A leaf type moringa tender bud aseptic rapid propagation of seedling culture medium and culture method

Abstract

The application discloses aseptic rapid propagation of leaf type moringa tender bud and a seedling culture medium and a culture method, relates to the agricultural technology field, and provides a culture medium combination and a cultivation method, which comprises the following steps of collecting and processing an explant; performing transfer culture to obtain aseptic axillary bud; differentiating the aseptic axillary bud into stable clump buds; inoculating and culturing the stable clump buds to obtain stable rooted seedlings; preparing a transplanting substrate; transplanting seedlings; and domesticating and managing seedlings. The application saves a large amount of time for propagation of leaf type moringa varieties. The seedling growth performance is uniform, and the whole-year factory batch seedling supply can be guaranteed, so that the production needs are met.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural technology, more particularly to a leaf type moringa tender bud aseptic rapid propagation of seedling culture medium and culture method. BACKGROUND

[0002] Moringa oleifera belongs to Moringaceae Moringa, alias oil moringa, horseradish tree, drum tree, and so on, is a multi-year fast-growing small trees in the tropics, originally from India, Africa and other countries, because its roots have a pungent flavor, named horseradish. Moringa contains rich nutrients, has unique edible and medicinal value, also known as "miracle tree". Moringa grows fast, drought tolerant, wide adaptability, at present in China's Yunnan, Hainan, Guangdong, Fujian, Hunan and Guangxi, etc. have large-scale planting, among which Yunnan has the largest planting area, accounting for about 40% of the country.

[0003] Moringa cultivation and medicinal history in 2000 years, has antipyretic, anti-inflammatory, stone, diuretic and other functions. India and African countries often use moringa to treat diabetes, hypertension, cardiovascular disease, obesity, skin disease and other diseases.

[0004] However, more than 90% of the production of moringa seedling is used by seed, which has the problems of unstable traits, poor disease resistance, low yield and so on, and a small part of it is used by cutting and high pressure seedling, but it needs a lot of materials and cannot be used and popularized in large quantities.

[0005] Therefore, it is an urgent problem for those skilled in the art to provide a leaf type moringa tender bud aseptic rapid propagation of seedling culture medium and culture method. SUMMARY

[0006] Therefore, the present application provides a leaf type moringa tender bud aseptic rapid propagation of seedling culture medium and culture method. The stability and quality of the excellent single plant can be guaranteed, which is of great significance for promoting the large-scale rapid propagation and popularization of moringa seedling factory.

[0007] In order to achieve the above purpose, the present application adopts the following technical scheme:

[0008] A leaf type moringa tender bud aseptic rapid propagation of seedling culture medium combination, comprising:

[0009] Aseptic axillary bud induction growth medium: 1 / 2MS, IBA 0.5-0.7 mg / L, NAA 0.15-0.25 mg / L, sucrose 20-25 g / L, agar 4.5-6.5 g / L, pH: 6.3-6.5;

[0010] Differentiation of cluster shoots induction medium: MS, 6BA 0.5-0.8 mg / L, NAA 0.05-0.1 mg / L, IAA 0.05-0.1 mg / L, TDZ 0.003-0.008 mg / L, sucrose 25-30 g / L + agar 4.5-6.5 g / L, pH: 6.3-6.5;

[0011] Stable differentiation of cluster shoots medium: MS, 6BA 0.5-0.7 mg / L, NAA 0.03-0.07 mg / L, sucrose 25-30 g / L, agar 4.5-6.5 g / L, pH: 6.3-6.5;

[0012] Rooting medium: 1 / 2MS, IBA 0.1-0.6 mg / L, NAA 0.05-0.2 mg / L, sucrose 15-20 g / L + agar 6-7 g / L, fresh seven-hole lotus root 8-12 g / L, pH: 5.8-6.0;

[0013] Stable rooting medium: 1 / 2MS, IBA 0.1-0.6 mg / L, NAA 0.05-0.2 mg / L, sucrose 15-20 g / L, agar 5-7 g / L, fresh seven-hole lotus root 8-12 g / L, carbon 0.5-0.6 g / L, pH: 5.6-5.8.

[0014] Preferred: includes:

[0015] Aseptic axillary bud induction growth medium: 1 / 2MS, IBA 0.6 mg / L, NAA 0.2 mg / L, sucrose 20 g / L, agar 5 g / L, pH: 6.5;

[0016] Differentiation of cluster shoots induction medium: MS, 6BA 0.6 mg / L, NAA 0.1 mg / L, IAA 0.1 mg / L, TDZ 0.003 mg / L, sucrose 25 g / L + agar 5 g / L, pH: 6.5;

[0017] Stable differentiation of cluster shoots medium: MS, 6BA 0.5 mg / L, NAA 0.03 mg / L, sucrose 30 g / L, agar 5 g / L, pH: 6.5;

[0018] Rooting medium: 1 / 2MS, IBA 0.2 mg / L, NAA 0.1 mg / L, sucrose 20 g / L + agar 6.5 g / L, fresh seven-hole lotus root 8-9 g / L, pH: 5.8;

[0019] Stable rooting medium: 1 / 2MS, IBA 0.2 mg / L, NAA 0.1 mg / L, sucrose 20 g / L, agar 6 g / L, fresh seven-hole lotus root 8-9 g / L, carbon 0.6 g / L, pH: 5.6.

[0020] The application also provides a culture method based on the culture medium combination, comprising the following steps:

[0021] (1) explant collection and treatment;

[0022] (2) subculture to obtain sterile axillary buds;

[0023] (3) differentiation of the sterile axillary buds into stable clumps;

[0024] (4) inoculation of the stable clumps to obtain stable rooted seedlings;

[0025] (5) preparation of transplanting substrate;

[0026] (6) hardening-off and transplanting;

[0027] (7) acclimatization and management of seedlings.

[0028] Preferably, step (1) specifically comprises: selecting tender bud stem segments as explants; removing leaves, cutting into 3-4 cm stem segments with axillary buds, washing, removing all single buds, cutting off 0.5-0.7 cm at the cut, disinfecting to obtain the disinfected stem segments with axillary buds, and reserving for use;

[0029] Washing: placing the stem segments with axillary buds into a small sieve basin, continuously rolling and washing for 20 min, rinsing for 3-5 min with 10 g of washing powder in 0.8 L of water, and washing for 40 min under tap water;

[0030] Disinfecting: sterilizing with 75% alcohol for 30 s, washing twice with sterile water, sterilizing with 0.1% mercury for 8 min, washing three times with sterile water, and filtering dry.

[0031] Further, the specific standard for selecting tender bud stem segments is: healthy growth, full axillary buds, and no pests and diseases.

[0032] Preferably, step (2) specifically comprises: inoculating the disinfected stem segments with axillary buds in step (1) onto the sterile axillary bud growth-inducing culture medium, and culturing under the conditions of 10 h of light per day, 25-27℃ of temperature, and 1500-2000 lx of light intensity to obtain sterile axillary buds after 20-23 days of inoculation and culture.

[0033] Preferably, step (3) specifically comprises: cutting the sterile axillary buds into 1-1.5 cm of one bud per segment, shallowly inserting the lower end of the morphological under the differentiation clump-inducing culture medium, culturing to obtain clumps, inoculating the clumps into the stable differentiation clump culture medium, and culturing to obtain stable clumps.

[0034] The culture conditions are all 10 h of light per day, 25-27℃ of temperature, and 1500-2000 lx of light intensity, and inoculation and culture are performed for 20-23 days.

[0035] Preferably, step (4) is specifically as follows: inoculating the stable clustered buds of more than 3 cm into the rooting medium to cultivate the rooted seedlings; when the rooted seedlings grow to 8-10 cm, taking one bud section of the middle and upper stem buds and inoculating them into the stable rooting medium, with multiple single bud sections per bottle, to cultivate the stable rooted seedlings;

[0036] The culture conditions are 10 hours of light per day, a temperature of 25-27°C, a light intensity of 1500-2000 lx, and a culture period of 20-23 days.

[0037] Preferably, step (5) is specifically as follows: 10 days before transplanting, a matrix is ​​prepared in a greenhouse according to a weight ratio of garden soil: fine sand: decomposed nut skin of 5:3:2, and mancozeb is mixed with the matrix at a weight ratio of 1:3000 to prepare a transplanting matrix, which is then sterilized and packed in nutrient bags. The matrix is ​​neatly arranged in rows on a seedbed in a light-transmitting and moisture-retaining shed and watered thoroughly for later use.

[0038] Preferably, step (6) is specifically as follows: the stably rooted seedlings are moved to an indoor seedling hardening rack for 4 days of natural light cultivation, wherein the bottle caps are loosened but not opened on the first day, and 1 / 3 of the caps are opened on the second, third, and fourth days for hardening; transplanting: 3 days before planting, the transplanting substrate in step (5) is first watered thoroughly, and the transplanting substrate is turned over and reserved one day before planting; transplanting time: after 17:00 in the afternoon, the hardened seedlings are planted to 100 plants, and 500 times diluted mancozeb solution is promptly watered for rooting disinfection, the amount being 100 ml / bag, and after planting, the moisturizing shed is promptly covered to obtain tissue culture seedlings;

[0039] Step (7) is specifically as follows: placing the tissue culture seedlings in a light-transmitting and moisture-retaining shed with the shed fully covered for 7 days and then half-opening the shed to 1 / 5, with an acclimation temperature of 22-34° C. and a relative humidity of 75%-90%;

[0040] For the first five days, spray the plants with clean water once daily between 8:00 AM and 9:00 AM and 5:00 PM and 6:00 PM. Water the bottom of the nutrient bag once on the sixth day. Ten days after transplanting, new roots begin to grow. After 15 days, new leaves begin to emerge, and old leaves tend to stabilize. The greenhouse should be fully opened to increase light transmittance and proper fertilizer and water management should be implemented. Planting should be done in the field 60 days after transplanting in the greenhouse.

[0041] The beneficial effect of step (6) is that the high humidity required for transplanting Moringa tissue culture seedlings is a major obstacle to the expansion of seedlings. If an appropriate seedling hardening method is not adopted, the leaves and stem tips of Moringa tissue culture seedlings will wilt and shrivel under low air humidity (<80%) conditions for as short as 30 minutes.

[0042] The beneficial effect of step (7) is that after transplanting, the plants must be sprayed immediately and sealed with plastic film to protect them from moisture, so as to improve the survival rate of the transplanted plants.

[0043] The present invention also provides applications of the above-mentioned culture medium combination or any of the above-mentioned culture methods in Moringa planting, Moringa new species cultivation, and Moringa processing.

[0044] As can be seen from the above technical solutions, compared with the prior art, the present invention provides a culture medium and method for the rapid aseptic propagation of Moringa oleifera buds into seedlings, which achieves the following technical effects: a significant amount of time is saved for the propagation of Moringa oleifera foliage varieties. The growth performance of the seedlings is uniform and consistent, and a factory-scale batch supply of seedlings can be guaranteed throughout the year to meet production needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] 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.

[0046] Figure 1 The accompanying drawings are diagrams of cleaning explants provided by the present invention.

[0047] Figure 2 The accompanying drawing is a diagram of the sterile axillary bud provided by the present invention.

[0048] Figure 3 The accompanying drawing is a diagram of the clustered buds provided by the present invention.

[0049] Figure 4 The accompanying drawing is a diagram of the stable bud cluster provided by the present invention.

[0050] Figure 5 The accompanying drawings are diagrams of rooted seedlings provided by the present invention.

[0051] Figure 6 The accompanying drawings are diagrams of the stable rooting seedlings provided by the present invention.

[0052] Figure 7 The accompanying drawing is a diagram of the moisturizing shed provided by the present invention.

[0053] Figure 8 The accompanying drawing is a diagram of the seedlings provided by the present invention after 60 days of transplantation.

[0054] Figure 9 The accompanying drawing is a growth diagram of Moringa oleifera planted for 2 years provided by the present invention. DETAILED DESCRIPTION

[0055] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of the present application.

[0056] The embodiment of the present application discloses a leaf type moringa tender bud aseptic rapid propagation of seedling culture medium and culture method.

[0057] The test material involved in the embodiment is a commercially available leaf type moringa (preferably a multi-oil type variety) planted for 10 years, and the tender bud stem segments of 7 to 21 days (generally 20 cm long below the top bud) are selected. No requirement is made on the variety; the experimental method not mentioned is a conventional experimental method, which will not be described here.

[0058] Embodiment 1

[0059] A leaf type moringa tender bud aseptic rapid propagation of seedling culture medium and culture method

[0060] (1) Explant collection and processing: select tender bud stem segments with healthy growth, full axillary buds, and no pests and diseases as explants. Remove the leaves, trim, and then cut into 3-4 cm stem segments with axillary buds using a sharp hand knife, and place them in a small sieve pot (the upper end of the edge of the pot has multiple water outlet small holes, see Figure 1 ) Use the water impact to continuously roll and wash for 20 min, 10 g of washing powder is diluted with 0.8 L of water, and the washing is gently rubbed for 3-5 min with fingers, and then the water is washed for 40 min under the tap, the whole single bud is taken out and cut at the incision for 0.5-0.7 cm, and the impurities infiltrated into the stem segments during the washing process are removed by cutting. Place the explants in a sterile bottle on a sterile operation table, sterilize, sterilize with 75% alcohol for 30 s, then wash twice with sterile water, then sterilize with 0.1% mercury for 8 min, wash three times with sterile water, and then filter dry water to obtain the sterilized stem segments with axillary buds, which are ready for use.

[0061] (2) Transfer culture: inoculate the sterilized stem segments with axillary buds in step (1) into the aseptic axillary bud induction growth medium (the sterilized stem segments are not cut at both ends and are placed flat on the culture medium).

[0062] Aseptic axillary bud induction growth medium: 1 / 2MS (purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., without agar and sugar, the same below) + IBA 0.6 mg / L + NAA 0.2 mg / L + (sucrose 20 g + agar 5 g) / L, pH: 6.5.

[0063] The culture conditions are 10 hours of light per day, a temperature of 25-27°C, and a light intensity of 1500-2000 lx. After 20-23 days of inoculation and culture, sterile axillary buds are grown (see Figure 2 ).

[0064] (3) Differentiation of sterile axillary buds into cluster buds: Cut the sterile axillary buds into 1-1.5 cm segments and insert the lower end of the buds shallowly into the differentiation cluster bud induction medium.

[0065] Differentiation cluster shoot induction medium: MS (purchased from Haibo Biotechnology Co., Ltd., Qingdao High-tech Industrial Park, without agar and sugar, the same below) + 6BA 0.6 mg / L + NAA 0.1 mg / L + IAA 0.1 mg / L + TDZ 0.003 mg / L (sucrose 25 g + agar 5 g) / L, pH: 6.5;

[0066] The culture conditions are 10 hours of light per day, a temperature of 25-27°C, and a light intensity of 1500-2000 lx. The buds are obtained after 20-23 days of inoculation and culture (see Figure 3 , beneficial effect: the bud proliferation coefficient can reach 8.6).

[0067] The cluster buds are inoculated into a stable differentiation cluster bud culture medium to obtain stable cluster buds.

[0068] Among them, the stable differentiation cluster bud culture medium: MS+6BA 0.5 mg / L+NAA0.03 mg / L+(sucrose 30 g+agar 5 g) / L, pH: 6.5.

[0069] The culture conditions are 10 hours of light per day, a temperature of 25-27°C, and a light intensity of 1500-2000 lx. Stable clustered buds are obtained after inoculation and culture for 20-23 days (see Figure 4 ).

[0070] (4) Inoculate the stable clustered buds larger than 3 cm into the rooting medium to obtain rooted seedlings:

[0071] Rooting medium: 1 / 2MS+lBA0.2 mg / L+NAA0.1 mg / L+(sucrose 20 g+agar 6.5 g+fresh seven-hole lotus root 8-9 g) / L, pH; 5.8.

[0072] The culture conditions are 10 hours of light per day, a temperature of 25-27°C, a light intensity of 1500-2000 lx, and rooting culture for 20-23 days (of which the rooting rate can reach more than 99% in 7-12 days, see Figure 5 ).

[0073] When the rooted seedlings grow to 8-10 cm, take the middle and upper stem buds and cut them into segments, and then inoculate them into the stable rooting medium (only 2-3 cm of the seedling stubs are left to sprout new buds for the next time), and multiple single buds per bottle. Repeat the process to continuously cut and inoculate the stem buds until the required amount of seedlings is obtained.

[0074] Stable rooting medium: 1 / 2MS + IBA 0.2 mg / L + NAA 0.1 mg / L + (sucrose 20 g + agar 6 g + fresh seven-hole lotus root 8-9 g + charcoal 0.6 g) / L, pH: 5.6.

[0075] The culture conditions are 10 hours of light per day, a temperature of 25-27°C, and a light intensity of 1500-2000 lx. The stable rooting culture is carried out for 20-23 days to obtain stable rooted seedlings (see Figure 6 ).

[0076] (5) Prepare the transplanting substrate: 10 days before transplanting, prepare the substrate in the greenhouse according to the ratio of garden soil: fine sand: decomposed nut skin = 5:3:2, and mix it with the manganese zinc fungicide at a weight ratio of 1:3000 to obtain the transplanting substrate. After sterilization, place the nutrient bags on the seedbed in the light-proof and moisture-proof shed (see Figure 7 , which is 110 cm high and 120 cm wide at the bottom) and pour water thoroughly.

[0077] (6) Acclimatize and transplant: move the stable rooted seedlings to the indoor acclimatization rack and culture them under natural light for 4 days (it is better to have 2 hours of morning light before 10 am each day). On the first day, fully loosen the bottle caps without opening them, and on the second, third, and fourth days, open the caps by 1 / 3 (the caps cannot be fully opened, as the seedlings will wilt due to dehydration).

[0078] Transplanting: 3 days before transplanting, pour water thoroughly into the transplanting substrate prepared in step (5), and 1 day before transplanting, use a 25 cm long obliquely opened 2 cm wide bamboo stick to prepare the soil for transplanting.

[0079] The transplanting time is after 17:00 in the afternoon. Transplant the acclimatized seedlings into the nutrient bags, and pour 500 times the amount of manganese zinc fungicide solution as a root fixation and sterilization water, with a volume of 100 ml per bag. After transplanting, cover the shed immediately to obtain the tissue culture seedlings (the roots of the moringa oleifera are fleshy and easily broken, especially the tissue culture seedlings, which have fleshy and callus roots. During the process of seedling removal and transplanting, the roots are easily broken, which affects the survival rate of the transplanted seedlings. Therefore, during transplanting, try to minimize human operation. Use a 15 cm long thin bamboo stick to gently lift the seedlings out of the culture medium, and try to minimize root breakage or damage to the tender roots. Remove as much of the culture medium as possible from the roots. To minimize root breakage, do not wash the roots with water. Directly transplant the seedlings into the nutrient bags prepared in step (5).

[0080] (7) Acclimatization and management of seedlings: after placing the tissue culture seedlings in the light-proof and moisture-proof shed for 7 days, open the shed by 1 / 5. The acclimatization temperature is 22-34°C, and the relative humidity is 75%-90%.

[0081] Among them, the first 7 days after transplanting is the most critical management stage. In the first 5 days, spray 1 time of clean water at 8:00-9:00 am and 17:00-18:00 pm respectively, and on the 6th day, pour water once at the bottom of the nutrient bag.

[0082] 10 days after transplanting, new roots begin to grow, 15 days later, new leaves begin to grow, and old leaves tend to be stable, and the plants begin to grow normally. The whole shed can be opened to increase the light transmittance and do well in the later fertilizer and water management, and the survival rate of transplanting can reach 90%. The plants are transplanted to the ground about 60 days after transplanting in the shed, and the survival rate of planting reaches 93% (see Figure 8 ) Among them, the growth of the leaf type (high oil) moringa of the 2-year-old cutting rod is seen Figure 9 It can be seen that the growth of the seedling of the field planting of moringa reaches uniformity, and can guarantee the whole year's factory batch supply of seedlings, and meets the production needs.

[0083] Comparative experiment 1

[0084] The present application, simply called tender bud aseptic rapid propagation, combined with previous research, a 10-year-old well-established mother tree with a diameter of 109 cm, a height of 3.2 meters, a crown width of 2.6 meters north-south and 2.8 meters east-west, is taken as the material at a distance of 100 cm from the ground.

[0085] Prior art: woody branch cutting, high-pressure woody branch.

[0086] With the passage of time, the results are compared in Table 1.

[0087]

[0088] The results show that: 1. The best time for woody branch cutting and high-pressure seedling is from March to October every year, and the cutting season is seasonal and has limitations. The tender bud can be cut all year round, and can be cut again 15 to 20 days after cutting. As long as the water and fertilizer are sufficient, the more you cut, the more you will sprout. 2. The cutting and high-pressure seedling require a large amount of material, and the amount of material used for cutting each time is small, which cannot meet the demand for large quantities of seedlings. The aseptic rapid propagation method uses less material, and the amount of seedlings is large after 100 days. After a stable aseptic system is established in the culture room, factory production can be carried out, and large quantities of seedlings can be supplied on a planned basis throughout the year. 3. The diameter of the seedlings obtained by cutting and high-pressure is not uniform, while the aseptic rapid propagation method has stronger uniformity. 4. The aseptic rapid propagation method is more convenient for transportation. 5. Taking 200 days as an example, the amount of seedlings obtained is compared: aseptic rapid propagation method 1500+3000+6000+12000+24000=46500; cutting: 30+30+30=90; high pressure: 40+40+40=120.

[0089] The various embodiments described in this specification are intended to be illustrative only and in no way limit the scope of the application. Changes and modifications can be made by those skilled in the art, which employ the principles of the application, without departing from the scope thereof. Accordingly, the application is not limited to the embodiments described herein, but instead has scope to encompass any changes and modifications that serve the same, equivalent, or similar purposes.

[0090] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to those skilled in the art and are within the scope of the application as defined by the appended claims. The particular implementations shown and described are shown as being representative alternatives only. As such, the application is not limited to the described implementations and alterations and modifications can be made to the described implementations without departing from the spirit or scope of the application in its broadest form.

Claims

1. A culture medium combination for aseptic rapid propagation of Moringa oleifera buds into seedlings, characterized in that: include: Sterile axillary bud induction growth medium: 1 / 2MS, IBA 0.5-0.7 mg / L, NAA 0.15-0.25 mg / L, sucrose 20-25 g / L, agar 4.5-6.5 g / L, pH: 6.3-6.5; Differentiation cluster shoot induction medium: MS, 6BA 0.5-0.8 mg / L, NAA 0.05-0.1 mg / L, IAA 0.05-0.1 mg / L, TDZ 0.003-0.008 mg / L, sucrose 25-30 g / L + agar 4.5-6.5 g / L, pH: 6.3-6.5; Stable differentiation cluster bud culture medium: MS, 6BA 0.5-0.7 mg / L, NAA 0.03-0.07 mg / L, sucrose 25-30 g / L, agar 4.5-6.5 g / L, pH: 6.3-6.5; Rooting medium: 1 / 2MS, lBA0.1~0.6mg / L, NAA0.05~0.2mg / L, sucrose 15~20g / L+agar 6~7g / L, fresh seven-hole lotus root 8~12g / L, pH; 5.8～6.0； And stable rooting medium: 1 / 2MS, IBA0.1-0.6mg / L, NAA0.05-0.2mg / L, sucrose 15-20g / L, agar 5-7g / L, fresh seven-hole lotus root 8-12g / L, charcoal 0.5-0.6g / L, pH: 5.6-5.8; The culture medium is combined with tender shoot stem segments as explants.

2. The culture medium combination according to claim 1, wherein include: Sterile axillary bud induction growth medium: 1 / 2MS, IBA 0.6 mg / L, NAA 0.2 mg / L, sucrose 20 g / L, agar 5 g / L, pH: 6.5; Differentiation cluster shoot induction medium: MS, 6BA 0.6 mg / L, NAA 0.1 mg / L, IAA 0.1 mg / L, TDZ 0.003 mg / L, sucrose 25 g / L + agar 5 g / L, pH: 6.5; Stable differentiation cluster shoot medium: MS, 6BA 0.5 mg / L, NAA 0.03 mg / L, sucrose 30 g / L, agar 5 g / L, pH: 6.5; Rooting medium: 1 / 2MS, 1BA0.2 mg / L, NAA0.1 mg / L, sucrose 20 g / L + agar 6.5 g / L, fresh seven-hole lotus root 8-9 g / L, pH; 5.8； And stable rooting medium: 1 / 2MS, IBA0.2 mg / L, NAA0.1 mg / L, sucrose 20g / L, agar 6g / L, fresh seven-hole lotus root 8-9g / L, charcoal 0.6g / L, pH: 5.

6.

3. A culture method using the culture medium combination according to claim 1 or 2, characterized in that: The following steps are involved: (1) Explant collection and processing: Moringa oleifera shoot stem segments were selected as explants; (2) Transfer culture to obtain sterile axillary buds; (3) Sterile axillary buds differentiate into clustered buds to obtain stable clustered buds; (4) inoculating and cultivating the stable clustered buds to obtain stable rooted seedlings; (5) Prepare transplanting medium; (6) Hardening and transplanting of seedlings; (7) Seedling domestication and management.

4. The culture method according to claim 3, wherein Step (1) specifically comprises: selecting a stem segment with tender buds as an explant; removing leaves, cutting into 3-4 cm stem segments with axillary buds, washing, taking out all single buds, and cutting off 0.5-0.7 cm at the incision, sterilizing, and obtaining the sterilized stem segment with axillary buds for later use; The cleaning method is as follows: put the stem segment with axillary buds into a small sieve basin and use the water to continuously roll and wash for 20 minutes, add 10g of washing powder to 0.8L of water and rinse for 3 to 5 minutes, and rinse under tap water for 40 minutes; The disinfection was as follows: sterilize with 75% alcohol for 30 seconds, rinse with sterile water twice, sterilize with 0.1% mercuric chloride for 8 minutes, rinse with sterile water three times and then filter dry.

5. The culture method according to claim 4, wherein Step (2) is specifically as follows: the stem segment with axillary buds disinfected in step (1) is inoculated onto a sterile axillary bud induction growth medium, the culture conditions are 10 hours of light per day, a temperature of 25-27°C, and a light intensity of 1500-2000lx, and sterile axillary buds are grown after inoculation and culture for 20-23 days.

6. The culture method according to claim 5, wherein Step (3) is specifically as follows: cutting the sterile axillary buds into 1-1.5 cm segments, shallowly inserting the lower end of the morphologically defined buds in a differentiation cluster bud induction medium at an angle; cultivating to obtain cluster buds; inoculating the cluster buds into a stable differentiation cluster bud medium, and cultivating to obtain stable cluster buds; The culture conditions are: 10 hours of light per day, a temperature of 25-27° C., a light intensity of 1500-2000 lx, and inoculation and culture for 20-23 days.

7. The culture method according to claim 6, wherein Step (4) is specifically as follows: inoculating the stable clustered buds of more than 3 cm into the rooting medium to cultivate rooted seedlings; when the rooted seedlings grow to 8 to 10 cm, taking a bud section of the middle and upper stem buds and inoculating them into the stable rooting medium, with multiple single bud sections per bottle, to cultivate stable rooted seedlings; The culture conditions are: 10 hours of light per day, a temperature of 25-27° C., a light intensity of 1500-2000 lx, and a culture period of 20-23 days.

8. The culture method according to claim 7, wherein Step (5) is specifically as follows: 10 days before transplanting, a matrix is ​​prepared in a greenhouse according to a weight ratio of garden soil: fine sand: decomposed nut skin of 5:3:2, mancozeb is mixed with the matrix at a weight ratio of 1:3000, and stirred evenly to prepare a transplanting matrix, which is then sterilized and packed in nutrient bags, and neatly arranged in rows on a seedbed in a light-transmitting and moisture-retaining greenhouse, and watered thoroughly for use.

9. The culture method according to claim 8, wherein Step (6) is specifically as follows: the stably rooted seedlings are moved to an indoor seedling hardening rack and cultured under natural light for 4 days, wherein the bottle caps are completely loosened and not opened on the first day, and 1 / 3 of the caps are opened on the second, third, and fourth days for hardening; Transplanting: 3 days before planting, water the transplanting medium in step (5) thoroughly, and turn the transplanting medium over one day before planting for later use; Transplanting time: After 17:00 in the afternoon, plant the seedlings to 100 plants, and promptly water them with 500 times diluted mancozeb solution for rooting disinfection, the amount being 100 ml / bag. After planting, cover the plants with a moisture-retaining shed to obtain tissue culture seedlings; Step (7) is specifically as follows: placing the tissue culture seedlings in a light-transmitting and moisture-retaining shed with the shed fully covered for 7 days and then half-opening the shed to 1 / 5, with an acclimation temperature of 22-34° C. and a relative humidity of 75%-90%; Among them, spray clean water once every day at 8:00-9:00 in the morning and 17:00-18:00 in the afternoon for the first 5 days, and irrigate the bottom of the nutrient bag with water once on the 6th day; 10 days after transplanting, new roots begin to grow, new leaves begin to grow after 15 days, and old leaves tend to stabilize. The greenhouse is fully opened to increase light transmittance and do a good job of fertilizer and water management in the later stage. Transplanting in the greenhouse is done 60 days before planting in the field.

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