CN109220801B - Plant tissue culture method - Google Patents
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- 238000004161 plant tissue culture Methods 0.000 title claims abstract description 13
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- 241000382455 Angelica sinensis Species 0.000 description 1
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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
- A01H4/001—Culture apparatus for tissue culture
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- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a plant tissue culture method, which mainly comprises the following steps: the preparation method comprises the steps of preparing a combined culture medium, namely alternately laying round filter paper sheets dipped with plant hormones and an MS agar culture medium in a layered mode; inducing callus, namely placing a sterile explant on a filter paper sheet, and performing dark culture to obtain the callus; and thirdly, the callus absorbs the bottom layer to induce the phytohormone to start to grow, so that adventitious buds and adventitious roots are formed. The invention also discloses a preparation method and an operation process of the culture medium, which are the combined culture medium and the operation process. The invention can realize high inductivity, needs no switching, has short growth period, convenient harvest, needs no culture medium separation, meets the market demand and has great popularization value.
Description
Technical Field
The invention belongs to the technical field of plant organ culture, and particularly relates to a plant tissue culture method.
Background
The plant tissue culture is also called in vitro culture, which is characterized in that the plant cell has totipotency theory, and in the sterile and proper artificial culture medium and external factors, the in vitro organs, tissues, cells and protoplasts of the plant are used for inducing to form callus, adventitious buds and adventitious roots, and finally the callus, the adventitious buds and the adventitious roots grow into a complete plant.
Under the rapid development environment of the traditional Chinese medicine industry in recent years, due to the deep research on medicinal plants, the medicinal plants are continuously developed, the demand is continuously increased, and compared with a seed propagation mode, the tissue culture regeneration mode has the advantages of short period, no limitation of climate all the year round, no pollution of harmful substances such as heavy metal, pesticide and the like, less investment and high economic benefit, so that the method is widely concerned by people and is considered to be an important way which is hopeful to relieve the shortage of land resources and the inconsistency of quality standards of the traditional Chinese medicines.
Disclosure of Invention
The invention aims to provide a plant tissue culture method. Compared with the traditional planting mode, the invention has the advantages of good repeatability, high inductivity, rapid growth, high content of effective components and convenient harvest.
A plant tissue culture method, which uses a filter paper layer containing plant hormone and a combined culture medium prepared by overlaying solid culture to culture plant tissues, comprises the following steps:
s1, induction of callus: placing the sterile explant root and young stem section of the plant about 2cm or young leaf on the top filter paper layer of the combined culture medium, and carrying out sterile culture in the dark at 24-28 ℃ for 10-15 days to obtain fresh callus;
wherein the combined culture medium is formed by alternately superposing a filter paper layer and a solid layer from bottom to top; the filter paper layer is 1 or more pieces of filter paper containing phytohormone, the solid layer is a solid culture medium, and the bottom layer and the top layer of the combined culture medium are both filter paper layers; the filter paper layer plays a role in slowly releasing plant hormone in the process of plant tissue culture;
s2, induction of plant tissues: after obtaining the callus, adding an aseptic liquid culture medium into the combined culture medium, enabling the liquid to submerge the top filter paper layer by about 1mm, and continuously inducing adventitious roots or adventitious roots and adventitious buds; wherein the adventitious root induction conditions are as follows: carrying out sterile culture in the dark for 15-20 days at the temperature of 24-28 ℃ to obtain adventitious roots with callus; the conditions for adventitious root and adventitious bud induction were: and performing sterile culture for 15-20 d at 24-28 ℃ and illumination for 12h/d to obtain a complete plant with adventitious roots and adventitious buds.
Preferably, the plant hormone in step S1 is one or more of 2, 4-dichlorophenoxyacetic acid (2,4-D), 6-benzylamino adenine (6-BA), indolebutyric acid (IBA), naphthylacetic acid (NAA), and Kinetin (KT).
Preferably, the thickness of the solid medium in step S1 is 0.5-1 cm.
Preferably, the solid culture medium in step S1 is MS solid culture medium.
Preferably, the combined culture medium of step S1 is formed by alternately stacking four filter paper layers and three solid layers from bottom to top.
Preferably, the combined culture medium in step S1 is formed by alternately stacking three filter paper layers and two solid layers from bottom to top.
Preferably, the combined culture medium of step S1 is formed by alternately stacking two filter paper layers and one solid layer from bottom to top.
Preferably, the culture medium in step S2 is MS liquid culture medium.
Preferably, the combined culture medium in step S1 is, from bottom to top, a 1 st filter paper layer, a 1 st solid layer, a 2 nd filter paper layer, a 2 nd solid layer and a top filter paper layer; the 1 st filter paper layer contains 2.5-5.0 mg/L IBA, 0.2-1.0 mg/L NAA, 0.5-4.0 mg/L6-BA and 0.5-1.0 mg/L KT; the 2 nd filter paper layer contains 1.0-2.0 mg/L IBA, 0.5-1.0 mg/L2,4-D, 0.5-2.0 mg/L6-BA and 0.2mg/L NAA; the top filter paper layer contains 0.5-1.0 mg/L2,4-D and 0.2-1.0 mg/L6-BA; the first layer of solid layer is an MS solid culture medium with the thickness of 0.5cm, the concentration of sucrose is 30-50 g/L, and the content of agar is 8g/L, and the second layer of solid layer is an MS solid culture medium with the thickness of 1cm, the concentration of sucrose is 30-50 g/L, and the content of agar is 8 g/L;
preferably, the preparation method of the combined culture medium in step S1 includes:
s11, cutting filter paper: cutting qualitative filter paper into 8cm circular filter paper sheet with size consistent with the bottom of the sterile tissue culture bottle, sealing with kraft paper in a clean culture dish, and sterilizing at 121 deg.C and 0.1MPa for 25 min;
s12, preparing a filter paper layer:
preparation of the 1 st filter paper layer: filtering and sterilizing the phytohormone in a superclean bench by using a filter membrane with the aperture of 0.22 mu m, and mixing the phytohormone in a small sterile beaker to obtain mixed phytohormone 1, wherein the mixed phytohormone 1 contains 2.5-5.0 mg/L IBA, 0.2-1.0 mg/L NAA, 0.5-4.0 mg/L6-BA and 0.5-1.0 mg/L KT, stacking 4-8 sterilized filter paper sheets obtained in the step S11 together, and soaking the stacked filter paper sheets in the mixed phytohormone 1 for 20S to obtain a layer 1 filter paper layer for inducing adventitious roots; preparation of the 2 nd filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker to obtain mixed phytohormone 2, wherein the mixed phytohormone 2 contains 1.0-2.0 mg/L IBA, 0.5-1.0 mg/L2,4-D, 0.5-2.0 mg/L6-BA and 0.2mg/L NAA, stacking 3-8 sterilized filter paper sheets obtained in the step S11 together, and soaking the stacked filter paper sheets in the mixed phytohormone 2 for 20S to obtain a 2 nd induced filter paper layer for inducing adventitious roots;
preparing a top layer induction filter paper layer: filtering and sterilizing phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker to obtain mixed phytohormone 3, wherein the mixed phytohormone 3 contains 0.5-1.0 mg/L2,4-D and 0.2-1.0 mg/L6-BA, stacking 3-5 filter paper sheets sterilized in the step S11 together, and soaking the filter paper sheets in the phytohormone 3 for 20S to obtain a top induction filter paper layer for inducing callus;
s13, preparation of a culture medium: accurately weighing MS solid powder, dissolving the MS solid powder in water, adding sucrose and agar, adjusting the pH to 5.8-6.0, sterilizing at 121 ℃ and 0.1MPa for 25min, preserving heat and preventing solidification, and preparing an MS agar culture medium with the concentration of sucrose of 30-50 g/L and the content of agar of 8 g/L;
s14, preparing a combined culture medium: flatly paving the 1 st filter paper layer obtained in the step S12 at the bottom of the sterile tissue culture bottle in the step S11, pouring the culture medium prepared in the step S13 on the 1 st filter paper layer, solidifying to form a 1 st solid layer with the thickness of 1cm, flatly paving the 2 nd filter paper layer obtained in the step S12 on the 1 st solid layer, pouring the culture medium prepared in the step S13 on the 2 nd filter paper layer, solidifying to form a 2 nd solid layer with the thickness of 0.5, flatly paving the top filter paper layer obtained in the step S12 on the 2 nd solid layer to obtain a combined culture medium; the combined culture medium comprises the following components from bottom to top: the filter paper comprises a 1 st filter paper layer, a 1 st solid layer, a 2 nd filter paper layer, a 2 nd solid layer and a top filter paper layer.
The invention has the beneficial effects that:
the invention can realize high inductivity, needs less raw materials and low contamination rate, the callus does not need to be transferred, the growth cycle is short, the harvest is convenient, the culture medium separation is not needed, the market demand is met, and the invention has great popularization value.
Drawings
FIG. 1 is a schematic view of a combined culture medium prepared in example 1 of the present invention,
wherein 1, the 1 st filter paper layer; 2. a 1 st solid layer; 3. a 2 nd filter paper layer; 4. a 2 nd solid layer; 5. a top filter paper layer; 6. tissue culture bottles; 7. a bottle cap; 8. and (5) filtering the membrane.
Detailed Description
The invention is further illustrated by the following specific examples.
The experimental procedures used in the following examples are conventional unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. MS solid powder was purchased from: chembase, cat No.: 27-11-2016.
Example 1
A method for culturing plant tissue comprises:
s1, induction of ginseng callus: placing the root segment with high growth activity of sterile ginseng about 2cm on the top filter paper layer of the combined culture medium, and performing sterile culture at 24 deg.C in the dark for 10 days to obtain fresh ginseng callus;
s2, induction of adventitious roots of ginseng: after obtaining the callus, adding a sterile MS liquid culture medium into the combined culture medium, wherein the liquid height is about 1mm higher than the top filter paper layer, because the callus absorbs the bottom layer induced phytohormone, carrying out sterile culture for 15d under the conditions of 24 ℃ and darkness, and the adventitious root starts to grow, so as to obtain the ginseng adventitious root with the callus, wherein the culture time is 35 days, the number of the adventitious root is 6, the length is 3.2cm, and the induction rate is 95.1%.
The combined culture medium in the step S1 is formed by alternately superposing three filter paper layers and two MS solid culture media from bottom to top, and the preparation method comprises the following steps:
s11, cutting filter paper: cutting qualitative filter paper into 8cm circular filter paper sheet with size consistent with the bottom of the sterile tissue culture bottle, sealing with kraft paper in a clean culture dish, and sterilizing at 121 deg.C and 0.1MPa for 25 min;
s12, preparing a filter paper layer:
preparation of the 1 st filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 1, wherein the mixed phytohormone 1 contains 2.5mg/L IBA, 0.2mg/L NAA, 0.5mg/L6-BA and 0.5mg/L KT, stacking 8 sterilized filter paper sheets obtained in the step S11 together, and soaking the filter paper sheets in the mixed phytohormone 1 for 20S to obtain a 1 st filter paper layer for inducing adventitious roots; preparation of the 2 nd filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super clean bench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 2, wherein the mixed phytohormone 2 contains 1.0mg/L IBA, 0.5mg/L2,4-D, 0.5mg/L6-BA and 0.2mg/L NAA, stacking 3 sterilized filter paper sheets obtained in the step S11, and soaking the stacked filter paper sheets in the mixed phytohormone 2 for 20S to obtain a 2 nd induced filter paper layer for inducing adventitious roots;
preparing a top layer induction filter paper layer: filtering and sterilizing phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 3, wherein the mixed phytohormone 3 contains 0.5mg/L2,4-D and 0.5mg/L6-BA, stacking 5 filter paper sheets sterilized in the step S11, and soaking the stacked filter paper sheets in the phytohormone 3 for 20S to obtain a top induction filter paper layer for inducing callus;
s13, preparation of a culture medium: accurately weighing MS solid powder, dissolving in water, adding sucrose and agar, adjusting pH to 5.8, sterilizing at 121 deg.C and 0.1MPa for 25min, keeping the temperature to prevent solidification, and preparing into MS agar culture medium with sucrose concentration of 30g/L and agar content of 8 g/L;
s14, preparing a combined culture medium: flatly paving the 1 st filter paper layer obtained in the step S12 at the bottom of the culture bottle in the step S11, pouring the culture medium prepared in the step S13 on the 1 st filter paper layer, solidifying to form a 1 st solid layer with the thickness of 1cm, flatly paving the 2 nd filter paper layer obtained in the step S12 on the 1 st solid layer, pouring the culture medium prepared in the step S13 on the 2 nd filter paper layer, and solidifying to form a 2 nd solid layer with the thickness of 0.5; flatly paving the top layer filter paper layer obtained in the step S12 on the 2 nd solid layer to obtain a combined culture medium; the combined culture medium comprises the following components from bottom to top: the filter paper comprises a 1 st filter paper layer, a 1 st solid layer, a 2 nd filter paper layer, a 2 nd solid layer and a top filter paper layer.
Example 2
A method for culturing plant tissue comprises:
s1, induction of salvia miltiorrhiza callus: placing the root segment with high growth activity of sterile Saviae Miltiorrhizae radix about 2cm on the top filter paper layer of the combined culture medium, and performing sterile culture at 28 deg.C in dark for 15d to obtain fresh Saviae Miltiorrhizae radix callus;
s2, induction of the adventitious roots of the salvia miltiorrhiza: after obtaining the callus, adding a sterile MS liquid culture medium into the combined culture medium, wherein the liquid height is about 1mm higher than the top filter paper layer, and since the callus absorbs the bottom layer induced phytohormone, the combined culture medium is aseptically cultured for 20 days under the conditions of 28 ℃ and darkness, the adventitious roots start to grow, so that the adventitious roots of the salvia miltiorrhiza with the callus are obtained, the total culture time is 35 days, the number of the adventitious roots is 5, the length is 2.9cm, and the induction rate is 97.3%.
The combined culture medium in the step S1 is formed by alternately superposing three filter paper layers and two MS solid culture media from bottom to top, and the preparation method comprises the following steps:
s11, preparing a filter paper layer: cutting qualitative filter paper into 8cm circular filter paper sheet with size consistent with the bottom of the sterile tissue culture bottle, sealing with kraft paper in a clean culture dish, and sterilizing at 121 deg.C and 0.1MPa for 25 min;
s12, preparing a filter paper layer:
preparation of the 1 st filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 1, wherein the mixed phytohormone 1 contains 5.0mg/L IBA, 0.4mg/L NAA, 1.0mg/L6-BA and 0.5mg/L KT, stacking 4 sterilized filter paper sheets obtained in the step S11 together, and soaking the filter paper sheets in the mixed phytohormone 1 for 20S to obtain a 1 st filter paper layer for inducing adventitious roots; preparation of the 2 nd filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super clean bench, mixing the phytohormone in a small sterile beaker to obtain mixed phytohormone 2, wherein the mixed phytohormone 2 contains 1.2mg/L IBA, 0.5mg/L2,4-D, 0.6 mg/L6-BA and 0.2mg/L NAA, stacking 3 sterilized filter paper sheets obtained in the step S11 together, and soaking the stacked filter paper sheets in the mixed phytohormone 2 for 20S to obtain a 2 nd induced filter paper layer for inducing adventitious roots;
preparing a top layer induction filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 3, wherein the mixed phytohormone 3 contains 0.5mg/L2,4-D and 0.5mg/L6-BA, stacking 3 filter paper sheets sterilized in the step S11, and soaking the stacked filter paper sheets in the phytohormone 3 for 20S to obtain a top induction filter paper layer for inducing callus;
s13, preparation of a culture medium: accurately weighing MS solid powder, dissolving in water, adding sucrose and agar, adjusting pH to 5.8, sterilizing at 121 deg.C and 0.1MPa for 25min, keeping the temperature to prevent solidification, and preparing into M agar culture medium with sucrose concentration of 30g/L and agar content of 8 g/L;
s14, preparing a combined culture medium: spreading the 1 st filter paper layer obtained in the step S12 on the bottom of the culture bottle in the step S11, pouring the culture medium prepared in the step S13 on the 1 st filter paper layer, and solidifying to form a 1 st solid layer with the thickness of 1 cm; spreading the 2 nd filter paper layer obtained in the step S12 on the 1 st solid layer, pouring the culture medium prepared in the step S13 on the 2 nd filter paper layer, and forming a 2 nd solid layer with the thickness of 0.5cm after solidification; flatly paving the top layer filter paper layer obtained in the step S12 on the 2 nd solid layer to obtain a combined culture medium; the combined culture medium comprises the following components from bottom to top: the filter paper comprises a 1 st filter paper layer, a 1 st solid layer, a 2 nd filter paper layer, a 2 nd solid layer and a top filter paper layer.
Example 3
A method for culturing plant tissue comprises:
s1, induction of liquorice callus: placing the root segment with high growth activity of aseptic licorice root about 2cm on the top filter paper layer of the combined culture medium, and carrying out aseptic culture at 26 ℃ in the dark for 12d to obtain fresh licorice root callus;
s2, inducing adventitious roots and adventitious buds of liquorice: after obtaining the callus, adding a small amount of sterile MS liquid culture medium into the combined culture medium, submerging the top filter paper layer by about 1mm, carrying out sterile culture for 18d under the conditions of 26 ℃ and illumination for 12h/d because the callus absorbs bottom induced phytohormone, starting to grow adventitious roots and adventitious buds to form a complete liquorice plant, and carrying out culture for 35 days, wherein the number of the adventitious roots is 6, the number of the adventitious buds is 11, the length of the adventitious roots is 3.0cm, the height of the adventitious buds is 5.5cm, and the induction rate is 96.9%.
The combined culture medium in the step S1 is formed by alternately superposing three filter paper layers and two MS solid culture media from bottom to top, and the preparation method comprises the following steps:
s11, cutting filter paper: cutting qualitative filter paper into 8cm circular filter paper sheet with size consistent with the bottom of the sterile tissue culture bottle, sealing with kraft paper in a clean culture dish, and sterilizing at 121 deg.C and 0.1MPa for 25 min;
s12, preparing a filter paper layer:
preparation of the 1 st filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 1, wherein the mixed phytohormone 1 contains 4.0mg/L IBA, 0.5mg/L NAA, 4.0 mg/L6-BA and 0.8mg/L KT, stacking 4 sterilized filter paper sheets obtained in the step S11 together, and soaking the filter paper sheets in the mixed phytohormone 1 for 20S to obtain a 1 st filter paper layer for inducing adventitious roots;
preparation of the 2 nd filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super clean bench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 2, wherein the mixed phytohormone 2 contains 1.5mg/L IBA, 0.8 mg/L2,4-D, 1.5 mg/L6-BA and 0.2mg/L NAA, stacking 5 sterilized filter paper sheets obtained in the step S11, and soaking the stacked filter paper sheets in the mixed phytohormone 2 for 20S to obtain a 2 nd induced filter paper layer for inducing adventitious roots;
preparing a top layer induction filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 3, wherein the mixed phytohormone 3 contains 0.8 mg/L2,4-D and 0.8mg/L6-BA, stacking 3 filter paper sheets sterilized in the step S11, and soaking the stacked filter paper sheets in the phytohormone 3 for 20S to obtain a top induction filter paper layer for inducing callus;
s13, preparation of a culture medium: accurately weighing MS solid powder, dissolving in water, adding sucrose and agar, adjusting pH to 5.9, sterilizing at 121 deg.C and 0.1MPa for 25min, keeping the temperature to prevent solidification, and preparing into MS agar culture medium with sucrose concentration of 50g/L and agar content of 8 g/L;
s14, preparing a combined culture medium: spreading the 1 st filter paper layer obtained in the step S12 on the bottom of the culture bottle in the step S11, pouring the culture medium prepared in the step S13 on the 1 st filter paper layer, and solidifying to form a 1 st solid layer with the thickness of 1 cm; flatly paving the 2 nd filter paper layer obtained in the step S12 on the 1 st solid layer, pouring the culture medium prepared in the step S13 on the 2 nd filter paper layer, and forming a 2 nd solid layer with the thickness of 0.5cm after solidification, wherein the 2 nd combined culture medium consists of the 2 nd filter paper layer and the 2 nd solid layer; flatly paving the top layer filter paper layer obtained in the step S12 on the 2 nd solid layer to obtain a combined culture medium; the combined culture medium comprises the following components from bottom to top: the filter paper comprises a 1 st filter paper layer, a 1 st solid layer, a 2 nd filter paper layer, a 2 nd solid layer and a top filter paper layer.
Example 4
S1, inducing astragalus membranaceus callus: placing young sterile radix astragali leaves on the top filter paper layer of the combined culture medium, and performing dark sterile culture at 25 deg.C for 10d to obtain fresh radix astragali callus;
s2, induction of adventitious roots of astragalus: after obtaining the callus, adding a sterile MS liquid culture medium into the combined culture medium, submerging the top filter paper layer by about 1mm, carrying out sterile culture at 25 ℃ for 16d under the dark condition because the callus absorbs bottom induced phytohormone, and starting to grow the adventitious root to obtain the astragalus adventitious root with the callus, wherein the total culture time is 35 days, the number of the adventitious root is 7, the length of the adventitious root is 3.2cm, and the induction rate is 98.3%.
The combined culture medium in the step S1 is formed by alternately superposing three filter paper layers and two MS solid culture media from bottom to top, and the preparation method comprises the following steps:
s11, cutting filter paper: cutting qualitative filter paper into 8cm circular filter paper sheet with size consistent with the bottom of the sterile tissue culture bottle, sealing with kraft paper in a clean culture dish, and sterilizing at 121 deg.C and 0.1MPa for 25 min;
s12, preparing a filter paper layer:
preparation of the 1 st filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 1, wherein the mixed phytohormone 1 contains 3.5mg/L IBA, 0.5mg/L NAA, 1.0mg/L6-BA and 0.5mg/L KT, stacking 6 sterilized filter paper sheets obtained in the step S11 together, and soaking the filter paper sheets in the mixed phytohormone 1 for 20S to obtain a 1 st filter paper layer for inducing adventitious roots;
preparation of the 2 nd filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super clean bench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 2, wherein the mixed phytohormone 2 contains 2.0mg/L IBA, 0.5mg/L2,4-D, 0.8mg/L6-BA and 0.2mg/L NAA, stacking 3 sterilized filter paper sheets obtained in the step S11, and soaking the stacked filter paper sheets in the mixed phytohormone 2 for 20S to obtain a 2 nd induced filter paper layer for inducing adventitious roots;
preparing a top layer induction filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain a mixed phytohormone 3, wherein the mixed phytohormone 3 contains 0.8 mg/L2,4-D and 0.5mg/L6-BA, stacking 4 filter paper sheets sterilized in the step S11, and soaking the stacked filter paper sheets in the phytohormone 3 for 20S to obtain a top induction filter paper layer for inducing callus;
s13 preparation of medium: accurately weighing MS solid powder, dissolving in water, adding sucrose and agar, adjusting pH to 5.8, sterilizing at 121 deg.C and 0.1MPa for 25min, keeping the temperature to prevent solidification, and preparing into MS agar culture medium with sucrose concentration of 30g/L and agar content of 8 g/L;
s14, preparing a layer 1 combined culture medium layer: the 1 st filter paper layer obtained in the step S12 is paved at the bottom of the culture bottle in the step S11, the culture medium prepared in the step S13 is poured on the 1 st filter paper layer, and a 1 st solid layer with the thickness of about 1cm is formed after solidification; spreading the 2 nd filter paper layer obtained in the step S12 on the 1 st solid layer, pouring the culture medium prepared in the step S13 on the 2 nd filter paper layer, and forming a 2 nd solid layer with the thickness of about 0.5cm after solidification; flatly paving the top layer filter paper layer obtained in the step S12 on the 2 nd solid layer to obtain a combined culture medium; the combined culture medium comprises the following components from bottom to top: the filter paper comprises a 1 st filter paper layer, a 1 st solid layer, a 2 nd filter paper layer, a 2 nd solid layer and a top filter paper layer.
Example 5
S1, induction of Psammosilene tunicoides callus: placing tender leaves of the psammosilene tunicoides on a top filter paper layer of a combined culture medium, and performing sterile culture at 27 ℃ in the dark for 12 days to obtain a fresh psammosilene tunicoides callus;
s2, inducing adventitious roots and adventitious buds of Psammosilene tunicoides: after obtaining the callus, adding a sterile MS liquid culture medium into the combined culture medium, submerging the top filter paper layer by about 1mm, performing sterile culture for 15d under the conditions of 27 ℃ and illumination for 12h/d because the callus absorbs bottom induced phytohormone, starting to grow adventitious roots and adventitious buds to form a complete psammosilene tunicoides plant, and culturing for 35 days, wherein the number of the adventitious roots is 7, the number of the adventitious buds is 9, the length of the adventitious roots is 3.5cm, the height of the adventitious buds is 5.1cm, and the induction rate is 95.9%.
The combined culture medium in the step S1 is formed by alternately superposing three filter paper layers and two MS solid culture media from bottom to top, and the preparation method comprises the following steps:
s11, cutting filter paper: cutting qualitative filter paper into 8cm circular filter paper sheet with size consistent with the bottom of the sterile tissue culture bottle, sealing with kraft paper in a clean culture dish, and sterilizing at 121 deg.C and 0.1MPa for 25 min;
s12, preparing a filter paper layer:
preparation of the 1 st filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 1, wherein the mixed phytohormone 1 contains 3.5mg/L IBA, 0.5mg/L NAA, 4.0 mg/L6-BA and 1.0mg/L KT, stacking 7 sterilized filter paper sheets obtained in the step S11 together, and soaking the filter paper sheets in the mixed phytohormone 1 for 20S to obtain a 1 st filter paper layer for inducing adventitious roots;
preparation of the 2 nd filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super clean bench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 2, wherein the mixed phytohormone 2 contains 1.5mg/L IBA, 1.0 mg/L2,4-D, 2.0 mg/L6-BA and 0.2mg/L NAA, stacking 4 sterilized filter paper sheets obtained in the step S11, and soaking the stacked filter paper sheets in the mixed phytohormone 2 for 20S to obtain a 2 nd induced filter paper layer for inducing adventitious roots;
preparing a top layer induction filter paper layer: filtering and sterilizing phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker to obtain mixed phytohormone 3, wherein the mixed phytohormone 3 contains 1.0 mg/L2,4-D and 1.0mg/L6-BA, stacking 3 filter paper sheets sterilized in the step S11, and soaking the stacked filter paper sheets in the phytohormone 3 for 20S to obtain a top induction filter paper layer for inducing callus;
s13, preparation of a culture medium: accurately weighing MS solid powder, dissolving in water, adding sucrose and agar, adjusting pH to 5.9, sterilizing at 121 deg.C and 0.1MPa for 25min, keeping the temperature to prevent solidification, and preparing into MS agar culture medium with sucrose concentration of 45g/L and agar content of 8 g/L;
s14, preparing a combined culture medium layer: the 1 st filter paper layer obtained in the step S12 is paved at the bottom of the culture bottle in the step S11, the culture medium prepared in the step S13 is poured on the 1 st filter paper layer, and a 1 st solid layer with the thickness of about 1cm is formed after solidification; spreading the 2 nd filter paper layer obtained in the step S12 on the 1 st solid layer, pouring the culture medium prepared in the step S13 on the 2 nd filter paper layer, and forming a 2 nd solid layer with the thickness of about 0.5cm after solidification; flatly paving the top layer filter paper layer obtained in the step S12 on the 2 nd solid layer to obtain a combined culture medium; the combined culture medium comprises the following components from bottom to top: the filter paper comprises a 1 st filter paper layer, a 1 st solid layer, a 2 nd filter paper layer, a 2 nd solid layer and a top filter paper layer.
Example 6
S1, induction of angelica sinensis callus: placing sterile young stem segments of radix Angelicae sinensis about 2cm on the top layer of filter paper layer of the combined culture medium, and performing dark sterile culture at 25 deg.C for 11 days to obtain fresh radix Angelicae sinensis callus;
s2, induction of adventitious roots of angelica: after obtaining the callus, adding a sterile MS liquid culture medium into the combined culture medium, submerging the top filter paper layer by about 1mm, carrying out sterile culture at 25 ℃ for 18d under the dark condition because the callus absorbs bottom induced phytohormone, and starting to grow the adventitious root to obtain the angelica adventitious root with the callus, wherein the total culture time is 35 days, the number of the adventitious root is 5, the length of the adventitious root is 2.7cm, and the induction rate is 97.6%.
The combined culture medium in the step S1 is formed by alternately superposing three filter paper layers and two MS solid culture media from bottom to top, and the preparation method comprises the following steps:
s11, cutting filter paper: cutting qualitative filter paper into 8cm circular filter paper sheet with size consistent with the bottom of the sterile tissue culture bottle, sealing with kraft paper in a clean culture dish, and sterilizing at 121 deg.C and 0.1MPa for 25 min;
s12, preparing a filter paper layer:
preparation of the 1 st filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 1, wherein the mixed phytohormone 1 contains 4.0mg/L IBA, 1.0mg/L NAA, 0.5mg/L6-BA and 1.0mg/L KT, stacking 6 sterilized filter paper sheets obtained in the step S11 together, and soaking the filter paper sheets in the mixed phytohormone 1 for 20S to obtain a 1 st filter paper layer for inducing adventitious roots;
preparation of the 2 nd filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super clean bench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 2, wherein the mixed phytohormone 2 contains 1.5mg/L IBA, 1.0 mg/L2,4-D, 0.5mg/L6-BA and 0.2mg/L NAA, stacking 2 sterilized filter paper sheets obtained in the step S11, and soaking the stacked filter paper sheets in the mixed phytohormone 2 for 20S to obtain a 2 nd induced filter paper layer for inducing adventitious roots;
preparing a top layer induction filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain a mixed phytohormone 3, wherein the mixed phytohormone 3 contains 1.0 mg/L2,4-D and 0.8mg/L6-BA, stacking 4 filter paper sheets sterilized in the step S11, and soaking the stacked filter paper sheets in the phytohormone 3 for 20S to obtain a top induction filter paper layer for inducing callus;
s13, preparation of a culture medium: accurately weighing MS solid powder, dissolving in water, adding sucrose and agar, adjusting pH to 6.0, sterilizing at 121 deg.C and 0.1MPa for 25min, keeping the temperature to prevent solidification, and preparing into MS agar culture medium with sucrose concentration of 30g/L and agar content of 8 g/L;
s14, preparing a combined culture medium: the 1 st filter paper layer obtained in the step S12 is paved at the bottom of the culture bottle in the step S11, the culture medium prepared in the step S13 is poured on the 1 st filter paper layer, and a 1 st solid layer with the thickness of about 1cm is formed after solidification; spreading the 2 nd filter paper layer obtained in the step S12 on the 1 st solid layer, pouring the culture medium prepared in the step S13 on the 2 nd filter paper layer, and forming a 2 nd solid layer with the thickness of about 0.5cm after solidification; flatly paving the top layer filter paper layer obtained in the step S12 on the 2 nd solid layer to obtain a combined culture medium; the combined culture medium comprises the following components from bottom to top: the filter paper comprises a 1 st filter paper layer, a 1 st solid layer, a 2 nd filter paper layer, a 2 nd solid layer and a top filter paper layer.
Example 7
S1, inducing callus of Yunnan rhizoma paridis: placing young and tender stem segments of rhizoma paridis Yunnanensis about 2cm on the top filter paper layer of the combined culture medium, and performing sterile culture at 26 deg.C in the dark for 10d to obtain callus of rhizoma paridis Yunnanensis;
s2, inducing adventitious roots of Paris polyphylla: after obtaining the callus, adding a sterile MS liquid culture medium into the combined culture medium, wherein the liquid height is about 1mm higher than the top filter paper layer, and since the callus absorbs the bottom layer induced phytohormone, the combined culture medium is subjected to sterile culture for 15d under the dark condition of 26 ℃, the adventitious root starts to grow, so that the adventitious root of the paris polyphylla with the callus is obtained, the total culture time is 35 days, the number of the adventitious root is 8, the length of the adventitious root is 3.1cm, and the induction rate is 96.9%.
Wherein the combined culture medium in the step S1 is formed by alternately overlapping two filter paper layers and one MS solid culture medium layer from bottom to top, and the preparation method comprises the following steps:
s11, cutting filter paper: cutting qualitative filter paper into 8cm circular filter paper sheet with size consistent with the bottom of the sterile tissue culture bottle, sealing with kraft paper in a clean culture dish, and sterilizing at 121 deg.C and 0.1MPa for 25 min;
s12, preparing a filter paper layer:
preparation of the 1 st filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super clean bench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 1, wherein the mixed phytohormone 1 contains 3.5mg/L IBA, 1.0 mg/L2,4-D, 0.5mg/L6-BA and 1.0mg/L NAA, stacking 8 sterilized filter paper sheets obtained in the step S11 together, and soaking the stacked filter paper sheets in the mixed phytohormone 1 for 20S to obtain a layer 1 filter paper layer for inducing adventitious roots;
preparing a top layer induction filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain a mixed phytohormone 3, wherein the mixed phytohormone 3 contains 1.0 mg/L2,4-D and 1.0mg/L6-BA, stacking 4 filter paper sheets sterilized in the step S11, and soaking the stacked filter paper sheets in the phytohormone 3 for 20S to obtain a top induction filter paper layer for inducing callus;
s13, preparation of a culture medium: accurately weighing MS solid powder, dissolving in water, adding sucrose and agar, adjusting pH to 5.8, sterilizing at 121 deg.C and 0.1MPa for 25min, keeping the temperature to prevent solidification, and preparing into MS agar culture medium with sucrose concentration of 30g/L and agar content of 8 g/L;
s14, preparing a combined culture medium: the 1 st filter paper layer obtained in the step S12 is paved at the bottom of the culture bottle in the step S11, the culture medium prepared in the step S13 is poured on the 1 st filter paper layer, and a 1 st solid layer with the thickness of about 1cm is formed after solidification; flatly paving the top layer filter paper layer obtained in the step S12 on the 1 st solid layer to obtain a combined culture medium; the combined culture medium comprises the following components from bottom to top: the filter paper comprises a 1 st filter paper layer, a 1 st solid layer and a top filter paper layer.
Example 8
S1, inducing American ginseng callus: placing sterile American ginseng root segment with 2cm around growth vigor on the top filter paper layer of the combined culture medium, and performing dark sterile culture at 25 ℃ for 10 days to obtain fresh American ginseng callus;
s2, inducing the American ginseng adventitious root: after obtaining the callus, adding a small amount of sterile MS liquid culture medium into the combined culture medium, wherein the height of the liquid submerged in the top filter paper layer is about 1mm, and since the callus absorbs the bottom induced phytohormone, the American ginseng adventitious root is cultured aseptically for 17 days at 25 ℃ in the dark, and the adventitious root begins to grow, so that the American ginseng adventitious root with the callus is obtained, the culture lasts for 35 days, the number of the adventitious root is 8, the length of the adventitious root is 2.9cm, and the induction rate is 98.4%.
The combined culture medium in the step S1 is formed by alternately superposing four filter paper layers and three MS solid culture media from bottom to top, and the preparation method comprises the following steps:
s11, cutting filter paper: cutting qualitative filter paper into 8cm circular filter paper sheet with size consistent with the bottom of the sterile tissue culture bottle, sealing with kraft paper in a clean culture dish, and sterilizing at 121 deg.C and 0.1MPa for 25 min;
s12, preparing a filter paper layer:
preparation of the 1 st filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 1, wherein the mixed phytohormone 1 contains 4.0mg/L IBA, 0.5mg/L NAA, 0.5mg/L6-BA and 0.5mg/L KT, stacking 4 sterilized filter paper sheets obtained in the step S11 together, and soaking the filter paper sheets in the mixed phytohormone 1 for 20S to obtain a 1 st filter paper layer for inducing adventitious roots;
preparation of the 2 nd filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super clean bench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 2, wherein the mixed phytohormone 2 contains 1.0mg/L IBA, 1.0 mg/L2,4-D, 1.0mg/L6-BA and 0.2mg/L NAA, stacking 3 sterilized filter paper sheets obtained in the step S11 together, and soaking the stacked filter paper sheets in the mixed phytohormone 2 for 20S to obtain a 2 nd induction filter paper layer;
preparation of a 3 rd filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super clean bench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain mixed phytohormone 3, wherein the mixed phytohormone 2 contains 2.0mg/L IBA, 1.5 mg/L2,4-D, 4.0 mg/L6-BA and 0.8mg/L NAA, stacking 3 sterilized filter paper sheets obtained in the step S11 together, and soaking the stacked filter paper sheets in the mixed phytohormone 3 for 20S to obtain a 2 nd induction filter paper layer;
preparing a top layer induction filter paper layer: filtering and sterilizing the phytohormone by adopting a filter membrane with the aperture of 0.22 mu m in a super-clean workbench, mixing the phytohormone in a small sterile beaker in an equal volume to obtain a mixed phytohormone 3, wherein the mixed phytohormone 3 contains 0.8 mg/L2,4-D and 0.5mg/L6-BA, stacking 4 filter paper sheets sterilized in the step S11, and soaking the stacked filter paper sheets in the phytohormone 3 for 20S to obtain a top induction filter paper layer for inducing callus;
s13, preparation of a culture medium: accurately weighing MS solid powder, dissolving in water, adding sucrose and agar, adjusting pH to 5.8, sterilizing at 121 deg.C and 0.1MPa for 25min, keeping the temperature to prevent solidification, and preparing into MS agar culture medium with sucrose concentration of 30g/L and agar content of 8 g/L;
s14, preparing a combined culture medium: spreading the 1 st filter paper layer obtained in the step S12 on the bottom of the culture bottle in the step S11, pouring the culture medium prepared in the step S13 on the 1 st filter paper layer, and solidifying to form a 1 st solid layer with the thickness of about 0.5 cm; spreading the 2 nd filter paper layer obtained in the step S12 on the 1 st solid layer, pouring the culture medium prepared in the step S13 on the 2 nd filter paper layer, and forming a 2 nd solid layer with the thickness of about 0.5cm after solidification; spreading the 3 rd filter paper layer obtained in the step S12 on the 2 nd solid layer, pouring the culture medium prepared in the step S13 on the 3 rd filter paper layer, and forming a 3 rd solid layer with the thickness of about 0.5cm after solidification; flatly paving the top layer filter paper layer obtained in the step S12 on the 3 rd solid layer to obtain a combined culture medium; the combined culture medium comprises the following components from bottom to top: the filter paper comprises a 1 st filter paper layer, a 1 st solid layer, a 2 nd filter paper layer, a 2 nd solid layer, a 3 rd filter paper layer, a 3 rd solid layer and a top filter paper layer.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (8)
1. A plant tissue culture method is characterized by comprising the following steps:
s1, induction of callus: placing 2cm of sterile explant roots, young stem segments or young leaves of plants on a top filter paper layer of a combined culture medium, and carrying out sterile culture in the dark at 24-28 ℃ for 10-15 days to obtain callus;
wherein the combined culture medium is formed by alternately superposing a filter paper layer and a solid layer from bottom to top; the filter paper layer is filter paper containing phytohormone, the solid layer is a solid culture medium, and the bottom layer and the top layer of the combined culture medium are both filter paper layers;
s2, induction of plant tissues: adding a liquid culture medium into the combined culture medium, and inducing adventitious roots or adventitious roots and adventitious buds when the liquid submerges the top filter paper layer by about 1 mm;
wherein the adventitious root induction conditions are as follows: carrying out sterile culture in the dark for 15-20 days at the temperature of 24-28 ℃ to obtain adventitious roots with callus; the conditions for adventitious root and adventitious bud induction were: and performing sterile culture for 15-20 d at 24-28 ℃ and illumination for 12h/d to obtain a complete plant with adventitious roots and adventitious buds.
2. The method for plant tissue culture according to claim 1, wherein the plant hormone of step S1 is one or more of 2,4-D, 6-BA, IBA, NAA, KT.
3. The method for culturing plant tissue according to claim 1, wherein the combined culture medium of step S1 comprises four filter paper layers and three solid layers stacked alternately from bottom to top.
4. The method for plant tissue culture according to claim 1, wherein the combined culture medium of step S1 is formed by alternately stacking three filter paper layers and two solid layers from bottom to top.
5. The method for plant tissue culture according to claim 1, wherein the combined culture medium of step S1 is formed by alternately stacking two filter paper layers and one solid layer from bottom to top.
6. The method for culturing plant tissue according to any one of claims 1 to 5, wherein the thickness of the solid layer in step S1 is 0.5 to 1 cm.
7. The method for plant tissue culture according to any one of claims 1 to 5, wherein the solid layer in step S1 is MS solid medium.
8. The method for culturing plant tissue according to any one of claims 1 to 5, wherein the liquid medium in step S2 is MS liquid medium.
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