Tissue culture medium for reducing folium ilicis cornutae and rapid propagation method

Abstract

The invention discloses a tissue culture medium for reducing folium ilicis cornutae and a rapid propagation method, and relates to the technical field of tissue culture of folium ilicis cornutae. The tissue culture medium comprises a proliferation subculture medium, and the proliferation subculture medium takes an improved WPM culture medium as a basic culture medium and is added with 1 mg / L of 6-benayl aminopurine, 0.5 mg / L of ethephon, 1 g / L of polyvinylpyrrolidone, 45 g / L of sucrose and agar. The method comprises the following steps: carrying out multiplication culture on an ilex cornuta aseptic seedling on a multiplication subculture medium to obtain a few-thorn multiplication seedling; and further culturing to obtain ilex cornuta rooting seedlings. By adjusting the components and concentration of the proliferation subculture medium, the proliferation seedlings with few thorns are obtained, the tissue culture operation difficulty caused by leaf thorns and the culture loss caused by operation damage are effectively reduced, then low-cost and efficient Chinese holly tissue culture is achieved, and the method is suitable for large-scale production of Chinese 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, is a plant of the genus Ilex of the Aquifoliaceae family. It has a compact plant shape, peculiar leaves, bright green, and is evergreen all year round. In autumn, the branches are full of red fruits, which are gorgeous and lovely even in winter. It is an excellent foliage and fruit-viewing tree species. It is often used for Christmas decorations in Europe and the United States, so it is also called the "Christmas tree". It is also the holly species with the largest cultivation area in my country. Common varieties include small-leaf holly, yellow-fruit holly, and multi-thorn holly.

[0003] There are two types of holly leaves. One is quadrangular oblong or ovate, with three sharp and hard thorns at the tip. The central thorn is often recurved, with 1-2 thorns on each side. The other is ovate, without thorns and entire margin. The thorns on the leaf margin are not only an important phenotypic shape of holly, but also a classification trait for the classification of holly plants. However, these sharp thorns are not conducive to daily management and harvesting in cultivation, so more labor costs are required.

[0004] Holly is usually propagated by seeds and cuttings, but there are problems such as "the characteristics of seedlings are severely separated, resulting in unstable commercial characteristics; the survival rate of cuttings is high, but it is greatly affected by the season and it is difficult to meet market demand." Therefore, tissue culture and rapid propagation are an important way to achieve factory-based seedling production of holly seedlings. So far, the only successful tissue culture of holly and its varieties are variegated holly (CN103392598B) and thornless holly (Jian Dawei, 2011), but the only research on holly tissue culture technology is reported by Zhou Xijun et al. (2008), which uses a rapid propagation system obtained through aseptic seed sowing, and its essence is still sexual reproduction.

[0005] Existing studies have shown that it is difficult for holly test tube seedlings to take root in a test tube, which is time-consuming and labor-intensive. Therefore, the rooting of holly tissue culture seedlings mainly depends on rooting outside the bottle (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. So far, there is no report on leaf thorns and related regulatory technologies in the process of 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 solution:

[0009] A tissue culture medium for reducing holly leaf thorns comprises a proliferation subculture medium, wherein 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;

[0010] Furthermore, the improved WPM medium is prepared by adjusting the concentration of thiamine hydrochloride in the conventional WPM 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 subjected to rooting culture using a test tube seedling micro-cutting method 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, the culture temperature is 25±1° C., the light intensity is 1200 Lux, and the proliferation culture is carried out for 50 days to obtain thornless proliferation seedlings.

[0014] More preferably, the few-thorn 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 1200 ppm ABT before cutting for 3 seconds.

[0016] Preferably, the holly sterile seedlings 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 in an induction medium, culturing them 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 obtains thorn-less proliferating seedlings by proliferating and culturing sterile holly seedlings on a proliferating and subculture medium; and further cultivates holly rooting seedlings. The present invention obtains thorn-less proliferating seedlings by adjusting the components and concentration of the proliferating and subculture medium, effectively reducing the difficulty of tissue culture operation caused by leaf thorns and tissue culture damage caused by operation damage, thereby realizing low-cost and efficient holly tissue culture, and is 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying 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 condition of the proliferated seedlings obtained by subculture in Comparative Example 13. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work 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 single plants that are healthy and free of diseases and insect pests.

[0026] (2) Explant treatment: Cut healthy young shoots of the current year from the mother plant as explants. Place the explant in a large beaker and add 1-2 drops of detergent, then rinse under running water for 30-60 minutes, and then place it on a clean bench for disinfection. It is advisable to soak it in 75% ethanol for 0.5 minutes, then rinse it twice with sterile water; after taking it out, place it in a sterile conical bottle, 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 the surface moisture.

[0027] (3) The explants that had been disinfected were cut off from the wound area that had been in contact with the disinfectant and cut into approximately 1.0 cm long stem segments with buds. The segments were then inoculated into a culture bottle containing an induction medium containing WPM + 0.2 mg·L -1 NAA+2.0mg·L -1 6-BA, pH 5.8. The culture temperature is 25℃, the light intensity is 1500Lux, and the light time is 14h / day. After 45 days of culture, it grows into sterile seedlings of 6-8cm 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 Appendix Figure 1 ).

[0030] The proliferation subculture medium was based on 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 preparation of the improved WPM medium is to change the thiamine hydrochloride (VB1) in the organic matter of the conventional WPM medium to 2 mg / L, and then add riboflavin (VB2) 28 mg / L, and the remaining macroelements, organic matter, iron salts and all trace elements are configured according to the conventional WPM medium formula. The conventional WPM medium components include macroelements: potassium sulfate (K 2 SO 4 )

[0032] 990mg / L, ammonium nitrate (NH 4 NO 3 )400mg / L, potassium dihydrogen phosphate (KH 2 PO 4 )170mg / L, magnesium sulfate (MgSO 4 7H 2 O) 370mg / L. Trace elements: calcium chloride (CaCl 2 )96mg / L, manganese sulfate (MnSO 4 ·4H 2 O) 22.5 mg / L, zinc sulfate (ZnSO 4 7H 2 O) 8.6 mg / L, copper sulfate (CuSO 4 ·5H 2 O) 0.25 mg / L, sodium molybdate (Na 2 MoO 4·2H 2 O) 0.25 mg / L, ferrous sulfate (FeSO 4 7H 2 O) 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-cutting of test tube seedlings: Take out the proliferated seedlings obtained in step 1 from the bottle, wash the culture medium sticking to the base of the plant, and separate it into single cuttings. Before cutting, dip it in rooting powder No. 1 with a concentration of 1200ppm ABT for 3 seconds, the cutting depth is 1cm, and the substrate is peat. After cutting, culture it at a temperature of 25℃, a light intensity of 2000Lux, and an air humidity of 80% for 35 days to obtain rooted seedlings.

[0034] Comparative Example 1

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

[0036] Comparative Example 2

[0037] In this comparative example, based on Example 2, 0.5 mg / L of ethephon in the proliferation subculture medium was removed to obtain the 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 cultured to obtain the proliferation seedlings. 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 the additives ethephon and polyvinyl pyrrolidone in the proliferation subculture medium were set to 0.2 mg / L and 0.5 g / L, respectively, and the proliferation seedlings were cultured to obtain the proliferation seedlings. The average number of leaf thorns on a single leaf of the proliferation seedlings was counted.

[0042] Comparative Example 5

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

[0044] Comparative Example 6

[0045] In this comparative example, based on Example 2, the concentrations of ethephon and polyvinyl pyrrolidone in the proliferation subculture medium were set to 0.8 mg / L and 0.5 g / L, respectively, and the proliferation seedlings were cultured to obtain the average number of leaf thorns on a single leaf of the proliferation seedlings.

[0046] Comparative Example 7

[0047] In this comparative example, based on Example 2, the concentrations of ethephon and polyvinyl pyrrolidone in the proliferation subculture medium were set to 0.2 mg / L and 1 g / L, respectively, and the proliferation seedlings were cultured to obtain the average number of leaf thorns on a single leaf of the proliferation seedlings.

[0048] Comparative Example 8

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

[0050] Comparative Example 9

[0051] In this comparative example, based on Example 2, the concentrations of ethephon and polyvinyl pyrrolidone in the proliferation subculture medium were set to 0.2 mg / L and 1.5 g / L, respectively, and the proliferation seedlings were cultured to obtain the average number of leaf thorns on a single leaf of the proliferation seedlings.

[0052] Comparative Example 10

[0053] In this comparative example, based on Example 2, the concentrations of ethephon and polyvinyl pyrrolidone in the proliferation subculture medium were set to 0.5 mg / L and 1.5 g / L, respectively, and the proliferation seedlings were cultured to obtain the average number of leaf thorns on a single leaf of the proliferation seedlings.

[0054] Comparative Example 11

[0055] In this comparative example, based on Example 2, the concentrations of ethephon and polyvinyl pyrrolidone in the proliferation subculture medium were set to 0.8 mg / L and 1.5 g / L, respectively, and the proliferation seedlings were cultured to obtain the average number of leaf thorns on a single leaf of the proliferation seedlings.

[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 improved WPM culture medium to obtain the proliferated seedlings. The average number of leaf thorns on a single leaf of the proliferated seedlings was counted.

[0058] Comparative Example 13

[0059] In this comparative example, based on Example 2, the basic culture medium was replaced by the conventional WPM culture medium without adding any additives, and the proliferated seedlings were obtained by culturing (see Appendix Figure 2 ). Count the average number of leaf thorns 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 the above Example 2 and Comparative Examples 1-13 was counted, and the reduction ratio of leaf thorns in the improved culture medium compared with the conventional culture medium (Comparative Example 13) was calculated, and the results are shown in Table 1. It can be seen that different additives and changes in additive ratios significantly affect the number of leaf thorns in the propagated seedlings of holly. The number of leaf thorns in the propagated seedlings of Example 1 is the least, and the reduction ratio of leaf thorns reaches 60%, indicating that the culture medium with this concentration ratio has 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 the test tube seedling in step 2 was removed, and the rooting culture in the bottle was adopted to separate the individual plants of the proliferated seedlings and transfer them to the 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 the rooting culture to the time when the hardening level was reached (when the roots grew to 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 rooting of holly

[0067]

[0068]

[0069] The results show that compared with Example 14, the average rooting rate, number of roots and average root length of the holly tissue culture seedlings in Example 1 were increased by 14.0%, 37.7% and 50.0% respectively compared with Comparative Example 14, indicating that the micro-cutting rooting method of the test tube seedlings can promote the occurrence and elongation of adventitious roots of the tissue culture seedlings compared with the conventional tissue culture bottle rooting. In terms of rooting culture time, Example 2 was shortened by 41.7% compared with Comparative Example 14, indicating that the micro-cutting rooting method of the test tube seedlings can improve the rooting rate of the tissue culture seedlings and greatly save the seedling raising 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 enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be 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 will not be limited to the embodiments shown herein, but rather 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 improved WPM medium is prepared by adjusting the concentration of thiamine hydrochloride in the conventional WPM medium to 2 mg / L, and then adding 28 mg / L of riboflavin.

2. A rapid propagation method for reducing holly leaf thorns, characterized in that: The tissue culture medium described in claim 1 is used to culture 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 thorn-less proliferation seedlings; the thorn-less proliferation seedlings are then separated into individual plants, which are rooted by micro-cutting in a test tube seedling manner 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, inoculated on proliferation subculture medium, cultured at 25±1°C, with a light intensity of 1200 Lux, and cultured for 50 days for proliferation to obtain thorn-less proliferation seedlings.

5. The rapid propagation method for reducing holly leaf thorns according to claim 3, characterized in that: The few-thorn propagation 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 proliferated seedlings are separated into individual plants, they are 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 holly sterile seedlings are obtained by the following method: the tender shoots of the current year are used as explants, sterilized, cut into stem segments with buds, and inoculated into an induction medium at a culture temperature of 25°C, a light intensity of 1500 Lux, and a light duration of 14 h / day. The culture is carried out for 45 days; The induction medium is WPM+0.2mg·L -1 NAA+2.0mg·L -1 6-BA, pH 5.8.

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