A method for inhibiting the occurrence of black wolfberry branch thorns and promoting stem and leaf growth

Abstract

The present invention discloses a method for inhibiting the occurrence of thorns on black wolfberry branches and promoting the growth of stems and leaves, comprising the following steps: step SS1: preparing salicylic acid solution; step SS2: cultivating and transplanting black wolfberry tissue culture seedlings; step SS3: dripping the salicylic acid solution prepared in step SS1 to the terminal buds of black wolfberry thorny plants. The present invention uses 0.01 mg / L exogenous SA treatment to significantly inhibit the occurrence of thorns on black wolfberry potted clones, and the thorn rate drops sharply from 76.64% to 12.74%, and the proportion of newly generated completely thornless stems reaches 50.89%. It is realized that SA inhibits the occurrence of thorns on branches and promotes the growth of stems and leaves at the same time, which is attributed to the synergistic effect of SA and IAA. This discovery provides a new perspective for in-depth exploration of the complex relationship between plant defense mechanism and growth and development, and provides a new and feasible method for cultivating black wolfberry varieties with thornless and excellent quality.

Description

Technical Field

[0001] The invention belongs to the field of forest biotechnology, and particularly relates to a method for inhibiting the occurrence of black wolfberry branch thorns and promoting the growth of stems and leaves. Background Art

[0002] The whole plant of wild black wolfberry is covered with thorns, which are an unfavorable trait that interferes with the production and management of economic forests. Referring to the excellent production performance of the seabuckthorn variety "Wu Ci Feng", it is speculated that thornless black wolfberry is more suitable for production.

[0003] The study found some interesting phenomena about the thorns of black wolfberry. First, drought promotes the production of thorns in potted black wolfberry plants. After the black wolfberry tissue culture clones are transplanted, they can remain thornless for a long time in the greenhouse under the conditions of sufficient water (≥80% field water holding capacity) and fertilizer (regular irrigation with Hogland nutrient solution); the soil

[0004] Drought (≤60% field water holding capacity) promotes the production of branch thorns in potted plants. However, the new branches of the above-mentioned thorny and thornless plants all have thorns after being transplanted into the field. Secondly, sucrose promotes the occurrence of branch thorns in potted plants of black wolfberry. Transplanted tissue culture plants (potted) of black wolfberry can also appear in two types, thorny and thornless, when placed under exactly the same maintenance conditions in the greenhouse; thorny and thornless plants of the same asexual line were used as experimental materials for treatment with sucrose, non-metabolizable sucrose analogs, and osmotic pressure regulators, and darkness combined with leaf removal. The results showed that high sucrose promotes the occurrence of branch thorns in black wolfberry through the dual effects of energy and signal, while low sucrose inhibits the occurrence of branch thorns in black wolfberry. High sucrose not only promotes the occurrence of branch thorns, but also promotes the occurrence of leaves and the growth of stems and leaves; low sucrose not only inhibits the occurrence of branch thorns, but also inhibits the growth of stems and leaves of plants, and even causes plant death. Third, the auxin IAA inhibits the occurrence of thorns on black wolfberry branches, but it also significantly inhibits the growth of its stems and leaves. In summary, sufficient water and fertilizer conditions can inhibit the occurrence of thorns on black wolfberry branches, but they cannot inhibit them for a long time; low sucrose inhibits the occurrence of thorns on black wolfberry branches, but it also inhibits the growth of plant stems and leaves, and even causes plant death; high IAA inhibits the occurrence of thorns on black wolfberry branches, but it also inhibits the growth of plant stems and leaves. Therefore, based on the above three methods of inhibiting the occurrence of thorns on black wolfberry branches, it is difficult to cultivate black wolfberry that is thornless and has excellent other traits.

[0005] Based on this, developing a method for inhibiting the occurrence of black wolfberry branch thorns while promoting the growth of stems and leaves is a technical problem that needs to be solved urgently. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for inhibiting the occurrence of thorns on the branches of black wolfberry and promoting the growth of stems and leaves. The present invention uses a salicylic acid solution of an appropriate concentration to treat transplanted tissue culture plants of black wolfberry at a certain development level. The results show that the salicylic acid solution significantly inhibits the occurrence of thorns on the branches of black wolfberry and promotes the growth of its stems and leaves.

[0007] Specifically, the method for inhibiting the occurrence of thorns on the branches of black wolfberry and promoting the growth of stems and leaves provided by the present invention comprises the following steps:

[0008] Step SS1: preparation of salicylic acid solution;

[0009] Step SS2: cultivating and transplanting black wolfberry tissue culture seedlings;

[0010] Step SS3: adding the salicylic acid solution prepared in step SS1 dropwise to the terminal buds of the thorny plants of Lycium ruthenicum.

[0011] Further, step SS1 salicylic acid solution preparation comprises the following steps:

[0012] Step SS11: weigh 0.03-0.005 g of salicylic acid powder in a beaker, then pour 2-10 ml of anhydrous ethanol and stir for 3-5 minutes to obtain solution A; at this time, the salicylic acid powder is completely dissolved;

[0013] Step SS12: Add distilled water to solution A until the total volume reaches 100 mL to complete the preparation of a 300-50 mg / L mother solution;

[0014] Step SS13: Take 1 mL of the mother liquor and transfer it to a new beaker, add ultrapure water to 100 mL to prepare a 3-0.5 mg / L solution;

[0015] Step SS14: Further dilute the solution prepared in step SS13 by 100 times to obtain a 0.03-0.005 mg / L salicylic acid solution, then dilute the solution by one time to obtain a 0.015-0.0025 mg / L salicylic acid solution, transfer the solution to a reagent bottle, and store it in a cool, dark place.

[0016] Further, step SS2 of cultivating and transplanting the black wolfberry tissue culture seedlings comprises the following steps:

[0017] Step SS21: The seeds of Lycium ruthenicum were disinfected with alcohol and mercuric chloride solution, and the treated seeds were inoculated in 1 / 2 MS medium for cultivation; the Lycium ruthenicum materials used in the experiment were grown in the Forestry College of Shenyang Agricultural University (Key Laboratory of Forest Genetics, Breeding and Cultivation of Liaoning Province).

[0018] Step SS22: After the seeds germinate, a single clone seedling is cut for micro-cutting culture. After the culture is completed, the leaf tip is cut and cultured in 1 / 2 MS culture medium. After the seedling grows out, subculture is completed;

[0019] Step SS23: performing closed bottle hardening and open bottle hardening on the seedlings in step SS22, and transplanting them into nutrient soil after hardening is completed;

[0020] Step SS24: After transplanting, the seedlings are periodically maintained and managed, and water and temperature are controlled to allow the plants to develop the thorny traits required for the test.

[0021] Further, step SS21 is specifically: soak the seeds in 75% alcohol in a clean bench for 30-60 seconds, then soak them in 0.1% mercuric chloride solution for 2 minutes, then rinse the seeds with sterile distilled water for 4-6 times, absorb excess water with sterile filter paper, and horizontally connect the seeds to 1 / 2 MS medium. The medium containing the seeds is placed in the tissue culture room of the Forestry College of Shenyang Agricultural University for culture (the light cycle is 12 h light / 12 h dark per day, and the temperature condition is 25±2°C), and after the seeds germinate, replace the new medium for single clone seedling culture.

[0022] Furthermore, during the cultivation of the single asexual seedling of step SS22, when the length of the upwardly growing stem is about 3-5 cm, the upper half of the stem is cut and micro-cutting culture is completed in 1 / 2 MS culture medium, and so on, at least ten generations of micro-cutting culture are completed. Subsequently, in the clean bench, the 7-10 mm part of the tip of the leaf is cut horizontally vertically from the main vein as an explant, and the explant is inoculated with the adaxial surface facing down in MS culture medium not containing PGR. When a complete plant is formed and the stem grows 5-7 cm upward, the upper half of the stem is cut and subculture is completed in 1 / 2MS culture medium, and so on, 3-4 subcultures are completed.

[0023] Furthermore, during the closed bottle hardening in step SS23, the black wolfberry seedlings are moved to an indoor ventilated environment with sunlight, and cultured at room temperature for 3 to 4 days. Subsequently, the lid of the culture bottle is opened and placed in the same growth environment for 3 to 4 days, i.e., the bottle is opened for hardening. Subsequently, the root culture medium of the plant after the hardening is completed is thoroughly rinsed with tap water, and the nutrient soil after high temperature sterilization is used for transplanting, and the carbendazim solution is poured to soak it, and a light-transmitting and breathable plastic cup is taken to cover the top of the plant.

[0024] Furthermore, in step SS24, the seedlings after transplanting were watered every 2 to 3 days in the morning according to the soil moisture conditions, with 130 to 150 mL of tap water per pot each time; susceptible pests such as aphids and red spiders were controlled, and different concentrations of imidacloprid and high-efficiency cypermethrin were sprayed every 2 days according to the scale of the pests; after the plants grew for about 30 to 40 days, the plastic cup cover was removed to allow the plants to grow normally under soil culture room conditions.

[0025] Further, step SS3 of dripping the salicylic acid solution prepared in step SS1 to the terminal buds of the thorny plants of Lycium barbarum includes the following steps:

[0026] Step SS31: Select the thorny stems of black wolfberry with the same growth potential as the test material, and mark the stem parts close to the terminal buds of the test group and the control group with black marks respectively;

[0027] Step SS32: taking equal amounts of clean water and salicylic acid solution to treat the thorny stem terminal buds of black wolfberry, respectively, and the blank control group was not treated;

[0028] Step SS33: Statistical analysis of experimental group and control group data.

[0029] Further, step SS31 specifically comprises: selecting black wolfberry thorny plants with similar growth under the same growth environment, and dividing them into three groups of salicylic acid solution treatment group, water control group and blank control group in equal amounts, and then conducting subsequent experiments; blackening the stem part close to the terminal bud with a black marker pen as a calibration of the same position before the experimental treatment, so as to compare and observe the subsequent growth and thorn emergence of the stems of plants in different groups;

[0030] Step SS32 is specifically as follows: the experiment was conducted in the soil culture room of the Forestry College of Shenyang Agricultural University at 9:00 am every day, 10 μL of clean water was taken with a pipette and dripped on the top buds of the completely thorny stems as the clean water control group; 10 μL of the prepared 0.01 mg / L salicylic acid solution was taken and dripped on the top buds of the completely thorny stems as the test group; the blank control group was not treated with any treatment except normal watering;

[0031] Step SS33 is specifically: repeat the above experiment for 14 days, and perform statistical work on the data of each group: including the determination of phenotypic data such as the presence or absence of branch thorns and branch elongation; the experiment is set up with 5 repetitions, and each repetition processes 25 stems from 5 pots of plants.

[0032] Furthermore, the components of the aforementioned culture medium are as follows: the composition of 1 / 2MS culture medium is: MS dry powder 2.37 g / L + sucrose 20 g / L + agar powder 4.5 g / L, pH = 5.8~6.2; the above components, except agar powder, are doubled to form MS culture medium. When preparing the culture medium, the three components weighed above are put into a pot, and 80% of the total volume of ultra-pure water is added. The culture medium is heated and boiled in an induction cooker until completely dissolved and the volume is adjusted to the accurate scale. Then, 4% NaOH is gradually added to the culture medium with a rubber-tipped dropper to adjust the pH value, and stirred evenly with a glass rod. A pH meter is used for real-time precision measurement. After the pH value adjustment is completed, the culture medium is sterilized in an autoclave (121°C, 20 min).

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] 1. The present invention uses 0.01 mg / L exogenous SA treatment to significantly inhibit the occurrence of branch thorns in the black wolfberry potted clones, and the thorn rate drops sharply from 76.64% to 12.74% (Table 1, Figure 2 ), the proportion of newly grown completely thornless stems reached 50.89%. Although SA can activate the plant's systemic resistance, it plays an inhibitory role in the formation of defense organ thorns.

[0035] 2. The method provided by the present invention for inhibiting the occurrence of branch thorns and promoting the growth of stems and leaves of black wolfberry realizes that SA inhibits the occurrence of branch thorns and promotes the growth of stems and leaves at the same time, which is attributed to the synergistic effect of SA and IAA. Specifically, in black wolfberry, the treatment with SA may activate the conduction of IAA signals, which is manifested as accelerated growth of stems and leaves; at the same time, SA effectively establishes a connection with the defense response and inhibits the production of thorns that induce the defense system. This discovery provides a new perspective for in-depth exploration of the complex relationship between plant defense mechanisms and growth and development, and provides new and feasible methodological tips for cultivating black wolfberry varieties that are thornless and of high quality.

[0036] 3. SA promotes the growth of stems and leaves of Lycium barbarum. This phenomenon may be related to the activity of SA in regulating cell division and expansion. SA plays an active role in the division of rice root cells. Therefore, the effect of SA on the growth of stems and leaves of Lycium barbarum is also inseparable from the pathway of cell division, thereby promoting the increase of the average elongation of the stem, the number of new stem nodes and the length of the leaf of the fifth stem node under the apical bud.

[0037] 4. The synthesis of SA is not limited to specific organs or tissues of plants, so the treatment of exogenous SA is mostly carried out by exogenous spraying. The present invention specifically treats the terminal buds of black wolfberry, and the results show that the treatment can stimulate the plant's response to SA and produce different phenotypes. This response may be related to the long-distance transport of SA in the plant vascular system. This transport mechanism enables SA to be widely distributed in the plant body, coordinate its stress resistance response and promote growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Graph of average branch elongation during treatment period;

[0039] Figure 2 The average number of new stem nodes during the treatment period;

[0040] Figure 3 Comparison of phenotypic images of thorny branches of Lycium barbarum before and after treatment, A: blank control group; B: water control group; C: salicylic acid treatment group, the red circle is the position of the black mark before treatment;

[0041] Figure 4 Leaf length diagram of the fifth stem node below the terminal bud. DETAILED DESCRIPTION

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0043] The salicylic acid powder in the present invention was purchased from Shanghai Merrill Biochemical Technology Co., Ltd., and the ultrapure water was obtained from the Asset and Laboratory Management Office of Shenyang Agricultural University. Example

[0044] This embodiment provides a method for inhibiting the occurrence of thorns on the branches of Lycium barbarum and promoting the growth of stems and leaves, comprising the following steps:

[0045] Step SS1: preparation of salicylic acid solution;

[0046] Step SS11: 0.02 g of salicylic acid powder was weighed in a beaker, and then 5 ml of anhydrous ethanol was poured in and stirred for 3 minutes to obtain solution A;

[0047] Step SS12: Add solution A to distilled water until the total volume reaches 100 mL to complete the preparation of a 200 mg / L mother solution;

[0048] Step SS13: Take 1 mL of the mother liquor and transfer it to a new beaker, add ultrapure water to 100 mL to prepare a 2 mg / L solution;

[0049] Step SS14: Further dilute the solution prepared in step SS13 by 100 times to obtain a 0.02 mg / L salicylic acid solution, then dilute the solution by one time to obtain a 0.01 mg / L salicylic acid solution, transfer the solution to a reagent bottle, and store it in a cool, dark place.

[0050] Step SS2: cultivating and transplanting black wolfberry tissue culture seedlings;

[0051] Step SS21: The black wolfberry material used in the experiment was grown in the Forestry College of Shenyang Agricultural University. The seeds were soaked in 75% alcohol for 30-60 seconds in a clean bench, then soaked in 0.1% mercuric chloride solution for 2 minutes, then rinsed with sterile distilled water 4-6 times, and the excess water was absorbed with sterile filter paper. The seeds were horizontally inoculated on 1 / 2 MS medium. The composition of 1 / 2MS medium is: MS dry powder 2.37 g / L + sucrose 20 g / L + agar powder 4.5 g / L, pH = 5.8-6.2.

[0052] Step SS22: After the seeds germinate, a single asexual seedling is cut for micro-cutting culture. During the culture of a single asexual seedling, when the length of the upward growing stem is 5 cm, the upper half of the stem is cut and placed in 1 / 2 MS medium for micro-cutting culture. This process is repeated for at least ten generations of micro-cutting culture. Subsequently, in a clean bench, a 10 mm portion of the tip of the leaf is cut transversely along the main leaf vein as an explant, and the explant is inoculated with the adaxial surface facing downward in MS medium without any plant growth regulators. When a complete plant is formed and the stem grows 7 cm upward, the upper half of the stem is cut and placed in 1 / 2 MS medium for subculture. This process is repeated for four subcultures.

[0053] Step SS23: The seedlings in step SS22 are subjected to closed bottle hardening and open bottle hardening in sequence, and are transplanted into nutrient soil after hardening is completed; during closed bottle hardening, the black wolfberry seedlings are moved to an indoor ventilated environment with sunlight, and cultured at room temperature for 4 days; then, the lid of the culture bottle is opened and placed in the same growth environment for 4 days, i.e., open bottle hardening; then, the root culture medium of the plant after hardening is washed off with tap water, and the nutrient soil sterilized by high temperature is used for transplanting, and the carbendazim solution is poured to soak it, and a light-transmitting and breathable plastic cup is taken to cover the top of the plant.

[0054] Step SS24: After transplanting, the seedlings were periodically maintained and managed, and water and temperature were controlled to make the plants have the thorny characteristics required for the test. Step SS24: After transplanting, the seedlings were watered every 3 days in the morning according to the soil moisture conditions, and each pot was watered with 150 mL of tap water each time; different concentrations of imidacloprid and high-efficiency cypermethrin were sprayed every 2 days according to the scale of insect pests; after the plants grew for 40 days, the plastic cups were removed to allow the plants to grow under soil culture room conditions.

[0055] Step SS3: adding the salicylic acid solution prepared in step SS1 dropwise to the terminal buds of the thorny plants of Lycium barbarum, comprising the following steps:

[0056] Step SS31: Select the black wolfberry thorny stems with the same growth potential as the test material, and mark the stem parts close to the terminal buds of the test group and the control group with black marks; that is, select black wolfberry thorny plants with the same growth potential under the same growth environment, and divide them into three groups of salicylic acid solution treatment group, water control group and blank control group in equal amounts for subsequent tests; paint the first stem node under the terminal bud with a black marker as the mark of the same position before the test treatment, so as to compare and observe the subsequent growth and thorn emergence of the stems of plants in different groups;

[0057] Step SS32: The experiment was conducted in the soil culture room of the Forestry College of Shenyang Agricultural University at 9:00 am every day. 10 μL of clean water was taken with a pipette and dripped on the top buds of the completely thorny stems as the clean water control group; 10 μL of the prepared 0.01 mg / L salicylic acid solution was taken and dripped on the top buds of the completely thorny stems as the test group; the blank control group was not treated with any treatment except normal watering;

[0058] Step SS33: Statistical analysis of the experimental and control group data. Repeat step SS32 for 14 days to perform statistical work on the data of each group, including the determination of the phenotypic data of the presence or absence of branch thorns and branch elongation. The experiment was repeated 5 times, and each repetition processed 25 stems from 5 pots of plants.

[0059] The phenotypic data statistics of the experimental results of the embodiment are as follows:

[0060] Phenotypic data statistics include the average branch elongation, the average number of newly generated stem nodes, the average number of newly generated branch thorns, the average thorn rate, and the average length of the leaves at the fifth node below the terminal bud during the treatment period. Among them, the average branch elongation was measured with a vernier caliper, and the measurement value was from the position of the black mark point before the test to the position of the terminal bud after the test; the average number of newly generated stem nodes was the number of newly generated stem nodes above the black mark point; the number of newly generated branch thorns was the number of branch thorns in the newly generated stem nodes; the average thorn rate was the average number of newly generated branch thorns / average number of newly generated stem nodes. Microsoft Office Excel was used for data collation and preliminary analysis, SPSS 23.0 was used for significant difference analysis, one-way analysis of variance was used, and LSD method was used for multiple comparisons (P <0.05).

[0061] 1. Average branch elongation during treatment

[0062] The test results showed that the average elongation of the branches in the blank control group was the lowest, at 45.84 mm, while the elongation of the branches in the water control group was slightly higher, at 51.16 mm. The average elongation of the branches treated with salicylic acid solution was significantly higher, at 59.20 mm. Figure 1 shown.

[0063] 2. Number of new stem nodes during treatment

[0064] The statistical results showed that the number of new stem nodes in the blank control group was 10, and the treatments of salicylic acid solution and clean water promoted the number of new stem nodes, which increased to 14. Figure 2 shown.

[0065] 3. Average number of new branches and thorns

[0066] After 14 days of treatment, no new completely thornless stems were produced in the blank control group ( Figure 3 A). After treatment with salicylic acid solution, the number of thorns in the newly grown stem nodes was significantly reduced. Compared with the blank control and the water control, the "thornless" phenotype first appeared, that is, no thorns were produced in the newly grown stem nodes above the black mark point under the treatment of salicylic acid solution, and the newly grown stems were completely thornless ( Figure 3 C) accounted for 50.89%, which was significantly different from the blank control group ( p <0.0001). This indicates that salicylic acid solution plays a key role in inhibiting the occurrence of branch thorns. In addition, in the water control group, we also found that the occurrence of branch thorns in the new stem nodes of black wolfberry was reduced, and there were new completely thornless stems ( Figure 3 B) appeared, accounting for 25.00%. In terms of probability, there was no significant difference compared with the blank control group. There was also a significant difference in the probability of completely thornless stems between the salicylic acid solution treatment group and the water control group ( p <0.001), in order to more clearly show the effect of salicylic acid solution in inhibiting the occurrence of thorns, this paper uses Figure 3 The representative results shown are used as an example. Please note that this result is not completely consistent with other indicators involved in the example. From the perspective of the number of thorns on new branches during the treatment period, there are significant differences between the salicylic acid solution treatment and the clear water control and blank control group treatments (Table 1).

[0067] Table 1 Effects of exogenous application of salicylic acid solution on the number of thorns on new branches of Lycium ruthenicum

[0068] Processing Type Number of new branch thorns Blank control <![CDATA[8.00±1.23 a ]]> Clear water control <![CDATA[6.29±1.00 a ]]> Salicylic acid treatment <![CDATA[2.21±0.46 b ]]>

[0069] Note: The data in the table are the mean ± standard error of five repeated experiments. The data in the same column marked with different lowercase letters are significantly different ( P <0.05, LSD)

[0070] In addition to the statistical data of black wolfberry thorns, we took the leaves of the fifth stem node below the terminal bud as the research object to explore its possible phenotypic changes. After two weeks of salicylic acid solution treatment, we found that the average leaf length increased slightly from 10.31 mm to 12.46 mm ( Figure 4 In addition, under the clean water treatment, the average leaf length increased from 10.31 mm to 10.71 mm ( Figure 4 )

[0071] In summary, the exogenous application of 0.01 mg / L salicylic acid solution significantly inhibited the occurrence of visible branch thorns in Lycium barbarum, and even made the new stems completely thornless. At the same time, the salicylic acid solution also promoted the growth of the stems and leaves of the plants. This increased our confidence in the "thornless" phenotype.

[0072] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for inhibiting the occurrence of thorns on wolfberry branches and promoting the growth of stems and leaves, characterized in that: The following steps are involved: step SS1: preparation of salicylic acid solution; Step SS2: cultivating and transplanting black wolfberry tissue culture seedlings; Step SS3: Add 10 μL of the prepared 0.01 mg / L salicylic acid solution to the terminal buds of the thorny plants of Lycium ruthenicum and repeat for 14 days.

2. The method for inhibiting the occurrence of thorns on the branches of Lycium barbarum and promoting the growth of stems and leaves according to claim 1, characterized in that: Step SS2: Cultivating and transplanting the black wolfberry tissue culture seedlings comprises the following steps: Step SS21: using alcohol and mercuric chloride solution to disinfect the black wolfberry seeds, and inoculating the treated seeds in 1 / 2 MS medium for cultivation; Step SS22: After the seeds germinate, a single clone seedling is cut for micro-cutting culture. After the culture is completed, the leaf tip is cut and cultured in 1 / 2 MS culture medium. After the seedling grows out, subculture is completed; Step SS23: performing closed bottle hardening and open bottle hardening on the seedlings in step SS22, and transplanting them into nutrient soil after hardening is completed; Step SS24: After transplanting, the seedlings are periodically maintained and managed, and water and temperature are controlled to allow the plants to develop the thorny traits required for the test.

3. The method for inhibiting the occurrence of thorns on the branches of Lycium barbarum and promoting the growth of stems and leaves according to claim 2, characterized in that: Step SS21 is specifically as follows: soak the seeds in 75% alcohol for 30-60 s in a clean bench, then soak them in 0.1% mercuric chloride solution for 2 min, then rinse the seeds with sterile distilled water 4-6 times, absorb excess water with sterile filter paper, and inoculate the seeds horizontally on 1 / 2 MS culture medium.

4. The method for inhibiting the occurrence of thorns on the branches of Lycium barbarum and promoting the growth of stems and leaves according to claim 2, characterized in that: During the cultivation of the single clone seedling of step SS22, when the length of the upwardly growing stem is 3-5 cm, the upper half of the stem is cut and micro-cutting culture is completed in 1 / 2 MS culture medium. By analogy, at least ten generations of micro-cutting culture are completed. Subsequently, in a clean bench, the 7-10 mm part of the tip of the leaf is cut transversely from the vertical main vein as an explant, and the explant is inoculated with the adaxial surface facing downward in MS culture medium without any plant growth regulator. When a complete plant is formed and the stem grows 5-7 cm upward, the upper half of the stem is cut and subculture is completed in 1 / 2 MS culture medium. By analogy, 3-4 subcultures are completed.

5. The method for inhibiting the occurrence of thorns on the branches of Lycium barbarum and promoting the growth of stems and leaves according to claim 2, characterized in that: During the closed bottle hardening in step SS23, the black wolfberry culture seedlings are moved to an indoor ventilated environment with sunlight, and cultured at room temperature for 3 to 4 days; then, the culture bottle lid is opened and placed in the same growth environment for 3 to 4 days, that is, the bottle is opened for seedling hardening; then, the root culture medium of the plant after the hardening is completed is rinsed with tap water, and the nutrient soil after high temperature sterilization is used for transplanting, and the carbendazim solution is poured to soak it, and a light-transmitting and breathable plastic cup is taken to cover the top of the plant.

6. The method for inhibiting the occurrence of thorns on the branches of Lycium barbarum and promoting the growth of stems and leaves according to claim 2, characterized in that: Step SS24: After transplanting, the seedlings are watered every 2 to 3 days in the morning according to the soil moisture conditions, with 130 to 150 mL of tap water per pot each time; different concentrations of imidacloprid and cypermethrin are sprayed every 2 days according to the scale of insect pests; after the plants grow for 30 to 40 days, the plastic cup cover is removed to allow the plants to grow under soil culture room conditions.

7. The method for inhibiting the occurrence of thorns on branches of Lycium barbarum and promoting the growth of stems and leaves according to any one of claims 3 or 4, characterized in that: The composition of 1 / 2MS culture medium is: MS dry powder 2.37 g / L + sucrose 20 g / L + agar powder 4.5 g / L, pH=5.8~6.2; except for agar powder, the above components are expanded by two times to obtain MS culture medium.

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