Efficient production method of passion fruit virus-free seedlings

Abstract

The present invention discloses an efficient production method for passion fruit virus-free seedlings, which relates to the field of plant virus-free technology, including the following steps: S1 virus-free seedling stock cultivation: after selecting passion fruit seeds for virus-free treatment, seed germination and stock cultivation are performed; S2 virus-free micro-bud scion cultivation: passion fruit plants are selected, treated with an antiviral agent solution under a maximum ambient temperature exceeding 35°C, and the axillary buds are grown to a predetermined length; S3 virus-free seedling production: the axillary bud stem tips cultivated in step S2 are peeled off and grafted onto the micro-bud grafting stock to form micro-bud grafted seedlings, the micro-bud grafted seedlings are cultivated as scion mother plants, the terminal buds or an axillary bud stem segment of the scion mother plant are used as scions, and the virus-free seedling production stock is grafted to form the passion fruit virus-free seedlings. The method provided by the present invention not only continues the excellent traits of the mother plant, but also has a certain improvement in the excellent fruit rate and fruiting period. The cultivation test performance fully meets the requirements of passion fruit virus-free commercial seedlings.

Description

Technical Field

[0001] The invention relates to the technical field of plant detoxification, in particular to a method for efficiently producing passion fruit detoxified seedlings. Background Art

[0002] Passion fruit (Passiflora spp.), also known as passion fruit, passion fruit, and egg fruit, is a perennial vine belonging to the Passifloraceae family and the genus Passiflora. This genus contains approximately 400 species, 60 of which are edible and six of which are commercially cultivated. Passion fruit has extremely high nutritional value and can be eaten fresh or processed into juice. Its rich, distinctive aroma is highly sought after by consumers, and high-quality fresh passion fruit is consistently in short supply on the market. Passion fruit is a vigorous perennial evergreen vine that typically takes only a few months from planting to harvest. With early production and proper management, it produces stable and high yields, resulting in high economic returns. In recent years, passion fruit has become a popular fruit prioritized for development in many regions of my country, with the cultivated area continuing to increase and the industry expanding.

[0003] With the continuous expansion of the passion fruit industry and the increase in the number of years of passion fruit cultivation in various producing areas, problems in the development of the passion fruit industry have gradually emerged.

[0004] Numerous documents and reports have mentioned the serious problem of viral diseases in the current development of the passion fruit industry, and emphasized the importance of virus-free and healthy seedlings to the development of the industry. However, the current system for breeding virus-free and healthy passion fruit seedlings is not perfect and immature, making it difficult to use in actual production. The commonly used tissue culture detoxification and rapid propagation technology still has many problems that are difficult to avoid: tissue culture detoxification requires multiple subcultures to obtain an ideal detoxification rate, and the detoxification agent needs to directly contact and act on the explants during the culture process. The explants, whose physiological and biochemical characteristics are already imperfect, are extremely susceptible to the influence of culture media or agents. The virus-free seedlings obtained by tissue culture detoxification are prone to vitrification and rigidification, and have poor growth and robustness. Moreover, after multiple generations of culture, it is difficult for the tissue culture seedlings to maintain the mature characteristics of the mother explants. The juvenile period is long, flowering and fruiting are difficult, and it takes a long time. Tissue culture detoxification is currently the main means of cultivating virus-free seedlings for horticultural plants. There have been a few research reports on the establishment of a tissue culture rapid propagation system for passion fruit. The reported primary survival rate, subculture bud differentiation rate, and proliferation coefficient of the tissue culture rapid propagation system are high and low, with large differences and are relatively unstable; the rooting efficiency is relatively high and stable, but the callus tissue is prone to excessive expansion during rooting culture, affecting the later seedling hardening and planting; in addition, during the subculture process, when the explant carries a large number of viruses, it will affect the elongation and growth of the buds. Although the buds differentiate normally, they are prone to rigidification when they grow to about 1mm, and even if they grow, they are prone to yellowing and vitrification. The seedlings bred by the existing passion fruit tissue culture rapid propagation system cannot stably continue the excellent traits of the mother plant. There are certain problems with the development of roots, stems and leaves, and the growth is weak. Subsequent planting has similar problems as seedlings. If the tree has a long infancy, slow development and maturity, and late flowering and fruiting, the commercial value is poor. At the same time, there are still relatively few research reports on the detoxification of passion fruit tissue culture, insufficient theoretical support, and key technologies need to be supplemented and improved. Therefore, passion fruit tissue culture virus-free rapid propagation technology is still difficult to be applied in the industrial production of healthy passion fruit seedlings. Summary of the Invention

[0005] In response to the problems existing in the prior art, the present invention provides a highly efficient production method for passion fruit virus-free seedlings, avoiding the currently immature tissue culture virus-free rapid propagation technology, and adopting the stem tip micro-bud grafting virus-free technology. Not only does it continue the excellent traits of the mother plant, but it also improves the high-quality fruit rate and fruiting period to a certain extent. The performance of the cultivation test fully meets the requirements of passion fruit virus-free commercial seedlings.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0007] The efficient production method of passion fruit virus-free seedlings comprises the following steps:

[0008] S1: cultivating virus-free seedling rootstocks: selecting passion fruit seeds for virus-free treatment, and then germinating the seeds and cultivating the rootstocks, wherein the virus-free seedling rootstocks include rootstocks for micro-bud grafting and rootstocks for virus-free seedling production;

[0009] S2 Non-toxic Microbud Scion Cultivation: Select passion fruit plants, treat them with antiviral solution under the condition of maximum ambient temperature exceeding 35°C, and wait for the axillary buds to grow to the predetermined length;

[0010] S3 virus-free seedling production: peel off the axillary bud stem tip cultivated in step S2, graft it onto the micro-bud grafting rootstock to form a micro-bud grafted seedling, cultivate the micro-bud grafted seedling as a scion mother plant, use the terminal bud or an axillary bud stem segment of the scion mother plant as a scion, and graft it onto the virus-free seedling production rootstock to form the passion fruit virus-free seedling.

[0011] Furthermore, the passion fruit variety selected for the rootstock for microbud grafting in step S1 is the same as the passion fruit variety selected for the non-toxic microbud scion cultivation in step S2.

[0012] Furthermore, in step S1, the detoxification method is specifically as follows:

[0013] Choose passion fruits that are evenly shaped, plump, mature, smooth-skinned, and healthy and free of disease. Remove the seeds, clean them, and dry them in the sun on a sunny day for 2-3 days. The maximum temperature on a sunny day should be ≥35°C.

[0014] The dried seeds are packed in envelopes, marked, and placed in a constant temperature drying oven for dry heat sterilization and detoxification. The treatment temperature is: preheat and dry at 50℃ for 4 hours, then heat to 80℃ and dry for 12 to 24 hours.

[0015] Furthermore, in step S1, the seed germination and rootstock cultivation method is specifically as follows:

[0016] Place the seeds in a holding cup, soak them in 300 mg / L gibberellin solution, and place them in a 35°C light incubator for 24 hours;

[0017] Choose a planting trough with a permeable bottom, evenly lay a 5cm thick moist seedling substrate on the bottom of the planting trough, evenly spread the soaked seeds on the substrate, then cover it with a 2-3cm thick moist substrate, and evenly water it with a sprinkler. When the bottom of the planting trough no longer seeps water, move the planting trough into a light incubator or germination room.

[0018] Environmental control during seed germination: 1-5 days after sowing, the temperature should be controlled between 32-38°C, and after the 6th day, it should be lowered to between 32-35°C. When more than 10% of the seeds have emerged from the soil, the temperature should be lowered to between 28-32°C. During the germination process, the air humidity should be maintained at ≥80%, and water should be sprayed regularly to keep the substrate moist.

[0019] When more than 50% of the seeds have germinated and emerged from the soil, the seedlings are transplanted into nutrient pots or hole trays. For seedlings used to cultivate rootstocks for micro-bud grafting, perforated cups are used as nutrient pots. When the seedlings grow to 1 to 3 true leaves, they can be used for step S3. For seedlings used for rootstocks for virus-free seedling production, when the seedling height grows to ≥15 cm and the maximum stem thickness is ≥0.4 cm, they can be used for step S3.

[0020] Furthermore, the non-toxic microbud scion cultivation method in step S2 is specifically as follows:

[0021] Passion fruit plants with high fruit setting rate, good traits, and healthy and disease-free appearance were selected as treatment objects;

[0022] Treatment conditions: Field treatment should be carried out when the maximum temperature in summer exceeds 35°C and the duration is more than 10 days; or the temperature in the greenhouse should be controlled in the range of 30-40°C and the maximum temperature per day should exceed 35°C;

[0023] Treatment method: 1) evenly spraying the passion fruit plant with an antiviral agent solution; 2) on the second day of spraying, removing all terminal buds, flower buds, flowers, and fruits of the passion fruit plant, while ensuring adequate water and fertilizer supply to promote rapid germination of axillary buds; 3) spraying the antiviral agent solution again when the axillary buds germinate to ≥1 cm, and then spraying again every 7 days; 4) when the axillary buds grow to >5 cm, they can be used for step S3.

[0024] Furthermore, each L of the antiviral agent solution contains 2 mL of 0.06% sterol as an active ingredient, 0.5 g of 30% chloranil as an active ingredient, and the remaining ingredients are water.

[0025] Furthermore, the virus-free seedling production in step 3 is specifically as follows:

[0026] Select the axillary buds with good growth from the S2 culture, peel off the 0.3mm-0.8mm stem tips of the axillary buds with a No. 11 blade, ensure that the stem tip bases are flush, and after the stem tip is peeled off, quickly cut off the buds upward from the base of the cotyledon petiole of the micro-bud grafting stock flush with the base, then use the exuded juice to align the peeled stem tip base with the incision and adsorb it, gently press the stem tip with the tip of the knife to adjust it to ensure that the incision is closely fitted, and finally seal it to complete the micro-bud grafting to form a micro-bud grafted seedling;

[0027] Micro-bud grafted seedling cultivation: 1) Move the micro-bud grafted seedling into the culture room, control the culture temperature between 26 and 28 ° C, and the light exposure time for 12 hours. Before the incision is healed, cover it with a white translucent plastic plate to avoid direct sunlight; 2) When the micro-bud grafted seedling grows to 2 to 3 true leaves, it can be moved out of the culture room and transferred to the growth greenhouse. Keep it sealed on the first day after being moved out. Start to uncover the plastic cup for ventilation for 4 hours on the second day. Increase the ventilation time by 2 hours every day from the 3rd to the 5th day. Start to completely uncover the plastic cup on the 6th day and The seedlings are transplanted into 11cm*7.5cm*8cm nutrient pots for conventional growth and cultivation. The temperature in the greenhouse is controlled between 20 and 35°C, a shade net is set up, and isolation measures are taken to prevent the introduction of diseases; 3) When the micro-bud grafted seedlings grow to 4 to 5 true leaves, virus testing is started on them, and seedlings with positive virus tests and weak growth are eliminated. The qualified micro-bud grafted seedlings are upgraded to scion mother plants, the shade net is removed and continued to be cultivated. When they grow to ≥15 cm, they are moved into an isolated field or an isolated greenhouse for conventional cultivation;

[0028] Production of virus-free seedlings: using the terminal bud or an axillary bud stem segment of the scion mother plant as the scion, adopting the conventional sleeve grafting method, and grafting the virus-free seedling production rootstock cultured in batches in step S1 to form the passion fruit virus-free seedlings.

[0029] Furthermore, the virus types detected by the virus detection include passion fruit lignification virus, cucumber mosaic virus, tuberose mosaic virus and East Asian passion fruit virus.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The passion fruit virus-free seedlings cultivated by the implementation method of the present invention not only continue the excellent traits of the female parent, but also can be further improved, and the excellent fruit rate and fruiting period also have a certain improvement. The cultivation test performance has fully met the requirements of the passion fruit non-toxic commercial seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a diagram of seeds being soaked and germinated in gibberellin solutions of different concentrations according to an embodiment of the present invention.

[0033] Figure 2 This is a diagram of a non-toxic seedling rootstock cultured at a variable temperature using a light incubator according to an embodiment of the present invention.

[0034] Figure 3 This is a diagram of a non-toxic seedling rootstock used for microbud grafting according to an embodiment of the present invention.

[0035] Figure 4 This is a picture of the non-toxic micro-bud scion directly peeled by a blade according to an embodiment of the present invention.

[0036] Figure 5 The micro-bud grafting of the embodiment of the present invention is completed Figure 1 .

[0037] Figure 6 The micro-bud grafting of the embodiment of the present invention is completed Figure 2 .

[0038] Figure 7 This is a sealed culture diagram after grafting is completed in an embodiment of the present invention.

[0039] Figure 8 This is a diagram of the healing interface of the micro-bud grafted seedling according to an embodiment of the present invention.

[0040] Figure 9 For the micro-bud grafted seedlings of the embodiment of the present invention Figure 1 .

[0041] Figure 10 For the micro-bud grafted seedlings of the embodiment of the present invention Figure 2 .

[0042] Figure 11 This is a diagram showing the expansion of 4 to 5 true leaves of a micro-grafted seedling according to an embodiment of the present invention.

[0043] Figure 12 This is a micro-grafting diagram of the scion mother plant according to an embodiment of the present invention being transplanted into a field or greenhouse for conventional cultivation.

[0044] Figure 13 Axillary buds induced by the comparative agent culture medium Figure 1 .

[0045] Figure 14 Axillary buds induced by the comparative agent culture medium Figure 2 .

[0046] Figure 15 This is a picture of callus tissue induced by the comparative example drug culture medium.

[0047] Figure 16 The embodiment of the present invention is a diagram of the micro-bud grafted virus-free seedlings in the peak fruiting period

[0048] Figure 17 The micro-bud grafted virus-free seedling fruit of the embodiment of the present invention Figure 1 .

[0049] Figure 18 The micro-bud grafted virus-free seedling fruit of the embodiment of the present invention Figure 2 . DETAILED DESCRIPTION

[0050] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0051] The efficient production method flow chart of passion fruit virus-free seedlings of the present invention is as follows Figure 1To illustrate the technical content, objectives and effects of the present invention in detail, the following embodiments are provided.

[0052] The efficient production method of passion fruit virus-free seedlings comprises the following steps:

[0053] S1: cultivating virus-free seedling rootstocks: selecting passion fruit seeds for virus-free treatment, and then germinating the seeds and cultivating the rootstocks, wherein the virus-free seedling rootstocks include rootstocks for micro-bud grafting and rootstocks for virus-free seedling production;

[0054] S2 Non-toxic Microbud Scion Cultivation: Select passion fruit plants, treat them with antiviral solution under the condition of maximum ambient temperature exceeding 35°C, and wait for the axillary buds to grow to the predetermined length;

[0055] Production of S3 virus-free seedlings: peel off the stem tips of the axillary buds cultivated in S2 and graft them onto the micro-bud grafting rootstock to form micro-bud grafted seedlings. Cultivate the micro-bud grafted seedlings as scion mother plants. Use the terminal bud or an axillary bud stem segment of the scion mother plant as a scion and graft it onto the rootstock for virus-free seedling production to form the passion fruit virus-free seedlings.

[0056] The production method is described in detail below in conjunction with specific embodiments.

[0057] Example 1

[0058] Seed detoxification

[0059] For example, we selected purple-fruited Tainong No. 1 passion fruit with uniform, plump, mature, smooth skin, and no disease. We then removed the seeds, cleaned them, and dried them in the sun for 2-3 days (on a sunny day with a maximum temperature of 35°C or higher). The dried seeds were packaged in envelopes, labeled, and placed in a constant-temperature drying oven for dry heat sterilization and detoxification. The treatments were preheated and dried at 50°C for 4 hours. The 12 dry heat sterilization treatments, as shown in Table 1, were followed by a blank control.

[0060] Table 1 Dry heat sterilization treatment comparison table

[0061]

[0062] After treatment, the seeds were cultured for germination, and the germination rate and status were calculated. The results are shown in Table 2. For the same treatment duration, germination initially increased and then rapidly decreased with increasing treatment temperature. For the same treatment temperature, the germination rate gradually decreased with increasing treatment duration, with little difference between 12-24 hours and 6-12 hours. At 80°C, the germination rate was not significantly different from the control group. However, when the treatment temperature was increased to 90°C, the temperature exceeded the tolerance limit of passion fruit seed dry heat sterilization. Considering that the most important factor in seed dry heat sterilization is virus removal, a longer treatment time is more beneficial for virus removal, while ensuring a moderate germination rate. Therefore, 80°C dry heat treatment for 12-24 hours is optimal.

[0063] Table 2 Effects of different dry heat sterilization treatments on germination rate and germination status

[0064]

[0065] Example 2

[0066] Seed germination

[0067] For example, the purple-fruited Tainong No. 1 passion fruit is selected. Fruits are uniformly shaped, plump, mature, smooth-skinned, and disease-free. The seeds are then removed, cleaned, and dried in the sun for 2-3 days (on a sunny day, with a maximum temperature of 35°C or higher). The dried seeds are then packaged in an envelope, labeled, and placed in a constant-temperature drying oven for dry heat sterilization and detoxification. The process involves preheating and drying at 50°C for 4 hours, then increasing the temperature to 80°C and drying for 12-24 hours.

[0068] Divide the processed seeds into five equal parts. Figure 1 As shown, 0 mg / L (K), 200 mg / L (N0), 300 mg / L (N1), 500 mg / L (N2), and 700 mg / L (N3) gibberellin solutions were placed in holding cups, immersed in a 35°C light incubator for 24 hours, and then planted in planting boxes and placed in a light incubator for cultivation.

[0069] After seed germination, the germination time and germination rate (30 days after sowing) for the five treatments were recorded and statistically analyzed. The results are shown in Table 3. After immersion in the gibberellin solution, the germination rate was significantly improved, and germination occurred earlier. With increasing gibberellin concentration, the germination rate gradually increased and then decreased, the germination time was first shortened and then delayed, and germination was initially improved and then gradually inhibited. Therefore, after dry heat sterilization, immersion in a 300 mg / L gibberellin solution for germination acceleration was the best treatment.

[0070] Table 3 Effects of different concentrations of gibberellin solution on germination time and germination rate

[0071]

[0072] Example 3

[0073] Cultivation of rootstock

[0074] For example, the purple-fruited Tainong No. 1 passion fruit is selected. Fruits are uniformly shaped, plump, mature, smooth-skinned, and disease-free. The seeds are then removed, cleaned, and dried in the sun for 2-3 days (on a sunny day, with a maximum temperature of 35°C or higher). The dried seeds are then packaged in an envelope, labeled, and placed in a constant-temperature drying oven for dry heat sterilization and detoxification. The process involves preheating and drying at 50°C for 4 hours, then increasing the temperature to 80°C and drying for 12-24 hours.

[0075] Place the seeds in a holding cup, soak them in 300 mg / L gibberellin solution, and place them in a 35°C light incubator for 24 hours;

[0076] like Figure 2 As shown, choose a planting trough with a permeable bottom, evenly lay a 5 cm thick moist seedling substrate on the bottom of the planting trough, evenly spread the soaked seeds on the substrate, then cover it with a 2-3 cm thick moist substrate, and evenly water it with a sprinkler. When the bottom of the planting trough no longer seeps water, move the planting trough into a light incubator or germination room;

[0077] Environmental control during seed germination: 1-5 days after sowing, the temperature should be controlled between 32-38°C, and after the 6th day, it should be lowered to between 32-35°C. When more than 10% of the seeds have emerged from the soil, the temperature should be lowered to between 28-32°C. During the germination process, the air humidity should be maintained at ≥80%, and water should be sprayed regularly to keep the substrate moist.

[0078] When the seeds germinate more than 50% of the way out of the soil, transplant the seedlings into nutrient pots or plug trays, select seedlings with normal cotyledons, intact and flat cotyledons, and well-developed root systems for transplanting, and directly eliminate deformed and unhealthy seedlings. Put the ungerminated ones back into the light incubator or germination room to continue germinating; Among them: seedlings used to cultivate rootstocks for micro-bud grafting, such as Figure 3 As shown, a perforated cup is used as a nutrient pot. When the seedling grows to 1 to 3 true leaves unfolded, it can be used for micro-bud grafting in step S3; for the seedlings used as rootstocks for the production of virus-free seedlings, a 32-hole seedling tray or a 9×9 model seedling nutrient pot is selected. When the seedling height grows to ≥15 cm and the maximum stem thickness grows to ≥0.4 cm, it can be used for the production of virus-free seedlings in step S3.

[0079] Example 4

[0080] Cultivation of non-toxic micro-bud scion

[0081] Passion fruit plants with high fruit setting rate, excellent traits, and healthy and disease-free appearance were selected as treatment objects.

[0082] Treatment conditions: The field treatment should be carried out when the maximum temperature in summer exceeds 35℃ and the duration is more than 10 days; or the temperature in the greenhouse should be controlled in the range of 30-40℃ during the treatment process, with the maximum daily temperature exceeding 35℃.

[0083] The purpose of choosing to carry out treatment under the above temperature conditions is to combine the high temperature latent symptoms of plant viral diseases and better cultivate non-toxic micro-bud scion.

[0084] Observations of daily passion fruit cultivation in orchards show that when summer temperatures exceed 35°C, passion fruit virus disease also exhibits heat-induced symptoms, with smooth, thick, and emerald green leaves, new axillary buds growing and expanding normally, and plump, smooth fruit with no lesions. This contrasts sharply with the autumn and winter symptoms of yellowing, wrinkling, and mosaic leaves, shrinking and stagnant branches, and deformed fruit with thickened and hardened skin and abnormal coloring. Therefore, the optimal treatment conditions are: "field treatment during summer temperatures exceeding 35°C for at least 10 days; and greenhouse treatment temperatures maintained between 30 and 40°C, with daily maximum temperatures exceeding 35°C."

[0085] Taking the purple-fruited Tainong No. 1 passion fruit as an example, cultivation was performed during summer periods with maximum temperatures exceeding 35°C and lasting for at least 10 days. Eight passion fruit planting plots of equal size and under the same cultivation conditions were divided and sprayed evenly on the passion fruit plants with seven antiviral solutions, as shown in Table 4, along with a blank solution. The day after the first spraying, the terminal buds, buds, flowers, and fruits of the passion fruit plants were removed. Adequate water and fertilizer supply was ensured to promote rapid axillary bud germination. A second spraying was performed when the majority of axillary buds in the planting plot had sprouted to 1 cm or greater. Seven days later, a third spraying was performed. After the three sprayings, the disease incidence, control efficacy, and growth of the newly sprouted axillary buds were analyzed 10 days apart. The results, as shown in Table 5, show that all seven antiviral solutions were effective against passion fruit virus diseases and did not affect axillary bud germination or normal growth. The Y5 antiviral solution had the lowest disease incidence and the best control efficacy. Therefore, it is best to use Y5 antiviral agent solution for treatment.

[0086] Table 4 Comparison table of antiviral agent solutions

[0087]

[0088]

[0089] Table 5 Effects of different antiviral solutions on newly germinated axillary buds

[0090]

[0091] Example 5

[0092] Microbud grafting

[0093] Taking purple fruit type Tainong No. 1 passion fruit, golden guava passion fruit, Qinmi No. 9 passion fruit and large fruit golden passion fruit as examples, the cultivation method of the non-toxic micro-bud scion in Example 4 (using Y5 antiviral agent solution) was adopted to cultivate non-toxic new axillary buds, and the micro-bud grafting stock (purple fruit type Tainong No. 1 passion fruit) cultivated in Example 3 was selected for standby use. Non-toxic new axillary buds with good growth that grew to >5cm were selected, and the 0.3mm~0.8mm stem tip of the axillary bud was peeled off with a 11# blade (using a direct peeling method). Figure 4 As shown, the base of the stem tip is guaranteed to be flush. After the stem tip is peeled off, the buds upward from the base of the cotyledon petiole of the rootstock are quickly cut off from the base. Then, using the exuded juice, the base of the peeled stem tip is aligned with the incision and adsorbed on it. The tip of the knife is used to gently press the stem tip to adjust it to ensure that the incision fits tightly. Finally, a plastic cup is turned upside down and clamped to the nutrient pot to seal it. The micro-bud grafting is completed to form a micro-bud grafted seedling. Figure 5 、 Figure 6 and Figure 7 300 plants of each variety were grafted and the wound healing was observed after 3 to 5 days. Figure 8 As shown in Table 6, the survival and growth of the scion were counted after 10 days. The results are shown in Table 6. The micro-grafted seedlings with the same variety of rootstock and scion have better interface healing, higher survival rate, rapid growth, and strong buds. Figure 9 、 Figure 10 As shown; the rootstock and scion are micro-grafted seedlings of different varieties, the grafting affinity is relatively low, the wound heals slowly, the buds grow slowly or even do not grow, and a large amount of callus tissue is easily differentiated at the rootstock interface in the later stage to wrap or exclude the scion, and then differentiate into new buds.

[0094] Therefore, for micro-bud grafting, it is best to choose the same variety for the rootstock and scion, and the grafting process should also be "fast, accurate and stable".

[0095] Table 6 Effects of different grafting stock / scion combinations on grafting survival rate and scion growth

[0096]

[0097] Comparative Example

[0098] Taking the purple-fruited Tainong No. 1 passion fruit as an example, the purple-fruited Tainong No. 1 passion fruit was selected for both the rootstock and the scion, and conventional stem tip micro-bud grafting was carried out, that is, the seedling rootstock was cultivated by tissue culture, the non-toxic stem tip scion was cultivated in vitro by tissue culture, and the micro-bud grafting was completed and then inoculated and cultured. The results are shown in Table 7. In the conventional stem tip micro-bud grafting of passion fruit, there were contamination losses in the three major steps, and the contamination rate of inoculation and culture after the micro-bud grafting was completed was the highest. The reason may be that the micro-bud grafting operation requires more precision and the operation takes a long time, resulting in the micro-grafted seedlings being exposed to pathogens for a long time, which ultimately causes higher contamination and a relatively low grafting survival rate. In addition, in the process of tissue culture in vitro cultivation of non-toxic stem tip scion, it was also found that the in vitro culture was detoxified, and the detoxification agent directly acted on the explant, and the bud growth would be inhibited, and the growth was extremely slow; after subculture, such as Figure 13 、 Figure 14 and Figure 15 As shown, the buds are very easy to become rigid and yellow, and the callus differentiation is abnormal, making it difficult to carry out the next step of culture.

[0099] The above results show that the innovative and improved passion fruit stem tip micro-bud grafting and virus-free method is significantly improved in operability, grafting survival rate and robust condition of grafted seedlings compared with the conventional passion fruit stem tip micro-bud grafting method.

[0100] Table 7 Contamination rate and grafting survival rate at each stage of conventional stem tip micro-bud grafting of passion fruit

[0101]

[0102]

[0103] Example 6

[0104] Cultivation Experiment of Micro-bud Grafted Seedlings and Virus-free Seedlings

[0105] Taking the purple-fruited Tainong No. 1 passion fruit as an example, on the basis of Example 5 (both the rootstock and the scion in the grafted rootstock-scion combination are the purple-fruited Tainong No. 1 passion fruit), the formed micro-bud grafted seedlings are further cultured as follows:

[0106] 1) The micro-bud grafted seedlings are moved into a culture room, the culture temperature is controlled between 26 and 28° C., the light exposure time is 12 hours, and before the incision is healed, it is covered with a white translucent plastic plate to avoid direct sunlight; 2) When the micro-bud grafted seedlings grow to 2 to 3 true leaves, they can be moved out of the culture room and transferred to a growth greenhouse. The plastic cup is kept sealed on the first day after being moved out. The plastic cup is opened and ventilated for 4 hours starting from the second day. The ventilation time is increased by 2 hours every day from the third to the fifth day. The plastic cup is completely opened on the sixth day, and the seedlings are transplanted into 11 cm*7.5 cm*8 cm nutrient pots for conventional growth and culture. The greenhouse temperature is controlled between 20 and 35 degrees Celsius, a sunshade net is set up, and isolation measures are taken to prevent the introduction of diseases; 3) When the micro-bud grafted seedlings grow to 4 to 5 true leaves, virus testing is started on them. The types of viruses tested include passion fruit lignification virus, cucumber mosaic virus, tuberose mosaic virus and East Asian passion fruit virus; virus-positive seedlings and seedlings with weak growth are eliminated, and the qualified micro-bud grafted seedlings are upgraded to scion mother plants, such as Figure 11 As shown, remove the shade net and continue to culture. When the growth reaches ≥15cm (as shown in Figure 12 As shown in the figure, 100 scion mother plants were taken as group A scion mother plants (micro-bud grafted seedlings), which were directly moved into field for cultivation and production. Conventional passion fruit cultivation and management methods were used for daily management and maintenance, and their growth and production conditions were recorded. The remaining scion mother plants were only used for scion production, without fruit production, and were moved into isolated field or isolated greenhouse for conventional cultivation.

[0107] The remaining scion plants are handled as follows:

[0108] Use high-density planting in separate areas, regularly guide vines and top them to promote branching, and regularly apply chemical protection to prevent plant virus infection. If the scion mother plant is infected with a virus or the plant ages, all the scion mother plants in the separate area must be eliminated and disinfected and left fallow;

[0109] Production of virus-free seedlings: The remaining scion mother plant terminal bud or an axillary bud stem segment is used as a scion, and the conventional sleeve grafting method is adopted to graft the virus-free seedling production rootstock cultured in batches in Example 3 to form the passion fruit virus-free seedlings.

[0110] 100 virus-free passion fruit seedlings were taken as virus-free seedlings of group B and transplanted into the field for cultivation and production. Conventional passion fruit cultivation and management methods were used for daily management and maintenance, and their growth and production conditions were recorded. The experimental results showed that the survival rate of virus-free seedlings of group B was higher than that of micro-bud grafted seedlings of group A. Micro-bud grafted seedlings of group A and virus-free seedlings of group B all showed well-developed root systems, rapid growth, strong plants, fast flower bud differentiation, early fruiting, and good fruit quality. Figure 16 、 Figure 17 and Figure 18As shown in Figure 8, fruit is large and full, smooth and has no lesions, is well colored, and has a high rate of excellent fruit. Cultivated in October of that year, began to bear fruit in April of the following year, entered the peak fruiting period in late May, and the fruiting period lasted until November, and the fruiting period was long. Therefore, the passion fruit virus-free seedlings cultivated through the implementation method of the present invention not only continued the excellent traits of the maternal parent, and realized improvement, and stress resistance also has a certain lifting, and the cultivation test performance has fully reached the requirement of the non-toxic commercial seedlings of passion fruit, and the passion fruit planting situation corresponding to the A group scion mother plant and the B group virus-free seedling is as shown in Table 8.

[0111] Table 8 Corresponding passion fruit planting conditions of scion mother plants of group A and virus-free seedlings of group B

[0112]

[0113] Although the present invention has been described in detail above using specific embodiments, it will be apparent to those skilled in the art that modifications or improvements may be made based on the present invention. Therefore, such modifications or improvements, which do not depart from the spirit of the present invention, are intended to fall within the scope of protection claimed herein.

Claims

1. The efficient production method of passion fruit virus-free seedlings is characterized in that, The steps include: S1: cultivating virus-free seedling rootstocks: selecting passion fruit seeds for virus-free treatment, and then germinating the seeds and cultivating the rootstocks, wherein the virus-free seedling rootstocks include rootstocks for micro-bud grafting and rootstocks for virus-free seedling production; The detoxification method is specifically: Choose passion fruits that are evenly shaped, plump, mature, smooth-skinned, and healthy and free of disease. Remove the seeds, clean them, and place them in the sun on a sunny day for 2-3 days. The maximum temperature on a sunny day should be ≥35°C. The dried seeds are packed in envelopes, marked, and placed in a constant temperature drying oven for dry heat sterilization and detoxification. The treatment temperature is: preheat and dry at 50℃ for 4 hours, then heat to 80℃ and dry for 12-24 hours. The seed germination and rootstock culture method is specifically as follows: Place the seeds in a holding cup, soak them in 300 mg / L gibberellin solution, and place them in a 35°C light incubator for 24 hours; Choose a planting trough with a permeable bottom, evenly lay a 5cm thick moist seedling substrate on the bottom of the planting trough, evenly spread the soaked seeds on the substrate, then cover it with a 2-3cm thick moist substrate, and evenly water it with a sprinkler. When the bottom of the planting trough no longer seeps water, move the planting trough into a light incubator or germination room. During the germination process, the temperature should be controlled between 32 and 38 degrees Celsius for 1 to 5 days after sowing. After the 6th day, the temperature should be lowered to between 32 and 35 degrees Celsius. When more than 10% of the seeds have emerged from the soil, the temperature should be lowered to between 28 and 32 degrees Celsius. During the germination process, the air humidity should be maintained at ≥80%. Water should be sprayed regularly to keep the substrate moist. When more than 50% of the seeds have germinated, the seedlings are transplanted into nutrient pots or plug trays. For seedlings used for micro-bud grafting, perforated cups are used as nutrient pots. When the seedlings have 1 to 3 true leaves unfolded, they can be used for step S3. For seedlings used for virus-free seedling production, when the seedling height is ≥15 cm and the maximum stem thickness is ≥0.4 cm, they can be used for step S3. S2 Non-toxic Microbud Scion Cultivation: Select passion fruit plants, treat them with antiviral solution under the condition of maximum ambient temperature exceeding 35°C, and wait for the axillary buds to grow to the predetermined length; The specific method of cultivating non-toxic micro-bud scion is as follows: Passion fruit plants with high fruit setting rate, good traits, and healthy and disease-free appearance were selected as treatment objects; Treatment conditions: Field treatment should be carried out when the maximum temperature in summer exceeds 35°C and the duration is more than 10 days; or the temperature in the greenhouse should be controlled in the range of 30-40°C and the maximum temperature per day should exceed 35°C; Treatment method: 1) evenly spray the passion fruit plant with an antiviral solution; 2) on the second day of spraying, remove all terminal buds, flower buds, flowers, and fruits of the passion fruit plant, while ensuring adequate water and fertilizer supply to promote rapid germination of axillary buds; 3) spray the axillary buds with the antiviral solution again when they germinate to ≥1 cm, and then spray again every 7 days; 4) when the axillary buds grow to >5 cm, they can be used for step S3; Each liter of the antiviral drug solution contains 2 mL of 0.06% sterol as an active ingredient, 0.5 g of 30% chloranil and morpholinoguanidine as an active ingredient, and the remaining ingredients are water; S3 virus-free seedling production: peel off the axillary bud stem tip cultivated in step S2, graft it onto the micro-bud grafting rootstock to form a micro-bud grafted seedling, cultivate the micro-bud grafted seedling as a scion mother plant, use the terminal bud or an axillary bud stem segment of the scion mother plant as a scion, and graft it onto the virus-free seedling production rootstock to form the passion fruit virus-free seedling.

2. the efficient production method of passion fruit virus-free seedlings according to claim 1, is characterized in that, The passion fruit variety selected for the rootstock for microbud grafting in step S1 is the same as the passion fruit variety selected for the non-toxic microbud scion cultivation in step S2.

3. the efficient production method of passion fruit virus-free seedlings according to claim 1, is characterized in that, The virus-free seedling production in step 3 is specifically as follows: Select axillary buds with good growth cultured in S2, and use a No. 11 blade to peel off the 0.3mm-0.8mm stem tip of the axillary bud, ensuring that the base of the stem tip is flush. After the stem tip is peeled off, the bud upward from the base of the cotyledon petiole of the micro-bud grafting rootstock is quickly cut off from the base flush, and then the base of the peeled stem tip is aligned with the incision using the exuded juice to absorb it. The stem tip is gently pressed with the tip of the knife to adjust it to ensure that the incision is tightly fitted, and finally sealed to complete the micro-bud grafting to form a micro-bud grafted seedling; Micro-bud grafted seedling cultivation: 1) Move the micro-bud grafted seedling into the culture room, control the culture temperature between 26 and 28 ° C, and the light exposure time for 12 hours. Before the incision is healed, cover it with a white translucent plastic plate to avoid direct sunlight; 2) When the micro-bud grafted seedling grows to 2 to 3 true leaves, it can be moved out of the culture room and transferred to the growth greenhouse. Keep it sealed on the first day after being moved out. Start to ventilate for 4 hours from the second day. Increase the ventilation time by 2 hours every day from the third to the fifth day. Start to completely remove the sealed plastic cup on the sixth day and move the seedling to the greenhouse. Planting in 11cm*7.5cm*8cm nutrient pots for conventional growth and cultivation; controlling the greenhouse temperature between 20 and 35°C, setting up a sunshade net, and taking isolation measures to prevent the introduction of diseases; 3) When the micro-bud grafted seedlings have 4 to 5 true leaves unfolded, they are tested for viruses, and virus-positive seedlings and seedlings with weak growth are eliminated. Qualified micro-bud grafted seedlings are upgraded to scion mother plants, and the sunshade net is removed for continued cultivation. When they grow to ≥15 cm, they are moved to an isolated field or isolated greenhouse for conventional cultivation; Production of virus-free seedlings: using the terminal bud or an axillary bud stem segment of the scion mother plant as the scion, adopting the conventional sleeve grafting method, and grafting the virus-free seedling production rootstock cultured in batches in step S1 to form the passion fruit virus-free seedlings.

4. the efficient production method of passion fruit virus-free seedlings according to claim 3, is characterized in that, The virus detection method can detect passion fruit lignification virus, cucumber mosaic virus, tuberose mosaic virus and East Asian passion fruit virus.

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