Tissue culture medium and rapid propagation method for reducing holly leaf thorns

Abstract

The present invention discloses a tissue culture medium and a rapid propagation method for reducing holly leaf thorns, and relates to the technical field of holly tissue culture. The tissue culture medium includes a proliferation subculture medium, which is based on a modified WPM medium and supplemented with 1mg / L 6-benzylaminopurine, 0.5mg / L ethephon, 1g / L polyvinylpyrrolidone, 45g / L sucrose and agar. The present invention obtains thorn-less proliferation seedlings by carrying out proliferation culture on the proliferation subculture medium; and further culture obtains holly rooting seedlings. The present invention obtains thorn-less proliferation seedlings by adjusting the components and concentration of the proliferation subculture medium, effectively reducing the difficulty of tissue culture operation caused by leaf thorns and the culture loss caused by operational damage, thereby realizing low-cost and efficient holly tissue culture, and is suitable for large-scale production of holly seedlings.

Description

Technical Field

[0001] The invention relates to the technical field of holly tissue culture, and more particularly to a tissue culture medium and a rapid propagation method for reducing holly leaf thorns. Background Art

[0002] Holly, also known as tiger thorn, cat thorn, and bird-free thorn, belongs to the genus Ilex in the Aquifoliaceae family. Its compact form and uniquely shaped, bright green leaves are evergreen year-round. In autumn, its branches are covered with red fruit, which endures the winter with its vibrant beauty. It is an excellent ornamental foliage and fruiting tree. It is often used in Christmas decorations in Europe and the United States, hence its nickname, "Christmas Tree." It is currently the most cultivated holly species in my country. Common varieties include small-leaved holly, yellow-fruited holly, and spiny holly.

[0003] Holly leaves have two types: one is quadrangular, oblong or ovate, with three sharp, hard thorns at the tip. The central thorn is often recurved, with one or two thorns on each side. The other is ovate, with an entire, spineless margin. The thorns on the leaf margin are not only an important phenotypic feature of the holly, but also a classification trait for the genus Ilex. However, these sharp thorns hinder daily management and harvesting in cultivation, increasing labor costs.

[0004] Holly is usually propagated through seeds and cuttings. However, there are problems such as "serious trait separation in seedlings, resulting in unstable commercial traits; while the survival rate of cuttings is relatively high, it is greatly affected by the season and cannot meet market demand." Therefore, tissue culture and rapid propagation are an important way to achieve factory-based production of holly seedlings. To date, the only holly varieties successfully cultured are variegated holly (CN103392598B) and thornless holly (Jian Dawei, 2011). However, the only research on holly tissue culture technology is reported by Zhou Xijun et al. (2008), which used a rapid propagation system achieved through sterile seed sowing, and its essence is still sexual reproduction.

[0005] Previous studies have shown that rooting holly seedlings in vitro is difficult, time-consuming, and labor-intensive. Therefore, rooting holly seedlings grown in tissue culture primarily relies on rooting outside the flask (Jian Dawei, 2011). However, during the holly tissue culture process, workers are often troubled by leaf thorns, which increases the difficulty of tissue culture operations and thus increases production labor costs. To date, there are no reports on leaf thorns and related control techniques during holly tissue culture rapid propagation.

[0006] Therefore, providing a tissue culture method for reducing the number of holly leaf thorns is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a tissue culture medium and a rapid propagation method for reducing holly leaf thorns.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A tissue culture medium for reducing holly leaf thorns comprises a proliferation and subculture medium, wherein the proliferation and subculture medium comprises the following components: a modified WPM medium as a basal medium, supplemented with 1 mg / L 6-benzylaminopurine, 0.5 mg / L ethephon, 1 g / L polyvinyl pyrrolidone, 45 g / L sucrose, and agar;

[0010] Furthermore, the modified WPM culture medium is prepared by adjusting the concentration of thiamine hydrochloride in the conventional WPM culture medium to 2 mg / L, and then adding 28 mg / L of riboflavin.

[0011] Another object of the present invention is to provide a rapid propagation method for reducing holly leaf thorns, wherein the holly sterile seedlings are cultured using the tissue culture medium to obtain holly tissue culture rooted seedlings.

[0012] Preferably, the sterile holly seedlings are separated into stem segments with buds, which are inoculated on a proliferation subculture medium for proliferation culture to obtain thornless proliferation seedlings; the thornless proliferation seedlings are then separated into individual plants, which are rooted by micro-cutting in vitro to obtain holly rooted seedlings.

[0013] More preferably, the sterile holly seedlings are separated into stem segments with single buds, inoculated on a proliferation subculture medium, cultured at a temperature of 25±1° C., with a light intensity of 1200 Lux, and cultured for 50 days for proliferation to obtain thornless proliferation seedlings.

[0014] More preferably, the few-thorned proliferated seedlings are taken out from the bottle, separated into individual plants for cuttings, and then cultured for 35 days under the conditions of a temperature of 25° C., a light intensity of 2000 Lux, and an air humidity of 80% to obtain rooted seedlings.

[0015] More preferably, after the few-thorn proliferated seedlings are separated into individual plants, they are dipped in rooting powder No. 1 with a concentration of 1200ppm ABT before cutting for 3 seconds.

[0016] Preferably, the sterile holly seedlings are obtained by the following method: using current year shoots as explants, sterilizing them, cutting them into stem segments with buds, and inoculating them into an induction medium at a temperature of 25° C., a light intensity of 1500 Lux, and a light duration of 14 h / day for 45 days;

[0017] The induction medium is WPM+0.2mg·L -1 NAA+2.0mg·L -1 6-BA, pH 5.8.

[0018] Beneficial Effects: The present invention proliferates and cultures sterile holly seedlings on a proliferation and subculture medium to obtain thornless proliferated seedlings; further culture yields rooted holly seedlings. By adjusting the composition and concentration of the proliferation and subculture medium, the present invention obtains thornless proliferated seedlings, effectively reducing the difficulty of tissue culture operations caused by leaf thorns and tissue culture damage caused by operational damage, thereby achieving low-cost and efficient holly tissue culture, suitable for large-scale production of holly seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0020] Figure 1 Example 2 Leaf thorns on the leaves of the proliferated seedlings obtained by subculture;

[0021] Figure 2 This is the leaf thorn situation of the proliferated seedlings obtained by subculture in Comparative Example 13. DETAILED DESCRIPTION

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0023] Example 1

[0024] 1. Preparation of sterile holly seedlings

[0025] (1) Mother plant selection: Select a single plant that is healthy and free of diseases and pests.

[0026] (2) Explant treatment: Cut healthy young shoots from the mother plant as explants. Place the explant in a large beaker with 1-2 drops of detergent, rinse under running water for 30-60 minutes, and then place it on a clean bench for disinfection. It is best to soak it in 75% ethanol for 0.5 minutes, then rinse it twice with sterile water. After removing it, place it in a sterile Erlenmeyer flask and disinfect it with 0.1% mercuric chloride for 8 minutes. Rinse it with sterile water 5 times, and place it on sterile filter paper to absorb surface moisture.

[0027] (3) After disinfection, the explants were cut to remove the wound part that was in contact with the disinfectant, and then cut into approximately 1.0 cm stem segments with buds. The segments were inoculated into culture bottles containing induction medium (WPM + 0.2 mg L -1 NAA+2.0mg·L -1 6-BA, pH 5.8. Cultivation temperature: 25°C, light intensity: 1500 Lux, photoperiod: 14 h / day. After 45 days of cultivation, sterile seedlings will grow to 6-8 cm in height.

[0028] Example 2

[0029] 1. Proliferation culture: The sterile holly seedlings obtained in Example 1 were separated into single bud stem segments of 0.8-1.0 cm, inoculated on a proliferation subculture medium, cultured at a temperature of 25±1°C and a light intensity of 1200 Lux, and cultured for 50 days for proliferation culture to obtain proliferated seedlings (see attached Figure 1 ).

[0030] The proliferation subculture medium was a modified WPM medium supplemented with 1 mg / L 6-benzylaminopurine, 0.5 mg / L ethephon, 1 g / L polyvinylpyrrolidone, 45 g / L sucrose, and 6 g / L agar, pH 5.8;

[0031] The modified WPM medium is prepared by changing the thiamine hydrochloride (VB1) content in the organic matter of the conventional WPM medium to 2 mg / L and adding riboflavin (VB2) 28 mg / L. The remaining macroelements, organic matter, iron salts and all trace elements are prepared according to the conventional WPM medium formula. The conventional WPM medium components include macroelements: potassium sulfate (K2SO4)

[0032] 990mg / L, ammonium nitrate (NH4NO3) 400mg / L, potassium dihydrogen phosphate (KH2PO4) 170mg / L, magnesium sulfate (MgSO4·7H2O) 370mg / L. Trace elements: calcium chloride (CaCl2) 96mg / L, manganese sulfate (MnSO4·4H2O) 22.5mg / L, zinc sulfate (ZnSO4·7H2O) 8.6mg / L, copper sulfate (CuSO4·5H2O) 0.25mg / L, sodium molybdate (Na2MoO4·2H2O) 0.25mg / L, ferrous sulfate (FeSO4·7H2O) 27.3mg / L. Vitamins: inositol 100mg / L, glycine 2mg / L, thiamine hydrochloride (VB1) 1mg / L, pyridoxine hydrochloride (VB6) 0.5mg / L, niacin (VB5) 0.5mg / L.

[0033] 2. Micro-Propagation of Test-Tube Seedlings: Remove the propagated seedlings from step 1 from the jar, clean the culture medium adhering to the base of the plant, and separate them into individual cuttings. Before cutting, dip the cuttings in 1200 ppm ABT Rooting Powder No. 1 for 3 seconds. The cuttings should be planted at a depth of 1 cm using peat as the substrate. After cutting, incubate at 25°C, 2000 Lux light intensity, and 80% humidity for 35 days to obtain rooted seedlings.

[0034] Comparative Example 1

[0035] In this comparative example, based on Example 2, both 0.5 mg / L ethephon and 1 g / L polyvinyl pyrrolidone in the proliferation subculture medium were removed, and the proliferated seedlings were cultured and the average number of leaf thorns on a single leaf of the proliferated seedlings was counted.

[0036] Comparative Example 2

[0037] In this comparative example, based on Example 2, 0.5 mg / L ethephon in the proliferation subculture medium was removed to obtain proliferation seedlings. The average number of leaf thorns on a single leaf of the proliferation seedlings was counted.

[0038] Comparative Example 3

[0039] In this comparative example, based on Example 2, 1 g / L polyvinyl pyrrolidone in the proliferation subculture medium was removed, and the proliferation seedlings were obtained by culture. The average number of leaf thorns on a single leaf of the proliferation seedlings was counted.

[0040] Comparative Example 4

[0041] In this comparative example, based on Example 2, the concentrations of ethephon and polyvinylpyrrolidone in the proliferation subculture medium were set to 0.2 mg / L and 0.5 g / L, respectively, to obtain proliferated seedlings. The average number of leaf thorns per leaf of the proliferated seedlings was counted.

[0042] Comparative Example 5

[0043] In this comparative example, based on Example 2, the concentrations of ethephon and polyvinylpyrrolidone in the proliferation subculture medium were set to 0.5 mg / L and 0.5 g / L, respectively, to obtain proliferated seedlings. The average number of leaf thorns per leaf of the proliferated seedlings was counted.

[0044] Comparative Example 6

[0045] In this comparative example, based on Example 2, the concentrations of ethephon and polyvinylpyrrolidone in the proliferation subculture medium were set to 0.8 mg / L and 0.5 g / L, respectively, to obtain proliferated seedlings. The average number of leaf thorns per leaf of the proliferated seedlings was counted.

[0046] Comparative Example 7

[0047] In this comparative example, based on Example 2, the concentrations of ethephon and polyvinylpyrrolidone in the proliferation subculture medium were set to 0.2 mg / L and 1 g / L, respectively, and the proliferated seedlings were cultured. The average number of leaf spines per leaf of the proliferated seedlings was counted.

[0048] Comparative Example 8

[0049] In this comparative example, based on Example 2, the concentrations of ethephon and polyvinylpyrrolidone in the proliferation subculture medium were set to 0.8 mg / L and 1 g / L, respectively, and the proliferated seedlings were cultured. The average number of leaf thorns on a single leaf of the proliferated seedlings was counted.

[0050] Comparative Example 9

[0051] In this comparative example, based on Example 2, the concentrations of ethephon and polyvinylpyrrolidone in the proliferation subculture medium were set to 0.2 mg / L and 1.5 g / L, respectively, to obtain proliferated seedlings. The average number of leaf spines per leaf of the proliferated seedlings was counted.

[0052] Comparative Example 10

[0053] In this comparative example, based on Example 2, the concentrations of ethephon and polyvinylpyrrolidone in the proliferation subculture medium were set to 0.5 mg / L and 1.5 g / L, respectively, to obtain proliferated seedlings. The average number of leaf thorns per leaf of the proliferated seedlings was counted.

[0054] Comparative Example 11

[0055] In this comparative example, based on Example 2, the concentrations of ethephon and polyvinylpyrrolidone in the proliferation subculture medium were set to 0.8 mg / L and 1.5 g / L, respectively, to obtain proliferated seedlings. The average number of leaf thorns per leaf of the proliferated seedlings was counted.

[0056] Comparative Example 12

[0057] In this comparative example, based on Example 2, the basic culture medium was replaced by the conventional WPM culture medium from the modified WPM culture medium to obtain the propagated seedlings. The average number of leaf thorns on a single leaf of the propagated seedlings was counted.

[0058] Comparative Example 13

[0059] In this comparative example, based on Example 2, the basic culture medium was changed from the improved WPM culture medium to the conventional WPM culture medium, and no additives were added to obtain the proliferated seedlings (see the attached Figure 2 ). Count the average number of leaf spines on a single leaf of the propagated seedlings.

[0060] Effect Example 1

[0061] The average number of leaf thorns on a single leaf of the propagated seedlings cultured in Example 2 and Comparative Examples 1-13 above was statistically analyzed, and the reduction ratio of leaf thorns in the modified culture medium compared to the conventional culture medium (Comparative Example 13) was calculated. The results are shown in Table 1. It can be seen that different additives and changes in the additive ratio significantly affected the number of leaf thorns in the propagated seedlings of holly. The propagated seedlings in Example 1 had the least number of leaf thorns, with a reduction ratio of thorns reaching 60%, indicating that the culture medium with this concentration ratio had the best effect in reducing the number of leaf thorns in the propagated seedlings of holly.

[0062] Table 1 Effects of different proliferation and subculture media on the number of leaf thorns in holly seedlings

[0063] Group Average number of thorns per leaf Reduction ratio (%) Example 2 5.27 60.08 Comparative Example 1 12.50 5.30 Comparative Example 2 8.28 37.27 Comparative Example 3 9.79 25.83 Comparative Example 4 8.35 36.74 Comparative Example 5 7.38 44.09 Comparative Example 6 6.15 53.41 Comparative Example 7 7.86 40.45 Comparative Example 8 5.96 54.85 Comparative Example 9 5.89 55.38 Comparative Example 10 6.45 51.14 Comparative Example 11 7.85 40.53 Comparative Example 12 7.88 40.30 Comparative Example 13 13.20 ——

[0064] Comparative Example 14

[0065] On the basis of Example 2, the micro-cutting link of step 2 test tube seedlings was removed, and rooting culture in the bottle was adopted to separate the proliferated seedlings into individual plants and transfer them to rooting medium to induce rooting. The rooting medium was 1 / 2MS+IBA 0.2mg / L+NAA0.2mg / L, the culture temperature was 25°C, and the light intensity was 1000Lux. After 35 days of cultivation, the rooting situation (rooting rate, number of roots, and average root length) was counted. In addition, the culture time from the beginning of rooting culture to the time when the hardening level was reached (when the root length was 2cm and the average number of roots was more than 10) was counted, and the results are shown in Table 2.

[0066] Table 2 Effects of different rooting methods on holly rooting

[0067]

[0068]

[0069] The results showed that, compared with Comparative Example 14, the average rooting rate, number of roots, and average root length of the holly tissue culture seedlings in Example 1 increased by 14.0%, 37.7%, and 50.0%, respectively, compared with Comparative Example 14. This indicates that the micro-cutting rooting method for test tube seedlings can promote the initiation and elongation of adventitious roots in tissue culture seedlings compared to conventional tissue culture bottle rooting. In terms of rooting culture time, Example 2 shortened the rooting culture time by 41.7% compared with Comparative Example 14, indicating that the micro-cutting rooting method for test tube seedlings can improve the rooting rate of tissue culture seedlings and greatly save seedling cultivation time.

[0070] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0071] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A tissue culture medium for reducing holly leaf thorns, comprising a proliferation and subculture medium, characterized in that: The proliferation subculture medium comprises the following components: a modified WPM medium as a basic medium, supplemented with 1 mg / L 6-benzylaminopurine, 0.5 mg / L ethephon, 1 g / L polyvinyl pyrrolidone, 45 g / L sucrose, and agar; Furthermore, the modified WPM culture medium is prepared by adjusting the concentration of thiamine hydrochloride in the conventional WPM culture medium to 2 mg / L and adding 28 mg / L of riboflavin.

2. A rapid propagation method for reducing holly leaf thorns, characterized in that: The tissue culture medium according to claim 1 is used to culture the holly sterile seedlings to obtain holly tissue culture rooted seedlings.

3. The rapid propagation method for reducing holly leaf thorns according to claim 2, characterized in that: The sterile holly seedlings are separated into stem segments with buds, which are inoculated on a proliferation subculture medium for proliferation culture to obtain thornless proliferation seedlings; the thornless proliferation seedlings are then separated into individual plants, which are rooted by using a test tube seedling micro-cutting method to obtain holly rooted seedlings.

4. The rapid propagation method for reducing holly leaf thorns according to claim 3, characterized in that: The sterile holly seedlings were separated into stem segments with single buds and inoculated on proliferation subculture medium at a culture temperature of 25±1°C and a light intensity of 1200 Lux. The proliferation culture was carried out for 50 days to obtain the few-thorned proliferation seedlings.

5. The rapid propagation method for reducing holly leaf thorns according to claim 3, characterized in that: The few-thorned proliferated seedlings were taken out from the bottle, separated into individual plants for cuttings, and cultured for 35 days under the conditions of temperature 25°C, light intensity 2000 Lux, and air humidity 80% to obtain rooted seedlings.

6. The rapid propagation method for reducing holly leaf thorns according to claim 5, characterized in that: After the few-thorn propagation seedlings were separated into individual plants, they were dipped in rooting powder No. 1 with a concentration of 1200 ppm ABT before cutting for 3 seconds.

7. The rapid propagation method for reducing holly leaf thorns according to claim 2, characterized in that: The sterile seedlings of holly are obtained by the following method: using the tender shoots of the current year as explants, sterilizing them, cutting them into stem segments with buds, inoculating them into an induction medium, culturing them at a temperature of 25° C., a light intensity of 1500 Lux, a light duration of 14 h / day, and culturing them for 45 days; The induction medium is WPM + 0.2 mg·L -1 NAA +2.0 mg•L -1 6-BA, pH 5.8.

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