hairy roots (Table 6).

Three days co-cultivation in the presence of acetosyringone was found optimum for induction of hairy roots. A promoting effect of acetosyringone has been reported in Salvia 09/09/03,sw13567spa,18 19militiorrhiza (Hu and Alfermann, 1993). The results showed that acetosyringone, an activator of the vir genes of Agrobacterium, increased the transformation frequency. Similarly, in this study, acetosyringone was required to induce hairy roots.

The transformed nature of the roots was confirmed by PCRTM amplification using a set of primers to amplify a portion of rol B gene. A diagnostic fragment of 645 bp was amplified in the four hairy root clones tested.

The hairy roots grown on liquid medium developed vigorously. Among the different basal media tested, MS medium was found best for hairy root growth (Table 8).

In a 125 mL flask, there was a 268-fold increase in growth in 4 weeks. With WPM and N and N medium, there was a 254-and 196-fold increase respectively. B5 and SH medium did not favor the optimal growth of hairy roots. Hairy roots slowly started browning on these two media. In one study, hairy roots were grown in different capacity flasks (125,250,500 and 1000 mL) with 20,50,100 and 400 MI MS medium, respectively. The growth kinetics are summarized in Table 7. Initially, the growth of hairy roots is vigorous and attained a 25.77-fold increase in 4 weeks in 125 mL flasks with a starting inoculum of 150 mg. As the flask capacity was increased, the growth of roots slightly decreased.

The growth of hairy roots can be sensitive to medium composition, especially mineral ions and carbon source (Wysokinska and Chmiel, 1997). For Acacia victoriae, five different basal media (MS, N and N, SH, WPM and Bs) were tested for effect on hairy root growth. MS medium was found best for growth. Sasaki et al. (1998) compared the growths of Coleus forskohlii hairy roots on various nutrient media and found that WPM was best for hairy root growth.

In this study, sucrose favored the growth of hairy roots compared to other carbon sources (fructose, glucose and mannose). The maximum growth (24.52-fold increase) was found in sucrose-containing medium. Glucose-containing medium was slightly inhibitory for growth, and mannose completely inhibited the growth (Table In Catharanthus roseus, catharanthine production could be doubled by the use of fructose as a carbon source in the medium. However, the authors reported that use of fructose resulted in an approximately 40% decrease in growth compared to sucrose (Jung et al., 1992).

Hairy roots do not require the addition of exogenous growth regulators for continued growth because genes that increase sensitivity to auxin are present in the Ri plasmid (Wysokinska and Chmiel, 1997). However, reports are available wherein exogenous hormones stimulate growth. The inventors tested the effect of gibberellic acid (0.1,0.5 and 1.0 mg/L) on hairy root growth. The growth of hairy roots was best in medium 09/09/03.swl3567spa,19 20 without GA3, as compared to GA3-containing medium (15.77-fold increase). Different levels of GA3 did not affect the growth significantly (Table 10). In Artemisia, GA3 did not enhance the overall biomass accumulation, but it facilitated reaching stationery phase sooner than cultures grown on medium without GA3 (Smith et al., 1997). Rhodes et al., (1994) found that the response of hairy roots of Brassica candida to GA3 depended largely on the clone examined. However, they observed that generally GA3 exerted a positive effect on growth and a reduction in the accumulation of alkaloids accompanied with changes in patterns of production. Ohkawa et al., (1989) reported GA3 at concentrations of 10 ng/L and 1 mg/L accelerated growth, enhanced elongation, and increased lateral branching in Datura innoxia hairy roots. Zobel (1989) suggested that GA3 acts as a C02 analog for root growth. For Acacia victoriae hairy roots, GA3 did not enhance the growth, which might indicate a differential response for various genotypes.

The use of hairy root cultures of Acacia victoriae will provide a suitable means for uniform culture of plant tissue from which the triterpene glycoside compositions of the parent invention, which include isolated mixtures or individual purified compounds, can be isolated.

Table 6: Agrobacterium rhizogenes Strain R1000 Infection of Embryo Axes of Acacia victoriae for Hairy Root Production Treatment *Media for co- No. of embryo No. explants a No. of roots cultivation axis infected with root with hairy root development morphology Control MS 20 (non infected) MS+Aceto. 21 Infected MS 33 5 MS+Aceto 38 .9 (23) 4 (17.3) *Acetosyringone (100, pM) was added after autoclaving into MS medium for co-cultivation a Number in paranthesis indicates percentage.

09/09/03,sw13567spa,20 -21 Table 7: Effect of Different Flask Sizes on the Growth of Hairy Roots of Acacia victoriae Flask size (mL) Initial Fresh weight Fresh Weight after Fold increase (mg) 4 weeks (mg)a 125 150 3866+0.569 25.77 250 300 6903+0.344 23.01 500. 1200 11817+0998 9.84 1000 2400 40080+ 3.479 16.70 a Data represents an average of 6 replicates S. 125.250,500 and 1000 mL capacity flasks with and 400 mL MS medium.

Table 8: Effect of Different Basal Media and Flask Size an the Growth of Hairy Roots of Acacia victoriae Media' Flask sizeb Initial Fresh.W. Fresh Weight Fold increase weight after 4 weeks (mL) (mg) weeks (mg) MS 125 10 2681 268 125 10 1933 193 N and N 125 10 196 196 SH 125 10 170 170 WPM 125 10 2549 254 MS 250 300 751

B

5 250 300 57 19 N and N 250 300 591 19.7 SH 250 300 54 18 WPM 250 300 v 21 a MS=Murashige and Skoog; B 5 Gamborg's; N and N= Nitsch and Nitsch; SH= Schenk and Hilderbrandt; WPM= Woody plant medium. b125 and 250 mL flasks with 25 and mL medium.

09/09/03,sw I 3567spa,21 22 Table 9: Effect of Various Carbon Sources in MS Medium on the Growth of Hairy Roots of Acacia victoriae Carbon source a Fresh weight after W/V) 4 weeks (gm) b Sucrose 7.356+0. 543c Glucose 2.87±0.53 Fructose 5.85±1.55 Mannose 0.305±0.065 a2% bThe initial F. W. for each treatment was 300 mg.

6 replicates S. E.

Fold increase 24.52 9.56 19.5 1.01 Data represents an average of Table 10: Effect of GA 3 on the Growth of Hairy Roots of Acacia victoriae GA3 Fresh weight after a Fold increase (mg/L) 4 weeks (gm) b 0 6.512+1.569b 21.70 0.1 4.732±0.086 15.77 4.634±0.088 15.44 1 4.310±0.344 15.44 aThe initial F. W. for each treatment was 300 mg. bData represents an average of 6 replicates S. E.

Different media were tested for growth of hairy roots. Best growth was obtained on MS medium containing 2% sucrose. The effect of different capacity flasks and gibberellic acid was tested on the growth of hairy roots. The hairy roots were also grown on MS liquid medium on gyratory shaker in a 125 ml conical flask with 20 ml medium. An increase in growth of 84 fold was noted in 4 weeks. The production of triterpene saponins corresponding to those identified in F035 was confirmed by HPLC analysis with a standard authentic sample.

All of the composition and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method 09/09103,swl3567spa.22 23 described herein without departing from the concept, spirit and scope of the invention.

More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the claims.

Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

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09/09/03,sw13567spa,32

Claims (4)

1. A hairy root tissue culture comprising cells of an Acacia victoriae plant which have been infected with Agrobacterium rhizogens R-1000 in a tissue culture medium.

2. The tissue culture of claim 1, wherein said tissue culture medium comprises from about 3% to about 4% sucrose by weight.

3. A method of continually harvesting an Acacia victoriae plant tissue comprising: a) cultivating an Acacia victoriae plant in a hydroponic growth system; and b) harvesting said tissue from said plant about 1 to about 4 times per year, wherein said harvesting does not kill said plant.

4. The method of claim 3, wherein said growth system is an aeroponic system. The method of claim 4, wherein said tissue is root tissue. Dated this 9 th day of September, 2003 RESEARCH DEVELOPMENT FOUNDATION By their Patent Attorneys: CALLINAN LAWRIE 09/09/03.sw 3567spa,33 SEQUENCE LISTING <110> ARN'rZEN, -HARLES J BLAKE, M1ARY 2. GUTTERMAN, JORDAN U. HOFFMAN, JOSEPH J. BAILEY. DAVID T. JAYATILAKE, GAMINI S. <120> TRITERPENE COMPOSITIONS AND METHODS FOR USE THEREOF <130> CLFR:006 <140> FILED HEREWITH <141> 2999-05-19 <150> 60/099,066 <151> 1998-09-03 <150> <151> 60/085,997

1998-05-19 <160> 9 <170> Patentln Ver. <210> 1 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial PRIMER Sequence: SYNTHETIC <400> 1 agttgagggg actttcccag gctcaactcc cctgaaaggg tccg <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: SYNTHETIC PRIMER <400> 2 ctaagcctgt tgttttgcag gac <210> 3 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: SYNTHETIC PRIMER <400> 3 catggcacta tactcttcta <210> 4 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: SYNTHETIC PRIMER <400> 4 catggcacta tactcttctt <210> <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: SYNTHETIC PRIMER <400> ccttggctaa gtgtgcttct cattgg 26 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: SYNTHETIC PRIMER <400> 6 acagcccacc tctggcaggt agg 23 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: SYNTHETIC PRIMER <400> 7 gaggggatcc gatttgcttt tg 22 <210> 8 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: SYNTHETIC PRIMER <400> 8 ctgatcaggc cccgagagtc <210> 9 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Primer <400> 9 ttgttacaag ggactttccg ctggggactt tccagggagg ctgg 44