NL8201937A - PROCESS FOR THE PREPARATION OF THE MULTIPLICATING MATERIAL OF FINGERED HERBS (DIGITALIS LANATA EHRH.) IN TISSUE CULTURE. - Google Patents

Description

N / 30937-Kp / Pf / vdM,? *> - 1 - i

Method for the preparation of the multiplicative material of foxglove (Digitalis Lanata EHRH.) In tissue culture.

The invention relates to a process for the preparation of the propagating material of foxglove (Digitalis Lanata Ehrh.) In tissue culture. More particularly, the invention relates to a process for the preparation of the sterile foxglove propagation material obtained in a sterile tissue culture, and to the preparation of non-sterile seedlings adapted to open-air conditions, which are prepared from the sterile multiplication material is obtained.

Foxglove is a valuable raw material for the pharmaceutical industry as it contains cardiac glycoside, and is grown by sowing seeds collected from the two-year-old plants and the 4-year-old 15 plants that have emerged from the seeds and are suitable for pharmaceutical purposes.

The pharmaceutical industry needs a vegetable raw material with a high active substance content and a constant composition. The plants with a suitable active ingredient content, which are currently cultivated, are already the result of a long-term breeding.

According to literature data, the genetic mutation capacity of the plant is very high. The morphological variations and the variation in yield and active substance content can be attributed to the fact that - the species is only grown for a relatively short period, - the chromosome number varies and - the plant has spontaneous hybrids.

30 ^ Lungenau, I., Lucrar. Cradin. Bone. Bucuresti 37 (1966);

Neczypor W., Pharmazie 9, 38 (1964); Schick, E.R., Reimann, P.R., Züchter 27, 7, 301 (1957) __ 7-

Based on measurements taken on. in the wild in the Budakalasz area (Hungary) 8201937 • »

V

- 2 - v Growing Foxglove Species A. Mathë observed significant differences in the average stem diameter and leaf veins of the families and found an equally large variation in the active ingredient content ^ Morphological, Chemical and 5 Oecological Properties of Plants with Cardiac Glycoside Contents; CSC-thesis, Budakalasz (1978) _7-

Inbreeding over several generations, which would promote homogeneity, is unfavorable since the plant's biopotential would be lost ^ Tétényi P., 10 Herba Hungarica S_, 3, 61, (1970)

Crossbreeding between unrelated species is difficult to perform ^ Michaela, A., Silva, P .: Herba Hungarica _ll, 1,29 (1972) J, while crossbreeding within the species is more efficient ^ Schwerdtfeger, G., Züchter _31,5 202 (196l) /. In this case, hybrid seeds with cytoplasmic and sterility must also be produced, making the highly labor-intensive castration unnecessary. As suggested by Schwerdt-feger, the yield and the internal composition are controlled by several genes and also strongly depend on the environmental conditions. Schwerdtfeger also mentions that when the plant is improved in terms of morphological homogeneity, its adaptability to the environment decreases and so the yield as a goal of the improvement becomes of secondary importance / Züchtungsprobleme bei Digitalis lanata Ehrh., 25 Referat: Arznei-pflanzencolloquium, Ranischholzhausen (1973 E. Schratz has shown that in places of culture the variability of the morphological characteristics of leaves and flowers is remarkably high / Kappert, H., Rudolf, W., Handbuch der Pflanzenzüchtung; Paray, Berlin-30 Hamburg, Vol. 5, p. 383 (1961) -7. G. Ligeti observed that the mutual ratio of the individual glycoside components in the plants varies widely. Pharmazie X4, 162 (1959) J. This is also confirmed by the data from P. Tétényi / intraspecific Chemi-35 cal Taxines and Improvement of Drug Plants? DSC-thesis, Budapest (1963) _7 · The latter author found a 1: 8 to 12: 1 fluctuation in the ratio of lanatoside A to lanatoside C in wild-growing plants, while a ratio of 8201937 * · ♦ - 3 - even 20: 1 was also found in cultured foxglove.

For example, the cultivation and improvement of foxglove presents several additional problems that could be solved by a vegetative multiplication method, since the offspring thus obtained are genetically identical to the starting progenies. However, the conventional methods of vegetative multiplication (stem splitting, cutting) have been found to be unsuitable for the multiplication of foxglove.

Based on experience from other plants, vegetative microreproduction by tissue culture seemed to be an appropriate route. However, according to literature data, the vegetative micro-reproduction of foxglove (Digita-15 lis lanata Ehrh.) Is still an unsolved problem. Lui and Staba / Phytochemistry ^ 18, 1915 (1979) J have inoculated foxglove on a Murashigo-Skoog culture medium, but they were only able to grow the plants for 12 weeks. These authors examined the dry matter content and digoxin level in 20 cell cultures, but they did not perform mass multiplication.

Several authors have attempted to produce the foxglove active substances by preparing a "callus" culture and then preparing a suspension of foxglove leaves culture and subjecting the culture to sterile cell culture under in vitro conditions / Helmbold, H., Planta Medica 33_, 3, 280 (1978)? Shyr, S.E. and Staba, H.

J., Planta Medica 0/86 (1976) 7- However, the digoxin content of the obtained plants was extremely low and cannot be isolated in practice. This mode of plant tissue culture in which undifferentiated individual cells are propagated in suspension on a liquid culture medium is not suitable for the production of genetically identical plants useful as seedlings for further cultivation.

As a result, the mass multiplication of homogeneous, genetically stable units with selected production capacities is still an unsolved problem.

The object of the invention is to elaborate a method for the vegetative micro-multiplication of foxglove (Digitalis lanata Ehrh.) In a sterile tissue culture, which yields genetically identical propagation material (seedlings) in 5 the open air in any quantity.

It has been found that the repeated multiplication of foxglove in sterile culture can be resolved both by using sterile seeds and by cultivating the sterile shoot tips of plants grown under open-air conditions, when a strictly defined number of stages in strictly determined sequence is applied for the multiplication of the plants, for the preparation of the planting in the open air and for the adaptation of the plant to the open-air conditions, observing strictly determined working parameters.

The invention relates to a process for the preparation of the propagation material (seedling) of foxglove (Digitalis lanata Ehrh.) In tissue culture, under vegetative micro-multiplication by subjecting the seeds or the stem tips of plants grown in the open air to surface sterilization . According to the invention, proceed as follows: a) a sterile plant with some, preferably 2-3, leaves is grown from the sterile seed or sterile stem tip of an open-grown plant on a known culture medium, b ) the sterile plant with some, preferably 2-3, leaves is uprooted and subsequently seeded (transferred) on a multiplication culture medium containing auxin (s), cytokinin (s) and possibly. gibberellic acid, the plant is grown there until a stage of 20-40 side shoots is reached, whereupon the plant is split into wreaths of preferably 2-3 leaves each, and if desired, the multiplication is repeated until the desired number plants, c) the sterile leaflets, preferably 2-3 leaves, are transplanted to a rooting culture medium containing an auxin or an auxin with a cytokinin and the 8201937c-5 rooting is induced until roots of length i 4-8 mm have developed, d) the plants with roots 4-8 mm long are further rooted on a culture medium, which is free of auxin (s) and cytokinin (s), until a root length of 4 -6 cm is reached and e) if desired, the obtained propagation material (seedling) is adapted to the open-air conditions.

In the first step of the above-mentioned method, sterile plants with preferably 2-3 leaves are grown from seeds which have been disinfected in a known manner or from disinfected stem tops of plants growing in the open air on a culture medium as described. in literature ^ Linsmaier and Skoog, Physiol. Plantarum, 18, 100-127 (1965); Nitsch and Nitsch, Science, 163, 85-87 (1969) J.

In the second step of the method according to the invention, the shoots of sterile plants, preferably 2-3 leaves, are transferred to a multiplication culture medium. The multiplication culture medium is to induce the formation of as many side shoots as possible, causing massive leaf sprout development. The multiplication culture medium is essentially one of the two media mentioned above, but supplemented with an appropriate combination of auxin (s), cytokinin (s) and, if desired, gibberellic acid. As auxin, for example, 0.1-1.0 ppm indole acetic acid or 0.01-0.05 ppm 2,4-dichlorophenoxyacetic acid can be used.

For example, the cytokinin to be combined with the auxin may be benzylaminopurine at a concentration of 0.1-1.0 ppm.

The ratio of the two components can vary between 1: 1 and 1. 10. When the auxin / cytokine ratio is 1: 1, the development of the plants multiplied in the tissue culture is also more favorable when gibberellic acid is also added to the multiplication culture medium 35 as a third additive at a concentration of 0.1-0.5 ppm.

The culture is incubated at 24 + 2 ° C under a relative humidity of 50-80%, under illumination with 9,000-14,000 lux for illumination periods of preferably 16 h daylight 8201937 - 6 - 8 h darkness. The cultivation takes about 4-6 weeks.

The branched stems with at least 50 side shoots are split under sterile conditions into shoots (leaves) with 2-3 leaves and a new multiplication step is started under the above parameters, the shoots being capable of further development on their own. Cleavage and multiplication can be repeated at will until the desired amount of multiplication material (seedlings) is produced.

When the required number of plants has been reached, rooting must be induced on the individual plants to facilitate their planting out. In this third step of the method according to the invention, the multiplied plants with 2-3 leaves are transplanted onto a rooting culture medium. This rooting medium may be a Nitsch-and-Nitsch type medium, or the Linsmeier-Skoog type discussed above, but suitably modified variants thereof containing only half of the macro elements of the original medium , can also be used advantageously. The culture media must always contain either an auxin or an auxin together with 10% of a cytokinin, based on the amount of auxin present. When indole butyric acid is used as auxin, its amount in the culture medium can be between 0.1 and 0.5 ppm. When a combination of an auxin and a cytokinin is used, the culture medium may contain, for example, 1 ppm of α-naphthyl acetic acid together with 0.1 ppm of kinetin. Root shooting proceeds faster and the roots formed are more vigorous when a low hormone concentration is employed and the macroelements of the culture medium are reduced to half the level used in the previous step. It is essential that the third stage be continued only until the plants begin to take root.

In the fourth step of the method according to the invention, the plants with roots of 4-8 mm in length are transplanted to a culture medium without hormones. The macro-elements content of this hormone-free culture medium is identical to that of the medium used in the previous step. When the cultures with induced rooting are further grown on a hormone-free medium, numerous strong roots are developed and the development of the vegetative parts of the plant is also suitable. The incubation conditions in the third and fourth stages are identical to those used in the previous one. The last two stages together take 4-6 weeks and at the end of this period rooted seedlings, which have developed properly from plants, are obtained.

In the fifth and final stage of the method of the invention, the seedlings are adapted to non-sterile open-air culture conditions. In this step, the seedlings are planted in pots filled with a mixture of sand and perlite, or peat and perlite. The mixture is wet up to 70% of its water adsorption level; when using a mixture of sand and perlite, the watering is carried out with a solution containing macro and micro elements (modified Knopp solution). The seedlings are incubated under semi-sterile conditions in a plant box or in a greenhouse with properly controlled temperature and humidity conditions. The adjustment period lasts approximately 4-5 weeks.

The main advantages of the vegetative micro-multiplication in tissue culture are as follows: - the homogeneity of the species (identity of order) can be maintained, - the multiplication of an individual plant can be carried out continuously, regardless of the season and weather conditions, any desired amount of homogeneous plants can be produced from a single individual plant.

On this basis, vegetative micro-multiplication can be used to maintain the foxglove species, to improve the plant and to produce a homogeneous seed stock required for cultivation.

The invention is illustrated in detail with 8201937-8 using the following non-limiting examples.

EXAMPLE I

Ten seeds of foxglove to be sown were subjected to surface sterilization in a 10% aqueous solution of sodium hypochlorite for 10 min and then the seeds were rinsed three times with sterile water. The sterile seeds were germinated on a culture medium of the Nitsch type / Nitsch and Nitsch, Science, 163, 85-87 (1969) J, and the germinating plants were allowed to develop until they reach the developmental stage of 2-3 leaves (3 weeks).

The culture medium used had the following composition: KNO3 950.0 mg / l NH4N03 720.0 mg / l

CaCl2.2H20 166.0 mg / 1 KH2PO4 68.0 mg / 1

MgS04.7H20 185.0 mg / 1

MnS04.H20 25.0 mg / 1 20 H3B ° 3 1O, 0 mg / 1

ZnS04.4H20 10.0 mg / 1

Na2MoO4.2H20 0.25 mg / 1

CuS04.5H20 0.025 mg / 1

Fe (II) -EDTA complex solution * 5.00 mg / 1 biotin 0.05 mg / 1 glycine 2.0 mg / 1 inositol 100.0 mg / 1 nicotinic acid 5.0 mg / 1 pyridoxine 0.5 mg / 1 30 thiamine 0.5 mg / 1 folinic acid 5.0 mg / 1 sucrose 2000.0 mg / 1 agar 1000.0 mg / 1

The pH of the above culture medium 35 was adjusted to 5.6 with 1N sodium hydroxide solution prior to sterilization.

* The Fe (II) -EDTA (ferric ethylenediamine tetraacetic acid) complex solution was prepared as follows: 8201937-9: 557 mg FeSO 4 .7H 2 O was mixed with 756 mg Na 2 EDTA and the volume of the mixture was adjusted to 100 ml with distilled water. 5 ml of the resulting solution is equivalent to 27.8 mg

FeSO 7 .7H 2 O, i.e., to 5.6 mg Fe (II).

20 ml aliquots of the above culture medium were placed in test tubes and sterilized in an autoclave for 20 min at 121 ° C.

The seeds were placed one by one under sterile conditions on the surface of the culture medium and then under air-conditioned conditions (temperature: 26 + 1 ° C, relative humidity: 65-75%, illumination intensity: 10,000-11,000 lux; night / daylight periods: 8/16} held for about 3 weeks, ie until the plants had reached the developmental stage of 2-3 leaves.

The roots of the 10 young plants thus obtained were removed and then the plants were transplanted onto a culture medium of the above composition 20 plus 1 ppm indole acetic acid, 1 ppm, benzylaminopurine and 0.1 ppm gibberellic acid as the plant growth regulators (100 ml culture medium was placed in a 250 ml Erlenmeier).

Indole acetic acid and benzylaminopurine were added to the culture medium for sterilization, while gibberellic acid sterilized by filtration was mixed with the culture medium when the latter had already cooled to about 40 ° C.

The plants were kept under the air-conditioned conditions described above until a strong induction of side shoots set in (about 4 weeks).

The side shoots were separated under sterile conditions and then individually seeded on a culture medium of the Nitsch type, which also contained 1 ppm α-naphthyl acetic acid and 0.1 ppm kinetin. After 2 weeks of incubation, the obtained plants with 4-8 mm roots were transplanted to a culture medium containing no plant growth regulators and the cultivation was continued. From 10 young plants, 145 rooted seedlings were grown in approx.

8201937 - 10 - 11-12 weeks obtained.

The seedlings with roots of 4-6 cm, grown under sterile conditions, were then planted in pots filled with a 1: 1 mixture of sand and perlite (the mixture was saturated to 70% of its water capacity) and in a plant box under controlled conditions adapted to the open air conditions. The relative humidity was set at 70% in the plant box and the illumination intensity was 16,000 lux. The temperature change from day to night was set at 21/15 ° C. The plants were watered with a modified Knopp solution (a solution of macro and microelements) to replenish the nutrients.

Since the plants initially need an environment with 100% humidity, the pots were covered with a glass clock for 4-6 days.

The seedlings kept in plant boxes under the above-mentioned conditions gained strength and could therefore be released later.

The morphological homogeneity of the offspring of one and the same young plant is characterized 2 by the X values of the number, the length and the width of the leaves. The results are listed in Table A. The numbers are lower than those given in Table 25 by E. Weber / See Svab, J .: Biometric Methods in Research Work (in Hungarian), Mezogazdasagi Kiadö, Budapest, 1973 / . This means that there are no significant differences between the individual offspring in terms of length and width of the leaves, i.e. the group is homogeneous.

30 8201937 - 11 -

TABLE A

length of the width of the number of leaves, cm leaves, cm plant leaves (average values for 11 leaves) 51 14 3.6 1.29 2 13 3.12 1.26 3 19 3.25 1.26 4 12 3.71 1.48 5 11 3.64 1.25 10 6 12 3.57 1.31 7 13 3.25 1.45 8 12 3.34 1.50 9 11 3.47 1.43.

X2 (calculated) 20.65 21.23 15 (given in Weber's table) 39.5 39.3

EXAMPLE II

Foxglove seeds were surface sterilized as follows: 20 - washing with a detergent, - rinsing with water, - soaking for 5 min in 70% alcohol, - washing with sterile water, - soaking for 5 min in a 5% aqueous sodium hypochlo- 25 cane solution, - wash three times with sterile water.

The sterilized seeds were germinated for 4-5 days on tap water containing 0.8% agar under the following conditions: temperature: 2l + 1 ° C, relative humidity: 70%, illumination intensity: 50 lux. The young plants were then seeded on a Linsmeier-Skoog culture medium / Physiol. 18, 100-127 (1965) J with the following composition: NH4N03 1650.0 mg / 1 KN03 1900.0 mg / 1 35 CaCl2.2H20 440.0 mg / 1 8201937 - 12 -

MgS04.7H20 370.0 mg / 1 KH2PO4 170.0 mg / 1

Fe (II) -EDTA complex solution * 5.0 ml H ^ BO ^ 6.2 mg / 1 5 MnS04.H20 22.3 mg / 1% ZnS04.4H20 8.6 mg / 1 KI 0.83 mg / 1

Na2MoO4.2H20 0.25 mg / 1

CuS04.5H20 0.025 mg / 1 10 CoC12.6H20 0.025 mg / 1 inositol 100.0 mg / 1 vitamin 1.0 mg / 1 sucrose 3000.0 mg / 1 agar 1000.0 mg / 1 15 De Fe (II) - EDTA complex solution was prepared as described in Example I.

The pH of the culture medium was adjusted to 5.6 prior to sterilization using an 1N sodium hydroxide solution.

20 80 ml portions of the culture medium were placed in 0.5 L jam jars and sterilized in an autoclave for 20 min at 121 ° C.

The plants were kept in an air-conditioned room until they reached a height of 8-10 cm. 25 The conditions in this room were: temperature: 26 + l ° C, relative humidity: 70%, light intensity: 10,000-12,000 lux (Airam Floralux I165 / 80-type electric discharge lamps with a spectrum rich in red and blue components and Tungsram 65 w F33 white-type electric discharge lamps with a 30 spectrum rich in white components were used in combination as light sources), exposure period: 8 h dark and 16 h exposure.

The sprouts of the developed young plants, which bore 2-3 leaves, were seeded on a culture medium of the above composition, which also contained 1 ppm of benzyl aminopurine and 0.1 ppm of indole acetic acid, and the plants were placed in an air-conditioned room. further 'bred. After 5-6 weeks of incubation, the thus obtained leaf wreaths (side shoots) were separated and the separate shoots were seeded on a culture medium of the above composition, which also contained 0.5 ppm indole butyric acid to effect root shoot. After 2 weeks, the plants with roots 5 of 4-8 mm in length were transplanted to a culture medium without the growth regulators and cultivated thereon until the rooting was completed (2-3 weeks).

20-40 viable individual plants were obtained on average from a single seed.

10 The seedlings with roots 4-6 cm in length were planted in a 1: 1 peat-perlite mixture covered with a transparent cold stretch film (LTE 5228, manufactured by Borsodi Vegyi Kombinat, Hungary) for one week, upon which the foil was removed and the plants were grown in a greenhouse under controlled conditions for about 4 weeks (natural illumination, 70% relative humidity, day / night temperature change 24/16 ° C).

The infested plants were then expanded and 95% of the seedlings started to grow.

The homogeneity of the micro-multiplied branches is characterized by the average values of plant height and weight per 1000 seeds, the standard deviations of these means (S) and the coefficient of variation (CV,%).

ark

The data is listed in Table B.

25 TABLE B

number of plant height, cm weight per 1000 seeds individual average in mg branch plants + S cv,% avg. + S cv,% 1 7 76.1 + 4.6 5.9 51 + 0.9 5.0 30 2 8 82 .2 + 5.5 6.6 41 + 0.9 6.1 3 7 82.4 + 3.0 3.6 45 + 1.2 7.2 4 7 76.8 + 4.0 5.2 504 ^ 1 6.0 control 7 73.4 + 7.2 10.1 47 + 2.7 15.4

The low standard deviation and variation coefficients, compared to the control, indicate complete homogeneity.

8201937 - 14 -

EXAMPLE III

The stem tip of an improved thimble herb plant, which was grown in the open air under conditions and tested for its active substance content, was cut 6-8 cm in length and sterilized in a 3% aqueous sodium hypochlorite solution for 20 min. . The shoot meristem of 3-5 mm in length was separated under sterile conditions and applied to a Nitsch-type culture medium of the composition described in Example 1. The 2-3 leaf shoots were placed on a multiplication culture medium further cultivated with the composition as described in Example I and under the conditions given in that Example. After one month of culture, the leaflets were separated and then further treated as described in Example I.

As shown by the analytical data, neither the total glycoside content nor the amount of C-series glycosides changed at micro-multiplication and no sign of degeneration could be observed.

EXAMPLE IV

Sterile stem meristems were obtained from mature foxglove plants grown on arable land, proceeding as follows: leaves longer than 60 mm were removed from the stem, the stem was washed with a copious amount of running water for 5 min. Soaked 70% alcohol, washed with sterile water and then soaked in 5% aqueous sodium hypochlorite solution for 10 min. The upper leaves were then removed and the stem tip was placed on a culture medium of the Linsmaier-Skoog type with the composition as described in Example II.

The 2-3 leaf stem was seeded on the same culture medium, which also contained 0.01 ppm 2,4-dichloro-phenoxyacetic acid and 0.1 ppm benzylaminopurine. After a culture period of 5-6 weeks, the leaf garlands were separated.

The leaf wreaths were inoculated on a modified variant of the culture medium described in Example II, the amount of certain components being reduced by half. These amounts and their components were the following: NH4N03 825.0 mg / 1 KN03 950.0 mg / 1 CaCl2.2H20 220.0 mg / 1

MgSO 4 .7H 2 O 185.0 mg / 1 KH 2 PO 4 85.0 mg / 1 0.1 ppm indole butyric acid was also added to the culture medium as an additive.

The leaf wreaths were grown on the culture medium for 2 weeks under the conditions indicated in Example II, and then after the appearance of roots, the plants were transplanted onto a modified culture medium of the above composition, which however did not contain indole butyric acid. The plants were then grown to full development, i.e., until the roots reached 5-6 cm in length. The plants were then adapted to arable conditions, as described in Example II.

Neither the total glycoside content nor the amount of C-series glycosides observed in the plants emerged from the micro-multiplied seedlings deviates significantly from those measured in the parent plant.

EXAMPLE V

The procedure was as described in Examples I and III, except that the leaf arcs formed on the culture medium which induced side shoot development (a culture medium containing 1 ppm indole acetic acid, 1 ppm ben-30-zylaminopurine and 0.1 ppm gibberellic acid) were not directly were rooted, but again placed on a propagation culture medium and incubated there for 4 weeks to form a next generation of garlands. This procedure was repeated three times, after which the 35 plants were rooted as described in Example 1. From 10 young plants, 3000 rooted seedlings were obtained by this method. The seedlings were adapted to arable land conditions, as described in Example I.

8201937 - 16 -

EXAMPLE VI

The procedure was as described in Examples II and IV, except that the wreaths formed on the culture medium of the Linsmaier-Skoog type, which also contained 1 ppm benzylaminopurine and 0.1 ppm indole acetic acid, were not rooted directly , but again were placed on a multiplication culture medium and after 5-6 weeks of culture the leafy wreaths formed were separated. This procedure was repeated until the desired amount of plants was obtained. In this way, starting from a single seed or shoot, approximately 27,000 plants were obtained within about 5 months.

15 8201937

Claims (17)

1. Process for the preparation of foxglove multiplication material (seedling) (Digitalis lanata Ehrh.) Under vegetative micro-multiplication in a tissue culture by subjecting the seeds or stem tips of open air plants to surface sterilization, characterized in that a) a sterile plant with some, preferably 2-3, leaves is produced from the sterile seed or sterile stem tip of an open-air plant on a known culture medium, b) the sterile plant with any, preferably 2-3, leaves uprooted and then is inoculated (transferred) onto a multiplication culture medium containing auxin (s), cytokinin (s) and possibly gibberellic acid, the plant is grown there until a stage of 20-40 side shoots is reached, then the plant is split into wreaths of preferably 2-3 leaves each and if desired the cultivation is repeated until the desired number of plants is obtained and, c) the sterile leaflets, preferably 2-3 leaves, are transplanted to a rooting culture medium containing an auxin or an auxin with a cytokinin and root shoots are induced until roots of 4-8 mm length are developed, d) the plants with roots of 4-8 mm in length are rooted on a culture medium without auxin (n) and cytokinin (n), until a root length of 4-6 cm is reached and 30 e) if desired, the obtained propagating material (seedling ) is adapted to outdoor conditions. 2. A method according to claim 1, characterized in that a young plant is produced from the sterile seed on aqueous agar, the young plant is then transplanted onto a culture medium of known composition and further cultivated until the development of some preferably 2-3, leaves. 8201937 r - 18 - A method according to claim 1, characterized in that the sterile plant with some, preferably 2-3, leaves is produced from the sterile seed or sterile stem tip of a plant which is in the open air under conditions cultured by inoculating the starting material at 24 + 2 ° C and a relative humidity of 50-80% under illumination at an intensity of 9,000-14,000 lux and preferably using an 8/16 h night / day illumination period. Method according to claim 2, characterized in that the young plant is produced from the sterilized seed at 24 + 2 ° C and a relative humidity of 50-80% under illumination with an intensity of 50-100 lux and preferably under using a night / 15 day exposure period of 8/16 h and the resulting young plant is seeded (transferred) on a culture medium of known composition. Method according to any one of claims 1-4, characterized in that the ratio of auxin 20 to cytokinin of the multiplication culture medium is set between 1: 1 and 1:10. 6. A method according to any one of claims 1-5, characterized in that 0.1-0.5 ppm gibberellic acid is also added to the culture medium containing an auxin and a cytokinin in a ratio of 1: 1. Process according to any one of claims 1 to 6, characterized in that indole acetic acid or 2,4-dichlorophenylacetic acid is used as auxin. 8. A method according to any one of claims 1-7, 30, characterized in that benzylaminopurine is used as cytokinin. Method according to any one of claims 1-8, characterized in that the root shoot is induced at 24 + 2 ° C and a relative humidity of 50-80% under illumination with an intensity of 9,000-14,000 lux and preferably under application of a night / day lighting period of 8/16 h. 10. A method according to any one of claims 1-9, 8201937-19 - characterized in that the culture medium inducing root-shooting contains an auxin or an auxin and a cytoquinine. A method according to claim 1 or 10, characterized in that the culture medium which induces root-shooting contains 0.1-0.5 ppm indole butyric acid or 0.8-1.5 ppm α-naphthylacetic acid as auxin. 12. A method according to claim 1 or 10, characterized in that when α-naphthylacetic acid 10 is used as auxin, 10% kinetin, calculated on the amount of α-naphthylacetic acid, is added to the culture medium as cytokinin. 13. A method according to any one of claims 1-12, characterized in that the culture medium for inducing root shoot and / or the culture medium for effecting root shoot preferably contains half the amount of macro elements present is in the known culture media. Method according to any one of claims 1-13, 20, characterized in that the rooting is effected at 24 + 2 ° C and a relative humidity of 50-80% under illumination with an intensity of 9,000-14,000 lux and preferably under application of a night / day lighting period of 8/16 h. Method according to any one of claims 1 to 14, characterized in that the propagating material (seedlings) is adapted to open air conditions in a peat / perlite mixture with a 60-80% moisture or in a sand / perlite mixture with a 60-80% 30 humidity and when a sand / perlite mixture is used, the nutrients are supplemented with a solution of macro and micro elements. A method according to any one of claims 1-15, characterized in that the multiplication material (seedlings) is adapted to the open air conditions in a plant box under illumination at an intensity of 12,000-16,000 lux, using a night / day exposure period of 8/16 h and a night / day temperature 8201937 ** V - 20 - temperature change of 15/21 ° C and setting the relative humidity for 1-8 d at 100% and then for a further 21 d at 50-80%. Method according to any one of claims 1-15, 5, characterized in that the propagating material (seedlings) is adapted to the open air conditions in a greenhouse under natural lighting for 1-8 d at 15-25 ° C and a relative humidity of 100% and then for a further 21 d at a relative humidity of 50-80%. 8201937