RU2807740C1 - Method of microclonal propagation of manchurian kirkazona (aristolochia manshuriensis kom.) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention can be used for the propagation of rare and medicinal tree species of Aristolochia L. genus, including Aristolochia L., including A. manshuriensis Kom. The developed method of microclonal propagation of Kirkazona Manchurian includes selection of explants and their sterilization, cultivation of sterile explants on a nutrient medium based on macro- and microsalts MS, propagation and rooting of microshoots on a nutrient medium based on macro- and microsalts MS, transfer of rooted microshoots to open soil conditions. The cultivation and propagation stages are carried out on a single nutrient medium based on macro- and microsalts MS, into which 0.1–0.7 mg/l 6-BAP and 0.5 mg/l ascorbic acid are additionally introduced, and for rooting microshoots a nutrient is taken MS medium with half the content of macro- and microsalts with the addition of 0.1–1.0 mg/l IBA. Sterilization of explants is performed by sequential treatment with a soap-alkaline solution for 10–15 minutes and a 0.1–0.2% diacid solution for 1–2 minutes with three times washing with sterile distilled water. Shoots of the current year 0.7–1.5 cm long with one or two axillary buds are taken as explants, and the rooting of each microshoot is carried out in a separate culture vessel.

EFFECT: obtaining a method of microclonal propagation of Manchurian kirkazona (Aristolochia manshuriensis Kom.)

1 cl, 1 dwg, 2 tbl

Description

The invention relates to plant biotechnology and can be used for propagation of rare and medicinal tree species of the genus Kirkazonaceae (AristolochiaL.), including for the Manchurian kirkazon (A. manshuriensis Kom.).

Kirkazon Manchurian is a woody vine of the genus Aristolochia (family Aristolochiaceae ), endemic to a small part of East Asia; its range is limited to the southwest of Primorsky Krai, the Korean Peninsula and the Mukden province of Eastern China. Vulnerable species on the border of its range. Natural populations of A. manshuriensis are in a depressed state, and self-renewal of the species is insignificant. The species is included in the Red Books of the RSFSR (1988), the Russian Federation (2008) and the Primorsky Territory (2008).

Scientists have determined that substances isolated from A. manshuriensis have medicinal properties [1]. Ariscanin A inhibits platelet aggregation; p -coumaric acid has antibacterial, antifungal, antihepatotoxic, antihypercholesterol, neuroprotective and choleretic effects. Ferulic acid shows antimitotic, antituberculosis, cytotoxic, antifungal, antiestrogenic, antihepatoxic properties, also inhibits platelet aggregation, and is an antioxidant; p -hydroxybenzoic acid activates the synthesis of prostaglandins, slows down the action of red blood cells, and is an antidote and antimutagen. Magnoflorin is a ganglion blocker that prevents an increase in blood pressure. Oleanoleic acid has antitumor, cardiotonic, diuretic, hypoglycemic, antituberculosis, antiplasmodium and antimalarial effects, reduces the permeability of blood capillaries, promotes the restoration and appearance of hepatocytes, inhibits platelet aggregation, triggers apoptosis, and is low-toxic. β-cytosterol has an antitumor, anti-inflammatory, antitussive effect, prevents the increase in cholesterol, inhibits platelet adhesiveness and tyrosinase. Stigmasterol exhibits antitumor, antileishmanial, antimalarial, and antiviral effects. Vanilla acid has antibacterial, antifungal, anti-inflammatory, anthelmintic properties, and is an antioxidant [1].

In Tibetan medicine, underground parts are used, wood is used in Korean and Chinese medicine as a diuretic; The infusion is drunk for rheumatism and radiculitis [2, 3, 4]. A drug was created from the stems of A. manshuriensis that affects heart rate in different doses [5]. Used for cardiac and renal edema, inflammation of the urinary tract. It is also prescribed to reduce fever and increase lactation [3]. Kirkazon Manchurian has long been used externally as an analgesic, and is also used for snake bites, theriotoxicosis, and also for stomatitis, diuretic phenomena, and hematuria [1,2].

Currently, the problem of preserving and restoring natural populations of the Manchurian plant is acute due to the uncontrolled collection of vines for medicinal raw materials. The selection of methods for mass propagation of plants for their further growing and planting in natural populations is the way to restore those that are depleted in numbers. The microclonal propagation method makes it possible to obtain genetically homogeneous regenerated plants in large quantities. Microplants can be used in pharmacology as a source of valuable biologically active substances and for introduction and reintroduction.

A known method for microclonal propagation of A. manshuriensis [6], including cultivation on a nutrient medium containing mineral macro- and microsalts according to the recipe of Murashige and Skoog (MS) [7], as well as various substances with cytokinin and auxin activity. The cytokinins used were 6-benzylaminopurine (6-BAP), N6-(2-isopentyl) adenine (2ip), 6-furfurylaminopurine (kinetin), and the drugs Dropp, Cytodef, Mycefit were also used. Of the substances with auxin activity, alpha-naphthylacetic acid (NAA), indolylbutyric acid (IBA), 3-indolylacetic acid (IAA) and 2,4-dichlorophenoxyacetic acid ( 2,4-D) were used. To reduce the phenolic load on microshoots, activated carbon was additionally introduced into the nutrient medium.

The disadvantage of this known method is the versatility of the substances used, which leads to an extension of the subcultivation period and an increase in material and labor costs. Despite the presence of activated carbon in all variants, browning of the medium was observed, probably due to a greater release of phenolic compounds. Thus, even the additional addition of substances to the nutrient medium did not allow the authors to obtain viable microplants - after 2-3 passages, the formed microshoots died.

There is also a known method for microclonal propagation of A. manshuriensis [8], including sterilization of primary explants with mercury (II) chloride, planting primary explants on 10 variants of nutrient media with macro- and microsalts according to the WPM recipe [9] and MS with the addition of vitamins: nicotinic acid - 0.5 and 1.0 mg/l, pyridoxine - 0.5 and 1.0 mg/l, glycine - 1.0 mg/l, ascorbic acid - 0.5 mg/l, thiamine - 0.5 mg/l, casein hydrolyzate - 80.0 mg/l, meso-inositol - 50.0 mg/l, kinetin - 0.2 mg/l; phytohormones: IBA - 0.5, 1.0 and 1.5 mg/l and IAA - 0.5, 1.1.5, 4 and 8 mg/l, 2ip - 4 and 8 mg/l. Daily passages were carried out for two weeks to reduce phenols released by explants in the medium.

The disadvantage of this method is the duration of treatment with an aggressive sterilizing agent, which leads to high mortality of primary explants. Daily transplants to a fresh nutrient medium lead not only to an increase in material and labor costs, but also to an increase in the duration of the period for obtaining a pure culture. In addition, additional transfers of sterile objects may increase the likelihood of contamination of the original explants.

Also The disadvantage of this known method is the use of nutrient media with multiple components and a relatively high concentration of phytohormones, which affects the genetic variability of plant cells, which is unacceptable for micropropagation and for further use of the resulting regenerated plants for medicinal purposes.

The closest technical solution for microcloning A. manshuriensis is the method of clonal micropropagation, including isolation of apical and lateral buds, sterilization with sodium hypochlorite (exposure 7-10 min). Planting on nutrient media containing macro- and microsalts according to the MS recipe with the addition of 0.8 mg/l 6-BAP and 0.05 mg/l IAA. As follows from the authors’ works, the propagation of microshoots and their subsequent rooting on a medium containing macro- and microsalts according to the MS recipe, with the addition of 20 mg/l sucrose and IBA at a concentration of 3-5 mg/l, was carried out by placing several cuttings in flasks.

Further adaptation of regenerated plants to ex vitro conditions was carried out by planting them on a mixture of sand, peat and turf leaf soil in a ratio of 1:1:1, previously sterilized at 85-90°C for 1-2 hours [10].

The disadvantages of this technical solution include:

- insufficiently high percentage of microshoots rooting (50%);

- the duration of treatment with an aggressive sterilizing agent can lead to high mortality of primary explants;

- a multi-stage period of subcultivation due to the use of different nutrient media for the development of adventitious buds, shoot growth and rooting of microplants, as a result of which the process of obtaining full-fledged microshoots is lengthened and material and labor costs increase. As a result, the process of obtaining regenerated A. manshuriensis plants becomes more complicated. This fact can lead to an increase in genetic (including chromosomal) variability of plant cells, which is unacceptable for the further use of the resulting regenerated plants as pharmacological raw materials;

- the viability of the resulting microplants may deteriorate due to the cultivation of several microshoots in one flask, since there is a high probability of intertwining of the resulting roots;

- there is a high probability of transmission of pathogens from one infected explant in a flask to all other explants, which reduces their viability.

The technical problem posed when creating the invention is the development of an effective method for obtaining viable microplants of A. manshuriensis for reintroduction and introduction while reducing the period of subcultivation of microplants for using the resulting plants as pharmacological raw materials and, as a result, reducing material and labor costs.

This technical problem is solved by the fact that in the known method of microclonal propagation of A. manshuriensis , including the selection of explants and their sterilization, cultivation of sterile explants on a nutrient medium based on macro- and microsalts MS, propagation and rooting of microshoots on a nutrient medium based on macro- and microsalts MS, transfer of rooted microshoots to open soil conditions, according to the invention, the cultivation and propagation stages are carried out on a single nutrient medium based on macro- and microsalts MS, into which 0.1-0.7 mg/l 6-BAP and 0.5 mg/l ascorbic acid are additionally added . In this case, MS medium with half the content of macro- and microsalts with the addition of 0.1-1.0 mg/l IBA is used as a nutrient medium for rooting microshoots.

Carrying out the stages of cultivation and propagation on a single nutrient medium contributes to the good development of adventitious buds, the growth and reproduction of shoots and the production of viable microshoots. Moreover, it is significant reduces the duration of the cycle for obtaining full-fledged microplants, since it eliminates the period of adaptation of shoots to the next version of the nutrient medium. This also allows you to reduce material and labor costs.

Additional administration of 6-benzylaminopurine at a concentration of 0.1-0.7 mg/l makes it possible to activate the dormant buds of the primary explant and stimulate shoot growth. The use of 6-BAP in a concentration greater than 0.7 mg/l leads to watering of microshoots, and the use of 6-BAP in a concentration less than 0.1 mg/l does not ensure the production of fully developed microshoots of A. manshuriensis .

The introduction of vitamin C (ascorbic acid), which is an antioxidant and takes part in the processes of division and photosynthesis, into the nutrient medium ensures the production of full-fledged regenerated plants while preserving the genotype of the original plants. The presence of ascorbic acid in the nutrient medium prevents the release of phenolic compounds by explants, which can lead to clouding of the nutrient medium, necrotization of the lower part of the shoot and deterioration in the conductivity of nutrients and, as a consequence, to a decrease in the quality of the resulting microshoots.

The introduction of ascorbic acid into the nutrient medium at a concentration of less than 0.4 mg/l has virtually no effect on the growth and development of microbes in vitro . Introducing it in a concentration of more than 0.6 mg/l is impractical, because At the same time, inhibition of growth processes in microplants was noted.

The use of a nutrient medium containing half the concentration of salts dissolved in distilled water according to the MS recipe with the additional introduction of IBA for rooting microshoots of A. manshuriensis allows one to obtain fully rooted, viable microplants with reduced material and labor costs.

The introduction of the phytohormone IBA into the nutrient medium in the range of 0.1-1.0 mg/l leads to an acceleration of the initiation of root primordia and the development of adventitious roots. An increase in the concentration of IBA to more than 1.0 mg/l leads to the formation of dense callus at the base of the cutting with deterioration of the rooting of microshoots in vitro . The use of an IBA concentration of less than 0.1 mg/l has virtually no effect on the rooting of microshoots and the development of the root system.

In order to reduce the influence of an aggressive sterilizing agent on the primary explant, it is advisable to sterilize explants using sequential treatment with a soap-alkaline solution for 10-15 minutes. and 0.1-0.2% diacide solution for 1-2 minutes. with washing three times with sterile distilled water.

Treatment with a soap-alkaline solution for less than 10 minutes did not lead to the death of the external infection, while treatment with a soap-alkaline solution for more than 15 minutes led to injury and necrosis of the primary explant.

The use of diacid solution promotes high-quality surface sterilization of explants. The use of a diacid solution in a concentration of less than 0.1% is not enough to obtain a sterile culture, and a solution concentration of more than 0.2% leads to necrosis of the primary explant tissue.

Treatment with the optimal diacid concentration for our task for less than 1 minute does not give the desired effect, and when treated for more than 2 minutes with a 0.2% diacid solution, necrosis of the treated surfaces is observed.

It has been experimentally established that in order to obtain viable microplants, it is necessary to take shoots of the current year 0.7-1.5 cm long with one or two axillary buds as explants.

Growing each microshoot in a separate culture vessel also helps achieve the stated technical result, since it does not allow the roots of microplants to intertwine and they are not injured when transferred to the soil. In addition, growing each microshoot in a separate culture vessel prevents the transfer of pathogens between explants.

The invention is illustrated by the following figures: FIG. the stages of microclonal reproduction of A. manshuriensis are shown, where: a) development of axillary buds; b) growth of microshoots; c) rooted microplants.

In table Table 1 shows the compositions of nutrient media for obtaining complete microplants of A. manshuriensis.

In table 2. indicators characterizing the viability of the resulting A. manshuriensis microplants are given.

The method is illustrated by the following examples.

To carry out the method of microclonal propagation of A. manshuriensis , we first prepare a nutrient medium for obtaining microshoots (cultivation and propagation stage) and a nutrient medium for rooting as follows (the contents of components are given in Table 1):

to prepare 1 liter of basic nutrient medium for cultivation and propagation, pour 50 ml of a mother solution containing macrosalts according to MS prescription into the first measuring cup: ammonium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, calcium nitrate tetrahydrate, potassium sulfate and 5 ml of mother solution , containing microsalts according to the MS prescription: boric acid, manganese sulfate dihydrate, copper sulfate pentahydrate, sodium molybdate dihydrate, zinc sulfate heptahydrate. Then add 5 ml of the mother solution: iron (II) sulfate heptahydrate, 2-water disodium salt of ethylenediaminetetraacetic acid, vitamins: ascorbic acid, glycine, thiamine and nicotinic acid; phytohormone: 6-BAP (in this case, the phytohormone is taken in different quantities - 0.5 and 1.2 mg/l), everything is mixed.

Dissolve sucrose in another measuring cup while stirring. The prepared sucrose solution is added to the first measuring cup and the total volume is adjusted to 500 ml with distilled water, the pH of the nutrient medium is adjusted to 4.6-4.8. In a third measuring cup, mix a sample of agar-agar with 500 ml of distilled water and heat until it is completely dissolved. Then all solutions are combined.

The prepared nutrient medium is poured into test tubes of 5 ml each, closed with cotton-gauze stoppers and sterilized by autoclaving at 0.8 atm for 20 minutes. After autoclaving, the nutrient medium is allowed to cool and settle for 1-2 days.

To prepare 1 liter of nutrient medium for rooting, pour 25 ml of a mother solution containing macrosalts according to the MS prescription into the first measuring cup: ammonium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, calcium nitrate tetrahydrate, potassium sulfate and 2.5 ml of a mother solution containing microsalts according to MS prescription: boric acid, manganese sulfate dihydrate, copper sulfate pentahydrate, sodium molybdate dihydrate, zinc sulfate heptahydrate. Then add 2.5 ml of the mother solution: iron (II) sulfate heptahydrate, 2-water disodium salt of ethylenediaminetetraacetic acid, vitamins: glycine, thiamine and nicotinic acid; phytohormone IMC in an amount of 0.5 mg/l, everything is mixed.

In a separate measuring glass, mix a sample of agar-agar with 500 ml of distilled water and heat until it is completely dissolved.

Dissolve sucrose in another measuring cup while stirring. The prepared sucrose solution is added to the first measuring cup and the total volume is adjusted to 500 ml with distilled water, the pH of the nutrient medium is adjusted to 4.6-4.8. Then combine all the solutions.

The prepared nutrient medium is poured into test tubes of 5 ml each, closed with cotton-gauze stoppers and sterilized by autoclaving at 0.8 atm for 20 minutes. After autoclaving, the nutrient medium is allowed to cool and settle for 1-2 days.

Example 1.

The primary explant - shoots of the current year 0.7 cm long with one or two axillary buds - is sequentially treated with a soap-alkaline solution for 10 minutes. and 0.2% diacid solution for 1 min with washing three times with sterile distilled water, after which the explant is placed vertically on a nutrient medium for cultivation and propagation containing 0.5 mg/l 6-BAP. Primary explants are taken in the amount of 10 pieces in triplicate. Explants are cultured for 3 weeks at a temperature of 24±1°C, a 16-hour photoperiod (16/8), and illumination with white fluorescent lamps with an intensity of 4 thousand lux. and relative air humidity of about 70%. After cultivation, the resulting microshoots are transferred to a fresh nutrient medium of the same composition for further propagation of microshoots, on which they are cultivated for three weeks. Then the microshoots are placed in separate culture vessels on a nutrient medium for rooting containing 0.5 mg/l IBA.

The cultivation results are presented in table. 2 and fig. a and b.

Example 2.

The prepared nutrient medium is poured into test tubes of 5 ml each, closed with cotton-gauze stoppers and sterilized by autoclaving at 0.8 atm for 20 minutes. After autoclaving, the nutrient medium is allowed to cool and settle for 1-2 days.

Before planting, sterilization of primary explants - shoots of the current year 1.5 cm long with one or two axillary buds - is carried out by sequential treatment with a soap-alkaline solution for 15 minutes and 0.1% diacid solution for 2 minutes with three times washing with sterile distilled water, after which the explant is placed vertically onto a nutrient medium for cultivation and propagation containing 1.2 mg/l 6-BAP. Primary explants are taken in the amount of 10 pieces in triplicate. Explants are cultured for 3 weeks at a temperature of 24±1°C, a 16-hour photoperiod (16/8), and illumination with white fluorescent lamps with an intensity of 4 thousand lux. and relative air humidity of about 70%. After cultivation, the resulting microshoots are transferred to a fresh nutrient medium of the same composition for further propagation of microshoots, on which they are cultivated for three weeks. Then the microshoots are placed in separate culture vessels on a nutrient medium for rooting containing 0.5 mg/l IBA.

The cultivation results are presented in table. 2 and fig. a and b.

Example 3.

The prepared nutrient medium for rooting is poured into culture vessels, in particular, using test tubes, into each of which 5 ml is poured, closed with cotton-gauze stoppers and sterilized by autoclaving at 0.8 atm for 20 minutes. After autoclaving, the nutrient medium is allowed to cool and settle for 1-2 days.

Before planting for rooting, the explant is placed vertically on a nutrient medium for rooting containing 0.5 mg/l IBA.

Explants are taken in quantities of 10 in triplicate, cultivated at a temperature of 24±1°C, a 16-hour photoperiod (16/8), and illuminated with white fluorescent lamps with an intensity of 4 thousand lux. and relative air humidity of about 70%. Root formation and growth occurs within 4 weeks. By this time, microplants reach 23.85±2.73 mm in height and have 3.0±0.41 pieces. leaf nodes and 8.33±2.19 pcs. roots with a root length of 23.33 mm.

After this, the resulting full-fledged microplants with a well-developed root system are planted in prepared soil for possible further use during reintroduction and introduction, or the use of the resulting plants as pharmacological raw materials.

The view of rooted microplants is shown in Fig. V.

As can be seen from the results presented in Table 2, the described method achieves the stated technical result and the resulting viable microplants of A. manshuriensis can be used for reintroduction and introduction while reducing the period of subcultivation of microplants and for using the resulting plants as pharmacological raw materials.

Literature

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2. Plant resources of the USSR. T. 1 Flowering plants, their chemical composition, use // resp. ed. A.A. Fedorov. L.: Nauka, 1984. 464 p.

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6. Doan T.T. Features of clonal micropropagation of rare and medicinal plants (Euonymus nana Bieb., Dioscorea nipponica Makino., Dioscorea caucasia Lipsky. and Aristolochia manshuriensis Kom.): dis. - Ross. state agrarian univ., 2013.

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10. clause of the Russian Federation No. 2662682, IPC A01H 4/00; A01H 5/00, publ. 06/21/2018.

Claims (1)

A method of microclonal propagation of Manchurian kirkazona (Aristolochia manshuriensis Kom.), including selection of explants and their sterilization, cultivation of sterile explants on a nutrient medium based on macro- and microsalts MS, propagation and rooting of microshoots on a nutrient medium based on macro- and microsalts MS, transfer of rooted microshoots in open soil conditions, characterized in that shoots of the current year 0.7-1.5 cm long with one or two axillary buds are taken as explants; explants are sterilized by sequential treatment with a soap-alkaline solution for 10-15 minutes and 0 ,1-0.2% diacid solution for 1-2 minutes with washing three times with sterile distilled water, the cultivation stage and the propagation stage are carried out on a single nutrient medium based on macro- and microsalts MS, into which 0.5- 1.2 mg/l 6-benzylaminopurine and 0.5 mg/l ascorbic acid, and as a nutrient medium for rooting microshoots, take a medium with half the content of macro- and microsalts MS and the addition of 0.5 mg/l indolylbutyric acid, and After rooting, each microshoot is placed in a separate culture vessel.