CN117084171B - Detoxification method of passion fruits - Google Patents
Detoxification method of passion fruits Download PDFInfo
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- CN117084171B CN117084171B CN202311134356.XA CN202311134356A CN117084171B CN 117084171 B CN117084171 B CN 117084171B CN 202311134356 A CN202311134356 A CN 202311134356A CN 117084171 B CN117084171 B CN 117084171B
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- 238000001784 detoxification Methods 0.000 title claims abstract description 152
- 244000288157 Passiflora edulis Species 0.000 title claims abstract description 84
- 235000000370 Passiflora edulis Nutrition 0.000 title claims abstract description 83
- 238000000034 method Methods 0.000 title claims abstract description 33
- 239000002609 medium Substances 0.000 claims abstract description 71
- 241000700605 Viruses Species 0.000 claims abstract description 24
- 239000001963 growth medium Substances 0.000 claims abstract description 11
- 238000005286 illumination Methods 0.000 claims description 42
- 229920001817 Agar Polymers 0.000 claims description 21
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 claims description 21
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 21
- 229930006000 Sucrose Natural products 0.000 claims description 21
- 239000008272 agar Substances 0.000 claims description 21
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 claims description 21
- 229960001669 kinetin Drugs 0.000 claims description 21
- 239000005720 sucrose Substances 0.000 claims description 21
- 101710134784 Agnoprotein Proteins 0.000 claims description 18
- 229920001542 oligosaccharide Polymers 0.000 claims description 18
- 150000002482 oligosaccharides Chemical class 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 235000011925 Passiflora alata Nutrition 0.000 claims description 6
- 235000011922 Passiflora incarnata Nutrition 0.000 claims description 6
- 235000013750 Passiflora mixta Nutrition 0.000 claims description 6
- 235000013731 Passiflora van volxemii Nutrition 0.000 claims description 6
- 241000724252 Cucumber mosaic virus Species 0.000 claims description 4
- 239000008223 sterile water Substances 0.000 claims description 4
- 241000219925 Oenothera Species 0.000 claims description 3
- 235000004496 Oenothera biennis Nutrition 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 229960002523 mercuric chloride Drugs 0.000 claims description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 3
- 239000006870 ms-medium Substances 0.000 claims description 3
- 201000010099 disease Diseases 0.000 abstract description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 8
- 238000001514 detection method Methods 0.000 abstract description 6
- 235000013399 edible fruits Nutrition 0.000 abstract description 5
- 230000035755 proliferation Effects 0.000 abstract description 4
- 238000012258 culturing Methods 0.000 description 38
- 239000000463 material Substances 0.000 description 25
- 230000004083 survival effect Effects 0.000 description 13
- 230000008859 change Effects 0.000 description 12
- 239000003443 antiviral agent Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000218996 Passiflora Species 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 1
- 235000012907 honey Nutrition 0.000 description 1
- 229940028444 muse Drugs 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- GMVPRGQOIOIIMI-DWKJAMRDSA-N prostaglandin E1 Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1CCCCCCC(O)=O GMVPRGQOIOIIMI-DWKJAMRDSA-N 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
Classifications
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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
- A01H4/002—Culture media for tissue culture
-
- 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
- Y02P60/14—Measures for saving energy, e.g. in green houses
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a detoxification method of passion fruits, and belongs to the technical field of detoxification and rapid propagation of fruit trees. Comprising the following steps: inoculating the stem tip of passion fruit on a detoxification medium for detoxification culture to obtain a first-generation tissue culture seedling; inoculating the stem tip of the obtained first-generation tissue culture seedling on a detoxification culture medium for detoxification culture to obtain a second-generation tissue culture seedling, wherein the total detoxification is carried out for 5 times. After the fifth detoxification, the stem tip is cultured for 30 days, and can be developed into tissue culture seedlings with the length of about 3.4 cm. The tissue culture seedlings are free of viruses through detection of main virus diseases. After the culture is transferred into a proliferation culture medium, the proliferation coefficient of the culture for 30d can reach 4.27.
Description
Technical Field
The invention relates to the technical field of detoxification and rapid propagation of fruit trees, in particular to a detoxification method of passion fruits.
Background
Passion fruit (Passiflora edulia Sims), also known as passion fruit, is a plant of the genus Passiflora of the family Passiflorae, and is a tropical, subtropical perennial evergreen vine berry-like fruit tree. The passion fruit planting has the characteristics of short time, quick response, high income and the like, and can bring better economic benefit to producers, so that the passion fruit planting is valued by governments in Guangdong, guangxi, yunnan and the like.
The prior main varieties of passion fruit include purple sweet No. 1, purple sweet No. 2, tainong No. 1, golden fruit series (muse taste and honey taste), qinmi No. 9 and the like. The variety is mixed and the variety of virus diseases which can infect passion fruits is various (tens of kinds), the asexual propagation such as cutting and grafting is mainly used in the production process, so that the virus diseases are seriously transmitted, and the occurrence frequency of passion fruit virus diseases is high in actual production, so that the yield is reduced and even absolute yield is realized. Therefore, the healthy passion fruit seedlings are produced by a nontoxic seedling detoxification rapid propagation technology and matched effective planting management measures are adopted, the stable yield and the yield increase of the passion fruit industry are realized, and the passion fruit seedling detoxification rapid propagation technology is a basic stone for realizing the healthy development of the passion fruit industry. The healthy development of the passion fruit industry is also an opportunity for realizing the countryside happiness in partial areas.
Tissue culture detoxification and rapid propagation are important means for producing plant virus-free seedlings. The method is simpler and more convenient to operate, has higher detoxification efficiency, and is more suitable for industrialized practice.
Disclosure of Invention
In order to solve the problems, the invention provides a detoxification method of passion fruits, which has high detoxification rate and provides technical and method support for detoxification and tissue culture rapid propagation production of passion fruits.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides a detoxification method of passion fruits, which comprises the following steps:
1) Inoculating the stem tip of passion fruit on a detoxification medium for detoxification culture to obtain a first-generation tissue culture seedling;
2) Inoculating the stem tip of the first-generation tissue culture seedling obtained in the step 1) on a detoxification culture medium for detoxification culture to obtain a second-generation tissue culture seedling;
3) Inoculating the stem tip of the second-generation tissue culture seedling obtained in the step 2) on a detoxification medium for detoxification culture to obtain a third-generation tissue culture seedling;
4) Inoculating the stem tip of the third-generation tissue culture seedling obtained in the step 3) on a detoxification medium for detoxification culture to obtain a fourth-generation tissue culture seedling;
5) Inoculating the stem tip of the fourth-generation tissue culture seedling obtained in the step 4) on a detoxification medium for detoxification culture to realize detoxification of passion fruits;
The detoxification medium is based on an MS medium and comprises kinetin 2.0mg/L, oligosaccharides 1.0mg/L, agNO 3 0.1.1 mg/L, sucrose 30g/L and agar 3.5g/L;
the conditions of the detoxification culture include: the time is 14d, the temperature is 28+/-3 ℃, the illumination time is 12 hours per day, and the illumination intensity is 40 mu mol.m -2·s-1.
Preferably, the length of the stem tip is 0.5-1.5 mm.
Preferably, the stem tip of the passion fruit in the step 1) is sterilized and then inoculated, and the sterilization comprises the following steps: the stem tip of passion fruit is washed clean by running water and then is soaked in 0.1 percent of mercuric chloride aqueous solution for 7 minutes, and is washed by sterile water for 3 to 5 times.
Preferably, the detoxified virus comprises one or more of passion flower virus, cucumber mosaic virus, evening primrose mosaic virus and passion flower lignified virus.
The invention provides a detoxification method of passion fruits, which starts from the disinfection of explants, the excision of stem tips and the detoxification culture, explores the proper method at each stage, perfects and optimizes the detoxification rapid propagation technology of passion fruits, and lays a material foundation and a technical support for the development of passion fruit industry. In the detoxification culture process, the inoculated terminal buds are not grown in a culture medium without any antiviral agent, and the survival rate is 0. Preferred first detoxification culture: the height of the excised terminal buds is 0.8-1.5 cm after the excised terminal buds grow in a culture medium for 14 days through effective antiviral agent combination; but the height is not obviously changed after the culture is continued for 7 days; and then the tissue culture seedlings are continuously cultured to turn yellow and die gradually. Second detoxification culture: re-performing stem tip detoxification on the tissue culture seedling subjected to the first detoxification culture for 14 days, and re-inoculating the tissue culture seedling into a culture medium containing an antiviral agent, and culturing for 14 days to a height of 1.2-1.8 cm; continuously culturing for 7 days, wherein the height is 1.5-2.3 cm; and the culture is continued again, and the height is not changed obviously. And (3) carrying out detoxification culture for the third time: re-performing stem tip detoxification on the tissue culture seedling obtained by the second detoxification culture, re-inoculating the tissue culture seedling into a culture medium containing an antiviral agent, and culturing for 14 days to a height of 1.5-2.0 cm; continuously culturing for 7 days, wherein the height is 2.3-2.6 cm; and culturing for 10 days again, wherein the small part of tissue culture seedlings can reach 3.4cm, and the height change of the large part of tissue culture seedlings is not obvious. Fourth detoxification culture: re-performing stem tip detoxification on the tissue culture seedling obtained by the third detoxification culture, re-inoculating the tissue culture seedling into a culture medium containing an antiviral agent, and culturing for 14 days to a height of 1.6-2.2 cm; continuously culturing for 7 days, wherein the height is 2.5-2.7 cm; and culturing for 10 days again, wherein most of the tissue culture seedlings can reach 3.4cm. After the fifth detoxification, the stem tip is cultured for 30 days, and can be developed into tissue culture seedlings with the length of about 3.4cm. The tissue culture seedlings are free of viruses through detection of main virus diseases. After the culture is transferred into a proliferation culture medium, the proliferation coefficient of the culture for 30d can reach 4.27.
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 view of selected explant material;
FIG. 2 is a pre-treatment of an explant material;
FIG. 3 is a microscopic view of the terminal bud of the explant material;
FIG. 4 is a flowchart of the apical bud removal of the explant material;
FIG. 5 is a photograph of a terminal bud culture without an antiviral agent added;
FIG. 6 shows tissue culture seedlings after 14 days of the first detoxification culture;
FIG. 7 shows tissue culture seedlings of the third detoxification culture for 14 days;
FIG. 8 shows a virus-free tissue culture seedling after virus disease detection.
Detailed Description
The invention provides a detoxification method of passion fruits, which comprises the following steps:
1) Inoculating the stem tip of passion fruit on a detoxification medium for detoxification culture to obtain a first-generation tissue culture seedling;
2) Inoculating the stem tip of the first-generation tissue culture seedling obtained in the step 1) on a detoxification culture medium for detoxification culture to obtain a second-generation tissue culture seedling;
3) Inoculating the stem tip of the second-generation tissue culture seedling obtained in the step 2) on a detoxification medium for detoxification culture to obtain a third-generation tissue culture seedling;
4) Inoculating the stem tip of the third-generation tissue culture seedling obtained in the step 3) on a detoxification medium for detoxification culture to obtain a fourth-generation tissue culture seedling;
5) Inoculating the stem tip of the fourth-generation tissue culture seedling obtained in the step 4) on a detoxification medium for detoxification culture to realize detoxification of passion fruits;
The detoxification medium is based on an MS medium and comprises kinetin 2.0mg/L, oligosaccharides 1.0mg/L, agNO 3 0.1.1 mg/L, sucrose 30g/L and agar 3.5g/L; the conditions of the detoxification culture include: the time is 14d, the temperature is 28+/-3 ℃, the illumination time is 12 hours per day, and the illumination intensity is 40 mu mol.m -2·s-1.
In the present invention, the length of the stem tip is preferably 0.5 to 1.5mm. In the present invention, the stem tip of passion fruit is preferably sterilized and then inoculated, the sterilization comprising: the stem tip of passion fruit is washed clean by running water and then is soaked in 0.1 percent of mercuric chloride aqueous solution for 7 minutes, and is washed by sterile water for 3 to 5 times. The invention preferably takes passion fruit buds as explants, performs stem tip excision under a split microscope after the explants are disinfected, and inoculates the stem tips on a detoxification medium. In the present invention, the virus that is detoxified preferably includes one or more of passion flower virus, cucumber mosaic virus, evening primrose mosaic virus, and passion flower lignified virus in east asia. In the present invention, it is preferable that the sterilization of the explant further comprises sucking the surface moisture of the explant with sterile filter paper, and cutting off the stem tip of the explant under a stereoscopic microscope to obtain a stem tip with a size of 0.5-1.5 mm. In the invention, when the stem tip is cut, the tendrils wrapping the stem tip are cut, and the stem tip meristem cannot be damaged.
The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.
Example 1
A detoxification method of passion fruit comprises the following steps:
Step 1) sterilizing the explant and cutting off the stem tip
Taking a purple fruit type passion fruit bud segment as an explant, removing leaves and tendrils of the explant by scissors with the length of the explant being 1.5-2.5 cm, washing the explant cleanly by flowing water, soaking and sterilizing the explant in HgCl 2 with the mass concentration of 0.1% for 7min, and finally washing the explant by sterile water for 3-5 times. The surface moisture of the explant was aspirated with sterile filter paper. The stem tip is excised under a stereomicroscope, the size of the stem tip is 0.5-1.5 mm, and the excision method is shown in figure 4.
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: kinetin (KT) 2.0mg/L, oligosaccharides 1.0mg/L, agNO 3 0.1.1 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the seedling height is 0.8-1.5 cm, and the survival rate is 12%; continuously culturing for 7 days, wherein the change of seedling height is not obvious; and then the cultivation is continued, and the leaves and stems of the material begin to yellow and die gradually.
Example 2
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: KT 1.0mg/L, oligosaccharides 0.5mg/L, agNO 3 0.1.1 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the seedling height is 0.2-0.3 cm, and the survival rate is 6%; continuously culturing for 5 days, wherein the change of seedling height is not obvious; and then the cultivation is continued, and the leaves and stems of the material begin to yellow and die gradually.
Example 3
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: KT 1.0mg/L, oligosaccharides 1.0mg/L, agNO 3 0.2.2 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the seedling height is 0.4-0.5 cm, and the survival rate is 8%; continuously culturing for 6 days, wherein the change of seedling height is not obvious; and then the cultivation is continued, and the leaves and stems of the material begin to yellow and die gradually.
Example 4
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: KT 1.0mg/L, oligosaccharides 1.5mg/L, agNO 3 0.3.3 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the seedling height is 0.4-0.6 cm, and the survival rate is 8%; continuously culturing for 6 days, wherein the change of seedling height is not obvious; and then the cultivation is continued, and the leaves and stems of the material begin to yellow and die gradually.
Example 5
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: KT 2.0mg/L, oligosaccharides 0.5mg/L, agNO 3 0.3.3 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the seedling height is 0.5-0.6 cm, and the survival rate is 9%; continuously culturing for 7 days, wherein the change of seedling height is not obvious; and then the cultivation is continued, and the leaves and stems of the material begin to yellow and die gradually.
Example 6
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: KT 2.0mg/L, oligosaccharides 1.5mg/L, agNO 3 0.2.2 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the seedling height is 0.7-0.8 cm, and the survival rate is 8%; continuously culturing for 6 days, wherein the change of seedling height is not obvious; and then the cultivation is continued, and the leaves and stems of the material begin to yellow and die gradually.
Example 7
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: KT 3.0mg/L, oligosaccharides 0.5mg/L, agNO 3 0.2.2 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the seedling height is 0.4-0.7 cm, and the survival rate is 8%; continuously culturing for 5 days, wherein the change of seedling height is not obvious; and then the cultivation is continued, and the leaves and stems of the material begin to yellow and die gradually.
Example 8
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: KT 3.0mg/L, oligosaccharides 1.0mg/L, agNO 3 0.3.3 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the seedling height is 0.5-0.9 cm, and the survival rate is 7%; continuously culturing for 6 days, wherein the change of seedling height is not obvious; and then the cultivation is continued, and the leaves and stems of the material begin to yellow and die gradually.
Example 9
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: KT 3.0mg/L, oligosaccharides 1.5mg/L, agNO 3 0.1.1 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the seedling height is 0.5-0.7 cm, and the survival rate is 10%; continuously culturing for 8 days, wherein the change of seedling height is not obvious; and then the cultivation is continued, and the leaves and stems of the material begin to yellow and die gradually.
Example 10
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) As in example 1
Step 3) passion fruit detoxification culture (second generation)
And 2) taking the bud seedlings cultured for 14 days in the step 2) as materials, re-cutting the stem tip, and re-inoculating the cut stem tip into a detoxification medium. The detoxification medium takes MS as a basic medium and further comprises: KT 2.0mg/L, oligosaccharides 1.0mg/L, agNO 3 0.1.1 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the height of the seedlings is 1.2-1.8 cm; continuously culturing for 7 days, wherein the seedling height is 1.5-2.3cm; and then culture is continued, and the height is not changed obviously.
Step 4) passion fruit detoxification culture (third generation)
And 3) taking the bud seedlings cultured for 14 days in the step 3) as materials, re-cutting the stem tip, and re-inoculating the cut stem tip into a detoxification medium. The detoxification medium takes MS as a basic medium and further comprises: KT 2.0mg/L, oligosaccharides 1.0mg/L, agNO 3 0.1.1 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the height of the seedlings is 1.5-2.0 cm; continuously culturing for 7 days, wherein the seedling height is 2.3-2.6cm; and then culturing is continued, and the height of a small part of seedlings can reach 3.4cm.
Step 5) passion fruit detoxification culture (fourth generation)
And 4) taking the bud seedlings cultured for 14 days in the step 4) as materials, re-cutting the stem tip, and re-inoculating the cut stem tip into a detoxification medium. The detoxification medium takes MS as a basic medium and further comprises: KT 2.0mg/L, oligosaccharides 1.0mg/L, agNO 3 0.1.1 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the height of the seedlings is 1.6-2.2 cm; continuously culturing for 7 days, wherein the seedling height is 2.5-2.7cm; culturing for another 10 days, and the height of most seedlings can reach 3.4cm.
Step 6) passion fruit detoxification culture (fifth generation)
And 5) taking the bud seedlings cultured for 14 days in the step 5) as materials, re-cutting the stem tip, and re-inoculating the cut stem tip into a detoxification medium. The detoxification medium takes MS as a basic medium and further comprises: KT 2.0mg/L, oligosaccharides 1.0mg/L, agNO 3 0.1.1 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
And after detoxification for 30 days, the heights of all buds can reach 3.4cm, and the final survival rate is 8%.
Step 7) passion fruit virus disease detection
Taking the detoxified seedlings obtained in the step 6) as materials, sampling and detecting the main virus diseases of passion fruits (passion fruit viruses, cucumber mosaic viruses and passion fruit lignified viruses), wherein the detection method is a passion fruit detoxication rapid propagation method (patent number: ZL 202010590046.9). Eventually all materials appeared negative by detection.
Comparative example 1
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: KT 1.0mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1. The stem tips are cultured for 14 days, all stem tips are not obviously increased, and all materials are continuously cultured to be yellow and dead.
Comparative example 2
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: KT2.0 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1. The stem tips are cultured for 14 days, all stem tips are not obviously increased, and all materials are continuously cultured to be yellow and dead.
Comparative example 3
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: KT 3.0mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1. The stem tips are cultured for 14 days, all stem tips are not obviously increased, and all materials are continuously cultured to be yellow and dead.
Comparative example 4
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: KT 0.1mg/L, oligosaccharides 0.1mg/L, agNO 3 0.05.05 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the seedling height is 0.2-0.5 cm, and the survival rate is 3%; continuously culturing for 5 days, wherein the change of seedling height is not obvious; and then the cultivation is continued, and the leaves and stems of the material begin to yellow and die gradually.
Comparative example 5
A detoxification method of passion fruit comprises the following steps:
Step 1) same as in example 1
Step 2) passion fruit virus-free culture (first generation)
Inoculating the stem tip (0.5-1.5 mm) into a detoxification medium for culturing for 30 days, and observing the growth condition of the bud tip; each treatment was inoculated 100 times and repeated 3 times. The detoxification medium takes MS as a basic medium and further comprises: KT 4.0mg/L, oligosaccharides 2.0mg/L, agNO 3 0.5.5 mg/L, sucrose 30g/L and agar 3.5g/L; the test culture temperature is 28+/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 40 mu mol.m -2·s-1.
Carrying out detoxification culture for 14 days, wherein the seedling height is 0.4-0.6 cm, and the survival rate is 4%; continuously culturing for 4 days, wherein the change of seedling height is not obvious; and then the cultivation is continued, and the leaves and stems of the material begin to yellow and die gradually.
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 (4)
1. A method for detoxification of passion fruit, comprising the steps of:
1) Inoculating the stem tip of passion fruit on a detoxification medium for detoxification culture to obtain a first-generation tissue culture seedling;
2) Inoculating the stem tip of the first-generation tissue culture seedling obtained in the step 1) on a detoxification culture medium for detoxification culture to obtain a second-generation tissue culture seedling;
3) Inoculating the stem tip of the second-generation tissue culture seedling obtained in the step 2) on a detoxification medium for detoxification culture to obtain a third-generation tissue culture seedling;
4) Inoculating the stem tip of the third-generation tissue culture seedling obtained in the step 3) on a detoxification medium for detoxification culture to obtain a fourth-generation tissue culture seedling;
5) Inoculating the stem tip of the fourth-generation tissue culture seedling obtained in the step 4) on a detoxification medium for detoxification culture to realize detoxification of passion fruits;
The detoxification medium is based on an MS medium and comprises kinetin 2.0mg/L, oligosaccharides 1.0mg/L, agNO 3 0.1.1 mg/L, sucrose 30g/L and agar 3.5g/L;
the conditions of the detoxification culture include: the time is 14d, the temperature is 28+/-3 ℃, the illumination time is 12 hours per day, and the illumination intensity is 40 mu mol.m -2·s-1.
2. The detoxification method according to claim 1, wherein the length of the stem tip is 0.5-1.5 mm.
3. The detoxification method according to claim 1, wherein the step 1) comprises sterilizing the stem tip of passion fruit and inoculating, wherein the sterilizing comprises: the stem tip of passion fruit is washed clean by running water and then is soaked in 0.1 percent of mercuric chloride aqueous solution for 7 minutes, and is washed by sterile water for 3 to 5 times.
4. The detoxification method of claim 1, wherein the detoxified virus comprises one or more of passion flower virus, cucumber mosaic virus, evening primrose mosaic virus and passion flower lignified virus.
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