CN112655561B - Method for in vitro preservation of red bud taro test-tube plantlet - Google Patents
Method for in vitro preservation of red bud taro test-tube plantlet Download PDFInfo
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Abstract
The invention discloses a method for in vitro preservation of test-tube seedlings of red-bud taro, and relates to the technical field of germplasm preservation. The in vitro preservation culture medium of the red bud taro test-tube plantlet is an MS culture medium containing 1-2mg/L paclobutrazol, 12-15g/L mannitol and 3-5g/L folium isatidis extract, and the preservation method comprises the steps of inoculating the red bud taro test-tube plantlet into the culture medium, keeping the temperature condition of 25 +/-1 ℃, controlling the relative humidity to be 60%, irradiating the red bud taro test-tube plantlet for 12h/d under the illumination condition of 2000Lx, and preserving the test-tube plantlet. The method can realize the in vitro preservation of the red-bud taro test-tube plantlet for 24 months, the survival rate is more than 85%, and the domestication survival rate of more than 98% can be realized after the preserved red-bud taro test-tube plantlet is domesticated and recovered to grow.
Description
Technical Field
The invention relates to the technical field of seed quality preservation, in particular to a method for in vitro preservation of test-tube seedlings of red bud taros.
Background
The red-bud taro is a traditional dominant product in the lead mountain county of Jiangxi province, and has a long planting history. The red-bud taro is bright in skin color, red in top bud, tender in meat quality, easy to boil and not burnt, rich in nutrition and rich in various trace elements, is a treasure in taro, is always a pretty robber and goods in markets at home and abroad, and has the advantage of rapid industrial development for over ten years, so that the red-bud taro becomes a leading industry for increasing income and becoming rich for farmers in the Shanxi county.
The method is one of the main methods for in vitro preservation of plant germplasm resources. The method for limiting growth and preservation is characterized in that the growth of test-tube plantlets is limited on the premise of ensuring the genetic integrity of plant germplasm, the number of subcultures is reduced, the purpose of preserving plant germplasm resources in the middle period is achieved, and in-vitro preservation is an important means for preserving the red-bud taro germplasm resources.
The growth inhibitor is a natural or artificially synthesized exogenous hormone and has strong physiological activity of inhibiting cell growth. Research shows that the proportion of the growth regulator in the culture medium is perfected and adjusted, particularly, the growth inhibitor is added, so that the storage time of the culture in a test tube can be prolonged, the quality of the test-tube plantlet and the transplanting survival rate can be improved, and the dual effects of storing and optimizing germplasm resources are achieved.
Paclobutrazol, also known as chloroconazole, is a typical growth inhibitor with a very wide application range and has a certain effect in short-medium-term in-vitro preservation of a plurality of plant germplasm resources. At present, a plurality of researchers discuss the effect of paclobutrazol in plant germplasm resource preservation, and the effect of paclobutrazol is different for different plant species. Although the paclobutrazol inhibitor plays an important role in the in vitro preservation of the red-bud taro test-tube plantlet and is also an inhibitor with a better effect aiming at the red-bud taro test-tube plantlet at present, the paclobutrazol inhibitor can only realize the in vitro preservation period of the red-bud taro test-tube plantlet for 12 months at most and the survival rate is about 60 percent at present, and still can not meet the requirement of germplasm resource preservation.
Therefore, how to improve the storage life and the survival rate of the paclobutrazol inhibitor to the in vitro preservation of the red-bud taro test-tube plantlet so as to ensure the good preservation effect of the germ plasm resource of the red-bud taro is a technical problem to be solved urgently in the in vitro preservation of the red-bud taro test-tube plantlet at present.
Disclosure of Invention
The invention aims to provide a method for in vitro preservation of test-tube plantlets of red bud taros, which solves the problems in the prior art, remarkably improves the in vitro preservation period and the survival rate of the test-tube plantlets of the red bud taros, and simultaneously ensures high-efficiency survival after domestication.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a culture medium for in vitro preservation of red-bud taro test-tube plantlets, which is an MS culture medium containing 1-2mg/L paclobutrazol, 12-15g/L mannitol and 3-5g/L folium isatidis extract.
Further, the pH of the medium is 5.8 to 6.0.
Further, the preparation method of the folium isatidis extract comprises the following steps:
washing folium Isatidis, drying, pulverizing, ultrasonic extracting, centrifuging, collecting supernatant, concentrating, and drying at 60-70 deg.C to obtain folium Isatidis extract.
Further, the ultrasonic extraction time is 4.5-5h, and the centrifugation time is 20-25 min.
Further, the drying time is 3-5 h.
The invention also provides an in vitro preservation method of the red-bud taro test-tube plantlet, which adopts the in vitro preservation culture medium for preservation and comprises the following steps:
and inoculating the red-bud taro test-tube plantlet in the culture medium, keeping the temperature condition of 25 +/-1 ℃, controlling the relative humidity to be 60%, and irradiating the red-bud taro test-tube plantlet for 12h/d under the illumination condition of 2000Lx for storing the test-tube plantlet.
The invention discloses the following technical effects:
the method extracts the effective components of the traditional Chinese medicine folium isatidis, adds the effective components into an MS culture medium containing paclobutrazol, simultaneously adds mannitol to adjust the osmotic pressure of the culture medium, can realize the in-vitro preservation of the test-tube plantlet of the red bud taro for 24 months by adjusting the concentrations of the paclobutrazol, the folium isatidis extract and the mannitol in the culture medium, has the survival rate of more than 85 percent, and can realize the domestication survival rate of more than 98 percent after the domestication and the recovery of the test-tube plantlet of the preserved red bud taro.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The "parts" in the present invention are all parts by mass unless otherwise specified.
The red bud taro test-tube plantlet used by the invention is a test-tube plantlet of the red bud taro in the Jiangxi Qianshan.
Example 1
1. Extracting the folium isatidis extract:
cleaning folium Isatidis, drying, pulverizing, sieving, adding distilled water, and ultrasonic extracting for 4.5 hr; centrifuging for 20min, collecting supernatant, concentrating, and drying at 60 deg.C for 5 hr to obtain folium Isatidis extract.
2. In-vitro preservation of the red-bud taro test-tube plantlet:
(1) preparing a culture medium of MS +1mg/L paclobutrazol +15g/L mannitol +5g/L folium isatidis extract, and adjusting the pH of the culture medium to 6.0 by using dilute hydrochloric acid;
(2) inoculating 100 red-bud taro test-tube plantlets in a culture medium, keeping the temperature condition of 25 +/-1 ℃, controlling the relative humidity to be 60%, irradiating the red-bud taro test-tube plantlets for 12h/d under the illumination condition of 2000Lx, and storing the test-tube plantlets.
Example 2
1. Extracting the folium isatidis extract:
cleaning folium Isatidis, drying, pulverizing, sieving, adding distilled water, and ultrasonic extracting for 5 hr; centrifuging for 25min, collecting supernatant, concentrating, and drying at 65 deg.C for 4 hr to obtain folium Isatidis extract.
2. In-vitro preservation of the red-bud taro test-tube plantlet:
(1) preparing a culture medium of MS +2mg/L paclobutrazol +12g/L mannitol +4g/L folium isatidis extract, and adjusting the pH of the culture medium to 5.8;
(2) inoculating 100 red bud taro test-tube plantlets in a culture medium, keeping the temperature condition of 25 +/-1 ℃, controlling the relative humidity to be 60%, irradiating the red bud taro test-tube plantlets for 12h/d under the illumination condition of 2000Lx, and storing the test-tube plantlets.
Example 3
1. Extracting the folium isatidis extract:
cleaning folium Isatidis, drying, pulverizing, sieving, adding distilled water, and ultrasonic extracting for 5 hr; centrifuging for 23min, collecting supernatant, concentrating, and drying at 68 deg.C for 3 hr to obtain folium Isatidis extract.
2. In-vitro preservation of the red-bud taro test-tube plantlet:
(1) preparing a culture medium of MS +2mg/L paclobutrazol +13g/L mannitol +3g/L folium isatidis extract, and adjusting the pH of the culture medium to 5.9;
(2) inoculating 100 red bud taro test-tube plantlets in a culture medium, keeping the temperature condition of 25 +/-1 ℃, controlling the relative humidity to be 60%, irradiating the red bud taro test-tube plantlets for 12h/d under the illumination condition of 2000Lx, and storing the test-tube plantlets.
Example 4
1. Extracting the folium isatidis extract:
cleaning folium Isatidis, drying, pulverizing, sieving, adding distilled water, and ultrasonic extracting for 4.8 hr; centrifuging for 24min, collecting supernatant, concentrating, and drying at 70 deg.C for 4 hr to obtain folium Isatidis extract.
2. In-vitro preservation of the red-bud taro test-tube plantlet:
(1) preparing a culture medium of MS +1mg/L paclobutrazol +14g/L mannitol +3g/L folium isatidis extract, and adjusting the pH of the culture medium to 6.0;
(2) inoculating 100 red bud taro test-tube plantlets in a culture medium, keeping the temperature condition of 25 +/-1 ℃, controlling the relative humidity to be 60%, irradiating the red bud taro test-tube plantlets for 12h/d under the illumination condition of 2000Lx, and storing the test-tube plantlets.
Comparative example 1
The difference from example 1 is that no folium Isatidis extract was added.
Comparative example 2
The difference from example 1 is that mannitol was not added.
Comparative example 3
The difference from example 1 is that the addition amount of the folium isatidis extract was adjusted to 6 g/L.
Comparative example 4
The difference from example 1 was that the amount of mannitol added was adjusted to 10 g/L.
Two months after inoculation, the plant height, stem thickness, leaf number and leaf SPAD value of the red bud taro test-tube plantlet in each example and comparative example are measured, and the average value is calculated, and the result is shown in Table 1; the survival rate of the test-tube plantlets was counted every four months at 4-24 months after inoculation, and the results are shown in table 2.
TABLE 1
| Average plant height (cm) | Average stem thickness (mm) | Average number of leaves (pieces) | Average SPAD value | |
| Example 1 | 7.32 | 0.92 | 15.36 | 36.21 |
| Example 2 | 7.31 | 0.91 | 15.42 | 36.12 |
| Example 3 | 7.33 | 0.92 | 15.46 | 36.25 |
| Example 4 | 7.32 | 0.92 | 15.52 | 36.51 |
| Comparative example 1 | 6.32 | 0.68 | 13.24 | 30.23 |
| Comparative example 2 | 6.34 | 0.71 | 13.25 | 30.22 |
| Comparative example 3 | 6.11 | 0.65 | 11.21 | 27.56 |
| Comparative example 4 | 6.01 | 0.64 | 11.03 | 26.59 |
TABLE 2
And (3) restoring the growth of the test-tube plantlet:
in the examples and the comparative examples, the test-tube plantlets of the red-bud taro which survived 24 months after being preserved in vitro and the test-tube plantlets which are cultured in 60d secondary bands are respectively inoculated into a culture medium (MS +5.5g/L agar +30g/L cane sugar) to restore the growth, the test-tube plantlets which restore the growth for 25d are subjected to seedling hardening treatment, the root culture medium is cleaned and potted, the plant height, the stem thickness, the leaf number and the SPAD value of the leaves are measured after 30d of conventional treatment, the average value is calculated, and the domestication survival rate is counted, and the results are shown in Table 3.
TABLE 3
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (3)
1. An in vitro preservation culture medium for red-bud taro test-tube plantlets is characterized in that the culture medium is an MS culture medium containing 1-2mg/L paclobutrazol, 12-15g/L mannitol and 3-5g/L folium isatidis extract;
the folium isatidis extract is obtained by one of the following extraction methods:
the first extraction method comprises the following steps: cleaning folium Isatidis, drying, pulverizing, sieving, adding distilled water, and ultrasonic extracting for 4.5 hr; centrifuging for 20min, concentrating the supernatant, and drying at 60 deg.C for 5 hr to obtain folium Isatidis extract;
and (2) an extraction method II: cleaning folium Isatidis, drying, pulverizing, sieving, adding distilled water, and ultrasonic extracting for 5 hr; centrifuging for 25min, concentrating the supernatant, and drying at 65 deg.C for 4 hr to obtain folium Isatidis extract;
the extraction method is three: cleaning folium Isatidis, drying, pulverizing, sieving, adding distilled water, and ultrasonic extracting for 5 hr; centrifuging for 23min, concentrating the supernatant, and drying at 68 deg.C for 3 hr to obtain folium Isatidis extract;
the extraction method is four: cleaning folium Isatidis, drying, pulverizing, sieving, adding distilled water, and ultrasonic extracting for 4.8 hr; centrifuging for 24min, collecting supernatant, concentrating, and drying at 70 deg.C for 4 hr to obtain folium Isatidis extract.
2. The in vitro preservation medium for the red-bud taro test-tube plantlets according to claim 1, wherein the pH of the medium is 5.8-6.0.
3. An in vitro preservation method of red bud taro test-tube plantlets, which is characterized in that the in vitro preservation culture medium of the red bud taro test-tube plantlets according to any one of claims 1-2 is adopted for preservation, and the preservation method comprises the following steps:
and inoculating the red bud taro test-tube plantlet in the culture medium, keeping the temperature condition of 25 +/-1 ℃, controlling the relative humidity to be 60%, and irradiating the red bud taro test-tube plantlet for 12h/d under the illumination condition of 2000Lx to store the test-tube plantlet.
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| CN104800271A (en) * | 2015-04-14 | 2015-07-29 | 南京多宝生物科技有限公司 | Preparation method of folium isatidis extract |
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