CN112273227A - Culture medium for improving survival rate of explants and preparation method and application thereof - Google Patents
Culture medium for improving survival rate of explants and preparation method and application thereof Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a culture medium for improving the survival rate of explants and a preparation method and application thereof, belonging to the technical field of plant culture. The culture medium comprises the following raw materials: b5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and hydrazine butyrate; the preparation method comprises the following steps: respectively weighing the above materials, heating, stirring, mixing, sterilizing at high temperature under high pressure, and cooling; the application is as follows: firstly, cutting an explant in a sterile box, and inoculating the explant into a culture medium for culture after the explant grows out a new bud. On the basis of the existing explant culture medium formula, substances such as phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine, butyrylhydrazine and the like are innovatively introduced, so that the disease resistance of an explant plant is improved, and a good improvement effect on the healthy growth of the explant is achieved; meanwhile, the preparation method and the application are simple to operate and strong in implementability, and the bud induction rate and the bud emergence time of the explant can be improved.
Description
Technical Field
The invention belongs to the technical field of plant culture, and particularly relates to a culture medium for improving the survival rate of explants, and a preparation method and application thereof.
Background
China rose (Rosa chinensis Jacq.) Generally, evergreen or semi-evergreen erect shrubs of the genus Rosa of the family Rosaceae are called Yueyuehong and Fangchun hong, and they can bloom many times each year. China rose is one of ten major flowers in China and is known as 'queen in flowers'. The Chinese rose has various types, various varieties, various flower types, rich colors, appropriate aroma, wide distribution and strong adaptability.
The conventional breeding approach of the Chinese rose is cuttage or grafting, and the development of the Chinese rose tissue culture technology can rapidly breed a large number of new varieties, accelerate the variety updating and promote the development of the Chinese rose industry. At present, a plurality of excellent Chinese rose varieties are potted in greenhouses, fungus pollution and plant diseases and insect pests are easy to occur in the greenhouses due to high temperature, high humidity, poor ventilation and the like, the Chinese rose cultivation process is particularly easy to cause germ infection and pest damage, once infected, the whole plant is infected, the phenomenon of carrying germs is difficult to change even if seedlings survive by using traditional propagation methods such as cuttage, grafting and the like, and therefore the quality of the survival seedlings is also influenced to a certain extent. One of the most important links of tissue culture is the disinfection and sterilization of explants, the surviving tissue culture seedlings are aseptic seedlings, and the tissue culture can provide the seedlings with proper temperature, humidity, illumination and hormones and carbohydrates required by plants, and carry out rapid mass propagation, thereby ensuring the quality of the seedlings and simultaneously having higher propagation speed.
Disclosure of Invention
1. Problems to be solved
Aiming at the problems in the prior art, the invention provides the culture medium for improving the survival rate of the explant as well as the preparation method and the application thereof.
2. Technical scheme
In order to solve the problems, the technical scheme provided by the invention is as follows:
a culture medium for improving the survival rate of explants comprises the following raw materials in parts by weight:
50-60 parts of B5 culture medium,
10 to 15 portions of cane sugar, and the like,
5 to 8 portions of agar, namely 5 to 8 portions of agar,
0.7 to 1.2 portions of phenylalanine,
0.05 to 0.08 portion of 1-naphthylacetic acid,
0.03 to 0.05 portion of 6-benzylaminopurine,
0.02 part to 0.04 part of butyryl hydrazine.
The culture medium for improving the survival rate of the explants comprises the following raw materials in parts by weight:
53-58 parts of B5 culture medium,
11 to 14 portions of cane sugar, 11 portions of cane sugar,
6 to 7 portions of agar, namely 6 portions of agar,
0.8 to 1.1 portions of phenylalanine,
0.06 to 0.07 portion of 1-naphthylacetic acid,
0.03 to 0.05 portion of 6-benzylaminopurine,
0.02 part to 0.04 part of butyryl hydrazine.
The culture medium for improving the survival rate of the explants comprises the following raw materials in parts by weight:
55 parts of a B5 culture medium,
13 parts of cane sugar, namely 13 parts of cane sugar,
7 parts of agar, namely 7 parts of agar,
0.9 part of phenylalanine, namely 0.9 part of phenylalanine,
0.07 part of 1-naphthylacetic acid,
0.04 part of 6-benzylaminopurine,
0.03 part of butyryl hydrazine.
A preparation method of a culture medium for improving the survival rate of explants comprises the following steps:
(1) preparing a B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine, and storing at a low temperature for later use;
(2) respectively weighing the B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine according to the proportion of the raw materials, adding water, heating, stirring and mixing, and then pouring into a culture dish for cooling;
(3) and placing the cooled culture dish into a sterilization pot for high-temperature and high-pressure sterilization, taking out after sterilization, and cooling at room temperature to obtain the culture dish.
In the preparation method of the culture medium for improving the survival rate of the explants,
in the step (1), the particle size ranges of the phenylalanine, the 1-naphthylacetic acid, the 6-benzylaminopurine and the butyryl hydrazine are 200 meshes to 400 meshes;
the temperature range of the low-temperature storage in the step (1) is 4-8 ℃.
In the preparation method of the culture medium for improving the survival rate of the explants,
the heating temperature in the step (2) is 95-100 ℃;
the rotating speed of stirring and mixing in the step (2) is 200rpm-400 rpm;
the temperature range of cooling in the step (2) is 10-25 ℃.
In the preparation method of the culture medium for improving the survival rate of the explants,
the temperature for high-temperature high-pressure sterilization in the step (3) is 121-126 ℃;
the pressure of high-temperature high-pressure sterilization in the step (3) is 0.15MPa-0.20 MPa;
the time of high-temperature high-pressure sterilization in the step (3) is 15min-20 min.
The application of the culture medium for improving the survival rate of the explants comprises the following steps:
firstly, cutting an explant in a sterile box, inoculating the explant into a culture medium for culture after the explant grows out a new bud, and controlling parameters of temperature, humidity and illumination conditions.
In the application of the culture medium for improving the survival rate of the explants,
the explant is a stem section with small young leaves, wherein the length range of the stem section is 2cm-3cm, and the number of the small young leaves is 1-2.
In the application of the culture medium for improving the survival rate of the explants,
the culture temperature is 26-28 ℃;
the culture humidity is 60% -80%;
the illumination condition is 8h-10h/day, 2000lux-3000 lux.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
compared with the prior art, the treatment methods of the embodiments 1 to 5 have good performance advantages, and the test parameters such as the bud induction rate, the average budding time and the survival rate show significant differences. On the basis of the existing explant culture medium formula, substances such as phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine, butyrylhydrazine and the like are innovatively introduced, and can generate a synergistic effect, so that the disease resistance of an explant plant is improved, and a good improvement effect on the healthy growth of the explant is achieved; meanwhile, the preparation method and the application of the invention have simple operation and strong feasibility, and can improve the bud induction rate and the bud emergence time of the explant.
Detailed Description
The invention is further described with reference to specific examples.
It should be reminded that the following related B5 culture medium was purchased from Haibo Biotechnology GmbH, a product code of Qingdao high-tech industrialisation park: HB 8487.
It should be reminded that the plant variety of the explant involved in the invention is China rose, which is purchased from the flower institute of agricultural academy of sciences in Yunnan province, and the variety is 'heart of red seed'.
Example 1
The culture medium for improving the survival rate of the explants comprises the following raw materials in parts by weight:
50 parts of a B5 culture medium,
15 parts of cane sugar, namely 15 parts of cane sugar,
5 parts of agar, namely adding 5 parts of agar,
1.2 parts of phenylalanine, namely amino acid,
0.05 part of 1-naphthylacetic acid,
0.05 part of 6-benzylaminopurine,
0.02 part of butyryl hydrazine.
The preparation method of the culture medium for improving the survival rate of the explants comprises the following steps:
(1) preparing a B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine, and storing at a low temperature for later use; in the step (1), the particle size ranges of the phenylalanine, the 1-naphthylacetic acid, the 6-benzylaminopurine and the butyryl hydrazine are 200 meshes; the temperature range of the low-temperature storage in the step (1) is 8 ℃;
(2) respectively weighing the B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine according to the proportion of the raw materials, adding water, heating, stirring and mixing, and then pouring into a culture dish for cooling; the heating temperature range in the step (2) is 95 ℃; the rotating speed of stirring and mixing in the step (2) is 400 rpm; the cooling temperature range in the step (2) is 10 ℃;
(3) placing the cooled culture dish in a sterilization pot for high-temperature and high-pressure sterilization, taking out after sterilization, and cooling at room temperature to obtain the culture dish; the temperature for high-temperature high-pressure sterilization in the step (3) is 121 ℃; the pressure of high-temperature high-pressure sterilization in the step (3) is 0.20 MPa; the time for high-temperature high-pressure sterilization in the step (3) is 15 min.
The application of the culture medium for improving the survival rate of the explants comprises the following steps:
firstly, cutting an explant in a sterile box, inoculating the explant into a culture medium for culture after the explant grows out a new bud, and controlling parameters of temperature, humidity and illumination conditions; the explant is a stem section with small young leaves, wherein the length range of the stem section is 2cm, and the number of the small young leaves is 2; the culture temperature is 26 ℃; the culture humidity is 80%; the illumination condition is 8h/day and 3000 lux.
Example 2
The culture medium for improving the survival rate of the explants comprises the following raw materials in parts by weight:
60 parts of a B5 culture medium,
10 parts of cane sugar, namely 10 parts of cane sugar,
8 parts of agar, namely 8 parts of agar,
0.7 part of phenylalanine, namely 0.7 part of phenylalanine,
0.08 portion of 1-naphthylacetic acid,
0.03 part of 6-benzylaminopurine,
0.04 part of butyryl hydrazine.
The preparation method of the culture medium for improving the survival rate of the explants comprises the following steps:
(1) preparing a B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine, and storing at a low temperature for later use; in the step (1), the particle size ranges of the phenylalanine, the 1-naphthylacetic acid, the 6-benzylaminopurine and the butyryl hydrazine are 400 meshes; the temperature range of the low-temperature storage in the step (1) is 4 ℃;
(2) respectively weighing the B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine according to the proportion of the raw materials, adding water, heating, stirring and mixing, and then pouring into a culture dish for cooling; the heating temperature range in the step (2) is 100 ℃; the rotation speed of stirring and mixing in the step (2) is 200 rpm; the cooling temperature range in the step (2) is 25 ℃;
(3) placing the cooled culture dish in a sterilization pot for high-temperature and high-pressure sterilization, taking out after sterilization, and cooling at room temperature to obtain the culture dish; the temperature for high-temperature high-pressure sterilization in the step (3) is 126 ℃; the pressure of high-temperature high-pressure sterilization in the step (3) is 0.15 MPa; and (4) performing high-temperature high-pressure sterilization in the step (3) for 20 min.
The application of the culture medium for improving the survival rate of the explants comprises the following steps:
firstly, cutting an explant in a sterile box, inoculating the explant into a culture medium for culture after the explant grows out a new bud, and controlling parameters of temperature, humidity and illumination conditions; the explant is a stem section with small young leaves, wherein the length range of the stem section is 3cm, and the number of the small young leaves is 1; the culture temperature is 26 ℃; the culture humidity is 80%; the illumination condition is 10h/day and 2000 lux.
Example 3
The culture medium for improving the survival rate of the explants comprises the following raw materials in parts by weight:
53 parts of B5 culture medium,
14 parts of cane sugar, namely 14 parts of cane sugar,
6 parts of agar, namely 6 parts of agar,
1.1 parts of phenylalanine, namely phenylalanine,
0.06 part of 1-naphthylacetic acid,
0.05 part of 6-benzylaminopurine,
0.02 part of butyryl hydrazine.
The preparation method of the culture medium for improving the survival rate of the explants comprises the following steps:
(1) preparing a B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine, and storing at a low temperature for later use; in the step (1), the particle size ranges of the phenylalanine, the 1-naphthylacetic acid, the 6-benzylaminopurine and the butyryl hydrazine are 200 meshes; the temperature range of the low-temperature storage in the step (1) is 4 ℃;
(2) respectively weighing the B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine according to the proportion of the raw materials, adding water, heating, stirring and mixing, and then pouring into a culture dish for cooling; the heating temperature range in the step (2) is 95 ℃; the rotation speed of stirring and mixing in the step (2) is 200 rpm; the cooling temperature range in the step (2) is 10 ℃;
(3) placing the cooled culture dish in a sterilization pot for high-temperature and high-pressure sterilization, taking out after sterilization, and cooling at room temperature to obtain the culture dish; the temperature for high-temperature high-pressure sterilization in the step (3) is 121 ℃; the pressure of high-temperature high-pressure sterilization in the step (3) is 0.15 MPa; the time for high-temperature high-pressure sterilization in the step (3) is 15 min.
The application of the culture medium for improving the survival rate of the explants comprises the following steps:
firstly, cutting an explant in a sterile box, inoculating the explant into a culture medium for culture after the explant grows out a new bud, and controlling parameters of temperature, humidity and illumination conditions; the explant is a stem section with small young leaves, wherein the length range of the stem section is 2cm, and the number of the small young leaves is 1; the culture temperature is 26 ℃; the culture humidity is 60%; the illumination condition is 8h/day and 2000 lux.
Example 4
The culture medium for improving the survival rate of the explants comprises the following raw materials in parts by weight:
58 parts of a B5 culture medium,
11 parts of cane sugar, namely 11 parts of cane sugar,
7 parts of agar, namely 7 parts of agar,
0.8 part of phenylalanine (I), 0.8 part of phenylalanine,
0.07 part of 1-naphthylacetic acid,
0.03 part of 6-benzylaminopurine,
0.04 part of butyryl hydrazine.
The preparation method of the culture medium for improving the survival rate of the explants comprises the following steps:
(1) preparing a B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine, and storing at a low temperature for later use; in the step (1), the particle size ranges of the phenylalanine, the 1-naphthylacetic acid, the 6-benzylaminopurine and the butyryl hydrazine are 400 meshes; the temperature range of the low-temperature storage in the step (1) is 8 ℃;
(2) respectively weighing the B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine according to the proportion of the raw materials, adding water, heating, stirring and mixing, and then pouring into a culture dish for cooling; the heating temperature range in the step (2) is 100 ℃; the rotating speed of stirring and mixing in the step (2) is 400 rpm; the cooling temperature range in the step (2) is 25 ℃;
(3) placing the cooled culture dish in a sterilization pot for high-temperature and high-pressure sterilization, taking out after sterilization, and cooling at room temperature to obtain the culture dish; the temperature for high-temperature high-pressure sterilization in the step (3) is 126 ℃; the pressure of high-temperature high-pressure sterilization in the step (3) is 0.20 MPa; and (4) performing high-temperature high-pressure sterilization in the step (3) for 20 min.
The application of the culture medium for improving the survival rate of the explants comprises the following steps:
firstly, cutting an explant in a sterile box, inoculating the explant into a culture medium for culture after the explant grows out a new bud, and controlling parameters of temperature, humidity and illumination conditions; the explant is a stem section with small young leaves, wherein the length range of the stem section is 3cm, and the number of the small young leaves is 2; the culture temperature is 28 ℃; the culture humidity is 80%; the illumination condition is 10h/day and 3000 lux.
Example 5
The culture medium for improving the survival rate of the explants comprises the following raw materials in parts by weight:
55 parts of a B5 culture medium,
13 parts of cane sugar, namely 13 parts of cane sugar,
7 parts of agar, namely 7 parts of agar,
0.9 part of phenylalanine, namely 0.9 part of phenylalanine,
0.07 part of 1-naphthylacetic acid,
0.04 part of 6-benzylaminopurine,
0.03 part of butyryl hydrazine.
The preparation method of the culture medium for improving the survival rate of the explants comprises the following steps:
(1) preparing a B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine, and storing at a low temperature for later use; in the step (1), the particle size ranges of the phenylalanine, the 1-naphthylacetic acid, the 6-benzylaminopurine and the butyryl hydrazine are 300 meshes; the temperature range of the low-temperature storage in the step (1) is 6 ℃;
(2) respectively weighing the B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine according to the proportion of the raw materials, adding water, heating, stirring and mixing, and then pouring into a culture dish for cooling; the heating temperature range in the step (2) is 98 ℃; the rotating speed of stirring and mixing in the step (2) is 300 rpm; the cooling temperature range in the step (2) is 18 ℃;
(3) placing the cooled culture dish in a sterilization pot for high-temperature and high-pressure sterilization, taking out after sterilization, and cooling at room temperature to obtain the culture dish; the temperature for high-temperature high-pressure sterilization in the step (3) is 124 ℃; the pressure of high-temperature high-pressure sterilization in the step (3) is 0.18 MPa; the time for high-temperature high-pressure sterilization in the step (3) is 18 min.
The application of the culture medium for improving the survival rate of the explants comprises the following steps:
firstly, cutting an explant in a sterile box, inoculating the explant into a culture medium for culture after the explant grows out a new bud, and controlling parameters of temperature, humidity and illumination conditions; the explant is a stem section with small young leaves, wherein the length range of the stem section is 2.5cm, and the number of the small young leaves is 2; the culture temperature is 27 ℃; the culture humidity is 70%; the illumination condition is 9h/day and 2500 lux.
Comparative example 1
The culture medium for improving the survival rate of the explants comprises the following raw materials in parts by weight:
55 parts of a B5 culture medium,
13 parts of cane sugar, namely 13 parts of cane sugar,
7 parts of agar, namely 7 parts of agar,
0.04 part of 6-benzylaminopurine,
0.03 part of butyryl hydrazine.
The preparation method of the culture medium for improving the survival rate of the explants comprises the following steps:
(1) preparing a B5 culture medium, sucrose, agar, 6-benzylaminopurine and butyryl hydrazine, and storing at a low temperature for later use; in the step (1), the particle size range of the 6-benzylaminopurine and the butyryl hydrazine is 300 meshes; the temperature range of the low-temperature storage in the step (1) is 6 ℃;
(2) respectively weighing the B5 culture medium, sucrose, agar, 6-benzylaminopurine and butyryl hydrazine according to the proportion of the raw materials, adding water, heating, stirring and mixing, and then pouring into a culture dish for cooling; the heating temperature range in the step (2) is 98 ℃; the rotating speed of stirring and mixing in the step (2) is 300 rpm; the cooling temperature range in the step (2) is 18 ℃;
(3) placing the cooled culture dish in a sterilization pot for high-temperature and high-pressure sterilization, taking out after sterilization, and cooling at room temperature to obtain the culture dish; the temperature for high-temperature high-pressure sterilization in the step (3) is 124 ℃; the pressure of high-temperature high-pressure sterilization in the step (3) is 0.18 MPa; the time for high-temperature high-pressure sterilization in the step (3) is 18 min.
The application of the culture medium for improving the survival rate of the explants comprises the following steps:
firstly, cutting an explant in a sterile box, inoculating the explant into a culture medium for culture after the explant grows out a new bud, and controlling parameters of temperature, humidity and illumination conditions; the explant is a stem section with small young leaves, wherein the length range of the stem section is 2.5cm, and the number of the small young leaves is 2; the culture temperature is 27 ℃; the culture humidity is 70%; the illumination condition is 9h/day and 2500 lux.
Comparative example 2
The culture medium for improving the survival rate of the explants comprises the following raw materials in parts by weight:
55 parts of a B5 culture medium,
13 parts of cane sugar, namely 13 parts of cane sugar,
7 parts of agar, namely 7 parts of agar,
0.9 part of phenylalanine, namely 0.9 part of phenylalanine,
0.07 part of 1-naphthylacetic acid,
0.03 part of butyryl hydrazine.
The preparation method of the culture medium for improving the survival rate of the explants comprises the following steps:
(1) preparing a B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid and butyrylhydrazine, and storing at low temperature for later use; in the step (1), the particle size ranges of the phenylalanine, the 1-naphthylacetic acid and the butyrhydrazide are 300 meshes; the temperature range of the low-temperature storage in the step (1) is 6 ℃;
(2) respectively weighing the B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid and butyrylhydrazine according to the proportion of the raw materials, adding water, heating, stirring and mixing, and then pouring into a culture dish for cooling; the heating temperature range in the step (2) is 98 ℃; the rotating speed of stirring and mixing in the step (2) is 300 rpm; the cooling temperature range in the step (2) is 18 ℃;
(3) placing the cooled culture dish in a sterilization pot for high-temperature and high-pressure sterilization, taking out after sterilization, and cooling at room temperature to obtain the culture dish; the temperature for high-temperature high-pressure sterilization in the step (3) is 124 ℃; the pressure of high-temperature high-pressure sterilization in the step (3) is 0.18 MPa; the time for high-temperature high-pressure sterilization in the step (3) is 18 min.
The application of the culture medium for improving the survival rate of the explants comprises the following steps:
firstly, cutting an explant in a sterile box, inoculating the explant into a culture medium for culture after the explant grows out a new bud, and controlling parameters of temperature, humidity and illumination conditions; the explant is a stem section with small young leaves, wherein the length range of the stem section is 2.5cm, and the number of the small young leaves is 2; the culture temperature is 27 ℃; the culture humidity is 70%; the illumination condition is 9h/day and 2500 lux.
Comparative example 3
The culture medium for improving the survival rate of the explants comprises the following raw materials in parts by weight:
55 parts of a B5 culture medium,
13 parts of cane sugar, namely 13 parts of cane sugar,
7 parts of agar, namely 7 parts of agar,
0.9 part of phenylalanine, namely 0.9 part of phenylalanine,
0.07 part of 1-naphthylacetic acid,
0.04 part of 6-benzylaminopurine.
The preparation method of the culture medium for improving the survival rate of the explants comprises the following steps:
(1) preparing a B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid and 6-benzylaminopurine, and storing at a low temperature for later use; in the step (1), the particle size ranges of the phenylalanine, the 1-naphthylacetic acid and the 6-benzylaminopurine are 300 meshes; the temperature range of the low-temperature storage in the step (1) is 6 ℃;
(2) respectively weighing the B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid and 6-benzylaminopurine according to the proportion of the raw materials, adding water, heating, stirring and mixing, and then pouring into a culture dish for cooling; the heating temperature range in the step (2) is 98 ℃; the rotating speed of stirring and mixing in the step (2) is 300 rpm; the cooling temperature range in the step (2) is 18 ℃;
(3) placing the cooled culture dish in a sterilization pot for high-temperature and high-pressure sterilization, taking out after sterilization, and cooling at room temperature to obtain the culture dish; the temperature for high-temperature high-pressure sterilization in the step (3) is 124 ℃; the pressure of high-temperature high-pressure sterilization in the step (3) is 0.18 MPa; the time for high-temperature high-pressure sterilization in the step (3) is 18 min.
The application of the culture medium for improving the survival rate of the explants comprises the following steps:
firstly, cutting an explant in a sterile box, inoculating the explant into a culture medium for culture after the explant grows out a new bud, and controlling parameters of temperature, humidity and illumination conditions; the explant is a stem section with small young leaves, wherein the length range of the stem section is 2.5cm, and the number of the small young leaves is 2; the culture temperature is 27 ℃; the culture humidity is 70%; the illumination condition is 9h/day and 2500 lux.
Comparative example 4
Chinese invention patent, application number: cn201710176228.x, publication No.: CN106818489B, which discloses a culture method of primordial tissue of stem explant of lilac daphne genkwa, as described in the specific implementation way, the specific steps are as follows:
"primary tissue culture medium: MS +0.1 mg.L-1 NAA +1.5 mg.L-1 ZT, sucrose 25g/L and agar 6.5g/L were added, and the pH was adjusted to 5.7.
The culture method comprises the following steps:
(1) selecting a lilac daphne flower bud explant: collecting current-year-growing branches of lilac daphne with strong growth, full axillary buds and no diseases and insect pests, removing all leaves, and cutting into 8cm stems with axillary buds as explants;
(2) treatment of lilac daphne flower bud explants: soaking the explant in a washing powder aqueous solution with the mass volume ratio of 0.2% for 20min, then washing the explant for 12h by running water, shearing the explant into 5cm stem sections under the aseptic environment of a clean bench of an inoculation chamber, disinfecting the stem sections by using an ethanol aqueous solution with the volume ratio of 70% for 45s, and soaking the stem sections in a mixed solution of 1 g.L < -1 > mercuric chloride and 3 drops of Tween 80 for 10min for disinfection treatment; sterilizing, washing with sterile water, cutting stem into 2.5cm length with upper cut 0.5cm from bud and lower cut 2cm from node, and inoculating into primary tissue culture medium;
(3) culturing a lilac daphne flower bud explant: and (3) inoculating the explant in the step (2) into a culture rack of an explant culture room in a primary tissue culture medium, performing illumination culture at the temperature of 25 ℃, the illumination intensity of 3000lx and the relative humidity of 75%, and culturing for 30d until cluster buds germinate in a photoperiod of 14h day/10 h night.
Comparative example 5
Chinese invention patent, application number: CN201810747357.4, publication No.: CN108668904A, which discloses a method for processing pinellia ternata explants, as described in the specific embodiment, the method specifically comprises the following steps:
the embodiment provides a method for processing a pinellia ternata explant, which comprises the following steps:
1) selection of explants: collecting tuber of pinellia tuber in dormancy stage as explant, cutting to 0.5cm × 0.5 cm;
2) pretreatment of explants: soaking in 10ppm washing powder water for 15min, and flushing with running water for 90 min;
3) and (3) disinfection of explants: soaking in 70% ethanol solution for 30s, soaking in 0.1% sodium hypochlorite solution for 720s, and rinsing with sterile water for 3min for 5 times.
4) Secondary disinfection: after the surface tissue is cut off, the surface tissue is soaked in a 75% ethanol solution for 30s, a 0.1% sodium hypochlorite solution for 700s, and then rinsed 7 times with sterile water for 3min each time, and then the contact part between the wound and the disinfectant is cut off.
Example 6
The treatments of examples 1 to 5 and comparative examples 1 to 5 were selected, respectively, and the survival rate, average germination induction rate, and average germination time were observed. Specifically, reference is made to "materials and methods" of the first prior art (Zhouyan, Huangchengling, Sambucus chinensis, etc.. cut flower China rose Kara tissue culture and rapid propagation research-screening of basic explants and primary culture medium [ J ]. seeds, 2011(11): 92-94.) and example 1 of the specification of the second prior art (Chinese invention patent, application No. CN201110314666.0, publication No. CN102422815A, disclosing a plant regeneration method using stems of a large flower aromatic China rose as explants).
TABLE 1 comparison of the test parameters
Compared with comparative examples 1-5, the treatment methods of examples 1-5 of the present invention have good performance advantages, and the test parameters such as the bud induction rate, the average budding time and the survival rate show significant differences. On the basis of the existing explant culture medium formula, substances such as phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine, butyrylhydrazine and the like are innovatively introduced, and can generate a synergistic effect, so that the disease resistance of an explant plant is improved, and a good improvement effect on the healthy growth of the explant is achieved; meanwhile, the preparation method and the application of the invention have simple operation and strong feasibility, and can improve the bud induction rate and the bud emergence time of the explant. Particularly, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and hydrazine butyryl are not used for inducing the germination of the explants, wherein phenylalanine is commonly used for rooting, 6-benzylaminopurine is commonly used as cytokinin, hydrazine butyryl is commonly used as a plant preservative, corresponding technical suggestions are not given in the prior art, the independent use of the four substances cannot improve the bud induction rate and shorten the bud emergence time of the explants of the Chinese rose stem segments, and even lead to the reduction of the survival rate of the Chinese rose stem segments, but when the four substances are jointly used, the survival rate, the bud induction rate and the average bud emergence time of the Chinese rose stem segments are remarkably improved, and unpredictable technical effects are brought.
The present invention and its embodiments have been described above schematically, without limitation to the description, and the actual structure is not limited to this. Therefore, if the person skilled in the art receives the teaching, it is within the scope of the present invention to design the embodiments similar to the technical solutions without the inventive concept.
Claims (10)
1. The culture medium for improving the survival rate of the explants is characterized by comprising the following raw materials in parts by weight:
50-60 parts of B5 culture medium,
10 to 15 portions of cane sugar, and the like,
5 to 8 portions of agar, namely 5 to 8 portions of agar,
0.7 to 1.2 portions of phenylalanine,
0.05 to 0.08 portion of 1-naphthylacetic acid,
0.03 to 0.05 portion of 6-benzylaminopurine,
0.02 part to 0.04 part of butyryl hydrazine.
2. The culture medium for improving the survival rate of explants according to claim 1, which comprises the following raw materials in parts by weight:
53-58 parts of B5 culture medium,
11 to 14 portions of cane sugar, 11 portions of cane sugar,
6 to 7 portions of agar, namely 6 portions of agar,
0.8 to 1.1 portions of phenylalanine,
0.06 to 0.07 portion of 1-naphthylacetic acid,
0.03 to 0.05 portion of 6-benzylaminopurine,
0.02 part to 0.04 part of butyryl hydrazine.
3. The culture medium for improving the survival rate of explants according to claim 2, which comprises the following raw materials in parts by weight:
55 parts of a B5 culture medium,
13 parts of cane sugar, namely 13 parts of cane sugar,
7 parts of agar, namely 7 parts of agar,
0.9 part of phenylalanine, namely 0.9 part of phenylalanine,
0.07 part of 1-naphthylacetic acid,
0.04 part of 6-benzylaminopurine,
0.03 part of butyryl hydrazine.
4. A preparation method of a culture medium for improving the survival rate of explants is characterized by comprising the following steps:
(1) preparing a B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine, and storing at a low temperature for later use;
(2) respectively weighing the B5 culture medium, sucrose, agar, phenylalanine, 1-naphthylacetic acid, 6-benzylaminopurine and butyrylhydrazine according to the proportion of the raw materials, adding water, heating, stirring and mixing, and then pouring into a culture dish for cooling;
(3) and placing the cooled culture dish into a sterilization pot for high-temperature and high-pressure sterilization, taking out after sterilization, and cooling at room temperature to obtain the culture dish.
5. The method for preparing a culture medium for improving the survival rate of explants according to claim 1,
in the step (1), the particle size ranges of the phenylalanine, the 1-naphthylacetic acid, the 6-benzylaminopurine and the butyryl hydrazine are 200 meshes to 400 meshes;
the temperature range of the low-temperature storage in the step (1) is 4-8 ℃.
6. The method for preparing a culture medium for improving the survival rate of explants according to claim 1,
the heating temperature in the step (2) is 95-100 ℃;
the rotating speed of stirring and mixing in the step (2) is 200rpm-400 rpm;
the temperature range of cooling in the step (2) is 10-25 ℃.
7. The method for preparing a culture medium for improving the survival rate of explants according to claim 1,
the temperature for high-temperature high-pressure sterilization in the step (3) is 121-126 ℃;
the pressure of high-temperature high-pressure sterilization in the step (3) is 0.15MPa-0.20 MPa;
the time of high-temperature high-pressure sterilization in the step (3) is 15min-20 min.
8. The application of the culture medium for improving the survival rate of the explants is characterized by comprising the following steps:
firstly, cutting an explant in a sterile box, inoculating the explant into a culture medium for culture after the explant grows out a new bud, and controlling parameters of temperature, humidity and illumination conditions.
9. The use of the culture medium for improving the survival rate of explants according to claim 8,
the explant is a stem section with small young leaves, wherein the length range of the stem section is 2cm-3cm, and the number of the small young leaves is 1-2.
10. The use of the culture medium for improving the survival rate of explants according to claim 8,
the culture temperature is 26-28 ℃;
the culture humidity is 60% -80%;
the illumination condition is 8h-10h/day, 2000lux-3000 lux.
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