CN116391623A - Induction medium, application thereof and induction method of mulberry explant callus - Google Patents
Induction medium, application thereof and induction method of mulberry explant callus Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
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- Engineering & Computer Science (AREA)
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Abstract
The invention belongs to the technical field of plant tissue culture, and particularly relates to an induction culture medium and application thereof, and an induction method of mulberry explant callus. The induction culture medium of the invention takes DKW as a basic culture medium and also comprises: 3.5mg/L of 6-benzylaminopurine, 1.5-3.0 mg/L, NAA 1.0.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar. The invention reasonably matches the composition of the culture medium, can meet the callus induction culture of a plurality of explants such as mulberry terminal buds, leaves, stem segments, petioles, stem segments with axillary buds and the like, and greatly improves the callus induction rate of various explants.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to an induction culture medium and application thereof, and an induction method of mulberry explant callus.
Background
The mulberry is a perennial deciduous tree or shrub with high economic value. Silkworm breeding is performed by mulberry in China, and silk production has been performed for thousands of years. The mulberry field is a dominant industry in China all the time and makes an excellent contribution to the national economy in China. In 2019, the area of the mulberry field in China is 1200 ten thousand mu, the annual output value of silk industry is 1500 hundred million yuan, and the silkworm cocoon and raw silk output accounts for more than 80% of the world. Besides the silkworm breeding of mulberry, the mulberry leaves and the mulberries have the characteristics of homology of medicine and food, not only have higher edible nutritive value, but also have the medicinal effects of bacteriostasis, blood sugar reduction, blood pressure reduction, tumor resistance, aging resistance and the like. According to the records of the Chinese pharmacopoeia of 2020 edition, sang Ye, sang Gu and sang Gen can be used as drugs. The mulberry leaves are rich in nutrition, have protein content higher than that of alfalfa of pasture, are rich in various functional active substances, can enhance the organism endurance and disease resistance of livestock and poultry, improve the meat quality and flavor, and can play a unique role in healthy cultivation and antibiotic substitution. As a native species in China, the mulberry has strong photosynthesis and large biological yield, has strong stress resistance such as barren resistance, salt resistance, drought resistance, waterlogging resistance and the like, is a whole body of mulberry, has wide application, and plays an increasingly important role in the fields of traditional mulberry, food, medicine, ecological restoration, livestock feed and the like.
With the industrial utilization of the mulberry multipurpose, the breeding of the mulberry suitable for different purposes meets the unprecedented challenges. As with traditional woody plant breeding, mulberry breeding is extremely slow and difficult due to high heterozygosity and long generation cycles. Along with the rapid development of biotechnology, molecular biology and bioinformatics, a large number of new technologies such as molecular breeding, transgenosis and gene editing technology are applied to plant breeding work, so that the plant breeding efficiency is greatly improved. At present, the combination of traditional breeding methods and novel breeding means for breeding excellent plant varieties has become a trend.
The agro-mulberry 14 is one of main mulberry varieties promoted in large areas in the main silkworm areas of China at present, and the excellent characters of the agro-mulberry are mainly represented by high leaf yield, better leaf quality, strong capability of resisting Sang Yibing, yellowing type atrophy, mulberry thrips, red spiders and the like, and high mulberry seedling breeding and emergence rate. However, the genetic background of the mulberry is complex, the callus induction rate of the mulberry applied to the mulberry 14 in the prior art is very low, and the requirements of the mulberry 14 regeneration and transgenic technology cannot be met, so that a large number of novel breeding technologies cannot be applied to subsequent breeding and character improvement of the mulberry 14.
Disclosure of Invention
The invention aims to make up the defects of the prior art, improve the induction rate of mulberry, especially the callus of the farmed mulberry No. 14, and improve the seedling rate of the tissue culture seedlings.
The invention provides an induction culture medium, which takes DKW as a basic culture medium and further comprises the following components: 3 to 3.5mg/L of 6-benzylaminopurine, 1.5 to 3.0mg/L, NAA mg/1.0 to 1.5mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 0.7 to 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar.
The invention also provides application of the induction culture medium in mulberry explant callus induction.
Preferably, the mulberry includes farmed mulberry No. 14 and its approximations.
The invention also provides an induction method of the mulberry explant callus, which comprises the following steps:
inducing the mulberry explant 10d under the dark condition by using the induction culture medium in the technical scheme to obtain an induction explant;
culturing the induced explant to obtain mulberry explant callus;
the temperature of the culture is 24-27 ℃, the humidity is 70-80%, the light culture time is 12h/d, and the illumination intensity is 1500-2000 lx; the time of the culture is 15-20 d.
Preferably, the mulberry explant is taken from a mulberry tissue culture seedling.
Preferably, the mulberry explant comprises one or more of leaves, petioles, stem segments, axillary bud-bearing stem segments and terminal buds of mulberry tissue culture seedlings.
Preferably, when the mulberry explant is a leaf, the induction medium is a DKW-based medium, further comprising: 3.5mg/L of 6-benzylaminopurine, 2.0mg/L, NAA 1.0.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar;
when the mulberry explant is petiole, the induction medium is a DKW-based medium, further comprising: 3.5mg/L of 6-benzylaminopurine, 1.5mg/L, NAA 1.0.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar;
when the mulberry explant is one or more of a stem segment, a stem segment with an axillary bud and a terminal bud, the induction medium is based on DKW, and further comprises: 3.5mg/L of 6-benzylaminopurine, 2.5mg/L, NAA 1.0.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar.
Preferably, the leaves are leaves with fully expanded 3-5 knots of mulberry tissue culture Miao Di; the petioles are petioles of leaves at 3-5 knots of mulberry tissue culture Miao Di.
Preferably, the preparation method of the mulberry tissue culture seedling comprises the following steps: inoculating the branch with the axillary bud of the mulberry propagation seedling to a tissue culture seedling propagation culture medium, and culturing for 20-25 d at 25 ℃ to obtain a mulberry tissue culture seedling;
the tissue culture seedling propagation medium takes DKW as a basic medium and further comprises: 1.5mg/L of 6-benzylaminopurine, 0.2mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar.
Preferably, the preparation method of the mulberry propagation seedling comprises the following steps: grafting mulberry branches with axillary buds to a tissue culture seedling propagation culture medium, and culturing for 20-30 d at 25 ℃ to obtain mulberry propagation seedlings;
the tissue culture seedling propagation medium takes DKW as a basic medium and further comprises: 1.5mg/L of 6-benzylaminopurine, 0.2mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar.
The invention provides an induction culture medium, which takes DKW as a basic culture medium and further comprises the following components: 3.5mg/L of 6-benzylaminopurine, 1.5-3.0 mg/L, NAA 1.0.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar. The invention reasonably matches the composition of the culture medium, can meet the callus induction culture of a plurality of explants such as mulberry terminal buds, leaves, stem segments, petioles, stem segments with axillary buds and the like, and greatly improves the callus induction rate of various explants.
Furthermore, the composition of the induction culture medium is further limited according to the specific type of the explant, so that the callus induction rate of different explants can be further improved; and by breeding the mulberry tissue culture seedlings, the stability, uniformity and easy acquisition of the explant mulberry leaves for callus induction can be ensured, the problem that the explant needs to be sterilized in the process of tissue culture of the mulberry is avoided, the pollution rate and the browning rate are reduced, and the successful induction rate is ensured.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a schematic diagram of a mulberry tissue culture seedling obtained in example 2;
FIG. 2-1 is a schematic illustration of an inoculated leaf explant of example 3-1;
FIG. 2-2 is a schematic diagram of example 3-2 for 15 days of callus induction;
FIG. 3-1 is a schematic representation of the inoculation of petiole explants of example 4-1;
FIG. 3-2 is a schematic representation of the induction of callus for 15 days in example 4-1;
FIG. 4-1 is a schematic representation of an inoculated stem segment explant of example 5-1;
FIG. 4-2 is a schematic diagram of example 5-3 for 15 days of callus induction;
FIG. 5-1 is a schematic illustration of the inoculation of a terminal bud explant of example 6-1;
FIG. 5-2 is a schematic representation of example 6-3 inducing callus for 15 days;
FIG. 6-1 is a schematic illustration of the inoculation of an axillary bud bearing stem segment explant of example 7-1;
FIG. 6-2 is a schematic representation of example 7-3 inducing callus for 15 days.
Detailed Description
The invention provides an induction culture medium, which takes DKW as a basic culture medium and further comprises the following components: 3 to 3.5mg/L of 6-benzylaminopurine, 1.5 to 3.0mg/L, NAA mg/1.0 to 1.5mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 0.7 to 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar.
In the present invention, the induction medium is DKW-based medium, and further comprises 3 to 3.5mg/L, preferably 3.5mg/L, of 6-benzylaminopurine (6-BA).
In the invention, the induction culture medium is based on DKW and further comprises 1.5-3.0 mg/L, preferably 1.5-2.5 mg/L or 3.0mg/L of 2, 4-dichlorophenoxyacetic acid (2, 4-D). In the specific implementation process, the value can be arbitrarily selected in the range of 1.5-3.0 mg/L, such as 1.5mg/L, 2.0mg/L, 2.5mg/L or 3.0mg/L.
In the invention, the induction medium is based on DKW and further comprises NAA 1.0-1.5 mg/L, preferably 1.0mg/L.
In the invention, the induction medium is based on DKW and further comprises 30.0g/L sucrose.
In the invention, the induction medium is based on DKW and further comprises 0.7-1.0 g/L polyvinylpyrrolidone (PVP), preferably 1.0g/L.
In the invention, the induction medium is based on DKW and further comprises 7.0g/L agar.
In the present invention, the pH of the induction medium is preferably 5.8.
The invention is reasonably matched, and can meet the requirements of callus induction culture of a plurality of explants such as mulberry terminal buds, leaves, stem segments, petioles, stem segments with axillary buds and the like by limiting the types and the dosage of components in an induction culture medium, thereby greatly improving the callus induction rate of various explants.
The invention also provides application of the induction culture medium in mulberry explant callus induction. In the present invention, the mulberry preferably includes farmmulberry No. 14 and its approximation, and further preferably farmmulberry No. 14.
The invention also provides an induction method of the mulberry explant callus, which comprises the following steps:
inducing the mulberry explant 10d under the dark condition by using the induction culture medium in the technical scheme to obtain an induction explant;
culturing the induced explant to obtain mulberry explant callus;
the temperature of the culture is 24-27 ℃, the humidity is 70-80%, the light culture time is 12h/d, and the illumination intensity is 1500-2000 lx; the time of the culture is 15-20 d.
In the present invention, the mulberry explant is preferably taken from a mulberry tissue culture seedling, and further preferably comprises one or more of a leaf, a petiole, a stem segment with an axillary bud and a terminal bud of the mulberry tissue culture seedling, and more preferably a leaf, a petiole, a stem segment with an axillary bud or a terminal bud of the mulberry tissue culture seedling. The invention takes mulberry terminal bud, leaf, stem segment, leaf stalk, stem segment with axillary bud and other materials of mulberry tissue culture seedling as explants for induction culture, which can greatly improve the induction rate of mulberry callus.
In the present invention, when the mulberry explant is a leaf, the induction medium is preferably a DKW-based medium, further comprising: 3.5mg/L of 6-benzylaminopurine, 2.0mg/L, NAA 1.0.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar. The leaves of the invention are preferably leaves with fully-expanded mulberry tissue culture Miao Di-5 knots.
In the present invention, when the mulberry explant is petiole, the induction medium is preferably a DKW-based medium, further comprising: 3.5mg/L of 6-benzylaminopurine, 1.5mg/L, NAA 1.0.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar. The petiole is preferably a petiole of mulberry tissue culture Miao Di-5-node leaves.
In the present invention, when the mulberry explant is one or more of a stem segment, a stem segment with an axillary bud and a terminal bud, the induction medium is preferably a DKW-based medium, further comprising: 3.5mg/L of 6-benzylaminopurine, 2.5mg/L, NAA 1.0.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar. The length of the stem segment is preferably 1cm; the stem segment is made of explant material only with the stem segment. In the specific implementation process, axillary buds of mulberry tissue culture seedlings are preferably used as centers, and cut into small sections with the length of 2-3 cm to obtain the stem explant with the axillary buds for culture. In the specific implementation process, the invention is preferably sheared at 0.5cm of the basal part of the terminal bud of the mulberry tissue culture seedling, so as to obtain the terminal bud explant for culture.
The invention further limits the composition of the induction culture medium according to specific explant types, and can further improve the callus induction rate of different explants. The example results show that the callus induction rate is 95.00% and the embryogenic callus induction rate is 93.33% by taking the leaves of the mulberry tissue culture seedling as explants; taking the petioles of the mulberry tissue culture seedlings as explants, wherein the callus induction rate is 100.00%, and the embryogenic callus induction rate is 91.67%; taking the stem segment of the mulberry tissue culture seedling as an explant, wherein the callus induction rate is 100.00%, and the embryogenic callus induction rate is 90.00%; taking the stem section with axillary buds of the mulberry tissue culture seedling as an explant, wherein the callus induction rate is 94.23%, and the embryogenic callus induction rate is 91.69%; taking terminal buds of the mulberry tissue culture seedlings as explants, wherein the callus induction rate is 100.00%, and the embryogenic callus induction rate is 87.92%.
In the invention, the preparation method of the mulberry tissue culture seedling preferably comprises the following steps: inoculating the branch with the axillary bud of the mulberry propagation seedling to a tissue culture seedling propagation culture medium, and culturing for 20-25 d at 25 ℃ to obtain a mulberry tissue culture seedling; the tissue culture seedling propagation medium is preferably a DKW-based medium, and further comprises: 1.5mg/L of 6-benzylaminopurine, 0.2mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar. The pH of the tissue culture seedling propagation medium is preferably 5.8. The length of the branch with axillary buds is preferably 2-3 cm. In the specific implementation process, preferably, the basal callus of the mulberry propagation seedling is cut off, leaves are left for 0.5cm, and the branch with the axillary buds is obtained by cutting with a single axillary bud as the center.
In the invention, the preparation method of the mulberry propagation seedling preferably comprises the following steps: grafting mulberry branches with axillary buds to a tissue culture seedling propagation culture medium, and culturing for 20-30 d at 25 ℃ to obtain mulberry propagation seedlings; the tissue culture seedling propagation medium takes DKW as a basic medium and further comprises: 1.5mg/L of 6-benzylaminopurine, 0.2mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar. In the invention, the pH of the tissue culture seedling propagation medium is preferably 5.8. The length of the mulberry twig with the axillary buds is preferably 2-3 cm. In the specific implementation process, the young mulberry branches with good growth state and no plant diseases and insect pests in the current year are preferably selected, the leaves are cut off, the leaves are left for 0.5cm, and the branches with the axillary buds are obtained by cutting with the single axillary buds as the center.
The invention preferably disinfects the tender mulberry twigs before the shearing. The disinfection treatment preferably comprises soaking tender mulberry twigs for 60 seconds by using an ethanol solution with the mass concentration of 75%, and soaking the tender mulberry twigs for 12 minutes by using mercury chloride with the mass concentration of 0.1%.
The invention can ensure the stability, uniformity and easy acquisition of the explant mulberry leaves for callus induction by breeding the mulberry tissue culture seedlings, avoid the problem that the explant needs to be sterilized in the process of tissue culture of the mulberry, reduce the pollution rate and the browning rate, and further ensure the successful induction rate.
After the induced explant is obtained, the induced explant is cultured to obtain the mulberry explant callus. In the present invention, the temperature of the culture is 24 to 27 ℃, preferably 25 ℃; the humidity of the culture is 70-80%, preferably 80%; the time of the light culture is 12h/d; the illumination intensity of the light culture is 1500-2000 lx, preferably 1600-1800 lx, and more preferably 1650lx; the time of the culture is 15 to 20 days, preferably 16 to 18 days.
For further explanation of the present invention, an induction medium and its use, a method for inducing callus of mulberry explants, provided by the present invention, will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
In the implementation process of the invention, if not specifically described, the technical means used in the examples are conventional means well known to those skilled in the art, and the used medicament is a conventional experimental medicament purchased from a commercial source.
Examples 1 to 4
The specific composition of the induction medium is shown in table 1.
TABLE 1 compositions of Induction Medium for examples 1 to 4
Example 2
The preparation method of the mulberry tissue culture seedling comprises the following steps:
(1) Collecting mulberry branches: the young mulberry branches of the agro-mulberry No. 14 which have good growth state in the current year and are free of plant diseases and insect pests are selected, leaves are cut off, and leaves stems are left for 0.5cm to be used as propagation materials of tissue culture seedlings;
(2) Sterilizing ramulus Mori: washing the mulberry twig in the step (1) with sterile water for 4 times, soaking the mulberry twig in an ethanol solution with the mass concentration of 75% for 60 seconds, washing the mulberry twig with sterile water for 4 times, soaking the mulberry twig in mercury chloride with the mass concentration of 0.1% for 12 minutes, and washing the mulberry twig with sterile water for 4 times;
(3) Culturing: cutting the mulberry branches sterilized in the step (2) to 2-3 cm long by taking a single axillary bud as a center, inoculating the mulberry branches to a tissue culture seedling propagation medium for culturing for 25 days at the culture temperature of 25 ℃ to obtain mulberry propagation seedlings; the tissue culture seedling propagation medium takes DKW as a basic medium, and is added with 1.5mg/L of 6-benzylaminopurine, 0.2mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar; the pH of the tissue culture seedling propagation medium is 5.8;
(4) Expanding propagation; cutting off the callus at the base part of the mulberry propagation seedling obtained by the culture in the step (3), cutting off leaves, reserving 0.5cm of petiole, cutting off branches to 2-3 cm long by taking a single axillary bud as the center, inoculating to a tissue culture seedling propagation culture medium for culture, and obtaining the mulberry tissue culture seedling (shown in figure 1); the tissue culture seedling propagation medium takes DKW as a basic medium and further comprises: 1.5mg/L of 6-benzylaminopurine, 0.2mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar; the pH of the tissue culture seedling propagation medium is 5.8.
Example 3-1
The induction method of the mulberry explant callus comprises the following steps:
(1) Taking mulberry tissue culture seedlings obtained by culturing for 20-25 d in the embodiment 2, and cutting young leaves which are completely unfolded at Miao Di-5 sections of the mulberry tissue culture seedlings on an ultra-clean workbench into squares of 1cm multiplied by 1cm to serve as leaf explants;
(2) Putting 30 leaf explants obtained in the step (1) into the induction culture medium obtained in the example 1 (shown in the figure 2-1), performing callus induction for 10 days under dark conditions, and culturing for 15 days under conditions of 25 ℃ and 80% humidity, 12h/d of illumination and 1500-2000 lx of illumination intensity;
(3) Repeating the step (2) for 2 times.
Example 3-2
The only difference from example 3-1 is that step (2): 30 leaf explants obtained in step (1) were placed transversely into the induction medium obtained in example 2.
Examples 3 to 3
The only difference from example 3-1 is that step (2): 30 leaf explants obtained in step (1) were placed transversely into the induction medium obtained in example 3.
Examples 3 to 4
The only difference from example 3-1 is that step (2): 30 leaf explants obtained in step (1) were placed transversely into the induction medium obtained in example 4.
Test example 1
The callus induction effects of examples 3-1 to 3-4 were measured with reference to the prior art (Wang Chao, hypersensitive, chen Qian, bao Ji, cao Mengqi, yang Caifeng, wu Fuan. Optimization study of conditions for callus induction and subculture of mulberry [ C ].2014 national mulberry disease and insect control academy of research, discussion, 2014:155-159.) and the results are shown in Table 2.
TABLE 2 Induction Effect of different Induction Medium on callus of Mulberry leaf explants
| Treatment mode | Callus induction rate | Embryogenic callus induction rate |
| Example 3-1 | 93.75％ | 65.63％ |
| Example 3-2 | 95.00％ | 93.33％ |
| Examples 3 to 3 | 87.50％ | 67.19％ |
| Examples 3 to 4 | 72.73％ | 47.73％ |
Note that: the values in the table are the average of 3 replicates.
As can be seen from Table 2, there was a significant difference between the induction effects of the different concentrations of 2, 4-dichlorophenoxyacetic acid induction medium when mulberry tissue culture seedling leaves were used as explants, and the callus induction rate and embryogenic callus induction rate were highest in the induction medium of example 2 (DKW-based medium, added with 3.5 mg/L6-benzylaminopurine, 2, 4-dichlorophenoxyacetic acid 2.0mg/L, NAA 1.0.0 mg/L, sucrose 30.0g/L, polyvinylpyrrolidone 1.0g/L and agar 7.0g/L, respectively, were 95.00% and 93.33% and significantly higher than the other three media).
Example 4-1
The induction method of the mulberry explant callus comprises the following steps:
(1) Taking the mulberry tissue culture seedlings obtained by culturing for 20-25 d in the embodiment 2, and cutting the petioles of leaves at the 3-5 sections of the mulberry tissue culture Miao Di on an ultra-clean workbench into 1cm sections to be used as petiole explants;
(2) Putting 30 petiole explants obtained in the step (1) into the induction culture medium obtained in the example 1 (shown in the figure 3-1), performing callus induction for 10 days under dark conditions, and culturing at 25 ℃ and 80% humidity under illumination for 12h/d and with illumination intensity of 1500-2000 lx for 15 days;
(3) Repeating the step (2) for 2 times.
Example 4-2
The only difference from example 4-1 is that step (2): 30 petiole explants obtained in step (1) were placed transversely into the induction medium obtained in example 2.
Examples 4 to 3
The only difference from example 4-1 is that step (2): 30 petiole explants obtained in step (1) were placed transversely into the induction medium obtained in example 3.
Examples 4 to 4
The only difference from example 4-1 is that step (2): 30 petiole explants obtained in step (1) were placed transversely into the induction medium obtained in example 4.
Test example 2
The callus induction effects of examples 4-1 to 4-4 were measured with reference to the prior art (Wang Chao, hypersensitive, chen Qian, bao Ji, cao Mengqi, yang Caifeng, wu Fuan. Optimization study of conditions for callus induction and subculture of mulberry [ C ].2014 national mulberry disease and insect control academy of research, proceedings, 2014:155-159.) and the results are shown in Table 3.
TABLE 3 Induction of callus effects of different Induction media on Mulberry petiole explants
| Treatment mode | Callus induction rate | Embryogenic callus induction rate |
| Example 4-1 | 100.00％ | 91.67％ |
| Example 4-2 | 100.00％ | 77.50％ |
| Examples 4 to 3 | 100.00％ | 73.64％ |
| Examples 4 to 4 | 96.67％ | 36.67％ |
Note that: the values in the table are the average of 3 replicates.
As can be seen from Table 3, when the leaves stalks of the mulberry tissue culture seedlings are used as explants, there is a significant difference between the induction effects of the induction media of different concentrations of 2, 4-dichlorophenoxyacetic acid, and the induction rate of the callus in the induction media of example 1 (based on DKW, added with 3.5mg/L of 6-benzylaminopurine, 1.5mg/L, NAA 1.0.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar) can reach 100%, and the induction rate of embryogenic callus is highest, 91.67%, and is significantly higher than that of the other three media.
Example 5-1
The induction method of the mulberry explant callus comprises the following steps:
(1) Taking the mulberry tissue culture seedling after 20-25 d culture in the example 2, cutting the stem of the mulberry tissue culture seedling into 1cm small segments on an ultra-clean workbench, and taking the stem as a stem segment explant;
(2) Putting 30 stem explants obtained in the step (1) into the induction culture medium obtained in the example 1 (shown in the figure 4-1), performing callus induction for 10 days under dark conditions, and culturing at 25 ℃ and 80% humidity under illumination for 12h/d and with illumination intensity of 1500-2000 lx for 15 days;
(3) Repeating the step (2) for 2 times.
Example 5-2
The only difference from example 5-1 is that step (2): 30 stem explants obtained in step (1) were placed transversely into the induction medium obtained in example 2.
Examples 5 to 3
The only difference from example 5-1 is that step (2): 30 stem explants obtained in step (1) were placed transversely into the induction medium obtained in example 3.
Examples 5 to 4
The only difference from example 5-1 is that step (2): 30 stem explants obtained in step (1) were placed transversely into the induction medium obtained in example 4.
Test example 3
The callus induction effects of examples 5-1 to 5-4 were measured with reference to the prior art (Wang Chao, hypersensitive, chen Qian, bao Ji, cao Mengqi, yang Caifeng, wu Fuan. Optimization study of conditions for callus induction and subculture of mulberry [ C ].2014 national mulberry disease and insect control academy of research, discussion, 2014:155-159.) and the results are shown in Table 4.
TABLE 4 Induction effects of different Induction Medium on Mulberry Stem explants callus
| Treatment mode | Callus induction rate | Embryogenic callus induction rate |
| Example 5-1 | 100.00％ | 68.13％ |
| Example 5-2 | 100.00％ | 66.67％ |
| Examples 5 to 3 | 100.00％ | 90.00％ |
| Examples 5 to 4 | 94.84％ | 56.13％ |
Note that: the values in the table are the average of 3 replicates.
As can be seen from Table 4, when the stem segments of the mulberry tissue culture seedlings are used as explants, there is a significant difference between the induction effects of the different concentrations of the 2, 4-dichlorophenoxyacetic acid induction medium, and the callus induction rate in the induction medium of example 3 (DKW-based medium, added with 3.5mg/L of 6-benzylaminopurine, 2.5mg/L of 2, 4-dichlorophenoxyacetic acid, NAA1.0mg/L, sucrose 30.0g/L, polyvinylpyrrolidone 1.0g/L and agar 7.0 g/L) can reach 100%, and the embryogenic callus induction rate is the highest, is 90.00%, and is significantly higher than that of the other three culture media.
Example 6-1
The induction method of the mulberry explant callus comprises the following steps:
(1) Taking the mulberry tissue culture seedling after 20-25 d culture in the example 2, and cutting out the top buds of the mulberry tissue culture seedling along the stem section with the base part of 0.5cm on an ultra-clean workbench to serve as a top bud explant;
(2) Taking 30 terminal bud explants obtained in the step (1), vertically placing the terminal bud explants into the induction culture medium obtained in the example 1 (shown in the figure 5-1), performing callus induction for 10 days under dark conditions, and culturing for 15 days under conditions of the temperature of 25 ℃, the humidity of 80%, the illumination of 12h/d and the illumination intensity of 1500-2000 lx;
(3) Repeating the step (2) for 2 times.
Example 6-2
The only difference from example 6-1 is that step (2): 30 terminal bud explants obtained in the step (1) are vertically placed in the induction culture medium obtained in the example 2.
Examples 6 to 3
The only difference from example 6-1 is that step (2): 30 terminal bud explants obtained in the step (1) are vertically placed in the induction culture medium obtained in the example 3.
Examples 6 to 4
The only difference from example 6-1 is that step (2): 30 terminal bud explants obtained in the step (1) are vertically placed in the induction culture medium obtained in the example 4.
Test example 4
The callus induction effects of examples 6-1 to 6-4 were measured with reference to the prior art (Wang Chao, hypersensitive, chen Qian, bao Ji, cao Mengqi, yang Caifeng, wu Fuan. Optimization study of conditions for callus induction and subculture of mulberry [ C ].2014 national mulberry disease and insect control academy of research, discussion, 2014:155-159.) and the results are shown in Table 5.
TABLE 5 Induction of callus effects of different Induction media on mulberry terminal bud explants
| Treatment mode | Callus induction rate | Embryogenic callus induction rate |
| Example 6-1 | 96.43％ | 61.43％ |
| Example 6-2 | 100.00％ | 76.67％ |
| Examples 6 to 3 | 100.00％ | 87.92％ |
| Examples 6 to 4 | 100.00％ | 65.45％ |
Note that: the values in the table are the average of 3 replicates.
As can be seen from Table 5, when the terminal buds of the mulberry tissue culture seedlings are used as explants, there is a significant difference between the induction effects of the induction media of different concentrations of 2, 4-dichlorophenoxyacetic acid, and the induction rate of the callus in the induction media of example 3 (based on DKW, added with 3.5mg/L of 6-benzylaminopurine, 2.5mg/L, NAA 1.0.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar) can reach 100%, and the induction rate of embryogenic callus is the highest, 87.92%, and is significantly higher than that of the other three media.
Example 7-1
The induction method of the mulberry explant callus comprises the following steps:
(1) Taking the mulberry tissue culture seedling after 20-25 d culture in the example 2, and cutting the stem with the axillary buds of the mulberry tissue culture seedling on an ultra-clean workbench to small segments with the length of 2-3 cm by taking each axillary bud as the center, and taking the stem with the axillary buds as an explant;
(2) Taking 30 axillary bud-bearing stem explants obtained in the step (1), vertically placing the stem explants into the induction culture medium obtained in the example 1 (shown in a figure 6-1), performing callus induction under dark conditions for 10 days, and culturing under conditions of the temperature of 25 ℃, the humidity of 80%, the illumination of 12h/d and the illumination intensity of 1500-2000 lx for 15 days;
(3) Repeating the step (2) for 2 times.
Example 7-2
The only difference from example 7-1 is that step (2): 30 axillary bud-bearing stem explants obtained in step (1) are taken and vertically placed into the induction culture medium obtained in example 2.
Examples 7 to 3
The only difference from example 7-1 is that step (2): 30 axillary bud-bearing stem explants obtained in step (1) are vertically placed in the induction culture medium obtained in example 3.
Examples 7 to 4
The only difference from example 7-1 is that step (2): 30 axillary bud-bearing stem explants obtained in step (1) are taken and vertically placed into the induction culture medium obtained in example 4.
Test example 5
The callus induction effects of examples 7-1 to 7-4 were measured with reference to the prior art (Wang Chao, hypersensitive, chen Qian, bao Ji, cao Mengqi, yang Caifeng, wu Fuan. Optimization study of conditions for callus induction and subculture of mulberry [ C ].2014 national mulberry disease and insect control academy of research, discussion, 2014:155-159.) and the results are shown in Table 6.
TABLE 6 Induction effects of different Induction Medium on callus of mulberry stem with axillary buds
| Treatment mode | Callus induction rate | Embryogenic callus induction rate |
| Example 7-1 | 91.67％ | 51.25％ |
| Example 7-2 | 98.21％ | 72.14％ |
| Examples 7 to 3 | 94.23％ | 91.69％ |
| Examples 7 to 4 | 93.75％ | 66.88％ |
Note that: the values in the table are the average of 3 replicates.
As can be seen from Table 6, when the mulberry tissue culture seedlings with axillary buds and stems are used as explants, there is a significant difference between the induction effects of the 2, 4-dichlorophenoxyacetic acid induction medium with different concentrations, and the callus induction rate is up to 98.21% in the induction medium of example 2 (DKW-based medium, 6-benzylaminopurine 3.5mg/L, 2, 4-dichlorophenoxyacetic acid 2.0mg/L, NAA 1.0.0 mg/L, sucrose 30.0g/L, polyvinylpyrrolidone 1.0g/L and agar 7.0g/L, added), and the callus induction rate is up to 94.23 in the induction medium of example 3 (DKW-based medium, 6-benzylaminopurine 3.5mg/L, 2, 4-dichlorophenoxyacetic acid 2.5mg/L, NAA 1.0.0 mg/L, sucrose 30.0g/L, polyvinylpyrrolidone 1.0g/L and agar 7.0g/L added); the induction rate of embryogenic callus was 91.69% and significantly higher than the other three media in example 3, and the induction effect of example 3 was best in combination.
Example 8
An induction method of mulberry leaf explant callus comprises the following steps:
(1) Collecting mulberry branches: the young mulberry branches of the agro-mulberry No. 14 which have good growth state in the current year and are free of plant diseases and insect pests are selected, leaves are cut off, and leaves stems are left for 0.5cm to be used as propagation materials of tissue culture seedlings;
(2) Sterilizing ramulus Mori: washing the mulberry twig in the step (1) with sterile water for 4 times, soaking the mulberry twig in an ethanol solution with the mass concentration of 75% for 60 seconds, washing the mulberry twig with sterile water for 4 times, soaking the mulberry twig with the mass concentration of 0.1% for 12 minutes, and washing the mulberry twig with sterile water for 4 times;
(3) Culturing: cutting the mulberry branches sterilized in the step (2) to 2-3 cm long by taking each axillary bud as a center, inoculating the mulberry branches to a tissue culture seedling propagation medium for culturing for 25 days at the culture temperature of 25 ℃ to obtain mulberry propagation seedlings; the tissue culture seedling propagation medium takes DKW as a basic medium, and is added with 1.5mg/L of 6-benzylaminopurine, 0.2mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar; the pH of the tissue culture seedling propagation medium is 5.8;
(4) Expanding propagation: cutting off basal callus, cutting off leaves, leaving leaf stalks 0.5cm, cutting off branches to 2-3 cm long by taking each axillary bud as a center, inoculating to a tissue culture seedling propagation culture medium for culture at the culture temperature of 25 ℃, wherein the tissue culture seedling propagation culture medium takes DKW as a basic culture medium, and adding 1.5 mg/L6-benzyl aminopurine, 0.2 mg/L2, 4-dichlorophenoxyacetic acid, 30.0g/L sucrose, 1.0g/L polyvinylpyrrolidone and 7.0g/L agar; the pH of the tissue culture seedling propagation medium is 5.8;
(5) Explant sampling: taking the mulberry tissue culture Miao Yuchao net workbench cultured for 25 days in the step (4), and cutting young leaves which are completely unfolded at Miao Di-5 knots of the mulberry tissue culture into the size of 1cm multiplied by 1cm to serve as leaf explants;
(6) Explant callus induction: transversely placing 30 leaf explants selected in the step (5) into an induction medium (specifically, DKW is taken as a basic medium, 3.5mg/L of 6-benzylaminopurine, 2.0mg/L, NAA 1.0.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar) in the embodiment 2, culturing the leaf explants in a dark condition for 15-20 days, wherein the culture temperature is 25 ℃;
(7) Repeating the steps (5) - (6) for 2 times.
Wherein the humidity during the culture in the steps (3), (4) and (7) is 80 percent, the illumination is 12h/d, and the illumination intensity is 1500-2000 lx.
Comparative example 1
The only difference is that the step of culturing mulberry group is not performed, and the plant leaves of the nong-mulberry No. 14 mother strain are directly selected for callus induction.
Example 9
The difference from example 8 is that:
when the explant in the step (5) is sampled, taking the mulberry tissue culture Miao Yuchao net bench cultured for 25 days in the step (4), and cutting the petiole of the mulberry tissue culture Miao Di-5 knots of leaves into 1cm sections to be used as the petiole explant;
in the step (6), the callus induction of the explant is performed by using the induction medium of example 1, and the petiole explant is transversely placed into the induction medium of example 1 for culture.
Comparative example 2
The only difference is that the step of culturing mulberry group is not performed, and the plant petiole of the parent plant of nong-mulberry No. 14 is directly selected for callus induction.
Example 10
The difference from example 8 is that:
when the explant in the step (5) is sampled, taking the mulberry tissue culture Miao Yuchao net bench cultured for 25 days in the step (4), cutting the stem of the mulberry tissue culture seedling into 1cm small segments to be used as a stem segment explant;
in the step (6), the callus induction of the explant is performed by using the induction medium of the example 3, and the stem explant is transversely placed into the induction medium of the example 3 for culture.
Comparative example 3
The only difference from example 10 is that the step of culturing mulberry group is not performed, and the stem of the parent plant of nong-mulberry No. 14 is directly selected for callus induction.
Example 11
The difference from example 8 is that:
when the explant is sampled, taking the mulberry tissue culture Miao Yuchao net bench cultured for 25 days in the step (4), and cutting the stem with the axillary buds of the mulberry tissue culture seedling to a small section with the length of 2-3 cm by taking each axillary bud as the center to serve as the explant of the stem section with the axillary buds;
in the step (6), the callus induction of the explant is performed by using the induction medium of the example 3, and the stem explant is transversely placed into the induction medium of the example 3 for culture.
Comparative example 4
The only difference from example 11 is that the step of culturing mulberry group is not performed, and the axillary buds of the parent plant of nong mulberry No. 14 are directly selected for callus induction.
Example 12
The difference from example 8 is that:
when the explant in the step (5) is sampled, taking the mulberry tissue culture Miao Yuchao net bench cultured for 25 days in the step (4), cutting out the top buds of the mulberry tissue culture seedlings along the stem sections with the base of 0.5cm, and taking the top buds as the top bud explant;
in the step (6), the callus induction of the explant is performed by using the induction medium of example 3, and the apical bud explant is vertically placed in the induction medium of example 3 for culture.
Comparative example 5
The only difference from example 10 is that the step of culturing mulberry group is not performed, and the top bud of the nong mulberry No. 14 mother strain is directly selected for callus induction.
Test example 6
The induction effect of each of the explants of examples 8 to 12 and comparative examples 1 to 5 was measured with reference to the prior art (Wang Chao, hypersensitive, chen Qian, bao Ji, cao Mengqi, yang Caifeng, wu Fuan. Optimization study of conditions for callus induction and subculture of mulberry [ C ].2014 national mulberry disease and insect control academy of study, discussion, 2014: 155-159.) and the results are shown in Table 7.
TABLE 7 callus induction effects of different mulberry explants
As can be seen from Table 7, each tissue of the plant stock plant is directly collected as an explant for callus induction, and compared with the collection of each tissue of the tissue culture seedling as the explant, the pollution rate and the death rate are obviously increased, and the induction rate of the callus is influenced.
According to the above, the technical scheme provided by the invention can meet the requirements of callus induction culture of a plurality of explants such as mulberry, especially mulberry No. 14 terminal bud, leaf blade, stem segment, leaf stalk, stem segment with axillary bud and the like, and greatly improves the callus induction rate of various explants.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. An induction medium, wherein the induction medium is based on DKW, further comprising: 3 to 3.5mg/L of 6-benzylaminopurine, 1.5 to 3.0mg/L, NAA mg/1.0 to 1.5mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 0.7 to 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar.
2. Use of the induction medium of claim 1 for the induction of callus of mulberry explants.
3. The use according to claim 2, wherein the mulberry comprises agronomic mulberry number 14 and its approximations.
4. A method for inducing callus of mulberry explant, comprising the following steps:
inducing the mulberry explant 10d under dark conditions using the induction medium of claim 1 to obtain an induced explant;
culturing the induced explant to obtain mulberry explant callus;
the temperature of the culture is 24-27 ℃, the humidity is 70-80%, the light culture time is 12h/d, and the illumination intensity is 1500-2000 lx; the time of the culture is 15-20 d.
5. The method of induction according to claim 4, wherein said mulberry explant is taken from a mulberry tissue culture seedling.
6. The induction method according to claim 5, wherein the mulberry explant comprises one or more of leaves, petioles, stem segments, axillary bud-bearing stem segments and terminal buds of mulberry tissue culture seedlings.
7. The method of claim 6, wherein the method comprises the steps of,
when the mulberry explant is a leaf, the induction medium is a DKW-based medium, further comprising: 3.5mg/L of 6-benzylaminopurine, 2.0mg/L of 2, 4-dichlorophenoxyacetic acid, NAA1.0mg/L, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar;
when the mulberry explant is petiole, the induction medium is a DKW-based medium, further comprising: 3.5mg/L of 6-benzylaminopurine, 1.5mg/L of 2, 4-dichlorophenoxyacetic acid, NAA1.0mg/L, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar;
when the mulberry explant is one or more of a stem segment, a stem segment with an axillary bud and a terminal bud, the induction medium is based on DKW, and further comprises: 6-benzylaminopurine 3.5mg/L, 2, 4-dichlorophenoxyacetic acid 2.5mg/L, NAA1.0mg/L, sucrose 30.0g/L, polyvinylpyrrolidone 1.0g/L and agar 7.0g/L.
8. The induction method according to claim 7, wherein the leaves are fully expanded leaves of 3-5 knots of mulberry tissue culture Miao Di; the petioles are petioles of leaves at 3-5 knots of mulberry tissue culture Miao Di.
9. The induction method according to claim 5, wherein the preparation method of the mulberry tissue culture seedling comprises the following steps: inoculating the branch with the axillary bud of the mulberry propagation seedling to a tissue culture seedling propagation culture medium, and culturing for 20-25 d at 25 ℃ to obtain a mulberry tissue culture seedling;
the tissue culture seedling propagation medium takes DKW as a basic medium and further comprises: 1.5mg/L of 6-benzylaminopurine, 0.2mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar.
10. The induction method according to claim 9, wherein the method for preparing the mulberry propagation seedling comprises: grafting mulberry branches with axillary buds to a tissue culture seedling propagation culture medium, and culturing for 20-30 d at 25 ℃ to obtain mulberry propagation seedlings;
the tissue culture seedling propagation medium takes DKW as a basic medium and further comprises: 1.5mg/L of 6-benzylaminopurine, 0.2mg/L of 2, 4-dichlorophenoxyacetic acid, 30.0g/L of sucrose, 1.0g/L of polyvinylpyrrolidone and 7.0g/L of agar.
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