CN111758569B - Method for inhibiting calli of fragrant fruit trees from browning - Google Patents

Method for inhibiting calli of fragrant fruit trees from browning Download PDF

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CN111758569B
CN111758569B CN202010645483.6A CN202010645483A CN111758569B CN 111758569 B CN111758569 B CN 111758569B CN 202010645483 A CN202010645483 A CN 202010645483A CN 111758569 B CN111758569 B CN 111758569B
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vitamin
callus
browning
subculture
inhibiting
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CN111758569A (en
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宋丽莉
张梦鸽
姜育龙
张婉萍
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Shanghai Institute of Technology
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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
    • 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
    • A01H4/008—Methods for regeneration to complete plants

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Abstract

The invention relates to a method for inhibiting calli of a fragrant fruit tree from browning, which comprises the following steps: (1) and (3) disinfection of explants: taking the petiole of the myrtle tree as an explant, and disinfecting to obtain a disinfected explant; (2) callus induction: inoculating the sterilized explant on a callus induction culture medium under an aseptic condition, and culturing to form callus on the explant; (3) anti-browning subculture: transferring the callus into a subculture medium, and performing subculture to achieve the effect of inhibiting the callus of the cone tree from browning. Compared with the prior art, the method takes the petiole of the fragrant fruit tree as an explant, adopts the special callus induction culture medium to induce the callus, and utilizes the browning-inhibiting subculture medium to perform subculture, so that the browning rate of the callus is reduced to 3.3 percent.

Description

Method for inhibiting calli of fragrant fruit trees from browning
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for inhibiting calli of fragrant fruit trees from browning.
Background
The myrtle tree is a tall deciduous tree of the genus Syzygium of the family Rubiaceae, originates from the middle-aged chalk era of about 1 hundred million years from today, is a quaternary glacier wiggling plant, is a unique single-species rare tree species in China, and has great value in the aspects of systematic development and morphological evolution of the Rubiaceae plant, the research of the geographical system of the Chinese plant, appreciation, medicinal use, material use and the like.
In a natural state, the fragrant fruit tree has typical intermittent fruit bearing characteristics, and blossoms and bears fruits once in 2-4 years; the seeds have the characteristics of dormancy, tiny seeds, low vitality, only 1 year of life, extremely low natural germination rate, declining population and endangered extinction, and are classified as national secondary protection plants. The method for developing the research on the seedling breeding technology of the wild fragrant fruit tree by using the plant tissue culture method has important significance for overcoming the difficulty in seed breeding of the fragrant fruit tree, saving the germplasm resources of the rare endangered wild fragrant fruit tree and realizing the sustainable utilization of the germplasm resources of the fragrant fruit tree.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for inhibiting the calli of the fragrant fruit trees from browning.
The purpose of the invention can be realized by the following technical scheme:
a method for inhibiting the calli of a myrtle tree from browning comprises the following steps:
(1) and (3) disinfection of explants: taking the petiole of the myrtle tree as an explant, and disinfecting to obtain a disinfected explant;
(2) callus induction: inoculating the sterilized explant on a callus induction culture medium under an aseptic condition, and culturing to form callus on the explant;
(3) anti-browning subculture: transferring the callus into a subculture medium, and performing subculture to achieve the effect of inhibiting the callus of the fragrant fruit tree from browning.
Further, the disinfection method comprises the following specific steps: sterilizing with ethanol and sodium hypochlorite, and washing with sterile water.
Further, the callus induction medium comprises the following components: calculated as per liter, from 495mg K2SO4、185mg MgSO4·7H2O、85mg KH2PO4、200mg NH4NO3、11.25mg MnSO4·4H2O、4.3mg ZnSO4·7H2O、3.1mg H3BO3、0.125mg CuSO4·5H2O、0.125mg Na2MoO4·2H2O、278mg Ca(NO3)2·4H2O、13.9mg FeSO4·7H2O、18.65mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 20g of cane sugar, 6g of agar and the balance of water.
Further, the temperature of the culture in the step (2) is 25 +/-2 ℃, and the time is 20 days; the temperature of the subculture in the step (3) is 25 +/-2 ℃ and the time is 20 days.
Further, the composition of the subculture medium is as follows: calculated as per liter, from 495mg K2SO4、185mg MgSO4·7H2O、85mg KH2PO4、200mg NH4NO3、11.25mg MnSO4·4H2O、4.3mg ZnSO4·7H2O、3.1mg H3BO3、0.125mg CuSO4·5H2O、0.125mg Na2MoO4·2H2O、278mg Ca(NO3)2·4H2O、13.9mg FeSO4·7H2O、18.65mg Na2EDTA, 50mg inositol, 1mg glycine0.5mg of vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 1-2g of polyvinylpyrrolidone, 20g of cane sugar, 6g of agar and the balance of water.
Further, the composition of the subculture medium is as follows: calculated as per liter, from 495mg K2SO4、185mg MgSO4·7H2O、85mg KH2PO4、200mg NH4NO3、11.25mg MnSO4·4H2O、4.3mg ZnSO4·7H2O、3.1mg H3BO3、0.125mg CuSO4·5H2O、0.125mg Na2MoO4·2H2O、278mg Ca(NO3)2·4H2O、13.9mg FeSO4·7H2O、18.65mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 2g of polyvinylpyrrolidone, 20g of sucrose, 6g of agar and the balance of water.
Further, the composition of the subculture medium is as follows: calculated as per liter, from 495mg K2SO4、185mg MgSO4·7H2O、85mg KH2PO4、200mg NH4NO3、11.25mg MnSO4·4H2O、4.3mg ZnSO4·7H2O、3.1mg H3BO3、0.125mg CuSO4·5H2O、0.125mg Na2MoO4·2H2O、278mg Ca(NO3)2·4H2O、13.9mg FeSO4·7H2O、18.65mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 0.5-1g of citric acid, 20g of cane sugar, 6g of agar and the balance of water.
Further, the composition of the subculture medium is as follows: calculated as per liter, from 495mg K2SO4、185mg MgSO4·7H2O、85mg KH2PO4、200mg NH4NO3、11.25mg MnSO4·4H2O、4.3mg ZnSO4·7H2O、3.1mg H3BO3、0.125mg CuSO4·5H2O、0.125mg Na2MoO4·2H2O、278mg Ca(NO3)2·4H2O、13.9mg FeSO4·7H2O、18.65mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 0.5g of citric acid, 20g of cane sugar, 6g of agar and the balance of water.
Further, the composition of the subculture medium is as follows: calculated as per liter, from 495mg K2SO4、185mg MgSO4·7H2O、85mg KH2PO4、200mg NH4NO3、11.25mg MnSO4·4H2O、4.3mg ZnSO4·7H2O、3.1mg H3BO3、0.125mg CuSO4·5H2O、0.125mg Na2MoO4·2H2O、278mg Ca(NO3)2·4H2O、13.9mg FeSO4·7H2O、18.65mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 0.01-0.2g of ascorbic acid, 20g of cane sugar, 6g of agar and the balance of water.
Further, the composition of the subculture medium is as follows: calculated as per liter, from 495mg K2SO4、185mg MgSO4·7H2O、85mg KH2PO4、200mg NH4NO3、11.25mg MnSO4·4H2O、4.3mg ZnSO4·7H2O、3.1mg H3BO3、0.125mg CuSO4·5H2O、0.125mg Na2MoO4·2H2O、278mg Ca(NO3)2·4H2O、13.9mg FeSO4·7H2O、18.65mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 0.01-0.1g of ascorbic acid, 20g of cane sugar, 6g of agar and the balance of water.
The callus browning mechanism of the fragrant fruit tree comprises the following steps: the activity of Phenylalanine Ammonia Lyase (PAL) and polyphenol oxidase (PPO) is improved by the higher concentration of 6-BA in the subculture medium, PAL promotes the synthesis and accumulation of phenolic substances in the callus, and the phenolic substances are oxidized under the catalysis of the high-activity PPO to generate a large amount of brown quinone substances, thereby causing the browning of the callus and causing the culture of the callus to be unable to continue.
Polyvinylpyrrolidone, citric acid and ascorbic acid inhibit the cause of browning: polyvinylpyrrolidone, citric acid and ascorbic acid with proper concentration are added into the subculture medium, so that PAL activity and PPO activity can be remarkably inhibited, the synthesis of phenolic substances and the formation of quinone substances are reduced, the browning rate of callus is reduced, and the subculture of the callus can be continued.
Compared with the prior art, the method takes the petiole of the fragrant fruit tree as an explant, adopts the specific callus induction culture medium to induce the callus, and utilizes the browning-inhibiting subculture medium to perform subculture, so that the browning rate of the callus can be obviously reduced, the subculture of the callus can be continued, and a usable material is provided for the subsequent bud differentiation and plant regeneration.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
A method for inhibiting calli of a fragrant fruit tree from browning comprises the following steps:
(1) and (3) disinfection of explants: taking the leaf stalks of the fragrant fruit trees as explants, sequentially disinfecting the leaf stalks by using 75% alcohol by volume percentage for 15 seconds and 0.1% sodium hypochlorite aqueous solution by volume percentage for 5 minutes, and then washing the leaf stalks by using sterile water to obtain disinfected explants;
(2) callus induction: inoculating the sterilized explant on a callus induction culture medium under an aseptic condition, controlling the temperature to be 25 +/-2 ℃ and culturing for 20 days to form callus on the sterilized explant;
callus induction medium consisting of 495mg K per liter2SO4、185mg MgSO4·7H2O、85mg KH2PO4、200mg NH4NO3、11.25mg MnSO4·4H2O、4.3mg ZnSO4·7H2O、3.1mg H3BO3、0.125mg CuSO4·5H2O、0.125mg Na2MoO4·2H2O、278mg Ca(NO3)2·4H2O、13.9mg FeSO4·7H2O、18.65mg Na2EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 20g of cane sugar, 6g of agar and the balance of water;
(3) subculturing on a browning-inhibiting medium: transferring the callus into a subculture medium, and performing proliferation subculture for 20 days at the temperature of 25 +/-2 ℃;
proliferation medium, calculated per liter, from 495mg K2SO4、185mg MgSO4·7H2O、85mg KH2PO4、200mg NH4NO3、11.25mg MnSO4·4H2O、4.3mg ZnSO4·7H2O、3.1mg H3BO3、0.125mg CuSO4·5H2O、0.125mg Na2MoO4·2H2O、278mg Ca(NO3)2·4H2O、13.9mg FeSO4·7H2O、18.65mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 2g of polyvinylpyrrolidone, 20g of sucrose, 6g of agar and the balanceAmount of water.
Example 2
The difference from example 1 is that the concentration of polyvinylpyrrolidone in the callus subculture medium in step (3) was 1 g/L.
Example 3
The difference from example 1 is that the concentration of polyvinylpyrrolidone in the callus subculture medium in step (3) was 1.5 g/L.
The anti-browning effects of examples 1-3 are shown in Table 1.
TABLE 1
Treatment of PVP concentration (g/L) Browning rate%
Control 0 44.3
Example 2 1 23.3
Example 3 1.5 14.0
Example 1 2 3.3
The result shows that the browning rate of the callus is only 3.3% when the addition amount of PVP is 2g/L, and the anti-browning effect is very obvious.
Example 4
A method for inhibiting calli of a fragrant fruit tree from browning comprises the following steps:
(1) and (3) disinfection of explants: taking the leaf stalks of the fragrant fruit trees as explants, sequentially disinfecting the leaf stalks by using 75% alcohol by volume percentage for 15 seconds and 0.1% sodium hypochlorite aqueous solution by volume percentage for 5 minutes, and then washing the leaf stalks by using sterile water to obtain disinfected explants;
(2) callus induction: inoculating the sterilized explant on a callus induction culture medium under an aseptic condition, controlling the temperature to be 25 +/-2 ℃ and culturing for 20 days to form callus on the sterilized explant;
callus induction medium, calculated per liter, from 495mg K2SO4、185mg MgSO4·7H2O、85mg KH2PO4、200mg NH4NO3、11.25mg MnSO4·4H2O、4.3mg ZnSO4·7H2O、3.1mg H3BO3、0.125mg CuSO4·5H2O、0.125mg Na2MoO4·2H2O、278mg Ca(NO3)2·4H2O、13.9mg FeSO4·7H2O、18.65mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 20g of cane sugar, 6g of agar and the balance of water;
(3) subculture on medium inhibiting browning: transferring the callus into a subculture medium, and performing proliferation subculture for 20 days at a controlled temperature of 25 +/-2 ℃;
proliferation medium, calculated per liter, from 495mg K2SO4、185mg MgSO4·7H2O、85mg KH2PO4、200mg NH4NO3、11.25mg MnSO4·4H2O、4.3mg ZnSO4·7H2O、3.1mg H3BO3、0.125mg CuSO4·5H2O、0.125mg Na2MoO4·2H2O、278mg Ca(NO3)2·4H2O、13.9mg FeSO4·7H2O、18.65mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 0.5g of citric acid, 20g of cane sugar, 6g of agar and the balance of water.
Comparative example 1
The difference from example 4 was that the concentration of citric acid in the callus subculture medium in step (3) was 0.2 g/L.
Example 5
The difference from example 4 was that the concentration of citric acid in the callus subculture medium in step (3) was 1 g/L.
Comparative example 2
The difference from example 4 was that the concentration of citric acid in the callus subculture medium in step (3) was 1.5 g/L.
The anti-browning effects of examples 4 to 5 and comparative examples 1 to 2 are shown in table 2.
TABLE 2
Treatment of Citric acid concentration (g/L) Browning rate%
Control 0 44.3
Comparative example 1 0.2 46.4
Example 4 0.5 24.5
Example 5 1 43.1
Comparative example 2 1.5 83.6
The result shows that the browning rate of the callus is only 24.5% when the addition amount of the citric acid is 0.5g/L, and the anti-browning effect is very obvious.
Example 6
A method for inhibiting calli of a fragrant fruit tree from browning comprises the following steps:
(1) and (3) disinfection of explants: taking the leaf stalks of the fragrant fruit trees as explants, sequentially disinfecting the leaf stalks by using 75% alcohol by volume percentage for 15 seconds and 0.1% sodium hypochlorite aqueous solution by volume percentage for 5 minutes, and then washing the leaf stalks by using sterile water to obtain disinfected explants;
(2) callus induction: inoculating the sterilized explant on a callus induction culture medium under an aseptic condition, controlling the temperature to be 25 +/-2 ℃ and culturing for 20 days to form callus on the sterilized explant;
callus induction medium, calculated per liter, from 495mg K2SO4、185mg MgSO4·7H2O、85mg KH2PO4、200mg NH4NO3、11.25mg MnSO4·4H2O、4.3mg ZnSO4·7H2O、3.1mg H3BO3、0.125mg CuSO4·5H2O、0.125mg Na2MoO4·2H2O、278mg Ca(NO3)2·4H2O、13.9mg FeSO4·7H2O、18.65mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 20g of cane sugar, 6g of agar and the balance of water;
(3) subculture on medium inhibiting browning: transferring the callus into a subculture medium, and performing proliferation subculture for 20 days at the temperature of 25 +/-2 ℃;
proliferation medium, calculated per liter, from 495mg K2SO4、185mg MgSO4·7H2O、85mg KH2PO4、200mg NH4NO3、11.25mg MnSO4·4H2O、4.3mg ZnSO4·7H2O、3.1mg H3BO3、0.125mg CuSO4·5H2O、0.125mg Na2MoO4·2H2O、278mg Ca(NO3)2·4H2O、13.9mg FeSO4·7H2O、18.65mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 0.1g of ascorbic acid, 20g of sucrose, 6g of agar and the balance of water.
Example 7
The difference from example 6 was that the ascorbic acid concentration in the callus subculture medium in step (3) was 0.01 g/L.
Example 8
The difference from example 6 was that the ascorbic acid concentration in the callus subculture medium in step (3) was 0.2 g/L.
Comparative example 3
The difference from example 6 was that the ascorbic acid concentration in the callus subculture medium in step (3) was 0.5 g/L.
The anti-browning effects of examples 6 to 8 and comparative example 3 are shown in table 3.
TABLE 3
Treatment of Ascorbic acid concentration (g/L) Browning rate%
Control 0 44.3
Example 7 0.01 21.9
Example 6 0.1 14.8
Example 8 0.2 41.9
Comparative example 3 0.5 94.4
The results show that the browning rate of the callus is only 14.8% when the addition amount of the ascorbic acid is 0.1g/L, and the anti-browning effect is very obvious.
In conclusion, the method for inhibiting calli of the fragrant fruit trees takes petioles of the fragrant fruit trees as explants, adopts the special callus induction culture medium to induce the calli, and utilizes the browning-inhibiting subculture medium to culture, so that the browning rate is only 3.3%. The obtained callus without browning and with good growth can be used for callus bud differentiation and plant regeneration of the passion fruit trees, and guarantee is provided for improving the propagation efficiency of the seedlings of the passion fruit trees.
The foregoing is merely an example of the embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims (7)

1. A method for inhibiting calli of a myrtle tree from browning is characterized by comprising the following steps:
(1) and (3) disinfection of explants: taking the petiole of the myrtle tree as an explant, and disinfecting to obtain a disinfected explant;
(2) callus induction: inoculating the sterilized explant on a callus induction culture medium under an aseptic condition, and culturing to form callus on the explant;
(3) anti-browning subculture: transferring the callus into a subculture medium, and performing subculture to achieve the effect of inhibiting browning of the callus of the fragrant fruit tree;
the subculture medium comprises the following components: calculated as per liter, from 495mg K2SO4、185 mg MgSO4·7H2O、85 mg KH2PO4、200 mg NH4NO3、11.25 mg MnSO4·4H2O、4.3 mg ZnSO4·7H2O、3.1 mg H3BO3、0.125 mg CuSO4·5H2O、0.125 mg Na2MoO4·2H2O、278 mg Ca(NO3)2·4H2O、13.9 mg FeSO4·7H2O、18.65 mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B6、2 mg 6-BA, 0.5mg 2,4-D, 1-2g polyvinylpyrrolidone, 20g sucrose, 6g agar and the balance of water;
or 495mg K per liter2SO4、185 mg MgSO4·7H2O、85 mg KH2PO4、200 mg NH4NO3、11.25 mg MnSO4·4H2O、4.3 mg ZnSO4·7H2O、3.1 mg H3BO3、0.125 mg CuSO4·5H2O、0.125 mg Na2MoO4·2H2O、278 mg Ca(NO3)2·4H2O、13.9 mg FeSO4·7H2O、18.65 mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 0.5-1g of citric acid, 20g of cane sugar, 6g of agar and the balance of water;
or 495mg K per liter2SO4、185 mg MgSO4·7H2O、85 mg KH2PO4、200 mg NH4NO3、11.25 mg MnSO4·4H2O、4.3 mg ZnSO4·7H2O、3.1 mg H3BO3、0.125 mg CuSO4·5H2O、0.125 mg Na2MoO4·2H2O、278 mg Ca(NO3)2·4H2O、13.9 mg FeSO4·7H2O、18.65 mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 0.01-0.2g of ascorbic acid, 20g of cane sugar, 6g of agar and the balance of water.
2. The method for inhibiting the calli of the myrtle tree from browning according to claim 1, wherein the disinfection is carried out by the following specific methods: sterilizing with ethanol and sodium hypochlorite, and washing with sterile water.
3. The method of claim 1, wherein the callus induction medium comprises: calculated as per liter, from 495mg K2SO4、185 mg MgSO4·7H2O、85 mg KH2PO4、200 mg NH4NO3、11.25 mg MnSO4·4H2O、4.3 mg ZnSO4·7H2O、3.1 mg H3BO3、0.125 mg CuSO4·5H2O、0.125 mg Na2MoO4·2H2O、278 mg Ca(NO3)2·4H2O、13.9 mg FeSO4·7H2O、18.65 mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 20g of cane sugar, 6g of agar and the balance of water.
4. The method for inhibiting the calli of the myrtle tree from browning according to claim 1, wherein the temperature of the culture in the step (2) is 25 ± 2 ℃ for 20 days; the temperature of the subculture in the step (3) is 25 +/-2 ℃ and the time is 20 days.
5. The method of claim 1, wherein the composition of the subculture medium is as follows: calculated as per liter, from 495mg K2SO4、185 mg MgSO4·7H2O、85 mg KH2PO4、200 mg NH4NO3、11.25 mg MnSO4·4H2O、4.3 mg ZnSO4·7H2O、3.1 mg H3BO3、0.125 mg CuSO4·5H2O、0.125 mg Na2MoO4·2H2O、278 mg Ca(NO3)2·4H2O、13.9 mg FeSO4·7H2O、18.65 mg Na2EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 2g of polyvinylpyrrolidone, 20g of sucrose, 6g of agar and the balance of water.
6. The method of claim 1, wherein the composition of the subculture medium is as follows: calculated as per liter, from 495mg K2SO4、185 mg MgSO4·7H2O、85 mg KH2PO4、200 mg NH4NO3、11.25 mg MnSO4·4H2O、4.3 mg ZnSO4·7H2O、3.1 mg H3BO3、0.125 mg CuSO4·5H2O、0.125 mg Na2MoO4·2H2O、278 mg Ca(NO3)2·4H2O、13.9 mg FeSO4·7H2O、18.65 mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B62mg of 6-BA, 0.5mg of 2,4-D, 0.5g of citric acid, 20g of cane sugar, 6g of agar and the balance of water.
7. The method of claim 1, wherein the composition of the subculture medium is as follows: calculated as per liter, from 495mg K2SO4、185 mg MgSO4·7H2O、85 mg KH2PO4、200 mg NH4NO3、11.25 mg MnSO4·4H2O、4.3 mg ZnSO4·7H2O、3.1 mg H3BO3、0.125 mg CuSO4·5H2O、0.125 mg Na2MoO4·2H2O、278 mg Ca(NO3)2·4H2O、13.9 mg FeSO4·7H2O、18.65 mg Na2-EDTA, 50mg inositol, 1mg glycine, 0.5mg vitamin B10.25mg vitamin B50.25mg vitamin B6、2 mg 6-BA、0.5 mg 2,4-D, 0.01-0.1g ascorbic acid, 20g sucrose, 6g agar and the balance water.
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