A method for the virus-free multiplication of guava plants

Abstract

The present application relates to the technical field of fruit tree virus-free rapid propagation, and particularly relates to a method for virus-free propagation of passion fruit. The method comprises the following steps: inoculating stem tips of passion fruit into a callus induction medium to induce callus, obtaining callus; inoculating the obtained callus into a bud differentiation induction medium to induce regenerated buds, obtaining regenerated buds; inoculating the obtained regenerated buds into a strong seedling culture medium to culture strong seedlings, and realizing virus-free propagation of passion fruit. The above steps are referred to as a virus-free propagation cycle, and the virus-free propagation efficiency is 69%. The diseased plants that are still positive after detection after one cycle of virus-free propagation are subjected to a second cycle of virus-free propagation according to the above method, and the virus-free propagation efficiency can reach 95%.

Description

Technical Field

[0001] This invention relates to the field of rapid propagation technology for virus-free fruit trees, and in particular to a method for virus-free passion fruit. Background Technology

[0002] Passion fruit, scientifically known as *Passiflora edulia* sims., is a plant belonging to the genus *Passiflora* in the family Passifloraceae. It is a tropical and subtropical perennial evergreen vine producing berries. It grows rapidly, taking only a few months from planting to harvest, resulting in early production and quick returns. Furthermore, the fruit is resistant to storage and transportation, has a wide market, and offers high economic benefits. Due to its unique aroma and taste, it is increasingly favored by consumers.

[0003] For decades, passion fruit cultivation has primarily relied on asexual reproduction methods such as cuttings and grafting, leading to the continuous accumulation of viral diseases in the stems. These viral diseases have also spread rapidly with the increase in planting area. In actual production, the high frequency of passion fruit viral diseases causes reduced yields and even complete crop failure. The continuous accumulation of viral diseases leads to varietal degeneration, weakened resistance, and decreased yield. Therefore, producing healthy passion fruit seedlings through seedling detoxification technology to achieve stable and increased production in the passion fruit industry is the cornerstone of its healthy development.

[0004] This invention provides a simpler and more efficient method for detoxifying passion fruit by combining stem tip induction, callus induction, organ regeneration, and the addition of antiviral agents during the cultivation process. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for detoxifying passion fruit, achieving a high detoxification rate and providing technical and methodological support for passion fruit detoxification.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

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

[0008] 1) The stem tips of passion fruit were inoculated into callus induction medium to induce callus and obtain callus tissue;

[0009] The callus induction culture medium consisted of MS medium containing 2,4-dichlorophenoxyacetic acid 2–6 mg / L, 1-naphthaleneacetic acid 0.1–0.3 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L, and agar 3.5 g / L.

[0010] 2) The callus tissue obtained in step 1) is inoculated into a bud differentiation induction medium to induce bud regeneration and obtain regenerated buds;

[0011] The bud differentiation induction medium is MS+6-benzylaminopurine 0.5-1.2 mg / L, thidiazuron 0.5-1.5 mg / L, kinetin 0.5-1.5 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L and agar 3.5 g / L;

[0012] 3) inoculating the regenerated buds obtained in the step 2) into a strong seedling culture medium for strong seedling culture, so as to realize the virus elimination of the passion fruit;

[0013] The strong seedling culture medium is MS+1-naphthaleneacetic acid 0.2 mg / L, kinetin 0.5 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L and agar 3.5 g / L.

[0014] Preferably, the step 1) callus induction medium is MS+2,4-dichlorophenoxyacetic acid 4 mg / L, 1-naphthaleneacetic acid 0.2 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L and agar 3.5 g / L.

[0015] Preferably, the step 2) bud differentiation induction medium is MS+6-benzylaminopurine 0.8 mg / L, thidiazuron 1 mg / L, kinetin 1.5 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L and agar 3.5 g / L.

[0016] Preferably, the length of the step 1) stem tip is 1.5-2.5 mm.

[0017] Preferably, the step 1) callus induction conditions include: dark culture for 3 d, and then light culture for 20 d, and the light culture conditions are: temperature is 28±3℃, light time is 12 h / d, and light intensity is 40 μmol·m -2 ·s -1 .

[0018] Preferably, the step 2) regenerated bud induction conditions include: temperature is 28±3℃, light time is 12 h / d, and light intensity is 40 μmol·m -2 ·s -1 , and the culture time is 40 d.

[0019] Preferably, the step 3) strong seedling culture conditions include: temperature is 28±3℃, light time is 12 h / d, and light intensity is 40 μmol·m -2 ·s -1 , and the culture time is 40 d.

[0020] Preferably, after the step 3) strong seedling culture, passion fruit seedlings are obtained, and the virus on the obtained passion fruit seedlings is detected.

[0021] When the passion fruit seedlings carry virus, the steps 1)-3) are repeated once.

[0022] Preferably, the virus comprises one or more of Passiflora edulis virus, cucumber mosaic virus, Ipomoea nil mosaic virus and Passiflora lignification virus.

[0023] Preferably, the passion fruit is of the yellow fruit type.

[0024] Advantages of the present application:

[0025] The present application provides a method for passion fruit virus elimination, which starts from stem tip excision, stem tip callus induction, callus bud differentiation induction, seedling culture and secondary virus elimination of positive plants, explores suitable methods at each stage, perfects and optimizes the virus elimination technology system, so as to provide technical support for high efficiency of passion fruit virus elimination. Preferably, the stem tip callus induction culture is based on MS medium, the excised top bud is cultured in the medium for 23 days through effective hormone and antiviral agent combination, the callus induction rate is 91.0%, the callus produced is mostly white granular and a small amount is yellow granular; preferably, the callus bud differentiation culture is based on MS medium, the above callus is cultured under illumination for 12 days through effective hormone and antiviral agent combination, the bud differentiation is obvious, the bud seedling is 1.0-1.5 cm high after 40 days of culture, the bud induction rate (the number of callus capable of inducing buds / the number of callus inoculation) is 74%, and the average number of buds per callus is 6.44; the regenerated bud is transferred into the seedling culture medium for illumination culture for 40 days, the seedling is 4-5 cm high, the leaf is tender green and strong in growth; the virus disease of each seedling is detected, and the virus elimination efficiency is 69% (the number of individuals detected as negative / the total number of individuals); the above process is called a virus elimination cycle. The individuals still positive after the first virus elimination culture are used as materials for stem tip excision for the second virus elimination culture, and the virus elimination efficiency can reach 95%. The method combines stem tip + callus induction + organ regeneration + addition of antiviral agent (oligosaccharide) in the culture process, and the virus elimination efficiency can reach 95% after the second virus elimination culture. If the oligosaccharide is not added, four virus elimination cultures are needed to achieve this effect. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced.

[0027] Figure 1 The selected explant material is stem tip;

[0028] Figure 2 The stem tip of the explant material is excised;

[0029] Figure 3 The callus induced from the stem tip is obtained;

[0030] Figure 4 regenerating shoots from the callus;

[0031] Figure 5 planting the regenerated shoots in a rooting medium. DETAILED DESCRIPTION

[0032] The application provides a method for detoxifying Passiflora edulis, which comprises the following steps:

[0033] 1) inoculating the stem tip of Passiflora edulis into a callus induction medium to induce callus, so as to obtain callus;

[0034] The callus induction medium is MS+2,4-dichlorophenoxyacetic acid 2-6 mg / L, 1-naphthaleneacetic acid 0.1-0.3 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L and agar 3.5 g / L.

[0035] 2) inoculating the callus obtained in the step 1) into a bud differentiation induction medium to induce regenerated shoots, so as to obtain regenerated shoots;

[0036] The bud differentiation induction medium is MS+6-benzylaminopurine 0.5-1.2 mg / L, thidiazuron 0.5-1.5 mg / L, kinetin 0.5-1.5 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L and agar 3.5 g / L.

[0037] 3) inoculating the regenerated shoots obtained in the step 2) into a strong seedling culture medium to culture strong seedlings, so as to realize detoxification of Passiflora edulis;

[0038] The strong seedling culture medium is MS+1-naphthaleneacetic acid 0.2 mg / L, kinetin 0.5 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L and agar 3.5 g / L.

[0039] The application inoculates the stem tip of Passiflora edulis into a callus induction medium to induce callus, so as to obtain callus; the callus induction medium is MS+2,4-dichlorophenoxyacetic acid 2-6 mg / L, 1-naphthaleneacetic acid 0.1-0.3 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L and agar 3.5 g / L.

[0040] In this invention, the preferred callus induction culture medium is MS + 2,4-dichlorophenoxyacetic acid 4 mg / L, 1-naphthaleneacetic acid 0.2 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L, and agar 3.5 g / L. In this invention, the preferred passion fruit is the yellow-fruited passion fruit. In this invention, the preferred stem tip length is 1.5–2.5 mm. In this invention, the preferred callus induction conditions include: dark culture for 3 days, followed by light culture for 20 days. The light culture conditions are: temperature 28 ± 3℃, light duration 12 h / d, and light intensity 40 μmol·m⁻¹. -2 ·s -1 .

[0041] In this invention, the obtained callus tissue is inoculated into a shoot differentiation induction medium to induce regeneration of shoots. The shoot differentiation induction medium consists of: MS + 6-benzylaminopurine 0.5–1.2 mg / L, thiazuron 0.5–1.5 mg / L, kinetin 0.5–1.5 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L, and agar 3.5 g / L. Preferably, the shoot differentiation induction medium contains: MS + 6-benzylaminopurine 0.8 mg / L, thiazuron 1 mg / L, kinetin 1.5 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L, and agar 3.5 g / L. The preferred conditions for shoot regeneration induction include: a temperature of 28 ± 3 °C, a photoperiod of 12 h / d, and a light intensity of 40 μmol·m⁻¹. -2 ·s -1 Cultured for 40 days.

[0042] This invention involves inoculating the obtained regenerated shoots into a seedling culture medium for vigorous seedling cultivation to achieve virus detoxification of passion fruit. The seedling culture medium comprises: MS + 1-naphthaleneacetic acid 0.2 mg / L, kinetin 0.5 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L, and agar 3.5 g / L. In this invention, the preferred conditions for seedling cultivation include: a temperature of 28 ± 3℃, a photoperiod of 12 h / d, and a light intensity of 40 μmol·m⁻¹. -2 ·s -1 Cultured for 40 days.

[0043] This invention preferably involves cultivating robust seedlings to obtain passion fruit seedlings, and then detecting viruses on the obtained passion fruit seedlings. When the passion fruit seedlings carry viruses, steps 1) to 3) are repeated once. This invention preferably uses the method described in patent number ZL202010590046.9, entitled "A Method for Rapid Propagation of Passion Fruit with Virus Detoxification," to detect the virus in the passion fruit seedlings and assess the detoxification effect. In this invention, the viruses preferably include one or more of East Asian passion fruit virus, cucumber mosaic virus, night-blooming jasmine mosaic virus, and passion fruit lignification virus.

[0044] In order to further illustrate the present application, the present application is described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0045] Example 1

[0046] A method for detoxifying guava, comprising the following steps:

[0047] Step 1) Stem tip cutting

[0048] Taking the guava tissue culture seedlings (not detoxified) of the yellow fruit type as the object, the terminal bud is cut off with a sterilized scissors, the cutting length is 1.5-2.5 mm, and the terminal bud is inoculated into the subsequent callus induction medium.

[0049] Step 2) Guava stem tip callus induction

[0050] The above stem tip (1.5-2.5 mm) is inoculated into the callus induction medium, dark culture for 3 days, light culture for 20 days, the callus induction rate is counted, and the phenotype of the formed callus is observed; 30 are inoculated for each treatment, and the operation is repeated for 3 times. The callus induction medium takes MS as the basic medium, and further comprises 2,4-dichlorophenoxyacetic acid (2,4-D) 4.0 mg / L, 1-naphthaleneacetic acid (NAA) 0.2 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature is 28±3℃, the light time is 12 h / d, and the light intensity is 40 μmol·m -2 ·s -1 .

[0051] After 23 days of callus induction culture, the callus induction rate is 91.0%; the callus produced is mainly white granular and a small amount is yellow granular.

[0052] Example 2

[0053] A method for detoxifying guava, comprising the following steps:

[0054] Step 1) Same as Example 1

[0055] Step 2) Guava stem tip callus induction

[0056] The stem tips (1.5-2.5 mm) are inoculated in callus induction medium, dark culture for 3 days, light culture for 20 days, the callus induction rate is counted, and the phenotype of the formed callus is observed; 30 are inoculated in each treatment, and the operation is repeated for 3 times. The callus induction medium takes MS as the basic medium, and further comprises 2,4-dichlorophenoxyacetic acid (2,4-D) 2.0 mg / L, 1-naphthaleneacetic acid (NAA) 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℃, the light time is 12h / d, and the light intensity is 40μmol·m -2 ·s -1 .

[0057] The callus induction culture is carried out for 23 days, and the callus induction rate is 62.0%; the callus produced is mainly green and massive, and a small amount is yellow and granular.

[0058] Example 3

[0059] A detoxification method of guava, the steps are:

[0060] Step 1) same as example 1

[0061] Step 2) guava stem tip callus induction

[0062] The stem tips (1.5-2.5 mm) are inoculated in callus induction medium, dark culture for 3 days, light culture for 20 days, the callus induction rate is counted, and the phenotype of the formed callus is observed; 30 are inoculated in each treatment, and the operation is repeated for 3 times. The callus induction medium takes MS as the basic medium, and further comprises 2,4-dichlorophenoxyacetic acid (2,4-D) 2.0 mg / L, 1-naphthaleneacetic acid (NAA) 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℃, the light time is 12h / d, and the light intensity is 40μmol·m -2 ·s -1 .

[0063] The callus induction culture is carried out for 23 days, and the callus induction rate is 71.0%; the callus produced is mainly green and massive, and a small amount is yellow and granular.

[0064] Example 4

[0065] A detoxification method of guava, the steps are:

[0066] Step 1) same as example 1

[0067] Step 2) guava stem tip callus induction

[0068] The stem tips (1.5-2.5 mm) are inoculated in the callus induction medium, dark culture for 3 days, light culture for 20 days, the callus induction rate is counted, and the phenotype of the formed callus is observed; 30 are inoculated in each treatment, and the operation is repeated for 3 times. The callus induction medium takes MS as the basic medium, and further comprises 2,4-dichlorophenoxyacetic acid (2,4-D) 2.0 mg / L, 1-naphthaleneacetic acid (NAA) 0.3 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature is 28±3℃, the light time is 12h / d, and the light intensity is 40 μmol·m -2 ·s -1 .

[0069] The callus induction culture is carried out for 23 days, and the callus induction rate is 78.0%; the callus produced is mainly green and massive, and a small amount is yellow and granular.

[0070] Example 5

[0071] A detoxification method of passion fruit, the steps are:

[0072] Step 1) same as example 1

[0073] Step 2) passion fruit stem tip callus induction

[0074] The stem tips (1.5-2.5 mm) are inoculated in the callus induction medium, dark culture for 3 days, light culture for 20 days, the callus induction rate is counted, and the phenotype of the formed callus is observed; 30 are inoculated in each treatment, and the operation is repeated for 3 times. The callus induction medium takes MS as the basic medium, and further comprises 2,4-dichlorophenoxyacetic acid (2,4-D) 2.0 mg / L, 1-naphthaleneacetic acid (NAA) 0.3 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature is 28±3℃, the light time is 12h / d, and the light intensity is 40 μmol·m -2 ·s -1 .

[0075] The callus induction culture is carried out for 23 days, and the callus induction rate is 74.0%; the callus produced is mainly green and massive, and a small amount is yellow and granular.

[0076] Example 6

[0077] A detoxification method of passion fruit, the steps are:

[0078] Step 1) same as example 1

[0079] Step 2) passion fruit stem tip callus induction

[0080] The stem tips (1.5-2.5 mm) are inoculated in the callus induction medium, dark culture for 3 days, light culture for 20 days, the callus induction rate is counted, and the phenotype of the formed callus is observed; 30 are inoculated in each treatment, and the operation is repeated for 3 times. The callus induction medium takes MS as the basic medium, and further comprises 2,4-dichlorophenoxyacetic acid (2,4-D) 4.0 mg / L, 1-naphthaleneacetic acid (NAA) 0.3 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature is 28±3℃, the light time is 12h / d, and the light intensity is 40 μmol·m -2 ·s -1 .

[0081] The callus induction culture is carried out for 23 days, the callus induction rate is 92.0%, and the callus produced is mainly in the form of milky white block and a small amount in the form of yellow granular.

[0082] Example 7

[0083] A detoxification method of guava, comprising the following steps:

[0084] Step 1) is the same as example 1

[0085] Step 2) callus induction of guava stem tips

[0086] The stem tips (1.5-2.5 mm) are inoculated in the callus induction medium, dark culture for 3 days, light culture for 20 days, the callus induction rate is counted, and the phenotype of the formed callus is observed; 30 are inoculated in each treatment, and the operation is repeated for 3 times. The callus induction medium takes MS as the basic medium, and further comprises 2,4-dichlorophenoxyacetic acid (2,4-D) 4.0 mg / L, 1-naphthaleneacetic acid (NAA) 0.3 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature is 28±3℃, the light time is 12h / d, and the light intensity is 40 μmol·m -2 ·s -1 .

[0087] The callus induction culture is carried out for 23 days, the callus induction rate is 81.0%, and the callus produced is mainly in the form of milky white block and a small amount in the form of yellow granular.

[0088] Example 8

[0089] A detoxification method of guava, comprising the following steps:

[0090] Step 1) is the same as example 1

[0091] Step 2) callus induction of guava stem tips

[0092] The stem tips (1.5-2.5 mm) are inoculated in callus induction medium, dark culture for 3 days, light culture for 20 days, and the callus induction rate is counted, and the phenotype of the callus formed is observed; 30 are inoculated in each treatment, and the test is repeated 3 times. The callus induction medium is MS basic medium, and further includes 2,4-dichlorophenoxyacetic acid (2,4-D) 6.0 mg / L, 1-naphthaleneacetic acid (NAA) 0.2 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L; the test culture temperature is 28±3℃, the light time is 12h / d, and the light intensity is 40μmol·m -2 ·s -1 .

[0093] The callus induction culture is carried out for 23 days, and the callus induction rate is 72.0%; the callus produced is mainly in the form of milky white blocks, and a small amount is in the form of yellow granules.

[0094] Example 9

[0095] A method for detoxifying Passiflora edulis, comprising the following steps:

[0096] Step 1) same as Example 1

[0097] Step 2) callus induction of Passiflora edulis stem tips

[0098] The stem tips (1.5-2.5 mm) are inoculated in callus induction medium, dark culture for 3 days, light culture for 20 days, and the callus induction rate is counted, and the phenotype of the callus formed is observed; 30 are inoculated in each treatment, and the test is repeated 3 times. The callus induction medium is MS basic medium, and further includes 2,4-dichlorophenoxyacetic acid (2,4-D) 6.0 mg / L, 1-naphthaleneacetic acid (NAA) 0.2 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L, and agar 3.5 g / L; the test culture temperature is 28±3℃, the light time is 12h / d, and the light intensity is 40μmol·m -2 ·s -1 .

[0099] The callus induction culture is carried out for 23 days, and the callus induction rate is 62.0%; the callus produced is mainly in the form of milky white blocks, and a small amount is in the form of yellow granules.

[0100] Example 10

[0101] A method for detoxifying Passiflora edulis, comprising the following steps:

[0102] Step 1) same as Example 1

[0103] Step 2) same as Example 1

[0104] Step 3) callus bud differentiation induction of Passiflora edulis

[0105] The callus cultured for 30 days in step 2) is used as material, which is inoculated into the callus bud differentiation induction medium. The callus bud differentiation induction medium takes MS as basic medium, and further comprises 6-BA 0.5 mg / L, TDZ 0.5 mg / L, KT 0.5 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature is 28±3℃, the illumination time is 12 h / d, and the illumination intensity is 40 μmol·m-2s-1. -2 -1 .

[0106] After illumination culture for 14 days, the bud differentiation is obvious, and after culture for 40 days, the bud seedlings are 1.5-2.0 cm high; the bud induction rate (the number of calli capable of inducing buds / the number of inoculated calli) is 47%, and the average number of bud sprouts per callus is 2.2.

[0107] Example 11

[0108] A detoxification method of Passiflora edulis Sims is provided, which comprises the following steps:

[0109] Step 1) is the same as that in Example 1

[0110] Step 2) is the same as that in Example 1

[0111] Step 3) is the bud differentiation induction of Passiflora edulis Sims callus

[0112] The callus cultured for 30 days in step 2) is used as material, which is inoculated into the callus bud differentiation induction medium. The callus bud differentiation induction medium takes MS as basic medium, and further comprises 6-BA 0.5 mg / L, TDZ 0.5 mg / L, KT 0.5 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature is 28±3℃, the illumination time is 12 h / d, and the illumination intensity is 40 μmol·m-2s-1. -2 -1 .

[0113] After illumination culture for 14 days, the bud differentiation is obvious, and after culture for 40 days, the bud seedlings are 1.5-2.0 cm high; the bud induction rate (the number of calli capable of inducing buds / the number of inoculated calli) is 47%, and the average number of bud sprouts per callus is 2.2.

[0114] Example 12

[0115] A detoxification method of Passiflora edulis Sims is provided, which comprises the following steps:

[0116] Step 1) is the same as that in Example 1

[0117] Step 2) is the same as that in Example 1​​

[0118] Step 3) Induction of bud differentiation of passion fruit callus

[0119] The callus cultured for 30 days in step 2) was inoculated into the induction medium for bud differentiation of callus. The induction medium for bud differentiation of callus was MS basic medium, further comprising 6-BA 0.5 mg / L, TDZ 1.5 mg / L, KT 1.5 mg / L, oligosaccharomyces 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature was 28±3℃, the light time was 12 h / d, and the light intensity was 40 μmol·m -2 ·s -1 .

[0120] After 14 days of illumination culture, the bud differentiation was obvious, and after 40 days of culture, the bud seedlings were 1.2-1.8 cm high; the bud induction rate (the number of calli capable of inducing buds / the number of callus inoculation) was 54%, and the average number of bud sprouting from each callus was 2.64.

[0121] Example 13

[0122] A detoxification method of passion fruit, comprising the following steps:

[0123] Step 1) The same as example 1

[0124] Step 2) The same as example 1

[0125] Step 3) Induction of bud differentiation of passion fruit callus

[0126] The callus cultured for 30 days in step 2) was inoculated into the induction medium for bud differentiation of callus. The induction medium for bud differentiation of callus was MS basic medium, further comprising 6-BA 0.5 mg / L, TDZ 1.5 mg / L, KT 1.5 mg / L, oligosaccharomyces 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature was 28±3℃, the light time was 12 h / d, and the light intensity was 40 μmol·m -2 ·s -1 .

[0127] After 14 days of illumination culture, the bud differentiation was obvious, and after 40 days of culture, the bud seedlings were 1.2-1.8 cm high; the bud induction rate (the number of calli capable of inducing buds / the number of callus inoculation) was 54%, and the average number of bud sprouting from each callus was 2.64.

[0128] Example 14

[0129] A detoxification method of passion fruit, comprising the following steps:

[0130] Step 1) The same as example 1

[0131] Step 2) same as Example 1

[0132] Step 3) induction of bud differentiation of passion fruit callus

[0133] The callus cultured for 30 days in Step 2) was used as material and inoculated into the induction medium for bud differentiation of callus. The induction medium for bud differentiation of callus was MS basic medium, further comprising 6-BA 0.8 mg / L, TDZ 1.0 mg / L, KT 1.5 mg / L, mannose 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature was 28±3℃, the light time was 12 h / d, and the light intensity was 40 μmol·m -2 ·s -1 .

[0134] After 12 days of illumination culture, the bud differentiation was obvious, and after 40 days of culture, the bud seedlings were 1.0-1.5 cm high; the bud induction rate (the number of calli capable of inducing buds / the number of callus inoculated) was 74%, and the average number of buds per callus was 6.44.

[0135] Example 15

[0136] A detoxification method of passion fruit, comprising the following steps:

[0137] Step 1) same as Example 1

[0138] Step 2) same as Example 1

[0139] Step 3) induction of bud differentiation of passion fruit callus

[0140] The callus cultured for 30 days in Step 2) was used as material and inoculated into the induction medium for bud differentiation of callus. The induction medium for bud differentiation of callus was MS basic medium, further comprising 6-BA 0.8 mg / L, TDZ 1.0 mg / L, KT 1.5 mg / L, mannose 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature was 28±3℃, the light time was 12 h / d, and the light intensity was 40 μmol·m -2 ·s -1 .

[0141] After 12 days of illumination culture, the bud differentiation was obvious, and after 40 days of culture, the bud seedlings were 1.0-1.5 cm high; the bud induction rate (the number of calli capable of inducing buds / the number of callus inoculated) was 74%, and the average number of buds per callus was 6.44.

[0142] Example 16

[0143] A detoxification method of passion fruit, comprising the following steps:

[0144] Step 1) same as Example 1

[0145] Step 2) same as Example 1

[0146] Step 3) bud differentiation induction of passion fruit callus

[0147] The callus cultured for 30 days in Step 2) was used as material and inoculated into the bud differentiation induction medium of callus. The bud differentiation induction medium of callus was MS basic medium, further comprising 6-BA 1.2 mg / L, TDZ 0.5 mg / L, KT 1.5 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature was 28±3℃, the light time was 12 h / d, and the light intensity was 40 μmol·m -2 ·s -1 .

[0148] After 12 days of light culture, the bud differentiation was obvious, and after 40 days of culture, the bud seedlings were 1.4-2.2 cm high; the bud induction rate (the number of calli capable of inducing buds / the number of callus inoculation) was 59%, and the average number of buds per callus was 4.13.

[0149] Example 17

[0150] A detoxification method of passion fruit, the steps are:

[0151] Step 1) same as Example 1

[0152] Step 2) same as Example 1

[0153] Step 3) bud differentiation induction of passion fruit callus

[0154] The callus cultured for 30 days in Step 2) was used as material and inoculated into the bud differentiation induction medium of callus. The bud differentiation induction medium of callus was MS basic medium, further comprising 6-BA 1.2 mg / L, TDZ 0.5 mg / L, KT 1.5 mg / L, oligosaccharide 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature was 28±3℃, the light time was 12 h / d, and the light intensity was 40 μmol·m -2 ·s -1 .

[0155] After 12 days of light culture, the bud differentiation was obvious, and after 40 days of culture, the bud seedlings were 1.4-2.2 cm high; the bud induction rate (the number of calli capable of inducing buds / the number of callus inoculation) was 59%, and the average number of buds per callus was 4.13.

[0156] Example 18

[0157] A method for detoxifying Passiflora edulis, comprising the following steps:

[0158] Step 1) same as Example 1

[0159] Step 2) same as Example 1

[0160] Step 3) induction of bud differentiation of Passiflora edulis callus

[0161] The callus cultured for 30 days in Step 2) is inoculated into a medium for induction of bud differentiation of callus. The medium for induction of bud differentiation of callus comprises MS as a basic medium, 6-BA 1.2 mg / L, TDZ 1.5 mg / L, KT 1.0 mg / L, oligosaccharomyces 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the culture temperature is 28±3℃, the light time is 12 h / d, and the light intensity is 40 μmol·m -2 ·s -1 .

[0162] After 12 days of illumination culture, bud differentiation is obviously observed, and after 40 days of culture, the height of the seedlings is 1.3-2.3 cm; the bud induction rate (the number of calli inducing buds / the number of calli inoculated) is 54%, and the average number of buds induced from each callus is 4.07.

[0163] Example 19

[0164] A method for detoxifying Passiflora edulis, comprising the following steps:

[0165] Step 1) same as Example 1

[0166] Step 2) same as Example 1

[0167] Step 3) same as Example 14

[0168] Step 4) seedling hardening culture

[0169] The seedlings with a height of more than 1 cm cultured for 40 days in Step 3) are transferred into a hardening medium for hardening culture, and each seedling is numbered. The hardening medium comprises MS as a basic medium, NAA 0.2 mg / L, KT 0.5 mg / L, oligosaccharomyces 2.0 mg / L, sucrose 30 g / L and agar 3.5 g / L; the culture temperature is 28±3℃, the light time is 12 h / d, and the light intensity is 40 μmol·m -2 ·s -1 .

[0170] After 40 days of illumination culture, the height of the seedlings is 4-5 cm, the leaves are tender green, and the growth is strong.

[0171] Step 5) Detection of major viral diseases of passion fruit

[0172] The leaves of the 40-day-old tissue culture seedlings in step 4) were cut, frozen with liquid nitrogen, and then each tissue culture seedling was detected for major viral diseases of passion fruit (East Asian Passiflora virus, Cucumber mosaic virus, Evening primrose mosaic virus, and Passiflora lignification virus) according to the method described in "A Passion Fruit Virus Elimination and Rapid Propagation Method (Patent No. ZL202010590046.9)". Finally, the virus elimination efficiency was 69% through detection.

[0173] Example 20

[0174] A passion fruit virus elimination method, the steps are:

[0175] Step 1) Passion fruit shoot tip cutting (second generation)

[0176] The tissue culture seedlings that were still positive for virus detection after the first virus elimination culture in steps 1) to 4) in Example 19 were used as materials, the top buds were cut off with sterilized scissors, the cutting length was 1.5-2.5 mm, and the top buds were inoculated into the subsequent callus induction medium.

[0177] Step 2) Passion fruit callus induction (second generation)

[0178] The same as Example 19

[0179] Step 3) Passion fruit callus bud differentiation culture (second generation)

[0180] The same as Example 19

[0181] Step 4) Passion fruit bud seedling strengthening culture (second generation)

[0182] The same as Example 19

[0183] Step 5) Detection of major viral diseases of passion fruit second generation bud seedlings

[0184] The same as Example 19, and the total virus elimination efficiency can reach 95% through detection.

[0185] Comparative Example 1

[0186] A passion fruit virus elimination method, the steps are:

[0187] Step 1) The same as Example 1

[0188] Step 2) Passion fruit shoot tip callus induction

[0189] The stem tips (1.5-2.5 mm) were inoculated in the callus induction medium, dark culture for 3 days, light culture for 20 days, the callus induction rate was counted, and the phenotype of the formed callus was observed; 120 were inoculated in each treatment, and repeated 3 times. The callus induction medium was MS basic medium, further comprising 2,4-dichlorophenoxyacetic acid (2,4-D) 4.0 mg / L, 1-naphthaleneacetic acid (NAA) 0.2 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature was 28±3℃, the light time was 12h / d, and the light intensity was 40μmol·m -2 ·s -1 .

[0190] The callus induction culture was 23 days, and the callus induction rate was 82.0%; the callus produced was mostly milky white granular, and a small amount was yellow granular.

[0191] Step 3) Guava callus bud differentiation induction

[0192] The callus cultured for 30 days in step 2) was used as material, which was inoculated into the callus bud differentiation induction medium. The callus bud differentiation induction medium was MS basic medium, further comprising 6-BA 0.8 mg / L, TDZ 1.0 mg / L, KT 1.5 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature was 28±3℃, the light time was 12h / d, and the light intensity was 40μmol·m -2 ·s -1 .

[0193] Light culture for 12 days, the bud differentiation began to be obvious, and the bud seedlings were 0.8-1.5 cm high after 40 days of culture; the bud induction rate (the number of calli that could induce buds / the number of calli inoculated) was 64%, and the average number of buds per callus was 4.86.

[0194] Step 4) Bud seedling hardening culture

[0195] The bud seedlings with a height of more than 1 cm in step 3) were transferred into the hardening medium for hardening culture, and each was numbered. The hardening medium was MS basic medium, further comprising NAA 0.2 mg / L, KT 0.5 mg / L, sucrose 30 g / L and agar 3.5 g / L; the test culture temperature was 28±3℃, the light time was 12h / d, and the light intensity was 40μmol·m -2 ·s -1 .

[0196] Light culture for 40 days, the seedlings were 2-5 cm high, the leaves were tender green, and the growth was strong.

[0197] Step 5) Main viral disease detection of passion fruit

[0198] The leaves of the 40-day-old tissue culture seedlings in step 4) were quickly frozen with liquid nitrogen, and each tissue culture seedling was subjected to main viral disease detection (East Asian passion fruit virus, cucumber mosaic virus, evening primrose mosaic virus and passion fruit woodiness virus). The detection method is the method specified in "A passion fruit virus-free rapid propagation method (Patent No. ZL202010590046.9)".

[0199] Comparative Example 2

[0200] A passion fruit virus-free method, the steps are:

[0201] Step 1) Passion fruit shoot tip excision (second generation)

[0202] The tissue culture seedlings that were still positive in virus detection after the first virus-free culture in steps 1) to 4) in Comparative Example 1 were used as materials, the terminal bud was cut off with sterilized scissors, the cutting length was 1.5-2.5 mm, and the terminal bud was inoculated into the subsequent callus induction medium.

[0203] Step 2) Passion fruit shoot tip callus induction (second generation)

[0204] The same as Comparative Example 1

[0205] Step 3) Passion fruit callus bud differentiation induction (second generation)

[0206] The same as Comparative Example 1

[0207] Step 4) Passion fruit bud seedling culture (second generation)

[0208] The same as Comparative Example 1

[0209] Step 5) Main viral disease detection of passion fruit

[0210] The same as Comparative Example 1, and the final detection showed that the virus-free efficiency was 55%.

[0211] Comparative Example 3

[0212] A passion fruit virus-free method, the steps are:

[0213] Step 1) Passion fruit shoot tip excision (third generation)

[0214] The tissue culture seedlings that were still positive in virus detection after the second virus-free culture in steps 1) to 4) in Comparative Example 2 were used as materials, the terminal bud was cut off with sterilized scissors, the cutting length was 1.5-2.5 mm, and the terminal bud was inoculated into the subsequent callus induction medium.

[0215] Step 2) Passion fruit shoot tip callus induction (third generation)

[0216] Example 1

[0217] Step 3) Induction of bud differentiation from passion fruit callus (the third generation)

[0218] Example 1

[0219] Step 4) Plantlet cultivation (the third generation)

[0220] Example 1

[0221] Step 5) Detection of main viral diseases of passion fruit

[0222] Example 1, and the final detection showed that the detoxification efficiency was 81%.

[0223] Example 4

[0224] A detoxification method of passion fruit, the steps are as follows:

[0225] Step 1) Stem tip cutting of passion fruit (the fourth generation)

[0226] The plantlets in Example 3 which were still positive in viral detection after the third detoxification cultivation of steps 1) to 4) were used as materials, the top buds were cut off with sterilized scissors, the cutting length was 1.5-2.5 mm, and the top buds were inoculated into the subsequent callus induction medium.

[0227] Step 2) Induction of passion fruit stem tip callus (the fourth generation)

[0228] Example 1

[0229] Step 3) Induction of bud differentiation from passion fruit callus (the fourth generation)

[0230] Example 1

[0231] Step 4) Plantlet cultivation (the fourth generation)

[0232] Example 1

[0233] Step 5) Detection of main viral diseases of passion fruit

[0234] Example 1, and the final detection showed that the detoxification efficiency was 96%.

[0235] Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. A method for detoxifying passion fruit, characterized in that, Includes the following steps: 1) The stem tips of passion fruit were inoculated into callus induction medium to induce callus and obtain callus tissue; The callus induction culture medium consisted of MS medium containing 2,4-dichlorophenoxyacetic acid 2–6 mg / L, 1-naphthaleneacetic acid 0.1–0.3 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L, and agar 3.5 g / L. 2) The callus tissue obtained in step 1) is inoculated into a bud differentiation induction medium to induce bud regeneration and obtain regenerated buds; The bud differentiation induction medium consisted of MS medium containing 0.5–1.2 mg / L of 6-benzylaminopurine, 0.5–1.5 mg / L of thiazuron, 0.5–1.5 mg / L of kinetin, 2 mg / L of oligosaccharide, 30 g / L of sucrose, and 3.5 g / L of agar. 3) The regenerated buds obtained in step 2) are inoculated into a seedling culture medium for seedling cultivation to achieve passion fruit detoxification; The seedling culture medium consisted of MS + 1-naphthaleneacetic acid 0.2 mg / L, kinetin 0.5 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L and agar 3.5 g / L. In step 1), the length of the stem tip is 1.5–2.5 mm; The conditions for callus induction in step 1) include: dark culture for 3 days, followed by light culture for 20 days. The light culture conditions are: temperature 28±3℃, light duration 12h / d, and light intensity 40μmol·m⁻¹. -2 ·s -1 ; The conditions for inducing regenerated shoots in step 2) include: a temperature of 28±3℃, a light duration of 12h / d, and a light intensity of 40μmol·m. -2 ·s -1 Cultured for 40 days; The conditions for seedling cultivation in step 3) include: a temperature of 28±3℃, a photoperiod of 12h / d, and a light intensity of 40μmol·m⁻². -2 ·s -1 Cultured for 40 days; After the seedling cultivation in step 3), passion fruit seedlings are obtained, and the viruses on the obtained passion fruit seedlings are detected. When the passion fruit seedlings are infected with the virus, repeat steps 1) to 3) once.

2. The method according to claim 1, characterized in that, The callus induction culture medium in step 1) is: MS + 4 mg / L 2,4-dichlorophenoxyacetic acid, 0.2 mg / L 1-naphthaleneacetic acid, 2 mg / L oligosaccharide, 30 g / L sucrose and 3.5 g / L agar.

3. The method according to claim 1, characterized in that, The bud differentiation induction medium in step 2) is: MS + 6-benzylaminopurine 0.8 mg / L, thidiazuron 1 mg / L, kinetin 1.5 mg / L, oligosaccharide 2 mg / L, sucrose 30 g / L and agar 3.5 g / L.

4. The method according to claim 1, characterized in that, The viruses include one or more of the following: East Asian passion fruit virus, cucumber mosaic virus, night-blooming jasmine mosaic virus, and passion fruit lignification virus.

5. The method according to claim 1, characterized in that, The passion fruit mentioned is the yellow-fruited type.

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