A virus-free method for passion fruit

Abstract

The present invention relates to the technical field of virus-free rapid propagation of fruit trees, and particularly to a virus-free method for passion fruit. The method includes: inoculating the axillary bud-bearing stem segments of passion fruit into a vitrification induction medium for inducing plant vitrification to obtain vitrified plants; inoculating the obtained vitrified plants into a recovery induction medium for recovery culture to achieve virus elimination of passion fruit. Using the obtained normal adventitious buds as the base number, cutting the leaves for detection of major virus diseases, and screening to obtain virus-free passion fruit seedlings, and the virus elimination efficiency can reach 95%.

Description

Technical Field

[0001] The present invention relates to the technical field of virus-free rapid propagation of fruit trees, and particularly to a method for detoxifying passion fruit. Background Art

[0002] Passion fruit, scientifically named Passiflora edulia sims, is a plant of the genus Passiflora in the family Passifloraceae, and is a tropical and subtropical perennial evergreen vine berry fruit tree. It grows rapidly, can be planted and harvested in the same year, has an early production start and quick returns; and its fruits have a wide market and high economic benefits. Due to its unique fragrance and taste, it is increasingly favored by consumers.

[0003] In the past few decades, the planting process of passion fruit has mainly relied on asexual propagation methods such as cutting and grafting, resulting in the continuous accumulation of virus diseases (such as Passiflora virus in East Asia, Passiflora woody virus, Cucumber mosaic virus, and Telosma cordata mosaic virus, etc.) in the stems. In actual production, the reduction or even failure of passion fruit planting due to the outbreak of virus diseases often occurs. The continuous accumulation of virus diseases will lead to the degradation of varieties, weakened resistance, and decreased yields. Therefore, producing healthy passion fruit seedlings through seedling virus-free technology to achieve stable and increased production in the passion fruit planting industry is the cornerstone for the healthy development of the passion fruit industry.

[0004] During the tissue culture proliferation and rapid propagation of yellow passion fruit, some seedlings will become vitrified. During an accidental detection of the main virus diseases of passion fruit using the PCR method, we found that the detection rate of virus diseases in vitrified seedlings is low. Therefore, it is speculated that after the plant seedlings become vitrified, their internal structure is damaged, which is not conducive to the development of viruses. As the vitrification time prolongs, the virus load in vitrified seedlings gradually decreases. Therefore, the present invention designs to induce the vitrification of passion fruit tissue culture seedlings, and after culturing for a certain period of time, then restore the vitrified seedlings through induction to detoxify passion fruit, thereby forming an effective virus-free rapid propagation technology for passion fruit. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a method for detoxifying passion fruit, which has a high detoxification rate and provides technical and method support for the detoxification of passion fruit.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for detoxifying passion fruit, comprising the following steps:

[0008] 1) Inoculate the axillary bud-containing stem segments of passion fruit into a vitrification induction medium to induce the vitrification of the plants, and obtain vitrified plants;

[0009] The vitrification induction medium is: MS + 6-benzylaminopurine 1-6 mg / L, indoleacetic acid 0.2-1.2 mg / L, chitosan oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.2-3.5 g / L;

[0010] 2) Inoculate the vitrified plants obtained in the step 1) into a recovery induction medium for recovery culture to achieve virus detoxification of passion fruit;

[0011] The recovery induction medium is: MS + 6-benzylaminopurine 0.5-1.0 mg / L, indoleacetic acid 0.1-0.2 mg / L, chitosan oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5-4.2 g / L.

[0012] Preferably, the vitrification induction medium in the step 1) is: MS + 6-benzylaminopurine 4 mg / L, indoleacetic acid 0.8 mg / L, chitosan oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.2-3.5 g / L.

[0013] Preferably, the recovery induction medium in the step 2) is: MS + 6-benzylaminopurine 0.5 mg / L, indoleacetic acid 0.1 mg / L, chitosan oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L.

[0014] Preferably, the passion fruit is yellow-fruited passion fruit.

[0015] Preferably, the virus includes one or more of Passiflora foetida virus, Cucumber mosaic virus, Telosma mosaic virus, and Passiflora woodiness virus.

[0016] Preferably, the conditions for inducing vitrification of the plants in the step 1) include: temperature 28 ± 3 °C, light duration 12 h / d, light intensity 40 μmol·m -2 ·s -1 , culture humidity 80-90%, and time 40 d.

[0017] Preferably, the conditions for the recovery culture in the step 2) include: temperature 28 ± 3 °C, light duration 12 h / d, light intensity 40 μmol·m -2 ·s -1 , culture humidity 60-80%, and time 40 d.

[0018] The beneficial effects of the present invention:

[0019] The present invention provides a virus - detoxification method for passion fruit. By taking advantage of the fact that the vitrification state is not conducive to virus reproduction, the vitrification of passion fruit tissue - cultured seedlings is actively induced. Then, the vitrified seedlings are restored to obtain adventitious buds, and finally virus - free seedlings are obtained through the detection of major virus diseases. Vitrification induction culture: Based on the MS medium, through the combination of effective hormones, agar concentration, and culture humidity, the stem segments with axillary buds are cultured under light in the medium for 16 days. The bases of some seedlings begin to show vitrification. After 30 days of culture, the vitrification is basically complete (the whole plant is vitrified). After 40 days of light culture, the vitrification induction rate is counted as 21%. Restoration induction culture of vitrified seedlings: Based on the MS medium, through the combination of effective hormones, agar concentration, and culture humidity, the vitrified seedlings cultured for 40 days above are inoculated into the vitrification restoration induction medium and cultured under light for 40 days. The restoration induction rate is 31.0%. The average number of healthy adventitious buds induced per restored individual is 2.04. The adventitious buds are robust, tender green, and 2 - 4 cm in height. Using all the adventitious buds obtained above as the base, the leaves are cut and divided for the detection of major virus diseases of passion fruit; finally, through the detection, the virus - detoxification rate is 95%. Brief Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0021] Figure 1 is the vitrified seedling induced and obtained;

[0022] Figure 2 is the vitrified seedling cultured in the restoration induction medium for 10 days, with normal adventitious buds formed. The red circle shows the induced normal adventitious bud points;

[0023] Figure 3 is the performance after the normal adventitious buds grow for 30 days. Detailed Embodiment

[0024] The present invention provides a virus - detoxification method for passion fruit, including the following steps:

[0025] 1) Inoculate the stem segments with axillary buds of passion fruit into the vitrification induction medium for inducing the vitrification of the plant to obtain vitrified plants;

[0026] The vitrification induction medium is: MS + 6 - benzylaminopurine 1 - 6 mg / L, indoleacetic acid 0.2 - 1.2 mg / L, chitosan oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.2 - 3.5 g / L;

[0027] 2) Inoculate the vitrified plants obtained in step 1) into the restoration induction medium for restoration culture to achieve the virus - detoxification of passion fruit;

[0028] The recovery induction medium is: MS + 6-benzylaminopurine 0.5 - 1.0 mg / L, indoleacetic acid 0.1 - 0.2 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 - 4.2 g / L.

[0029] In the present invention, the axillary bud-bearing stem segments of passion fruit are inoculated into a vitrification induction medium for plant vitrification induction to obtain vitrified plants; the vitrification induction medium is: MS + 6-benzylaminopurine 1 - 6 mg / L, indoleacetic acid 0.2 - 1.2 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.2 - 3.5 g / L. In the present invention, the vitrification induction medium is preferably: MS + 6-benzylaminopurine 4 mg / L, indoleacetic acid 0.8 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.2 - 3.5 g / L. In the present invention, the passion fruit is preferably the yellow fruit type passion fruit. In the present invention, the conditions for plant vitrification induction preferably include: temperature of 28 ± 3 °C, light duration of 12 h / d, light intensity of 40 μmol·m -2 ·s -1 , culture humidity of 80 - 90%, and time of 40 d.

[0030] In the present invention, the obtained vitrified plants are inoculated into a recovery induction medium for recovery culture to achieve virus elimination of passion fruit; the recovery induction medium is: MS + 6-benzylaminopurine 0.5 - 1.0 mg / L, indoleacetic acid 0.1 - 0.2 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 - 4.2 g / L. In the present invention, the recovery induction medium is preferably: MS + 6-benzylaminopurine 0.5 mg / L, indoleacetic acid 0.1 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L. In the present invention, the conditions for recovery culture preferably include: temperature of 28 ± 3 °C, light duration of 12 h / d, light intensity of 40 μmol·m -2 ·s -1 , culture humidity of 60 - 80%, and time of 40 d.

[0031] In the present invention, the virus preferably includes one or more of Passiflora suberosa virus, Cucumber mosaic virus, Passiflora foetida mosaic virus, and Passiflora lignification virus.

[0032] In the present invention, the obtained vitrified plants are inoculated into a recovery induction medium for recovery culture, and the obtained passion fruit seedlings are detected for the virus by the method described in the patent No. ZL202010590046.9, titled "A Method for Virus Elimination and Rapid Propagation of Passion Fruit", to detect the virus elimination effect.

[0033] To further illustrate the present invention, the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] A virus - detoxification method for passion fruit, the steps are as follows:

[0036] Step 1) Vitrification induction of passion fruit tissue - cultured seedlings

[0037] Taking the tissue - cultured seedlings (not virus - detoxified) of yellow - fruit passion fruit as the object, using sterilized scissors to cut them into stem segments with axillary buds, and then inoculating them into the vitrification induction medium, and culturing them under light in a light incubator for 40 days, and counting the vitrification induction rate (the number of individuals with vitrification / the number of inoculations); 30 are inoculated for each treatment, and repeated 3 times. The vitrification induction medium uses MS as the basic medium, and also includes: 6 - benzylaminopurine (6 - BA) 1.0 mg / L, indole - 3 - acetic acid (IAA) 0.2 mg / L, chitosan oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L; the test culture temperature is 28 ± 3 °C, the light time is 12 h / d, the light intensity is 40 μmol·m -2 ·s -1 , and the culture humidity is 80%.

[0038] After culturing under light for 40 days, the vitrification induction rate is 3.0%.

[0039] Example 2

[0040] A virus - detoxification method for passion fruit, the steps are as follows:

[0041] Step 1) Vitrification induction of passion fruit tissue - cultured seedlings

[0042] Taking the tissue - cultured seedlings (not virus - detoxified) of yellow - fruit passion fruit as the object, using sterilized scissors to cut them into stem segments with axillary buds, and then inoculating them into the vitrification induction medium, and culturing them under light in a light incubator for 40 days, and counting the vitrification induction rate (the number of individuals with vitrification / the number of inoculations); 30 are inoculated for each treatment, and repeated 3 times. The vitrification induction medium uses MS as the basic medium, and also includes: 6 - benzylaminopurine (6 - BA) 2.0 mg / L, indole - 3 - acetic acid (IAA) 0.4 mg / L, chitosan oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L; the test culture temperature is 28 ± 3 °C, the light time is 12 h / d, the light intensity is 40 μmol·m -2 ·s -1 , and the culture humidity is 80%.

[0043] After culturing under light for 40 days, the vitrification induction rate is 6.0%.

[0044] Example 3

[0045] A virus - free method for passion fruit, the steps are as follows:

[0046] Step 1) Vitrification induction of passion fruit tissue - cultured seedlings

[0047] Using yellow - fruit passion fruit tissue - cultured seedlings (not virus - free) as the object, cut them into stem segments with axillary buds using sterilized scissors, and then inoculate them into the vitrification induction medium. Incubate them in a light incubator for 40 days with light, and count the vitrification induction rate (the number of individuals with vitrification / the number of inoculations); inoculate 30 for each treatment, and repeat 3 times. The vitrification induction medium uses MS as the basic medium, and also includes: 6 - benzylaminopurine (6 - BA) 4.0 mg / L, indoleacetic acid (IAA) 0.8 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L; the test culture temperature is 28 ± 3 °C, the light time is 12 h / d, the light intensity is 40 μmol·m -2 ·s -1 , and the culture humidity is 80%.

[0048] After 40 days of light culture, the vitrification induction rate is 9.0%.

[0049] Example 4

[0050] A virus - free method for passion fruit, the steps are as follows:

[0051] Step 1) Vitrification induction of passion fruit tissue - cultured seedlings

[0052] Using yellow - fruit passion fruit tissue - cultured seedlings (not virus - free) as the object, cut them into stem segments with axillary buds using sterilized scissors, and then inoculate them into the vitrification induction medium. Incubate them in a light incubator for 40 days with light, and count the vitrification induction rate (the number of individuals with vitrification / the number of inoculations); inoculate 30 for each treatment, and repeat 3 times. The vitrification induction medium uses MS as the basic medium, and also includes: 6 - benzylaminopurine (6 - BA) 6.0 mg / L, indoleacetic acid (IAA) 1.2 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L; the test culture temperature is 28 ± 3 °C, the light time is 12 h / d, the light intensity is 40 μmol·m -2 ·s -1 , and the culture humidity is 80%.

[0053] After 40 days of light culture, the vitrification induction rate is 8.0%, and most of the seedlings turn yellow and die.

[0054] Example 5

[0055] A virus - free method for passion fruit, the steps are as follows:

[0056] Step 1) Vitrification induction of passion fruit tissue culture seedlings

[0057] Using yellow passion fruit tissue culture seedlings (not detoxified) as the object, cut them into stem segments with axillary buds using sterilized scissors, and then inoculate them into the vitrification induction medium. Incubate them in a light incubator for 40 days with light, and count the vitrification induction rate (the number of individuals with vitrification / the number of inoculations); inoculate 30 for each treatment and repeat 3 times. The vitrification induction medium uses MS as the basic medium and also includes: 6-benzylaminopurine (6-BA) 4.0 mg / L, indoleacetic acid (IAA) 0.8 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.2 g / L; the test culture temperature is 28 ± 3 °C, the light time is 12 h / d, the light intensity is 40 μmol·m -2 ·s -1 、and the culture humidity is 80%.

[0058] After 40 days of light culture, the vitrification induction rate is 12%.

[0059] Example 6

[0060] A method for detoxifying passion fruit, the steps are:

[0061] Step 1) Vitrification induction of passion fruit tissue culture seedlings

[0062] Using yellow passion fruit tissue culture seedlings (not detoxified) as the object, cut them into stem segments with axillary buds using sterilized scissors, and then inoculate them into the vitrification induction medium. Incubate them in a light incubator for 40 days with light, and count the vitrification induction rate (the number of individuals with vitrification / the number of inoculations); inoculate 30 for each treatment and repeat 3 times. The vitrification induction medium uses MS as the basic medium and also includes: 6-benzylaminopurine (6-BA) 4.0 mg / L, indoleacetic acid (IAA) 0.8 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.2 g / L; the test culture temperature is 28 ± 3 °C, the light time is 12 h / d, the light intensity is 40 μmol·m -2 ·s -1 、and the culture humidity is 90%.

[0063] After 16 days of light culture, the base of some seedlings begins to show vitrification, and after 30 days of culture, the vitrification is basically complete (the whole plant is vitrified); after 40 days of light culture, the vitrification induction rate is counted as 21%.

[0064] Example 7

[0065] A method for detoxifying passion fruit, the steps are:

[0066] Step 1) The same as Example 6

[0067] Step 2) Recovery induction of Passiflora edulis vitrified seedlings

[0068] The vitrified seedlings cultured for 40 days above were inoculated into the recovery induction medium for vitrified seedlings and cultured under light for 40 days. The recovery induction rate (the number of individuals that can re-develop into normal adventitious buds / the inoculation quantity) was counted, and the number of normal adventitious buds induced was calculated; 30 were inoculated for each treatment, with 3 replicates. The recovery induction medium for vitrified seedlings used MS as the basic medium and also included: 6-benzylaminopurine (6-BA) 1.0 mg / L, indoleacetic acid (IAA) 0.2 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L; the test culture temperature was 28 ± 3 °C, the light time was 12 h / d, the light intensity was 40 μmol·m -2 ·s -1 , and the culture humidity was 80%.

[0069] After culturing under light for 40 days, the recovery induction rate was 13.0%; the average number of healthy adventitious buds induced per recovered individual was 1.92, and the adventitious buds were strong, tender green, and 2 - 4 cm in height.

[0070] Step 3) Detection of main virus diseases of Passiflora edulis

[0071] Using all the adventitious buds obtained above as the basis, the leaves were cut and separated for the detection of main virus diseases of Passiflora edulis (East Asian Passiflora virus, Cucumber mosaic virus, Tuberose mosaic virus, and Passiflora woody virus), and the detection method was the method specified in "A method for rapid propagation of virus-free Passiflora edulis (Patent No.: ZL202010590046.9)". Finally, through detection, the virus-free rate was 94%.

[0072] Example 8

[0073] A virus-free method for Passiflora edulis, the steps are as follows:

[0074] Step 1) The same as Example 6

[0075] Step 2) Recovery induction of Passiflora edulis vitrified seedlings

[0076] The vitrified seedlings cultured for 40 days above were inoculated into the recovery induction medium for vitrified seedlings and cultured under light for 40 days. The recovery induction rate (the number of individuals that can re-develop into normal adventitious buds / the inoculation quantity) was counted, and the number of normal adventitious buds induced was calculated; 30 were inoculated for each treatment, with 3 replicates. The recovery induction medium for vitrified seedlings used MS as the basic medium and also included: 6-benzylaminopurine (6-BA) 1.0 mg / L, indoleacetic acid (IAA) 0.2 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 4.2 g / L; the test culture temperature was 28 ± 3 °C, the light time was 12 h / d, the light intensity was 40 μmol·m-2 ·s -1 The culture humidity is 80%.

[0077] After 40 days of light culture, the recovery induction rate is 14.0%; the average number of healthy adventitious buds induced per recovered individual is 1.95, and the adventitious buds are strong, tender green, and 2 - 4 cm in height. There is no significant difference from Example 7.

[0078] Step 3) is the same as Example 7. Finally, through detection, the virus elimination rate is 93%.

[0079] Example 9

[0080] A virus elimination method for passion fruit, the steps are as follows:

[0081] Step 1) is the same as Example 6

[0082] Step 2) Recovery induction of vitrified passion fruit seedlings

[0083] The above-mentioned vitrified seedlings cultured for 40 days are inoculated into the vitrified seedling recovery induction medium and cultured under light for 40 days. The recovery induction rate (the number of individuals that can re-develop into normal adventitious buds / the inoculation quantity) is counted, and the number of normal adventitious buds induced is calculated; 30 are inoculated for each treatment, and repeated 3 times. The vitrified seedling recovery induction medium uses MS as the basic medium and also includes: 6-benzylaminopurine (6-BA) 0.5 mg / L, indoleacetic acid (IAA) 0.1 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L; the test culture temperature is 28 ± 3 °C, the light time is 12 h / d, and the light intensity is 40 μmol·m -2 ·s -1 The culture humidity is 80%.

[0084] After 40 days of light culture, the recovery induction rate is 24.0%; the average number of healthy adventitious buds induced per recovered individual is 2.36, and the adventitious buds are strong, tender green, and 2 - 4 cm in height.

[0085] Step 3) is the same as Example 7. Finally, through detection, the virus elimination rate is 95%.

[0086] Example 10

[0087] A virus elimination method for passion fruit, the steps are as follows:

[0088] Step 1) is the same as Example 6

[0089] Step 2) Recovery induction of vitrified passion fruit seedlings

[0090] The vitrified seedlings cultured for 40 days above were inoculated into the vitrified seedling recovery induction medium, and cultured under light for 40 days. The recovery induction rate (the number of individuals that can re-develop into normal adventitious buds / the inoculation quantity) was counted, and the number of induced normal adventitious buds was calculated; 30 were inoculated for each treatment, with 3 replicates. The vitrified seedling recovery induction medium was based on MS medium, and also included: 6-benzylaminopurine (6-BA) 0.5 mg / L, indoleacetic acid (IAA) 0.1 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L; the test culture temperature was 28 ± 3 °C, the light time was 12 h / d, the light intensity was 40 μmol·m -2 ·s -1 , and the culture humidity was 70%.

[0091] After culturing under light for 40 days, the recovery induction rate was 31.0%; the average number of healthy adventitious buds induced per recovered individual was 2.04, and the adventitious buds were robust, tender green, and 2 - 4 cm in height.

[0092] Step 3) was the same as Example 7. Finally, through detection, the detoxification rate was 95%.

[0093] Example 11

[0094] A detoxification method for passion fruit, the steps are as follows:

[0095] Step 1) was the same as Example 6

[0096] Step 2) Recovery induction of passion fruit vitrified seedlings

[0097] The vitrified seedlings cultured for 40 days above were inoculated into the vitrified seedling recovery induction medium, and cultured under light for 40 days. The recovery induction rate (the number of individuals that can re-develop into normal adventitious buds / the inoculation quantity) was counted, and the number of induced normal adventitious buds was calculated; 30 were inoculated for each treatment, with 3 replicates. The vitrified seedling recovery induction medium was based on MS medium, and also included: 6-benzylaminopurine (6-BA) 0.5 mg / L, indoleacetic acid (IAA) 0.1 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L; the test culture temperature was 28 ± 3 °C, the light time was 12 h / d, the light intensity was 40 μmol·m -2 ·s -1 , and the culture humidity was 60%.

[0098] After culturing under light for 40 days, the recovery induction rate was 30.0%; the average number of healthy adventitious buds induced per recovered individual was 1.81, and the adventitious buds were robust, tender green, and 2 - 3 cm in height.

[0099] Step 3) was the same as Example 7. Finally, through detection, the detoxification rate was 93%.

[0100] Comparative Example 1

[0101] A virus - free method for passion fruit, the steps are as follows:

[0102] Step 1) Vitrification induction of passion fruit tissue - cultured seedlings

[0103] Using yellow - fruited passion fruit tissue - cultured seedlings (not virus - free) as the object, cut them into stem segments with axillary buds using sterilized scissors, and then inoculate them into the vitrification induction medium, and carry out light culture in a light incubator for 40 days, and count the vitrification induction rate (the number of vitrified individuals / the number of inoculations); 30 are inoculated for each treatment, and repeated 3 times. The vitrification induction medium uses MS as the basic medium, and also includes: 6 - benzylaminopurine (6 - BA) 0.25 mg / L, indole - 3 - acetic acid (IAA) 0.05 mg / L, chitosan oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L; the test culture temperature is 28 ± 3 °C, the light time is 12 h / d, the light intensity is 40 μmol·m -2 ·s -1 , and the culture humidity is 80%.

[0104] After 40 days of light culture, the growth of axillary buds is slow, the induction rate of adventitious buds is very low, and the vitrification induction rate is 0.

[0105] Comparative Example 2

[0106] A virus - free method for passion fruit, the steps are as follows:

[0107] Step 1) Vitrification induction of passion fruit tissue - cultured seedlings

[0108] Using yellow - fruited passion fruit tissue - cultured seedlings (not virus - free) as the object, cut them into stem segments with axillary buds using sterilized scissors, and then inoculate them into the vitrification induction medium, and carry out light culture in a light incubator for 40 days, and count the vitrification induction rate (the number of vitrified individuals / the number of inoculations); 30 are inoculated for each treatment, and repeated 3 times. The vitrification induction medium uses MS as the basic medium, and also includes: 6 - benzylaminopurine (6 - BA) 8.0 mg / L, indole - 3 - acetic acid (IAA) 1.6 mg / L, chitosan oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L; the test culture temperature is 28 ± 3 °C, the light time is 12 h / d, the light intensity is 40 μmol·m -2 ·s -1 , and the culture humidity is 80%.

[0109] After 40 days of light culture, all axillary buds turn yellow and die, the induction rate of adventitious buds is 0, and the vitrification induction rate is 0.

[0110] Comparative Example 3

[0111] A virus - free method for passion fruit, the steps are as follows:

[0112] Step 1) The same as Example 6

[0113] Step 2) Recovery induction of vitrified seedlings of Passiflora edulis

[0114] The above-mentioned vitrified seedlings cultured for 40 days were inoculated into the recovery induction medium for vitrified seedlings, and cultured under light for 40 days. The recovery induction rate (the number of individuals that can re-develop into normal adventitious buds / the inoculation quantity) was counted, and the number of normal adventitious buds induced was calculated; 30 were inoculated for each treatment, with 3 repetitions. The recovery induction medium for vitrified seedlings used MS as the basic medium, and also included: 6-benzylaminopurine (6-BA) 0.5 mg / L, indoleacetic acid (IAA) 0.1 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 4.5 g / L; the test culture temperature was 28 ± 3 °C, the light time was 12 h / d, the light intensity was 40 μmol·m -2 ·s -1 , and the culture humidity was 70%.

[0115] After culturing under light for 40 days, the recovery induction rate was 31.5%; the average number of healthy adventitious buds induced per recovered individual was 1.86. The adventitious buds were robust, tender green, and 2 - 4 cm in height. Its recovery induction rate had no significant difference from that of Example 10, and the average number of healthy adventitious buds induced per recovered individual decreased significantly.

[0116] Comparative Example 4

[0117] A virus-free method for Passiflora edulis, the steps are as follows:

[0118] Step 1) The same as Example 6

[0119] Step 2) Recovery induction of vitrified seedlings of Passiflora edulis

[0120] The above-mentioned vitrified seedlings cultured for 40 days were inoculated into the recovery induction medium for vitrified seedlings, and cultured under light for 40 days. The recovery induction rate (the number of individuals that can re-develop into normal adventitious buds / the inoculation quantity) was counted, and the number of normal adventitious buds induced was calculated; 30 were inoculated for each treatment, with 3 repetitions. The recovery induction medium for vitrified seedlings used MS as the basic medium, and also included: 6-benzylaminopurine (6-BA) 0.5 mg / L, indoleacetic acid (IAA) 0.1 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L; the test culture temperature was 28 ± 3 °C, the light time was 12 h / d, the light intensity was 40 μmol·m -2 ·s -1 , and the culture humidity was 50%.

[0121] After 40 days of light culture, the recovery induction rate was 13.5%; the average number of healthy adventitious buds induced per recovered individual was 1.08, and the adventitious buds were yellowish, shrunken, and 1-2 cm in height.

[0122] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detoxifying passion fruit, characterized in that: The following steps are involved: 1) inoculating passion fruit stem segments with axillary buds into a vitrification induction medium to induce plant vitrification to obtain vitrified plants; The vitrification induction medium is: MS+6-benzylaminopurine 1-6 mg / L, indoleacetic acid 0.2-1.2 mg / L, oligochitosan 2.0 mg / L, sucrose 30 g / L and agar 3.2-3.5 g / L; 2) inoculating the vitrified plants obtained in step 1) into a recovery induction medium for recovery culture to achieve detoxification of passion fruit; The recovery induction culture medium comprises: MS+6-benzylaminopurine 0.5-1.0 mg / L, indoleacetic acid 0.1-0.2 mg / L, oligochitosan 2.0 mg / L, sucrose 30 g / L and agar 3.5-4.2 g / L.

2. The detoxification method according to claim 1, characterized in that: The vitrification induction medium of step 1) is: MS+6-benzylaminopurine 4 mg / L, indoleacetic acid 0.8 mg / L, oligochitosan 2.0 mg / L, sucrose 30 g / L and agar 3.2-3.5 g / L.

3. The detoxification method according to claim 1, characterized in that: The step 2) recovers the induction medium comprising: MS+0.5 mg / L 6-benzylaminopurine, 0.1 mg / L indoleacetic acid, 2.0 mg / L oligochitosan, 30 g / L sucrose and 3.5 g / L agar.

4. The detoxification method according to claim 1, characterized in that: The passion fruit is a yellow fruit type passion fruit.

5. The detoxification method according to claim 1, characterized in that: The virus includes one or more of East Asian passion fruit virus, cucumber mosaic virus, tuberose mosaic virus and passion fruit lignification virus.

6. The detoxification method according to claim 1, characterized in that: The conditions for inducing plant vitrification in step 1) include: temperature of 28±3°C, light duration of 12h / d, light intensity of 40μmol·m -2 ·s -1 , the culture humidity is 80-90%, and the time is 40 days.

7. The detoxification method according to claim 1, characterized in that: The conditions for the recovery culture in step 2) include: a temperature of 28±3°C, a light exposure time of 12h / d, and a light intensity of 40μmol·m -2 ·s -1 , the culture humidity is 60-80%, and the time is 40 days.

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