DD207731A1 - METHOD FOR THE VIRUS-FREE VEGETATIVE REPRODUCTION AND CONSERVATION OF DIGITALIS HIGH PERFORMANCE PLANTS - Google Patents

Abstract

The invention relates to a method for the non-viral vector growth and preservation of digital high-performance plants in Vitro, which over a long period produces an equally high content of cortisol in its composition. THEREFORE, AFTER AN INTERMITTENT TEMPERATURE AND LIGHTING EFFECT THAT LEADS TO ELIMINATE VIRUSES FROM THE MERISTEMIC WOVEN FABRICS, FROM MERISTEMEN OR MERISTEMANAGENTS BZW. BRANCH TIPS STENGELSTUECKEN, PAGE buds or flowers WITH meristems OR MERISTEMANLAGEN UNDER STERILE CONDITIONS ON USUAL nutrient media cytokinins AND / OR auxins INCLUDED CAN, RUNG AND meristem tissue ATTRACTED THOSE OF THE AUSGANGSEXPLATATEN DISCONNECTED, NEXT MUSHROOMS AND CLUB TENT AND broth containing ROCK FLOUR ALUMINUM SILICATE OF CERTAIN KORNGROESSE BROUGHT TO ROOTING. THE PLANTS THUS OBTAINED WILL BE ISOLATED, PICTURED IN ROCKSTICK AND UNDER FOLIETUNNEL AT GEWAECHSHAUS- BZW. FREELAND CONDITIONS ADAPTED.

Description

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Title of the invention

Method for virus-free vegetative propagation and preservation of digitalis high performance plants

Field of application of the invention

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Daia for the production of cardiac glycosides used plant material from Digitalia lanata ⁷ and Digitalis purpurea is produced field-moderately using elite seed, which must be produced annually by a complex cross-cutting and SeLektionsprozeß. The invention makes it possible to obtain healthy and uniform starting material for production cultivation without the annual cycle of holding cultivation.

Characteristic of the known technical solutions

For the recovery of cardiac glycosides usually defined varieties of Digitalis lanata and Digitalis purpurea are agriculturally grown · Since die.Digitalispflanzen die after seed maturity, to preserve -the varieties as well as for seed production for growing a regular breeding process is required.! Due to the cross-pollination in the genus Digitalis it comes to an undesirable range of variation in the characteristics of the offspring, which must be eliminated by, an extremely high analytical and cross-technical effort · To meet these difficulties, have been so far

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In principle, two ways are taken, namely: 1 · the formation of cardenolides in cell cultures of Digitalis, z. B. according to BD-PS Ir · 151 451, or 2 · the Verklonung of genetically high quality Outer lances, ζ · 'Β · on the basis of embryogenic cell cultures according to BD-PS Mrw 151 473.

The ridiculous parts of the former method are mainly in too low Glykosidausbetiten, undying cardenolide spectrum and in a long and elaborate Submerskultur. According to the second method, there is the danger that in embryogenic cell cultures the genetically valuable properties of the parent plant may be lost or adversely affected.

In Plant Cell Reports (1981) 2 »34-35, Erdei, I ·, Kistf, Z» 'and Maliga, P. reported on a method for the clonal propagation of digitalis lanata according to which seed cultures are obtained by inoculation in aseptic culture from RH-Hedium to linsmaier and Skoog, supplemented with 1 mg 6-BAP and 0.1 mg IES / 1, with 3 - 5, at the long shoot tips of germinated seeds or field crops.The formation of up to The proportion of root-forming shoots is increased by lowering the salt and auxin concentration. The transmission of the rooted shoots into the greenhouse occurs in 85-98 % of the cases when the growth takes place in hormone-free medium. This method has several drawbacks, namely that seeds are poorly suited as starting material for cloning seed plants for technical reasons, and that only a relatively low rate of yield can be achieved in this way It has also been shown that although the rooting of the meristem culture succeeds under certain conditions, the survival rate in the transition to greenhouse and, in particular, field conditions remains low, since the seedlings are not sufficiently resistant

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From the literature it is still known that digitalis. often infected by viruses, the z. In the Pail τοπ Digitalis lanata have been identified as a strain of tobacco mosaic virus and as field bean wilt virus (Schumann, Phytopath, Z * 48 (1963) 1-28). According to Silva and Pop (Pharmacia 2Q (1965) 110 - 112), the glycoside content of such virus-infected plants may decrease by 32-46 % . Therefore, an intensive vegetative propagation, as represented by the in vitro fertilization, is vimeless starting material and the M Possibility of a reliable Yirustest an indispensable prerequisite * The task of the virus-free vegetative propagation of high-performance plants, which are superior with respect to vitality and active substance content of the usually available breeding material, has not yet been solved, '

Object of the invention '. > '"',

The aim of the invention is the more effective use of high-performance digitalis plants with a simultaneous qualitative improvement of the germinal material and reduction of the cultivation effort.

Explanation of the essence of the invention

The object of the invention was to overcome the shortcomings of the previously known methods for the preservation and propagation of high-quality digitalis plant material and to develop a method which

- the preservation of the genetic material allows for the extraction of seed of selected high-performance digital plants for a long period beyond natural age,

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allowing the vegetative propagation of these plants in a very high number and with practical suitability for field cultivation, the resulting plants forming an XLon with uniformly high carbohydrate content uniform in composition; '- the elimination of virus infestation and testing

allowed on Tira freedom in the clone plants. By cultivating suitable virus-free clones in a mixture, a hybrid seed can be obtained, from which plants develop which, by heteroai effect, are superior to the cloned starting material in the mentioned characteristics. "

Thus, a method has been found for virus-free vegetative propagation and maintenance of genetically high-quality individuals from digit species, after which the plants are exposed to intermittent temperature and light effects for a certain time in the first or second growing year, in order to practically extant viruses from the meristems and meristem plants of the plants eliminate. Thereafter, meristematic tissues are transferred under sterile conditions to cvtokinis and optionally auxin-containing medium and cultured until sprouts or net meristem tissue are formed. These hay formations are separated from the initial explants, isolated and propagated in a manner known per se, whereby the augmentation cycle can be repeated as required. The rooting of the shoots is carried out under likewise sterile conditions by culturing on rock flour and Bewuraäungsmedium. The resulting plants are isolated, transferred from the sterile environment in Geateinsmehl and adapted under Folietunneln to greenhouse or field conditions. In the following, the freedom from viruses of the plants is checked by sensitive Hachweisverfahren. The

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virus-free plants can in turn be propagated vegetatively as described above or used for seed extraction. The preservation of the material is carried out by preservation, either in the form of sterile plants as canned labor or in the form of sterile meristems as a long-term depot.

Far method of the invention the virus · freedom is achieved by the plant ^ 4 to 10 weeks 16 hours intermittently about 37 ⁰ C and about 5000 Lux, and subjected to 8 hours about 24 ⁰ C and darkness. According to the invention, the starting materials for the sterile culture are meristems or meristem plants or shoot tips, stalk pieces, side buds or flowers with meristems or meri-breathing plants.

For the formation or propagation of sprouts or meristems, the culture medium according to Hurashige and Skoog (MS medium, Table 1) in original or ζ is preferred for the method according to the invention. B. by addition of various cytokinins and auxins modified composition used. , \\ ' ..', '· ·'. For rooting the attracted shoots are in accordance with the invention in tube. With rock flour from aluminum silicates a grain size of 0.5-3 mm and a nutrient solution with reduced or no cytokinin or increased auxin activity transferred; it turns out after leaving the cytokines z. However, rooting is also possible in ausin-free media The rock flour used for the adaptation after the in vitro phase corresponds to that used for rooting.

The detection of virus freedom is carried out according to the invention by specific tests on a serological basis. For the preservation of the material according to the erfinduigsgemäßen process either the sterile plants on a

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Auxin free substrate at 0 to 4 ⁰ O 500 - kept 1000 lux or sterile meristems after Torbehandlung with cryoprotectants, in particular mixtures of dimethylsulfoxide and water or dimethyl sulfoxide, polyhydric alcohols, preferably glycerol, and sucrose with water slowly or very quickly temperatures below - 100 ⁰ C exposed, stored in this area and quickly thawed for recultivation.

The method according to the invention thus enables the preservation of genetically valuable breeding material of digitalis species over a long period of time, leads to virus-free, genetically uniform plants (clones) with high vitality and qualitatively and quantitatively uniform cardenol content and ensures the reproducible propagation of this plant material over long periods , in any amount and regardless of the natural vegetation conditions. In accordance with the process of the invention, several tens of thousands of virus-free daughter plants with mutually identical properties can be obtained under production-related conditions from a digitalis soybean plant.

The invention will be explained in more detail with reference to the embodiments.

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embodiments

example 1

(Virus for re fl ecting meristems)

Digitalis plants in 1 * or 2 * vegetation are exposed for 8-10 weeks daily for Τβ hours a temperature of about 37 C and irradiated simultaneously with white light of about 5000 lux, they are zwisehen the individual heat treatments each 8 hours stored at about 24 ⁰ C in the dark.

Example 2 ',

(Cultivation of sterile shoots)

Meristems or Meristemanlagen or shoot tips, stalks, side buds or flowers with meristems or Meristemanlagen according to Example 1 treated plants are externally disinfected and brought to a liquid or solid Uährmedium according to Table 1, which additionally 0.02 mg kinetin / 1 or other cytokinins and possibly other auxins, e.g. B. 1.0 mg 2,4-dichlorophenosyacetic acid / l contains (1 explant to 10 - 15 ml of nutrient solution in roll edge bottles with dimensions of 80 χ 30 mm). The cultivation is carried out at 22 ⁰ C at a daily I6stündigen exposure, which is interrupted by 8-hour dark periods at 20 ⁰ C. After six weeks sprouts form and new meristems and buds develop.

Example 3

(Proliferation of sterile shoots), shoots obtained in Example 2 are placed on a medium according to Example 2 (1 shoot on 10-15 ml of medium in rolled-edge bottles with dimensions of 80 × 30 mm) and

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cultured in the light-dark-change as described in Example 2. In two to three holes, it forms solitary sprouts, which can be used singly and either for further propagation or regenerated to plants, as well as new meristems and buds. "

Example 4

(Rooting of sterile shoots) ^[

Sprouts obtained according to Example 3 are used in a medium according to Table 1, which is used to promote rooting Ausine, preferably 1.0 mg of 2,4-dichlorophenoxyacetic acid / l, and rock flour of aluminum silicates with a grain size of 1 - 2 mm, z. B "Perlite, contains, cultivated. Within about 8 weeks she has formed a sufficient root mass for the transfer from the sterile environment.

Example 5 "

(Adaptation of sterile propagated plants to field conditions and checking the freedom from viruses) The sterile plants prepared according to Example 4 are in rock meal, which corresponds to that in Example 4, piqued and cultured under Folietunneln. By constantly extending ventilation times, the humidity under the film tunnels is reduced until the plants are adapted to greenhouse conditions and later to outdoor conditions. The greenhouse plants are tested for their freedom from viruses using highly sensitive Hachwels methods. For this purpose, the viruses occurring on digitalis plants (eg broad bean wilt virus, strains of the tobacco mosaic virus) are isolated. These viruses are purified for antigen recovery by the method of Leiser and Richter (Arch. Phytopathol and Pflanzenschutz H (1978), pp. 337-350). Haeh setting up an immunization scheme

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too rabbit anti-sera are obtained · As a highly sensitive, specific test, the agar gel double diffusionateat is used, which is particularly suitable for routine examinations.

Example 6

Sterile plants produced (preservation of virus-free sterile mother plants) Each example 4 are on a hormone-free, for solidification with agar * or rock powder of aluminum silicates having a particle size of 1 to 2 mm (e.g., B · perlite) added medium at 0 to 4 ⁰ C and an illumination of ea 500 LuS "The sprouts of these plants can be used for a period of up to 1.2 months for re-propagation according to Example 3 · '

Example 7 '. ,

(Preservation of meristems), -. Sterile meristems obtained according to Example 2 or 3 are treated with cryoprotectants in the form of mixtures of 5-10 % dimethyl sulfocide and 90-95 % or 5-10 % mixtures . Dimethylsulfoxid, 5 - 10 % glycerol and 5 - 10 % sucrose and 70 - 85 % water and after constant action of these cryoprotectants slowly (0.5 to 1 ⁰ C / min) cooled to about - 100 ⁰ C. The meristems are then transferred to liquid nitrogen and stored in or over liquid nitrogen for any length of time. If necessary, the meristems are quickly thawed in a water bath at + 40 ⁰ G and cultured as described in Example ² . The developing rungs can be used according to Example 3 for further propagation. '' ·.

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Table 1

Modified Häiirmediuia after IURASHIGB and SKOOG (Phyaiol. Plant · Ί5 (1962) 479-497)

mg / 1

CaCl ₀ . 6-HoO Cm Cm 661 KH ₂ PQ ₄ 170 Ha ₂ BDTA. 2H ₂ O 37.3 H ₃ BO ₃ . , 6.2 MnSO ₄ 15.1 ZnSO ₄ . 7H ₂ O 8.6 KJ '0.83 Sa ₂ MoO ₄ ; _{# 2} yy ₂ o 0.25 M ₄ IO ₃ 1650.0 MO ₃ SIgSO ₄ , 7 H ₂ Q 1900.0 370.0 FeSO ₄ .7H ₂ O 27.8 thiamine 0.5 pyridoxine 0.5 licotinsäureamid 1.0 m-Inoait 100.0 sucrose 30000 Amino 3ätjregemiach (a "Table 2) 10.0 ml

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Modified amino acid composition according to KOBLITZ (H, KOBUTZ,

 J. HAGEI, Flora J ^ 2 (1962) 447).

fflg /. 1

! -Alanine 2970

! -Arginine,. 310

Asparagine. "380

! Aspartic acid 170

! -Glutamine 30

! -Glutamic acid 175

Glycine ^. 470

! -Histidine 5 '

! -Hydroxyproline 125

! -! eucin 495

! -lysine / HCl. 195

! -Methionine .5

! -Phenylalanine 5

L- »Proline 195

! -Serin,. 1275

! -Threonin 265 '

1-Tyroain 5

i-Yalin .230

Claims (10)

- 12 Invention claims 740 5 1. A method for virus-free vegetative propagation and preservation of digitalis high-performance plants by cloning meristematic tissue in vitro in a customary for tissue culture Hährmedium, characterized in that subjecting the starting plants intermittent temperature and light effects, atis the plants Meristeme or Heristemanlagen or, sprouting tips, Stem pieces, side buds or flowers with meristems or Heristemanlagen wins, these sterilized and cultured on a cytokinin and optionally containing auxin Hährmedium until new growth of sprouts and meristem tissue, this separated from the Ausgangssexplantaten, isolated and in a conventional manner on a Gytokinine and optionally auxins propagated, then, on rock meal with rooting nutrient solution with reduced or missing cytokinin content, preferably cultivated in the presence of auxins to rooting, which thus obtains isolated plants, adapted by propagation in rock flour under Folietunneln to greenhouse or * field conditions and checks for freedom from viruses and sterile plants at minimal development conditions or sterile meristems in the presence of cryoprotectant: preserved· Cryoprotectants at temperatures below -100 ⁰ C 2 * Method according to item 1, characterized in that the temperature and light over a period of 4 - 10, preferably 6 - 8 weeks intermittently for 16 hours at 37 ⁰ C and 5000 lux and 8 hours at 24 ⁰ C and darkness. 3 · Method according to item 1, characterized in that 23 974 0 for the rooting and adaptation phase rock flour of aluminum silicates with a particle size of 0.5 3 mm, preferably 1-2 mm, such as. B · The Altiminiuasilikatmehl perlite, applies. 4. The method according to item 1, characterized in that the greenhouse plants are tested for their freedom from viruses using highly sensitive detection methods on the basis of a specific serological test by means of antisera, preferably against the field bean wilt and tobacco mosaic virus. 5. A method for the preservation of high performance plants according to item 1, characterized in that in vitro attracted plants in the sterile state at 0 to 4 ⁰ C and 500 - 1000 lux on a hormone-free with agar or rock flour of aluminum silicates, preferably perlite, solidified Bahrmedium sealed, d · H · stored at a relative humidity close to 100 % , become· ' . - ·. 6. A method for preserving high-altitude crops according to item 1, characterized in that in vitro attracted meristems after cryoprotectant treatment either very slowly (approx. 0.5 to 5 ° C / min) to - 100 ⁰ C or   frozen very quickly by immersion in liquid nitrogen and then stored in or over liquid nitrogen, thawed again as quickly as possible and further cultivated on a suitable nutrient medium. 7 · retracts, according to item 1 and 6 characterized in that as cryoprotectors mixtures of 5 - 10% dimethyl sulfoxide and from 90 to 95% water, or from 5% -.10 Dimethylsulfosid, 5 - 10% glycerol, and 5 - 10% Saccharomyces 23 97 4 - 14 rose and 70 - 85 % water are used. 8, method according to. Item 6 dadureh indicated that the cryoprotectants act on the meristems for up to 48 hours to initiate the preservation. 9. The method according to item 6 dadureh characterized in that the building is carried out by heating with water clay about 40 ⁰ G. J 10. The method according to item 6 dadureh characterized in that the eryoprotector after thawing from the meristems slowly diffused into the surrounding medium and removed by replacement of the medium.