Passiflora edulis explant anti-browning tissue culture medium and tissue culture method thereof

Abstract

The invention relates to a passionflower explant anti-browning tissue culture medium and a tissue culture method thereof, and relates to the technical field of passionflower tissue culture. The invention provides an anti-browning tissue culture medium for a passionflower explant. The tissue culture medium comprises the following components: an MS culture medium, passionflower juice, 6-BA, IBA, sucrose and agar. According to the method, the culture medium is improved, the explant is subjected to low-temperature treatment, and the explant is subjected to secondary disinfection, so that the pollution rate and browning rate of the explant are reduced, the survival rate of the passionflower explant in the tissue culture process is increased, the defect of high browning death rate in the passionflower tissue culture process is overcome, and the tissue culture efficiency of the passionflower is improved.

Description

Technical Field

[0001] The invention relates to the technical field of passion fruit tissue culture, and in particular to a passion fruit explant anti-browning tissue culture medium and a tissue culture method thereof. Background Art

[0002] Passionflower is a perennial evergreen woody vine of the Passiflora genus of the Passifloraceae family. Guangxi Zhuang Autonomous Region, Guizhou Province, Guangdong Province, Fujian Province, Yunnan Province and other provinces in my country are the main planting areas. It has edible, medicinal and ornamental uses. It has a strong flavor, pleasant aroma, rich in vitamin C, minerals, amino acids, and has high nutritional value. It has become popular in the market in recent years.

[0003] Traditional passion fruit propagation mainly adopts vine cuttings and grafting. These two propagation methods are slow due to climate and origin restrictions, and there are many problems in production, such as variety degeneration, quality decline and yield reduction. These problems restrict the development of the passion fruit industry. Passion fruit tissue culture has the advantages of fast speed and high quality, and is an important technical means to promote the development of the passion fruit industry. However, there are problems such as high browning mortality and high contamination rate in passion fruit tissue culture rapid propagation, which seriously affect the efficiency of tissue culture. Summary of the invention

[0004] In order to solve the above-mentioned technical problems, the object of the present invention is to provide a kind of passion fruit explant anti-browning tissue culture medium and tissue culture method thereof. The present invention reduces the contamination rate and browning rate of the explant by improving the culture medium, low temperature treatment of the explant and secondary disinfection of the explant, improves the survival rate of the passion fruit explant in the tissue culture process, overcomes the high shortcoming of browning mortality rate in the passion fruit tissue culture process, and improves the tissue culture efficiency of the passion fruit.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The first object of the present invention is to provide a passion fruit explant anti-browning tissue culture medium, the tissue culture medium comprises the following components: MS culture medium, passion fruit juice, 6-BA, IBA, sucrose, agar.

[0007] The beneficial effects of adopting the above further scheme are as follows: the present invention adds passion fruit juice into the tissue culture medium, and the passion fruit juice is rich in antioxidant components such as vitamin C and flavonoid compounds, which reduces enzyme activity, reduces oxidation of polyphenols, and reduces the occurrence of browning during culture; at the same time, the passion fruit juice contains a variety of rich nutrients such as amino acids, vitamins, minerals, sugars, organic acids, etc., which can supplement the nutrients needed in the passion fruit culture process.

[0008] Based on the above technical solution, the present invention can also be improved as follows.

[0009] Furthermore, the components of the tissue culture medium include the following components in the following amounts: 1 / 2MS culture medium, 150ml / L to 200ml / L of passion fruit juice, 0.5mg / L to 1.5mg / L of 6-BA, 0.05mg / L to 0.15mg / L of IBA, 25g / L to 35g / L of sucrose, and 6g / L to 8g / L of agar.

[0010] The beneficial effect of adopting the above further scheme is that the present invention reduces the amount of MS culture medium components, making Mg 2+ , Ca 2+ , Cu 2 + The content of divalent ions is reduced, which inhibits the oxidation of phenols and reduces browning.

[0011] Furthermore, the preparation method of the passion fruit juice is specifically as follows: squeezing passion fruit pulp, filtering with gauze, and obtaining the passion fruit juice.

[0012] Furthermore, the passion fruit is Tainong passion fruit.

[0013] Furthermore, the gauze has a mesh size of 60 to 80.

[0014] A second object of the present invention is to provide a tissue culture method for preventing browning of passion fruit explants, comprising the following steps:

[0015] (1) selecting a passion fruit stem segment with buds as an explant, cleaning and sterilizing the explant to obtain a sterilized explant, and storing the sterilized explant at a low temperature to obtain a low-temperature treated explant;

[0016] (2) The explants treated at low temperature are taken out, soaked in disinfectant and then rinsed, and inoculated into the tissue culture medium for tissue culture.

[0017] The invention has the beneficial effects that the invention reduces the contamination rate and browning rate of the explants through improvement of the tissue culture medium, cold treatment of the explants and secondary disinfection of the explants, thereby improving the survival rate of the passion fruit explants in the tissue culture process and overcoming the disadvantage of high browning mortality rate in the passion fruit tissue culture process.

[0018] Furthermore, the low temperature preservation in step (1) is specifically as follows: the sterilized explant is immersed in a polyvinyl pyrrolidone sterile solution for low temperature preservation.

[0019] The beneficial effects of adopting the above further scheme are as follows: the invention reduces enzyme activity and alleviates the occurrence of browning reaction by treating the explant at low temperature; the combination of polyvinyl pyrrolidone and phenolic substances reduces the amount of phenolic substrates that can be oxidized by polyphenol oxidase and alleviates the occurrence of browning reaction; the secondary disinfection after eluting the phenolic substances effectively reduces the contamination rate and improves the survival rate and the tissue culture efficiency of passion fruit.

[0020] Furthermore, the low-temperature storage temperature is 2°C to 5°C; the time is 1 day to 3 days; and the mass concentration of the polyvinyl pyrrolidone sterile solution is 0.25% to 1%.

[0021] Furthermore, the explants are cleaned and disinfected in step (1) as follows: the explants are soaked in a 2% to 4% detergent solution for 10 to 20 minutes, rinsed with running water for 1 hour to 3 hours, the branches of the explants are cut into small sections, disinfected with 70% to 80% alcohol for 30 seconds, disinfected with 0.1% to 1% HgCl2 for 5 minutes, and rinsed with sterile water for 3 to 5 times.

[0022] Furthermore, the soaking in the disinfectant in step (2) is specifically: soaking in a HgCl2 with a mass concentration of 0.1% to 1% for 2 minutes to 10 minutes. DETAILED DESCRIPTION

[0023] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0024] Embodiment 1: passion fruit explant anti-browning tissue culture one

[0025] (1) Low temperature treatment of explants: Select passion fruit branches with buds, cut off the leaves, soak in 2% detergent solution for 15 minutes, rinse with running water for 2 hours, cut the passion fruit branches with buds into 5 cm small segments, disinfect with 75% alcohol for 30 seconds on a clean bench, disinfect with 0.1% HgCl2 for 5 minutes, and rinse with sterile water 4 times. Soak the disinfected explants in 0.5% polyvinyl pyrrolidone sterile solution and store at 4°C for 2 days to obtain low temperature treated explants;

[0026] (2) Preparation of tissue culture medium: dig out the pulp of Tainong passion fruit, crush and blend it with a juicer, filter it with 3 layers of 80-mesh gauze to obtain passion fruit juice, and set it aside; mix 1 / 2MS culture medium (Beijing Bell Biotechnology Co., Ltd.), 175 ml / L passion fruit juice, 1.0 mg / L 6-BA, 0.10 mg / L IBA, 30 g / L sucrose, and 7 g / L agar, adjust the pH to 5.9, and dispense it into triangular bottles, 20 bottles per liter, sterilize it under high pressure, and cool it to obtain tissue culture medium, and set it aside;

[0027] (3) Re-sterilization and inoculation of explants: Soak the cryogenically treated explants in 0.1% HgCl2 for 5 min, rinse with sterile water 4 times, and inoculate the cryogenically treated explants on tissue culture medium, with one flask inoculated with one stem segment. Place the flask in a culture room at 26°C to 28°C and a humidity below 40%, with a light intensity of 2000 lx and a lighting time of 12 h / day.

[0028] Embodiment 2: Passiflora edulis explant anti-browning tissue culture II

[0029] (1) Low temperature treatment of explants: Select passion fruit branches with buds, cut off the leaves, soak in 2% detergent solution for 15 minutes, rinse with running water for 2 hours, cut the passion fruit branches with buds into 5 cm small segments, disinfect with 75% alcohol for 30 seconds on a clean bench, disinfect with 0.1% HgCl2 for 5 minutes, and rinse with sterile water 4 times. Soak the disinfected explants in 0.5% polyvinyl pyrrolidone sterile solution and store at 4°C for 2 days to obtain low temperature treated explants;

[0030] (2) Preparation of tissue culture medium: dig out the pulp of Tainong passion fruit, crush and blend it with a juicer, filter it with 3 layers of 80-mesh gauze to obtain passion fruit juice, and set it aside; mix 1 / 2MS culture medium (Beijing Bell Biotechnology Co., Ltd.), 150 ml / L passion fruit juice, 0.5 mg / L 6-BA, 0.05 mg / L IBA, 25 g / L sucrose, and 6 g / L agar, adjust the pH to 5.8, and dispense it into Erlenmeyer bottles, 20 bottles per liter, sterilize it under high pressure, and cool it to obtain tissue culture medium, and set it aside;

[0031] (3) Re-sterilization and inoculation of explants: Soak the cryogenically treated explants in 0.1% HgCl2 for 5 min, rinse with sterile water 4 times, and inoculate the cryogenically treated explants on tissue culture medium, with one flask inoculated with one stem segment. Place the flask in a culture room at 26°C to 28°C and a humidity below 40%, with a light intensity of 2000 lx and a lighting time of 12 h / day.

[0032] Embodiment 3: Passiflora edulis explant anti-browning tissue culture three

[0033] (1) Low temperature treatment of explants: Select passion fruit branches with buds, cut off the leaves, soak in 2% detergent solution for 15 minutes, rinse with running water for 2 hours, cut the passion fruit branches with buds into 5 cm small segments, disinfect with 75% alcohol for 30 seconds on a clean bench, disinfect with 0.1% HgCl2 for 5 minutes, and rinse with sterile water 4 times. Soak the disinfected explants in 0.5% polyvinyl pyrrolidone sterile solution and store at 4°C for 2 days to obtain low temperature treated explants;

[0034] (2) Preparation of tissue culture medium: dig out the pulp of Tainong passion fruit, crush and blend it with a juicer, filter it with 3 layers of 80-mesh gauze to obtain passion fruit juice, and set it aside; mix 1 / 2MS culture medium (Beijing Bell Biotechnology Co., Ltd.), 200 ml / L passion fruit juice, 1.5 mg / L 6-BA, 0.15 mg / L IBA, 35 g / L sucrose, and 8 g / L agar, adjust the pH to 6.0, and dispense it into Erlenmeyer bottles, 20 bottles per liter, sterilize it by high pressure, and cool it to obtain tissue culture medium, and set it aside;

[0035] (3) Re-sterilization and inoculation of explants: Soak the cryogenically treated explants in 0.1% HgCl2 for 5 min, rinse with sterile water 4 times, and inoculate the cryogenically treated explants on tissue culture medium, with one flask inoculated with one stem segment. Place the flask in a culture room at 26-28°C and a humidity below 40%, with a light intensity of 2000 lx and a lighting time of 12 h / day.

[0036] Comparative Example 1: Tissue Culture Method for Anti-Browning of Passiflora Explants

[0037] The difference between this comparative example and Example 1 is that the raw materials of the tissue culture medium in step (2) are different, and the remaining steps, conditions, and raw materials are the same as those in Example 1. Step (2) is specifically as follows:

[0038] (2) Preparation of tissue culture medium: MS medium, 1.0 mg / L 6-BA, 0.10 mg / L IBA, 30 g / L sucrose, and 7 g / L agar were adjusted to pH 5.9, dispensed into Erlenmeyer flasks, 20 bottles per liter, sterilized by high pressure, and cooled to obtain tissue culture medium for later use.

[0039] Comparative Example 2: Anti-browning tissue culture of passion fruit explants

[0040] The difference between this comparative example and Example 1 is that the explant treatment in step (1) is different, and the explant does not need to be sterilized twice in step (3). The remaining steps, conditions, and raw materials are the same as those in Example 1. Steps (1) and (3) are specifically as follows:

[0041] (1) Explant treatment: Select passion fruit branches with buds, cut off the leaves, soak in 2% detergent solution for 15 min, rinse with running water for 2 h, cut the branches into 5 cm small segments, disinfect with 75% alcohol for 30 s on a clean bench, disinfect with 0.1% HgCl2 for 5 min, and rinse with sterile water 4 times;

[0042] (3) Explant inoculation and culture: The explants disinfected in step (1) are inoculated on tissue culture medium, with one flask inoculated with one stem segment, and the flask is placed in a culture room at a temperature of 26-28° C. and a humidity of less than 40%, with a light intensity of 2000 lx and a lighting time of 12 h / day.

[0043] Comparative Example 3: Anti-browning tissue culture of passion fruit explants

[0044] The difference between this comparative example and Example 1 is that the explant treatment in step (1) and the preparation of tissue culture medium in step (2) are different, and the explant in step (3) does not require secondary sterilization. The remaining steps, conditions, and raw materials are the same as those in Example 1. Steps (1), (2), and (3) are specifically as follows:

[0045] (1) Explant treatment: Select passion fruit branches with buds, cut off the leaves, soak in 2% detergent solution for 15 min, rinse with running water for 2 h, cut the branches into 5 cm small segments, disinfect with 75% alcohol for 30 s on a clean bench, disinfect with 0.1% HgCl2 for 5 min, and rinse with sterile water 4 times;

[0046] (2) Preparation of tissue culture medium: MS medium (Beijing Bell Biotechnology Co., Ltd.), 1.0 mg / L 6-BA, 0.10 mg / L IBA, 30 g / L sucrose, and 7 g / L agar were adjusted to pH 5.9 and dispensed into Erlenmeyer flasks, 20 bottles per liter, sterilized by high pressure, and cooled to obtain tissue culture medium for later use.

[0047] (3) Explant inoculation and culture: The explants disinfected in step (1) were inoculated on tissue culture medium, with one stem segment inoculated in one Erlenmeyer flask, and cultured in a culture room at a temperature of 26-28° C. and a humidity of less than 40%, with a light intensity of 2000 lx and a lighting time of 12 h / day.

[0048] Test example:

[0049] 360 "Zhuangmi 05" passion fruit branches with bud stem segments were selected as explants and randomly divided into 6 groups, each with 60. Tissue culture was carried out according to the culture method of Examples 1 to 3 and Comparative Examples 1 to 3, and the passion fruit explants of Examples 1 to 3 and Comparative Examples 1 to 3 after 20 days of tissue culture were taken out, and the contamination rate, browning mortality rate, and survival rate of the explants in each group were observed and counted, wherein the contamination rate = the number of contaminated stem segments / the number of inoculated stem segments; the browning mortality rate = the number of browning dead stem segments / the number of inoculated stem segments; the survival rate = the number of surviving stem segments / the number of inoculated stem segments, and the experiment was repeated three times, and the results are shown in Table 1.

[0050] Table 1 (Unit: %)

[0051]

[0052]

[0053] From Table 1, we can get:

[0054] (1) The explant contamination rate and browning mortality rate of the passion fruit explants after tissue culture in Example 1 were significantly lower than those of the passion fruit explants in Comparative Examples 1 to 3. The survival rate of Example 1 was 88.3%. The culture medium of Comparative Example 1 did not add passion fruit juice, and the amount of MS culture medium added was not halved compared with that in Example 1. The survival rate was 73.3%, which was lower than that in Example 1. The reason may be that Mg in MS culture medium 2+ , Ca 2+ , Cu 2+ The content of divalent ions is high, the degree of phenolic oxidation and browning is high; at the same time, no passion fruit juice was added in Comparative Example 1, the antioxidant components in the culture medium are less, the enzyme activity is high, the polyphenols are oxidized quickly, and the degree of browning in the culture medium is high.

[0055] (2) In comparative example 2, the explants were not subjected to low temperature treatment during tissue culture, and the survival rate was 71.7%. It can be seen that the present invention reduces the enzyme activity and alleviates the occurrence of browning reaction by low temperature treatment of the explants; the combination of polyvinyl pyrrolidone and phenolic substances reduces the amount of phenolic substrates that can be oxidized by polyphenol oxidase and alleviates the occurrence of browning reaction; the secondary disinfection after elution of the phenolic substances effectively reduces the contamination rate and improves the survival rate.

[0056] (3) The survival rate of Comparative Example 3 was only 48.3%. It can be seen that the low temperature treatment of the explants and the improvement of the culture medium cooperated with each other, which reduced the browning mortality rate and contamination rate during the tissue culture process of Passiflora edulis, and the explant survival rate was increased to more than 80%.

[0057] In summary, the tissue culture method of the present invention reduces the contamination rate and browning rate of explants, improves the survival rate of passion fruit explants in the tissue culture process, overcomes the disadvantage of high browning mortality rate in the passion fruit tissue culture process, and improves the passion fruit tissue culture efficiency.

[0058] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A tissue culture medium for preventing browning of passion fruit explants, characterized in that: The tissue culture medium comprises the following components: MS culture medium, passion fruit juice, 6-BA, IBA, sucrose and agar.

2. a kind of passion fruit explant anti-browning tissue culture medium according to claim 1, is characterized in that, The tissue culture medium comprises the following components in the following amounts: 1 / 2MS culture medium, 150ml / L-200ml / L of passion fruit juice, 0.5mg / L-1.5mg / L of 6-BA, 0.05mg / L-0.15mg / L of IBA, 25g / L-35g / L of sucrose, and 6g / L-8g / L of agar.

3. a kind of passion fruit explant anti-browning tissue culture medium according to claim 2, is characterized in that, The preparation method of the passion fruit juice is specifically as follows: squeezing passion fruit pulp, filtering with gauze, and obtaining the passion fruit juice.

4. a kind of passion fruit explant anti-browning tissue culture medium according to claim 3, is characterized in that, The passion fruit is Tainong passion fruit.

5. a tissue culture medium for preventing browning of passion fruit explant according to claim 3, characterized in that, The gauze has 60 to 80 meshes.

6. A tissue culture method for preventing browning of passion fruit explants, characterized in that: The tissue culture method comprises the following steps: (1) selecting a passion fruit stem segment with buds as an explant, cleaning and sterilizing the explant to obtain a sterilized explant, and storing the sterilized explant at a low temperature to obtain a low-temperature treated explant; (2) The explant treated at low temperature is taken out, soaked in a disinfectant, rinsed, and inoculated onto the tissue culture medium according to any one of claims 1 to 5 for tissue culture.

7. a kind of passion fruit explant anti-browning tissue culture method according to claim 6, is characterized in that, The low temperature preservation in step (1) is specifically as follows: the sterilized explant is immersed in a polyvinyl pyrrolidone sterile solution for low temperature preservation.

8. a kind of tissue culture method for anti-browning of passion fruit explant according to claim 7, it is characterized in that, The temperature of the low-temperature storage is 2° C. to 5° C., and the time is 1 day to 3 days; the mass concentration of the polyvinyl pyrrolidone sterile solution is 0.25% to 1%.

9. a kind of tissue culture method for anti-browning of passion fruit explant according to claim 6, is characterized in that, The explants are cleaned and disinfected in step (1) as follows: the explants are soaked in a 2% to 4% detergent solution for 10 to 20 minutes, rinsed with running water for 1 hour to 3 hours, the branches of the explants are cut into small sections, disinfected with 70% to 80% alcohol for 30 seconds, disinfected with 0.1% to 1% HgCl2 for 5 minutes, and rinsed with sterile water for 3 to 5 times.

10. A tissue culture method for preventing browning of passion fruit explants according to claim 6, characterized in that, The soaking in the disinfectant in step (2) is specifically: soaking in 0.1% to 1% HgCl2 for 2 minutes to 10 minutes.

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