CN115843691B - Induction method for regulating garlic test tube bulb by using multiwall carbon nanotubes - Google Patents
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Abstract
The invention discloses an induction method for regulating and controlling garlic test tube bulbs by utilizing multiwall carbon nanotubes. Mainly adopts six-petal red garlic bulbs as explants, and multi-wall carbon nanotubes as regulators to promote callus induction, test tube seedling differentiation and test tube bulb induction. Through statistical analysis, when the multiwall carbon nano tube with the concentration of 50mg/L is added in the induction regulation of the garlic callus, the biomass of the garlic callus is up to 72g; when 75 mg/L multiwall carbon nanotubes are added in the regulation and control of garlic callus differentiation, the differentiation rate of the test tube plantlet reaches up to 133%; when the multi-wall carbon nano tube with the concentration of 25 mg/L is added in the induction regulation of the garlic test tube bulbs, the induction rate of the test tube bulbs is up to 92 percent, and the average diameter and the weight of the test tube bulbs are up to 6.21 mm g and 0.253g respectively. The method provides a potential new starting point for the industrialized and large-scale development of garlic detoxification.
Description
The invention obtains the conversion of the Tianjin city agricultural commission technological achievement and the promotion key project fund (project number: 202101060).
Technical Field
The invention belongs to the technical field of agricultural bioengineering, relates to a garlic callus cell growth regulation and control process, and particularly discloses a method for promoting rapid induction and differentiation of garlic callus and formation of test tube bulbs by utilizing multi-wall carbon nanotubes.
Background
Garlic is an important economic crop and plays an important role in agricultural production in China. Currently, garlic detoxification and rapid propagation technologies have successfully established indirect organogenesis mainly by the callus pathway, direct organogenesis mainly by the inflorescence axis pathway, and the like. Among these, many reports have been made, and the technical maturation is an indirect organogenesis pathway using organs with low degrees of differentiation such as root tips and bulb pieces as explants. However, the indirect organogenesis approach still has the realistic problems of low propagation coefficient, long period and the like in experiments and production. Carbon nanotubes are recently discovered as novel nanoscale materials having various good properties, and have been widely used in research fields such as energy storage, composite materials, and biology. After the carbon nano tube is modified, the carbon nano tube can enter cells through passive phagocytosis, active penetration and other ways after being functionalized, and plays a role in cytoplasm or nucleus; furthermore, the functionalized carbon nano tube can be used as a protein carrier to carry macromolecular substances into cells, and can also be used as a single-stranded DNA and double-stranded RNA carrier to transfer the macromolecular substances into cells, thereby playing a corresponding biological role. However, researches for regulating and controlling the growth of garlic callus cells and inducing test tube bulbs based on multi-wall carbon nanotubes are rarely reported.
In view of the above requirements, the invention constructs a method for promoting the induction and growth of garlic callus cells, improving the propagation coefficient of garlic, shortening the culture period and improving the quality of garlic test tube bulbs based on the multi-wall carbon nanotube technology. The method overcomes the defects of the existing garlic detoxification and rapid propagation technology, improves the production efficiency, reduces the production cost, and improves the quality of garlic bulb garlic seeds in garlic test tubes.
Disclosure of Invention
The invention aims at disclosing a method for regulating and controlling the growth and differentiation of garlic callus cells and inducing test tube bulbs by utilizing multiwall carbon nanotubes, which is characterized by comprising the following steps:
(1) Garlic callus induction regulation and control technology based on multiwall carbon nano tube:
selecting bulbs which have no disease spots, good growth vigor and uniform size as explants, and inoculating the explants to an induced callus culture medium: MS+0.8 mg/L2, 4-D+0.3 mg/L KT+25 g/L sucrose+7.0 g/L agar, pH=6.2, adding multiwall carbon nanotubes with the concentration of 50mg/L as a regulator, placing the mixture into a culture chamber with the temperature of 25+/-1 ℃ for garlic callus induction, culturing 15D to obtain yellow or light yellow granular callus, and carrying out 3 times of subculture;
(2) Garlic callus differentiation regulation and control technology based on multiwall carbon nano tube:
transferring the induced garlic callus to a differentiation regulating culture medium: MS+2. mg/L6-BA+35 g/L sucrose+6.5 g/L agar, pH=5.5, adding multi-wall carbon nano tube with the concentration of 75 mg/L as a regulator, placing in a culture room with the temperature of 25+/-1 ℃ for culturing 20 d to obtain garlic regenerated plant test tube plantlets;
(3) Garlic test tube bulb induction regulation and control technology based on multiwall carbon nanotubes:
transferring the differentiated garlic test tube plantlets to a test tube bulb induction regulation culture medium: MS+150 g/L sucrose+5.5 g/L agar, pH=7.2, adding multiwall carbon nanotube with concentration of 25 mg/L as regulator, placing in a culture chamber with temperature of 25+ -1deg.C, culturing at 25+ -1deg.C, and harvesting garlic test tube bulb.
(4) Multi-wall carbon nanotube concentration contrast test:
in the regulation and control process, comparison tests are set with the concentration gradients of the multiwall carbon nanotubes of 0, 25, 50, 75 and 100mg/L, and the biomass of the garlic callus, the emergence rate of adventitious buds, the induction rate of test tube bulbs and quality parameters are calculated. Experimental results show that the multi-wall carbon nanotubes with different concentrations can promote the induction of garlic callus, and the biomass of the garlic callus shows a trend of rising and then reducing along with the increase of the concentration of the multi-wall carbon nanotubes, wherein when the concentration of the multi-wall carbon nanotubes is 50mg/L, the biomass of the garlic callus is up to 72g; the influence of the multi-wall carbon nanotubes with different concentrations on the garlic callus differentiation regeneration plant test tube plantlet is complex, the differentiation rate of the test tube plantlet shows a bimodal curve trend of rising and then falling, rising and then falling along with the increase of the multi-wall carbon nanotubes, wherein when the concentration of the multi-wall carbon nanotubes is 75 mg/L, the differentiation rate of the test tube plantlet is up to 133 percent; the multi-wall carbon nanotubes with different concentrations have promotion effect on the induction of garlic test tube bulbs, and as the concentration of the multi-wall carbon nanotubes increases, the induction rate of the test tube bulbs rises rapidly and enters a steady state, the induction rate of the test tube bulbs cannot be obviously improved by continuously increasing the concentration of the multi-wall carbon nanotubes, and conversely, the multi-wall carbon nanotubes have a reduction trend, wherein when the concentration of the multi-wall carbon nanotubes is 25 mg/L, the induction rate of the test tube bulbs is up to 92%, and the average diameter and the weight of the test tube bulbs are also up to 6.21 mm g and 0.253g respectively.
The 2,4-D is 2,4-dichlorophenoxyacetic acid (2, 4-Dichlorophenoxyacetic acid), which is called 2,4-D for short; KT is Kinetin (Kinetin), abbreviated KT; multiwall carbon nanotube outer diameter: 10-20nm, length:<2 μm, specific surface area: 100-160 m 2 /g。
The MS basic culture medium comprises the following formula:
each 1L of the culture medium contains: KNO (KNO) 3 1900mg，MgSO 4 •7H 2 O 370mg，NH 4 NO 3 1650mg，KH 2 PO 4 170mg，CaCl 2 ·2H 2 O 440mg，ZnSO 4 ·7H 2 O 8.6mg，H 3 BO 3 6.2mg，KI 0.83mg，MnSO 4 ·4H 2 O 22.3mg，Na 2 MoO 4 ·2H 2 O 0.25mg，FeSO 4 ·7H 2 O 27.8mg，CuSO 4 ·5H 2 O 0.025mg，CoCl 2 ·6H 2 O 0.025mg，Na 2 EDTA·2H 2 37.7mg of O, 0.5mg of pyridoxine hydrochloride, 0.4mg of thiamine hydrochloride, 2.0mg of glycine, 100mg of inositol and 0.5mg of nicotinic acid.
The invention further discloses a method for regulating and controlling the growth and differentiation of garlic callus cells and inducing test tube bulbs by utilizing the multiwall carbon nanotubes. The experimental results show that: the biomass of the garlic callus is up to 72g, the differentiation rate of the test tube plantlet is up to 133%, the induction rate of the test tube bulb is up to 92%, and the average diameter and weight of the test tube bulb are also up to 6.21 mm g and 0.253g respectively.
Compared with the prior art, the method for regulating and controlling the growth and differentiation of garlic callus cells and inducing test tube bulbs by utilizing the multiwall carbon nanotubes disclosed by the invention has the key points that:
(1) The invention can obviously improve the induction proliferation of garlic callus by adopting the specific multiwall carbon nanotube as the cell growth metabolism regulator, and the induction rate and quality of the callus differentiated test tube plantlet and test tube bulb.
(2) The constructed multi-walled carbon nanotube-based garlic callus cell growth and test tube bulb induction regulation and control process system can not only meet the requirements of high-efficiency and high-quality induction of garlic callus, but also achieve the purposes of improving the propagation coefficient of garlic test tube bulbs and improving the detoxification and rapid propagation quality of garlic.
(3) There is no report on the induction and control of garlic callus by using multi-wall carbon nanotube technology, differentiation and formation of test tube bulb. The patent carries out systematic optimization on key processes by analyzing gradient concentration effect of the multiwall carbon nanotube regulator, and constructs a complete process scheme for promoting garlic callus induction, test tube seedling differentiation and test tube bulb induction. The invention can obviously improve the biomass of garlic callus, increase the propagation coefficient and improve the quality of test tube bulbs. The method of the invention has not been reported at home and abroad.
Drawings
FIG. 1 is a drawing of garlic callus induced based on multiwall carbon nanotubes as a modulator;
FIG. 2 shows the growth state of garlic test tube plantlets based on multiwall carbon nanotubes as a regulator;
FIG. 3 is a garlic tube bulb morphology based on multiwall carbon nanotubes as modulators.
Detailed Description
The present invention is described below with reference to examples, which are not limited to the embodiments described herein, but may be modified and varied by those skilled in the art in light of the spirit of the present invention, and all such modifications and variations are to be regarded as being within the scope of the invention, which is defined in the following claims. Wherein, the various chemical reagents such as the multi-wall carbon nano tube, the 2,4-D, the KT, the 6-BA and the like used in the invention are all conventional medicines and are all commercially available.
Example 1
(1) Garlic callus induction regulation and control technology based on multiwall carbon nano tube:
selecting bulbs which are free of disease spots, good in growth vigor and uniform in size as explants, peeling off the outer skin, washing for 10min by using distilled water, soaking in 0.3% (w/w) NaClO solution in a sterile ultra-clean workbench for 15min, washing for 5min by using sterile distilled water, soaking in 75% (w/w) ethanol for 15min, and washing by using sterile distilled water for 5min for later use; transversely cutting garlic bulbs into bulb pieces with the thickness of 0.5-1.0 cm, and inoculating the bulb pieces to an induced callus culture medium: MS+0.8 mg/L2, 4-D+0.3 mg/L KT+25 g/L sucrose+7.0 g/L agar, pH=6.2. Taking a multiwall carbon nanotube as a regulator, setting a concentration gradient of 0, 25, 50, 75 and 100mg/L for comparison test, placing the multiwall carbon nanotube in a culture chamber with the temperature of 25+/-1 ℃ for garlic callus induction, culturing 15 d to obtain yellow or yellowish granular callus, calculating the biomass of the callus, and performing 3 times of subculture.
The experimental results show that: the multi-wall carbon nano tube regulator with different concentrations can promote the induction of garlic callus, and the biomass of the garlic callus shows a trend of rising and then reducing along with the increase of the concentration of the multi-wall carbon nano tube, wherein the biomass of the garlic callus is up to 72g when the concentration of the multi-wall carbon nano tube is 50mg/L (see table 1).
(2) Garlic callus differentiation regulation and control technology based on multiwall carbon nano tube:
transferring the induced garlic callus to a differentiation regulating culture medium: MS+2. mg/L6-BA+35 g/L sucrose+6.5 g/L agar, pH=5.5, adding multi-wall carbon nanotube with concentration of 75 mg/L as regulator, placing in a culture room with temperature of 25+ -1deg.C, culturing at 20 d to obtain regenerated plant test-tube plantlet of garlic.
(3) Garlic test tube bulb induction regulation and control technology based on multiwall carbon nanotubes:
transferring the differentiated garlic test tube plantlets to a test tube bulb induction regulation culture medium: MS+150 g/L sucrose+5.5 g/L agar, pH=7.2, adding multiwall carbon nanotube with concentration of 25 mg/L as regulator, placing in a culture chamber with temperature of 25+ -1deg.C, culturing at 25+ -1deg.C, and harvesting garlic test tube bulb;
TABLE 1 callus biomass
Example 2
(1) Garlic callus induction regulation and control technology based on multiwall carbon nano tube:
selecting bulbs which are free of disease spots, good in growth vigor and uniform in size as explants, peeling off the outer skin, washing for 10min by using distilled water, soaking in 0.3% (w/w) NaClO solution in a sterile ultra-clean workbench for 15min, washing for 5min by using sterile distilled water, soaking in 75% (w/w) ethanol for 15min, and washing by using sterile distilled water for 5min for later use; transversely cutting garlic bulbs into bulb pieces with the thickness of 0.5-1.0 cm, and inoculating the bulb pieces to an induced callus culture medium: MS+0.8 mg/L2, 4-D+0.3 mg/L KT+25 g/L sucrose+7.0 g/L agar, pH=6.2, adding multiwall carbon nanotubes with the concentration of 50mg/L as a regulator, placing the mixture into a culture chamber with the temperature of 25+/-1 ℃ for inducing garlic callus, culturing 15D to obtain yellow or light yellow granular callus, and carrying out 3 times of subculture.
(2) Garlic callus differentiation regulation and control technology based on multiwall carbon nano tube:
transferring the induced garlic callus to a differentiation regulating culture medium: MS+2. mg/L6-BA+35 g/L sucrose+6.5 g/L agar, pH=5.5. Taking multi-wall carbon nano tube as regulator, setting concentration gradient of 0, 25, 50, 75 and 100mg/L for comparison test, placing in culture room with temperature of 25+ -1deg.C, culturing 20 d to obtain garlic regenerated plant test tube plantlet, and calculating test tube plantlet differentiation rate.
The experimental results show that: the influence of the multi-wall carbon nanotubes with different concentrations on the test tube plantlet of the garlic callus differentiation regeneration plant is complex, the differentiation rate of the test tube plantlet shows a bimodal curve trend of rising and then falling, rising and then falling along with the increase of the multi-wall carbon nanotubes, wherein the differentiation rate of the test tube plantlet is up to 133 percent when the concentration of the multi-wall carbon nanotubes is 75 mg/L (see table 2).
(3) Garlic test tube bulb induction regulation and control technology based on multiwall carbon nanotubes:
transferring the differentiated garlic test tube plantlets to a test tube bulb induction regulation culture medium: MS+150 g/L sucrose+5.5 g/L agar, pH=7.2, adding multiwall carbon nanotube with concentration of 25 mg/L as regulator, placing in a culture chamber with temperature of 25+ -1deg.C, culturing at 25+ -1deg.C, and harvesting garlic test tube bulb.
TABLE 2 test tube plantlet differentiation rate
Example 3
(1) Garlic callus induction regulation and control technology based on multiwall carbon nano tube:
selecting bulbs which are free of disease spots, good in growth vigor and uniform in size as explants, peeling off the outer skin, washing for 10min by using distilled water, soaking in 0.3% (w/w) NaClO solution in a sterile ultra-clean workbench for 15min, washing for 5min by using sterile distilled water, soaking in 75% (w/w) ethanol for 15min, and washing by using sterile distilled water for 5min for later use; transversely cutting garlic bulbs into bulb pieces with the thickness of 0.5-1.0 cm, and inoculating the bulb pieces to an induced callus culture medium: MS+0.8 mg/L2, 4-D+0.3 mg/L KT+25 g/L sucrose+7.0 g/L agar, pH=6.2, adding multiwall carbon nanotubes with the concentration of 50mg/L as a regulator, placing the mixture into a culture chamber with the temperature of 25+/-1 ℃ for inducing garlic callus, culturing 15D to obtain yellow or light yellow granular callus, and carrying out 3 times of subculture.
(2) Garlic callus differentiation regulation and control technology based on multiwall carbon nano tube:
transferring the induced garlic callus to a differentiation regulating culture medium: MS+2. mg/L6-BA+35 g/L sucrose+6.5 g/L agar, pH=5.5, adding multi-wall carbon nanotube with concentration of 75 mg/L as regulator, placing in culture room with temperature of 25+ -1deg.C, culturing 20 d to obtain garlic regenerated plant test-tube plantlet
(3) Garlic test tube bulb induction regulation and control technology based on multiwall carbon nanotubes:
transferring the differentiated garlic test tube plantlets to a test tube bulb induction regulation culture medium: ms+150 g/L sucrose+5.5 g/L agar, ph=7.2. Taking multi-wall carbon nano tube as regulator, setting concentration gradient of 0, 25, 50, 75 and 100mg/L comparison test, placing in culture room with temperature of 25+ -1deg.C, culturing for 25+ -1deg.C, harvesting garlic test tube bulb, calculating induction rate of test tube bulb, and counting average diameter and weight of test tube bulb.
The experimental results show that: the multi-wall carbon nanotubes with different concentrations have promotion effect on the induction of garlic test tube bulbs, and as the concentration of the multi-wall carbon nanotubes increases, the induction rate of the test tube bulbs rises rapidly and enters a steady state, the continuous increase of the concentration of the multi-wall carbon nanotubes cannot obviously promote the induction rate of the test tube bulbs, but has a decreasing trend, wherein when the concentration of the multi-wall carbon nanotubes is 25 mg/L, the induction rate of the test tube bulbs reaches 92 percent at most, and the average diameter and the weight of the test tube bulbs reach 6.21 mm g and 0.253g (see table 3) respectively.
TABLE 3 test tube bulb induction and average diameter and weight
Claims (2)
1. The induction method for regulating and controlling garlic test tube bulbs by utilizing the multiwall carbon nanotubes is characterized by comprising the following steps of:
(1) Garlic callus induction regulation and control based on multiwall carbon nanotubes:
the bulbs which have no disease spots, good growth vigor and uniform size are selected as explants, and inoculated to an induced callus culture medium: MS+0. mg/L2, 4-D+0.3 mg/L KT+25 g/L sucrose+7.0 g/L agar, pH=6.2, adding multiwall carbon nanotubes with the concentration of 50mg/L as a regulator, performing garlic callus induction, culturing 15D to obtain yellow or light yellow granular callus, and performing 3 times of subculture;
(2) Garlic callus differentiation regulation and control based on multiwall carbon nanotubes:
transferring the induced garlic callus to a differentiation regulating culture medium: MS+2. mg/L6-BA+35 g/L sucrose+6.5 g/L agar, pH=5.5, adding multi-wall carbon nano tube with the concentration of 75 mg/L as a regulator, and culturing 20 d to obtain garlic regeneration plant test tube plantlets;
(3) Garlic test tube bulb induction regulation and control based on multiwall carbon nanotubes:
transferring the differentiated garlic test tube plantlets to a test tube bulb induction regulation culture medium: MS+150 g/L sucrose+5.5 g/L agar, pH=7.2, and adding multi-walled carbon nanotubes with a concentration of 25 mg/L as a regulator, culturing 25 d, and harvesting garlic test tube bulbs.
2. Use of the method of claim 1 for garlic tube bulb induction.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102792887A (en) * | 2012-07-27 | 2012-11-28 | 天津师范大学 | Method for inducing garlic test tube bulb |
| CN104642109A (en) * | 2015-02-04 | 2015-05-27 | 天津师范大学 | Method for constructing garlic micropropagation by taking bulb sheets as explants |
| CN112237142A (en) * | 2020-11-02 | 2021-01-19 | 江苏省中国科学院植物研究所 | Tissue culture medium for lycoris, callus culture method and method for establishing lycoris regeneration system |
| CN114615882A (en) * | 2019-08-27 | 2022-06-10 | 雷利卡基因组公司 | Transformed plants and methods of making and using the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017025967A1 (en) * | 2015-08-13 | 2017-02-16 | Forrest Innovations Ltd. | Formulations and compositions for delivery of nucleic acids to plant cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102792887A (en) * | 2012-07-27 | 2012-11-28 | 天津师范大学 | Method for inducing garlic test tube bulb |
| CN104642109A (en) * | 2015-02-04 | 2015-05-27 | 天津师范大学 | Method for constructing garlic micropropagation by taking bulb sheets as explants |
| CN114615882A (en) * | 2019-08-27 | 2022-06-10 | 雷利卡基因组公司 | Transformed plants and methods of making and using the same |
| CN112237142A (en) * | 2020-11-02 | 2021-01-19 | 江苏省中国科学院植物研究所 | Tissue culture medium for lycoris, callus culture method and method for establishing lycoris regeneration system |
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