CN113100064A - Culture medium for tissue culture of broussonetia papyrifera and preparation method - Google Patents
Culture medium for tissue culture of broussonetia papyrifera and preparation method Download PDFInfo
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- CN113100064A CN113100064A CN202110506572.7A CN202110506572A CN113100064A CN 113100064 A CN113100064 A CN 113100064A CN 202110506572 A CN202110506572 A CN 202110506572A CN 113100064 A CN113100064 A CN 113100064A
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- paper mulberry
- culture medium
- tissue culture
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- tender leaf
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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
Abstract
The invention provides a culture medium for tissue culture of paper mulberry and a preparation method thereof, wherein the culture medium is prepared by mixing an MS culture medium and tender leaf extracting solution of paper mulberry according to the volume ratio of (10-20) to 1. The culture medium for tissue culture of paper mulberry can accelerate the rooting of tissue culture seedlings of the primary paper mulberry, the bud seedling cracking and the growth of the subculture seedling, the seedlings are strong in the seedling training stage, the root system is developed, the survival rate is as high as more than 90%, and the culture medium has strong adaptability to the change of illumination, temperature and humidity in the nature.
Description
Technical Field
The invention relates to the technical field of culture media, in particular to a culture medium for tissue culture of broussonetia papyrifera and a preparation method thereof.
Background
The paper mulberry, Moraceae, Broussonetia, which is adopted by the invention, is named as: the paper mulberry, paper mulberry and sand paper tree have fast growth speed, strong adaptability and wide distribution in China and south east Asia countries. Because the leaves and the twigs of the broussonetia papyrifera are high in protein content and are good raw materials of livestock and poultry feed, the feed prepared by taking the broussonetia papyrifera as a raw material can obviously improve the quality of livestock and poultry meat food, and branches and barks of the broussonetia papyrifera are good raw materials for papermaking, in recent years, broussonetia papyrifera seedlings are artificially cultured, broussonetia papyrifera is planted in a large area, and the development of the broussonetia papyrifera industry is highly valued and concerned by govern. In order to obtain a large amount of paper mulberry seedlings, the seedling culture by adopting a tissue culture technical method is the best method for obtaining a large amount of paper mulberry seedlings in a short time. However, most of the culture mediums adopted by the tissue culture seedling of paper mulberry are standard plant tissue culture mediums, namely MS culture mediums, or the dosage of vitamins, trace elements and growth regulating substances is properly adjusted on the basis of the MS culture mediums. However, the MS culture medium cannot be completely consistent with the nutrients required by the tissue and cell development of the paper mulberry, so the results expressed in the tissue culture seedling raising practice are as follows: the buds in the culture bottle have slow division speed and are easy to yellow; the seedlings are not vigorous in growth and even withered to death when transferred into subculture; even in the stage of training and culturing in a nutrition cup, due to insufficient and incomplete nutrition and healthy seedling congenital development, the survival rate is low (30-60%), and serious or even all death is caused, which is a real loss for seedling culturing enterprises and planting enterprises.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a culture medium for tissue culture of paper mulberry, which can accelerate the rooting of tissue culture seedlings of the original paper mulberry, the splitting and planting of tooth seedlings and the growth of subculture seedlings, and has strong seedlings in the seedling training stage, developed root systems, high survival rate of more than 90 percent and strong adaptability to the change of illumination, temperature and humidity in the nature.
The second purpose of the invention is to provide a preparation method of the culture medium for paper mulberry tissue culture, which is simple to operate and low in cost.
In order to solve the technical problem, the invention provides a culture medium for tissue culture of paper mulberry, which is prepared by mixing an MS culture medium and a tender leaf extracting solution of paper mulberry according to the volume ratio of (10-20) to 1.
The culture medium of the invention consists of an MS culture medium and a tender leaf extracting solution of a paper mulberry variety corresponding to tissue culture, wherein the MS culture medium is a universal plant tissue culture medium at home and abroad, is sold in markets at home and abroad, and generally does not need to be prepared by self. The extract of the tender leaves of the paper mulberry contains rich total nutrient substances required by the division and development of paper mulberry tissues and cells, including more than 20 amino acids, trace elements such as calcium, phosphorus, potassium, sodium, iron, manganese, zinc, molybdenum, copper, cobalt and the like, and a plurality of vitamins such as B vitamins, VC, biotin, calcium pantothenate, inositol and the like, and the balance degree of the natural nutrient substances is far higher than that of an artificially prepared MS culture medium, so that the extract has unexpected effects on making up the deficiency of the nutrition of the plant MS culture medium and solving the defect of low nutrition balance degree of the MS culture medium, can accelerate the rooting of tissue culture seedlings of the primary paper mulberry, can realize the rapid split planting of dental seedlings, can realize the rapid growth of subculture seedlings, can promote the health of seedlings in the seedling training stage, has developed root systems, has the survival rate of more than 90 percent, and has strong adaptability to the change of illumination, temperature and humidity in the nature.
Preferably, the MS culture medium and the tender leaf extracting solution of the broussonetia papyrifera are mixed according to the volume ratio of 15: 1. The inventor obtains the best seedling culture effect of the culture medium for tissue culture of the broussonetia papyrifera after proportioning the MS culture medium and the tender leaf extracting solution of the broussonetia papyrifera according to the volume ratio of 15:1 through a great amount of practices.
Preferably, the tender leaves of the tender leaf extract are tender leaves within 5 leaves from the top of the branches of the paper mulberry, because the tender leaves contain the most abundant nutrients favorable to plant cells, for example, the content of biotin is 5 to 10 times that of the leaves of the annual old trees.
In addition, the invention also provides a preparation method of the culture medium for paper mulberry tissue culture, which comprises the following steps:
and uniformly stirring and mixing the MS culture medium and the tender leaf extracting solution of the paper mulberry according to a proportion to obtain the culture medium for tissue culture of the paper mulberry.
Preferably, the MS culture medium is sterilized at the constant temperature of 120 ℃ for 15-25 minutes and then is stirred and mixed with the tender leaf extracting solution uniformly according to the proportion.
Preferably, the preparation method of the tender leaf extract comprises the following steps:
(1) rinsing tender leaves of paper mulberry, soaking in hydrogen peroxide, rinsing, draining, cutting and crushing into pulp;
(2) and then deslagging, sterilizing and disinfecting to obtain the tender leaf extracting solution.
Preferably, the soaking time in the step (1) is 30 to 60 minutes.
Preferably, the concentration of the hydrogen peroxide in the step (1) is 0.5%.
Preferably, the step (2) is performed with filtration by a filter for sterilization, and the pore diameter of the filter element of the filter is less than 100 nm.
Preferably, pasteurization is adopted in the step (2), wherein the pasteurization temperature is 80-85 ℃, and the pasteurization time is 20-30 minutes.
Compared with the prior art, the invention has the beneficial effects that:
1. can accelerate the tissue culture seedling of the primary broussonetia papyrifera to take root quickly, the time for the primary root is 4-5 days, and the average is 3-5 days ahead.
2. The bud seedlings are fast to crack, the time for the second bud seedling to crack and expose on the surface of the culture medium is 10-12 days, and the average time is 5-7 days earlier.
3. The growth speed of the subculture seedlings is high, the seedlings only need 20-25 days when growing to 3-4cm and 3 small leaves, the time for the seedlings to be 6-8 seedlings from single buds to split is 30-35 days, and the subculture time is shortened by 5-10 days compared with the method of simply using a standard plant tissue culture MS culture medium.
4. In the seedling training stage, the seedlings are strong, the leaves are dark green, the root systems are developed, the adaptability to the change of illumination, temperature and humidity in the nature is strong, and the survival rate of the tissue culture seedlings of the paper mulberry reaches more than 90 percent.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
Picking 1000 g of tender leaves of a paper mulberry which grows vigorously and is within 5 leaves from the top end of a branch, rinsing the tender leaves of the paper mulberry with clear water for three times, then rinsing the tender leaves of the paper mulberry twice in an ultraclean workbench in a sterile room by using sterile normal saline, soaking the tender leaves of the paper mulberry in 0.5% of hydrogen peroxide for 30 minutes, rinsing the tender leaves of the paper mulberry twice in sterile pure water, taking out the tender leaves of the paper mulberry, draining the water, cutting the tender leaves into pieces by using a sterile blade, then placing the cut tender leaves of the paper mulberry in a sterilized high-speed homogenizer to crush the tender leaves of the paper mulberry into pulp, filtering the pulp by using sterile filter cloth to remove slag, and filtering the filtrate by using a filter, wherein the aperture of a filter element of the filter is less than 100nm, so as to achieve the purpose of completely removing the slag and sterilizing, performing pasteurization on the filtrate filtered by the.
And sterilizing 1000mL of MS culture medium at constant temperature of 120 ℃ for 15 minutes, adding 100mL of broussonetia papyrifera tender leaf extracting solution, and uniformly stirring and mixing to obtain the culture medium for tissue culture of the broussonetia papyrifera.
Example 2
Picking 1000 g of tender leaves of a paper mulberry which grows vigorously and is within 5 leaves from the top end of a branch, rinsing the tender leaves of the paper mulberry with clear water for three times, then rinsing the tender leaves of the paper mulberry twice in an ultraclean workbench in a sterile room by using sterile normal saline, soaking the tender leaves of the paper mulberry in 0.5% of hydrogen peroxide for 60 minutes, rinsing the tender leaves of the paper mulberry twice in sterile pure water, taking out the tender leaves of the paper mulberry, draining the water, cutting the tender leaves into pieces by using a sterile blade, then placing the cut tender leaves of the paper mulberry in a sterilized high-speed homogenizer to crush the tender leaves of the paper mulberry into pulp, filtering the pulp by using sterile filter cloth to remove slag, and filtering the filtrate by using a filter, wherein the aperture of a filter element of the filter is less than 100nm, so as to achieve the purpose of completely removing the slag and sterilizing, performing pasteurization on the filtrate filtered by the.
And (3) sterilizing 1000mL of MS culture medium at constant temperature of 120 ℃ for 25 minutes, adding 50mL of broussonetia papyrifera tender leaf extracting solution, and uniformly stirring and mixing to obtain the culture medium for tissue culture of the broussonetia papyrifera.
Example 3
Picking 1000 g of tender leaves of a paper mulberry which grows vigorously and is within 5 leaves from the top end of a branch, rinsing the tender leaves of the paper mulberry with clear water for three times, then rinsing the tender leaves of the paper mulberry twice in an ultraclean workbench in a sterile room by using sterile normal saline, soaking the tender leaves of the paper mulberry in 0.5% of hydrogen peroxide for 45 minutes, rinsing the tender leaves of the paper mulberry twice in sterile pure water, taking out the tender leaves of the paper mulberry, draining the water, cutting the tender leaves into pieces by using a sterile blade, then placing the cut tender leaves of the paper mulberry in a sterilized high-speed homogenizer to crush the tender leaves of the paper mulberry into pulp, filtering the pulp by using sterile filter cloth to remove slag, and filtering the filtrate by using a filter, wherein the aperture of a filter element of the filter is less than 100nm, so as to achieve the purpose of completely removing the slag and sterilizing, performing pasteurization on the filtrate filtered by the.
And (3) sterilizing 1000mL of MS culture medium at constant temperature of 120 ℃ for 25 minutes, adding 75mL of broussonetia papyrifera tender leaf extracting solution, and uniformly stirring and mixing to obtain the culture medium for tissue culture of the broussonetia papyrifera.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and such modifications or alterations do not depart from the essence of the corresponding technical solution.
Claims (10)
1. A culture medium for tissue culture of paper mulberry is characterized in that the culture medium is prepared by mixing an MS culture medium and a tender leaf extracting solution of paper mulberry according to the volume ratio of (10-20) to 1.
2. The medium for tissue culture of broussonetia papyrifera according to claim 1, wherein the MS medium and the tender leaf extract of broussonetia papyrifera are mixed in a volume ratio of 15: 1.
3. The medium for tissue culture of paper mulberry according to claim 1, wherein the tender leaf of the tender leaf extracting solution is the tender leaf within 5 sheets from the top of the paper mulberry branch.
4. The method for preparing culture medium for paper mulberry tissue culture according to any one of claims 1 to 3, comprising the following steps:
and uniformly stirring and mixing the MS culture medium and the tender leaf extracting solution of the paper mulberry according to a proportion to obtain the culture medium for tissue culture of the paper mulberry.
5. The preparation method according to claim 4, wherein the MS culture medium is sterilized at 120 ℃ for 15-25 minutes and then is mixed with the tender leaf extract in proportion and stirred uniformly.
6. The method as set forth in claim 4, wherein the method for preparing the young leaf extract comprises the steps of:
(1) rinsing tender leaves of paper mulberry, soaking in hydrogen peroxide, rinsing, draining, cutting and crushing into pulp;
(2) and then deslagging, sterilizing and disinfecting to obtain the tender leaf extracting solution.
7. The method according to claim 6, wherein the soaking time in the step (1) is 30 to 60 minutes.
8. The preparation method according to claim 6, wherein the concentration of hydrogen peroxide in the step (1) is 0.5%.
9. The preparation method according to claim 6, wherein the step (2) is performed by filtering with a filter having a filter element pore size of less than 100nm for sterilization.
10. The method according to claim 6, wherein the step (2) comprises pasteurization, wherein the pasteurization temperature is 80-85 ℃ and the pasteurization time is 20-30 minutes.
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Citations (5)
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| CN107129365A (en) * | 2017-05-18 | 2017-09-05 | 中科天华生物科技有限公司 | A kind of culture medium for paper mulberry nursery |
| CN107347641A (en) * | 2017-07-25 | 2017-11-17 | 贵州海铭巍杂交构树产业开发有限公司 | A kind of paper mulberry tissue culture nutrient solution |
| CN108834904A (en) * | 2018-08-21 | 2018-11-20 | 云南兴构农业产业发展有限公司 | The culture medium and breeding method of paper mulberry are cultivated for root tissue |
| CN109566421A (en) * | 2019-02-20 | 2019-04-05 | 贵州神龙百草科技有限公司 | A kind of rapid propagation method for the tissue cultures hybridizing paper mulberry |
| CN109618935A (en) * | 2019-02-20 | 2019-04-16 | 贵州神龙百草科技有限公司 | A kind of hybridization paper mulberry efficient in vifro culture method |
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2021
- 2021-05-10 CN CN202110506572.7A patent/CN113100064A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107129365A (en) * | 2017-05-18 | 2017-09-05 | 中科天华生物科技有限公司 | A kind of culture medium for paper mulberry nursery |
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| CN108834904A (en) * | 2018-08-21 | 2018-11-20 | 云南兴构农业产业发展有限公司 | The culture medium and breeding method of paper mulberry are cultivated for root tissue |
| CN109566421A (en) * | 2019-02-20 | 2019-04-05 | 贵州神龙百草科技有限公司 | A kind of rapid propagation method for the tissue cultures hybridizing paper mulberry |
| CN109618935A (en) * | 2019-02-20 | 2019-04-16 | 贵州神龙百草科技有限公司 | A kind of hybridization paper mulberry efficient in vifro culture method |
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