Passion fruit detoxification rapid propagation method combining micro stem tips with chemical treatment and application of passion fruit detoxification rapid propagation method

Abstract

The invention discloses a detoxification and rapid propagation method for passion fruits by combining micro stem tips with chemical treatment and application of the detoxification and rapid propagation method, and relates to the technical field of tissue culture detoxification. The method comprises the following steps: using 0.5-1mm micro stem tips, taking MS + 1mg / L 6-BA + 0.3 mg / L NAA as an optimal micro stem tip induction culture medium, adding 25mg / L virazole for treatment, taking MS + 1.5 mg / L 6-BA + 0.1 mg / L NAA as an optimal multiplication culture medium, and taking 1 / 2MS + 1.5 mg / L IBA + 0.5 mg / L NAA as an optimal rooting culture medium. The virus-free seedling rates of TeMV and EAPV before virus azole treatment are respectively 11.78 + / -1.39% and 13.04 + / -2.24%, and the virus-free seedling rates of TeMV and EAPV after virus azole treatment are respectively 21.38 + / -2.08% and 20.40 + / -3.02%. The method provided by the invention can provide a basis for production of nontoxic seedlings of passion fruits.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue culture detoxification, and in particular relates to a passion fruit detoxification and rapid propagation method using a micro-stem tip combined with chemical treatment and an application thereof, specifically including passion fruit micro-stem tip detoxification and the application of ribavirin in chemical detoxification, as well as the establishment of a rapid propagation method. Background Art

[0002] Passion fruit (Passiflora edulis), also known as passion fruit, passion fruit, and Brazil nut, is a perennial vine native to South America. It belongs to the genus Passiflora in the family Passifloraceae. As a tropical fruit, it possesses a unique flavor and aroma. Nutrientally, passion fruit is rich in various vitamins, amino acids, phenolic and flavonoid active substances, minerals, and other essential nutrients.

[0003] In recent years, passion fruit cultivation has been vigorously promoted in southern my country. However, viral diseases have severely hampered the development of the passion fruit industry. Plant viruses can stunt the growth and development of their hosts, reduce yields, degrade quality, and in severe cases, even lead to total crop failure. Furthermore, plant viruses are known to infect and infect for life, making conventional control methods difficult to eradicate. Against this backdrop, plant virus removal techniques, through in vitro culture to produce virus-free seedlings, have become an important approach to combating viral diseases. Micro-tip virus removal and chemical virus removal are two key techniques (Fu Hongqi, 2005).

[0004] Micro-tip detoxification is economical, effective, and widely applicable, making it the most commonly used detoxification technique. Viruses are unevenly distributed throughout plants and must propagate through the vascular system. However, meristems have not yet formed vascular bundles, and cell division is much faster than the speed of viral movement. Consequently, the virus load in the meristem of the apex is low or even absent. Therefore, culturing the apex can yield virus-free tissue culture seedlings (He Jihong, 2012). The key to micro-tip detoxification lies in the size of the apex. Smaller apex sizes increase the detoxification rate but decrease the survival rate. Excessively large apex sizes yield higher survival rates but are ineffective in removing viruses (Fu Hongqi, 2005). Micro-tip detoxification can remove Potato Virus S (PVS). The highest detoxification rate, at 12.08%, is achieved with apex sizes between 0.14 and 0.19 mm, while the lowest, at only 1%, is achieved with apex sizes between 0.47 and 0.58 mm (Lu Chunyan, 2023). The number of stem tip culture cycles also affects the virus-free rate. For ginger (Zingiber officinale), the virus-free rate reached 100% after two stem tip culture cycles, significantly higher than that achieved with a single stem tip culture (Liu Chengcai et al., 2021). In garlic (Allium sativum) virus-free seedlings obtained through stem tip culture, it was found that stem tip size was inversely proportional to the virus-free rate, but directly proportional to the callus formation rate and survival rate (Lei Yuming et al., 2023).

[0005] Chemical detoxification is the process of obtaining virus-free seedlings by adding antiviral agents such as ribavirin, cypermethrin, salicylic acid, and dihydrolipoic acid to the culture medium. Antiviral agents can stimulate systemic resistance in plants or inhibit the movement and proliferation of plant viruses (Zhao Xiwu et al., 2013). Previous studies have often combined chemical treatment with micro-tip detoxification to achieve even better detoxification results. In one study, a combination of tip detoxification and chemical treatment was used to remove three latent viruses from apples. The antiviral agents used were acyclovir, guanidine hydrochloride, and cypermethrin, with concentrations ranging from 5mg to 60mg. The results showed that different concentrations and agents resulted in different detoxification rates. At appropriate concentrations, all three antiviral agents were effective in removing latent viruses from apples (Yan Na, 2009). A study using a combination of chemical treatment and micro-tip culture to detoxify potato (Solanum tuberosum) carrying potato virus Y (PVY) found that chemical treatment alone inhibited viral proliferation but did not eliminate any viruses. After micro-tip culture, the detoxification rate reached 61.54% (Yin Yan, 2023). Currently, there are no reports on the detoxification of passion fruit using micro-tip culture combined with chemical treatment. Establishing a tissue culture detoxification method for passion fruit could provide a theoretical approach for the industrial production of virus-free seedlings. Summary of the Invention

[0006] At present, the passion fruit industry is developing rapidly, forming a complete industrial chain covering planting, processing and other links, and has become an important pillar industry. However, in recent years, the prevalence of passion fruit virus disease has become more and more serious, causing quality and output to decline and leading to serious economic losses. In order to solve this major problem in the industry, the present invention uses passion fruit as material, and cultivates virus-free seedlings through micro-stem tip detoxification and chemical detoxification, screens proliferation and rooting culture medium, establishes a stable and efficient virus-free rapid propagation system, and provides a basis for the production of passion fruit non-toxic seedlings.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] One of the purposes of the present invention is to provide a method for the virus-free rapid propagation of passion fruit combined with a micro-shoot tip and chemical treatment. The method comprises: using a micro-shoot tip with a diameter of 0.3 to 1.2 mm, using MS+0.8 to 1.2 mg / L 6-BA+0.1 to 0.5 mg / L NAA as a micro-shoot tip induction medium, adding 20 to 30 mg / L of ribavirin for treatment, using MS+1.3 to 1.7 mg / L 6-BA+0.05 to 0.15 mg / L NAA as a proliferation medium, and using 1 / 2 MS+1.3 to 1.7 mg / L IBA+0.3 to 0.7 mg / L NAA as a rooting medium.

[0009] Furthermore, the micro-stem tip retains 2 to 4 leaf primordia and has a length of 0.5 to 1 mm.

[0010] Furthermore, the micro-stem tip is obtained by carefully peeling off the axillary buds and / or the young leaves of the terminal bud from the stem segment with buds using a scalpel and a dissecting needle in a clean bench.

[0011] Furthermore, the stem segment with buds is 1 to 2 cm long and is cut from young branches of the current year with leaves removed and cut to 5.5 to 6.5 cm in length. The stem segment is then disinfected with a 500-fold carbendazim solution, 75% alcohol, and 20% NaClO in sequence.

[0012] Furthermore, the micro-shoot tip induction medium is preferably MS+1 mg / L 6-BA+0.3 mg / L NAA.

[0013] Furthermore, the added concentration of ribavirin is preferably 25 mg / L.

[0014] Furthermore, the proliferation culture medium is preferably MS+1.5 mg / L 6-BA+0.1 mg / L NAA.

[0015] Furthermore, the rooting medium is preferably 1 / 2MS+1.5mg / LIBA+0.5mg / LNAA.

[0016] The second object of the present invention is to provide the application of any of the passion fruit virus-free and rapid propagation methods in passion fruit planting.

[0017] Furthermore, the application includes application in preventing and controlling passion fruit tuberose mosaic virus (TeMV) and / or East Asian passion fruit virus (EAPV) diseases.

[0018] Compared with the prior art, the present invention has the following technical effects:

[0019] 1. The present invention screens out a micro-tip induction culture medium and an optimal chemical treatment method by analyzing the seedling rate, the virus-free rate and the virus-free seedling rate (seedling rate × virus-free rate): 0.5-1 mm micro-tip is peeled off for culture, MS+1 mg / L 6-BA+0.3 mg / L NAA is the optimal micro-tip induction culture medium, the seedling rate is 53.33±3.30%, the virus-free rates for tuberose mosaic virus (TeMV) and East Asian passion fruit virus (EAPV) are 22.22±3.85% and 24.44±3.85%, respectively. The virus-free seedling rate is used to evaluate the virus-free effect, and it is found that the virus-free seedling rates of TeMV and EAPV are 11.78±1.39% and 13.04±2.24%, respectively. We continued to explore the detoxification effect of ribavirin treatment combined with micro-shoot tip treatment. The results showed that after 25 mg / L ribavirin treatment, the TeMV virus-free seedling rates and EAPV virus-free seedling rates were 21.38±2.08% and 20.40±3.02%, respectively, indicating that ribavirin treatment combined with micro-shoot tip detoxification can effectively remove viruses from passion fruit.

[0020] 2. The virus-free seedling stem segments obtained by combining ribavirin treatment with micro-shoot tip treatment were used as explants. The present invention found that the proliferation coefficient of MS+1.5mg / L6-BA+0.1mg / LNAA could reach 5.57±0.18, which is the optimal proliferation medium; the rooting rate of 1 / 2MS+1.5mg / LIBA+0.5mg / LNAA was 65.96%±1.34%, which is the optimal rooting medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the Small RNA sequencing result of the diseased passion fruit leaves in embodiment 1 of the present invention.

[0022] Figure 2 The present invention is implemented in the 1st passion fruit passion fruit two viruses RT-PCR detection results.

[0023] Figure 3 The present invention relates to a process in which passion fruit micro-stem tips germinate into seedlings in embodiment 1 of the present invention.

[0024] Figure 4 The present invention relates to a process in which a passion fruit stem segment with buds develops into a rootless seedling in embodiment 1 of the present invention.

[0025] Figure 5 The present invention implements the rooting culture of the rootless tissue culture seedling in 1. DETAILED DESCRIPTION

[0026] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the inventive method, steps or conditions are intended to fall within the scope of the present invention. The reagents, products and instruments used in the following examples are all commercially available, and the methods used in the examples are consistent with conventional methods unless otherwise specified.

[0027] The technical solution of the present invention is further elaborated in detail below in conjunction with embodiments.

[0028] Example 1

[0029] 1.1 Experimental Materials

[0030] 1.1 Plant materials and culture

[0031] The passion fruit used in the present invention is taken from the passion fruit test base of the Anshun Academy of Agricultural Sciences in Guizhou Province. The variety is "Qinmi No. 9". It is transplanted into a sterilized cultivation substrate and then moved into an artificial climate chamber for cultivation. The environmental parameters are set to a constant temperature of 26°C, a day and night photoperiod of 14h / 8h, and a constant relative humidity of 70%.

[0032] 2 Identification of the main viruses infecting passion fruit

[0033] 2.1 Total RNA extraction and cDNA synthesis from diseased passion fruit leaves

[0034] Total RNA was extracted from diseased passion fruit leaves using the TRIzol method. The specific steps are as follows:

[0035] (1) 100-200 mg of fresh diseased leaf samples were cut and placed in a 2 mL grinding tube filled with sterilized steel balls, and then quickly frozen in liquid nitrogen.

[0036] (2) Place the mixture in a grinder and grind thoroughly until it becomes powdery (50 Hz, 30 s). Add 1 mL of TRIzol, shake to mix, and let it stand on ice for 5 min.

[0037] (3) Add 200 μL RNA extraction solution, shake to mix, and let stand on ice for 5 min.

[0038] (4) Centrifuge at 12000 rpm for 10 min at 2°C and transfer 500 μL of the supernatant into a new 1.5 mL enzyme-free centrifuge tube.

[0039] (5) Add 500 μL of isopropanol (pre-cooled at -20°C), invert the tube 20 times, and place it in a -20°C refrigerator for 20 min.

[0040] (6) Centrifuge at 12,000 rpm for 10 min at 2°C, discard the supernatant, and immediately add 800 μL of enzyme-free 70% alcohol to wash the precipitate.

[0041] (7) Centrifuge at 12,000 rpm for 5 min at 2°C, carefully discard the supernatant, briefly centrifuge, and aspirate the remaining liquid. Open the lid and let it dry for 5–8 min. Add 50 μL of DEPC water and store at -80°C until use.

[0042] The total RNA was used as a template to synthesize cDNA. The first-strand cDNA was synthesized according to the instructions of HiScript IV 1st Strand cDNA Synthesis Kit. 50 μL ddH2O was added to dilute the cDNA and stored at -20°C until use.

[0043] 2.2 Smail RNA sequencing

[0044] Total RNA was extracted from diseased passion fruit leaves, and the degree of RNA degradation was verified by 1% agarose gel electrophoresis (190V, 25min). The RNA concentration and contamination degree (OD) were detected by NanoDrop microspectrophotometer. 260 / 280=1.8-2.2). RNA samples with acceptable quality were subjected to small RNA sequencing. Small RNA sequencing was performed using the Illumina HiSeq sequencing platform. Off-line data were filtered for quality control, with low-quality bases at the 3' end removed. Adaptor sequences and low-quality reads (length <18 nt or N ratio >10%) were removed from the raw data to generate clean reads. After obtaining high-quality sequencing results, clean reads were assembled using Velvet 1.2.10 to generate contigs and singleton sequences. The assembled results were compared with the DPV plant virus database (http: / / www.dpvweb.net / ) using blastn, and the results were statistically annotated.

[0045] The results are as follows Figure 1 As shown in the figure, clean reads were counted by length, among which 21nt clean reads were the largest, accounting for 32.74%, followed by 22nt and 19nt, accounting for 18.59% and 17.67% respectively ( Figure 1 A). Classification annotation was performed in the DPV plant virus database, among which EAPV and TeMV had the highest matching rates, accounting for 43.61% and 35.61% respectively ( Figure 1 B).

[0046] 2.3 Primer synthesis

[0047] The passion fruit virus detection specific primers of the present invention were commissioned to be synthesized by Beijing Qingke Biotechnology Co., Ltd. (Table 1).

[0048] Table 1 Passion fruit virus detection specific primers

[0049]

[0050] 2.4 RT-PCR detection of viruses

[0051] The RT-PCR system and procedures are shown in Tables 2 and 3. Figure 2 As shown, the detection rate of EAPV was 18.75%, while the detection rate of TeMV reached 79.17%. The passion fruit plants infected with the two viruses were used as explant materials for the next step of detoxification treatment.

[0052] Table 2 RT-PCR reaction system

[0053]

[0054] Table 3 PCR reaction program

[0055]

[0056] Note: The reaction was performed for 35 cycles.

[0057] 2.5 Tissue culture conditions

[0058] 4.41g / L MS basal salt, 30g / L sucrose, and 6.5g / L agar. Adjust the pH to 6.0±0.1 and sterilize by autoclaving at 121°C, 0.1MPa for 20 minutes. Culture conditions: ambient temperature set at 24±1°C, daily 12-hour photoperiod, and light intensity of approximately 1800 lx.

[0059] 3. Optimal Micro-shoot Tip Induction Culture and Chemical Agent Concentration Screening

[0060] 3.1 Calculation formula and data processing

[0061] The following calculation formula is used to quantify the various indicators:

[0062] Seedling rate (%) = (total number of micro-stem tips that germinated into seedlings / total number of inoculated micro-stem tips) × 100%;

[0063] Virus-free rate (%) = (total number of virus-free tissue culture seedlings / total number of tested tissue culture seedlings) × 100%;

[0064] Virus-free seedling rate (%) = seedling rate × virus-free rate;

[0065] Proliferation coefficient = total number of adventitious bud proliferation / total number of initial inoculated explants;

[0066] Rooting rate (%) = (number of explants induced to root / total number of inoculated explants) × 100%.

[0067] 3.2 Explant disinfection

[0068] On a sunny afternoon during the passion fruit growing season (March to September), cut young branches of the current year from passion fruit plants with good growth conditions, close to the main vine, and remove the leaves. Cut the branches to about 6 cm and use them as explant materials. Soak the explants in a 500-fold carbendazim solution for 30 minutes, then rinse with running water for more than 3 hours. Transfer the explants to a clean bench, first completely immerse them in 75% alcohol for 30 seconds, then rinse them three times with sterile water, each time for more than 2 minutes. Use 20% NaClO as a disinfectant and disinfect for 10 minutes. After disinfection, rinse with sterile water three times, each time for more than 2 minutes, and shake the bottle containing the explants continuously during the process. Use filter paper to absorb the moisture on the surface of the explant, cut off its ends, and cut off a stem segment with buds of 1 to 2 cm in length.

[0069] 3.3 Passion fruit micro-shoot tip culture

[0070] In a clean bench, use a scalpel and a dissecting needle to carefully peel off the young leaves of the axillary buds or terminal buds, and obtain a micro-stem tip (length: 0.5-1mm) with 2-4 leaf primordia. Transfer the micro-stem tip to the micro-stem tip induction medium. Add 6-BA and NAA to the MS medium, set different concentration levels, and screen the optimal micro-stem tip induction medium for passion fruit. Inoculate one micro-stem tip per bottle, 30 bottles per group, repeat 3 times, and count the seedling rate after 60 days. The results are as follows. Figure 3 and as shown in Table 4.

[0071] Table 4 Passion fruit micro-shoot tip induction culture medium screening

[0072]

[0073]

[0074] Note: The data in the table are presented in the form of "mean ± error", and different lowercase letters represent the difference comparison results at the significance level of P < 0.05 (the following tables are marked in the same way).

[0075] The results showed that when the 6-BA concentration was 1 mg / L and the NAA concentration was 0.3 mg / L, the micro-tip seedling rate was significantly higher than that of the other treatment groups. Therefore, the T6 treatment group: MS + 1 mg / L 6-BA + 0.3 mg / L NAA was the most suitable micro-tip induction medium.

[0076] 3.4 Chemical detoxification of passion fruit

[0077] Different concentrations of ribavirin were added to MS medium, and 6-9 cm tall passion fruit tissue culture seedlings were transferred to this medium. Twenty-one days after inoculation, the shoot tips were removed using the same method as in 3.2. Untreated micro-shoot tips served as blank controls. Thirty micro-shoot tips were inoculated for each treatment, repeated three times. Sixty days after inoculation, the seedling survival rate, virus-free rate, and virus-free seedling rate were calculated. The results are shown in Tables 5 and 6.

[0078] Table 5 Effect of ribavirin treatment on seedling rate and virus-free rate

[0079]

[0080] The results showed that among all treatments, 50 mg / L ribavirin had the highest detoxification rate, with TeMV and EAPV detoxification rates of 57.78±3.85% and 55.56±3.85%, respectively, both significantly higher than the blank control. Comparing the detoxification effect by comparing the detoxification seedling rate, when 25 mg / L ribavirin was added, the TeMV and EAPV detoxification rates were the highest, at 21.38±2.08% and 20.40±3.02%, respectively. Therefore, 25 mg / L ribavirin was the optimal detoxification treatment.

[0081] 3.5 Passion fruit proliferation culture

[0082] Using the virus-free seedlings obtained in 3.4 as the explant source, cut 1-2 cm stem segments with buds and transfer them to the proliferation culture medium for proliferation culture. Cytokinin 6-BA and auxin NAA were added to the MS medium. By setting different concentration levels, the optimal proliferation culture medium was screened. One explant was inoculated in each bottle. Each experimental treatment was inoculated in 30 bottles, repeated 3 times, and the proliferation rate was calculated after 60 days of inoculation. The results are shown in Table 6 and Figure 4 shown.

[0083] Table 6 Passion fruit proliferation culture medium screening

[0084]

[0085]

[0086] The results showed that when the 6-BA concentration was 1.5 mg / L and the NAA concentration was 0.1 mg / L, the proliferation coefficient was 5.57 ± 0.18, significantly higher than that of the other groups, indicating that Z6 had the best proliferation effect. Therefore, MS + 1.5 mg / L 6-BA + 0.1 mg / L NAA is the optimal proliferation medium for passion fruit.

[0087] 3.6 Passion Fruit Rooting Culture

[0088] The rootless tissue culture seedlings obtained in 3.5 were used as materials. Rootless tissue culture seedlings with a height of more than 5 cm and 2 to 3 nodes that grew well were selected for rooting induction. The callus tissue at the base was removed and transferred to the rooting medium. The optimal rooting medium for passion fruit was screened by setting different MS and IBA concentrations. One rootless tissue culture seedling was inoculated in each bottle. Each experimental treatment was inoculated with 20 bottles, repeated 3 times, and the rooting rate was calculated 60 days after inoculation. The results are shown in Table 7 and Table 7. Figure 5 shown.

[0089] Table 7 Effects of MS and IBA on the rooting rate of passion fruit

[0090]

[0091] The results showed that NS6 was significantly better than the other treatment groups, with a rooting rate of 65.96% ± 1.34%. Therefore, 1 / 2MS + 1.5mg / L LIBA + 0.5mg / L NAA was the most suitable rooting medium.

[0092] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for the rapid propagation of passion fruit by combining micro-stem tip with chemical treatment, characterized in that: The method comprises: using a micro-shoot tip of 0.3 to 1.2 mm, using MS+0.8 to 1.2 mg / L 6-BA+0.1 to 0.5 mg / L NAA as a micro-shoot tip induction medium, adding 20 to 30 mg / L of ribavirin for treatment, using MS+1.3 to 1.7 mg / L 6-BA+0.05 to 0.15 mg / L NAA as a proliferation medium, and using 1 / 2 MS+1.3 to 1.7 mg / L IBA+0.3 to 0.7 mg / L NAA as a rooting medium.

2. passion fruit detoxification and rapid propagation method according to claim 1, is characterized in that, The micro-stem tip retains 2 to 4 leaf primordia and has a length of 0.5 to 1 mm.

3. passion fruit detoxification and rapid propagation method according to claim 2, is characterized in that, The micro-stem tip is obtained by carefully peeling off the axillary buds and / or the young leaves of the terminal bud from the stem segment with buds in a clean bench using a scalpel and a dissecting needle.

4. passion fruit detoxification and rapid propagation method according to claim 3, is characterized in that, The bud stem segment is 1 to 2 cm long and is cut from young branches of the current year with leaves removed and cut to 5.5 to 6.5 cm in length. The stem segment is disinfected in sequence with a 500-fold carbendazim solution, 75% alcohol and 20% NaClO.

5. passion fruit detoxification and rapid propagation method according to claim 4, is characterized in that, The micro-shoot tip induction culture medium is MS+1 mg / L 6-BA+0.3 mg / L NAA.

6. passion fruit detoxification and rapid propagation method according to claim 5, is characterized in that, The added concentration of ribavirin is 25 mg / L.

7. passion fruit detoxification and rapid propagation method according to claim 6, is characterized in that, The proliferation culture medium is MS+1.5 mg / L 6-BA+0.1 mg / L NAA.

8. passion fruit detoxification and rapid propagation method according to claim 7, is characterized in that, The rooting medium is 1 / 2MS+1.5mg / L IBA+0.5mg / L NAA.

9. Application of the passion fruit virus-free rapid propagation method according to any one of claims 1 to 8 in passion fruit planting.

10. The use according to claim 9, characterized in that The application includes application in preventing and controlling passion fruit tuberose mosaic virus (TeMV) and / or East Asian passion fruit virus (EAPV) diseases.

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