A method for virus-free cultivation of purple passion fruit stem tip

Abstract

The present invention discloses a method for detoxification culture of purple passion fruit stem tip. The present invention breaks through the original limitation of only being able to remove one virus, successfully removes all the main viruses existing in the main passion fruit producing areas in Guizhou, and achieves a technical breakthrough. The present invention is simple to operate and can be used for the culture of detoxified mother plants. After testing, the detoxification effect is that the detoxification rates of TeMV, PLV, and EAPV are all 100%.

Description

Technical Field

[0001] The invention relates to the technical field of agriculture, in particular to a method for virus-free cultivation of purple passion fruit stem tips. Background Art

[0002] The yield of passion fruit is relatively high, and the yield data can exceed 2,000 kg / mu. In 2018, the national yield of passion fruit per mu was 1,146.2 kg, while the average yield of passion fruit per mu in Guizhou Province was less than 500 kg, less than 1 / 2 of the domestic average. There are other reasons such as management level, but the poor quality of seedlings is the most fundamental reason. More than 90% of the seedlings purchased in Guizhou are not produced from seedlings bred from regular production channels. Not only are they of poor quality, they also carry various viruses. Through high-throughput sequencing of 105 passion fruit leaves from the main passion fruit producing areas in Guizhou, it was found that the detection rates of TeMV (Evening Jasmine Mosaic Virus), EAPV (East Asian Passion Fruit Virus) and PLV (Passion Fruit Latent Virus) were 100%, 81.9% and 74.3% respectively, indicating that the passion fruit in the main production areas of Guizhou are all infected with viruses to varying degrees, and the virus infection is serious in some areas.

[0003] After the passion fruit is infected with the virus, the leaves will show mosaic, deformity, and yellowing, and the fruit will show deformity and lignification. The passion fruit virus disease will seriously affect the growth of the passion fruit. The infection of seedlings will cause the plants to grow slowly, and the infected plants will have fewer fruits, a high rate of fruit deformity, and a decrease in fruit water content. In severe cases, the fruit will die.

[0004] Therefore, if Guizhou wants to continue to develop the passion fruit industry, it must first solve the bottleneck and fundamental problem of high-quality seedlings, and the promotion of high-quality and healthy seedlings is imperative. Summary of the invention

[0005] The object of the present invention is to provide a method for virus-free cultivation of purple passion fruit stem tips, which can effectively remove TeMV, PLV and EAPV present in passion fruit seedlings and improve yield and quality.

[0006] The technical solution of the present invention is a method for virus-free cultivation of purple passion fruit stem tips, comprising the following steps:

[0007] (1) Axillary bud disinfection: The axillary buds of purple passion fruit were first immersed in 75% ethanol for 30 seconds, then washed twice with sterile water, each time for 2 minutes, and then disinfected with 0.1% HgCl2 solution for 15 minutes, and then thoroughly washed with sterile water;

[0008] (2) Axillary bud culture: After the axillary buds are sterilized and dried, they are cultured in a medium containing MS + 2.0 mg / L 6-BA and 0.1 mg / L NAA to obtain axillary buds with strong growth;

[0009] (3) Peeling off the stem tip: cutting off the newly grown tender tip of the axillary bud, and then sterilizing it in the manner of step (1), peeling off the stem tip of 0.6 mm; the smaller the peeled stem tip, the better the detoxification effect, but it is not easy to survive. The stem tip size of the predecessors was 1 mm, but the present invention reduces the stem tip size to 0.6 mm while ensuring the survival rate, which is more conducive to removing the virus from the tissue culture seedlings.

[0010] (4) Induction culture: The peeled stem tip is inoculated into the induction culture medium for induction culture. After 30 days of culture, a cluster of green buds appears;

[0011] (5) Proliferation culture: When the stem tip grows to 5-7 cm, cut the stem tip into segments, each segment is 0.4-0.6 cm long and has one internode, and inoculate into MS+1.5 mg / L 6-BA and 0.1 mg / L IBA medium for subculture;

[0012] (6) Rooting culture: Select proliferating shoots with a length of 2 to 3 cm, cut the shoots from the base, and inoculate them into 1 / 2MS + IBA 0.1 mg / L medium for rooting culture;

[0013] (7) Secondary stem tip virus-free culture: Take a healthy tissue culture seedling, cut off a 3 cm stem segment with a terminal bud, and culture it according to the steps (3) to (6); secondary virus-free culture often improves the virus-free effect, but significantly reduces the survival rate. The secondary virus-free culture of the present invention can increase the virus-free rate to 100%, while maintaining the survival rate above 70%. Compared with the current conventional secondary virus-free culture, the survival rate is increased by about 30%.

[0014] (8) Hardening and transplanting: When the roots of the tissue culture seedlings grow to 2-3 cm, select tissue culture seedlings with uniform growth, 5-7 cm plant height and 4-5 main roots, and place their tissue culture bottles indoors in a place not exposed to direct sunlight for 5-7 days. After the hardening is completed, take out the tissue culture seedlings and gently clean the residual culture medium on the roots with running water. Then transplant them into the seedling pot and irrigate them with enough water for rooting. After 10-15 days after transplanting, new roots and new leaves will grow to obtain virus-free seedlings.

[0015] The culture conditions are as follows: the culture temperature is 25±2°C, the light intensity is 2 000 lx, the light duration is 12 h / d, and the relative humidity is 85-90%.

[0016] The composition of the induction medium is MS as the basic medium, to which 4.74 g / L MS powder, 30 g / L sucrose, 7 g / L agar, 1.0 mg / L 6-BA and 0.1 mg / L NAA are added, and the pH value is adjusted to 6.0.

[0017] The proliferation medium is composed of MS as the basic medium, to which 4.74 g / L MS powder, 30 g / L sucrose, 7 g / L agar, 1.5 mg / L 6-BA and 0.1 mg / L IBA are added, and the pH value is adjusted to 6.0.

[0018] The rooting medium is composed of 1 / 2MS as the basic medium, to which 2.47 g / L MS powder, 30 g / L sucrose, 7 g / L agar, and 0.1 mg / L IBA are added, and the pH value is adjusted to 6.0.

[0019] The seedling raising medium is a mixture of peat soil and perlite in a mass ratio of 4:1.

[0020] The beneficial effects of the present invention are as follows: compared with the prior art background, the present invention breaks through the original limitation of only being able to remove one virus, successfully removes all the main viruses existing in the main passion fruit producing areas in Guizhou, and achieves a technical breakthrough. The present invention is simple to operate and can be used for the cultivation of detoxified mother plants. After testing, the detoxification effect is that the detoxification rates of TeMV, PLV, and EAPV are all 100%. Compared with the current conventional detoxification, not only can three viruses be removed at the same time, but the removal rates of the three viruses all reach 100%, which can be said to be a very important breakthrough. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The virus detection result after the first detoxification of the present invention;

[0022] Figure 1 Middle: M: DNA standard molecular weight; 1-9: first shoot tip detoxification tissue culture seedlings; 10-12: shoot tip detoxification tissue culture seedlings; 13: blank control

[0023] Figure 2 This is the virus detection effect after the second detoxification of the present invention;

[0024] Figure 2 Middle: A: TeMV, B: EAPV, C: PLV, M: DNA marker; 1-5: shoot tip detoxified tissue culture seedlings; 6: tissue culture seedlings without shoot tip detoxification; 7: blank control. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0026] Test location: Laboratory of Guizhou Fruit Science Research Institute, Lurong Township, Zhenfeng County, Qianxinan Prefecture

[0027] Test time: 2019

[0028] Experimental materials: Purple passion fruit materials selected from the Zhenning base of Guizhou Fruit Science Research Institute

[0029] Experimental method: The method of virus-free cultivation of the stem tip of "Qianxiang No. 1" purple passion fruit.

[0030] (1) Axillary bud disinfection: The axillary buds of "Qianxiang No. 1" purple passion fruit were first fully immersed in 75% ethanol for 30 seconds, washed twice with sterile water, each time for 2 minutes, and then disinfected with 0.1% HgCl2 solution. During the disinfection, the beaker was constantly shaken to allow the disinfectant to fully contact the material. After 15 minutes, it was rinsed with sterile water for 6 times, each time for 2 minutes;

[0031] (2) Axillary bud culture: After the axillary buds are sterilized and dried, they are cultured in MS+2.0 mg / L 6-BA+0.1 mg / L NAA medium to obtain axillary buds with strong growth;

[0032] (3) Peeling the stem tip: Cut the newly grown tender tip of the axillary bud, disinfect it in (1), and peel off the 0.6 mm stem tip under a dissecting microscope;

[0033] (4) Induction culture: The peeled stem tip was inoculated into MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA medium for induction culture. After 30 days of culture, a cluster of green buds appeared;

[0034] (5) Proliferation culture: When the stem tip grows to 5-7 cm, cut it into segments for subculture. Each segment is about 0.5 cm long and has one internode. Inoculate it into MS+1.5 mg / L 6-BA+0.1 mg / L IBA medium for subculture.

[0035] (6) Rooting culture: Select proliferating shoots with a length of 2 to 3 cm, cut the shoots from the base, and inoculate them into 1 / 2MS + IBA 0.1 mg / L medium for rooting culture;

[0036] (7) Secondary stem tip virus-free culture: Take a healthy test tube seedling, cut off a 3 cm stem segment with a terminal bud, and culture it according to steps (3) to (6);

[0037] (8) Hardening and transplanting: When the roots of the tissue culture seedlings grow to 2-3 cm, select tissue culture seedlings with uniform growth, plant height of 5-7 cm and 4-5 main roots, and place their tissue culture bottles indoors in a place not exposed to direct sunlight for about 5-7 days. After the hardening is completed, take out the tissue culture seedlings from the bottles, gently clean the residual culture medium on the roots with running water, and then transplant them into seedling pots. Use a matrix of peat soil: perlite = 4:1 as the transplanting medium, irrigate with enough water for rooting, and wait 10-15 days after transplanting for new roots and new leaves to obtain virus-free seedlings.

[0038] (9) Planting: In July 2020, the virus-free seedlings of purple passion fruit "Qianxiang No. 1" obtained in the laboratory of Guizhou Fruit Science Research Institute were planted in the mother garden in Lurong Township, Zhenfeng County, Qianxinan Prefecture.

[0039] (10) Field management: Use a trellis-type frame and apply 10 kg of farmyard manure and 0.5 kg of compound fertilizer to each plant. Passion fruit is a waterlogged species, so just keep the soil moist.

[0040] Culture conditions: culture temperature is 25±2℃, light intensity is 2 000lx, light duration is 12h / d, relative humidity is 85-90%.

[0041] The composition of the induction medium includes: MS as the basic medium, to which 4.74g / L MS powder, 30g / L sucrose, 7g / L agar, 1.0mg / L 6-BA, 0.1mg / L NAA are added, and the pH value is adjusted to 6.0.

[0042] The composition of the proliferation medium includes: MS as the basic medium, to which 4.74g / L MS powder, 30g / L sucrose, 7g / L agar, 1.5mg / L 6-BA, 0.1mg / L IBA are added, and the pH value is adjusted to 6.0.

[0043] The composition of the rooting medium includes: 1 / 2MS as the basic medium, to which 2.47g / L MS powder, 30g / L sucrose, 7g / L agar, 0.1mg / L IBA are added, and the pH value is adjusted to 6.0.

[0044] This embodiment adopts the basic culture medium as follows:

[0045] Table 1 Basic culture medium formula

[0046]

[0047] The Chinese name of BA is 6-benzylaminoadenine, and its chemical formula is C12H11N5.

[0048] The Chinese name of NAA is naphthylacetic acid, and its chemical formula is C12H10O2.

[0049] The chemical name of IBA is indolebutyric acid, and its chemical formula is C12H12NO2.

[0050] The results of PCR are shown in the figure to explain the effect of secondary detoxification in detail.

[0051] according to Figure 1-2 It can be seen that after the first detoxification, TeMV and PLV were not completely removed, especially PLV, but after the second detoxification, all three viruses were removed, and the removal rate reached 100%, indicating that the detoxified seedlings after the second detoxification can be used as mother plants for seedling cultivation.

[0052] Table 2 Specific primers for RT-PCR detection of three viruses

[0053]

[0054] In February 2020, the applicant randomly took 10 leaves and sent them to the Institute of Biotechnology, Chinese Academy of Tropical Agricultural Sciences for analysis and testing. The test method was to extract RNA from the sample leaves, reverse transcribe and synthesize cDNA, which served as a template for detecting the target virus nucleotide sequence; using designed conservative primers, the cDNA of the passion fruit suspected virus samples was PCR amplified and sequenced for identification.

Claims

1. A method for virus-free cultivation of purple passion fruit stem tip, characterized in that: The steps include: (1) Disinfection of axillary buds: The axillary buds of purple passion fruit were first immersed in 75% ethanol for 30 seconds, then washed twice with sterile water, each time for 2 minutes, and then disinfected with 0.1% HgCl2 solution for 15 minutes, and then thoroughly washed with sterile water; (2) Axillary bud culture: After the axillary buds are sterilized and dried, they are cultured in MS + 2.0 mg / L 6-BA and 0.1 mg / L NAA medium to obtain strong axillary buds. (3) Peeling the stem tip: Cut the newly grown tender tip of the axillary bud, and then sterilize it in the manner of step (1) to peel off the 0.6 mm stem tip; (4) Induction culture: The peeled stem tip is inoculated into the induction culture medium for induction culture. After 30 days of culture, a cluster of green buds will appear; (5) Proliferation culture: When the stem tip grows to 5-7 cm, cut the stem tip into segments, each 0.4-0.6 cm long with one internode, and inoculate them into the proliferation medium for subculture; (6) Rooting culture: Select proliferating shoots 2 to 3 cm in length, cut off the shoots from the base, and inoculate them into rooting medium for rooting culture; (7) Secondary stem tip virus-free culture: Take a robust test tube tissue culture seedling, cut off a 3 cm long stem segment with a terminal bud, and culture it according to steps (3) to (6); (8) Hardening and transplanting: When the roots of the tissue culture seedlings grow to 2-3 cm, select tissue culture seedlings with uniform growth, 5-7 cm plant height and 4-5 taproots, and place their tissue culture bottles indoors in a place not exposed to direct sunlight for 5-7 days. After the hardening is completed, take out the tissue culture seedlings and gently clean the residual culture medium on the roots with running water. Then transplant them into the seedling pot and water them with enough water for rooting. After 10-15 days after transplanting, new roots and new leaves will grow, and the virus-free seedlings will be obtained; The composition of the induction medium is MS as the basic medium, to which 4.74 g / L MS powder, 30 g / L sucrose, 7 g / L agar, 1.0 mg / L 6-BA and 0.1 mg / L NAA are added, and the pH value is adjusted to 6.0; The composition of the proliferation medium is MS as the basic medium, to which 4.74 g / L MS powder, 30 g / L sucrose, 7 g / L agar, 1.5 mg / L 6-BA and 0.1 mg / L IBA are added, and the pH value is adjusted to 6.

0.

2. The method for virus-free cultivation of purple passion fruit stem tip according to claim 1, characterized in that: Cultivation conditions: The cultivation temperature of steps (4) to (7) is 25±2°C, the light intensity is 2,000 lx, the illumination duration is 12 h / d, and the relative humidity is 85-90%.

3. The method for virus-free cultivation of purple passion fruit stem tip according to claim 1, characterized in that: The composition of the rooting medium is 1 / 2MS as the basic medium, to which 2.47 g / L MS powder, 30 g / L sucrose, 7 g / L agar, and 0.1 mg / L IBA are added, and the pH value is adjusted to 6.

0.

4. The method for virus-free cultivation of purple passion fruit stem tip according to claim 1, characterized in that: The seedling medium is a mixture of peat soil and perlite in a mass ratio of 4:1.

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