CN112753578A - Method for inducing and rapidly proliferating Chinese wolfberry callus - Google Patents
Method for inducing and rapidly proliferating Chinese wolfberry callus Download PDFInfo
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- CN112753578A CN112753578A CN202110097023.9A CN202110097023A CN112753578A CN 112753578 A CN112753578 A CN 112753578A CN 202110097023 A CN202110097023 A CN 202110097023A CN 112753578 A CN112753578 A CN 112753578A
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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
- A01H4/008—Methods for regeneration to complete 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
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses a method for inducing and rapidly proliferating Chinese wolfberry callus, and relates to the technical field of plant tissue culture. Selecting strong-growing Chinese wolfberry stem segments; selecting a stem section of the medlar as an explant, inoculating the stem section after disinfection to an induction culture medium by using a small section which is cut into a length of 2-3cm, wherein the insertion depth of the stem section is 1-2 cm. Inoculating to induction medium, culturing at 22-24 deg.C under illumination of 2000Lux for 16 hr and culturing in dark for 8 hr. The stem explant callus tissue produced by taking the medlar as a material can be used for cell suspension culture to obtain metabolites, and can also be used for obtaining regenerated plants through callus embryogenesis or organ genesis, so that the aim of rapid propagation is fulfilled; the material has high activity and the multiplication factor can reach 3-7 times.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing and rapidly proliferating Chinese wolfberry callus.
Background
Fructus Lycii (Lycium barbarum L.) of Ningxia is shrub, or cultivated into large shrub with height of 0.8-2 m and stem diameter of 10-20 cm; the branches are fine and dense, the branches are mostly developed and slightly lifted or bent when the branches are wild, the branches are bent and the crowns are mostly round when the branches are cultivated, and the branches have longitudinal ridges, are gray white or gray yellow, have no hair and are slightly glossy, and have short thorns without leaves and long thorns with leaves and flowers. The leaves are intertillage or fasciculate, the shape of the needle is scaly, the top end is short, tapered or sharp, the base part is wedge-shaped, the length is 2-3cm, the width is 4-6 mm, the length is 12 cm, the width is 1.5-2 cm during cultivation, the leaves are slightly fleshy, and the veins are not obvious.
The berry is red or orange in cultivation type, the pulp of the peel and the juice are rich, the shape and the size are variable due to long-term artificial cultivation or different plant ages and habitats, and the plant is in a wide elliptical shape, a rectangular circle shape, an oval shape or a nearly spherical shape, the top end of the plant is provided with a short tip or a frustum, sometimes a little dent, the length of the plant is 8-20 mm, and the diameter of the plant is 5-10 mm. The seeds usually have more than 20 grains, are shaped like kidney, are flat and pressed, are brownish yellow and are about 2 mm long. The flower and fruit period is long, the fruits usually bloom and grow at the same time from 5 months to 10 months, and the fruits are ripe and picked in one batch.
The prior published technology about the medlar is still blank. The method is characterized in that the secondary metabolites can be cultured on a large scale by using the medlar through plant cells, and the explant is dedifferentiated and induced into callus tissues and proliferated in a large amount by using a cell culture technology, but the method is not practically applied to the medlar.
Disclosure of Invention
The invention provides a method for inducing and rapidly proliferating Chinese wolfberry callus.
The method comprises the following steps:
a, selecting strong-growing Chinese wolfberry stem segments;
b, selecting a stem section of the Chinese wolfberry as an explant, and selecting the lateral bud of the branch collected in the step A and a tender tip of the stem section of the Chinese wolfberry with a meristem and a length of 5-10cm from the top;
c, cleaning the stem section material of the Chinese wolfberry before disinfection, and washing the stem section explant for 20 minutes after the stem section explant is cleaned by dipping a brush in a detergent solution;
d, washing with tap water for 20-30 min, and sucking surface water with filter paper;
e, disinfection: sterilizing with 70-75% alcohol for 30s, washing with sterile water for 2 times, sterilizing with 0.1% mercuric chloride for 5-10 min, washing with sterile water for 3 times, and drying with sterile filter paper;
f, inoculating the sterilized stem segments into an induction culture medium by using small segments which are cut into lengths of 2-3cm, wherein the insertion depth of the stem segments is 1-2 cm;
g, inoculating the strain to an induction culture medium, and culturing at 22-24 ℃ under illumination of 2000Lux for 16 hours and 8 hours in the dark every day;
inoculating fresh and glossy cell clusters from the callus tissues obtained by H induction to an induction culture medium for dark culture;
and I, after the cells are subcultured for 20 days, transferring the subcultured and proliferated cell mass to a differentiation medium for culturing to obtain the proliferated tissue of the medlar callus tissue.
Further, the formula of the induction medium is as follows: NH was included in 1L medium4NO3 1025mg、KNO31300mg、CaCl2·2H2O 440mg、MgSO4·7H2O 370mg、KH2PO4 195mg、Na2·EDTA 37.3mg、FeSO4·7H2O 27.8mg、H3BO3 6.2mg、MnSO4·4H2O 22.3mg、ZnSO4·7H2O 8.6mg、KI 0.83mg、Na2MoO4·2H2O 0.25mg、CuSO4·5H2O 0.025mg、CoCl2·6H2O0.025 mg, inositol 100mg, nicotinic acid 0.5mg, glycine 2.0mg, VB60.5 mg, VB10.1mg, BA 1.5mg, NAA 0.5mg, Vc 5 mg.
Further, the formula of the culture medium for subculture is as follows: NH was included in 1L medium4NO3 1025mg、KNO3 1300mg、CaCl2·2H2O 440mg、MgSO4·7H2O 370mg、KH2PO4 195mg、Na2·EDTA 37.3mg、FeSO4·7H2O 27.8mg、H3BO3 6.2mg、MnSO4·4H2O 22.3mg、ZnSO4·7H2O 8.6mg、KI 0.83mg、Na2MoO4·2H2O 0.25mg、CuSO4·5H2O 0.025mg、CoCl2·6H2O0.025 mg, inositol 100mg, nicotinic acid 0.5mg, glycine 2.0mg, VB60.5 mg, VB10.1mg, BA 1.5mg, NAA 1.5mg, Vc 5 mg.
Further, the formula of the culture medium for the differentiation culture is that NH is contained in 1L of the culture medium4NO3 1025mg、KNO3 1300mg、CaCl2·2H2O 440mg、MgSO4·7H2O 370mg、KH2PO4 195mg、Na2·EDTA 37.3mg、FeSO4·7H2O 27.8mg、H3BO3 6.2mg、MnSO4·4H2O 22.3mg、ZnSO4·7H2O 8.6mg、KI 0.83mg、Na2MoO4·2H2O 0.25mg、CuSO4·5H2O 0.025mg、CoCl2·6H2O0.025 mg, inositol 100mg, nicotinic acid 0.5mg, glycine 2.0mg, VB60.5 mg, VB10.1mg, BA 3mg, NAA 1.5mg, Vc 5 mg.
The invention has the beneficial effects that:
the stem explant callus tissue produced by taking the medlar as a material can be used for cell suspension culture to obtain metabolites, and can also be used for obtaining regenerated plants through callus embryogenesis or organ genesis, so that the aim of rapid propagation is fulfilled; the material has high activity and the multiplication factor can reach 3-7 times.
Drawings
FIG. 1 shows the growth of callus cultured by induction after 20 days of subculture;
FIG. 2 adventitious bud germination on T76 medium by day 20;
FIG. 3 adventitious bud development by day 18 when grown on T76 medium;
FIG. 4 is a photograph showing callus proliferation culture for 20 days;
FIG. 5 is a photograph showing callus proliferation culture for 20 days.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
The method comprises the following steps:
a, selecting strong-growing Chinese wolfberry stem segments;
b, selecting a stem section of the Chinese wolfberry as an explant, and selecting the lateral bud of the branch collected in the step A and a tender tip of the stem section of the Chinese wolfberry with a meristem and a length of 5-10cm from the top;
c, cleaning the stem section material of the Chinese wolfberry before disinfection, and washing the stem section explant for 20 minutes after the stem section explant is cleaned by dipping a brush in a detergent solution;
d, washing with tap water for 20-30 min, and sucking surface water with filter paper;
e, disinfection: sterilizing with 70-75% alcohol for 30s, washing with sterile water for 2 times, sterilizing with 0.1% mercuric chloride for 5-10 min, washing with sterile water for 3 times, and drying with sterile filter paper;
f, inoculating the sterilized stem segments into an induction culture medium by using small segments which are cut into lengths of 2-3cm, wherein the insertion depth of the stem segments is 1-2 cm;
g, inoculating the strain to an induction culture medium, and culturing at 22-24 ℃ under illumination of 2000Lux for 16 hours and 8 hours in the dark every day;
inoculating fresh and glossy cell clusters from the callus tissues obtained by H induction to an induction culture medium for dark culture;
and I, after the cells are subcultured for 20 days, transferring the subcultured and proliferated cell mass to a differentiation medium for culturing to obtain the proliferated tissue of the medlar callus tissue.
Material
The test material is high-yield and high-quality variety Ningqi No. 7 of Ningxia wolfberry fruit (Lycium barbarum L.) in China.
Culture medium formula
Results of the experiment
Induction (T75)
As shown in FIG. 1, when stem segments were inoculated on T75 medium for 5-7 days, the lower end of the stem segment of the explant began to swell and grow larger, a small amount of pale yellow callus appeared around day 15 at the edge of the swollen and enlarged part, and the pale yellow callus proliferated and changed into a large mass up to day 20-25
Statistics of induction rate
Sub-generation (T76)
As shown in figure 2, the loose granular callus appears along with the proliferation of the callus, the whole callus cells are distributed in a lump shape and a loose shape, the cell state of the loose granular callus is best after 20 days of subculture, the fresh weight of the callus can be increased by nearly 10 times after 20 days of culture, the fresh weight of the callus is increased by one time after 2 days on average, after 25 days, the proliferation speed of the cell line begins to be slowed down, and obviously enters a stationary phase at 35-40 days, and part of the callus begins to become grayish brown.
Proliferation fold statistics
Differentiation (T77)
As shown in figure 3, the subcultured proliferated cell mass is transferred to a differentiation medium T76 to induce the germination of adventitious buds, about 2g of callus is inoculated in each bottle, the callus is spread on the surface of a T76 medium, a plurality of small bulges appear on the surface of the callus in succession for about one week, the top end of each small bulge is changed into purple when the culture is continued until the 10 th day and then gradually grows into a leaf, the leaf gradually grows up and grows into a second leaf at the base of the leaf when the 17 th day, the leaf gradually turns green when the 20 th day as the two leaves continue to grow, until the 25 th day to the 28 th day, the small bulges become a bud with 3-4 leaves, each small bulge grows into a cluster of buds, and each cluster bud has about 12-13 buds per square centimeter.
Statistics of germination quantity
As shown in Table 1, the callus of Lycium barbarum collected in the manner described in example 1 is inoculated and cultured, after the induction culture time is over, the callus induction rate of stem explant is higher than that of the traditional induction method, and after the stem explant is transferred to the enrichment medium for culture for one period, the multiplication multiple can reach nearly 14 times, as shown in FIGS. 4 and 5, it can be seen that the method for soaking, sterilizing, inoculating, inducing and proliferating is the most optimal, and is most beneficial to the induction and growth of callus of Lycium barbarum stem explant.
TABLE 1 Effect of different media formulations on the callus proliferation characteristics of Lycium barbarum
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 considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (4)
1. A method for inducing and rapidly proliferating Chinese wolfberry callus is characterized by comprising the following steps:
a, selecting strong-growing Chinese wolfberry stem segments;
b, selecting a stem section of the Chinese wolfberry as an explant, and selecting the lateral bud of the branch collected in the step A and a tender tip of the stem section of the Chinese wolfberry with a meristem and a length of 5-10cm from the top;
c, cleaning the stem section material of the Chinese wolfberry before disinfection, and washing the stem section explant for 20 minutes after the stem section explant is cleaned by dipping a brush in a detergent solution;
d, washing with tap water for 20-30 min, and sucking surface water with filter paper;
e, disinfection: sterilizing with 70-75% alcohol for 30s, washing with sterile water for 2 times, sterilizing with 0.1% mercuric chloride for 5-10 min, washing with sterile water for 3 times, and drying with sterile filter paper;
f, inoculating the sterilized stem segments into an induction culture medium by using small segments which are cut into lengths of 2-3cm, wherein the insertion depth of the stem segments is 1-2 cm;
g, inoculating the strain to an induction culture medium, and culturing at 22-24 ℃ under illumination of 2000Lux for 16 hours and 8 hours in the dark every day;
inoculating fresh and glossy cell clusters from the callus tissues obtained by H induction to an induction culture medium for dark culture;
and I, after the cells are subcultured for 20 days, transferring the subcultured and proliferated cell mass to a differentiation medium for culturing to obtain the proliferated tissue of the medlar callus tissue.
2. The method for inducing and rapidly proliferating medlar callus according to claim 1, wherein the formula of the induction medium is as follows: NH was included in 1L medium4NO3 1025mg、KNO3 1300mg、CaCl2·2H2O 440mg、MgSO4·7H2O 370mg、KH2PO4 195mg、Na2·EDTA 37.3mg、FeSO4·7H2O 27.8mg、H3BO36.2mg、MnSO4·4H2O 22.3mg、ZnSO4·7H2O 8.6mg、KI 0.83mg、Na2MoO4·2H2O 0.25mg、CuSO4·5H2O 0.025mg、CoCl2·6H2O0.025 mg, inositol 100mg, nicotinic acid 0.5mg, glycine 2.0mg, VB60.5 mg, VB10.1mg, BA 1.5mg, NAA 0.5mg, Vc 5 mg.
3. The method of claim 1, wherein the culture medium for subculture is prepared fromThe method comprises the following steps: NH was included in 1L medium4NO3 1025mg、KNO3 1300mg、CaCl2·2H2O 440mg、MgSO4·7H2O 370mg、KH2PO4 195mg、Na2·EDTA 37.3mg、FeSO4·7H2O 27.8mg、H3BO3 6.2mg、MnSO4·4H2O 22.3mg、ZnSO4·7H2O 8.6mg、KI 0.83mg、Na2MoO4·2H2O 0.25mg、CuSO4·5H2O 0.025mg、CoCl2·6H2O0.025 mg, inositol 100mg, nicotinic acid 0.5mg, glycine 2.0mg, VB60.5 mg, VB10.1mg, BA 1.5mg, NAA 1.5mg, Vc 5 mg.
4. The method of claim 1, wherein the culture medium for differentiation culture comprises NAA at a concentration of 1.5mg/L and BA at a concentration of 3 mg/L. NH was included in 1L medium4NO3 1025mg、KNO3 1300mg、CaCl2·2H2O 440mg、MgSO4·7H2O 370mg、KH2PO4 195mg、Na2·EDTA 37.3mg、FeSO4·7H2O 27.8mg、H3BO3 6.2mg、MnSO4·4H2O 22.3mg、ZnSO4·7H2O 8.6mg、KI 0.83mg、Na2MoO4·2H2O 0.25mg、CuSO4·5H2O 0.025mg、CoCl2·6H2O0.025 mg, inositol 100mg, nicotinic acid 0.5mg, glycine 2.0mg, VB60.5 mg, VB10.1mg, BA 3mg, NAA 1.5mg, Vc 5 mg.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BG1457U1 (en) * | 2011-02-09 | 2011-06-30 | "Био Трии" Оод | Nutrition medium for in vitro proliferation of lycium barbarum |
| CN103609451A (en) * | 2013-11-29 | 2014-03-05 | 山东省农作物种质资源中心 | Medlar sterile seedling and method for obtaining induced healing of medlar sterile seedling |
| CN104273034A (en) * | 2014-10-21 | 2015-01-14 | 甘肃农业大学 | Induction and differentiation culture technique of Lycium ruthenicum Murr. |
| CN107135950A (en) * | 2017-06-19 | 2017-09-08 | 南京晓庄学院 | A kind of breeding method of quick acquisition black fruit fructus lycii regrowth |
| CN111226797A (en) * | 2020-03-19 | 2020-06-05 | 内蒙古自治区林业科学研究院 | Lycium ruthenicum tissue culture method |
-
2021
- 2021-01-25 CN CN202110097023.9A patent/CN112753578A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BG1457U1 (en) * | 2011-02-09 | 2011-06-30 | "Био Трии" Оод | Nutrition medium for in vitro proliferation of lycium barbarum |
| CN103609451A (en) * | 2013-11-29 | 2014-03-05 | 山东省农作物种质资源中心 | Medlar sterile seedling and method for obtaining induced healing of medlar sterile seedling |
| CN104273034A (en) * | 2014-10-21 | 2015-01-14 | 甘肃农业大学 | Induction and differentiation culture technique of Lycium ruthenicum Murr. |
| CN107135950A (en) * | 2017-06-19 | 2017-09-08 | 南京晓庄学院 | A kind of breeding method of quick acquisition black fruit fructus lycii regrowth |
| CN111226797A (en) * | 2020-03-19 | 2020-06-05 | 内蒙古自治区林业科学研究院 | Lycium ruthenicum tissue culture method |
Non-Patent Citations (1)
| Title |
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| 李冬杰: "植物生长调节剂对黑果枸杞愈伤组织诱导、增殖及分化的影响", 《河北林业科技》 * |
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