African cherry tissue culture rapid propagation medium and method

Abstract

The application discloses a tissue culture and rapid propagation medium and method for African marula fruits, which is composed of lateral bud germination medium, cluster bud induction medium, cluster bud growth medium and rooting medium; 1L of the lateral bud germination medium is composed of MS medium, 0.05-0.07 mg of TIBA, 0.1-0.8 mg of GA3 and 3-10 g of agar; 1L of the cluster bud induction medium is composed of MS medium, 0.03-0.15 mg of TIBA, 0.2-1.2 mg of ZT, 0.3-0.8 mg of NAA, 20-40 g of sucrose and 3-10 g of agar; and the cluster bud growth medium is added with phosphate fertilizer on the basis of the cluster bud induction medium. The propagation coefficient of the method can reach 6.8 at most, the propagation effect is good, and the rooting rate of the marula fruit tissue culture seedlings is up to 95% or above. The application solves the problem of local planting of the African marula fruits.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture and rapid propagation medium and method for African cherry. BACKGROUND

[0002] African cherry is a medicinal plant of the tree of the Apocynaceae family, and its main producing area is in Ghana. The main bioactive component of African cherry seeds is indole alkaloid, and the content of which is high in the seeds. African cherry alkaloids can be divided into two chemical types: one is ibogaine type alkaloid, including ibogaine and voacangine; the other is ibogamine type alkaloid, including ibogamine and vobtusine. Among them, ibogamine and voacangine are the most demanded raw medicinal materials in the plant medicine market. Voacangine can be used as a precursor for the semi-synthesis of ibogaine, and ibogamine can be used as a precursor for the semi-synthesis of vinpocetine, which is a cardiovascular drug widely used in the market. Both of them are in short supply in the market. Especially, the content of ibogamine in African cherry seeds is very high, about 2-3%, so due to the huge demand for voacangine and ibogamine in the plant medicine market, African cherry seeds have become a product that is fiercely competed for in the plant medicine market. At present, domestic pharmaceutical enterprises import the seeds of African cherry from Africa back to China, and then extract the effective compounds of the plant as raw medicinal materials and sell them to the European and American markets. Due to the high cost of purchasing wild medicinal plants from Africa and transporting them back to China, it is difficult to solve the problem of raw material sources.

[0003] So far, there is no cultivation report in China, mainly because the propagation technology is difficult, the seeds imported from abroad do not germinate, and it is difficult to obtain African cherry plants, which is a plant variety that is strangling the neck of European and American countries.

[0004] African cherry cutting propagation has the disadvantages of long seedling period, great influence of season, and unsuitable growth conditions, and it is difficult to propagate seedlings with uniform growth state in a short time. As a rapid propagation technology, tissue culture can effectively solve these problems and propagate a large number of excellent tree species in a short time. Although plant tissue culture technology is becoming mature, the improvement of tissue culture effect of woody plants is still affected by multiple factors. So far, there is no report on the establishment of a tissue culture and rapid propagation system for African cherry. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the deficiencies and defects mentioned in the above background, and to provide a tissue culture and rapid propagation medium and method for African cherry.

[0006] To solve the above technical problems, the technical solution provided by the present application is:

[0007] A tissue culture rapid propagation medium for African cherry fruit, which is composed of lateral bud germination medium, cluster bud induction medium, cluster bud growth medium and rooting medium;

[0008] The lateral bud germination medium is composed of MS medium, 0.05-0.07 mg of TIBA, 0.1-0.8 mg of GA3 and 3-10 g of agar per 1 L.

[0009] The cluster bud induction medium is composed of MS medium, 0.03-0.15 mg of TIBA, 0.2-1.2 mg of ZT, 0.3-0.8 mg of NAA, 20-40 g of sucrose and 3-10 g of agar per 1 L.

[0010] The cluster bud growth medium is added with phosphorus fertilizer on the basis of the cluster bud induction medium.

[0011] The rooting medium is composed of 1 / 2MS medium, 0.3-0.8 mg of ZT, 0.1-0.25 mg of NAA, 0.1-0.25 mg of IAA, 10-30 g of sucrose and 3-10 g of agar per 1 L.

[0012] The pH value of the lateral bud germination medium, the cluster bud induction medium, the cluster bud growth medium and the rooting medium is controlled at 5.5-6.5.

[0013] As a further improvement, the lateral bud germination medium contains 0.4-0.6 mg of GA3 per 1 L, and the mass ratio of GA3 to TIBA contained in the medium is 9-10:1.

[0014] As a further improvement, the lateral bud germination medium is composed of MS+0.06 mg / L TIBA+0.6 mg / L GA3+6 g / L agar.

[0015] As a further improvement, the cluster bud induction medium contains 0.06-0.1 mg of TIBA and 0.5-1 mg of ZT per 1 L.

[0016] As a further improvement, the cluster bud induction medium is composed of MS+0.06 mg / L TIBA+1.0 mg / L ZT+0.5 mg / L NAA+30 g / L sucrose+6 g / L agar.

[0017] As a further improvement, the phosphorus fertilizer in the cluster bud growth medium is KH2PO4, and the concentration is 0.5-3 mmol / L.

[0018] As a further improvement, the rooting medium consists of 1 / 2MS+0.5mg / L ZT+0.2mg / L NAA+0.2mg / L IAA+20g / L sucrose+6g / L agar.

[0019] The application also provides a method for tissue culture and rapid propagation of African cherry using the medium, comprising the following steps:

[0020] (1) collecting African cherry branches, and cutting stem segments with lateral buds as explants;

[0021] (2) inoculating the explants obtained in step (1) on a lateral bud germination medium, and performing lateral bud germination culture, wherein the lateral bud germination culture conditions are as follows: day temperature 35±2℃, night temperature 15±2℃, light intensity 2000-3000Lx, and daily light time 15-17h;

[0022] (3) transferring to a cluster bud induction medium, and performing cluster bud induction culture, wherein the cluster bud induction culture conditions are as follows: day temperature 25±2℃, night temperature 15±2℃, light intensity 2000-3000Lx, and daily light time 15-17h;

[0023] (4) transferring to a cluster bud growth medium, and performing bud subculture to form more cluster buds;

[0024] (5) cutting the cluster buds obtained in step (4) and inoculating on a rooting medium, and performing rooting culture to obtain rooted seedlings, wherein the rooting culture conditions are as follows: day temperature 25±2℃, night temperature 15±2℃, light intensity 2000-3000Lx, and daily light time 13-15h;

[0025] (6) transplanting the rooted seedlings obtained in step (5) to a culture substrate for culture.

[0026] Compared with other woody plants, the medium, added components and component concentration at each stage are quite different when African cherry is subjected to tissue culture, and each factor has a more complex influence on the culture effect, so a large number of optimizations need to be made on each factor to obtain the best culture effect.

[0027] The medium and method for tissue culture and rapid propagation of African cherry provided by the application have a proliferation coefficient of up to 6.8, good proliferation effect, and a rooting rate of African cherry tissue culture seedlings of up to 95% or more. The application solves the problem of local planting of African cherry and solves the problem of raw material sources mentioned in the background art, and provides technical support for large-scale factory production of seedlings. DETAILED DESCRIPTION

[0028] For the purpose of understanding the present application, the present application will be described in further detail below with reference to preferred embodiments. However, the scope of the present application is not limited to the below specific embodiments.

[0029] Unless otherwise defined, all terms used in the description herein, including technical terms, scientific terms, and scientific symbols, have the same meanings as those generally used and understood by one of ordinary skill in the art. The terms used in the description of the present application are only used to describe particular embodiments and are not intended to limit the scope of the present application.

[0030] Unless otherwise specified, each of the various materials, reagents, instruments and apparatuses used in the present application can be purchased from the market or can be prepared by the existing method.

[0031] In some embodiments, the tissue culture and rapid propagation medium for African cherry fruit of the present application is composed of lateral bud germination medium, cluster bud induction medium, cluster bud growth medium and rooting medium.

[0032] The lateral bud germination medium is composed of MS medium, 0.05-0.07 mg of TIBA, 0.1-0.8 mg (preferably 0.4-0.6 mg) of GA3 and 3-10 g of agar per 1 L of the lateral bud germination medium. In some preferred embodiments, the lateral bud germination medium is composed of MS+0.06 mg / L TIBA+0.6 mg / L GA3+6 g / L agar. The ratio of GA3 to TIBA affects the germination rate, and the preferred ratio of GA3 to TIBA is 9-10:1. When the ratio of GA3 to TIBA is 10:1, the germination time is shortened, and the germination rate is the highest.

[0033] The cluster bud induction medium is composed of MS medium, 0.03-0.15 mg (preferably 0.06-0.1 mg) of TIBA, 0.2-1.2 mg (preferably 0.5-1 mg) of ZT, 0.3-0.8 mg of NAA, 20-40 g of sucrose and 3-10 g of agar per 1 L of the cluster bud induction medium. In some preferred embodiments, the cluster bud induction medium is composed of MS+0.06 mg / L TIBA+1.0 mg / L ZT+0.5 mg / L NAA+30 g / L sucrose+6 g / L agar. TIBA has a significant effect on cluster bud induction, and the induction rate increases by more than 20% when 0.06-0.1 mg / L of TIBA is added, and the induction multiplication coefficient can be more than 6. At the same time, the optimal effect can be achieved by the mutual cooperation of the amounts of TIBA and ZT, and the preferred amounts of TIBA and ZT are 0.06 mg / L TIBA+1.0 mg / L ZT and 0.1 mg / L TIBA+0.5 mg / L ZT.

[0034] Bud growth medium: the bud growth medium is added with phosphorus fertilizer, preferably KH2PO4, at a concentration of 0.5-3 mmol / L, preferably 2.5 mmol / L, on the basis of the bud induction medium.

[0035] Rooting medium: 1 L of the rooting medium is composed of 1 / 2MS medium, ZT 0.3-0.8 mg, NAA 0.1-0.25 mg, IAA 0.1-0.25 mg, sucrose 10-30 g, and agar 3-10 g. In some preferred embodiments, the rooting medium is composed of 1 / 2MS+0.5 mg / L ZT+0.2 mg / L NAA+0.2 mg / L IAA+20 g / L sucrose+6 g / L agar.

[0036] The pH values of the lateral bud germination medium, the bud induction medium, the bud growth medium and the rooting medium of the application are controlled at 5.5-6.5, and a conventional pH regulator in the medium field can be used to adjust the pH.

[0037] In the formula, MS refers to the conventional medium MS component in the existing plant tissue culture field. 1 / 2MS refers to a conventional medium MS component in which the amount of elements is halved, and the rest of the components remain unchanged.

[0038] TIBA is 2,3,5-triiodobenzoic acid, which is an auxin transport inhibitor.

[0039] GA3 is commercially available gibberellin GA3, which is a plant growth regulator.

[0040] ZT is zeatin, which is a cytokinin.

[0041] NAA is commercially available naphthalene acetic acid, which is a plant growth regulator for promoting plant root growth.

[0042] IAA is commercially available indole acetic acid, which is a plant growth hormone.

[0043] In some specific embodiments, the African cherry tissue culture and rapid propagation method of the application comprises the following steps:

[0044] (1) Collecting the current year's new branches of African cherry and cutting the stem segments with lateral buds as explants.

[0045] (2) Inoculating the obtained explants on the lateral bud germination medium for lateral bud germination culture.

[0046] In some embodiments, the side bud germination culture condition is: temperature (day temperature / night temperature) 35±2℃ / 15±2℃, light intensity 2000-3000Lx, daily light time 15-17h. The culture time is 3-7d.

[0047] (3) Transfer to the cluster bud induction medium, and perform cluster bud induction culture (the side bud induces the cluster bud).

[0048] In some embodiments, the cluster bud induction culture condition is: temperature (day temperature / night temperature) 25±2℃ / 15±2℃, light intensity 2000-3000Lx, daily light time 15-17h. The culture time is 25-30d.

[0049] (4) Transfer to the cluster bud growth medium, and perform bud subculture to form more cluster buds.

[0050] In some embodiments, the culture condition is: temperature (day temperature / night temperature) 25±2℃ / 15±2℃, light intensity 2000-3000Lx, daily light time 15-17h.

[0051] (5) Cut the cluster buds obtained in step (4) and inoculate to the rooting medium to perform rooting culture to obtain the rooted seedlings.

[0052] In some embodiments, the rooting culture condition is: temperature (day temperature / night temperature) 25±2℃ / 15±2℃, light intensity 2000-3000Lx, daily light time 13-15h. The culture time is 18-25d.

[0053] (6) Transfer the obtained rooted seedlings to the cultivation substrate for culture.

[0054] The tissue culture medium and the tissue culture method provided by the application have high proliferation coefficient (up to 6.8 at most) and high rooting rate (up to more than 95%).

[0055] Embodiment

[0056] 1. Selection and treatment of explants

[0057] The side buds of the newly grown branches of the main stem of the Malus hallings are selected as the tissue culture explant materials; the newly grown branches cut on the day are cut into 1-3 sections with side buds in an ultraclean workbench, the stem sections are oscillated in a 75% alcohol solution for 10-20s, taken out, washed with sterile water for 3 times, dried with sterilized water-absorbing paper, the side buds are cut, the base of the side buds is left with 0.5cm stem section, the upper part is left with 0.2-0.3cm stem section, the cut side buds are oscillated in a 0.1% mercury solution for 10min, taken out, washed with sterile water for 5 times, and the time for each washing with sterile water is 25-30s.

[0058] 2. Lateral bud germination culture

[0059] The obtained stem segment lateral buds were inoculated on lateral bud germination medium (MS + 0.06 mg / L TIBA + 0.6 mg / L GA3 + 6 g / L agar, pH 6.2) for lateral bud germination culture, and the culture conditions were temperature (day / night temperature) 35±2℃ / 15±2℃, illumination 2000Lx, light cycle 16h / 8h (day / night), lateral bud germination after 3d, lateral bud grew to 1cm length after 7d. The culture germination situation is shown in Table 1, Table 2.

[0060] The lateral bud germination rate of Malan fruit was significantly different at different culture temperatures, and appropriate high day temperature was beneficial to germination. The effect of adding TIBA was better than that of TDZ.

[0061] Table 1. Effect of different culture temperatures on lateral bud germination of Malan fruit

[0062]

[0063] Table 2. Effect of different hormones on lateral bud germination of Malan fruit

[0064]

[0065] 3. Cluster bud induction culture

[0066] Transfer to cluster bud induction medium (MS + 0.06 mg / L TIBA + 1.0 mg / L ZT + 0.5 mg / L NAA + 30 g / L sucrose + 6 g / L agar, pH 5.7) for cluster bud induction culture, and the culture conditions were temperature (day / night temperature) 25±2℃ / 15±2℃, illumination 2000Lx, light cycle 16h / 8h (day / night), cluster bud formation after 25d, and the proliferation coefficient was 6.8;

[0067] TIBA had a significant effect on cluster bud induction, and 0.06-0.1 mg / L TIBA induced a proliferation coefficient of more than 6.

[0068]

[0069] 4. Cluster bud growth

[0070] Phosphorus had a significant promoting effect on the growth of Malan fruit cluster buds.

[0071] KH2PO4 as phosphate fertilizer, set 0.5 (P1), 1.0 (P2), 1.5 (P3), 2.0 (P4), 2.5 (P5) and 3.0 mmol / L (P6) a total of six levels of phosphorus, added to the cluster bud induction medium (MS+0.06 mg / L TIBA+1.0 mg / L ZT+0.5 mg / L NAA+30 g / L sucrose+6 g / L agar, pH 5.7), to form more clusters of buds. The culture conditions are temperature (day / night temperature) 25±2℃ / 15±2℃, light 2000Lx, photoperiod 16h / 8h (day / night).

[0072] The bud height is increased by 1.25~2.60 times than that without phosphorus. The bud height is increased by 2.6 times with 2.5 mmol / L of phosphorus.

[0073] 5. Rooting culture

[0074] The cluster buds obtained in step 4 are cut and inoculated into the rooting medium (1 / 2MS+0.5 mg / L ZT+0.2 mg / L NAA+0.2 mg / L IAA+20 g / L sucrose+6 g / L agar, pH 6.1) for rooting culture, and the culture conditions are temperature 25±2℃, light 2000Lx, photoperiod 14h / 10h (day / night), and roots grow after 18 days.

[0075]

[0076] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Therefore, any simple modification, equivalent change and modification of the above embodiment according to the technical essence of the present application, which does not deviate from the technical scheme of the present application, shall fall within the scope of protection of the technical scheme of the present application.

Claims

1. A tissue culture medium for rapid propagation of Aframomum melegueta, characterized in that, It consists of lateral bud germination medium, cluster bud induction medium, cluster bud growth medium and rooting medium; Each 1L of the lateral bud germination medium consists of MS medium, TIBA 0.05-0.07 mg, GA3 0.1-0.8 mg and agar 3-10 g; Each 1L of the cluster bud induction medium consists of MS medium, TIBA 0.03-0.15 mg, ZT 0.2-1.2 mg, NAA 0.3-0.8 mg, sucrose 20-40 g and agar 3-10 g; The cluster bud growth medium adds phosphorus fertilizer on the basis of the cluster bud induction medium; the phosphorus fertilizer is KH2PO4, and the concentration is 0.5-3 mmol / L; Each 1L of the rooting medium consists of 1 / 2MS medium, ZT 0.3-0.8 mg, NAA 0.1-0.25 mg, IAA 0.1-0.25 mg, sucrose 10-30 g and agar 3-10 g; The pH values of the lateral bud germination medium, cluster bud induction medium, cluster bud growth medium and rooting medium are all controlled in 5.5-6.5; The explant is a stem section with lateral buds.

2. The in vitro culture medium for tissue culture propagation of Aframomum melegueta according to claim 1, characterized in that, Each 1L of the lateral bud germination medium consists of MS medium, TIBA 0.05-0.07 mg, GA3 0.4-0.6 mg and agar 3-10 g.

3. The in vitro culture medium for tissue culture propagation of Aframomum melegueta according to claim 1, characterized in that, The lateral bud germination medium consists of MS+0.06 mg / L TIBA+0.6 mg / L GA3+6 g / L agar.

4. The tissue culture medium for rapid propagation of African cherry according to any one of claims 1 to 3, characterized in that, Each 1L of the cluster bud induction medium consists of MS medium, TIBA 0.06-0.1 mg, ZT 0.5-1 mg, NAA 0.3-0.8 mg, sucrose 20-40 g and agar 3-10 g.

5. The tissue culture medium for rapid propagation of African cherry according to any one of claims 1 to 3, characterized in that, The cluster bud induction medium consists of MS+0.06 mg / L TIBA+1.0 mg / L ZT+0.5 mg / L NAA+30 g / L sucrose+6 g / L agar.

6. The tissue culture medium for rapid propagation of African cherry according to any one of claims 1 to 3, characterized in that, The rooting medium consists of 1 / 2MS+0.5 mg / L ZT+0.2 mg / L NAA+0.2 mg / L IAA+20 g / L sucrose+6 g / L agar.

7. A method for tissue culture propagation of African cherry using the medium according to any one of claims 1 to 6, characterized in that, The steps include the following: (1) collecting African cherry branches, and cutting stem sections with lateral buds as explants; (2) inoculating the explants obtained in step (1) on the lateral bud germination medium, and performing lateral bud germination culture, wherein the lateral bud germination culture conditions are as follows: day temperature 35±2℃, night temperature 15±2℃, light intensity 2000-3000 Lx, and daily light time 15-17 h; (3) transferring to the cluster bud induction medium, and performing cluster bud induction culture, wherein the cluster bud induction culture conditions are as follows: day temperature 25±2℃, night temperature 15±2℃, light intensity 2000-3000 Lx, and daily light time 15-17 h; (4) transferring to the cluster bud growth medium, and performing bud subculture to form more cluster buds; and (5) transferring to the rooting medium, and performing rooting culture. (5) The cluster buds obtained in the culturing of step (4) are inoculated onto a rooting culture medium to carry out rooting culture to obtain rooted seedlings, and the rooting culture conditions are as follows: day temperature 25±2℃, night temperature 15±2℃, light intensity 2000-3000Lx, and daily light time 13-15h; (6) The rooted seedlings obtained in step (5) are transplanted into a culture medium for culture.

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