Rapid propagation method of guangnong sterile seedling of malus domestica

Abstract

The application belongs to the technical field of plant tissue culture and particularly relates to a rapid propagation method of Guoguang aseptic seedlings of apple. The propagation method is realized through the following steps: after the stem segments after cutting and cleaning are sterilized, the stems are subjected to bud peeling, post-sterilization treatment and cutting, and then germination, the germinated buds are subjected to proliferation and rooting culture. The Guoguang aseptic seedling system and the tissue culture and rapid propagation system thereof are established for the first time, which lays an important foundation for scientific research and agricultural production development of Guoguang apple, promotes the sustainable development of Guoguang apple, and provides important support for apple seed industry safety. The rapid propagation method can improve the germination rate, reduce the browning rate in the culture process, shorten the time required for propagation, realize the rapid propagation of Guoguang apple in a short time, realize the rapid improvement of Guoguang, accelerate the breeding speed of new varieties, and improve the breeding efficiency and success rate.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant tissue culture, and particularly relates to a rapid propagation method of Guoguang aseptic seedlings of apples. BACKGROUND

[0002] Apple is a deciduous tree of Rosaceae, Maloideae and Malus. Apple is known as the king of fruits, and it is rich in minerals and vitamins. It is recorded in Dian Nan Ben Cao that apple can relieve dryness, moisten the lungs, relieve heat, stimulate appetite, and sober up, and can treat muscle and bone pain. Apple is one of the most important fruits in China, and its yield and planting area rank first in the world, and it occupies an important position in the Chinese agricultural economy.

[0003] Guoguang apple is an apple cultivar originally from Virginia, USA, and was bred in the late 1700s. It was introduced into China around 1905. Guoguang fruit is round or slightly oblate, with smooth skin, usually light green and with mottling, and beautiful color. The flesh is firm, juicy and crisp, with excellent taste, and is deeply loved by people. Guoguang also has a unique fruit aroma, with a rich sweet taste and a slightly sour taste, giving people a sour and sweet experience. Guoguang apple has strong adaptability, and has strong resistance to cold, drought, and barren conditions, and can adapt to harsh growing environments. In addition, Guoguang apple has strong disease resistance and strong continuous yield capacity. In addition, Guoguang apple also has a long shelf life and storage period, and is very suitable for commercial planting and sales.

[0004] However, it is gradually eliminated by the market due to its small fruit size. In order to realize the rapid improvement of Guoguang, speed up the breeding speed of new varieties, and improve the breeding efficiency and success rate, it is urgent to establish a Guoguang genetic transformation platform. However, at present, there is no related record of Guoguang tissue culture and rapid propagation system. SUMMARY

[0005] In view of the blank in the prior art, the application provides a rapid propagation method of Guoguang aseptic seedlings of apples. The method provides a suitable culture environment under sterile conditions, induces the germination of lateral buds of apple branches, and provides better sterile materials for subsequent tissue culture.

[0006] The technical scheme adopted by the application to achieve the above purpose is as follows:

[0007] The application provides a rapid propagation method of Guoguang aseptic seedlings of apples, comprising the following steps:

[0008] (1) Prepare culture medium I, sterilize and use;

[0009] (2) Shearing and cleaning of stem segments: take healthy branches, cut them into stem segments, ensure one bud per segment, clean the stem segments with laundry detergent water, and then rinse with flowing clean water;

[0010] (3) Pre-sterilization treatment: on the clean bench, the cleaned stem segments are treated with 75% ethanol for a period of time, washed with sterile water; then treated with 3% sodium hypochlorite, washed with sterile water;

[0011] (4) Bud stripping: bud stripping is performed on the clean bench;

[0012] (5) Post-sterilization treatment: treated with 75% ethanol, washed with sterile water; then treated with 3% sodium hypochlorite, washed with sterile water;

[0013] (6) Bud cutting: the branches or buds on the branches treated in step (5) are cut off and placed on the prepared medium I;

[0014] (7) Bud germination: the medium I inoculated in step (6) is treated in the dark, and the medium I needs to be replaced in time when browning occurs, and then subcultured after 4 weeks and placed in a light incubator;

[0015] (8) Proliferation and rooting culture: the germinated aseptic seedlings are subjected to proliferation and rooting culture.

[0016] Further, in step (1), the formula of the medium I is: MS+1 mg / L 6-BA+0.05 mg / L NAA+0.5 mg / L GA.

[0017] Further, in step (2), the selection of healthy branches is: 1-3 months for normally growing and dormant healthy branches, and 6-9 months for vigorously growing healthy new shoot branches; the mass concentration of the washing powder water is 0.5%.

[0018] Further, in step (2), the length of the stem segments is 2-3 cm; the washing time of the washing powder water is 30 min; and the washing time of the flowing clean water is 3-5 h.

[0019] Further, in step (3), the treatment time of the 75% ethanol is 3-5 min; and the sterilization treatment time of the 3% sodium hypochlorite is 8-10 min.

[0020] Further, in step (4), the specific procedure of bud stripping is:

[0021] For the stem segments obtained from the healthy branches in 1-3 months: the epidermis around the bud is removed clean, and then the bud is peeled off layer by layer to expose the growth point;

[0022] Or

[0023] For the stem segments obtained from the new shoot branches in 6-9 months: the petiole around the bud is removed clean.

[0024] Further, in step (5), the treatment time of the 75% ethanol is 3-5 min; the disinfection treatment time of the 3% sodium hypochlorite is 8-10 min.

[0025] Further, in step (6), the specific procedure for cutting the bud is as follows:

[0026] For the stem segments obtained from the healthy branches in January-March: the bud on the branch treated in step (5) is cut off and placed on the prepared medium I, and 80% of the bud is inserted into the medium.

[0027] Or

[0028] For the stem segments obtained from the new shoot branches in June-September: the branch treated in step (5) is inserted into the medium I.

[0029] Further, in step (7), the dark treatment is carried out in an incubator at 18-25 DEG C; the temperature of the light incubator is 25 DEG C.

[0030] Further, in step (8), the composition of the proliferation culture medium used in the proliferation culture is: MS+6-BA 0.8 mg / L+NAA 0.2 mg / L; the composition of the rooting culture medium used in the rooting culture is: 1 / 2MS+IBA 0.75 mg / L.

[0031] The beneficial effects of the present application are as follows:

[0032] (1) The present application first establishes the Guoguang sterile seedling system and the tissue culture and rapid propagation system thereof, which lays an important foundation for the scientific research and agricultural production development of Guoguang apples, promotes the sustainable development of Guoguang apples, and provides important support for the safety of apple seed industry;

[0033] (2) The rapid propagation method provided by the present application can improve the germination rate, reduce the browning rate in the culture process, shorten the time required for propagation, realize the rapid propagation of Guoguang apples in a short time, realize the rapid improvement of Guoguang, accelerate the breeding speed of new varieties, and improve the breeding efficiency and success rate. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 It is the flow chart of the rapid propagation of Example 4;

[0035] Figure 2 It is the propagation and proliferation culture process chart of Example 5; wherein, A is a sterile seedling, B is subculture, C is subculture for 3 times, and D is after the propagation and proliferation;

[0036] Figure 3 It is the rooting culture chart of Example 6. DETAILED DESCRIPTION

[0037] The application will be further described in connection with the specific embodiments. The experimental reagents, instruments and equipment used in the experiment are conventional reagents, instruments and equipment.

[0038] The laundry used in the application is ordinary commercially available laundry detergent.

[0039] The apple variety used in the application is Guoguang.

[0040] Example 1

[0041] (1) Prepare medium I and sterilize for later use.

[0042] The medium I formula is: MS + 1 mg / L 6-BA + 0.05 mg / L NAA + 0.5 mg / L GA.

[0043] The main components of the MS medium are MS nutrients (Beijing Coolabos Technology Co., Ltd.) 4.4 g / L, sucrose (Tianjin Kaitong Chemical Reagent Co., Ltd.) 30 g / L, and agar powder (Beijing Solaybao Technology Co., Ltd.) 8 g / L.

[0044] 6-BA is 6-benzylaminopurine, which belongs to the cytokinin class and can promote cell division and induce new shoots to form; NAA is naphthalene acetic acid, which can promote rooting, especially the growth of adventitious roots; GA is gibberellin, which can promote seed germination and stem growth.

[0045] (2) Cutting and cleaning of stem segments: In January-March, take normal and healthy dormant branches, cut them into 2-3 cm long stem segments, and ensure that each stem segment has a bud. Put the stem segments into 0.5% laundry detergent water, stir with a magnetic stirrer for 30 min, and then rinse the treated stem segments with flowing clean water for 3 h.

[0046] (3) Pre-sterilization treatment of stem segments: In the clean bench, soak in 75% ethanol for 3 min, then rinse with sterile water three times, each time for 2 min, then soak in 3% sodium hypochlorite twice, each time for 8 min, then rinse with sterile water five times, each time for 2 min.

[0047] (4) Bud peeling: In the clean bench, use a scalpel to remove the epidermis around the bud, and then peel the bud layer by layer to the growth point.

[0048] (5) Post-sterilization treatment of stem segments: In the clean bench, soak in 75% ethanol for 20 s, then rinse with sterile water three times, each time for 2 min, then soak in 3% sodium hypochlorite twice, each time for 3-5 min, then rinse with sterile water five times, each time for 2 min.

[0049] (6) Cutting bud: the peeled bud with a part of xylem was cut down and placed on prepared medium I, and most of the bud was inserted into medium I.

[0050] (7) The medium I inoculated in step (6) was placed in a 25 °C incubator in the dark, and the brown color appeared during the period, and the medium I needed to be replaced in time, and then it was placed in a 25 °C light incubator after 4 weeks.

[0051] Example 2

[0052] (1) Prepare medium I and sterilize for later use.

[0053] The formula of medium I is: MS + 1 mg / L 6-BA + 0.05 mg / L NAA + 0.5 mg / L GA.

[0054] The main components of MS medium are MS nutrients (Beijing Coolab Technology Co., Ltd.) 4.4 g / L, sucrose (Tianjin Kaitong Chemical Reagent Co., Ltd.) 30 g / L, and agar powder (Beijing Solabio Technology Co., Ltd.) 8 g / L.

[0055] 7-BA is 6-benzylaminopurine, which belongs to the class of cytokinins, and can promote cell division and induce new bud formation of plants; NAA is naphthalene acetic acid, which can promote rooting, especially the growth of adventitious roots; GA is gibberellin, which can promote seed germination and stem growth.

[0056] (2) Cutting and cleaning of stem segments: in January-March, take normal and healthy dormant branches, cut them into 2-3 cm long stem segments, and ensure that each stem segment has a bud. Put the stem segments into 0.5% laundry detergent water, stir with a magnetic stirrer for 30 min, and then rinse the treated stem segments with flowing clean water for 3 h.

[0057] (3) Pre-sterilization of stem segments: in the clean bench, soak in 75% ethanol for 3 min, then rinse with sterile water three times, each time for 2 min, then soak in 3% sodium hypochlorite for two times, each time for 8 min, then rinse with sterile water five times, each time for 2 min.

[0058] (4) Peeling bud: in the clean bench, use a scalpel to remove the epidermis around the bud, and then peel the bud layer by layer until it is close to the growth point.

[0059] (5) Post-sterilization of stem segments: in the clean bench, soak in 75% ethanol for 20 s, then rinse with sterile water three times, each time for 2 min, then soak in 3% sodium hypochlorite for two times, each time for 3-5 min, then rinse with sterile water five times, each time for 2 min.

[0060] (6) Cutting bud: the peeled bud with a part of xylem is cut down and placed on prepared medium I, and most of the bud is inserted into medium I.

[0061] (7) The inoculated medium I in step (6) is placed in a 18°C incubator for dark treatment, and the medium I needs to be replaced in time during the appearance of browning, and is placed in a 25°C light incubator after 4 weeks.

[0062] Example 3

[0063] (1) Prepare medium I and sterilize for later use.

[0064] (2) Cutting, cleaning and sterilizing of stem segments: in June-September, take healthy new shoots with vigorous growth and cut them into 3cm-long stem segments, ensuring that each stem segment has a bud. Dip the stem segments in 5% sodium hypochlorite for 5min, then put them in 0.5% laundry detergent water and stir with a magnetic stirrer for 30min, then rinse the treated stem segments with flowing clean water for 3h.

[0065] (3) Pre-sterilization treatment of stem segments: soak in 75% ethanol for 3min, then rinse with sterile water three times, each time for 2min, then soak in 3% sodium hypochlorite for 8min, then rinse with sterile water five times, each time for 2min.

[0066] (4) Peeling: on the clean bench, remove the petiole around the bud carefully without damaging the bud.

[0067] (5) Post-sterilization treatment of stem segments: soak in 75% ethanol for 20s, then rinse with sterile water three times, each time for 2min, then soak in 3% sodium hypochlorite for 3-5min, then rinse with sterile water five times, each time for 2min.

[0068] (6) Water absorption: place the stem segments treated according to the above steps in a culture dish covered with filter paper to remove excess water, and place them on the prepared medium, and insert the part without the outer skin into the medium.

[0069] (7) Place the inoculated medium in step (6) in a 25°C incubator for dark treatment, and replace the medium in time during the appearance of browning, and subculture after two weeks and place in a 25°C light incubator.

[0070] Example 4

[0071] (1) Prepare medium I and sterilize for later use.

[0072] (2) Cutting, cleaning and sterilization of stem segments: Take vigorous and healthy new shoots from June to September and cut them into stem segments of about 3 cm, ensuring that each stem segment has a bud. Soak the stem segments in 5% sodium hypochlorite for 5 minutes, then put them into clean water with laundry detergent and stir with a magnetic stirrer for 30 minutes. Then rinse the treated stem segments with running water for 3 hours.

[0073] (3) Pre-sterilization treatment of stem segments: Soak in 75% ethanol for 3 minutes, then rinse three times with sterile water for 2 minutes each time, then soak in 3% sodium hypochlorite twice for 8 minutes each time, and then rinse five times with sterile water for 2 minutes each time.

[0074] (4) Peeling: On a clean bench, remove the petioles around the buds, being careful not to damage the buds.

[0075] (5) Post-sterilization treatment of stem segments: Soak in 75% ethanol for 20 seconds, then rinse three times with sterile water for 2 minutes each time, then soak in 3% sodium hypochlorite twice for 3-5 minutes each time, and then rinse five times with sterile water for 2 minutes each time.

[0076] (6) Water absorption: Place the stem segments processed according to the above steps into a petri dish lined with filter paper to remove excess water, place them on the prepared culture medium, and insert the part with the outer skin removed into the culture medium.

[0077] (7) Place the culture medium that was inoculated in step (6) in an 18°C ​​incubator in the dark. If browning occurs during this period, the culture medium should be replaced in time. After two weeks, subculture and place it in a 25°C light incubator.

[0078] In Example 4, the specific flowchart is as follows: Figure 1 As shown.

[0079] Example 5: Proliferation and Culture of *Gynostemma pentaphyllum* Tissue Culture Seedlings

[0080] Cut off the 1-2cm germinated, sterile seedlings of *Gastrodia elata*. Figure 2 A), placed in subculture medium II (MS + 0.5 mg / L 6-BA) for subculture ( Figure 2 B). After three generations of subculturing ( Figure 2 C), *Gynostemma pentaphyllum* tissue culture seedlings with similar growth, leaf size, and height were selected and placed in culture media with different hormone concentrations for propagation. After 30 days, the proliferation coefficient of the seedlings was calculated. The results showed significant differences in proliferation effects among seedlings in different hormone concentrations. With other hormones remaining constant, the proliferation coefficient first increased and then decreased with increasing cytokinin 6-BA concentration, reaching its highest value in D3 medium containing 0.8 mg / L 6-BA. Figure 2D) and the seedling morphology is normal. Under low concentration of cell division factor, the Guoguang tissue culture seedling morphology is normal, but the plant growth is slow and the increment coefficient is low; under high concentration of cell division factor, the Guoguang tissue culture seedling leaf morphology is normal, slender, curled or growth is abnormal. Through comparative analysis, the medium of MS+6-BA 0.8 mg / L+NAA 0.2 mg / L is finally selected as the Guoguang tissue culture seedling proliferation culture medium.

[0081] Table 1, effect of different concentrations of hormones on Guoguang tissue culture seedling proliferation culture

[0082]

[0083] Example 6, rooting culture of Guoguang tissue culture seedlings

[0084] Take Guoguang tissue culture seedlings with consistent growth, consistent leaf size and consistent height after subculture for 30 days, and place them in rooting culture medium with different concentrations of auxin for rooting test. After 30 days, the rooting coefficient of Guoguang tissue culture seedlings is counted. The results show that there is a significant difference in the rooting effect of Guoguang tissue culture seedlings in culture medium with different concentrations of auxin. With the increase of auxin concentration, the rooting coefficient first increases and then decreases. The Guoguang tissue culture seedlings have the highest rooting coefficient in the E4 culture medium with 0.75 mg / L IBA Figure 3 ), and the root morphology is normal. Under low concentration of auxin, Guoguang tissue culture seedlings develop normally, but the rooting coefficient is low; under high concentration of auxin, Guoguang tissue culture seedlings do not root or have short roots or grow callus. Through comparative analysis, the medium of 1 / 2MS+IBA 0.75 mg / L is finally selected as the Guoguang tissue culture seedling rooting culture medium.

[0085] Table 2, effect of different concentrations of hormones on Guoguang tissue culture seedling rooting culture

[0086]

[0087] Effect example

[0088] (1) The method provided in Examples 1-4 is used in the culture process, and the pollution rate, browning rate, survival rate and germination rate of each method are counted. The specific results are shown in Table 3.

[0089] Table 3

[0090]

Claims

1. A method for rapid propagation of Guotong disease-free seedlings of Malus domestica, characterized in that, The method comprises the following steps: (1) preparing medium I and sterilizing it for later use; (2) cutting and cleaning the stem segments: taking healthy branches, cutting them into stem segments, ensuring that each segment has one bud, cleaning the stem segments with laundry detergent water, and then rinsing them with flowing clean water; (3) pre-sterilization treatment: on the clean bench, treating the cleaned stem segments with 75% ethanol for a period of time, rinsing them with sterile water, then sterilizing them with 3% sodium hypochlorite, and rinsing them with sterile water again; (4) bud peeling: peeling the buds on the stem segments on the clean bench; (5) post-sterilization treatment: treating the stem segments with 75% ethanol, rinsing them with sterile water, then sterilizing them with 3% sodium hypochlorite, and rinsing them with sterile water again; (6) bud cutting: cutting the buds from the branches or the buds on the branches treated in step (5) and placing them on the prepared medium I; (7) bud germination: placing the medium I inoculated in step (6) in a dark environment, replacing the medium I in time when browning occurs, subculturing after 2 weeks, and placing it in a light incubator; (8) proliferation and rooting culture: proliferating and rooting the germinated aseptic seedlings; In step (1), the formula of the medium I is: MS+1 mg / L 6-BA+0.05 mg / L NAA+0.5 mg / L GA; In step (4), the specific procedure for bud peeling is as follows: For stem segments obtained from new shoot branches in June-September: remove the petioles around the buds; In step (7), the dark treatment is performed in an 18°C incubator; In step (8), the proliferation medium used for proliferation culture comprises: MS+6-BA 0.8 mg / L+NAA 0.2 mg / L; and the rooting medium used for rooting culture comprises: 1 / 2MS+IBA 0.75 mg / L.

2. The rapid propagation method according to claim 1, characterized by, In step (2), the healthy branches are selected as follows: taking healthy new shoot branches growing vigorously in June-September; and the mass concentration of the laundry detergent water is 0.5%.

3. The rapid propagation method according to claim 2, characterized by, In step (2), the length of the stem segments is 2-3 cm; the cleaning time with laundry detergent water is 30 min; and the rinsing time with flowing clean water is 3-5 h.

4. The rapid propagation method according to claim 1, characterized by, In step (3), the treatment time of 75% ethanol is 3-5 min; and the sterilization treatment time of 3% sodium hypochlorite is 8-10 min.

5. The rapid propagation method according to claim 1, characterized by, In step (5), the treatment time of 75% ethanol is 3-5 min; and the sterilization treatment time of 3% sodium hypochlorite is 8-10 min.

6. The rapid propagation method according to claim 1, 2 or 5, characterized by, In step (6), the specific procedure for bud cutting is as follows: For stem segments obtained from new shoot branches in June-September: inserting the branches treated in step (5) into the medium I.

7. The rapid propagation method according to claim 1, characterized by, In step (7), the temperature of the light incubator is 25°C.

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