A promoter for in vitro culture of stevia rebaudiana bertoni leaves and its use

Abstract

The application discloses a kind of Schwendener ironland leaf in vitro culture accelerant and its application.The accelerant includes the mass ratio of TDZ, 2, 4-D and IAA, and ZT or BR, the mass ratio of ZT and TDZ is 0.2-0.5:0.5-2.0, and the mass ratio of BR and TDZ is 0.2-0.5:0.1, and the mass ratio is 0.1-2.0:0.2-2.0:0.2-0.5.The application applies the above-mentioned accelerant in Schwendener ironland leaf in vitro rapid propagation, and the specific method is as follows: cutting Schwendener ironland young leaves are disinfected;induced disinfection after explant forms callus;callus is differentiated and cultured to obtain cluster buds;after taking cluster buds, seedling culture is carried out, and seedling transplanting is completed to complete propagation;accelerant is contained in callus culture medium, differentiation medium and seedling culture medium.The method effectively improves the success rate and efficiency of Schwendener ironland leaf in vitro tissue culture propagation.

Description

TECHNICAL FIELD

[0001] The present application relates to a plant tissue culture promoter and its application, in particular to a Tillandsia straminea leaf in vitro culture promoter and its application. BACKGROUND

[0002] Tillandsia straminea is a perennial herb of Tillandsia in Bromeliaceae, and its branches and leaves are umbrella-shaped, and it can grow normally without soil. Its propagation mainly depends on seed propagation and stolon propagation, but the propagation speed of these two ways is relatively slow, which limits its popularization and application. Tissue culture and rapid propagation technology can accelerate its propagation speed. At present, the reports on tissue culture and rapid propagation of Tillandsia plants mainly involve seeds as explants, and a small amount of literature about daughter plants as explants, but there is no report on the in vitro culture of young leaves. In addition, different varieties of Tillandsia have different growth habits, and the process of tissue culture conditions and growth regulation is difficult to be mutually referenced. The tissue culture and propagation method of Tillandsia straminea cannot directly refer to the tissue culture method of other varieties of Tillandsia, so it is necessary to gradually explore its optimal conditions.

[0003] The plant growth regulators for promoting Tillandsia tissue culture in the prior art include gibberellin, indole-3-acetic acid, benzylaminoadenine, etc. For example, a culture method for inducing air pineapple by tissue culture is disclosed in Chinese patent CN 104094844 A. The method adds gibberellin, indole-3-acetic acid, benzylaminoadenine and other plant growth regulators in the MS culture medium, thereby improving the tissue culture effect of Tillandsia minor daughter plants. However, the method does not disclose how to culture the young leaf tissue of other varieties of Tillandsia, and it is not known how to select and match plant growth regulators to improve the tissue culture and propagation effect of Tillandsia straminea. SUMMARY

[0004] The purpose of the present application is to provide a Tillandsia straminea leaf in vitro culture promoter, and to solve the problem of how to promote the tissue culture and propagation effect of Tillandsia straminea. Another purpose of the present application is to provide a method for improving the in vitro rapid propagation of Tillandsia straminea leaf by using the promoter, and to solve the problem of how to in vitro rapidly propagate Tillandsia straminea.

[0005] Technical solution: A kind of Svermyna iron leaf in vitro culture accelerator described in the application, comprising basic component and adaptive component, the basic component is the mass ratio of 0.1-2.0:0.2-2.0:0.2-0.5 of phenyl thiazole base urea, 2,4-dichlorophenoxyacetic acid and indole acetic acid, the adaptive component is zeatin or brassinolide, the mass ratio of zeatin and phenyl thiazole base urea is 0.2-0.5:0.5-2.0, and the mass ratio of brassinolide and phenyl thiazole base urea is 0.2-0.5:0.1.

[0006] The application is directed to the method for in vitro tissue culture of tender leaves of Svermyna iron, and five plant growth regulators, phenyl thiazole base urea, 2,4-dichlorophenoxyacetic acid, indole acetic acid, brassinolide and zeatin, are screened and combined, which can improve the in vitro tissue culture propagation effect and speed of Svermyna iron leaves at appropriate working concentration.

[0007] The accelerator is applied to in vitro rapid propagation of Svermyna iron, and a method for in vitro rapid propagation of Svermyna iron leaves is constructed, comprising the following steps:

[0008] (1) picking tender leaves of Svermyna iron as explants, and disinfecting the explants;

[0009] (2) inoculating the disinfected explants on callus culture medium to induce callus;

[0010] (3) inoculating the callus on differentiation culture medium to produce multiple shoots;

[0011] (4) inoculating the multiple shoots on strong seedling culture medium for strong seedling culture to obtain tissue culture seedlings, and transplanting the tissue culture seedlings after hardening can complete the propagation; at least one of the callus culture medium, the differentiation culture medium and the strong seedling culture medium contains the accelerator of claim 1.

[0012] The accelerator is reasonably added to the callus culture medium, the differentiation culture medium or the strong seedling culture medium, the induction and differentiation efficiency of callus is effectively improved, and the strong seedling effect is further improved, and a method for in vitro rapid propagation of Svermyna iron leaves is successfully constructed.

[0013] Preferably, in step (1), the disinfection method is: first, soaking in 75% alcohol for 10-30s, and repeatedly washing and oscillating the explants in sterile water for at least 3 times, each time for not less than 5min; then, transferring into 10% NaClO solution for disinfection for 10-20min, and repeatedly washing and oscillating the explants in sterile water for at least 3 times, each time for not less than 5min; finally, transferring into 0.1% HgCl2 for disinfection for 4-6min, and after repeatedly washing and cleaning with sterile water, absorbing the water on the surface of the explants with sterilized paper.

[0014] Preferably, in step (2), the concentration of the promoting agents in the callus culture medium is 0.5-1.5 mg / L phenylthiazole urea, 0.2 mg / L zeatin, 1.0-1.5 mg / L 2,4-dichlorophenoxyacetic acid and 0.5 mg / L indoleacetic acid.

[0015] Preferably, in step (3), the concentration of the promoting agents in the differentiation medium is 0.5-2.0 mg / L phenylthiazole urea, 0.2-0.5 mg / L zeatin, 0.2-1.0 mg / L 2,4-dichlorophenoxyacetic acid and 0.5 mg / L indoleacetic acid.

[0016] Preferably, in step (4), the concentration of the promoting agents in the seedling culture medium is 0.1 mg / L phenylthiazole urea, 0.2-0.5 mg / L brassinolide, 0.2-1.5 mg / L 2,4-dichlorophenoxyacetic acid and 0.2-0.5 mg / L indoleacetic acid.

[0017] Preferably, the callus culture medium is MS medium containing 50-80 g / L sucrose and 2-4 g / L activated carbon; the differentiation medium is MS medium containing 2-4 g / L activated carbon, 25-30 g / L sucrose and 100-120 g / L coconut juice; and the seedling culture medium is MS medium containing 2-4 g / L activated carbon, 25-30 g / L sucrose and 120-150 g / L coconut juice.

[0018] Preferably, in step (2), the method for inducing callus is to culture under sterile room conditions after inoculation, with the culture conditions being temperature 22±2℃ and dark culture; and in step (3), the method for culturing to produce clusters is to culture under sterile room conditions after inoculation, with the culture conditions being temperature 25±2℃, light intensity 1500-2000 lx and light time 12 h / d.

[0019] Preferably, in step (4), the method for seedling culture is to cut the clusters on the differentiation medium into individual small buds, select uniform small buds and inoculate them on different seedling culture media, and then culture under sterile room conditions after inoculation, with the culture conditions being temperature 25±2℃, light intensity 2000-2500 lx and light time 14 h / d; and the method for hardening and transplanting is to place the seedlings in a greenhouse with temperature 20-25℃ for 10 d with loosened bottle caps, remove the solid culture medium on the seedlings for transplanting, place the seedlings in a tray with sphagnum moss for management, spray water once a day under the condition of 75% shading, control the temperature at 20-25℃, spray nutrient solution once every 7 d, and keep good ventilation.

[0020] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages: the present application can effectively improve the callus induction effect, callus differentiation effect and seedling strengthening effect in the in vitro rapid propagation of S. sempervirens leaf, which is obviously superior to the existing plant regulators or their compositions. The in vitro rapid propagation technology of S. sempervirens leaf based on the present application can effectively improve the propagation success rate and efficiency of S. sempervirens, reduce the propagation cost, and is suitable for batch efficient propagation of S. sempervirens. DETAILED DESCRIPTION

[0021] The technical solutions of the present application are further described below.

[0022] Example 1: A S. sempervirens leaf in vitro culture promoter, including formula one and formula two, both of which contain the basic components phenylthiadiazolyl urea, 2,4-dichlorophenoxyacetic acid and indole acetic acid, the difference is that:

[0023] Formula one also contains zeatin, and the mass ratio of phenylthiadiazolyl urea, 2,4-dichlorophenoxyacetic acid, indole acetic acid and zeatin is 0.5-2.0:0.2-2.0:0.2-0.5:0.2-0.5. Formula one is mainly used in the callus induction stage and differentiation stage.

[0024] And formula two also contains brassinolide, and the mass ratio of phenylthiadiazolyl urea, 2,4-dichlorophenoxyacetic acid, indole acetic acid and brassinolide is 0.1:0.2-1.5:0.2-0.5:0.2-0.5. Formula two is mainly used in the seedling strengthening stage.

[0025] The specific ratio and concentration of the two formulas are adaptively selected according to actual application requirements.

[0026] The above-mentioned promoter is used for in vitro rapid propagation of S. sempervirens leaf, and the method is as follows:

[0027] 1. Disinfection and sterilization

[0028] Take 1-2 tender leaves at the top of freshly picked S. sempervirens as explants, wash the explants under running water for 30 min, soak with an appropriate amount of detergent for 60 min, and shake while soaking, then wash clean and move to the clean bench for the next disinfection treatment.

[0029] The disinfection treatment is as follows: first, immerse in 75% alcohol for 20s, repeatedly wash and shake the explants in sterile water for at least 3 times, each time for no less than 5 min; then, transfer into 10% NaClO solution for 15 min, repeatedly wash and shake the explants in sterile water for at least 3 times, each time for no less than 5 min; finally, transfer into 0.1% HgCl2 solution for 5 min, and then wash repeatedly in sterile water until clean, and then absorb the water on the surface of the explants with sterile paper. Place the disinfected leaves in a culture dish, cut into small pieces with a length of 0.5 cm and an upper and lower width of 0.2-0.4 cm with scissors, and inoculate on the callus culture medium.

[0030] 2. Callus induction

[0031] Different callus culture medium formulations are set up for comparison. Each medium formulation is based on MS medium containing 2-4 g / L activated carbon and 50-80 g / L sucrose, and adding corresponding types and concentrations of promoters. In this embodiment, the activated carbon content is 3 g / L, the sucrose content is 65 g / L, the pH value is 5.6-5.8, and the medium formulation is shown in Table 1. Among them, TDZ is phenylthiazolyl urea, ZT is zeatin, 2,4-D is 2,4-dichlorophenoxyacetic acid, IAA is indole acetic acid, NAA is naphthalene acetic acid, BA is benzyl amino adenine, and GAs is gibberellin. After inoculation, the culture conditions are as follows: temperature 22±2℃, dark culture. After about 30d, callus appears at the wound of the leaf, and the callus induction rate is counted after 60d. Callus induction rate (%)=(number of explants inducing callus / number of inoculated explants)×100. The explants of S. sempervirens are placed in a triangular flask, covered with gauze, and repeatedly washed under running water for 3-4h; then, add an appropriate amount of detergent and 2 / 3 tap water, and repeatedly shake for 0.5h; then, wash with pure water until no foam is generated; after the explants are washed, move to the clean bench for the next disinfection treatment.

[0032] Table 1 Callus induction under different medium

[0033]

[0034]

[0035] Note: TDZ 0.5+ZT 0.2+2,4-D 0.5+IAA 0.5 means that the concentration of TDZ is 0.5 mg / L, the concentration of ZT is 0.2 mg / L, the concentration of 2,4-D is 0.5 mg / L, and the concentration of IAA is 0.5 mg / L. Other formulations are similar.

[0036] From Table 1, it can be seen that the callus induction rates of formulations A4, A5, A6 and A7 are relatively high, being 92%, 90%, 100% and 89% respectively, and the calli have good appearance, compact texture and healthy light yellow-green or green color. Although the callus induction rates of formulations A8, A9, A10 and A11 are not low, being 63%-89%, the calli have poor appearance, waterlogged or hard texture and yellow (black) brown or yellowish white color, which seriously affects the differentiation of the later-stage multiple shoots. The callus induction effects of formulations A12-A25 are obviously inferior to those of formulations A8, A9, A10 and A11.

[0037] It is found that the medium suitable for inducing callus from S. verticillata leaves is MS medium + TDZ (0.5-1.0 mg / L) + ZT 0.2 mg / L + 2,4-D (1.0-1.5 mg / L) + IAA 0.5 mg / L + sucrose (50-80 g / L) + activated carbon 3 g / L. On the preferred medium, the callus induction rate from S. verticillata leaves as explants is over 89%, and the calli have good appearance, compact texture and healthy color.

[0038] 3. Differentiation culture of callus

[0039] The good calli are cut into small pieces of 2-4 mm and inoculated into different differentiation media for differentiation culture, and the conditions of the media are compared. Each formulation is MS medium containing activated carbon (2-4) g / L, sucrose (25-30) g / L and coconut juice (100-120) g / L, to which growth regulators of corresponding types and concentrations are added. In this example, the activated carbon content is 3 g / L, the sucrose content is 28 g / L, the coconut juice content is 110 g / L, the pH value is 5.6-5.8, and the medium formulations are shown in Table 2. After inoculation, the culture is carried out in a sterile room under the following conditions: temperature 25±2°C, light intensity 1500-2000 lx and light time 12 h / d. After culture in the differentiation media, a certain number of multiple shoots are sprouted from the calli. After 60 d, the differentiated multiple shoots are observed and counted, and the differentiation rate and differentiation coefficient are calculated. The differentiation rate = (the number of inoculated materials from which shoots are differentiated / the number of inoculated materials) x 100, and the differentiation coefficient = the number of differentiated adventitious shoots / the number of inoculated materials.

[0040] Table 2 Differentiation of callus in different media

[0041]

[0042] Note: Vigor+, indicates weak; Vigor++, indicates general; Vigor+++, indicates robust. Color, indicates yellow-green; Color**, indicates green. Glassiness+, indicates no glassiness; Glassiness++, indicates partial glassiness; Glassiness+++, indicates severe glassiness.

[0043] As shown in Table 2, different media for differentiation culture of callus have different differentiation conditions. In several media, the differentiation rate and differentiation coefficient of formula B3, B4, B8 and B9 are higher, which are 87%, 98%, 99% and 95% respectively. The cluster shoots grow well, have high height, most of which are robust and green without glassiness. The second is formula B7, which has higher differentiation rate and differentiation coefficient. The cluster shoots grow in poor state, which show severe growth failure, weak and yellow-green color. In summary, the excellent formula for differentiation of cluster shoots from callus is: MS medium + TDZ (1.5-2.0 mg / L) + ZT 0.5 mg / L + 2,4-D (0.2-0.5 mg / L) + IAA 0.5 mg / L + coconut juice (100-120) g / L + sucrose (25-30 g / L) + activated carbon 3 g / L. After differentiation culture of callus in the excellent differentiation medium, the differentiation rate reaches 87%-99%, the average height of differentiated cluster shoots is 0.8-1.0 cm, the shoots are green, robust and have no glassiness. Subculture is carried out once every 50 d, and the medium is still the differentiation medium. After 3 times of differentiation and subculture, the propagation coefficient reaches 6-8 times. The differentiation effect of callus in formula B11-B24 is obviously inferior to that in formula B3, B4, B8 and B9.

[0044] 4. Seedling culture

[0045] The cluster shoots on the differentiation medium are cut into individual small shoots, and the uniform small shoots are inoculated on different seedling culture media to compare the growth of seedlings under different media. Each formula is based on MS medium containing activated carbon (2-4) g / L, sucrose (25-30) g / L and coconut juice (120-150) g / L, and adding corresponding types and concentrations of growth regulators. In this embodiment, the activated carbon content is 3 g / L, the sucrose content is 28 g / L, the coconut juice content is 135 g / L, the pH value is 5.6-5.8, and the medium formula is shown in Table 3. After inoculation, the culture is carried out in a sterile room under the following conditions: temperature 25±2℃, light intensity 2000-2500 lx, and light time 14 h / d. TDZ is phenylthiazolyl urea, BR is brassinolide, 2,4-D is 2,4-dichlorophenoxyacetic acid, IAA is indole acetic acid, NAA is naphthalene acetic acid, BA is benzyl amino adenine, and GAs is gibberellin. After 50 d of culture on the seedling culture medium, the growth of seedlings is observed and recorded, including height, robustness and color.

[0046] Table 3: Growth of seedlings in different seedling culture media

[0047]

[0048]

[0049] Note: The growth of seedlings is indicated by height, robustness, and color; robustness: +++, robust; ++, average growth; +, weak and tender; color: +++, dark green; ++, green; +, yellow-green.

[0050] As shown in Table 3, different seedling culture media have different effects on the growth of seedlings. In the four media of formulations C5, C6, C8, and C9, the seedlings grow well, with high height, robust plants, and dark green color. In the two media of formulations C7 and C10, although the seedlings have high height of 3.0 cm and 3.2 cm, respectively, they are weak and tender, with unhealthy yellow-green color. Therefore, when the Spathiphyllum sp. tissue culture shoots are cultured in the seedling medium of MS medium + TDZ 0.1 mg / L + BR (0.2-0.5 mg / L) + 2,4-D (0.5-1.0 mg / L) + IAA 0.5 mg / L + coconut juice (120-150 g / L) + sucrose (25-30 g / L) + activated carbon 3 g / L, the seedlings grow robustly, with green color, and the height can reach 4.0-4.6 cm. The seedling effect of formulations C11-C24 is obviously inferior to that of formulations C5, C6, C8, and C9.

[0051] 5. Acclimatization and transplantation of tissue culture seedlings

[0052] Acclimatization: First, place the tissue culture seedlings in a greenhouse with a temperature of 20-25°C for 10 days, with the bottle cap loosened. Then, clean the solid medium on the tissue culture seedlings, and then transplant them.

[0053] Transplantation and management: Transplantation is carried out in a well-ventilated and shady shaded area. The transplantation substrate is moss, which is fully soaked in water and then controlled to not drip water. Then, place the moss in the holes in the plug tray, filling it to 2 / 3 of the height. Place the treated Spathiphyllum sp. tissue culture seedlings in the plug tray with moss, and manage them. Light management: in a greenhouse with a shading rate of 75%. Water management: spray water once a day at noon until the plants are completely wet. Temperature management: control the temperature at 20-25°C through an automatic temperature control device. Fertilization management: spray nutrient solution once every 7 days in the afternoon from 4-5 pm. The nutrient solution is based on Hoagland's nutrient solution, with N, P, and K concentrations of 120, 80, and 100 mg / L, respectively. Ventilation conditions: good ventilation, with the ventilation curtain and water curtain opened for 1-2 hours at noon every day.

[0054] After one month of transplantation, the survival rate of the tissue culture seedlings reaches more than 95%, with good growth

[0055] Example 2: Using the preferred formula and tissue culture method in Example 1 to carry out tender leaf tissue culture of different varieties of iron orchid:

[0056] The medium for inducing callus from tender leaves is MS medium + TDZ 1.0 mg / L + ZT 0.2 mg / L + 2,4-D 1.0 mg / L + IAA 0.5 mg / L + sucrose 65 g / L + activated carbon 3 g / L;

[0057] The medium for inducing differentiation of the cluster buds is: MS medium + TDZ 1.5 mg / L + ZT 0.5 mg / L + 2,4-D 0.2 mg / L + IAA 0.5 mg / L + sucrose 28 g / L + activated carbon 3 g / L + coconut juice 110 g / L;

[0058] The medium for tissue culture of strong seedlings is: MS medium + TDZ 0.1 mg / L + BR 0.5 mg / L + 2,4-D 0.5 mg / L + IAA 0.5 mg / L + sucrose 28 g / L + activated carbon 3 g / L + coconut juice 135 g / L.

[0059] The iron orchid varieties are T. ionantha, T. bulbosa, T. velutina, and T. kolbii, and the tissue culture results are as follows:

[0060] Table 4: Effect of the preferred formula on tissue culture of different varieties of iron orchid

[0061]

[0062] As can be seen from the results in Table 4, the promoting agent in the present application has a large difference in the effect on tissue culture of different varieties of iron orchid, and the promoting agent in the present application has a significantly better promoting effect on tissue culture and propagation of T. swinhoei than the rest of the varieties, and is more suitable for tissue culture and rapid propagation of T. swinhoei.

Claims

1. A method for in vitro culture of Swida delavayi leaf blades, characterized in that, The method comprises the following steps: (1) picking the tender leaves of S. verticillata as explants, and sterilizing the explants; (2) inoculating the sterilized explants on a callus culture medium to induce callus, wherein the callus culture medium is prepared from MS medium, 0.5-1.0 mg / L phenylthiazolyl urea, 0.2 mg / L zeatin, 1.0-1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L indole acetic acid, 50-80 g / L sucrose and 3 g / L activated carbon; (3) inoculating the callus on a differentiation medium to generate cluster buds, wherein the differentiation medium for inducing the callus to generate cluster buds is prepared from MS medium, 1.5-2.0 mg / L phenylthiazolyl urea, 0.5 mg / L zeatin, 0.2-0.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L indole acetic acid, 100-120 g / L coconut juice, 25-30 g / L sucrose and 3 g / L activated carbon; (4) inoculating the cluster buds on a seedling culture medium to culture seedlings and obtain tissue culture seedlings, and transplanting the tissue culture seedlings to complete the propagation, wherein the seedling culture medium is prepared from MS medium, 0.1 mg / L phenylthiazolyl urea, 0.2-0.5 mg / L brassinolide, 0.5-1.0 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L indole acetic acid, 120-150 g / L coconut juice, 25-30 g / L sucrose and 3 g / L activated carbon.

2. The ex vivo culture method according to claim 1, wherein, In step (1), the sterilization method is as follows: first, soaking the explants in 75% alcohol for 10-30 s, and repeatedly washing and oscillating the explants in sterile water for at least 3 times, each time for no less than 5 min; then, transferring the explants into a 10% NaClO solution for sterilization for 10-20 min, and repeatedly washing and oscillating the explants in sterile water for at least 3 times, each time for no less than 5 min; finally, transferring the explants into a 0.1% HgCl2 solution for sterilization for 4-6 min, and then repeatedly washing the explants in sterile water until clean, and finally absorbing the water on the surface of the explants with sterilized paper.

3. The ex vivo culture method according to claim 1, wherein, In step (2), the method for inducing callus is as follows: culturing the inoculated explants in a sterile room under the following conditions: temperature 22±2 ℃ and dark culture; in step (3), the method for culturing to generate cluster buds is as follows: culturing the inoculated explants in a sterile room under the following conditions: temperature 25±2 ℃, light intensity 1500-2000 lx and light time 12 h / d.

4. The ex vivo culture method according to claim 1, wherein, In step (4), the method for seedling culture is: cutting the cluster buds on the differentiation culture medium into single small buds, selecting uniform small buds and inoculating them on different seedling culture media, and then culturing them in a sterile room after inoculation, with the culture conditions being: temperature 25±2℃, light intensity 2000-2500lx, and light time 14h / d; the method for seedling hardening and transplanting is: first placing the bottle cap in a greenhouse at a temperature of 20-25℃ for 10d, removing the solid culture medium on the tissue culture seedlings for transplanting, placing the tissue culture seedlings in a hole tray covered with moss for management, spraying water once a day under the condition of 75% shading rate, controlling the temperature at 20-25℃, spraying nutrient solution once every 7d, and keeping good ventilation.

5. Application of the culture method of claim 1 to in vitro rapid propagation of S. verticillata leaves.

Images