A tissue culture medium and a tissue culture method for preventing browning of passion fruit explants

Abstract

The present application relates to a kind of passion fruit explant brown prevention tissue culture medium and its tissue culture method, it is related to passion fruit tissue culture technical field.The present application provides a kind of passion fruit explant brown prevention tissue culture medium, the tissue culture medium includes the following components:MS culture medium, passion fruit juice, 6-BA, IBA, sucrose, agar.The present application is improved by culture medium, explant low-temperature treatment and explant secondary disinfection, and the pollution rate of explant, browning rate is reduced, improves the survival rate of passion fruit explant in the process of tissue culture, overcomes the shortcoming that browning mortality is high in the process of passion fruit tissue culture, improves the tissue culture efficiency of passion fruit.

Description

A tissue culture medium for preventing browning of passionflower explants and its tissue culture method Technical Field

[0001] This invention relates to the field of passion fruit tissue culture technology, specifically to a tissue culture medium for preventing browning of passion fruit explants and its tissue culture method. Background Technology

[0002] Passionflower is a perennial evergreen woody vine belonging to the genus Passiflora (Passiflora Linn.) of the family Passifloraceae. Major cultivation areas in my country include Guangxi Zhuang Autonomous Region, Guizhou Province, Guangdong Province, Fujian Province, and Yunnan Province. It has uses for food, medicine, and ornamental purposes. Due to its rich flavor, pleasant aroma, and high nutritional value (including vitamin C, minerals, and amino acids), it has become increasingly popular in the market in recent years.

[0003] Traditional passion fruit propagation primarily relies on vine cuttings and grafting. These methods are limited by climate and geographical location, resulting in slow propagation rates and numerous problems such as varietal degeneration, quality decline, and reduced yield. These issues hinder the development of the passion fruit industry. Passion fruit tissue culture offers advantages in terms of speed and quality, making it a crucial technology for enhancing the passion fruit industry. However, rapid propagation through tissue culture suffers from high rates of browning mortality and contamination, severely impacting its efficiency. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a tissue culture medium for preventing browning of passion fruit explants and a method for its tissue culture. This invention reduces the contamination and browning rates of explants by modifying the culture medium, subjecting explants to low-temperature treatment, and performing secondary sterilization of the explants. This improves the survival rate of passion fruit explants during tissue culture, overcomes the high mortality rate due to browning during passion fruit tissue culture, and enhances the efficiency of passion fruit tissue culture.

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

[0006] The first objective of this invention is to provide a tissue culture medium for preventing browning of passion fruit explants, the tissue culture medium comprising the following components: MS medium, passion fruit juice, 6-BA, IBA, sucrose, and agar.

[0007] The beneficial effects of adopting the above-mentioned further scheme are as follows: In this invention, passion fruit juice is added to the tissue culture medium. Passion fruit juice is rich in antioxidants such as vitamin C and flavonoids, which reduces enzyme activity, reduces the oxidation of polyphenols, and reduces the occurrence of browning during culture. At the same time, passion fruit juice contains a variety of nutrients such as amino acids, vitamins, minerals, sugars, and organic acids, which can supplement the nutrients required during the cultivation of passion fruit.

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

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

[0010] The beneficial effects of adopting the above-mentioned further scheme are: the present invention reduces the amount of MS culture medium components, thereby increasing the amount of Mg... 2+ Ca 2+ Cu 2 + The reduced content of divalent ions inhibits phenol oxidation and reduces browning.

[0011] Furthermore, the preparation method of the passion fruit juice is as follows: the pulp of the passion fruit is juiced, filtered through gauze, to obtain the passion fruit juice.

[0012] Furthermore, the passion fruit mentioned is the Taiwanese passion fruit.

[0013] Furthermore, the gauze is 60-80 mesh.

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

[0015] (1) Select passionflower stem segments with buds as explants, clean and disinfect the explants to obtain disinfected explants, and preserve the disinfected explants at low temperature to obtain low-temperature treated explants.

[0016] (2) Take out the explants that have been treated at low temperature, soak them in disinfectant and rinse them, and then inoculate them onto the tissue culture medium for tissue culture.

[0017] The beneficial effects of this invention are: by improving the tissue culture medium, cold treatment of explants and secondary disinfection of explants, this invention reduces the contamination rate and browning rate of explants, improves the survival rate of passion fruit explants in tissue culture, and overcomes the disadvantage of high browning mortality rate in passion fruit tissue culture.

[0018] Furthermore, the low-temperature preservation in step (1) is specifically as follows: the disinfected explants are immersed in a sterile polyvinylpyrrolidone solution for low-temperature preservation.

[0019] The beneficial effects of adopting the above-mentioned further scheme are as follows: the present invention treats explants at low temperature, thereby reducing enzyme activity and mitigating the browning reaction; the binding of polyvinylpyrrolidone with phenolic substances reduces the amount of phenolic substrates that can be oxidized by polyphenol oxidase, thus mitigating the browning reaction; the secondary disinfection after elution of phenolic substances effectively reduces the contamination rate and improves the survival rate and tissue culture efficiency of passion fruit.

[0020] Furthermore, the cryopreservation temperature is 2℃~5℃; the time is 1d~3d; and the mass concentration of the polyvinylpyrrolidone sterile solution is 0.25%~1%.

[0021] Furthermore, the cleaning and disinfection of the explants in step (1) are as follows: the explants are soaked in a 2% to 4% detergent solution for 10 to 20 minutes, rinsed with running water for 1 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 to 10 minutes, and rinsed with sterile water 3 to 5 times.

[0022] Furthermore, the soaking in the disinfectant in step (2) specifically involves soaking in 0.1% to 1% HgCl2 for 2 to 10 minutes. Detailed Implementation

[0023] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0024] Example 1: Tissue culture for preventing browning of passion fruit explants

[0025] (1) Low-temperature treatment of explants: Select passion fruit stem segments with buds, remove the leaves, soak in 2% detergent solution for 15 min, rinse with running water for 2 h, cut the passion fruit stem segments with buds into 5 cm 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. Soak the disinfected explants in 0.5% polyvinylpyrrolidone sterile solution and store at 4℃ for 2 days to obtain low-temperature treated explants;

[0026] (2) Preparation of tissue culture medium: The pulp of the passion fruit was scooped out and crushed and blended using a juicer. The pulp was then filtered through three layers of 80-mesh gauze to obtain passion fruit juice for later use. 1 / 2 MS 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 were mixed and the pH was adjusted to 5.9. The mixture was dispensed into Erlenmeyer flasks, 20 flasks per liter, and autoclaved. After cooling, the tissue culture medium was obtained for later use.

[0027] (3) Re-disinfection and inoculation culture of explants: The explants treated with low temperature were soaked in 0.1% HgCl2 for 5 min and rinsed 4 times with sterile water. The explants treated with low temperature were inoculated on tissue culture medium, one stem segment was inoculated in one Erlenmeyer flask, and the Erlenmeyer flasks were placed in a culture room with a temperature of 26℃~28℃ and a humidity of less than 40% for culture, with a light intensity of 2000 lx and a light duration of 12 h / day.

[0028] Example 2: Tissue culture for preventing browning of passionflower explants (Part II)

[0029] (1) Low-temperature treatment of explants: Select passion fruit stem segments with buds, remove the leaves, soak in 2% detergent solution for 15 min, rinse with running water for 2 h, cut the passion fruit stem segments with buds into 5 cm 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. Soak the disinfected explants in 0.5% polyvinylpyrrolidone sterile solution and store at 4℃ for 2 days to obtain low-temperature treated explants;

[0030] (2) Preparation of tissue culture medium: The pulp of the passion fruit was scooped out and crushed and blended using a juicer. The pulp was then filtered through three layers of 80-mesh gauze to obtain passion fruit juice, which was set aside. 1 / 2 MS 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 were mixed and the pH was adjusted to 5.8. The mixture was dispensed into Erlenmeyer flasks, 20 flasks per liter, and autoclaved. After cooling, the tissue culture medium was obtained and set aside.

[0031] (3) Re-disinfection and inoculation culture of explants: The explants treated with low temperature were soaked in 0.1% HgCl2 for 5 min and rinsed 4 times with sterile water. The explants treated with low temperature were inoculated on tissue culture medium, one stem segment was inoculated in one Erlenmeyer flask, and the Erlenmeyer flasks were placed in a culture room with a temperature of 26℃~28℃ and a humidity of less than 40% for culture, with a light intensity of 2000 lx and a light duration of 12 h / day.

[0032] Example 3: Tissue culture for preventing browning of passion fruit explants (Part 3)

[0033] (1) Low-temperature treatment of explants: Select passion fruit stem segments with buds, remove the leaves, soak in 2% detergent solution for 15 min, rinse with running water for 2 h, cut the passion fruit stem segments with buds into 5 cm 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. Soak the disinfected explants in 0.5% polyvinylpyrrolidone sterile solution and store at 4℃ for 2 days to obtain low-temperature treated explants;

[0034] (2) Preparation of tissue culture medium: The pulp of the passion fruit was scooped out and crushed and blended using a juicer. The pulp was then filtered through three layers of 80-mesh gauze to obtain passion fruit juice, which was set aside. 1 / 2 MS 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 were mixed and the pH was adjusted to 6.0. The mixture was dispensed into Erlenmeyer flasks, 20 flasks per liter, autoclaved, and cooled to obtain tissue culture medium, which was set aside.

[0035] (3) Re-disinfection and inoculation culture of explants: The explants treated with low temperature were soaked in 0.1% HgCl2 for 5 min and rinsed 4 times with sterile water. The explants treated with low temperature were inoculated on tissue culture medium, one stem segment was inoculated in one Erlenmeyer flask, and the Erlenmeyer flasks were placed in a culture room with a temperature of 26-28℃ and a humidity of less than 40% for culture, with a light intensity of 2000 lx and a light duration of 12 h / day.

[0036] Comparative Example 1: Tissue culture method for preventing browning of passionflower explants

[0037] The only difference between this comparative example and Example 1 is the raw material of the tissue culture medium in step (2). All other steps, conditions, and raw materials are the same as in Example 1. Step (2) is as follows:

[0038] (2) Preparation of tissue culture medium: MS medium, 6-BA 1.0 mg / L, IBA 0.10 mg / L, sucrose 30 g / L, agar 7 g / L, adjust the pH to 5.9, dispense into Erlenmeyer flasks, dispense 20 flasks per liter, autoclave, cool to obtain tissue culture medium for later use.

[0039] Comparative Example 2: Tissue culture for preventing browning of passionflower explants

[0040] The only difference between this comparative example and Example 1 is the explant treatment in step (1). In step (3), the explant does not require secondary sterilization. All other steps, conditions, and raw materials are the same as in Example 1. Steps (1) and (3) are as follows:

[0041] (1) Treatment of explants: Select passion fruit branches with buds, remove the leaves, soak in 2% detergent solution for 15 minutes, rinse with running water for 2 hours, cut the branches into 5cm sections, 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.

[0042] (3) Explant inoculation and culture: The explants that have been disinfected in step (1) are inoculated on tissue culture medium. One stem segment is inoculated into one Erlenmeyer flask. The Erlenmeyer flasks are placed in a culture room with a temperature of 26-28℃ and a humidity of less than 40% for culture. The light intensity is 2000 lx and the light duration is 12h / day.

[0043] Comparative Example 3: Tissue culture for preventing browning of passionflower explants

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

[0045] (1) Treatment of explants: Select passion fruit branches with buds, remove the leaves, soak in 2% detergent solution for 15 minutes, rinse with running water for 2 hours, cut the branches into 5cm sections, 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.

[0046] (2) Preparation of tissue culture medium: MS medium (Beijing Bell Biotechnology Co., Ltd.), 6-BA 1.0 mg / L, IBA 0.10 mg / L, sucrose 30 g / L, agar 7 g / L, adjust the pH to 5.9, dispense into Erlenmeyer flasks, dispense 20 flasks per liter, autoclave, cool to obtain tissue culture medium for later use.

[0047] (3) Explant inoculation and culture: The explants that have been disinfected in step (1) are inoculated on tissue culture medium. One stem segment is inoculated in one Erlenmeyer flask and cultured in a culture room with a temperature of 26-28℃ and a humidity of less than 40%, with a light intensity of 2000 lx and a light duration of 12 h / day.

[0048] Experimental example:

[0049] Thirty-sixty passion fruit stem segments with buds of the 'Zhuangmi 05' variety were selected as explants and randomly divided into six groups of 60 segments each. Tissue culture was performed according to the methods used in Examples 1-3 and Comparative Examples 1-3. After 20 days of tissue culture, the passion fruit explants from Examples 1-3 and Comparative Examples 1-3 were removed, and the contamination rate, browning mortality rate, and survival rate of each group were observed and statistically analyzed. The contamination rate was calculated as: number of contaminated stem segments / number of inoculated stem segments; browning mortality rate was calculated as: number of browned and dead stem segments / number of inoculated stem segments; and survival rate was calculated as: number of surviving stem segments / number of inoculated stem segments. 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 obtain:

[0054] (1) The explants of *Passiflora edulis* cultured in Example 1 showed significantly lower explant contamination and browning mortality rates compared to those in Comparative Examples 1-3. The survival rate in Example 1 was 88.3%. In Comparative Example 1, no *Passiflora edulis* juice was added to the culture medium, and the amount of MS medium added was not halved compared to Example 1; the survival rate was 73.3%, lower than in Example 1. This may be due to the presence of Mg in the MS medium. 2+ Ca 2+ Cu 2+ The content of divalent ions was high, the degree of phenolic oxidation was high, and the degree of browning was high. Meanwhile, in Comparative Example 1, no passion fruit juice was added, the antioxidant components in the culture medium were less, the enzyme activity was high, the polyphenols were oxidized quickly, and the degree of browning in the culture medium was 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 enzyme activity and alleviates browning reaction by treating the explants with low temperature; the combination of polyvinylpyrrolidone and phenolic substances reduces the amount of phenolic substrates that can be oxidized by polyphenol oxidase, thus alleviating browning reaction; the secondary disinfection after elution of 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%, which shows that the low temperature treatment of explants and the improvement of culture medium synergistically reduced the browning mortality rate and contamination rate of passion fruit tissue culture, and increased the survival rate of explants 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 during tissue culture, overcomes the disadvantage of high browning mortality during passion fruit tissue culture, and improves the tissue culture efficiency of passion fruit.

[0058] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A tissue culture medium for preventing browning of passionflower explants, characterized in that, The tissue culture medium consists of the following components in the following amounts: 1 / 2 MS medium, passion fruit juice 150ml / L~200ml / L, 6-BA 0.5mg / L~1.5mg / L, IBA 0.05mg / L~0.15mg / L, sucrose 25g / L~35g / L, and agar 6g / L~8g / L.

2. The tissue culture medium for preventing browning of passionflower explants according to claim 1, characterized in that, The specific method for preparing the passion fruit juice is as follows: the pulp of the passion fruit is juiced, filtered through gauze, to obtain the passion fruit juice.

3. The tissue culture medium for preventing browning of passionflower explants according to claim 2, characterized in that, The passion fruit mentioned is the Taiwanese passion fruit.

4. The tissue culture medium for preventing browning of passionflower explants according to claim 2, characterized in that, The gauze is 60-80 mesh.

5. A tissue culture method for preventing browning of passionflower explants, characterized in that, The tissue culture method includes the following steps: (1) Select passionflower stem segments with buds as explants, clean and disinfect the explants to obtain disinfected explants, and preserve the disinfected explants at low temperature to obtain low-temperature treated explants; (2) Take out the low-temperature treated explants, soak them in disinfectant solution and rinse them, and inoculate them onto the tissue culture medium according to any one of claims 1 to 4 for tissue culture; The low-temperature preservation in step (1) is specifically as follows: Soak the disinfected explants in polyvinylpyrrolidone solution. The explants are stored at low temperature in a sterile polyvinylpyrrolidone solution. The temperature for low temperature storage is 2℃~5℃ and the time is 1d~3d. The mass concentration of the sterile polyvinylpyrrolidone solution is 0.25%~1%. The specific steps for cleaning and disinfecting the explants in step (1) are as follows: the explants are soaked in a 2%~4% detergent solution for 10min~20min, rinsed with running water for 1h~3h, the branches of the explants are cut into small sections, disinfected with 70%~80% alcohol for 30s, disinfected with a 0.1%~1% HgCl2 solution for 5min~10min, and rinsed with sterile water 3~5 times. The specific steps for soaking in the disinfectant solution in step (2) are as follows: soaking in a 0.1%~1% HgCl2 solution for 2min~10min.

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