Method for culturing jujube witches broom tissue culture seedlings by using liquid culture medium

Abstract

The invention relates to the technical field of jujube witches broom tissue culture seedling culture, and discloses a method for culturing jujube witches broom tissue culture seedlings by using a liquid culture medium, which realizes efficient culture of jujube witches broom tissue culture seedlings through specific steps of material preparation, early-stage material treatment, liquid culture medium preparation, high-temperature sterilization treatment, subculture of tissue culture seedlings and the like. Compared with a traditional solid culture medium culture method, the method for culturing the jujube witches broom tissue culture seedlings by using the liquid culture medium can significantly improve the propagation rate of the jujube witches broom tissue culture seedlings, reduce the labor cost, reduce the contamination probability of the tissue culture seedlings, and improve the survival rate of the jujube witches broom tissue culture seedlings. And a more effective test material is provided for scientific research and prevention and control technology research and development of jujube witches broom.

Description

Technical Field

[0001] The invention relates to the technical field of culturing tissue culture seedlings of jujube madness, and in particular to a method for culturing tissue culture seedlings of jujube madness by utilizing a liquid culture medium. Background Art

[0002] Jujube (Ziziphus jujuba Mill.) is a plant of the genus Ziziphus (Ziziphus Mill.), family Rhamnaceae. Jujube trees are cold-resistant and drought-tolerant, making them particularly well-suited to arid mountainous regions. Jujubes are native to my country and are a uniquely economical tree species. Jujubes are highly nutritious and a tonic, and can also be processed into a variety of products, making them popular both domestically and internationally. In China, there are over 1,000 varieties of jujube, which are primarily categorized into five categories based on their intended use: those specifically for fresh consumption, those for drying, those for both fresh and dried consumption, ornamental varieties, and those used for candied processing.

[0003] Jujube witches' broom (JWB), caused by phytoplasmas, is a devastating infectious disease affecting jujube production. It is widespread in nearly all jujube cultivation areas. Once JWB strikes, it manifests as endogenous hormone imbalances and physiological disturbances in the tree, resulting in arbuscular fasciitis, small leaves, flower-leaf malformation, fruit deformities, and yellowing leaves. This leads to the tree's failure to shed leaves normally in winter, resulting in death from freezing. For this reason, JWB is often referred to as the "cancer" of the jujube tree. JWB is an infectious disease that poses a significant threat to jujube production, severely impacting the highly economically valuable tree. In jujube-growing areas, the disease is almost inevitable, and once it breaks out, it is often difficult to effectively control. It is considered one of the most destructive diseases to the jujube tree and its industry. From an agricultural production perspective, the continued spread of JWB not only significantly increases production costs but also severely damages the quality and yield of jujube fruit, reducing farmers' economic returns and posing a direct threat to the sustainability of the entire industry. Therefore, jujube madness is a serious obstacle to the sustainable development of the jujube industry, and it is also an important difficulty that needs to be overcome.

[0004] In the early studies, most of the focus was on observing the morphological changes of the jujube trees that were the hosts of the disease. After the disease, the growth of the flower organs, buds, branches, leaves, stipules, and fruits of the jujube trees was abnormal: the flower organs reverted to their original form and turned into twigs, among which the calyx tube of the calyx turned into leaflets with axillary buds that were whorled on the first node of the twig; the petals turned into 5 leaflets that alternated with the sepals on the first node of the twig; the stamens turned into leaflets on the first node of the twig that were opposite to the petal leaflets, or branches with 2 leaflets opposite each other; the pistil disappeared, and 2 leaflets were opposite each other on the second node of the twig; the disc and ovules atrophied, leaving traces of the ovary; the buds and hidden buds germinated abnormally (the main buds were located on the first and second branches of development, and the main buds in the main buds of the year were not normal). Generally, buds do not germinate, forming latent buds in the second year. These latent buds do not germinate, while secondary buds are located on perennial branches and may germinate in the same year. Diseased primary and latent buds germinate to form developmental branches, and the primary and secondary buds formed on these branches germinate again to form developmental branches. Jujube branches infected with jujube madness extend and grow into developmental branches. Old leaves in the diseased leaves turn yellow and curl, while the veins of young leaves turn yellow and curl into a spoon-shaped shape. After inoculation with the pathogen, the main veins of the diseased young leaves separate from the mesophyll at the leaf tip. Spines develop abnormally and become stipules after infection. Fruit development is abnormal, manifested by growth cessation and the formation of warty protrusions with red and light green mottled areas on the fruit surface. The appearance of diseased jujube trees varies at different stages of the disease, divided into five stages: leaf change, flower change, branch change, tree madness, and tree death. Related studies have shown that the leaf and branch change stages are favorable periods for preventing and controlling jujube madness.

[0005] Currently, jujube madness is rapidly spreading and has become a major factor hindering the healthy development of the jujube industry. Its presence poses a significant threat to this species, which holds great economic potential. However, due to the difficulty of culturing phytoplasmas in vitro, tissue culture of jujube madness seedlings has become a highly effective test material for jujube madness research.

[0006] However, there are many shortcomings in the existing solid culture medium for culturing jujube madness tissue culture seedlings, such as the slow growth of jujube madness tissue culture seedlings, high labor costs and a high chance of infection, and solid culture medium is not conducive to the screening of anti-jujube madness agents.

[0007] Therefore, it is necessary to provide a method for culturing jujube madness tissue culture seedlings using liquid culture medium to solve the above problems. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for culturing jujube madness tissue culture seedlings using liquid culture medium, so as to increase the proliferation rate, reduce labor costs and reduce the probability of infection of jujube madness tissue culture seedlings.

[0009] The method for culturing jujube madness tissue culture seedlings using a liquid culture medium provided by the present invention comprises the following steps:

[0010] Step 1: Material preparation:

[0011] 20ml MS1, 5ml MS2, 2ml MS3, 10ml MS4, 10ml MS5, 20ml MS6, 10ml KH2PO4, 30g sucrose, 1.5ml KOH, a heat-resistant sponge, a marker, scissors, a ruler, a beaker, and 350ml canning jars with matching lids.

[0012] Step 2: Preliminary material processing:

[0013] First, use a marker to measure a 6cm diameter circle on a sponge cloth and cut it out. Then, cut three 5mm holes 1.5cm from the edge of the circle. Once the holes are cut, place them in a canning jar, cover tightly, and set aside.

[0014] Step 3: Preparation of liquid culture medium for Jujube Madness tissue culture seedlings:

[0015] First, pour 20ml of MS1, 5ml of MS2, 2ml of MS3, 10ml of MS4, 10ml of MS5, 20ml of MS6, 10ml of KH2PO4, and 1L of sterile water into a pot and stir thoroughly with a glass rod. Then, heat. Add 30g of sucrose and stir thoroughly with a glass rod. Once the mixture is fully homogenized, stop heating. Adjust the pH by adding approximately 1.5ml of KOH to 1L of MS0 medium. Stir thoroughly with a glass rod and check the pH with pH paper. A pH of approximately 5.8 is recommended. Finally, transfer the mixture to a beaker.

[0016] Step 4: High-temperature sterilization: Place the cans containing the sponges in the autoclave for sterilization. At the same time, place the beaker containing the MS0 culture medium solution in the autoclave for high-temperature sterilization at 121°C for 20 minutes. Allow to cool before use.

[0017] Step 5. Take out the canned bottle after high-temperature sterilization and place it in the fume hood. Open the lid and use tweezers to insert the jujube madness tissue culture seedlings that need to be subcultured into the three small holes. Then pour the liquid culture medium into the canned bottle. Pour 40-50ml of liquid culture medium into the 350ml canned bottle. After pouring the liquid culture medium, you can find that the sponge inserted into the jujube madness tissue culture seedlings floats on the liquid culture medium, and then close the lid tightly.

[0018] Compared with the related art, the method of culturing jujube madness tissue culture seedlings using liquid culture medium provided by the present invention has the following beneficial effects:

[0019] 1. The present invention significantly increases the propagation rate of jujube madness tissue culture seedlings. Normal solid culture medium requires about a month for jujube madness tissue culture seedlings to grow by about 1-1.5 cm. Liquid culture medium has high nutrient mobility, which is more conducive to nutrient absorption by jujube madness tissue culture seedlings, thereby accelerating the propagation rate of jujube madness tissue culture seedlings, allowing jujube madness tissue culture seedlings to grow by about 1.5-2 cm every month, resulting in an increased propagation rate of jujube madness tissue culture seedlings.

[0020] 2. The present invention reduces labor costs. Since solid culture medium for tissue culture seedlings of jujube madness requires manual subculture and replacement of new culture medium every month to maintain the supply of nutrients for the tissue culture seedlings, a lot of labor costs are consumed. In contrast, the culture medium in the canning bottle is automatically consumed every three months or so. Therefore, by directly pouring the liquid culture medium into the canning bottle, the number of manual subcultures is shortened and the growth time of the jujube madness tissue culture seedlings is prolonged. In addition, the present invention eliminates the need for tedious subculture processing. Instead, directly pouring the liquid culture medium into the canning bottle is convenient and quick, reducing manual processing time and greatly reducing labor costs.

[0021] 3. The present invention can reduce the probability of infection of tissue culture seedlings with jujube madness. Since solid tissue culture propagation requires manual operation and processing every month, there is a risk of infection as long as manual contact is made. In addition, in order to avoid infection during the operation, the hands must be sterilized with alcohol multiple times and new sterilization tools must be replaced multiple times, which greatly increases the labor time cost. However, once the jujube madness tissue culture seedlings are infected, they cannot be used. For liquid culture medium, just pour the liquid culture medium directly into the canning bottle every 3 months. This method can reduce the number of times manual contact is made with the jujube madness tissue culture seedlings, and pouring the liquid culture medium is quick and easy to avoid the risk of infection of the jujube madness tissue culture seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of culturing jujube madness tissue culture seedlings using liquid culture medium in the present invention;

[0023] Figure 2 The figures are comparative diagrams of culturing jujube madness tissue culture seedlings in liquid culture medium and solid culture medium respectively in the present invention.

[0024] Figure numerals: 1. canning bottle cap; 2. canning bottle; 3. jujube madness tissue culture seedlings; 4. sponge; 5. liquid culture medium; 6. small hole. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0026] Example 1: Investigate the effect of solid culture medium and liquid culture medium with the same composition on the propagation rate of the same variety of jujube mad disease tissue culture seedlings after 30 days:

[0027] 1. Test methods

[0028] Step 1. Prepare the following materials: 20ml MS1, 5ml MS2, 2ml MS3, 10ml MS4, 10ml MS5, 20ml MS6, 10ml KH2PO4, 5.5g agar, 30g sucrose, 1.5ml KOH, a heat-resistant sponge, a marker, scissors, a ruler, a beaker, a 350ml canning jar, and a lid of the same size.

[0029] Step 2: Preliminary material processing: First, use a marker to measure a 6cm diameter circle on a sponge cloth and cut it out. Then, cut three 5mm holes 1.5cm from the edge of the circle. After cutting, place the circle in a can, cover the bottle tightly, and set aside.

[0030] Step 3: Prepare the liquid culture medium for the jujube madness seedlings: First, pour 20ml of MS1, 5ml of MS2, 2ml of MS3, 10ml of MS4, 10ml of MS5, 20ml of MS6, 10ml of KH2PO4, and 1L of sterile water into a pot and stir thoroughly with a glass rod. Then, heat the mixture. Add 30g of sucrose and stir thoroughly with a glass rod. Once the mixture is fully homogenized, stop heating. Adjust the pH by adding approximately 1.5ml of KOH to 1L of MS0 medium, stirring thoroughly with a glass rod, and then test the pH with pH paper. A pH of approximately 5.8 is recommended. Finally, place the mixture into a beaker.

[0031] In addition to the above solvents, the solid culture medium also needs to add 5.5g of agar, which will solidify after cooling before use.

[0032] Step 4: High-temperature sterilization: Place the cans containing the sponges in the autoclave for sterilization. At the same time, place the beaker containing the MS0 culture medium solution in the autoclave for high-temperature sterilization at 121°C for 20 minutes. Allow it to cool before use. The same procedure applies to solid culture medium.

[0033] Step 5. Take out the canned bottle after high-temperature sterilization and put it into the fume hood. Open the lid and use tweezers to insert the jujube madness tissue culture seedlings that need to be subcultured into the three small holes. Then pour the liquid culture medium into the canned bottle. Pour 40-50ml of liquid culture medium into the 350ml canned bottle. After pouring the liquid culture medium, find that the sponge inserted into the jujube madness tissue culture seedlings floats on the liquid culture medium, then close the lid tightly; after taking out the solid culture medium, put it into the fume hood and subculture the jujube madness tissue culture seedlings normally.

[0034] Among them, MS0 is a common solid culture medium for date madness; the components and concentrations of MS1-MS6 are shown in Table 1:

[0035] Table 1: Components and contents of MS1-MS6

[0036]

[0037] 2. Index detection

[0038] Photos of the same variety of jujube seedlings with mad jujube disease were taken at the same time and the same number of jujube seedlings with mad jujube disease were taken 30 days later for comparison to observe the changes in their growth.

[0039] 3. Results Analysis

[0040] like Figure 2 The following figures show tissue cultured jujube seedlings cultured on liquid and solid culture medium, respectively. The jujube variety is Lizao. A was taken on the first day after subculture, and B was taken 30 days later. The white line represents a length of 1 cm. Comparing the figures, we can see that liquid culture has a higher propagation rate and grows faster than solid culture.

[0041] Compared with the related art, the method of culturing jujube madness tissue culture seedlings using liquid culture medium provided by the present invention has the following beneficial effects:

[0042] Generally, jujube madness tissue culture seedlings are cultured using solid culture medium. The present invention culture jujube madness tissue culture seedlings using liquid culture medium, and uses a sponge for fixation. The sponge can float on the liquid culture medium and has absorption performance. It can not only fix and support the shape of the jujube madness tissue culture seedlings in the liquid culture medium, but also allow the liquid culture medium to fully flow into the jujube madness tissue culture seedlings so that they absorb nutrients. The jujube madness tissue culture seedlings cultured in liquid have a higher reproduction rate and grow faster than those cultured in solid culture.

[0043] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent process transformations made using the contents of the present invention specification, or any direct or indirect application in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for culturing jujube madness tissue culture seedlings using liquid culture medium, characterized in that: The following steps are involved: Step 1: Material preparation: Prepare MS1, MS2, MS3, MS4, MS5, MS6, KH2PO4, sucrose, KOH, a heat-resistant sponge, a marker, scissors, a ruler, beakers, canning jars, and lids of matching sizes; Step 2: Preliminary material processing: Use a marker to measure a 6cm diameter circle on a sponge cloth and cut it out. Then, cut three 5mm holes 1.5cm from the edge of the circle. Once the holes are cut, place them in a canning jar, cover tightly, and set aside. Step 3: Preparation of liquid culture medium for Jujube Madness tissue culture seedlings: Pour MS1, MS2, MS3, MS4, MS5, MS6, KH2PO4 and 1L sterile water into a pot and stir thoroughly with a glass rod. After heating, add sucrose and stir evenly, then stop heating. Add KOH solution to 1L of MS0 culture medium, adjust the pH value, stir evenly with a glass rod, and test the pH with pH paper, and finally put it into a beaker; Step 4: High temperature sterilization: Place the canning bottle containing the sponge and the beaker containing the MS0 culture medium solution into the autoclave for high-temperature sterilization and wait for them to cool; Step 5: Take out the canned bottle after high-temperature sterilization and place it on a sterilized clean bench. Open the lid and use tweezers to insert the jujube madness tissue culture seedlings that need to be subcultured into the three small holes respectively. Then pour the liquid culture medium into the canned bottle so that the sponge (4) inserted with the jujube madness tissue culture seedlings floats on the liquid culture medium, and close the lid tightly.

2. The method for culturing jujube madness tissue culture seedlings using liquid culture medium according to claim 1, wherein: The dosage of MS1, MS2, MS3, MS4, MS5, MS6, KH2PO4 and KOH are 20ml, 5ml, 2ml, 10ml, 10ml, 20ml, 10ml and 1.5ml respectively, and the dosage of sucrose is 30g.

3. The method for culturing jujube madness tissue culture seedlings using liquid culture medium according to claim 1, wherein: The can has a capacity of 350 ml and is made of transparent glass, so that the growth status of the jujube madness tissue culture seedlings inside can be observed.

4. The method for culturing jujube madness tissue culture seedlings using liquid culture medium according to claim 1, wherein: The prepared amount of KOH is 1.5 ml, which is added dropwise to the MSO culture medium and adjusted to a pH value of about 5.

8.

5. The method for culturing jujube madness tissue culture seedlings using liquid culture medium according to claim 1, wherein: The temperature of the high temperature sterilization treatment in step 4 is 121° C. and the time is 20 minutes.

6. The method for culturing jujube madness tissue culture seedlings using liquid culture medium according to claim 1, wherein: In step 5, the amount of liquid culture medium added to the canning bottle is 40-50 ml.

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