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CN117158317A - Formula for promoting proliferation of saffron somatic embryos - Google Patents

Formula for promoting proliferation of saffron somatic embryos Download PDF

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Publication number
CN117158317A
CN117158317A CN202311191885.3A CN202311191885A CN117158317A CN 117158317 A CN117158317 A CN 117158317A CN 202311191885 A CN202311191885 A CN 202311191885A CN 117158317 A CN117158317 A CN 117158317A
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Prior art keywords
saffron
proliferation
chain protein
somatic embryos
oligosaccharide chain
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CN202311191885.3A
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CN117158317B (en
Inventor
查萍
李海滨
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Fujian Sanan Sino Science Photobiotech Co Ltd
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Fujian Sanan Sino Science Photobiotech Co Ltd
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Abstract

The application discloses an application of oligosaccharide chain protein in preparing a formula for promoting proliferation of saffron somatic embryos and a related formula thereof, wherein the formula comprises the following components: oligosaccharide chain protein, anti-browning substance, cytokinin, auxin, silver nitrate and vitamin H. By combining with anti-browning substances in tissue culture and plant hormones and substances commonly used for callus induction proliferation, the somatic embryos can be proliferated in a large amount and have high activity, the later differentiation is facilitated, and the industrialization of a saffron somatic embryo balling system can be promoted and induced.

Description

Formula for promoting proliferation of saffron somatic embryos
Technical Field
The application belongs to the cell proliferation technology, and particularly relates to a formula for promoting proliferation of saffron somatic embryos.
Background
Stigma croci is also called stigma croci, belongs to subtropical medicinal plants, is used as a medicament by filaments (flower columns and column heads) thereof, and has the effects of detumescence, analgesia, blood circulation promoting, blood stasis removing, blood nourishing, menstruation dredging and the like. The cultivation and production of saffron in China mainly adopts an indoor cultivation method for harvesting fresh flowers. The seed cannot be set under the cultivation condition, and the seed is mainly propagated by the bulb. In the cultivation process, the smaller the bulb, the less and less flowers and even no flowers of the small bulb, so that the medical value is lost.
Along with the wide application of the tissue culture technology on medicinal plants, the callus induction technology of the saffron is also developed, and a research basis is provided for culturing high-quality saffron germplasm resources in a large quantity. The influence of hormone conditions on the formation and growth of callus of crocus sativus flower columns in the conventional literature (influence of hormone conditions on the formation and growth of callus of crocus sativus flower columns in Wang Hailing, li Lingwei, chen Yuanyuan, in the construction of university of Suzhou, architecture and gardening system of urban environment university) is mainly studied, wherein crocus sativus flower columns are used as explants, NAA, 2,4-D, IBA and 6-BA with different concentration ratios are added, and influence of different hormone conditions on the formation and growth of callus is studied.
The application mainly adopts oligosaccharide chain protein to promote the proliferation of the saffron somatic embryo, improve the proliferation coefficient and activity of the somatic embryo, promote the differentiation of the embryo at the next stage and accelerate the progress of the differentiation of the saffron somatic embryo into a ball system. In order to improve the proliferation coefficient and embryo activity of the saffron somatic embryos, facilitate the differentiation of later-stage embryos and promote the industrialized development of a saffron embryoid balling system, the application provides a formula for promoting the proliferation of the saffron somatic embryos.
Disclosure of Invention
The application aims to provide a formula for promoting proliferation of saffron somatic embryos, and the ingredients have synergistic effect.
In order to achieve the above purpose, the application adopts the following specific technical scheme:
a formulation for promoting proliferation of saffron somatic embryos, comprising the following components: oligosaccharide chain protein, activated carbon, potassium dihydrogen phosphate, anti-browning substance, cytokinin, auxin, silver nitrate and vitamin H.
Preferably, the auxin is 2.4-D and IAA, the cytokinin is TDZ, and the anti-browning substance is citric acid.
The preferable proposal is that the oligosaccharide chain protein is 0.1-1.0 g/L, activated carbon is 0.5g/L, monopotassium phosphate is 0.2g/L, citric acid is 0.01g/L, auxin IAA is 5mg/L, cytokinin TDZ is 1mg/L, silver nitrate is 0.01-0.05 g/L, and vitamin H is 1mL/L.
An application method for promoting proliferation of stigma croci Sativi somatic embryo comprises adding the above formula into MS culture medium, sterilizing under high pressure, and cooling.
The beneficial effects of the application are as follows: the inventor has unexpectedly found that the antiviral agent oligosaccharide chain protein plays a role in promoting cell proliferation in tissue culture, and through combining with anti-browning substances commonly used in tissue culture and plant growth regulators and substances commonly used in callus induction proliferation, a formula combination which can promote the proliferation of saffron somatic embryos more efficiently and can improve the activity of the saffron somatic embryos is screened out, so that the industrialized development of saffron embryoid balling systems is promoted.
Drawings
FIG. 1 is a growth state diagram of test 1-2 saffron somatic embryo proliferation;
FIG. 2 is a growth state diagram of test 3-6 saffron somatic embryo proliferation;
FIG. 3 is a growth state diagram of test 7-10 saffron somatic embryo proliferation;
FIG. 4 is a growth state diagram of the proliferation of somatic embryos of safflower carthamus in experiments 11-16.
Detailed Description
In order to describe the technical content, constructional features, achieved objects and effects of the technical solution in detail, the following description is made in connection with the specific embodiments in conjunction with the accompanying drawings.
The experimental methods used in the following examples are all conventional.
Materials, reagents and the like used in the following examples were obtained commercially unless otherwise specified.
Example 1
1. Test materials: poison-free callus of saffron
2. Culture conditions: culturing on an illumination culture rack with light intensity of 20-30 umol/m 2 S, photoperiod 10h/d. The temperature is 20 to 24 O C。
3. Test treatment:
1、MS
2. MS+oligosaccharide chain protein 0.1g/L
4. Culturing steps and conditions:
preparing a culture medium according to the designed test treatment, sterilizing at 121 ℃ for 25min under high pressure, and cooling for later use. On an ultra-clean workbench, taking about 1cm of stigma croci Sativi detoxification callus, inoculating 3-4 clusters on a prepared culture medium, and placing the inoculated culture medium on an illumination culture rack for culture.
5. Analysis of test results
Table 1: effect of different treatments on proliferation of saffron somatic embryos
Test treatment Proliferation coefficient Growth state
1 0.1 The callus was not substantially altered, but no death occurred
2 1.0 The callus surface has faint yellow granular somatic embryo formation, and a small amount of somatic embryo is formed
As can be seen from the table 1 and the figure 1, the growth of the stigma croci Sativi detoxification callus on the MS culture medium is basically unchanged, the surface of the detoxification callus is formed by light yellow granular somatic embryo after the oligosaccharide chain protein is added, the amount of the oligosaccharide chain protein is small, the formation and the proliferation of the stigma croci Sativi detoxification callus embryo can be promoted, and the proliferation coefficient is 1.0.
Example 2
1. Test materials: poison-free callus of saffron
2. Culture conditions: culturing on an illumination culture rack with light intensity of 20-30 umol/m 2 S, photoperiod 10h/d. The temperature is 20 to 24 O C。
3. Test treatment:
3. MS+oligosaccharide chain protein 0.1g/L+50g/L banana
4. MS+oligosaccharide chain protein 0.1g/L+50g/L banana+0.5 g/L tryptone
5. MS+oligosaccharide chain protein 0.1g/L+50g/L banana+0.5 g/L tryptone+activated carbon 0.5g/L
6. MS+50g/L banana+oligosaccharide chain protein 0.1g/L+0.5g/L tryptone+activated carbon 0.5 g/L+potassium dihydrogen phosphate 0.2g/L
4. Culturing steps and conditions:
preparing a culture medium according to the designed test treatment, sterilizing at 121 ℃ for 25min under high pressure, and cooling for later use. On an ultra-clean workbench, taking about 1cm of stigma croci Sativi detoxification callus, inoculating 3-4 clusters on a prepared culture medium, and placing the inoculated culture medium on an illumination culture rack for culture.
5. Analysis of test results
Table 2: effect of different treatments on proliferation of saffron somatic embryos
Test treatment Proliferation coefficient Growth state
3 0.1 The callus is brown and dead, and the yellowish granular somatic embryo grows out on the surface
4 0.1 The callus is brown and dead, and the yellowish granular somatic embryo grows out on the surface
5 0.2 The callus is brown and dead, and the yellowish granular somatic embryo grows out on the surface
6 0.1 The callus is brown and dead, and a tiny amount of faint yellow granular somatic embryo grows on the surface
As can be seen from the above Table 2 and FIG. 2, after banana juice, tryptone, activated carbon and potassium dihydrogen phosphate are added into MS culture medium containing oligosaccharide chain protein, the treated stigma croci detoxification callus is browned and dead, but a small amount of pale yellow granular somatic embryo is formed on the surface, and the proliferation coefficient is lower and is 0.1-0.2.
Experimental example:
1. test materials: poison-free callus of saffron
2. Culture conditions: culturing on an illumination culture rack with light intensity of 20-30 umol/m 2 S, photoperiod 10h/d. The temperature is 20 to 24 O C。
3. Test treatment:
7. MS+50g/L banana+oligosaccharide chain protein 0.1g/L+0.5g/L tryptone+activated carbon 0.5 g/L+potassium dihydrogen phosphate 0.2 g/L+citric acid 10mg/L
8. MS+50g/L banana+oligosaccharide chain protein 0.1g/L+0.5g/L tryptone+activated carbon 0.5 g/L+potassium dihydrogen phosphate 0.2 g/L+10 mg/L citrate+5 mg/L IAA
9. MS+50g/L banana+oligosaccharide chain protein 0.1g/L+0.5g/L tryptone+0.5 g/L activated carbon+0.2 g/L potassium dihydrogen phosphate+10 mg/L citric acid+IAA 5mg/L+TDZ1mg/L
10. MS+50g/L banana+oligosaccharide chain protein 0.1g/L+0.5g/L tryptone+0.5 g/L activated carbon+0.2 g/L potassium dihydrogen phosphate+10 mg/L citric acid+IAA 5mg/L+TDZ1mg/L+2.4-D0.5mg/L
11. MS+50g/L banana+oligosaccharide chain protein 0.1g/L+0.5g/L tryptone+0.5 g/L activated carbon+0.2 g/L monopotassium phosphate+10 mg/L citric acid+IAA 5mg/L+TDZ1mg/L+2.4-D0.5 mg/L+silver nitrate 10mg/L
12. MS+50g/L banana+oligosaccharide chain protein 0.1g/L+0.5g/L tryptone+0.5 g/L activated carbon+0.2 g/L potassium dihydrogen phosphate+10 mg/L citric acid+IAA 5mg/L+TDZ1mg/L+2.4-D0.5 mg/L+silver nitrate 10 mg/L+vitamin H1ml/L
13. MS+50g/L banana+0.5 g/L tryptone+0.5 g/L activated charcoal+0.2 g/L potassium dihydrogen phosphate+10 mg/L citric acid+5 mg/L IAA+1 mg/L TDZ+2.4-D0.5 mg/L silver nitrate+10 mg/L vitamin H1ml/L oligosaccharide chain protein 0.5g/L
15. MS+50g/L banana+0.5 g/L tryptone+0.5 g/L activated charcoal+0.2 g/L potassium dihydrogen phosphate+10 mg/L citric acid+5 mg/L IAA+1 mg/L TDZ+2.4-D0.5 mg/L silver nitrate+10 mg/L vitamin H1 ml/L+1.0 g/L oligosaccharide chain protein
16. MS+50g/L banana+0.5 g/L tryptone+0.5 g/L activated charcoal+0.2 g/L potassium dihydrogen phosphate+10 mg/L citric acid+5 mg/L IAA+1 mg/L TDZ+2.4-D0.5 mg/L silver nitrate+10 mg/L vitamin H1ml/L
4. Culturing steps and conditions:
preparing a culture medium according to the designed test treatment, sterilizing at 121 ℃ for 25min under high pressure, and cooling for later use. On an ultra-clean workbench, taking about 1cm of stigma croci Sativi detoxification callus, inoculating 3-4 clusters on a prepared culture medium, and placing the inoculated culture medium on an illumination culture rack for culture.
5. Analysis of test results
Table 3: effect of different treatments on proliferation of saffron somatic embryos
Test treatment Proliferation coefficient Growth state
7 1.0 The callus surface has faint yellow granular somatic embryo formation, and a small amount of somatic embryo is formed
8 1.0 The callus surface has faint yellow granular somatic embryo formation, and a small amount of somatic embryo is formed
9 1.5 The callus surface has faint yellow granular somatic embryo formation, and a small amount of somatic embryo is formed
10 3.0 The callus surface has faint yellow granular somatic embryo formation, and a small amount of somatic embryo is formed
11 2.0 The callus surface has faint yellow granular somatic embryo formation, and a small amount of somatic embryo is formed
12 6.0 The callus surface has faint yellow granular somatic embryo formation
13 2.0 The callus surface has faint yellow granular somatic embryo formation
14 3.0 The callus surface has faint yellow granular somatic embryo formation
15 3.0 The callus surface has faint yellow granular somatic embryo formation
16 2.5 The callus surface has faint yellow granular somatic embryo formation
As can be seen from the above Table 3 and FIGS. 3 and 4, based on the formulation of treatment 6, no browning occurred in the stigma croci Sativi detoxification callus, a small amount of somatic embryos formed on the surface of the callus, and the amount of somatic cells proliferated more with the addition of IAA, TDZ, 2.4-D in treatments 8 to 10, and when silver nitrate and vitamin H were continuously added in the culture medium, a large amount of somatic embryos formed on the surface of the stigma croci Sativi detoxification callus, and the proliferation coefficient could reach more than 6.0. The concentration of the oligosaccharide chain protein was adjusted based on the treatment 12, and the proliferation of somatic embryos decreased with the increase in concentration.
Thus, a preferred formulation suitable for the proliferation of saffron somatic embryos is treatment 12: MS+50g/L banana+oligosaccharide chain protein 0.1g/L+0.5g/L tryptone+0.5 g/L activated carbon+0.2 g/L potassium dihydrogen phosphate+10 mg/L citric acid+5 mg/L IAA+1 mg/L TDZ+2.4-D0.5 mg/L+10 mg/L silver nitrate+1 ml/L vitamin H.
In the formulation of comparative 16, the proliferation factor was not as high as that of the formulation of treatment 12, although the saffron somatic embryos proliferated under treatment 16. In conclusion, the oligosaccharide chain protein can promote the proliferation of the saffron somatic embryos, the proliferation effect of a single oligosaccharide chain protein is not obvious, and under the combined action of a plurality of substances, the proliferation of the saffron somatic embryos can be more effectively promoted, and the activity of the somatic embryos is stronger.
While the embodiments have been described above, other variations and modifications will occur to those skilled in the art once the basic inventive concepts are known, and it is therefore intended that the foregoing description and drawings illustrate only embodiments of the application and not limit the scope of the application, and it is therefore intended that the application not be limited to the specific embodiments described, but that the application may be practiced with their equivalent structures or with their equivalent processes or with their use directly or indirectly in other related fields.

Claims (5)

1. The application of oligosaccharide chain protein in preparing a formula for promoting proliferation of saffron somatic embryos.
2. A formulation for promoting proliferation of saffron somatic embryos, which is characterized by comprising the following components: oligosaccharide chain protein, activated carbon, potassium dihydrogen phosphate, anti-browning substance, cytokinin, auxin, silver nitrate and vitamin H.
3. The formulation of claim 2, wherein the auxin and the mitogen are IAA, 2.4-D and TDZ, respectively, and the anti-browning agent is citric acid.
4. A formulation according to claim 3, wherein the oligosaccharide chain protein is 0.1-1.0 g/L, activated carbon is 0.5g/L, potassium dihydrogen phosphate is 0.2g/L, citric acid is 0.01g/L, auxin IAA is 5mg/L and 2.4-d0.5mg/L, cytokinin TDZ1mg/L, silver nitrate is 0.01-0.05 g/L, vitamin H1mL/L.
5. An application method for promoting proliferation of saffron somatic embryos is characterized in that the formulation of claim 4 is applied and added into an MS culture medium, and the mixture is cooled for standby after autoclaving.
CN202311191885.3A 2023-09-15 2023-09-15 Formula for promoting proliferation of saffron somatic embryos Active CN117158317B (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2198870A1 (en) * 1994-09-02 1996-03-14 Drexel University A plant promoter useful for directing the expression of foreign proteins to the plant epidermis
CN102858984A (en) * 2010-02-26 2013-01-02 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and a method for making the same
CN104762316A (en) * 2009-06-19 2015-07-08 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and a method for making the same
CN115885853A (en) * 2022-12-21 2023-04-04 福建省中科生物股份有限公司 Tissue culture method for promoting embryonic callus induction and budding of crocus sativus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2198870A1 (en) * 1994-09-02 1996-03-14 Drexel University A plant promoter useful for directing the expression of foreign proteins to the plant epidermis
CN104762316A (en) * 2009-06-19 2015-07-08 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and a method for making the same
CN102858984A (en) * 2010-02-26 2013-01-02 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and a method for making the same
CN115885853A (en) * 2022-12-21 2023-04-04 福建省中科生物股份有限公司 Tissue culture method for promoting embryonic callus induction and budding of crocus sativus

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
董峰丽;饶君凤;毛碧增;: "西红花组织培养研究进展", 药物生物技术, no. 01, 15 February 2013 (2013-02-15), pages 91 - 94 *
陈书安;王晓东;赵兵;王玉春;: "藏红花胚性愈伤组织的发生及其调控", 过程工程学报, no. 01, 28 February 2007 (2007-02-28) *

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