A method for meristem tip detoxification of jujube tree

Abstract

The application discloses a method for detoxification of jujube tree stem tips, which comprises the steps of material pre-culture, secondary cutting of stem tips, loading, vitrification of ultra-low temperature treatment, thawing and recovery culture. The application effectively improves the survival rate of stem tip explants by optimizing the pre-culture in the medium of the formula and secondary cutting of stem tips, and to some extent, solves the contradiction among the size of stem tips, survival rate and detoxification rate. The phloem-associated virus is detoxified by the treatment of ultra-low temperature freezing therapy, and the survival rate of the explants reaches 50%, and the detoxification rate is 100%. Therefore, the jujube stem tip ultra-low temperature freezing detoxification technology can effectively remove the phloem-associated virus of winter jujube.

Description

Technical Field

[0001] This invention belongs to the field of plant pathology applications, specifically relating to a method for detoxifying the stem tips of jujube trees. Background Technology

[0002] Jujubes can be eaten fresh or dried, and there are many varieties with a wide distribution. However, jujube witches' broom (JWB), caused by phytoplasma, has been a persistent problem. Infected trees produce fewer jujubes, have lower quality, or even fail to produce any fruit. Generally, after infection, young trees die completely within 1-2 years, and mature trees within 5-6 years. Currently, it can be controlled through surgical treatment, drug treatment, and the selection of resistant varieties. However, in most affected areas, this disease has not been effectively controlled, hence its nickname "jujube tree cancer." Asexual reproduction methods (root suckers and grafting) are the main reasons for the disease's spread from infected areas to disease-free areas, particularly in seedling and young tree populations in most jujube-producing regions.

[0003] Currently, the main method for virus removal in jujube trees is shoot tip tissue culture combined with heat treatment. This method has drawbacks such as long processing time, difficulty in controlling the heat treatment time and temperature, and high requirements for the size of the shoot tip cuttings. Improper processing can easily affect the survival rate and virus removal rate of the shoot tips. Summary of the Invention

[0004] To address the above problems, this invention provides a method for virus removal from the stem tips of jujube witches'-broom disease. The method includes the following steps 1)-5):

[0005] 1) Material Pre-culture: In spring, terminal or axillary buds were removed from diseased main branches of jujube trees. The stem tips were then peeled and disinfected, peeled to a depth of 2.5-3.5 mm, and inoculated onto pre-medium 1. Cultured under conditions of 2000-2500 Lux light intensity, 25℃ temperature, 60% humidity, and 12 hours of light per day for 20-25 days. Then, regenerated buds with a height of 1.0-2.0 cm were taken, separated into single buds on a clean bench, and inoculated onto pre-medium 2 for another 20-25 days to obtain well-cultured explants. Pre-medium 1 was a solid medium obtained by adding 2 mg / L 6-BA, 0.2 mg / L NAA, 7 g / L agar, and 30 g / L sucrose to WPM medium, adjusting the pH to 5.8, and sterilizing. Pre-medium 2 was an MS medium containing 2 mg / L 6-BA, 0.2 mg / L NAA, and 0.2 mg / L NAA. The solid culture medium was prepared by adjusting the pH to 5.8 and sterilizing NAA, 0.4 mol / L sucrose, 0.4 mol / L glycerol and 7 g / L agar.

[0006] 2) Loading

[0007] After pre-culturing the explants in step 1), shoot tips of approximately 2-6 mm in size containing 1-3 layers of leaf primordia were taken under sterile conditions. These shoot tips were then placed in a loading solution and treated at 25°C for 30 minutes. The loading solution was a mixture of vitrification solution 2 and MS liquid medium at a ratio of 6:4, adjusted to pH 5.8. Vitrification solution 2 was formulated as follows: a solution containing 30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.4 mol / L sucrose.

[0008] 3) Vitrification cryogenic treatment

[0009] After removing the loaded stem tips from 25°C in step 2), continue to treat them in the loading solution at 0°C for 24 hours. Then, immerse the stem tips in vitrification solution 2 and quickly put them into a liquid nitrogen tank (-196°C) containing liquid nitrogen for 40-80 minutes.

[0010] 4) Thawing

[0011] Remove the stem tip from liquid nitrogen and quickly immerse it in a 40°C water bath for 60-100 seconds to thaw. Then wash it 2-4 times with a 1.2M sucrose solution for about 10 minutes each time until the stem tip floats on the liquid surface.

[0012] 5) Restore culture

[0013] The washed stem tips were transferred to MS solid medium supplemented with 2 mg / L 6-BA, 0.2 mg / L NAA and 30 g / L sucrose. After 7 days of dark culture, the samples were transferred to conditions of 2000-2500 Lux light intensity, 25℃ temperature, 60% humidity and 12 h light per day to obtain the virus-free regenerated line.

[0014] Preferably, the stem tip loaded in step 2) has a length of 6 mm.

[0015] Preferably, in step 3), the stem tip is immersed in vitrification solution 2 and then placed in a liquid nitrogen tank containing liquid nitrogen for 40 minutes.

[0016] Compared with other jujube detoxification technologies, the advantage of this invention is that it reduces the impact of stem tip size on survival rate and detoxification efficiency. Generally speaking, the stem tip size for jujube detoxification is 1-10mm, and the stem tip size is directly proportional to the survival rate and inversely proportional to the detoxification rate.

[0017] This invention improves the survival rate of stem tip explants by pre-culturing in an optimized culture medium and then harvesting the stem tips a second time, thus resolving to some extent the contradiction between stem tip size, survival rate, and detoxification rate. Furthermore, phytoplasm detoxification is achieved through cryotherapy, resulting in a 50% explant survival rate and a 100% detoxification rate. Therefore, this cryotherapy detoxification technique for jujube stem tips can effectively remove phytoplasms from winter jujubes. Attached Figure Description

[0018] Figure 1 Status of explant growth

[0019] Figure 2 This image shows the growth of virus-free jujube seedlings after cryotherapy treatment.

[0020] Figure 3 Gel electrophoresis images of PCR products for detecting phytoplasma jujuba blight before and after cryotherapy.

[0021] Lanes 1-4 contain explants treated with cryotherapy; P1 and P2 are positive controls from sampled branches exhibiting fasciculation symptoms. The arrows indicate the target band for PCR amplification of a partial fragment of phytoplasma 16S rRNA. Detailed Implementation

[0022] Example 1. Detoxification method and efficacy verification of ultra-low temperature cryotherapy for stem tips of jujube witches' broom disease.

[0023] I. Detoxification of Jujube Blight Stem Tips via Ultra-Low Temperature Cryotherapy

[0024] 1. Preparation of culture medium

[0025] The basic culture media for jujube plant tissue culture are MS and WPM media.

[0026] 1) Stock solution required for MS medium 1

[0027] 20 mL (50×) of macroelements, 1 mL (1000×) of microelements, 10 mL (1000×) of calcium salt, 1 mL (1000×) of potassium iodide, 1 mL (1000×) of organic elements, 5 mL (200×) of iron salt, 0.1 g of inositol, and appropriate amounts of plant hormones are added. The formulations of various solutions are shown in Table 1.

[0028] Table 1. Stock solution formulations required for MS culture medium

[0029]

[0030]

[0031] 2) Preparation of culture medium

[0032] WPM woody plant culture medium (sucrose-free agar) was purchased from Mumu Biotechnology Co., Ltd. When using it, weigh 2.78g, add an appropriate amount of plant hormone (hormone ratio is shown in Table 2), 7g / L agar, 30g / L sucrose, adjust the pH to 5.8, and make up to 1L of distilled water.

[0033] Table 2. Concentration ratio of hormones 6-BA and NAA

[0034] combination 6-BA (mg / L) NAA (mg / L) 1 0 0 2 0.5 0.2 3 1.5 0.2 4 2 0.3 5 2 0.2

[0035] 2. Ultra-low temperature detoxification

[0036] 1) Material pre-culture

[0037] a. Peeling stem tips: In spring, from March to April, remove terminal or axillary buds from the diseased main branches of jujube trees suffering from witches' broom disease. Wash them with clean water, then peel off the scales from the terminal or axillary buds in a clean bench, and place the stem tips in sterile water. Rinse them with sterile water 3-4 times to perform a sterile operation.

[0038] b. Disinfecting the stem tips: First, soak the stem tips in a 75% alcohol solution for 40-60 seconds, then rinse with sterile water 3-4 times. Next, soak them in a 0.1% mercuric chloride solution for 10 minutes, then rinse with sterile water 5-7 times.

[0039] c. Pre-culture: After sterilization, the shoot tips were peeled to 3mm and inoculated onto sterilized culture medium (WPM + 2mg / L 6-BA + 0.2mg / L NAA, 7g / L agar, 30g / L sucrose, pH 5.8). The plants were placed in an intelligent light incubator (light intensity 2000-2500 Lux, temperature 25℃, humidity 60%, daily light duration 12hrs) for 20-25 days. Then, regenerated shoots with a height of 1.0-2.0cm were taken, separated into single shoots on a clean bench, and inoculated onto sterilized culture medium (MS + 2mg / L 6-BA + 0.2mg / L NAA + 0.4mol / L sucrose + 0.4mol / L glycerol + 7g agar, pH 5.8) for another 20-25 days to obtain well-cultured explants.

[0040] 2) Loading

[0041] After pre-culturing the explants in step 1), 30 shoot tips of approximately 2 mm, 4 mm, and 6 mm in size were taken under sterile conditions. These shoot tips were placed in a loading solution and treated at 25°C for 30 min. The loading solution was a mixture of vitrification solution 2 (PVS2) and MS liquid medium at a ratio of 6:4, adjusted to pH 5.8. The formulation of vitrification solution 2 (PVS2) was: a solution containing 30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.4 mol / L sucrose.

[0042] 3) Vitrification cryogenic treatment

[0043] After removing the loaded stem tips from 25°C in step 2), continue to treat them in the loading solution at 0°C for 24 hours. Then, place the stem tips into centrifuge tubes and add PVS2 solution (a solution containing 30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.4 mol / L sucrose) to the centrifuge tubes. Immediately transfer the solution into a liquid nitrogen tank (-196°C) containing liquid nitrogen for storage. The cryogenic treatment time was set at three gradients: 40 min, 60 min, and 80 min.

[0044] 4) Thawing

[0045] Remove the stem tips from liquid nitrogen and quickly immerse them in a 40°C water bath for 60-100 seconds to thaw. Then wash them 2-4 times with 1.2M sucrose culture medium (prepared by adding sucrose to distilled water), each time for about 10 minutes, until the stem tips float on the surface of the liquid.

[0046] 5) Restore culture

[0047] The washed shoot tips were transferred to MS solid medium containing 2 mg / L 6-BA + 0.2 mg / L NAA (MS liquid medium + 2 mg / L 6-BA + 0.2 mg / L NAA, agar 7 g / L, sucrose 30 g / L, pH 5.8), and cultured in the dark for 7 days. Then, they were transferred to an intelligent light incubator (light intensity 2000-2500 Lux, temperature 25℃, humidity 60%, light duration 12 h per day) for further culture, and the regenerated lines were subcultured.

[0048] II. Experimental Results

[0049] 1. Survival rate of jujube stem tip explants in pre-culture

[0050] Shoot tips were used as explants, and WPM medium with different concentrations of 6-BA and NAA was prepared. A total of 495 explants were cultured. The different medium formulations and the number of explants are shown in Table 3.

[0051] Explant culture results: The survival rate of explants cultured on medium without 6-BA and NAA was 80.9%; the survival rate of explants cultured on medium with 0.5 mg / L 6-BA and 0.2 mg / L NAA was 83.3%; the survival rate of explants cultured on medium with 1.5 mg / L 6-BA and 0.2 mg / L NAA was 81.3%; the survival rate of explants cultured on medium with 2.0 mg / L 6-BA and 0.3 mg / L NAA was 73.3%; and the survival rate of explants cultured on medium with 2.0 mg / L 6-BA and 0.2 mg / L NAA was 84.7%, which was the highest among the five batches of explants (see Table 3).

[0052] Table 3 Explant survival rate

[0053] Combinations of different concentrations of 6-BA and NAA Number of explants Number of surviving explants (units) Survival rate (%) 0 84 68 80.9 6-BA 0.5 mg / L + NAA 0.2 mg / L 96 80 83.3 6-BA 1.5 mg / L + NAA 0.2 mg / L 112 91 81.3 6-BA 2.0 mg / L + NAA 0.3 mg / L 105 77 73.3 6-BA 2.0 mg / L + NAA 0.2 mg / L 18 83 84.7

[0054] Explants were cultured in WPM medium for 20-25 days. Then, regenerated shoots with a height of 1.0-2.0 cm were collected, separated into single shoots on a clean bench, and inoculated onto sterilized medium (MS + 2 mg / L 6-BA + 0.2 mg / L NAA + 0.4 mol / L sucrose + 0.4 mol / L glycerol + 7 g agar, pH 5.8) for further culture. The explants were then cultured in an incubator for 20-25 days to obtain the cultured explants (see...). Figure 1 ).

[0055] 2. Results of ultra-low temperature virus detoxification treatment of jujube stem tips

[0056] The cultured jujube stem tip tissue was subjected to ultra-low temperature detoxification treatment. The stem tips were cut twice and three treatments were set: 2 mm, 4 mm and 6 mm. The ultra-low temperature liquid nitrogen freezing time was set to three treatments: 40 min, 60 min and 80 min. The recovery culture medium after ultra-low temperature treatment was set to different concentrations of 6-BA and NAA: 0.2 mg / L 6-BA + 0.2 mg / L NAA and 0.5 mg / L 6-BA + 0.2 mg / L NAA.

[0057] Survival rate (%) = (Number of surviving shoot tips / Number of treated shoot tips) * 100%

[0058] Virus elimination rate (%) = (Number of shoot tips without detected phytoplasma / Number of surviving shoot tips) * 100%

[0059] Twenty shoot tips were collected for each treatment. After pre-culture, cryogenic treatment, and recovery culture, phytoplasm detection was performed. The results are shown in Table 4. The survival rate of shoot tips after cryogenic treatment at 2 mm was very low, and shoot tip size was positively correlated with survival rate. The highest shoot tip survival rate was achieved when the cryogenic treatment time was 40 min, but the survival rate gradually decreased with increasing time, indicating a negative correlation between cryogenic treatment time and shoot tip survival rate. The best combination for shoot tip survival and growth potential in the recovery culture medium screening was 2 mg / L 6-BA + 0.2 mg / L NAA, which is consistent with the optimal formula for shoot tip pre-culture. Based on the above results, the treatment combination with the highest survival rate was 6 mm shoot tips, cryogenic liquid nitrogen freezing for 40 min, and a hormone ratio of 2 mg / L 6-BA + 0.2 mg / L NAA in the recovery culture medium after cryogenic treatment, which was the optimal condition, achieving a shoot tip regeneration survival rate of 50%.

[0060] After 30-60 days of cultivation, the surviving shoot tips after virus elimination grew well, producing normal branches and leaves. Some plants were able to differentiate roots, forming healthy, virus-free seedlings (see...). Figure 2 ).

[0061] Table 4. Effects of shoot tip cutting length, cryogenic liquid nitrogen freezing time, and hormone ratio in the recovery culture medium on shoot tip regeneration.

[0062]

[0063]

[0064] 3. Results of phytoplasma detection in jujube witches' broom disease before and after cryotherapy treatment

[0065] Total DNA was extracted from explants after cryopreservation treatment. Partial fragment detection of 16S rRNA from phytoplasmas was performed using universal primers R16mF2 and R16mR1 (R16mF2: CATGCAAGTCGAACGG and R16mR1: CTTAACCCCAATCATCGAC). Branches exhibiting wilting symptoms were selected as positive controls, and healthy jujube branches were used as negative controls. PCR products were subjected to 1% agarose gel electrophoresis. Results for some samples are shown below. Figure 3 The results showed that phytoplasma could be detected in the branches and leaves of jujube trees before treatment. All surviving shoot tips after recovery culture were subjected to PCR detection using universal primers for phytoplasma. The results showed that all shoot tips were virus-free, with a detoxification rate of 100%. This indicates that cryogenic freezing treatment can effectively remove phytoplasma from the plants and achieve detoxification of jujube tree phytoplasma pathogens.

[0066] The embodiments described above are merely illustrative of several implementations of the present invention, and are described in detail, but are not intended to limit the scope of the invention. Those skilled in the art will recognize that many modifications, variations, and improvements can be made without departing from the spirit and scope defined by the appended claims, and these all fall within the protection scope of the present invention.

Claims

1. A method for detoxifying jujube shoot tips and removing jujube witches'-broom phytoplasma, comprising the following steps 1)-5): 1) Material pre-culture: In spring, terminal or axillary buds are picked from diseased main branches of jujube trees, and stem tips are peeled off. The stem tips are disinfected and peeled to about 3 mm. They are then inoculated onto pre-medium 1 and cultured under conditions of 2000-2500 Lux light intensity, 25℃ temperature, 60% humidity, and 12 hours of light per day for 20-25 days. Then, regenerated buds with a height of 1.0-2.0 cm are taken, separated into single buds on a clean bench, and inoculated onto pre-medium 2 for another 20-25 days to obtain well-cultured explants. The pre-medium 1 is a solid culture medium obtained by adding 2 mg / L 6-BA, 0.2 mg / L NAA, 7 g / L agar, and 30 g / L sucrose to WPM medium, adjusting the pH to 5.8, and then sterilizing. The pre-medium 2 is a solid culture medium obtained by adding 2 mg / L 6-BA, 0.2 mg / L NAA, 0.4 mol / L sucrose, 0.4 mol / L glycerol, and 7 g / L agar to MS medium, adjusting the pH to 5.8, and then sterilizing. 2) Loading After pre-culturing the explants in step 1), take stem tips with a length of 2-6 mm containing 1-3 layers of leaf primordia under sterile conditions. Place these stem tips in the loading solution and treat them at 25°C for 30 min. The loading solution was obtained by mixing vitrification solution 2 and MS liquid culture medium at a ratio of 6:4 and adjusting the pH to 5.

8. The formulation of vitrification solution 2 is as follows: a solution containing 30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.4 mol / L sucrose; the loaded stem tip length is 6 mm. 3) Vitrification cryogenic treatment After the stem tips loaded in step 2) are removed from 25°C, they are treated in the loading solution at 0°C for 24 h. Then, the stem tips are immersed in vitrification solution 2 and quickly placed into a liquid nitrogen tank containing liquid nitrogen for 40 min. 4) Thawing Remove the stem tip from liquid nitrogen and quickly immerse it in a 40°C water bath for 60-100 seconds to thaw. Then wash it 2-4 times with a 1.2 M sucrose solution for about 10 minutes each time until the stem tip floats on the liquid surface. 5) Restore culture The washed shoot tips were transferred to MS solid medium supplemented with 2 mg / L 6-BA, 0.2 mg / L NAA and 30 g / L sucrose. After 7 days of dark culture, they were transferred to a culture environment with a light intensity of 2000-2500 Lux, a temperature of 25℃, a humidity of 60% and a daily light duration of 12 h to obtain the virus-free regenerated line.

Images