CN115413579A - Wild kiwi fruit tissue culture method and catechin preparation method - Google Patents

Wild kiwi fruit tissue culture method and catechin preparation method Download PDF

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CN115413579A
CN115413579A CN202211143727.6A CN202211143727A CN115413579A CN 115413579 A CN115413579 A CN 115413579A CN 202211143727 A CN202211143727 A CN 202211143727A CN 115413579 A CN115413579 A CN 115413579A
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tissue culture
wild kiwi
wild
culture
mol
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韦桂丽
韦坤华
梁莹
韦范
秦双双
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Guangxi Botanical Garden of Medicinal Plants
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/002—Culture media for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
    • C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/14—Measures for saving energy, e.g. in green houses

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  • Life Sciences & Earth Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention discloses a wild kiwi tissue culture method, which comprises the following steps: taking the wild kiwi fruit tissue culture seedling for rooting culture, wherein a rooting culture medium comprises 1/2MS, indolebutyric acid, sucrose and agar powder; wherein, in the rooting culture stage, the light intensity is 180-220 mu mol/(m) 2 S) light irradiation. The method can improve the catechin content of wild kiwi plant seeds, and is convenient for preparing catechin.

Description

Wild kiwi fruit tissue culture method and catechin preparation method
Technical Field
The invention relates to the technical field of tissue culture. More specifically, the invention relates to a wild kiwi fruit tissue culture method.
Background
Catechin has antitumor, antioxidant, antibacterial, and heart and brain organ protecting effects. The wild kiwi fruit plants contain catechin, but the content is not high, the wild kiwi fruit resources in China are various in variety, large in quantity and extremely wide in regional distribution, and if the catechin content in the wild kiwi fruit plants can be increased, the medicinal value of the wild kiwi fruit plants can be increased and the economic benefit can be enhanced.
Disclosure of Invention
The invention aims to provide a tissue culture method of wild kiwi fruits and a catechin preparation method, which can improve the catechin content of wild kiwi fruit plants and further facilitate the preparation of catechin.
To achieve these objects and other advantages and in accordance with the purpose of the invention, as embodied and broadly described herein, the present invention provides a method for tissue culture of wild kiwi, comprising: taking the wild kiwi fruit tissue culture seedling for rooting culture, wherein a rooting culture medium comprises 1/2MS, indolebutyric acid, sucrose and agar powder; wherein, in the rooting culture stage, the light is irradiated by light with the light intensity of 180 to 220 mu mol/(m 2 s).
Further, the concentration of the indolebutyric acid is 0.5-0.7 mL/L.
Furthermore, the concentration of the sucrose is 25-35 g/L.
Furthermore, the concentration of the agar powder is 4-6 g/L.
Further, rooting culture is carried out for 25-40 days.
Further, the rooting culture medium is composed of 1/2MS, 0.6mL/L indolebutyric acid, 30g/L sucrose and 5g/L agar powder, rooting culture is carried out for 30 days, and the light intensity is 200 mu mol/(m) mol in the rooting culture stage 2 S) LED light irradiation, with a temperature of 25 ℃ and a humidity of 40%.
Further, the method also comprises propagation culture, wherein the propagation culture comprises MS, 6-benzylaminopurine and indoleacetic acid.
Furthermore, the concentration of the 6-benzylaminopurine is 0.8-1.2 mL/L.
Furthermore, the concentration of the indoleacetic acid is 0.15-0.25 mL/L.
According to another aspect of the present invention, there is also provided a catechin preparing method comprising:
obtaining a wild kiwi plant by using the wild kiwi tissue culture method;
extracting wild kiwi plants to obtain catechin.
The invention at least comprises the following beneficial effects:
the invention carries out rooting culture on the wild kiwi fruit tissue culture seedling, and the culture process is 180-220 mu mol/(m) 2 S) light and rooting MediumThe method improves the catechin content in the wild kiwi plant and improves the medicinal value and the economic benefit of the wild kiwi.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Embodiments of the present application provide a wild kiwi tissue culture method, comprising: taking wild kiwi fruit tissue culture seedlings for rooting culture, wherein a rooting culture medium comprises 1/2MS, indolebutyric acid, sucrose and agar powder, and the steps of inducing, proliferating and propagating the tissue culture seedlings and the like can refer to the prior art; wherein, in the rooting culture stage, the light intensity is 180-220 mu mol/(m) 2 S) light irradiation; preferably 200. Mu. Mol/(m) 2 S); through detection, the content of the active carbon is 180 to 220 mu mol/(m) 2 S) and rooting culture medium, the concentration of catalase is higher, the high concentration of catalase shows that the organism self-defense capability is strong, toxic product hydrogen peroxide generated in the in vivo metabolic process can be removed in time, the activity of superoxide dismutase is higher than that of M1, the total protein content is also higher, the wild kiwi fruit grows faster, and the catechin content in the wild kiwi fruit plant is higher.
In other embodiments, the concentration of indolebutyric acid is 0.5-0.7 mL/L, preferably 0.6mL/L.
In other embodiments, the concentration of sucrose is 25 to 35g/L, preferably 30g/L.
In other embodiments, the concentration of agar powder is 4 to 6g/L, preferably 5g/L.
In other embodiments, rooting culture is carried out for 25 to 40 days, preferably 30 days.
In other embodiments, the rooting medium is composed of 1/2MS, 0.6mL/L indolebutyric acid, 30g/L sucrose and 5g/L agar powder, and is cultured for 30 days in rooting medium stage with light intensity of 200 μmol/(m) mol 2 S) LED light irradiation, with a temperature of 25 ℃ and a humidity of 40%.
In other embodiments, the method further comprises propagation culture, wherein the propagation culture comprises MS, 6-benzylaminopurine and indoleacetic acid; preferably, the temperature is kept at 25 ℃, the humidity is kept at about 40%, sterile operation is carried out on an ultra-clean workbench, wild kiwi fruit tissue culture seedlings are clamped into a sterile tray from a tissue culture seedling bottle by using forceps, stems with tender buds and axillary buds are selected and cut off in whole sections by using scissors, two tender bud nodes of one section of tissue culture seedlings are suitable, the whole length is 2-3cm, and if leaves of the wild kiwi fruit plants are too long, so that the bottom ends of the wild kiwi fruit plants cannot contact with a propagation medium, some leaves can be cut off properly; inserting the stem of the tender bud of the wild kiwi fruit into a culture medium by using tweezers, wherein the depth is one third to one half of the culture medium, the nutrition absorption of plant tissues is facilitated, and the method is suitable for grafting 4-5 plants in one bottle.
In other embodiments, the concentration of 6-benzylaminopurine is 0.8 to 1.2mL/L; preferably, it is 1mL/L.
In other embodiments, the concentration of indole acetic acid is from 0.15 to 0.25mL/L, preferably 0.2mL/L.
Embodiments of the present application also provide a catechin preparation method, comprising: obtaining a wild kiwi plant by using the wild kiwi tissue culture method; extracting wild fructus Actinidiae chinensis to obtain catechin, for example, extracting with phosphoric acid buffer solution such as PBS buffer solution; alternatively, the following method may be used for extraction: cutting a wild kiwi fruit plant sample, putting the cut wild kiwi fruit plant sample into a mortar, adding liquid nitrogen, keeping the enzymatic activity of various biological enzymes of plant cells, adding a proper amount of phosphate buffer (pH = 6.8), grinding the sample until homogenate is obtained, pouring the homogenate into a centrifuge tube, adding a proper amount of phosphate buffer (pH = 6.8) to wash the mortar, pouring the solution in the mortar into the centrifuge tube, putting the centrifuge tube into a low-temperature centrifuge, and setting the rotation speed of 14000r.p.m and the time of 5min; the supernatant was taken out and put into a 10mL centrifuge tube, and the volume was determined with 100mmol/L phosphate buffer (pH = 6.8), and the solution was stored at a low temperature of 4 ℃ for future use.
The following examples are given by way of illustration.
Example (b):
taking a wild kiwi fruit tissue culture seedling, and performing rooting culture on the same wild kiwi fruit rooting culture medium: 1/2MS +0.6IBA +30.0 g.L －1 Sucrose +5.0 g. L －1 Agar powder, under the same temperature (25 +/-3) DEG C, the LED light intensity is 200 mu mol/(m) 2 S) culturing the tissue culture seedling of the wild kiwi fruit for 30 days;
comparative example:
the light intensity is reduced to 110 mu mol/(m) 2 S), the other conditions and parameters are the same as in the examples.
And (4) conclusion:
the wild kiwi plants obtained in the examples and the comparative examples were tested, and the results are as follows.
1. In physiological and biochemical experiments, the determination data of peroxidase activity (POD) is very obvious, and the peroxidase concentration of wild kiwi fruits under the light intensity of 200 mu mol/(m 2. S) is more than 110 mu mol/(m 2 s) 2 S) high, high peroxidase concentration indicates a strong self-protection and cold-resistance capability of the organism, i.e. a light intensity of 200 [ mu ] mol/(m) 2 S) specific light intensity of 110. Mu. Mol/(m) for wild kiwi fruit grown in 2 S) good stress resistance. The determination data of the activity of the peroxygenase (CAT) has obvious difference, and the light intensity is 200 mu mol/(m) 2 S) catalase concentration ratio light intensity 110. Mu. Mol/(m) 2 S) is high, the concentration of catalase is high, which indicates that the organism has strong self-defense capability and can timely eliminate toxic product hydrogen peroxide generated in the metabolic process in vivo, namely the light intensity is 200 mu mol/(m) 2 S) specific light intensity of 110. Mu. Mol/(m) for wild kiwi fruit grown in 2 S) good stress resistance. The activity determination data of the superoxide dismutase (SOD) enzyme has obvious difference, and the light intensity is 200 mu mol/(m) 2 S) the wild kiwi fruit grown under the conditions has higher superoxide dismutase activity than M1 and light intensity of 200 mu mol/(M) 2 S) the stress resistance of the wild kiwi fruit growing under the conditions is stronger, namely the light intensity is 200 mu mol/(m) 2 S) to promote the SOD predominance. General assemblyThe protein content is also determined by the light intensity of 200 mu mol/(m) 2 S) the wild kiwifruit grown in the culture medium has a higher content, indicating that the cells have strong water retention capacity.
2. In the five endogenous hormone indexes, the light intensity is 110 mu mol/(m) in table 1 2 S) abscisic acid content ratio intensity of 200. Mu. Mol/(m) 2 S) while low concentration ABA promotes growth and enhances stress resistance of plants to stress environment, i.e. light intensity is 200 [ mu ] mol/(m) 2 S) the wild kiwifruit grown well. Light intensity is 110 mu mol/(m) in gibberellin content determination 2 S) specific intensity of 200. Mu. Mol/(m) 2 S) the wild kiwifruit grown under the condition is high, and the GA content is high, so that the growth of the plants is promoted. The light intensity of the brassinosteroid content determination is 110 mu mol/(m) 2 S) specific intensity of 200. Mu. Mol/(m) 2 S) is high, the BR content is high, high active substances promoting plant stem elongation and cell division are suggested, and the defense against environmental stress is strong. Except that the intensity of light was 200. Mu. Mol/(m) in the assay of zeatin and methyl jasmonate 2 S) higher than 110. Mu. Mol/(m) of light intensity 2 S), high ZA content, promoting callus growth, high JA-ME content, suggesting that growth has reached equilibrium, insufficient nutrients in the culture medium, gradually inhibiting growth from beginning to age. Compared with 110 [ mu ] mol/(m) 2 S) overall, the intensity is again 200. Mu. Mol/(m) 2 S) the growth vigor of wild kiwifruit grown under the conditions is relatively fast.
TABLE 1 treatment effect of different LED plant lighting lamp illumination intensities on physiological and biochemical indexes of wild kiwi fruit tissue culture seedling
Figure BDA0003854750190000041
Figure BDA0003854750190000051
3. The light intensity is 110. Mu. Mol/(m), see Table 2 2 S) the catechin content of the wild kiwi tissue culture seedling is 0.221mg/g, the light intensity is high200μmol/(m 2 S) the catechin content of the wild kiwi tissue culture seedlings is 0.235mg/g, the epicatechin content is 0.851mg/g and 0.967mg/g, respectively, and obviously, the light intensity is 200 mu mol/(m 2 s) compared with the light intensity is 110 mu mol/(m 2 s) 2 S) the treatment favoured the accumulation of catechins and epicatechins, and the resulting plants had significantly higher contents of catechins and epicatechins.
TABLE 2 results of treatment of catechin and epicatechin contents of wild kiwi fruits by different illumination intensities of LED plant illumination lamps
Figure BDA0003854750190000052
The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. The application, modification and variation of the wild kiwi tissue culture method and the catechin preparation method of the present invention will be apparent to those skilled in the art.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the embodiments shown and described without departing from the generic concept as defined by the claims and their equivalents.

Claims (10)

1. The wild kiwi fruit tissue culture method is characterized by comprising the following steps:
taking the wild kiwi fruit tissue culture seedling for rooting culture, wherein a rooting culture medium comprises 1/2MS, indolebutyric acid, sucrose and agar powder;
wherein, in the rooting culture stage, the light intensity is 180-220 mu mol/(m) 2 S) light irradiation.
2. The method for tissue culture of wild kiwi of claim 1, wherein the concentration of indolebutyric acid is 0.5-0.7 mL/L.
3. The tissue culture method of wild kiwi fruit of claim 2, wherein the concentration of sucrose is 25-35 g/L.
4. The tissue culture method of wild kiwi fruit of claim 3, wherein the concentration of agar powder is 4-6 g/L.
5. The tissue culture method of wild kiwi fruit of claim 4, wherein rooting culture is carried out for 25-40 days.
6. The method for tissue culture of wild kiwi as claimed in claim 5, wherein the rooting medium is composed of 1/2MS, 0.6mL/L indolebutyric acid, 30g/L sucrose and 5g/L agar powder, rooting culture is carried out for 30 days, and the light intensity used in the rooting culture stage is 200 μmol/(m) mol 2 S) LED light irradiation, with a temperature of 25 ℃ and a humidity of 40%.
7. The method for tissue culture of wild kiwi of claim 1, further comprising propagation culture, wherein the propagation culture comprises MS, 6-benzylaminopurine and indoleacetic acid.
8. The method for tissue culture of wild kiwi of claim 7, wherein the concentration of 6-benzylaminopurine is 0.8-1.2 mL/L.
9. The tissue culture method of wild kiwi fruit of claim 7, wherein the concentration of indoleacetic acid is 0.15-0.25 mL/L.
10. The preparation method of catechin is characterized by comprising the following steps:
obtaining a wild kiwi plant by using the wild kiwi tissue culture method according to any one of claims 1-9; extracting wild kiwi plant to obtain catechin.
CN202211143727.6A 2022-09-20 2022-09-20 Wild kiwi fruit tissue culture method and catechin preparation method Pending CN115413579A (en)

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CN107549014A (en) * 2017-09-27 2018-01-09 重庆市天沛农业科技有限公司 A kind of mating system of red magnificent Kiwifruit Tissue Culture seedling
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CN108271691A (en) * 2018-01-29 2018-07-13 宝鸡松良农业科技有限公司 A method of carrying out tissue cultures using Kiwi berry stem
CN109392715A (en) * 2018-11-22 2019-03-01 中国科学院武汉植物园 A kind of tissue culture and rapid propagation method of Kiwi berry gold and jade
CN110881408A (en) * 2019-12-27 2020-03-17 福建省农业科学院果树研究所 Method for establishing tissue culture rapid propagation system of Donghong kiwi fruit

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