Establishment method of protein mulberry stem regeneration system

Abstract

The invention belongs to the technical field of genetic engineering and plant tissue culture, and particularly relates to a method for establishing a protein mulberry stem regeneration system. The invention provides an establishment method of a protein mulberry stem regeneration system. The establishment method comprises the following steps: (1) cleaning, disinfecting and germinating protein mulberry seeds; (2) inducing calluses and adventitious buds by utilizing the stem segments; (3) culturing adventitious buds; and (4) hardening and transplanting the protein mulberry saplings. According to the method, the economic cost required by protein mulberry regeneration is saved to a great extent, the survival efficiency of protein mulberry regenerated plants is improved, and the method has a certain economic value and thus has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and plant tissue culture, and particularly relates to a method for establishing a protein mulberry stem segment regeneration system. Background Art

[0002] Mulberry originated from China and has high economic value. Protein mulberry belongs to a relatively new strain. Protein mulberry is also called feed mulberry. It is a new strain obtained by hybridization and optimization of 28 mulberry varieties collected from all over the country. After long-term selection and improvement, compared with other feed crops, protein mulberry has a relatively high protein content, with the protein content of leaves and branches reaching 36% and 28% respectively. Its leaves can be used as edible fresh vegetables, mulberry tea is also a very popular pure natural product for reducing "three highs", mulberry powder is a good product for beauty and skin care, and mulberry strips can be used as feed plant protein. Therefore, protein mulberry is an efficient and high-yield economic crop. In addition, protein mulberry has the characteristics of drought resistance, wind and sand resistance, cold resistance, and barrenness resistance, so it can be planted in saline-alkali land, arid land, and barren mountainous areas, which plays a very positive role in reducing soil desertification and preventing soil erosion.

[0003] Therefore, it is of great significance to study the establishment method of protein mulberry regeneration system.

[0004] However, since protein mulberry is a relatively new species, as a woody plant, callus induction and differentiation are difficult, and mulberry virus diseases also affect its growth and development. Therefore, there are few studies on the establishment of protein mulberry regeneration system at home and abroad.

[0005] In 2016, researchers including Xiong Yi from the School of Life Sciences of Shihezi University introduced a method for constructing a protein mulberry regeneration system in the article "Establishment of a protein mulberry leaf regeneration system and determination of its sensitivity to glufosinate". This method uses petioles to induce adventitious buds. The technical problems are that firstly, a sterile protein mulberry seedling has fewer petioles, and the processing process is complicated. Secondly, the number of adventitious buds induced is small, and the induction time is about four weeks, which is a long time.

[0006] In 2022, Cui Jihao and other researchers from the School of Life Sciences and Technology of Mudanjiang Normal University introduced a method for constructing a protein mulberry regeneration system in the study of protein mulberry tissue culture. This method uses the stem segments of one-year-old protein mulberry with axillary buds as explant experimental materials to establish a protein mulberry regeneration system. However, this method has the problems of high contamination rate, low proliferation of adventitious buds, and low survival rate during seedling hardening and transplanting. Summary of the invention

[0007] In order to overcome the technical problems existing in the above-mentioned prior art, the present invention provides a method for establishing a protein mulberry stem segment regeneration system, in order to provide a new idea for solving the above-mentioned technical problems.

[0008] The present invention adopts genetic engineering and plant tissue culture technology, separates a part of the plant body (ie, explant) through aseptic operation, inoculates it on a culture medium, and cultivates it under artificially controlled conditions to generate a complete plant.

[0009] The technical solution provided by the present invention is a method for establishing a protein mulberry stem segment regeneration system, which comprises the following steps:

[0010] (1) Cleaning, disinfection and germination of protein mulberry seeds

[0011] Select protein mulberry seeds with complete shape and full grains, wash and disinfect them, and then inoculate them on the culture medium for germination;

[0012] (2) Using stem segments to induce callus and adventitious buds

[0013] Select the complete stem segments of new seedlings as explants, cut them into appropriate lengths, inoculate them into induction medium, and then place them in a culture room with appropriate temperature, light intensity and light duration;

[0014] (3) Cultivation of adventitious buds

[0015] When the adventitious buds obtained by the above steps have grown to a suitable length, the buds are inserted into a container containing a rooting medium, the container mouth is sealed, and the buds are placed in a culture room for culture;

[0016] (4) Hardening and transplanting of protein mulberry seedlings

[0017] The protein mulberry seedlings with good root growth and fibrous roots obtained through the above steps are screened out, and an appropriate amount of sterile water is added to the container in which the seedlings are grown, and the container mouth is not completely opened. After several days, the container mouth is completely opened; after several days, the culture medium is dispersed with running water, the protein mulberry seedlings are taken out of the container, the culture medium at the roots is washed, and then the whole seedlings are immersed in a carbendazim solution. After several minutes, the seedlings are transplanted into a mixed cultivation medium, and the seedlings are covered with a plastic transparent cup, and then normal cultivation management is carried out.

[0018] The method for establishing the protein mulberry stem segment regeneration system of the present invention comprises the following specific steps:

[0019] (1) The cleaning and disinfection operation of protein mulberry seeds is to place them in a clean container and perform it in a clean bench. First, use sterile water to wash several times, then soak them in 75% alcohol for several minutes, and then wash them with sterile water for several times to wash away the residual alcohol; then soak them in a 5% NaClO solution for several minutes for disinfection; wash away the residual NaClO with sterile water, and finally use tweezers to evenly place them in a suitable container containing 1 / 2MS culture medium;

[0020] (2) The induction medium contains MS induction medium, to which agar strips and sucrose of appropriate concentrations and quantities are added, the pH is adjusted to an appropriate value with NaOH, and then TDZ of appropriate concentrations and quantities is added. The culture room temperature is 25±2°C, the light intensity is about 3000 lx, the light duration is 16 h / d, and the culture is carried out for about one month;

[0021] (3) Placing the protein mulberry adventitious buds in a culture dish for cultivation. When the protein mulberry adventitious buds extend to 2-3 cm, cut off the induced adventitious buds and insert them into a wide-mouth bottle containing 1 / 2MS, 8 g / L agar strips, 15 g / L sucrose, and a rooting medium adjusted to pH 5.8-6.0 with NaOH. Place one bud in each bottle, tighten the bottle cap, and place it in a culture room with a culture temperature of 25±2°C, a light intensity of about 3000 lx, and a light duration of about 16 h / d;

[0022] (4) The container containing the protein mulberry seedlings is a wide-mouth bottle. Add an appropriate amount of sterile water to it, loosen the bottle cap and place it on the bottle mouth but not completely open it. After leaving it for 2-3 days, completely open the bottle cap. After 2-3 days, use running water to disperse the culture medium, take the plant out of the bottle, wash the culture medium at the root, soak the whole seedling in the carbendazim solution for 2-3 minutes, and then transplant it to V 泥炭土 :V 蛭石 :V 珍珠岩 =3:1:2 mixed cultivation medium, use plastic transparent cups to cover the newly transplanted seedlings, and then carry out normal cultivation management; cultivation conditions: temperature 28±2℃, relative humidity 70±5%, light intensity about 8000lx, lighting time about 16h / d; remove the cups after 3-4 days.

[0023] The method for establishing the protein mulberry stem segment regeneration system of the present invention is preferably that the specific operation of cleaning and disinfecting the protein mulberry seeds in step (1) is as follows: placing the seeds in a clean 150mL glass conical flask, washing them with sterile water for 3 times to wash away dust and impurities on the surface of the seeds, and then soaking them in 75% alcohol for 30s to 1min; then washing them with sterile water for 3 times to wash away the residual alcohol; pouring 20 to 80mL of a 5% NaClO solution into the conical flask, soaking and shaking for 7 to 9min, then rinsing with sterile water for multiple times to wash away the residual NaClO solution, and evenly placing the seeds in a 150mL conical flask containing 1 / 2MS culture medium with tweezers, and culturing them in a culture room with a temperature of 25±2°C, a light intensity of 3000lx, and a light duration of 16h / d.

[0024] The method for establishing the protein mulberry stem segment regeneration system of the present invention is preferably performed as follows in step (2): selecting new seedlings with a height of 8 to 10 cm, clamping them out of a triangular flask with tweezers, placing them horizontally in a culture dish containing an induction culture medium, placing three to five seedlings neatly together, cutting off the roots and cotyledons of the seedlings, cutting the stem segments into 1.0 to 2.0 cm in length, scattering them in the culture dish, and then gently clamping them into a new 90 mm culture dish containing an induction culture medium with tweezers, covering it with a lid, and sealing it for culture; the culture medium for inducing callus tissue and adventitious buds is: MS+2.0 to 12.0 mg / L TDZ; the temperature of the culture room is 25±2° C., the light intensity is 3000 lx, and the light duration is 16 h / d; and the culture medium is replaced with a new one every half a month until adventitious buds are induced.

[0025] The method for establishing a protein mulberry stem segment regeneration system of the present invention preferably comprises the following steps (3): when the protein mulberry adventitious buds extend to 2-3 cm, the culture dish is opened, the adventitious buds are cut off from the callus tissue, a small amount of callus tissue is retained at the bottom of the adventitious buds, the stems of the adventitious buds are not cut, and too many large pieces of callus tissue are not taken; the cut adventitious buds are inserted into a wide-mouth bottle containing 1 / 2MS rooting medium at an angle of 45-60 degrees, one bud is placed in one bottle, the bottle cap is tightened, and the buds are cultured in a culture room at a temperature of 25±2°C, a light intensity of 3000lx, and a light duration of 16h / d.

[0026] The method for establishing the protein mulberry stem segment regeneration system of the present invention is preferably performed in step (4) as follows: adding 1 / 4 volume of sterile water to a wide-mouth bottle, loosening the bottle cap and placing it on the bottle mouth but not completely opening it, leaving it for 2 days and then completely opening the bottle cap, after 2 days, using running water to disperse the culture medium at the root of the seedling, taking the plant out of the bottle, washing the culture medium at the root, soaking the whole seedling in a carbendazim solution for 2 minutes and then transplanting it to a V 泥炭土 :V 蛭石 :V 珍珠岩 =3:1:2 mixed cultivation medium, use plastic transparent cups to cover the newly transplanted seedlings and carry out normal cultivation management; cultivation conditions: temperature 28±2℃, relative humidity 70±5%, light intensity 8000lx, light time 16h / d; remove the cups after 4 days and let them grow naturally.

[0027] Technical Effects

[0028] The protein mulberry tissue culture disclosed in the prior art uses the stem segments of one-year-old protein mulberry with axillary buds as explant experimental materials to establish a protein mulberry regeneration system. The results show that the suitable disinfection combination for the protein mulberry stem segments is 75% ethanol 20s + mercuric chloride 7min, the germination rate reaches 89.47%, and the contamination rate is 24%; in terms of adventitious bud proliferation, the suitable culture medium is MS + 3mg / L 6BA, and the adventitious bud proliferation coefficient reaches 4.50 (generally about 3 to 5); in terms of rooting culture, the suitable rooting culture medium for protein mulberry is 1 / 2MS + 0.1mg / L NAA, and the rooting rate is 96%; in terms of seedling hardening and transplanting, the best number of days for seedling hardening is 4d, and the survival rate reaches 88%. This method has the problems of high contamination rate, low adventitious bud proliferation, and low survival rate during seedling hardening and transplanting.

[0029] The invention uses a protein mulberry seed disinfection combination of 75% ethanol for 30 seconds to 1 minute and 5% NaClO for 8 to 9 minutes, and a suitable culture medium of 1 / 2MS, and a germination rate of 100% (20 seeds are planted each time, and all of them germinate); selects new seedling stem segments with vigorous growth to induce adventitious buds, and a suitable culture medium of MS+2.0 to 12.0 mg / L TDZ, and a differentiation rate of more than 95% (the new seedling stem segments are cut into 1.5 to 2.0 cm long, and 50 segments are cut, among which 47 stem segments are induced to differentiate), breaks off the adventitious buds, and places them in a regeneration culture medium, and the buds are basically free of deformity; in terms of rooting culture, the suitable rooting culture medium of the protein mulberry is 1 / 2MS, and the rooting rate is 100% (47 regenerated protein mulberry buds are cut off, inserted into the rooting culture medium, and all of them are induced to root); the optimal number of days for hardening the seedlings is 4 days, and the survival rate is 100% (all of the 47 transplanted regenerated protein mulberry seedlings survive).

[0030] In conclusion, the present invention saves the economic cost required for protein mulberry regeneration to a great extent and improves the survival efficiency of protein mulberry regenerated plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Protein mulberry sterile seedlings

[0032] Figure 2 Diagram of callus induction in stem segment

[0033] Figure 3 Diagram showing callus differentiation into adventitious buds

[0034] Figure 4 Adventitious bud growth diagram

[0035] Figure 5 Rooting diagram for adventitious buds

[0036] Figure 6 Schematic diagram of regenerated plants DETAILED DESCRIPTION

[0037] The present invention is further explained in detail below through the description of embodiments, so as to help those skilled in the art to have a more complete, accurate and in-depth understanding of the inventive concept and technical solution of the present invention.

[0038] Plant tissue culture is an asexual reproduction technology developed based on the theory that plant cells are omnipotent. In a broad sense, it is also called in vitro culture, which refers to the technology of separating tissues, organs or cells, protoplasts, etc. that meet the needs from the plant body, and inoculating them on a culture medium containing various nutrients and plant hormones under sterile conditions to obtain regenerated complete plants.

[0039] The present invention utilizes plant tissue culture technology to carry out in vitro culture on new seedling stem segments generated by germination of protein mulberry seeds, establishes an efficient protein mulberry regeneration system, and quickly obtains protein mulberry regenerated plants.

[0040] Example 1

[0041] Experimental materials: The protein mulberry seed variety used was "Fengchi Sang", which was purchased from a seed company on the market.

[0042] Experimental reagents: 75% alcohol, 5% NaClO

[0043] Experimental consumables: Purified water is packaged in reagent bottles, each bottle is 500mL or 150mL Erlenmeyer flask, covered with sealing film and tied with cotton thread. Tweezers, scalpels, culture media, culture dishes, etc. need to be wrapped with newspaper and placed in an autoclave for sterilization at 121℃ for 20 minutes.

[0044] Preparation and packaging of MS culture medium: weigh 4.7g / L of Best MS culture medium powder, add 8g / L agar strips, 15g / L sucrose, add 700mL of water and heat to dissolve, dilute to 1L, adjust to pH 5.8-6.0, and dispense into 250mL tissue culture bottles while hot, 200mL per bottle. Cover with sealing film and tie with cotton thread, then sterilize in an autoclave at 121℃ for 20min. Take out the tissue culture bottle and place it in a clean bench to cool down for use. When the temperature of the culture medium drops to 60-80℃, remove the sealing film, add TDZ to the culture medium with a pipette, the final concentration is 2.0-12.0mg / L TDZ, shake well, pour the culture medium into a 90mm culture dish about 0.5cm, cool and seal for use.

[0045] Preparation and packaging of 1 / 2MS culture medium: weigh 2.35g / L of Best MS culture medium powder, add 8g / L agar strips, 15g / L sucrose, add 700mL water, heat and dissolve, dilute to 1L, adjust to pH 5.8-6.0, and dispense into 150mL Erlenmeyer flasks and wide-mouth bottles while hot, 50mL per bottle. Cover the Erlenmeyer flask with sealing film and tie it with cotton thread, screw the lid on the smooth-mouth bottle, and then sterilize it in an autoclave at 121℃ for 20min. Take it out and cool it for later use.

[0046] Example 1 The entire process is completed in the LUMI clean bench, including the following steps:

[0047] (1) Cleaning, disinfection and germination of protein mulberry seeds; (2) Inducing callus and adventitious buds using stem segments;

[0048] (3) Cultivation of adventitious buds; (4) Hardening and transplanting of protein mulberry seedlings.

[0049] The specific steps are as follows:

[0050] (1) Disinfection and germination of protein mulberry seeds

[0051] The protein mulberry seeds with complete morphology and full grains were selected, placed in a clean 150 mL glass conical flask, washed with sterile water for 3 times to remove dust and impurities on the seed surface, then soaked in 75% alcohol for 30s to 1min to disinfect the seed surface; then washed with sterile water for 3 times to wash away the residual alcohol; 20 to 80 mL of a 5% NaClO solution was poured into the conical flask, soaked and shaken for 7 to 9 minutes for disinfection, then rinsed with sterile water for several times to wash away the residual NaClO solution, and the seeds were evenly placed in a 150 mL conical flask containing 1 / 2MS culture medium with tweezers, and cultured in a culture room (temperature of 25±2°C, light intensity of 3000lx, and light time of 16h / d).

[0052] (2) Induction of callus and adventitious buds from stem segments

[0053] Select new seedlings that are 8 to 10 cm tall, use tweezers to clip them out of the triangular bottle, and place them horizontally in a culture dish containing induction culture medium. Three or five seedlings are neatly placed together. Use tweezers in one hand to pick up the seedlings, and use scissors in the other hand to cut off the roots and cotyledons of the seedlings, pinch off the heads and tails, cut the stem segments into 1.0 to 2.0 cm in length, scatter them in the culture dish, and then use tweezers to gently clip them into a new 90 mm culture dish containing induction culture medium, cover it with a lid, and seal it for culture. The culture medium for inducing callus tissue and adventitious buds is: MS + 2.0 to 12.0 mg / L TDZ. The temperature in the culture room is 25 ± 2 ° C, the light intensity is 3000 lx, and the light time is 16 h / d. Replace the stem segments with new culture medium every half a month until adventitious buds are induced.

[0054] (3) Root induction of adventitious buds

[0055] When the protein mulberry adventitious buds extend to 2-3cm, open the culture dish, hold tweezers in one hand and a scalpel in the other hand, and cut the adventitious buds from the callus. In this step, it is necessary to keep a small amount of callus at the bottom of the adventitious buds, do not cut the stems of the adventitious buds, and do not take too many large pieces of callus. Use tweezers to tilt the cut adventitious buds 45-60° and insert them into a wide-mouth bottle containing 1 / 2MS rooting medium. Place one bud in one bottle, tighten the bottle cap, and place it in the culture room (temperature 25±2℃, light intensity 3000lx, light time 16h / d) for culture.

[0056] (4) Hardening and transplanting of regenerated protein mulberry plants

[0057] The protein mulberry regeneration plants with good root growth and fibrous roots were hardened and transplanted. First, 1 / 4 volume of sterile water was added to the wide-mouth bottle, and the bottle cap was loosened and placed on the bottle mouth without being completely opened. The bottle cap was completely opened after 2 days to allow the seedlings to adapt to the external environment. After 2 days, the culture medium at the root of the seedlings was dispersed with running water, the plant was taken out of the bottle, the culture medium at the root was washed, and the whole seedling was soaked in a carbendazim solution for 2 minutes and then transplanted to a mixed cultivation matrix of V (peat soil): V (vermiculite): V (perlite) = 3:1:2. The newly transplanted seedlings were buckled with a plastic transparent cup and normal cultivation management was performed; the cultivation conditions were: temperature 28±2°C, relative humidity 70±5%, light intensity 8000lx, and light time 16h / d; the cup was removed after 4 days to allow it to grow vigorously.

Claims

1. A method for establishing a protein mulberry stem segment regeneration system, characterized in that: It includes the following steps: (1) Cleaning, disinfection and germination of protein mulberry seeds Select protein mulberry seeds with complete shape and full grains, wash and disinfect them, and then inoculate them on the culture medium for germination; (2) Using stem segments to induce callus and adventitious buds Select the complete stem segments of new seedlings as explants, cut them into appropriate lengths, inoculate them into induction medium, and then place them in a culture room with appropriate temperature, light intensity and light duration; (3) Cultivation of adventitious buds When the adventitious buds obtained by the above steps have grown to a suitable length, the buds are inserted into a container containing a rooting medium, the container mouth is sealed, and the buds are placed in a culture room for culture; (4) Hardening and transplanting of protein mulberry seedlings The protein mulberry seedlings with good root growth and fibrous roots obtained through the above steps are screened out, and an appropriate amount of sterile water is added to the container in which the seedlings are grown, and the container mouth is not completely opened. After several days, the container mouth is completely opened; after several days, the culture medium is dispersed with running water, the protein mulberry seedlings are taken out of the container, the culture medium at the roots is washed, and then the whole seedlings are immersed in a carbendazim solution. After several minutes, the seedlings are transplanted into a mixed cultivation medium, and the seedlings are covered with a plastic transparent cup, and then normal cultivation management is carried out.

2. The method for establishing the protein mulberry stem segment regeneration system according to claim 1, characterized in that: The step (1) of cleaning and disinfecting the protein mulberry seeds is to place them in a clean container in a clean bench, first wash them several times with sterile water, then soak them in 75% alcohol for several minutes, and then wash them several times with sterile water to wash away the residual alcohol; then soak and disinfect them in a 5% NaClO solution for several minutes; wash away the residual NaClO with sterile water, and finally place them evenly in a suitable container containing 1 / 2MS culture medium with tweezers; the induction culture medium of step (2) contains MS induction culture medium, and adds agar strips and sucrose of appropriate concentration and quantity thereto, adjusts the pH to an appropriate value with NaOH, and then adds TDZ of appropriate concentration and quantity, the temperature of the culture room is 25±2°C, the light intensity is about 3000lx, the light exposure time is 16h / d, and the culture is about one month; step (3) is specifically as follows: The protein mulberry adventitious buds are placed in a culture dish for cultivation. When the protein mulberry adventitious buds are elongated to 2-3 cm, the induced adventitious buds are cut off and inserted into a wide-mouth bottle containing 1 / 2MS, 8g / L agar strips, 15g / L sucrose, and a rooting medium adjusted to pH 5.8-6.0 with NaOH. One bud is placed in one bottle, the bottle cap is tightened, and the bottle is placed in a culture room with a culture temperature of 25±2°C, a light intensity of about 3000lx, and a light time of about 16h / d. Step (4) is specifically as follows: the container containing the protein mulberry seedlings is a wide-mouth bottle, an appropriate volume of sterile water is added thereto, the bottle cap is loosened and placed on the bottle mouth but not completely opened, and the bottle cap is completely opened after being placed for 2-3 days, and the culture medium is dispersed with running water after 2-3 days, the plant is taken out of the bottle, the culture medium at the root is washed, the whole seedling is soaked in a carbendazim solution for 2-3 minutes, and then transplanted to V 泥炭土 :V 蛭石 :V 珍珠岩 =3:1:2 mixed cultivation medium, use plastic transparent cups to cover the newly transplanted seedlings, and then carry out normal cultivation management; cultivation conditions: temperature 28±2℃, relative humidity 70±5%, light intensity about 8000lx, lighting time about 16h / d; remove the cups after 3-4 days.

3. The method for establishing the protein mulberry stem segment regeneration system according to claim 2, characterized in that: The specific operation of cleaning and disinfecting the protein mulberry seeds in step (1) is as follows: placing the seeds in a clean 150mL glass conical flask, washing them with sterile water for 3 times to wash away dust and impurities on the surface of the seeds, and then soaking them in 75% alcohol for 30s to 1min; washing them with sterile water for 3 times to wash away the residual alcohol; pouring 20 to 80mL of a 5% NaClO solution into the conical flask, soaking and shaking for 7 to 9min, and then rinsing them with sterile water for several times to wash away the residual NaClO solution, and evenly placing the seeds in a 150mL conical flask containing 1 / 2MS culture medium with tweezers, and culturing them in a culture room with a temperature of 25±2°C, a light intensity of 3000lx, and a light duration of 16h / d.

4. The method for establishing the protein mulberry stem segment regeneration system according to claim 3, characterized in that: The specific operation of step (2) is as follows: selecting new seedlings with a height of 8 to 10 cm, taking them out of the triangular bottle with tweezers, placing them horizontally in a culture dish containing an induction culture medium, placing three to five seedlings neatly together, cutting off the roots and cotyledons of the seedlings, cutting the stem segments into 1.0 to 2.0 cm in length, scattering them in the culture dish, and then gently clamping them with tweezers into a new 90 mm culture dish containing an induction culture medium, covering it with a lid, and sealing it for culture; the culture medium for inducing callus tissue and adventitious buds is: MS+2.0 to 12.0 mg / L TDZ; the temperature of the culture room is 25±2° C., the light intensity is 3000 lx, and the light duration is 16 h / d; and the culture medium for the stem segments is replaced with a new one every half a month until adventitious buds are induced.

5. The method for establishing the protein mulberry stem segment regeneration system according to claim 4, characterized in that: The specific operation of step (3) is as follows: when the adventitious buds of the protein mulberry grow to 2 to 3 cm, the culture dish is opened, and the adventitious buds are cut off from the callus tissue, a small amount of callus tissue is retained at the bottom of the adventitious buds, the stems of the adventitious buds are not cut, and too many large pieces of callus tissue are not taken; the cut adventitious buds are inserted into a wide-mouth bottle containing 1 / 2MS rooting medium at an angle of 45 to 60 degrees, one bud is placed in one bottle, the bottle cap is tightened, and the buds are cultured in a culture room at a temperature of 25±2°C, a light intensity of 3000lx, and a light duration of 16h / d.

6. The method for establishing a protein mulberry stem segment regeneration system according to claim 5, characterized in that: The specific operation of step (4) is as follows: add 1 / 4 volume of sterile water to the wide-mouth bottle, loosen the bottle cap and place it on the bottle mouth but not completely open it, leave it for 2 days and then completely open the bottle cap, wash the culture medium at the root of the seedling with running water after 2 days, take the plant out of the bottle, wash the culture medium at the root, soak the whole seedling in the carbendazim solution for 2 minutes and then transplant it to V 泥炭土 :V 蛭石 :V 珍珠岩 =3:1:2 mixed cultivation medium, use plastic transparent cups to cover the newly transplanted seedlings and carry out normal cultivation management; cultivation conditions: temperature 28±2℃, relative humidity 70±5%, light intensity 8000lx, light time 16h / d; remove the cups after 4 days and let them grow naturally.

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