CA1045067A - Cultivation of plant cells - Google Patents

Abstract

Abstract of the Disclosure Plant cells starting from species of plants classed in the branch of phanerogamic plants are cultivated in vitro on a culture medium comprising as principal source of organic carbon, lactose or one of its natural origin forms. Further plant cells capable of growing normally in vitro on lactose-containing media may be obtained by adaption or selection.

Description

~1~)4S~i7 The present invention relates to carbon nutrition of in yi_r_ cultures of plant cells using lactose and its natural origin forms, in particular residues from the dairy industry. The plant cells in question are those of the group ~
of species classified in the branch of phanerogamic plants. - -It is known that plant cells can live and multiply in vitro in undifferentiated form and indefinite fashion. These cells in dividing give rise either to isolated elements or to -small cellular masses (of about 2 to 100 cells) or again to larger forms currently called "cals" which have variable morphological characteristics. The plant cell cultures require a nutritive solution which can be liquid or solidified by agar-agar and which should contain mineral salts, growth substances, vitamins etc. and at least one source of organic carbon. This is necessary since generally plant cells cul-tivated in vitro, even chlorophyl-containing ones, do not achieve sufficient photosynthesis for their carbon nutrition.
To mitigate this situation one uses principally carbohydrates, an acid or an alcohol being sometimes usable. The most commonly used organic carbon sources are glucose or saccharose.
In any case whatever the source of carbon it only permits growth if the cultured cells possess, and by environmental induction derepress, the necessary genetic information for !,;"'.' its translation into a form compatible with its incorporation into the general metabolic cycle. The use of any new source of organic carbon is thus conditioned by the presence and ;~
derepression of the necessary information for this transformation. -Certain researchers have tried to use lactose but their efforts have remained practically without success. One may for example note among others:
1) According to the work of R. ~. GAUTHBRET (La ~y~h ~'~

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S~367 culture ~e tissus vegetaux, M~SSON, Paris 1959), - E. BALL, on Sequoia sempervirens, showing that "when the medium comprises mannose or lactose, these sugars simply add on to those contained in the colonies", - GAUTHERET himself working on normal carrot tissue (Daucus carota L.) and concluding "the saccharose constitutes the best source of carbon; it is followed closely by glucose, maltose and raffinose; then come fructose and galactose, then mannose and lactose which are clearly less efficacious"~
2) On the other hand in a recent publication, M.~.
MAT~ES and his coworkers (Plant and Cell Physiology 14, 797-801 (1973)) only found limited growth of cells of maple (Acer pseudoplatanus L.) in the presence of lactose.
Furthermore all the researchers who obtained weak growth on lactose observed their cultures after a single planting on this substrate. It is thus probable that the tissues spread using reserves accumulated elsewhere.
Obtaining substantial growth of plant cells on lactose media is thus only possible if one derepresses the 20 genetic information necessary for the utilisation of lactose -~
and in particular that permitting its hydrolysis that is to say the operon of ~-galactosidase. This derepression can be envisaged by adapting or by selecting sources.
The present invention can thus be realised by means of creating strains capable of growing normally on lactose media. This creation can be carried out either by obtaining primary cals directly on lactose or by progressive selection or not starting from strains growing on other principal carbon sources.
According to the present invention there is provided a method of obtaining a substantial yield of plant cells which 5~i7 comprises cultiYating in Yitro plant cells of a species of the branch of phanerogamic plants capable of growth based upon a lactose carbon source, said cells being obtained by adaption or selection of the plant cells of said branch, wherein said selected or adapted plant cells are administered lactose or a natural origin form of lactose as a principal source of organic carbon to obtain said substantial yield of plant cells.
The culture medium may comprise as principal organic carbon source the sugar lactose itself or a residue of the dairy industry such as lactose juice or milk ultrafiltrate or a lactose serum. Preferably the culture medium comprises the conventional nutritive elements with the exception of ~-the principal source of organic carbon which is constituted by lactose or one of its natural origin forms. The culture medium may be solidified e.g. witn agar-agar or in liquid ; form. In the case of a liquid culture medium the cultivation is suitably carried out in a fermenter.
The invention also provides a method of obtaining, by adaptation or selection strains of plant cells starting from species of plants classified in the branch of phanero-gamic plants, plant cells capable of growing normally in vitro on lactose-containing media.
Strains of plant cells starting from species of Phanerogamae plants which are capable of growing normally in vitro on lactose-containing media may be obtained by adaption or selection. Thus a strain of plant cells which are capable of growing normally in vitro on lactose-containing media may be obtained by adaption starting from a species of Phanerogamae plants by cultivating a primary cal of Phanerogamae plant cells in vitro directly on a lactose-containing medium. Alternatively, .
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5~67 a strain of plant cells which are capable of ~rowing normally in vitro on lactose-containing media may be obtained from a species of Phanerogamae plants by cultivating Phanerogamae plant cells in vitro on media of increasing lactose content.
This method may be carried out progressive or non-progressive selection. [Progressive selection comprises the successive pricking out of the strain on media in which the proportion of lactose is each time (i.e. progressively) increased. ~on-progressive selection comprises the direct pricking out of the strains from one medium to another.] In these ways new strains may be formed by modification of the genetic regulation of cells which new strains are able to be grown normally on lactose-containing media. The modification of the genetic information, obtained by adapting or selecting strains on,the lactose-containing medium, is the derepression of the genetic information necessary to use lactose. In other words, it is necessary to raise the blockade on ~-galactosidase operon which is present in the cells but which does not work) to enable cells capable of growing on lactose to be formed.
Industrial usage of cells obtained by the methods of the invention can be as diverse as that of cells or tissues of the same species cultivated in vitro in conventional manner (generally on glucose or sucrose) or of the plants from which they issued. Thus these methods can be used in the production of materials for exarnple starting from leguminous materials, proteins for example starting from lucerne or soya, alkaloids starting in particular from belladona, periwinkle or poppy, blucosides for example starting from fox glove and in general any product of vegetable origin comrnonly obtained starting 30 from a plant classified in the branch of phanerogamic plants: , crude vegetable material, proteins, enzymes, essences, ~; . . .

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5~7 flayours, pigments, steroids, alkaloids, flavones~ ~lucosides, antibacterial, antiviral or antitumoral compounds.
The invention is further illustrated by way of example in the following Examples.
The cultures which are described are carried out on nutrient media composed of conventional nutrient elements with the exception of the principal source of organic carbon. Thev contain (compare Examples 1 to 15):
- a solution of mineral macro-elements such as the HELLER solution or that of MURASHIGUE and SKOOG, - a solution of mineral oligo-elements such as that of HELLER or of MURASHIGUE and SKOOG, - a source of iron for example ferric chloride or ~-the well known compound FeEDTA, - a vitamin complex for example the solution indicated as Bl , - growth promoting substances (auxins, kinetins), - optionally other substances which have been less frequently used such as amino acids, immature coconut milk (cocos nucifera L.), yeast extracts, casein hydrolysates etc.
- a principal source of organic carbon which is according to the invention, pure lactose or lactose-containing composition obtained from natural sources, noteably the lac- ;
toserums, the lactose-containing juices or ultrafiltrates of milk and other residues from the dairy industry.
The relative proportions of the ingredients noted ~;
above and the culture conditions vary over a wide range as set out in the experimental details reported in the Examples.
The proportions of nutritient elements given therein are not 30 limitative and can be modified. In most cases poorly defined ;~
substances such as immature coconut milk which can entrain a 10~5')6'^~
sugar source interfering with the lactose should be avoided.
EXA~LE 1 Obtaining cals of ~ledicago sativa L.
(Papilionacea) and multiplication of the cals obtained Grains of lucerne of variety Europe (INRA No. G 1748) are sterilised in a solution of calcium hypochlorite (40 g/l) for 45 minutes in the presence of a wetting agent and under reduced pressure. Tlle grains are then subjec-ted to three successive rinsings with sterile water. Starting from this stage of the experiment all the operations on the vegetable material are carried out under rigorously aseptic conditions.
The treated grains are placed in sterile culture tubes containing one of the two following nutrient media:
1) KNOP medium Concentration in Components m~l of medium Calcium nitrate tetrahydrate500 Potassium nitrate 125 Magnesium sulphate heptahydrate 125 20 Monopotassium phosphate 125 Agar-Agar 7000 2) "Germination" medium Solution of mineral macro-elements, HELLER solution Concentration in mg/l of medium Potassium chloride 750 .; .
Sodium nitrate 600 Magnesium sulphate heptahydrate 250 ; 30 Monosodium phosphate monohydrate 125 Calcium chloride dihydrate 75 ;,',' .:
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HELLER solution of oligo-elements Concentration in Components mg/l of medium Zinc sulphate heptahydrate 1 Boric acid Magnesium sulphate tetrahydrate 0.1 Copper sulphate pentahydrate 0.03 Aluminium chloride hexahydrate 0.05 Potassium iodide 0.01 ; 10 Nickel chloride hexahydrate 0.03 Iron source Ferric chloride hexahydrate 1 mg/l of medium Carbon source Glucose 30 t 000 mg/l of medium Solidification agent Agar-Agar- 7,000 mg/l of medium.
The media are sterilised in an autoclave under a vapour pressure of 0.7 bar for 20 minutes.
After having deposited the grains on the surface of 20 the medium contained in the tubes, the tubes were sealed with Parafilm (Trade Mark - American Can Company, Neenah, Wisconsin) and placed in daylight in a room at normal temperature.
About 3 weeks after their having been germinated one ., f can see in the tubes small plants which have reached the two--~; leaf stage.
There are then removed aseptically from the plants ~' either root fragments or fragments of stalk, free of any primary or secondary bud or shoot, or of the cotyledons.
` These fragments are put in tubes on the following nutritive media denoted "lucerne starter medium".
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1~)45~7 Medium (1) A solution of mineral macro-elements of MURASHIGUE
and SKOOG
Concentration in Components mg/l of medium Ammonium nitrate 1650 Potassium nitrate 1900 Calcium chloride dihydrate 440 Magnesium sulphate heptahydrate 370 10 Monopotassium phosphate 170 Oligo-element HELLER solution identical to that for the germination medium Reference source of iron Fe EDTA
Concentration in Components mg/l oE medium Ethylene-diamino-tetra-acetic acid dihydrate 37.3 mg Ferrous sulphate heptahydrate 27.8 mg ~; Vitamin solution denoted Bl Concentration in : 20 Components mg/l_of medium .; Calcium panthotenate Pyridoxine ~ 1 Nicotinic acid 1 Thiamine Meso-inositol 10 ;~ Biotin 0.01 ~''' ''.
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45~7 - Growth modifiers 2-(214-DiGhloro-phenoxy)-acetic acid 1 mg/l of m~di~m 6-(Furfurylamino) purine (Kinetin ) 1 mg/l of medium Carbon source: Glucose~0,000 mg/l or medium 5 Solidification agent: Agar-Agar6J000 m~/1 of mediumO

There is added immature coconut milk at a concentr~tion of 10%, i.e. 100 ml/l of medium.
The medium was adjusted to pE 6.5 and sterilised in an autoclave under vapour pressure of 0.7 bar for 20 minutes.

M i ~
~ he second medium used for obtaining the primary lucern cals was strictly identical to the prec~ding medium with the ex-ception of the source of organic carbon.
~ he glucose o~ medium (1) was replaced ln medium (2) by lactose. Quantitatively, medium (2) had a concentration of lactose such that after total hydrolysis one would obtain theo-retically the same concentration of hexose as that in medium (1) from glucose.
~here are-thus introduced into the medium (2) ~0,000 mg/l of lactose monohydrate.
After i~planting on these media the different frag~
men~s of plant1 the tubes sealed with Parafilm are placed in perfectl~ ho~ogenous condi-tions at a temperature of abou-t 23'G and under a defined ~ umination as follows:
25 - luminous spectrum lamp type Sylvania Gro~L~, - luminous intensity of the order of 5000 lux, - light period : 16 h/day.

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~he.se conditions of temperature and illumination are those to which reference is made in t-he following Examples;
Abo-ut 4 to 6 weeks after putting the plant fragrnents on theculture, substantial spreading can be observed~
~atever the type of organ;(root, ste~ or cotyledon) used for obtaining the spreading or primary cals, equivalent success percentages are found on media (1) and (2).
Furthe~more the shape of these spreadings is substan-tially identical on the two ~edia. The appearance and the growth xhytl~ of the primar~ luce.rn cals is thus not modified upon replacing glucose by lactose in the nutrient media.
After having aseptically separated the primary growth from the fragment of the organ ~hich spawned it, the cals are multiplied and the strains obtained maintained on media re-spectively identical to those which served for obtaining the primary cal.
~he strains obtained on medium (2) had gro~h rates substantially equivalent to those of the sbrains maintained on medium (1).
~gain the form in which the organic carbon i5 brought to t~e lucern cell~ (glucose or lactose) does not seem to have had a direct influence on the speed of growth of these cells.

~XAMPIE 2 ~ cation , f. cals >

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In this second series of tests,experiments were carried out strictly identically to those of Example 1 with the excep tion of a change si~lultaneously made to media (1) and (2).
Liquid albumen from the immature ~ut of the cocopalm (cocos nucifera ~) or "coconut milk" is is now studied as growth inducer in nutrient media employed for vegetable tissue cultures.
Its composition and its role being poorly de~ined, the experiments of Example 1 were carried out again eliminating it from the nutrient media (1) and (2).
In these new conditions there was observed a rate of obtaining primary cals and the shape thereof substantially identical o~ media (1) and (2) themsel~es.
In the two cases the appearance of the primary cals was later than in the two tests of Example l; at the same time their growth appeared less rapid.
~ hese observations are confirmed after replanting and maintaining the strains.
Omitting the coconut milk has thus not led to any noteable difference between the cultures on medium (1) and those on medium (2). It has it seems si~ply and simultaneously diminished their efficaciousnessO

XAMPLE
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~Pa~ilioneacea ) on different mo mult~plicati~r o' tb al~

.' ' ' ' ~', , ` . . '' ' . ', ' . '. ' 5~67 ~, In this third series of tests,e~periments w~Ie carried out strictly identical to -those described in ExarQples 1 and 2 with the exception of a new modification made to the medium (2) of Examples 1 and 2.
~actose, used as the organic carbon source of medium (2) was replaced by,"lactose jui~,e" obtained b~ ultrafiltra-tion of milk, the kno~m composition of which ~asas follows:
water 2.3%
` ash 6.0%
tot-al nitrogen 0.68%
lactose ~8.6%
Qua~titatively to obtain the same concentration of ,,, lactose i~ th~se media as in the medillm (2) of th~ preceding Examples~ 33.86 g of lactose ~uice were introduced.
In these new conditions the observations of ~xamples 1 and 2 have beerl con~irmed i.e.~
- the medium (1) (glucose) and (2) (lactose juice) gave si~ilar results both for obtaining primary cals and for maintaining the strains iSSUil~g from these primary cals, - - th~ absence of coconut milk in media (1) and (2) simultaneousl~ diminishes their effectiveness but has not given ;
rise to any dif~erence between the results obtained with o~e , or the other.
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~XA~3 Ll j, Ada~tation of a strain of Me~ g~ iva I,.
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~P~ilionaceQ ) to a lactose ~medium ~here had previously been obtained and multiplied a ~'.

0~67 ~trai~ of lucern~ This strain,denoted re~erence R18,had the following characteristics:
It grows in sterile Pe~ri boxes placed in to.tal a~d permanent darkness in an enclosure tne temperature of which is 25~C.
Mo.rphologicall~ these cultuI~es are ~ello~rish and dis-socia-ted in very small cal~.
~ he growths are not morphogenic.
~ he nut~itive medium.used for maintaining it i~ referenced ~.E.l~ It has the following composition:
Solution of macro-elements of ~ SHIGUE and SKOOG (cf.
Example 1) Solu~ion of oligo-element of MURASHIGUE and SEOOG
Concentration in Components ~E~ iu .
i5 Boric acid 6.2 Mangnesium sulphate mono~ydrate 19.6 Zinc sulphate tetrahydrate 8.6 Potassiu~ iodide 0.83 - Sodium Molybdate dihydrate 0.025 20 Cobalt chlorlde hexahydrate 0~025 Iron source So~ tion denoted Fe EDTA (cf. Example 1) Vitamin solution referenced Bl (cf. Example 1) Growth modifiers ----. 25 2-(2,4-~ichloro-phenoxy)~acetic acid0.5 mg/l of medium 6-Furfurylamino-purine0.1 mg/l of medium Orga~ic carbon source .~.
Saccharose 30~000 mg/l of medium .

~ 14 04S~36~
Solidif:ication ag~nt Agar Agar 60,000 mg/l of medium ~ he medium was adjusted to pH 6 and sterilised in an auto-elave under vapour pressure of 0~7 bar for 20 minutes. The re-plantin~ ,rh~thm was 6 weeks~
~ he pree~ding medium (L.E~l) was changed into medium L.E.2 by replacing the saccharose by lactoseO ~anti-tatively the mediu~ L.E. 2 has a eoncentration of lactose strictly iden-tical to that of saccharose of the medium l.,E.l. '~here is thus introdueed 31~600 m~ of laetose monohydrate per litre of medium L.E.2.
After 6 weeks growth i~ these conditions it was noted that strain R 18 had undergo~e substantial grow-th on medium ~o~2 but sli~htly less than that of the same strain on medium 15 [J~
Despite this differenee the two strains were regularly replanted on their respective medium. After 4 OX 5 replanting eycles, this differenee in the growing rates was no longer observable. me R 18 strain of lueern has thus modified and 20 adapted progressively'in such fashion that it ean consume the -''~
laetose with a real equivalence to that of its previous eonsumption of saccllarose.
~ EX~MPLE 5 Obtain n~ Gl~cina hlspids ~ __cea ) and ~ ir~ b~ eal~ .b ~a1n ed In the same eonditions as for the lucern seeds of Example 1, INRA origin soya grains were placed in sterile germinatio~.

5~S7 About 3 weeks later very.developed plants l~e:ce ob-served occupying the whole of the f~ volume of the tubes.
The cotyledons of these small plants were removed 5 aseptically and pl.aced in the tubes on the following nutritive media:
1) Medium Sl Solution of mineral macro-elements Concentration in Components ~Dm Potassium nitrate~ 2500 ~ :
Ammonium sulphate 134 Monosodium phosphate monohydrate 150 Mag~nesium sulphate heptahydrate 250 Calcium chloride dihydrate 150

3 15 ~olution of mineral oligo-elements .
Concentration in Gomponents m~ f medium Manganese sulphate monohydrate 10 I Bor~c acid 3 J Zinc sulphate heptahydrate 2 . 20 Sodium molybdate dihydrate . 0.250 : Copper sulphate pentahydrate 0.025 : - Cobalt chloride heptah~drate 0.025 Potassium iodide 0.750 - I~on source Solution de~oted Fe EDTA (cf. ~xa~ple 1) o~n~ 4 Vita~in soluti . . Conce~tra~ion in ,~
Co~p,onents, , ~ d_ m Mesoinositol 100 Nicotinic acid 5 Thiamine . 10 Pyridoxine Growth modifi.er 2-(2-Dichloro-phenox~)-acetiC acid5 ~g/l of m~dium Source of organic carbon Saccharose 20~000 mg/l of medium Solidification.agent7~000 mg/l of medium, The pE was adjusted to 6 if necessary and the mediu~ .
sterilised in an autoclav~ under a vapour pressure of 0~7 bars for 20 minutes ., ~:) Medium S2 .:
Medium S2 is strictly identical to medium Sl with the .
exceptio~ of the source of organic carbon. . ' The saccharose of medium Sl was re~laced b~ laetose iD
medium S2.
Quantitati~ely the medium S2 has a lactose concentra~
tion strictly identical to that of sacch~rose of the medium S1.
Ihere was therefore introduced 21tO50 mg of lactose mo~ohydrate per litre of mediu~ S2. ~ u,, - ~he tubes were placed in culture conditions similar to those of Example 1 for the lucern cultures.
About 4 to 6 weeks after they were put to culture '.' primary growths developed at the edge o~ the cotyledons are ,.... . . - 17 - .
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1~5¢~6~7 observedO Th~ speed of the appearanc~ of these growths a~ld their shap~ are substant~ly equivalent on media Sl and S2.
~he rate of appearance and the growth rhythm of the primary cals of soya thus do not seem to be modified if the saccharose is replaced by lactose in the mltritive media~

Adaptation of a stxain of Vinca minor ea ) to the lactose medium There was obtained and kept a substan-tial population of strains of small periwi~kle. ~here strains are dlfferent by various morphological or physiological characteristics.
One of these strains,denoted 3g has the following r characteristics:
It was grown in tubes on a semi-solid medium in the temperature and illlmination- conditions defined in Example 1~
Morphologicall~ the cals have the following appearance:
- they are white and thus containing absolutely no chloroph~l, - they are not morphogènic9 _ they are strictly not dissociated, they are relatively little friable~
The nutrient medium used for the Maintenance of this strain,referenced VOl,had the following composition.
Solution of mineral macro-elements o~ ~R (cf. Example 1) 25 Solution of mineral oligo-elements of ~ELIER (cf~ Example 1).
Iron source Ferric chloride hexahydrate 1 mg/l of medium . ~ , ..

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Vitamin solutlon denoted ~1 (cf. Example 1) Growth modifier 2-(2,4-Dichloro-phenxoy)-acetic acid0.1 mg/l of medium Carbon source Glucose 30.000 m~/l of medium Solidification agent Agar-Agar 7~000 mg/l of medium The medium is adjusted to pH 5.6 if necessary and it is sterilised in an autoclave under a vapour pressure o~ 0.7 bar ~or 20 minutes.
The replanting r~m is of 6 weeks.
The V01 medium was modified to a V02 medium by replacing the glucose by lactose. Quantitatively the V02 medium has a lactose concentration such that after total hydrolysis one ob tains theoretically a concentration of hexose iden-tical to that from the glucose of medium V01.
There was thus introduced into medium V02 30tO00 mg/l of lactose monohydrate.
After the noxmal growth cycle,i.e. 6 weeks, the following facts were observed:
- no cal of the culture of V02 had a necrose, - no cal cultivated on V02 was morphologically distinguished from those cultivated on VOl, - - the average growth of the cals cultivated on V2 seemed slightly less than that of cals cultivated on VOl. ~he increase in fresh weight with respect to the starter implant is of the order of 150~o in the first case and 200% in the second.

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5'~67 ` ' ~he strains were repl~n~ed on the same media and in the same conditions for several growth cycles. After ~ or .-5 successive replantings there is no longer a notable differ-'ence between the gro~7th rates of the strain 3 cultivated on . 5 medium V01 and that of the same strain adapted to medium V02.
Strain 3 has thus become modified in such a way that it can consume lactose as a source of organic carbon with a yield equivale~t to that of its previous consumption of ~lucose.

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~XhMP~_7 Adaptation b,y select~on of a strain an,acea) to lactose med.ium Among the population o~ strains of small periwinkle mentioned in Example ~ the strain referenced 13 was retained to carry out new use tests for lac~ose for vegetable tissue cul-turesO
This strain has the following characteristics:
It is gro~n in the same conditions as strain. 3 of ~xample 6.
Morphologically the cals have the appearance described belo~: ' - they are very intense green and thus strongly chlorophyl~contai~ing, - they are not morphogenic, ---25 - they are stri~tly non dissociated9 - they are much more friable than those of strain 3 of Example 6.

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5'~7 The nu~rient medium on which the~ are maintained is exactly tha sarne as that used for strain 3 of ~xample 6~
~he replanting ,rhythm o~ this strain 13 is ~ weeks.
As for the tests described in ~xample 6, cals of strain 13 are placed simultaneousl~ on meclia VOl and V02.
After 6 weeks growth the follo~ing ~acts are observed - 80% of the cals grown on V02 have necroses, - certain of the cals cultivated on V02 are morpho-' logically different fro~ those cultivated on VOl (as concerns the chlorophyl state,for example), ~ - the growth of non-necrotic cals cultivated on ,, V2 is very clearly less than that of cals cultivated on VO~
Despite these results the non-necrotic cals cultiva~ed on V02 were replanted on the same medium for se~eral growth ,;~
cycles. Figure 1 shows the evolution of the necrosii~i rates of the cals cultivated on V02 in dependence on successive re- `
plantings. One can observe o~ this graph that from the fifth "' growth cycle on V02 the percentage of necrosis is substantiall~ ' identical to that;observed at the same time for strain 13 cul-ti~ated on VOl i~e. of the order of 3%.
At the end of the third growth cycle,it was possible to separate on morphological criteria (the chlorophyl state) and physiological criteria (average growth rate) the cals issuing from strain 13 cultivated on V02 into three new strains which were ~'' denoted 13(1), 13(2) and 13(3). '''~''~
- 13(1) was a chlorophyl-containing source of high growth rate, - 13(2) was a chlorophyl-containiDg source of average growth rate, , -' ' S~367 . ~
- 13(3) was a non-chloroph~l-containing source of low ~rowth rate.
Study of the growth rates in the course of the fourth replanting cycle of strain 13 cultivated on VOI and of strain 13(1) cultivated on V02 gave the followin~ results:
- l~e average result- i~ fresh weight during a replanting cycle ~Ias with respect to the implant of:
265% for the strain 13 cultivated on VOl and 310% for the strain 13(1)cultivated on V02.
~he evolution of the water content of the cals in the course of the growth cycle was identical in these two cases and furthermore practically negligible.
A classical statistical test to compare averages, calledthe "t" test , showed that these t~o average growth percentages were significantly different with a probability ~ = 0.01.
~On the other hand ~igure 2 shows ~hat the distribution of growth rates as a function of the weight of the implant was subs~antia].l~ different for the strain~l3(1) cultivated on V02 from thatfcr strain 13 cultivated on medium VOl.
By a selection.o~ a five replanting cycle one has therefore obtained a small periwinkle strain (13(1~) capable of utilising lactose as a pri~cipal source of organic carbo~
with a yield superior to that of its previous use o.f glucose.

EXAMPI~
~ tation O.r sources of Vinca minor L.
~ ed lactose medium ___ _ I~ Example 7 strain 13 was defined by growing on medium VOl and strain 13(1)by growing on medium V02.

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~5~167 . ., An at-tempt was made -to cultivate these two st-rains on the following medium,referenced V03.
Medium V03 is exactly identical to media V01 and Y02 with the exception of the source of organic carbon. The glucose of V01 or lactose of V02 ~ras replaced in the m~dium V03 by lactose duice that is to say by an ultrafiltrate of milk~the known composition of which was described i~ Example 3.
Quantitatively to obtain in V0~ a concentration of theoretically available hexose identical to that in V01 or to that in V02 after the potential hydrolysis of the lactose ~here were introduced 33~860 mg of lactose juice per ~itre of medium V03.
~or the two strains 1~ and 13(1) there~Yere observed selection phenomena (evolution of the percentage of necrosis) substantially identical to those described in Exa~ple 7~
The only not~ble difference was that the return to the necrosis rates of strain 1~ on medlum ~l (about 3%
j ~las more rapid during the adaptation of strain 13(1) to V03 `~
than during the adaptation of strain 13 to the sa~e ~edium.
After 4 or 5 replanting cycles the average rates of growth of the strains 13 and 13(1) on V03 were equivalent to those of strain 13 on V01 or of strain 13(1) on Y02 re-respectively.
, 25 The replaceme~t of glucose or lactose by lactose juice ; in the nutrient media V01 and V02 is thus5 after selection, without effect on the utilisation yield of the source of organic carbon b~ the strains of small periwinkie defined and classi~
fied under ref`erence 13.

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SC~67 " ~XAI`lPI~ 9 _ Adaptation of strains of Vinca ~inor ~.
medium dlfferentl~ modified In Examples 7 and 8 the strain 13 was defined growing on medium VOl~the strain 13(1) growing on medium VO2sand strains 13 and 13(1) growing on medium V03.
A-ttempts were made to cultivate these strains on the following medium reference V.04.
Medium V04 is exactly identical to media V01, V02 and V03 with the exception of the principal source of organic carbon. ~he glucose of VOl, the lactose of V02 and the lactose juice of V03 were replaced in V04 by a lactose serum the main composition of which is the following:
Iactose from 7~.7 to 81.6%
Salts from 8.65 to 9.6%
Acetic acid from 2.4 to 3.84%
Nitrogen materials from 1-9? to 2.88%
~at materials from 0.144 to 0.173%
Water from 4 to 5~
Quantitatively to obtain, in V04, a concentration of theoretically available hexose identical to that in V01 or to that in V02 and V03 after substantial hydrolysis of the lactose there was introduced 37~190 mg of lactose ser.um per litre of .
medium V04. The concentration was established on the basis f an average content of 80.65% of lactose in the samples of lactose serum.
~or all the four strains (13 on V01~ 13(1) on - 24 _ ";

- - -0~5~1~7 V02, 13 and 13(1) on V0~) cultivated o~ V04~ there was observed selectio~ phenomena (evolution of the percentage of necrosis) similar to those observed in ~xamples 7 and 8 It is however ~ecessary to note t~lo points:
In the experimen-tal conditions used the direct adapta-tion of strain 13 on V01 to medium V04 was i.mpossible;
As in ~xample 8 the return to -the necrosis rates of the strain 13 on V01 (about 3%) was more rapid during the adaptation of 13(1) on V03 to VOL~ than during the adaptation of 13(1) on V02 to YC4.
After L~ or 5 replanting cycl~s~the average growth rate of strain 13(1) on medium V0~ were substantially iden-tical to those of strain 13(1) on V02 or V03.
Replace~ent of lactose or lactose juiGe by lactose .
sexum in the nutricnt media V02 or V03 is thuslafter selection of the strains,without consequence on the utilisation ~ield of the ¦ :~
organic carbon source from the strain of small periwir~le de-fined and classified under reference 13(1). ~

. EXI~PIE 10 :~.
Adap ation of a strain of Vinca mlnor L.
~ eclium In ~xample 7 the obtaining and maintenance o~ strain 13(1~ of sm~l periwinkle was described the charactexistics of !1 .:
which are the following:
Growth in the same conditions as strain 13 of Example 7.
- Morphologically the cals have the following appearance:

~ 25 -1~4S~P~7 - they have a very intense green and thus strongly chlorophyl-containing, - they are not morphogenic, - they are strictly non-dissociated, - they are relatively friable.
This strain is maintained on the medium V02 defined in the course of Example 7.
Its replanting rhythm is 5 weeks i.e. one week lcss than that of strain 13. '~his fact is justified b~ growth of ~he strain 1~(1) being more rapid than that of strain 13 (compare Figure 2 in Example 7).
~his strain was cultivated on the two following media :
-V2 defined in E~ample 7 which was sterilised by autoclaving, -the same medium V02 but sterilised by membrane filtration.
After the normal culture cycle, ie. five weeks, it was ; impossible to distinguish the cals cultivated on the auto-claved medium V02 from those cultivated on the filtered V02, all their morphological and growth characteristics being iden-tical~

EX~MPLE 11 __ ____on of a strain o~ Vinca minor L. (Apoclynacea ) In the course of Example 7 the obtaining of strain 13 was defined,the characteristics of which are described in Example 10 .

.

,, . , , ~ ... ... ... . .... . .
, . . . . . ... . . .

~5~ ~7 ~ his strain l3(l) was replanted in tubes containi~g sterile V02 medium. ~he tubes were placed at about 25 in total and permanent darkness.
~ 'i~e weeks after culturing the following facts were observed:
- a~out 50% of the cals were necrotic, - the non-necrotic &als had a morphological aspect different from those of starting strain l~
they were whitish and thus practically nonrehloro phyl-containing and more dissociated~
The non-necrotic cals were then replanted regularly.
After 4 or 5 replanting cycles the percentage of necrosis ; was of the order vf 3%,i.e. substantially identical to that observed on original strain l3(l).
', 15 Cals of small periwinkle,defined and classifi~d as ;1 a reference strain l3(l),can thus,after adaptation,be culti-vated on a lactose medium in total a~d permanent darkness. ;~
~ he same test is carried out startin$ from strain 13 , maintained on medium V0l. It was possible on this strain ~ 20 and in the conditions of the medium to make the same observa-`-~ tions as were made in the preceding case for strain l3(l).' ~he adap~ation of cells of small periwinkle to growth in total and permanent darkness thus seems to promote ; identical phenomena whether it i5 a question of cells of strain 13 kept on a glucose medium or cells of-strain l3 kept on a lactose medium.
. .

., .
... . ..

~45~67 EXAMPI~ 12 Culture in li~uid l ctose medium of a strein of Vlnca minor I~ 7~ a_l--In ~amples 7 and 10 strain 13(1) was defined and characterised growing on a semi-solid lactose medium.
'~here was removed aseptically the lower parts of cals of strain 1~(1) that is to say those parts which were in con-tact with the semi-solid medium V02. ~hey are ~airly friable.
~hey are introduced into an ERLENMEYER flask of 250 ml capacity containing 50 ml of medium (V02)1, strictly identiGal to medium V2 but with the exception of the absence of agaI-agar. It is accordingly a liquid ~edium.
After inoculation by dissociated fragments of these lower parts of the cals of strain 13(1), these flask were placed in the following conditions:
- circular~ horizontal and continuous agitation of about 120 revolutions per minute, - ambient temperature of about 23~, - residual ambient light at an`intensity of 250 to 500 lux and for 16 hours per 24 hours.
In the first 24 hours of culture there was observed a dissociation of the tissue masses inoculated into elements of reduced shape and of ~luffy appearance~
- The cultures were replanted each week. In the cou~5e `f the first replantings the nutrient medium was simpl~ renewed with a view to progressively increasing the density of the cul-ture.

.~

::: : ' ''. .. ' - i ,~., ' ." , . ..
, ~ ., . , . ;;
, ... .. , ~ ; . . .. . . .

5~67 . , .
When this appeared to be sufficient, one o~1~ further part of the replanted culture was reinoculated in the ERLENMEYER
fl~ask containin~ the new mediumO
In general the i~oculum represented between a third and a half of the culture obtained in 7 days.
The grow-th in fresh weight of the cultures over the course of a period of 7 days was thus of the order of 100%
to 200% with reference to the starting inoculum. ~his re-sult is substantially identical to that observed fro~ a culture in V01 liquid medium (glucose) obtained in the same cnnditions starting from strain 13.
The fact of cultivat~g the cells of th~ strain of small periwinkle defined and classified under reference 13~1) on the semi-solid medium V02 containing lactose as a principal organic carbon source is thus not absolutely incompatible wi~h a dis-sociation and culture in liquid medium of this source-On the other hand the strain 1~ cultivated in theliquid ~edium V02 has a ut;l;sation yield of lactose equivalent to that of the utilisation of glucose by strain 1~ cultivated in the liquid medium VO10 ...
XAMP ~13 Progre~ssive adaptation b selection of a strain of Atropa belladona ~. (Solanacea ) in a lactose medium.
! ' ' There was obtained and maintained a population of strains -~
f Belladona. These strains are different according to various morphological and ph~siological characteristics.

:

0 ~S~ 67 One of these strains,reference Ab 709,possesses the - ~ following characteristics:
- it gro~s in tubes on a semi-solid medium in conditions of temperature and illumination defined in Example 1, - morphologically,the cals are cleax green and thus weakly chlorophyI-co~taining, the~ are very weakly morphogenic, strictly non-dissociated, very c~osed and non-friablec - the nutrient medium uséd for maintaining these strains? referenced H G A5, possesses the fol-lowing composition:
Mineral macro-element solution of HE~LER (cf. ~xample 1 Mineral oligo-element solution of H~IIER (cf. Example 1) Iron source ~erric chloride hexahydrate 1 mg/l of medium Vitamin Source refere~ced Bl (cf. Example 1) -~ Growth modifier 2-(2,~-Dichloro-phenoxy)_acetic acld 5 mg/l of medium i 20 Carbon source /1 Glucose 30,000 mg/l of medium ~¦ Solidification agent Agar-agar 7,000 m~/l of medium Immature coconut milk100 ml/l of medium The medium is adjusted to pH 6 and sterilised in an ;~ , - autoclave under a vapour pressure of 0.7 bar for 20 mi~utes.
; ~he replanting rh~thm for this strain is 7 weeks.
The X G A5 medium was progressively modified by partial replacement and then total replacement of the glucose b~ lactose.

.
l ~ ~ 30 -, , , . , . , . :; . ~,: . .

` ` 10~5~;7 Quantitatively four types of new media were produced:
Medium (HGA5)l = 75~ of the source of organic carbon was provided by glucose (22500 mg/l) and 25%
by lactose i.e. (7500 mg of lactose mono-hydrate per litre of medium) Medium (~GA5)2 = 50% of glucose and 50% of lactose Medium (HGA5)3 = 25% of glucose and 75% of lactose , Medium (HGA5)4 = 100% of lactose i~e. 30,000 mg of lactose monohydrate per litre of medium~
~he strain of ~elladona referenced Ab 709 was adapted progressively to the medium (~GA5)~ by cultivating it successively and chronologically on media (HGA5)l, (HGA5)2, (HGA5~3 and finally on (HG~s)4-The number of replantings on each of these media was a function of the speed of the adaptationto the same.
As soon as adaptation appeared to have taken place on one of these media, the strain was replanted on the following medium and this continued until a culture of strain Ab 709 on ~ medium (HGA5)4 was obtained.
'' 20 By this technique there was obtained a strain of Belladona Ab 709 modified a~d adapted to consume lactose as ;
a principal source of organic carboIl with a yield equivalent to that of its previous consumpton of glucose.
.' , ' ,:
~XAMPLE 14 .
A~t~_ion of a strain of Papaver Somniferum L.
.. .. . .. ~
~Papaveracea) to a_lactose medium - A population of strains of poppies was obtai~ed and maintained.

-~ 31 -..

. .:, . ., , . ~ .. . . , . . . .. ~ .. . , ,:

S~67 One of these s-trains referenced Ps 501 had the fol-lowing characteristics:
- it grows in tubes on a semi-solid medium inthe conditions of illumination and temperature de-fined in Example 1, - morphologically the cals have the following appearance:
they are greenish and thus weakly chlorophyl-con-taining, they are not morphogenic, they are ver~ dissociated in small elements they are very friable, - the nutrient medium used for maintaining this strain referenced Gl had the following composition Mineral macro-element solution of Heller (cf. Exa~ple l) Mineral oligo-element solution of Heller (cf. Example l) Iron source Ferric chloride hexah~drate l (mg/l of medium) Solution of vitamins referenced Bl (cf~ ~xample 1) Growth modifiers ~ 2-(2,4-Dichloro-phenoxy)-acetic acid 10 mg/l of medium 6~ rfurylamino)-purinelO mg/l of medium Carbon source Glucose 30,000 mg/l Soldification agent Agar-agar 7,000 mg/l ~ he medi~ is adjusted to pH 5.6 and sterilised in an autoclave under a vapour pressure of 0.7 bar for 20 minutes.

~, - ~2 .

- '. . ' . ` ' ' .' ' ` ~ ' `

S~7 The replanting r~ythm of this strain is 5 weeks.
The medium Gl was modified into a medium G2 by re-placement of the glucose b~ lactose.
Quantitatively the medium G2 is at a concentration of lactose monohydrate of 3t mg/l with the object of pro-viding after the total theoretical hydrolysis of the lactose the same concentration i~ hexose as that of medium Gl from the glucose.
After 5 weeks growth of strain Ps 501 0~ medium G2 the following facts are observed:
- no cal of culture G2 was necrotic - no cal cultivated on G2 wa~ morphologi.cally distinguishable from those cultivated on Gl - the average growth of cals cultivated on medium . G2 is not significantly different from that of . -the cals cultivated on medium Gl.
- As a.result during the first c~cle of growth on lactose medium (G2) one can state that the popp~ cells of strain PS501 used the lactose as a source of organic carbon with an equivalent yield to that of their previous utilisation of glucose.

XAMPLE_15 .
Ada~tation o~ a_strain of Digi~alis purpurea ~.
5s crofulariacea) to a lactose_medium.
A population of strains of fox glove was obtained and mainteined.
One of th~se strains referenced ~pl has the following characteristic6:

-r; : ~ " . ~ "

45~'7 - it grows either in Petri boxes or in tubes on a semi-~olid medium in the conditions of tempera-ture and illummation~ described in Example 1, - morphologically the cals have the appearance des-cribed above, - they have a very clear green and thus are ver~
slightly chlorophylic, - they are not morphogenic, - they are very dissociated in small granular ele-ments, - the~ are very friable.
~ he nutrient medium utilised for maintaining this strain re~erence M1 ha,s the following composition:
Mineral macro-element solution of Murashigue and Skoog (cf.
~xample 1) Mineral oligo-element solution of Heller (cf ~xample 1) Iron source Ferric chloride hexahydrate 1 mg/l of medium Vitamin sou~ce reference Bl (cf. Example 1) Growth modifier ~-Naph~halene acetic acid1 mg/l of medium Various compounds ~lycine 3 mg/l of medium Source of organic carbon Glucose30,000 mg/l of medium So~dification agent Agar-agar7,000 mg/l of medium .
, 5~367 ~he medium is adjusted to pH 6 and sterilised in an ~-autoclave under a vapour pressure o~ 0.7 bar for 20 mi~utes.
The replanting rh~thm of the strain ~ 1 is 6 weeks.
~ he medium Ml was modified into medium M2 by replacin~
the glucose b~ lactose.
There was introduced into the medium M2 30,000 mg/1 of lactose monohydrate with the ob~ect of having after the theoretical total hydrolysis thereof a concentration o~ hexose identical to that of medium Ml from glucose.
10After a normal replanting c~cle that is to say after 6 weeks growing the strain Dpl on the medium M2 the following fact~ are observed:
- no cal in culture on medium ~ was necrotic, ~ - none of the cals cultivated on medium M2 were j 15 morphologically distinguishable from those ¦ cultivated on medium Ml, ~, - the average growth of the cals cultivated on I ~edium M2 was clearly less than that of the cals cultivated on medium Ml. In fact about j 20 25% of the cals cultivated on medium M2 had `, a growth identical to those cultivated on medium Ml but about 75% had a lower growth to a more o~
less great extentO
Despite these average results obtained a~ter the first replanting one ~aintained on medium M2 the cals ~lhich had good growth.

,, .

,~
. .
, . . . .

1~3~S~J~7 AEter 4 or 5 culture cycles the average growth of the cals cul-tivated on medium M2 were substantially identical to that of the cals cultivated on Ml.
After adaptation the cells of fox glove of the strain referenced Dpl can thus use the lactose as a source of organic carbon with a yield equivalent to that of their previous u~ilisation of glucose.
Starting from the results described in the preceding Examples one can formulate several hypotheses explaining the growths of superior plant cells on media only containing lactose as a principal source of organic carbon.
First hypothesis The lactose is not consumed by the cells, these finding the source of organic carbon which is necessary for them in the immature coconut milk. The results obtained and described in Examples 2 and 3 in particular and the other Examples where the nutrient media did not contain coconut milk permit this hypothesis to be eliminated.
Second hypothesis The lactose is not consumed by the cells; these com-pensate for the lack of organic carbon by an increased photo-synthesis. The results obtained and described in Examples 4 and 11 in particular (culture in the dark) and all the Examples where the cals were strictly non-chlorophyll-containing permits this hypothesis to be elimina-ted.
Third hypothesis The lactose is consumed by the plant cells; but these are not those which assure the necessary hydrolysis for its use, the passage in the autoclave of the medium bringing about modifications in its composition and among others the transformation of lactose into hexose. The results ~ ~ .

.. , . ~ . . . . . . .

5'r~7 described in Exa~ple 10 permit this hypothesis to be eliminated.
Fourth hypothesls The lactose is consumed by the cells which are capable of hydrolysing it. This hypothesis can be verified by demonstrating that the plant cells adapted or selected were effectively capable of hydrolysing lactose. One can see very clearly in evidence the enzyme ~-galactosidase in the cals of the strains adapted or selected and in the medium on which they have grown.
For this demonstration the following technique is used:
After obtaining extracts of the enzymes of the cals and of the culture media, they were put to incubate at 37~C in the presence of a specific substrate (~-phenyl-galactoside obtained from the Societe SIGMA) and buffered to pH 4.7 (sodium acetate - acetic acid 0.10 M). The reaction is stopped by the addition of sodium hydroxide. The phenol liberated was then subjected to spectrophotometry at 285 nm, conditions in which the substrate does not absorb.
The free phenol thus demonstrated to be present implies a ~-galactosidase activity on the substrate given and thus the presence of the enzyme ~-galactosidase in the extracts of cals and the culture media used.
By adaptation or by selection the genetic regulation of the strains has been modified as regards the expression of the operon of ~-galactosidase and as concerns other character-istics. Thus Example 7 describes the obtaining of three strains references 13(1)~ 13(2) and 13(3) the chlorop~lyll characteristics and growth characteristics of which are different. Their characters are likewise different from that of the original strain 13.

~``! j;~

J~Sf~;7 Other examples confirm this result showing an improved growth of the strains by adaptation to the lactose medium.
All the selected or adapted strains behave conven-tionally. I'hey can grow on a liquid or solid medium as set out in the preceding Examples and all industrial culture tecnniques for example fermentation techniques adapted for plant cells are applicable thereto.

- 38 - :

Claims (14)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of obtaining a substantial yield of plant cells which comprises cultivating in vitro plant cells of a species of the branch of phanerogamic plants capable of growth based upon a lactose carbon source, said cells being obtained by adaption or selection of the plant cells of said branch, wherein said selected or adapted plant cells are administered lactose or a natural origin form of lactose as a principal source of organic carbon to obtain said substantial yield of plant cells.

2. A method according to claim 1 wherein said plant cells capable of growth based upon a lactose carbon source are obtained by adaption by cultivating a primary cal of phanerogamic plant cells in vitro directly on a lactose-containing medium.

3. A method according to claim 1 wherein said plant cells capable of growth based upon a lactose carbon source are obtained by selection by cultivating phanerogamic plant cells in vitro on media of increasing lactose content.

4. A method according to claim 1 wherein the culture medium comprises the sugar lactose itself as principal organic carbon source.

5. A method according to claim 1 wherein the culture medium comprises a residue of the dairy industry as principal organic carbon source.

6. A method according to claim 5 wherein the residue of the dairy industry is a lactose juice or milk ultrafiltrate.

7. A method according to claim 5 wherein the residue of the dairy industry is a lactose serum.

8. A method according to claim 1 wherein the culture medium for the vegetable cells comprises the conventional nutritive elements with the exception of the principal source of organic carbon which is constituted by lactose or one of its natural origin forms.

9. A method according to claim 8 wherein the culture medium is solidified with agar-agar.

10. A method according to claim 8 wherein the culture medium is in liquid form.

11. A method according to claim 8 wherein the culture medium is in liquid form and which is carried out in a fermenter.

12. A method of obtaining plant cell strains which are capable of normal growth in vitro on a lactose containing medium, which comprises the adaption or selection of such plant cells starting from species of plants classified in the branch of phanerogamic plants, whereby there is obtained said vegetable cell strains.

13. A method according to claim 12 wherein said plant cells capable of growth based upon a lactose carbon source are obtained by adaption by cultivating a primary cal of phanerogamic plant cells in vitro directly on a lactose-containing medium.

14. A method according to claim 12 wherein said plant cells capable of growth based upon a lactose carbon source are obtained by selection by cultivating phanerogamic plant cells in vitro on media of increasing lactose content.