FR2548523A1 - Process for producing wormwood having a high content of aromatic oils - Google Patents

Abstract

The process is characterised in that: One or more wormwood clones having a high content of aromatic oils are selected; A fragment of wormwood belonging to a selected clone is collected; There is performed a vegetative propagation from this fragment by in vitro culture on a nutrient medium in the presence of a substance promoting the development of axillary buds, separation of the buds, of culture of the separated buds on a similar medium and repetition of the latter operations as many times as necessary; and A final culture of the small plants is performed in vitro on a nutrient medium containing a sugar and practically free of substances promoting the development of axillary buds.

Description

 The present invention relates to a process for obtaining génépi having a high content of aromatic essences.

 The génépi is a rare plant that grows spontaneously in some areas of the Alps and whose ecological requirements are apparently very strict. It is forbidden to pick it because it is an endangered plant. There are several species of génépi: Artemisia genépi, A. mutellina, A.

umbelliformis, A. spicata. These plants possess the peculiarity of containing very appreciated aromatic principles which go into the manufacture of certain liqueurs.

 The object of the present invention is to make it possible to obtain, on an industrial scale, genépi having a high content of aromatic essences by in vitro culture according to the method of vegetative propagation.

 Methods for obtaining plants by the method of vegetative propagation have already been proposed.

Thus, the patent FR 2,278,267 describes a method for multiplication of strawberry plants by in vitro micropropagation consisting in taking axillary buds and depositing them on a nutrient medium containing a cytokinin, separating the buds formed, transplanting them. these buds in a similar nutrient medium, repeat as many times as necessary these operations, to root the buds by removing the cytokinin and to plant in the ground the small plants obtained.

 By in vitro culture of génépi, one could expect to obtain plants practically devoid of aromatic essences.

 However, it has been found that by the method according to the invention using the method of vegetative propagation in vitro it was possible to obtain seedlings of génépi having high levels of aromatic essences. In addition, it was found that by in vitro culture on a culture medium containing certain percentages of sugar, it was possible to further increase the content of aromatic essences.

The process for obtaining génépi according to the present invention is characterized in that
a selection of one or more génépi clones having a high content of aromatic essences is carried out,
a fragment of génépi belonging to a selected clone is taken,
vegetative propagation is carried out from this fragment by in vitro culture on a nutritive medium in the presence of a substance promoting the development of axillary buds, separation of the buds, culture of the separated buds on a similar medium and repetition of these latter operations as much many times it is necessary,
and final cultivation of the small plants in vitro is carried out on a nutrient medium containing a sugar and practically free of substance promoting the development of the axillary buds.

 As the sugar present in the final culture, glucose or sucrose can be used.

 The sugar doses are advantageously between 5 and 12% relative to the weight of the culture medium.

The selection of high aromatic content clones among different genepi clones can be made by determining the total amount of aromatic species on small plants grown on a nutrient medium. This determination can be carried out according to the methods described by H. Thieme and TT Nguyen, 1972, Pharmazie. Dtsch. 27 (4), 255-265;
AK Picman, RL Ranieri, GHN Towers, J. Lam, 1980, J. Chromatogr. 189, 187; and G. Appendino, F.

Belliardo, G. M. Nano, S. Stefenelli, 1982, J. Agric.

Food Chem. 30, 518-521.

 Vegetative propagation is advantageously carried out from apices taken from seedlings of génépi.

 This vegetative multiplication can also be done by removing apex from adult plants or from floral stem fragments.

 These samples and the multiplication are performed under sterile conditions.

 The detailed description which follows will make it easier to understand how the method according to the invention can be realized.

Genépi seeds are disinfected by the action of a solution obtained after filtration of a mixture of 20 g of chlorometric calcium hypochlorite and 1 liter of water. After two hours soaking in this solution, the seeds are placed in petri dishes containing sterile wet filter paper. The seeds germinate in the light and at a temperature of 230C; after 6 days they give seedlings that can be used for apex sampling; these are isolated and placed on an agaric nutrient medium under sterile conditions. The medium used has the following composition:
KNO3 950 mg.l
NH4NO3 825
KH2PO4 85
MgSO 4, 7H 2 O 185
CaCl2, 2H2O 220
Na2EDTA 37.3
FeSO4, 7H20 27.8 mg.l 1
ZnSO4, 7H2O 8.6
H2BO3 6.2
MnSO4, 4H2O 22.3
CuSO4, 5H2O 0.025
CoCl2, 6H2O 0.025
Na2MoO4, 2H2O 0.25
KI 0.83
Glucose 20 g / l
The cultures are placed in an air-conditioned room at a temperature of 22 C, during the day, and 180C, at night, with a photoperiod of 16 hours of light and 8 hours of darkness (quality Manda Fluor, Daylight and Sylvania Grolux F 40 W). During the first passage on this medium, the apex elongates, forms a leafy stem; the neoformation of roots is also observed. Apices which are placed on a medium identical to the preceding one, but to which a vitamin mixture is added under aseptic conditions (inositol 5 mg / l, nicotinic acid 250 mg / l, thiamine 500 μg / l, biotin 25 Ag / l, folic acid 50 μg / l, pyridoxine 250 Ag / l) glycine 1 mg / l, naphthalene acetic acid 10 7 g / ml and benzyladenine g / ml. ml.

 On this medium, many axillary buds develop; there are about forty developed from an initial bud, after 20 days of culture. By separating the stems resulting from the development of the axillary buds, we can isolate apical and axillary buds again and ensure an intense and rapid multiplication.

 The dose of benzyladenine used was determined to be essentially the minimum dose that would result in the formation of many buds. Below this dose, there is a development of roots.

Table I below shows the influence of the dose of benzyladenine
TABLE I

<tb><SEP> Doses <SEP> of <SEP> Observations <SEP> on
<tb> Benzyladenine
<tb> in <SEP> the <SEP> middle
<tb><SEP> the <SEP> training <SEP> the <SEP> training <SEP> the <SEP> content
<tb><SEP> of <SEP> buds <SEP> of <SEP> roots <SEP> in <SEP> water
<tb><SEP> 0 <SEP> 1 <SEP> bud <SEP> very <SEP> many <SEP> not
<tb><SEP> roots <SEP> of hyperhydria
<tb><SEP> 10-7 <SEP> g.ml-1 <SEP> many <SEP> roots <SEP> not
<tb><SEP> buds <SEP> of hyperhydria
<tb> 5.10 <SEP> 7 <SEP> g.ml-1 <SEP> very <SEP> many <SEP> not <SEP> of <SEP> roots <SEP> slight
<tb><SEP> bud <SEP> s <SEP> hyperhydria
<tb><SEP> 10-6 <SEP> g.ml-1 <SEP> very <SEP> many <SEP> not <SEP> of <SEP> roots <SEP> hyperhydria
<tb><SEP> buds <SEP> ~ <SEP> ~ <SEP> ~ <SEP> ~ <SEP> ~ <SEP>
<Tb>
When operating as described above from seeds of various origins, seedlings are available on which a selection test can be made from the point of view of the content of aromatic essences.

 As an indication, Table II gives the results obtained with seeds of various origins and culture on media similar to that indicated above, but containing instead of 2 by weight of glucose respectively 2% and 8% by weight. of sucrose.

TABLE II
Variations of the total amount of aromatic essences of different clones

<SEP> identification of <SEP> clones
<tb> a <SEP> b <SEP> c <SEP> d <SEP> e <SEP> f <SEP> g <SEP> h
<tb> Quantity <SEP> total
<tb> of petrol <SEP> in <SEP> mg / g
<tb> 3.6 <SEP> 2.4 <SEP> 7.7 <SEP> 2.0 <SEP> 6.1 <SEP> 4.6 <SEP> 3.0 <SEP> 2.9
<tb> fresh <SEP> material
<tb> on <SEP> sucrose <SEP> 2 <SEP>%
<tb> Quantity <SEP> total
<tb> of petrol <SEP> in <SEP> mg / g
<tb> 10.2 <SEP> 6.8 <SEP> 21.8 <SEP> 5.6 <SEP> 17.3 <SEP> 13.0 <SEP> 8.5 <SEP> 8.2
<tb> fresh <SEP> material
<tb> on <SEP> sucrose <SEP> 10 <SEP>%
<Tb>
In view of these results, the clone (c) is particularly interesting and can be selected for subsequent vegetative propagation. These results also highlight the influence of the sugar content on the content of aromatic essences.

 We then proceed with the clone (s) chosen (s) for vegetative propagation, as described above with the same medium until the desired degree of multiplication.

 In the final phase, a small plant culture is carried out on a nutrient medium which differs from that used for vegetative propagation only by the absence of benzyladenine and a sucrose content of 10% by weight (instead of 2% of glucose ).

 This sucrose content appears to be decisive, as evidenced by the results obtained by varying the sucrose content in the nutrient medium.

TABLE III
Influence of sucrose concentration in
the culture medium on the amount of material
estimated after three weeks of culture and
on the aromatic content.

<Tb>

Dose <SEP> of <SEP> sucrose <SEP> Percentage <SEP> of <SEP> Content <SEP> in <SEP> lactones
<tb><SEP> in <SEP> weight <SEP> material <SEP> dry <SEP> sesquiterpenic
<tb><SEP>.<SEP> MF) <SEP>
<tb><SEP> 2 <SEP>% <SEP> 13.8 <SEP>% <SEP> ~ <SEP> 828
<tb><SEP> 7.5 <SEP>% <SEP> 15.3 <SEP>% <SEP> 972
<tb><SEP> 10 <SEP>% <SEP> 17.2 <SEP>% <SEP> 1026
<tb><SEP> 12.5 <SEP>% <SEP> 24.1 <SEP>% <SEP> 609
<Tb>
It should be noted that when the sugar dose is increased, growth is also strongly stimulated.

 A comparative study of the sesquiterpene lactone content of the clones and their aromatic value was carried out. There is good agreement between the percentage of sesquiterpene lactones of the various clones analyzed and their organoleptic value.

 Similar results can be obtained by taking apex from selected adult plants or by growing floral stem fragments of selected plants. The disinfection conditions with calcium hypochlorite are then slightly modified: an aqueous solution containing 150 g / l of calcium hypochlorite is used; the sterilization time is 15 minutes. This is followed by several rinsing with sterile water before culturing on the first medium.

Claims (6)

 A process for obtaining génépi having a high content of aromatic essences, characterized in that  a selection of one or more génépi clones having a high content of aromatic essences is carried out,  a fragment of génépi belonging to a selected clone is taken,  vegetative propagation is carried out from this fragment by in vitro culture on a nutrient medium in the presence of a substance promoting the development of axillary buds, separation of the buds, culture of the separated buds on a similar medium and repetition of these latter operations as much many times it is necessary,  and final culture of the small plants in vitro is carried out on a nutrient medium containing a sugar and practically free of substances promoting the development of the axillary buds.  2. Method according to claim 1, characterized in that the sugar present in the final culture is selected from glucose and sucrose.  3. Method according to claim 1 or claim 2, characterized in that the sugar represents from 6 to 12% of the weight of the culture medium.  4. Method according to any one of claims 1 to 3, characterized in that the fragment used for vegetative propagation is an apex.  5. Method according to any one of claims 1 to 4, characterized in that the vegetative propagation is carried out in the presence of benzyladenine at a concentration of at least 10 g / l.  6. Method according to any one of claims 1 to 5, characterized in that the vegetative propagation is carried out in the presence of benzyladenine at a concentration of 10 7 to 10 6 g / l.