WO2004033673A2

WO2004033673A2 - Production process for plant biomass and its use

Info

Publication number: WO2004033673A2
Application number: PCT/IT2002/000645
Authority: WO
Inventors: Roberto Dal Toso; Giovanna Pressi; Renzo Dal Monte
Original assignee: I.R.B. Istituto Di Ricerche Biotecnologiche S.R.L.
Priority date: 2002-10-10
Filing date: 2002-10-10
Publication date: 2004-04-22
Also published as: WO2004033673A3; AU2002349829A1; AU2002349829A8; WO2004033673A8

Abstract

The present invention relates to a production process for plant biomass and the subsequent use thereof in the production of substances of industrial interest, in particular biologically active substances. More in particular, the present invention relates to a method for the cultivation of a plant cell line capable of producing metabolites of interest, comprising a selection phase for the cell line which includes the following cycle of operations: a) cultivating on solid medium a predetermined stabilized plant cell line for a time sufficient to obtain the multiplication of said cell line to give substantially distinct cellular clusters; b) taking from said solid medium said substantially distinct cellular clusters and placing each of them into a distinct liquid culture medium; c) cultivating each of said substantially distinct cellular clusters in said liquid culture medium for a time sufficiently long to allow the multiplication of said cellular clusters and the analytical determination of the primary and/or secondary metabolites produced by it; d) operating a qualitative and quantitative determination of the primary and/or secondary metabolites produced by each of said cellular clusters in said liquid culture medium; e) selecting the cellular cluster capable of producing the greatest quantity of said metabolite of interest; f) repeating the cycle of operations according to steps a), b), c), d) and e) on said cellular cluster selected according to step e) for a sufficient number of times until the quantity of said metabolite of interest produced by a selected cellular cluster and by the cellular cluster derived from a successive cycle of the selection operation remains substantially unchanged.

Description

DESCRIPTION

"Production process for plant bio ass and its use"

The present invention refers to a plant biomass production process and its subsequent use for the production of substances of industrial interest, in particular biologically active substances.

The use of plant cell cultures for the production of biomass for use in bioreactors is known. In higher plants, both gymnosperms and angiosperms, tissues exist which are characterised by undifferentiated cells in rapid multiplication. These tissues, known as meristematic, are characterised by small-sized cells, with thin cell walls and devoid of vacuoles. The cytoplasm is rich in ribosomes, protoplasts and mitochondria, whilst the endoplasmic reticulum is poorly developed Their nucleus is very voluminous with respect to the size of the cells. In higher plants the meristematic cells, following multiple mitotic divisions, give rise to morphologically and physiologically differentiated cells, according to the location and the tissue to which they will belong and also lose their capacity for active proliferation. Meristematic cells have, therefore, embryonal type stem cell characteristics with "totipotent" potential for differentiation and development.

These embryonal characteristics can be newly acquired by differentiated cells when, for example, the tissue becomes damaged and a "scar" is formed. In this case the differentiated cells return to having an activity similar to the meristematic cells to repair the wound caused in the tissue. In this case, a repair tissue, known commonly as "callus" is produced.

This phenomenon, which normally takes place through natural causes, can be however induced for scientific purposes by creating incisions in the plant tissues so as to stimulate the formation of "calli" .

Current techniques allow one to cultivate meristematic cells, impeding differentiation so as to maximally exploit their embryonal characteristics. Furthermore, by varying the composition of the culture media, it is possible to select cell lines with different biochemical and metabolic characteristics. The maintenance of said embryonal characteristics is brought about principally through the action of hormonal substances, known as "growth factors", which are able to keep the meristematic activity alive. These hormones are produced by the plant and are capable of provoking very wide and complex physiological responses, from fruit maturation, to the induction of abscission tissue in leaves, to the induction of bud dormancy.

In particular, meristematic activity is maintained thanks to the action of two groups of substances : the auxins such as indole acetic acid and the cytokinins such as kinetin.

The mechanism of action of these plant hormones is manifested above all at the ribosomal level, both by increasing the synthesis of RNA, and inducing the synthesis of specific proteins. With auxins, induction has been observed of the synthesis of the enzyme cellulase, which determines the elasticity of the cell walls and therefore favours stretching and cellular growth. The action of the cytokinins however is manifested at the level of the cytodieresis, inducing proteins whose low concentration is a limiting factor for growth speed.

The combination of auxin effects on cellular growth and the effect on cellular multiplication caused by ^" cytokinins is sufficient to achieve both the maintenance of the undifferentiated state and the proliferation activity of the meristematic cells.

In addition to the presence of growth factors, it is however necessary to supply the cells with an adequate culture medium and the appropriate environmental conditions. The culture medium must contain the substances necessary for the maintenance of cellular metabolism, first and foremost a source of organic carbon, which is generally constituted by sucrose. The cells in culture, in fact, loose their photosynthetic capacity and so require an exogenous source of energy and carbon. The macroelements necessary for cell growth: nitrogen, phosphorous, sulphur, both in organic and inorganic forms, and a series of mineral macroelements such as iron, manganese, copper, zinc and cobalt in salt form in addition to some vitamins must further be supplied.

The callus tissue induction procedure is a known technology, although it is necessary from time to time to make adjustments and adaptations for the particular type of plant and tissue selected for the generation of the callus. These adaptations are however definable through routine experiments achievable by those of ordinary skill in this field. .

Depending on the chosen cell line, one can therefore obtain, at the end of the entire culture process, a biomass capable of producing, through fermentation, substances of industrial interest, typically biologically active compounds, not easily obtainable through synthesis or by means of extraction. It is however known, that a cell culture process such as that summarised above is characterised by certain critical steps which limit the final biomass productivity to a point which renders the entire process poorly advantageous from the economic point of view. The intrinsic characteristics of the cell line, the method by which it is cultured and the conditions under which the fermentation of the biomass is carried out influence greatly the final yield of the desired product.

The basic problem of the present invention is therefore that of providing a plant cell culture method and a successive procedure of the biomass thus obtained to give a product of industrial interest, typically a biologically active product, which overcomes the inherent disadvantages in the state of the art methods. That problem is solved by a cell culture method and by a procedure for the fermentation of the biomass thus obtained, as set out in the enclosed claims.

The cell culture method according to the invention involves various steps which will be described in detail below.

FORMATION OF THE CALLUS TISSUE

The callus tissue induction procedure is the first step of the present method. It follows on from an essentially known procedure, but requires adjustments and adaptations for the particular plant and tissue type selected for callus production. These adjustments however are part of the normal tools of those skilled in this sector.

By way of example, a procedure, generally used for this phase, will be herein described.

The collected tissue is firstly cleaned and sterilised, to reduce the amount of explants contaminated by bacteria and saprophytic yeasts. Normally the plants, following collection, are hence washed in running water for a sufficiently long time

(generally around two hours) . After the washing, leaves, stems and roots are cut into segments of 2-5 cm and placed in sterile Petri dishes. Next, the plant fragments are sterilised, for example by treatment in sequence with 70% ethanol for around 15 minutes, with 2% sodium hypochlorite for around 5 minutes and finally with 0.05% HgCl₂ for around 1 minute. Between each treatment, the plant fragments are washed, typically three times or more, with sterile distilled water, preferably working under a sterile laminar flow hood.

Each fragment is again then cut with sterile scalpels or forceps into minute fragments (called "explants") and then placed onto Petri dishes containing nutrient medium enriched with growth hormones, solidified with Agar and without antibiotics. The number of explants completed in this phase substantially influences the success of the following phases in the cell cultivation method. For example, for some Taxus cell lines it is important to prepare at least 15000 explants, because, notwithstanding the sterilisation procedure, a major part of the explants (on average from 70% to 80%) are discovered as being contaminated and must be discarded. The successive cell line selection phase, proceeds therefore, on the about 3000 remaining explants. Analogous explant numbers have been used also with asteraceae and caprifoliaceae cell lines for antioxidant metabolites. In general from 2000 to 5000 non contaminated explants are sufficient to proceed with the successive selection phase. The formation of undifferentiated callus tissue takes place following storage of the non contaminated explants in the dark, generally at around 28°C.for about 21 days. The tissue is then multiplied following transfer onto a larger area surface with fresh medium. Following a further 14 days under the same conditions, the sufficiently developed callus parts are transferred onto fresh medium.

CELL LINE STABILISATION

The phase following on from the formation of the undifferentiated callus tissue is the stabilisation of the plant cell line.

A "stable cell line" is defined as a culture which presents the following characteristics: i) high and constant proliferation rate throughout time; ii) preservation of the same phenotypic characteristics throughout various subcultures (cell colour, aggregate, friability, size, etc.); iii) constant levels of secondary metabolites, per unit of mass, during the course of the various subcultures (secondary metabolite content is measured by chemical analysis of the extracts) ; iv) constant content, per unit of mass, of the primary metabolites (protein, lipids and polysaccharides) .

The cell culture undergoes a certain number of transfers onto fresh culture medium, each transfer originating a subculture which will be analysed for the above mentioned parameters. It has been observed that, to obtain a stable plant cell line, it- is important to perform at least ten subcultures and to measure the above described parameters .

Each subculture is carried out on solid medium. That solid medium may be advantageously constituted of 0.8-1% agar in a standard culture medium to which has been added plant peptone, which allows an equilibrated contribution of aminoacids and guarantees the maintenance of good cell wall integrity. The addition of plant peptone is therefore important, inasmuch that plant cells, characterised by high structural fragility, could otherwise result in lysis in the successive operations of the procedure. The culture medium will contain therefore a biologically efficacious amount of plant peptone, where the term "biologically efficacious amount" means an amount capable of causing the above described effects on the cells. Preferably, the plant peptone will be added in quantities comprised between 500 and 4000 mg/L of culture medium. More preferably, that culture medium will have the following composition: agar 0.8-1% of the total volume of the medium Ammonium sulphate 100-180 mg/L Calcium chloride.2H₂0 110-190 mg/L Magnesium .sulphate .7H₂0 200-300 mg/L Magnesium sulphate.H₂0 . 0.5-1.5 mg/L Potassium nitrate 1500-5000 mg/L

Monobasic Sodium phosphate 80-190 mg/L Cobalt chloride 0.008-0.018 mg/L Copper(III) sulphate .5H₂0 0.050-0.200 mg/L Sodium molybdate .2H₂0 0.05-0.20 mg/L Potassium iodide 0.20-0.60 mg/L

Zinc sulphate .7H₂0 0.5-1.5 mg/L Boric acid 0.5-3 mg/L

Myo-inositol 10-100 mg/L

Nicotinic acid 0.1-2.0 mg/L

Pyridoxine.HC1 0.1-2.0 mg/L

Thyamine.HCl . 2-10 mg/L Sodium EDTA.2H₂0 5-50 mg/L

Iron (II) sulphate.7H₂0 5-50 mg/L Naphthalenacetic acid 0.1-5 mg/L Kinetin 0.1-5 mg/L

Indolacetic acid 0.01-1 mg/L Sucrose 5000-40000 mg/L

Plant peptone 500-4000 mg/L

Phenylalanine 50-600 mg/L

PH 5.0-6.4

It is to be noted that a . culture medium containing plant peptone (preferably 500-4000 mg of peptone per litre of medium) will also be preferably used, naturally without the addition of agar, as a liquid culture medium in the applications which will be described following in the present description. Also in this case, more preferably, the culture medium will have the composition outlined in the previous paragraph.

A typical example of solid culture medium used is : 0.9% agar in Gamborg B5 + 20 g/1 of sucrose + 2 g/1 of plant peptone + 1 mg/1 of kinetin + 1 mg/1 of naphthalenacetic acid + 0.2 mg/1 of indolacetic acid.

The subculture is made to multiply on solid medium for 10-15 days, then is transferred onto new medium giving rise to the successive subculture.

As mentioned above, prior to each subculture it is ' important to perform controls on a series of parameters. In particular, the secondary metabolite content, which constitutes a substance of industrial interest of the plant cell culture, it is a characterising parameter of the entire procedure and must therefore be measured with the utmost attention. To that end, the sample of subculture must be extracted with appropriate solvents, such as ethanol, methanol or dichloromethane, which provoke the rupture of the plant cells. The organic extracts obtained are analysed by ELISA or HPLC assays to quantify the secondary metabolite content.

In the experimental section will be reported some examples of the extraction and the determination of the secondary metabolite content on various plant cell types. Also the primary metabolite content must be constant from one subculture to the successive one. The determination of their contents is carried out using normal analytical techniques. The determination of the water content of the stable cell culture is also very important which must be constant throughout the course of the subcultures of the cells. In fact, the water content in non stable cultures is very variable, also as a function of the degree of differentiation of the cells. More differentiated cells have a lower water content with respect to cells under proliferating conditions .

The phenotypic characteristics of a stable cell line must also remain unaltered through time. By the term "phenotypic characteristics" is intended the cell colour, the friability, the size and cellular morphology. Verification of the last two parameters is performed using an electron microscope . CLONAL SELECTION OF THE CELL LINES Further to the stabilisation phase, the cell line is subjected to "clonal selection". This phase is extremely important in order to increase the productivity of the cell line with respect to the substance of industrial interest (typically, a primary or secondary metabolite produced by the cell line) which it is desired to obtain. The clonal selection phase is carried out starting from the stable plant cell line, obtained in the previous phase, cultivated on solid medium described previously, typically 0.8-1% agar in Gamborg B5 with added plant peptone, sucrose, naphthalenacetic acid^', indolacetic acid and kinetin as described above 'for the stabilisation phase. Following an appropriate time (typically, 10-15 days of culture) , isolated cell clusters are removed from the solid culture medium and each of said cell clusters is inoculated into the liquid culture medium described above. Preferably, approx. 1 g of cellular cluster are inoculated into around 20 ml of liquid medium. A typical liquid medium is Gamborg B5 + 20 g/1 of sucrose + 2 g/1 of plant peptone + 1 mg/1 of kinetin + 1 mg/1 of naphthalenacetic acid + 0.2 mg/1 of indolacetic acid. Following fermentation for such time necessary to obtain an adequate multiplication of the cell cluster (from now on referred to as "clone"), a time generally comprised of between 10 and 15 days, the amounts of the metabolite of interest are determined for each clone. The analytical methods used will be generally these described in the previous phase and will preferably be rapid methods, such as HPLC analysis and ELISA tests. The analytical determination allows the ^■ selection of the clone with the highest content of the metabolite of interest.

A portion, typically 1 ml, of the selected ^" clone suspension in liquid medium is removed and placed on solid medium. The deposition must be carried out in a uniform way preferably by covering the whole available surface. The solid medium is the same as that used above .

Following an adequate culture time, . generally comprised of between 10 and 15 days, the new cell clusters (genotypically and phenotypically homogeneous clones) formed will be removed and inoculated into individual liquid media where the fermentation' takes place. Also in this case, following an adequate time, the levels of metabolites of interest will be determined, so as to select the clone which allows the greatest production of the same.

The operations described above can be repeated until a clone of the plant cell line, in which the productivity of the metabolite of interest is the highest, is selected.

It is to be noted that the alternation of culturing on solid and liquid media is essential for the clonal selection procedure of the present invention. In fact, culture on solid medium allows one to obtain cell clusters (clones) distinct and easily separable. These clones contain predominantly cells originating from a phenotypically homogeneous group, which thing allows the selection of the desired clone. Vice versa, the culture of the clone in liquid medium allows one to obtain a sufficient quantity of cells to carry out the chemical analysis of the metabolite content - in other words, performing an "amplification" of the metabolic content of the cell line - and therefore to select the most suitable clones for the production phase. It is also to be noted that the undifferentiated plant cells rapidly modify their metabolism due to the single gene mutations and changes in chromosomal euploidy which occur during culture. It is therefore essential that the clonal selection process described above does not end' with the identification of the most active clone, but is constantly repeated so as to keep the selected clone phenotypically homogeneous.

From what has been said, it is also clear that the increase of the metabolic content of the clone does not just allow the easy evaluation of the metabolic content of interest, but also allows the determination of . the presence of metabolites, either new or known, but of minor quantity in cell products. The amplification in liquid medium can in fact raise beyond the threshold of analytical detection, the content of these metabolites. It is therefore evident that the same steps described above for clonal selection can at the same time be used to define a screening method for cell lines suitable for producing metabolites of industrial interest. Therefore, a screening method for plant cell lines suitable for producing metabolites of industrial interest, constitutes a further object of the present invention.

FERMENTATION PHASE The plant, cell line selected as described above is therefore multiplied to obtain a sufficient quantity of biomass to carry out the productive fermentation phase. Such quantity will depend on specific production requirements, on the characteristics of the plant cell line typology used and on the type of metabolite it is desired for production.

The growth of the selected plant line is carried out in successive stages working^' in suspension in liquid medium. A suitable culture medium is that described above and more preferably is constituted of Gamborg B5 + 20 g/1 of sucrose + 2 g/1 of plant peptone + 1 mg/1 of kinetin + 1 mg/1 of naphthalenacetic acid + 0.2 mg/1 of indolacetic acid. The weight ratio of the initial inoculum with respect to the volume of the liquid medium is preferably comprised of between 1:15 and 1:35, more preferably around 1:25.

The biomass thus obtained can be passed directly into the final fermenter, or can be subjected to one or more further growth phases in liquid medium, working with intermediate volumes .

The final passage into the fermenter takes place with an inoculum of the cell line suspension equal to 5- 30% of the total volume of the starting medium, typically around 15%. The total volume of the biomass thus formed will depend, as stated above, on the specific requirements and will^' be generally comprised of between 10 L and 100 L.

The conditions under which the fermentation is performed are extremely critical. The fermentation will be normally carried out at a temperature comprised of between 15°C and 35°C, typically around 25°C and for a time normally comprised of between- 7 and 40 days, preferably between 14 and 21 days. It is essential that the biomass is adequately aerated and that at the same time is stirred by means of stirring means external to the fermenter. It has been in fact noted that plant biomass is comprised of cells with walls poorly resistant to rupture. A stirrer submerged into the biomass acts mechanically on the cells and can easily cause their lysis. It is nevertheless necessary that the stirring, albeit delicate, is efficient, above all in the final stage of the fermentation when the biomass greatly increases its density. Stirring means particularly appropriate for the purposes of the present invention are orbital stirring means . These stirring means operate preferably at 40-200 rpm, more preferably at around 120 rpm.

It is appropriate that the volume of the container (fermenter) in which the fermentation is carried out is considerably greater than the volume of the biomass. Typically, the volume of the reactor will be from 50% to 200% greater than the volume of the biomass.

As said, an efficient fermentation development requires adequate oxygenation. The oxygenation is normally carried out with sterile air with a flow rate of 0.5-4 L/minute, more preferably 2-2.5 L/minute, for a volume of 10 L of biomass. Alternatively, gas mixtures can be used containing from 10% to 100% v/v of oxygen.

As said above regarding stirring, also an oxygenation with over violent bubbling can provoke the rupture of the cell walls. It is therefore necessary to ensure that the oxygenation is carried out in a delicate manner, for example by applying appropriate bubble diffusers. It will be preferable to use means of diffusion of air or oxygen with a flow rate at the nozzle exit comprised of between 10 m/min and 600 m/min, more preferably between 50 m/min and 350 m/min.

Also the shape of the fermentation chamber assumes notable importance. It is in fact recommendable that it has a smooth and uniform surface, that is does not contain sharp edges, corners or parts which can provoke the rupture of the biomass cell walls.

As the fermentation proceeds, the biomass increases its nutritional requirements . It has been seen that an efficient biomass productivity can be obtained by adding a solution of sucrose portionwise during the course of the fermentation process. Typically, 70 to 130 ml are added, preferably 100 ml, of a sterile solution of 60% w/v sucrose per day for each 10 L of biomass. The first addition will take place preferably on the third or fourth day from the beginning of the fermentation process.. Six sucrose additions will normally be carried out.

According to a particular embodiment of the present invention, additives to augment the solubility of oxygen in water will -be added to the biomass. Such additives will be preferably selected from these substances defined as "artificial blood" , for example the perfluorinated hydrocarbons (PFC) . PFCs, introduced for clinical use at the beginning of the ^x 80s, are aromatic or aliphatic chemical compounds in which all the hydrogen atoms are substituted by fluorine. They are biologically inert and virtually insoluble in aqueous solutions, whilst the solubility of 0₂ in PFC results as being from 10 to 20 fold greater than in water. Of course, other types of additives which perform the same function can also be used. It will be preferable to effect the addition of such additives in the final stage of the fermentation, typically in. the last 2-3 days, i.e. when the biomass occupies a preponderant part of the total volume of the suspension and the reduced amount of water is not capable of ensuring the correct oxygenation of the biomass .

In the last days of the fermentation phase, in fact, a critical parameter is the oxygen solubility in the biomass, in as much as the percentage by weight of fresh cells reaches^" values as high as around 75-85%. To increase the solubility of oxygen in the cellular suspension on the tenth or twelfth day of fermentation (preferably on the twelfth day) one can hence add substances with elevated oxygen solubility, such as for example:

-HO (CH₂CH₂0) ₇₅ (CH (CH₃) CH₂OH) _so (CH₂CH₂0) ₇₅H (PLURONIC F68) at concentrations of 0.005%-0.05% (preferably at 0.01%); -octadecafluoro decahydronaphthalene

(PERFLUORODECALIN) at concentrations of 0.0005%-0.005% (preferably at 0.001%).

The addition of these substances increases the 5 oxygen dissolved and available for biomass growth in the last days of the fermentation process and favours the increase of secondary metabolite productivity.

It is evident that the fermentation procedure herein outlined includes such characteristics to render

10_. it successfully useable also on classical plant cell lines, i.e. not selected by the clonal selection procedure of the present invention.

A further object of the present invention is therefore a fermentation procedure and a fermentation 15 reactor as outlined in the preceding description.

The present invention will now be described in a non limiting exemplifying manner by the following examples .

EXPERIMENTAL PART 20 A . Determination of the content of secondary metabolites in .plant cell cultures

Example Al - Content of phenylpropanoids A and B in plant cultures of Ajuga reptans

The secondary metabolites are extracted from the 25 cell culture using a solution of 50% ethanol and homogenised for 2-5 minutes with an ultraturax.

The ethanolic extract obtained is analysed by HPLC with a gradient system of water/phosphoric acid 0.01 N and acetonitrile (flow rate 1 ml/min) . The phenylpropanoids have retention times : 4.8 min. for FpA 6.2 min. for FpB

Example A2 - Content of verbascoside in cell cultures of Olea europea The verbascoside is extracted using a solution of 50% ethanol and homogenising the cells for 2-5 minutes with an ultraturax. The ethanolic extract is analysed by HPLC with a gradient system of water/phosphoric acid 0.01 N and acetonitrile (flow rate of 1 ml/min) . The retention time of verbascoside is 5.4 min.

Example A3 - Content of verbascoside in cell cultures of Siringa vulgar is

The verbascoside is extracted using a solution of 50% ethanol and homogenising the cells for 2-5 minutes with an ultraturax. The ethanolic extract is analysed by HPLC with a gradient system, water/phosphoric acid 0.01 N and acetonitrile (flow rate of 1 ml/min) . The retention time of verbascoside is 5.4 min.

Example A4 - Content of verbascoside in cell cultures of Appia citrobara The verbascoside is extracted using a solution of 50% ethanol and homogenising the cells for 2-5 minutes with an ultraturax. The ethanolic extract is analysed by HPLC with a gradient system, water/phosphoric acid 0.01 N and acetonitrile (flow rate of 1 ml/min) . The retention time of verbascoside is 5.4 min. B . Stabilisation of a plant cell line Bl. Stabilisation of cell cultures of Ajuga reptans Ajuga reptans cells with high rates of cellular proliferation (obtained as described previously in the ^•paragraph regarding the induction of callus tissue) have been stabilised using the following procedure: high growth rate soft calli (around 2 g) have been transferred to Petri dishes with a diameter of 90 mm, containing solid medium comprised of 0.9% agar in Gamborg B5 + 20 g/1 of sucrose + 2 g/1 of plant peptone + 1 mg/1 of kinetin +' 1 mg/1 of naphthalenacetic ^•acid + 0.2 mg/1 of indolacetic acid. The Petri dishes have been incubated at 25°C for 12-15 days, the well grown and piiable cells -have been transferred onto fresh solid culture medium and incubated under the same conditions as described previously. After 10-15 repeated transfers, the cells presented a phenotypic appearance which remained constant with time. Following extraction of the cellular preparation with 50% (v/v) ethanol, homogenisation with an ultraturax and centrifugation to separate the supernatant, the ethanolic extract was analysed by HPLC to determine the ' phenylpropanoid content (500-600 μg/g fresh cells) , which was substantially unchanged with respect to the previous transfer. The total phenylpropanoid content is expressed as μg/g of fresh cells, in as much as the extraction is performed directly from the cells grown on solid medium. The chromatographic profile also remained constant indicating the maintenance of a characteristic secondary metabolism.

B2. Stabilisation of a cell culture of Olea europeae Olea europeae cells with a high rate of cellular proliferation have been stabilised following the procedure described in example Bl.

Following 15-20 repeated transfers the cells presented a constant phenotypic appearance and constant HPLC analysis profile. The verbascoside content was equal to 300-400 μg/g of fresh cells. The verbascoside concentration is expressed as μg/g in as much as the extraction has been performed directly on the cells grown on solid medium. B3. Stabilisation of a cell culture of Siringa vulgar is

Siringa vulgaris cells with high rates of cell proliferation have been stabilised following the procedure described in example Bl. Following 25-30 transfers the cells presented a constant phenotypic appearance and constant HPLC analysis profile. The verbascoside content was equal to 400-500 μg/g_. of fresh, cells. The verbascoside concentration is expressed as μg/g in as much as the extraction has been performed directly on the cells grown on solid medium.

B4. Stabilisation of a cell culture of Appia ci trobara

Appia citrobara cells with high rate of cell proliferation have been stabilised following the procedure described in example Bl .

Following 8-12 transfers the cells presented a constant phenotypic appearance and constant HPLC analysis profile. The verbascoside content was equal to 1200-1500 μg/g of fresh cells. The verbascoside concentration is expressed as μg/g in as much as the extraction has been performed directly on the cells grown on solid medium.

C . Example of clonal selection CI. Clonal selection of a cell culture stabilised from Ajuga reptans

The stabilised cell line obtained according to the procedure in example Bl has been subjected to clonal selection in the following way: from a Petri dish containing cells of 10-15 days growth have been removed a quantity equal to 0.4-0.8 g of cells, which have then been inoculated into 25 ml of liquid medium constituted of Gamborg B5 + 20 g/1 of sucrose + 2 g/1 of plant peptone + 1 mg/1 of kinetin + 1 mg/1 of naphthalenacetic acid + 0.2 mg/1 of indolacetic acid, contained in a 100 ml flask.

From each Petri dish 20 flasks containing different cells from Ajuga reptans originating from the same Petri dish have been set up. In this way the cell population of the dish was subdivided into 20 groups.

The flasks were then incubated at 25°C for 14 days. At the end of' the fermentation period, from each flask, 1 ml of suspension was removed which was later deposited onto the surface of a Petri dish containing solid culture medium. The dish was incubated at 25°C for 14 days .

The remaining suspension was used for the extraction of the phenylpropanoids . The sample was extracted with 5 volumes of 50% (v/v) ethanol, homogenised with ^' an ultraturax and centrifuged to separate the supernatant. The ethanolic extract was analysed by HPLC to quantify the concentration of phenylpropanoids from the various clones (the clones analysed presented concentrations varying from 200 to 1100 μg/ml of phenylpropanoids) . The concentration of the total phenylpropanoids is expressed as μg/ml in as much as the extraction was performed on the cell suspensions.. The clone containing, the highest phenylpropanoid concentration (1100 μg/ml) was used to establish a further 20 cell suspensions and to perform a further cycle of clonal selection. The cells to establish the 20 suspensions were removed from the Petri dish inoculated with 1 ml of the corresponding clone.

Following 6 cycles of clonal selection, the titre of the most productive clone had reached 4000 μg/ml of total phenylpropanoids .

C2. Clonal selection of a cell culture stabilised from Olea europeae

The stabilised cell line derived from the procedure described in example B2 has been subjected to clonal selection as described in example CI .

Following 3 cycles of clonal selection, the titre of the most productive clone had reached values equal to

1500 μg/ml of verbascoside (the concentration is expressed as μg/ml in as much as the extraction has been performed directly on cell suspensions) .

C3. Clonal selection of a cell culture stabilised from Siringa vulgaris

The stabilised cell line derived from the procedure described in example B3 has been subjected to clonal selection as described in example CI .

Following 2 cycles of clonal selection the titre of the most productive clone had reached values equal to 900 μg/ml of verbascoside. C4. Clonal selection of a' cell culture stabilised from Appia citrobara

The stabilised cell line derived from the procedure described in example B4 has been subjected to clonal selection as described in example CI. Following 3 cycles of clonal selection the titre of the most productive clone had reached values equal to 1800 μg/ml of verbascoside.

D . Examples of plant biomass fermentation

DI. Fermentation procedure of a clone selected from Ajuga reptans for the production of phenylpropanoids

The most productive Ajuga reptans clones selected according to the procedure described in example CI are used to inoculate suspensions to be utilised in the scaled up fermentative processes The scaling up process is set up in the following manner:

10 g of cells (usable interval of between 8 and 15 g) are inoculated into 250 ml of liquid culture medium constituted by Gamborg B5 + 20 g/1 of sucrose + 2 g/1 of plant peptone + 1 mg/1 of kinetin + 1 mg/1 of naphthalenacetic acid + 0.2 mg/1 of indolacetic acid, contained in a 1 litre flask. The suspension is incubated at 25°C (usable interval 25-28°C) for 7 days and placed over an orbital shaker at 120 rpm (usable interval 100-120. rpm) . The suspension is used to inoculate 1.5 litres of liquid medium as defined above, contained in a 5 litre flask. The cell culture is then incubated under the conditions described previously and used to inoculate 10 litres of liquid culture medium contained in a fermenter with a capacity of 20 litres. The fermenter is maintained at 25°C, for 14 days, over an orbital shaker at 120 rpm, with aeration by gentle bubbling (obtained by use of -a diffuser) and with the addition of 50% sucrose solutions (in quantities equal to 100 ml for each addition) from the fourth to the tenth day of fermentation.

The biomass is homogenised with a homogeniser, filtered and from the filtrate is isolated the main active ingredient by absorption on absorption resin XA D4. The resin is eluted with ethanol. The ethanolic solution so obtained is evaporated under reduced pressure, the sediment is redissolved with water and lyophilised. One obtains 25 g of a mixture of phenylpropanoids with a purity of 70-80%. D2. Fermentation procedure of a clone selected from Olea europeae for the production of verbascoside

The most productive clones obtained through clonal selection described in example C2 are used for the scaled up fermentation process as described in example DI.

The biomass is homogenised using a homogeniser, filtered and from the filtrate is isolated the active ingredient by absorption on absorption resin XA D4. The resin is eluted with ethanol . The ethanolic solution obtained is evaporated under reduced pressure, retaken up in water and lyophilised. One obtains 10 g of a mixture of verbascoside with a purity of 65-75%.

D3. Fermentation procedure of a clone isolated from Siringa vulgaris for the production of verbascoside The most productive clones obtained through clonal selection described in example C3 are used for the scaled up fermentation process as described in example DI.

The verbascoside extraction is performed as described in example D2. At the end of the extraction process one obtains 9 g of verbascoside with a purity comprised of between 62% and 70%.

D4 - Fermentation procedure of a clone selected from Appia citrobara for the production of verbascoside

The most productive clones obtained through clonal selection described in example B4 are used for the scaled up fermentation process as described in example DI. The verbascoside extraction is performed as described in example D2.

At the end of the extraction process one obtains 16 g of verbascoside with a purity comprised of between 68% and 75%.

Claims

1. Method of cultivation of a plant cell line capable of producing metabolites of interest, comprising a cell line selection phase which includes the following cycle of operations: a) cultivating on solid medium, a predetermined stabilised plant cell line for a sufficient time to obtain the multiplication of said cell line to give substantially distinct cell clusters; b) taking from said solid medium said substantially distinct cell clusters and place each of them in a separate liquid culture medium; c) cultivating each of the said substantially distinct cellular clusters in said liquid culture medium for a time sufficiently long to allow the multiplication of said cellular cluster and the analytical determination of the primary and/or secondary metabolites produced by it; d) operating a qualitative and quantitative determination of the primary and/or secondary metabolites produced by each of said cellular clusters in said liquid culture medium; e) selecting the cellular cluster capable of producing the greatest quantity of said metabolite of interest; f) repeating . the cycle of operations according to steps a) , b) , c) , d) and e) on said cellular cluster selected according to step e) for a sufficient number of times until the quantity of said metabolite of interest produced by a selected cellular cluster and by the cellular cluster derived from a further cycle of operation of selection results substantially unchanged.

2. The procedure according to claim 1, wherein said solid and liquid culture media comprise a biologically efficacious amount of plant peptone.

3. The procedure according to claim 2 , wherein said plant peptone is present in said solid and liquid culture media in quantities comprised of between 500 and 4000 mg/1 of medium.

4. A procedure according to any of the claims from 1 ^'to 3, wherein said liquid culture medium has the following composition per litre of medium:

Ammonium sulphate 100-180 mg/L Calcium chloride .2H₂0 110-190 mg/L

Magnesium sulphate.7H₂0 200-300 mg/L

Magnesium sulphate.H₂0 0.5-1.5 mg/L

Potassium nitrate 1500-5000 mg/L Monobasic Sodium phosphate 80-190 mg/L Cobalt chloride 0.008-0.018 mg/L Copper (III) sulphate .5H₂0 0.050-0.200 mg/L Na molibdate.2H₂0 0.05-0.20 mg/L

Potassium iodide 0.20-0.60 mg/L

Zinc sulphate .7H₂0 0.5-1.5 mg/L Boric acid 0.5-3 mg/L

Myo-inositol 10-100 mg/L

Nicotinic acid 0.1-2.0 mg/L

Pyridoxine .HC1 0.1-2.0 mg/L

Thyamine.HCl 2-10 mg/L Sodium EDTA.2H₂0 5-50 mg/L

Iron (II) sulphate .7H₂0 5-50 mg/L Naphthalenacetic acid 0.1-5 mg/L Kinetin 0.1-5 mg/L

Indolacetic acid 0.01-1 mg/L Sucrose 5000-40000 mg/L

Plant peptone 500-4000 mg/L

Phenylalanine 50-600 mg/L

PH 5.0-6.4

5. A procedure according to any of the claims from 1 to 4, wherein said liquid medium is

Gamborg B5 supplemented by 20 g/1 of sucrose, 2 g/1 of plant peptone, 1 mg/1 of kinetin, 1 mg/1 of naphthalenacetic acid and 0.2 mg/1 of indolacetic acid.

6. A procedure according to any of the claims from 1 to 5, in which said solid medium comprises agar at 0.8-1% w/v of the medium as defined in any of the claims from 2 to 5.

7. A procedure according to any of the claims from 1 to 6, in which said culture time in said steps a) or c) is comprised of between 10 and 15 days.

8. A procedure according to any of the claims from 1 to 7, said procedure further comprising, prior to said step a) , a stabilisation phase of the plant cell line.

9. The procedure according to claim 8, in which said stabilisation phase comprises the following subphases :

(i) providing a plant material with an undifferentiated callus structure; (ii) cultivating said plant callus material on solid medium for a time sufficient to obtain the multiplication of said plant material and the analytical determination of the primary and/or secondary metabolites produced by it; (i_.ii) operating a qualitative and quantitative determination of the primary and/or secondary metabolites produced by said plant material and a quantitative determination of the water content;

(iv) transferring of said plant material onto a solid culture^' medium for a time sufficient to obtain the multiplication of said plant material to give a cellular subculture and the analytical determination of the primary and/or secondary metabolites produced by it; (v) operating a qualitative and quantitative determination of the primary and/or secondary metabolites produced by said cellular subculture and a quantitative determination of the water content; (vi) comparing the amounts of said primary and/or secondary metabolites and of said water with the values determined in the preceding step (iii) and comparing the phenotypic characteristics of the plant materials obtained in steps (ii) and (iv) ; (vii) repeating the operation of the subphases (iv) , (v) and (vi) for a sufficient number of times to obtain a substantially constant content of said primary and/or • secondary metabolites and' of said water and a substantial invariability of the phenotypic characteristics of the cellular subculture, so as to obtain a stabilised plant cell line.

10. The procedure according to claim 9, in which said solid culture medium is as defined in claim 6.

11. A procedure according to claims 9 or 10, in which the number of said cellular subcultures is at least 10.

12. A procedure according to any of the claims from 9 to 11, in which said culture time in said subphases (ii) and (iv) is comprised of between 10 and 15 days.

13. A plant cell line obtainable by the procedure outlined in any of the claims from 1 to 12.

14. A fermentation procedure of a plant cell line for the production of at least one metabolite of interest, wherein said plant cell line is a clone obtainable according to the process outlined in any of the claims from 1 to 12.

15. The procedure according to claim 14, comprising the following steps :

A) inoculation of said plant clone into liquid medium and its multiplication for a time sufficient to obtain an increase of the cellular mass of at least 300% of its weight; B) optionally, transfer of the suspension obtained in step A) into fresh liquid culture medium and its multiplication for a time sufficient to obtain an increase i the cellular mass of at least 300% of its weight; C) optionally, repetition of step B) at least one more time;

D) transfer of the suspension obtained in steps A) , B) or C) into fresh liquid culture medium in a fermenter to give a biomass and conduct the fermentation in such conditions and for a time sufficient to obtain in said biomass the production of at least one said metabolite of interest;

E) separation of said at least one metabolite of interest from said biomass.

16. The procedure according to claim 15, wherein said steps A) , B) , C) and/or D) are carried out under stirring by stirring means external to said fermenter.

17. The procedure according to claim 16, wherein said external stirring means are of a orbital type.

18. The procedure according to claims 16 or 17, wherein said stirring means operate at 40-200 rpm, preferably between around 100 and around 120 rpm.

19. The procedure according to any of the claims from 14. to 18, wherein at least said step D) is carried out with aeration by bubbling through appropriate means of diffusion.

20. The procedure according to claim 19, wherein said aeration is carried out with sterile air and with a flow rate of 0.5-4 l/minute, preferably 2-2.5 l/minute, calculated on a volume of 10 litres of biomass.

21. The procedure according to any of the claims from 14 to 20, wherein said fermentation time in step D) is comprised of between 7 and 40 days, preferably between 14 and 21 days.

22. The procedure according to any of the claims from 14 to 21, wherein said step D) of fermentation is carried out at a temperature comprised of between 15° and 35°C.

23. The procedure according to any of the claims from 14 to 22, wherein the percentage in volume of said cellular suspension with respect to the liquid culture medium in said step D) is comprised of between 5% and 35%, preferably around 15%.

24. The procedure . according to. any of ^" the claims from 14 to 23, in which said liquid culture medium is as defined in any of the claims from 2 to 5.

25. The procedure according to any of the claims from 14 to 24, wherein the initial volume of said biomass in said step D) is comprised of between 10 1 and 100 1.

26. The procedure according to any of the claims from 14 to 25, wherein said step D) is carried out in a fermentation chamber with smooth and uniform surfaces and devoid of asperities, corners or other parts which can provoke the rupture of the cell walls of the biomass. 5

27. The procedure according to any of the claims from 14 to 26, wherein in said step D) said biomass is supplemented with from 70 to 130 ml, preferably around 100 ml, _tof a sterile 60% w/v sucrose solution per day for each 10 litres of biomass, or with 0 a quantity of pure sucrose or in solution to the same equivalent.

28. The procedure according to claim 27, wherein said sucrose addition begins not prior to the third day from the beginning of said fermentation and 5 continues for 6 days.

29. The procedure according to any of the claims from 14 to 28, wherein in said step D) said biomass is supplemented with an efficacious quantity of an additive which increases the oxygen solubility in the 0 water.

30. The procedure according to claim 29, wherein said addition is initiated not prior to the third day from the end of said fermentation.

31. The procedure according to claim 29 or _5 30, wherein said additive is a perfluorinated hydrocarbon.

32. The procedure according to any of the claims from 29 to 31, wherein said additive is selected from: -HO(CH₂CH2θ)₇₅(CH(CH₃)CH₂OH)₃o(CH₂CH₂θ)₇5H (PLURONIC F68) at a concentration of 0.005-0.05%, preferably 0.01%, with respect to the volume of the biomass, and

-octadecafluoro decahydronaphthalene

(PERFLUORODECALIN) at a concentration of 0.0005-0.005%, preferably 0.001%, with respect to the volume of the biomass .

33. A plant cell line according to claim 13, wherein said plant cell line is a clone selected from a stabilised cell line of Ajuga reptans, Olea europeae, Siringa vulgaris or Appia citrobara .

34. A procedure according to any of the claims from 14 to 32, wherein said at least one metabolite of interest is a phenylpropanoid or verbascoside .

35. An apparatus for the fermentation of a plant biomass • comprising a fermenter fitted with a fermentation chamber with smooth and uniform surfaces and devoid of sharp edges, corners or other parts which can cause the rupture of the cell walls of the biomass and of stirring means external to said fermentation chamber, said stirring means being preferably of orbital type; said fermentation chamber comprising means for the bubbling of air or oxygen into the biomass, said means for the bubbling of air or oxygen being preferably fitted with means of diffusion of said air or oxygen with the flow rate at the exit of the nozzle comprised of between 10 m/min and 600 m/min, preferably between 50 m/min and 350 m/min.

36. A method of screening of plant cell lines capable of producing at least one metabolite of interest, comprising the following operative steps: a) cultivating on solid medium a predetermined stabilised plant cell line for a time sufficient to obtain the multiplication of said cell line to give substantially distinct cellular clusters; b) taking from said solid medium said substantially distinct cellular clusters and placing each of them in- a distinct liquid culture- medium; c) cultivating each of said substantially distinct cellular clusters in said liquid culture medium for a time sufficiently long to allow the multiplication of said cellular cluster and the analytical determination of the primary and/or secondary metabolites produced by it; d) operating a qualitative and quantitative determination of the primary and/or secondary metabolites produced by each of said cellular clusters in said liquid culture medium; e) selecting the cellular cluster capable of producing said at least one metabolite of interest different from the primary and/or secondary metabolites known for said plant cell line.

37. The screening method according to claim 36, in which said steps a), b) , c) and d) are carried out as described in any of the claims from 2 to 7.

38. A liquid culture medium for plant cell lines characterised by the fact of comprising a biologically efficacious amount of plant peptone, as described in any of the claims from 2 to 5.

39. A solid culture medium for plant cell lines as described in claim 6.

40. A fermentation procedure for a plant cell line for the production of at least one metabolite of interest, comprising the following steps: A) providing an apparatus for the fermentation of plant biomass as described in claim 35;

B) inoculating said plant cell line into a liquid medium and its multiplication for a time sufficient to obtain an increase in the cellular mass of at least 300% of its weight; C) optionally, transferring the suspension obtained in step B) into fresh liquid culture medium and its multiplication for a time sufficient to obtain an increase in the cellular mass of at least 300% of its weight;

D) optionally, repetition of step C) at least one more time;

E) transfer . of the suspension obtained in steps B) , C) or D) into fresh liquid culture medium in said fermentation apparatus to give a biomass and conduct the fermentation under such conditions and for a time sufficient to obtain in said biomass the production of said at least one metabolite of interest;

F) separation of said at least one metabolite of interest from said biomass.

41. The procedure according to claim 40, said procedure being carried out under conditions described in any of the claims from 15 to 32.