CN114617062B

CN114617062B - Tissue culture and rapid propagation method for crocodile flower

Info

Publication number: CN114617062B
Application number: CN202210113530.1A
Authority: CN
Inventors: 王文莉; 王鹏; 陈丹凌; 沈向
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2022-01-30
Filing date: 2022-01-30
Publication date: 2022-11-01
Anticipated expiration: 2042-01-30
Also published as: ZA202202662B; CN114617062A

Abstract

The invention discloses a tissue culture and rapid propagation method of crocodile flowers, and belongs to the technical field of plant tissue culture. The method comprises the following steps: (1) explant selection and sterilization: sterilizing the regenerated tender branches serving as explants; and (2) axillary bud growth induction: cutting the disinfected twig into stem sections, inoculating the stem sections into an induction culture medium, and inducing the growth of axillary buds; (3) cluster bud induction and subculture: cutting lateral branches obtained by axillary bud growth into small segments, inoculating the small segments into a differentiation culture medium, inducing the growth of cluster buds, and inoculating the obtained cluster buds into the differentiation culture medium for subculture; (4) rooting culture: cutting off cluster buds obtained by subculture, and inoculating the cluster buds into a rooting culture medium for culturing to obtain rooted seedlings; (5) hardening and transplanting seedlings: and (4) hardening the obtained rooted seedlings for 3d, transplanting to a sand bed, and culturing. The invention establishes a rapid, stable and effective crocodile flower rapid propagation technical system, can be effectively applied to production, and provides scientific basis and technical guarantee for development and utilization of crocodile flowers in large-scale production in the future.

Description

Tissue culture and rapid propagation method for crocodile flower

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a crocodile tissue culture rapid propagation method.

Background

Alligator flower [ Clinacanthus nutans (burm.f.) Lindau ] is a plant of the Acanthaceae alligator genus of the Acanthaceae family (Acanthaceae family) which is a tall and erect herb, also known as clinaca nutgrass, sambucus chinensis, and is distributed in south China tropical and south east asia countries. The crocodile flower contains higher flavonoid substances, including betulin, pentacyclic triterpene compound lupeol, isovitexin, etc. The whole plant can be used as a medicine, has the effects of regulating menstruation, reducing swelling, removing blood stasis, diminishing inflammation, relieving pain and setting bone, has the effects of treating traumatic injury, anemia, jaundice, rheumatism, reducing blood fat and the like, and has a good anticancer effect. In addition, in the areas of Hainan, yunnan and the like in China, tender stems and leaves of alligator crocodile are generally eaten as leaf vegetables, and the alligator crocodile is a plant with homology of medicine and food.

With the continuous deepening of the understanding of the medicinal value and the edible value of the crocodile mouth, the commercial development of the crocodile mouth as a traditional medicinal plant and food material, the wild resources are far from meeting the current requirements, and the artificial propagation is urgently needed to meet the corresponding market requirements. At present, the propagation of alligator crocodile flowers mainly depends on cutting propagation, but the cutting propagation coefficient is low, cutting materials conflict with development and use parts, and the requirements of large-scale planting are difficult to meet due to the limitation of growth conditions such as low temperature resistance and the like. The tissue culture and the rapid propagation of the plants can provide a large number of excellent seedlings with consistent properties and growth in a short time, and a convenient way is provided for the large-scale production of the crocodile flower. In recent years, bihua Chen (2015), wangqiang (2018) and the like establish an in vitro rapid propagation system of alligator crocodile flowers by taking stem segments of the alligator crocodile flowers as explants. However, in the seedling strengthening link, organic matters need to be added, and in repeated experiments, cluster buds cannot be well generated in the cluster bud generation stage due to different seedling sources, so that the phenomena of callus generation and uneven growth of the cluster buds (or lateral buds) on the base parts are common. Therefore, a new crocodile flower tissue culture rapid propagation system is very necessary to establish.

Disclosure of Invention

The tissue culture rapid propagation method established by the invention can ensure that the transplanting survival rate of the alligator rosettes reaches 100%, can provide a large number of high-quality seedlings in a short period, and provides scientific basis and technical guarantee for development and utilization of the alligator rosettes in the future and large-scale production.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a tissue culture and rapid propagation method of crocodile flower, which comprises the following steps:

(1) Explant selection and sterilization: taking the new tender branches as explants, cleaning and then disinfecting;

(2) Axillary bud growth induction: cutting the disinfected twig into stem sections, inoculating the stem sections into an induction culture medium, and inducing the growth of axillary buds; the induction medium comprises: MS +6-BA0.5mg/L + NAA0.05mg/L;

(3) Cluster bud induction and subculture: cutting lateral branches obtained by axillary bud growth into small segments, inoculating the small segments into a differentiation culture medium, inducing the growth of cluster buds, and inoculating the obtained cluster buds into the differentiation culture medium for subculture; the differentiation medium comprises: MS + CPPU0.1mg/L + KT0.2mg/L + NAA0.05mg/L;

(4) Rooting culture: cutting off cluster buds obtained by subculture, and inoculating the cluster buds into a rooting culture medium for culturing to obtain rooted seedlings; the rooting medium comprises: 1/2MS + IBA0.2mg/L +2% sucrose or MS +6-BA0.1mg/L + KT0.05mg/L + NAA0.05mg/L +3% sucrose;

(5) Hardening and transplanting seedlings: and (4) hardening the obtained rooted seedlings for 3d, transplanting to a sand bed for culturing.

Further, in the step (1), before the cleaning operation, the tender branches are cut into 7-8cm stem segments.

Further, the cleaning in the step (1) is specifically that tap water is firstly washed, then the washing powder is used for soaking for 3-5 minutes, and then the running water is washed for 40min.

Further, the disinfection treatment in the step (1) is to wash the cleaned explant with sterile water for 2 times, absorb the moisture, disinfect the explant with 75% alcohol by volume fraction for 35-40s, then wash the explant with sterile water for 2 times, absorb the moisture, disinfect the explant with 2% sodium hypochlorite solution by mass percentage for 20-25min, and finally wash the explant with sterile water for 3 times, and absorb the moisture.

Further, the stem section in the step (2) is a small section with axillary buds, and the length of the small section is 1.5-2 cm.

Further, in the step (3), before the obtained lateral branches are inoculated into the culture medium, the lateral branches are cut into stem segments or top tips with the height of 0.6-1.5cm and at least one stem node.

Further, in step (4), before the rooting medium is inoculated, the subcultured multiple shoots are firstly divided into stem segments or top tips with the height of 1-1.5 cm.

Further, the rooting medium for stem segment access comprises: MS +6-BA0.1mg/L + KT0.05mg/L + NAA0.05mg/L +3% sucrose, the rooting culture medium for apical tip inoculation comprises: 1/2MS + IBA0.2mg/L +2% sucrose.

Further, the culture conditions in the step (5) are that 50% of shading is kept, the humidity is 85%, and the temperature is 20-30 ℃.

Further, the tissue culture conditions in the steps (1) to (4) are that the temperature is 26 +/-1 ℃, the illumination time is 13.5h/d, and the illumination intensity is 2500lx.

The invention discloses the following technical effects:

the method takes the regenerated tender branches of the crocodile flower as explants, hormones with different types and concentrations are added into different combined culture media to carry out tissue culture and rapid propagation tests, and the crocodile flower tissue culture and rapid propagation method is researched and optimized by researching the influence of different plant growth regulators on the axillary bud induction, the cluster bud differentiation, the proliferation and the rooting culture of the stem segments of the crocodile flower. The result shows that the culture medium of MS +6-BA0.5mg/L + NAA0.05mg/L is most beneficial to the induction and growth of axillary buds of the stem segment, and the induction rate is 90 percent; the culture medium MS + CPPU0.1mg/L + KT0.2mg/L + NAA0.05mg/L is the best culture medium for inducing differentiation of axillary buds of alligator buds, the differentiation rate is 100%, and the propagation coefficient is 5.02; the optimal culture medium of the top shoot and the stem section is different, the optimal culture medium of the top shoot rooting is 1/2MS + IBA0.2mg/L +2% sucrose, the rooting rate reaches 100%, the optimal culture medium of the stem section rooting is MS +6-BA0.1mg/L + KT0.05mg/L + NAA0.05mg/L +3% sucrose, the rooting rate reaches 100%, and the root system is developed; the clean river sand is used as a substrate for hardening seedlings and transplanting, and the survival rate of the crocodile flower is 100 percent. Therefore, the rapid propagation method of the crocodile flower establishes a rapid, stable and effective crocodile flower rapid propagation technical system, can be effectively applied to production, solves the problem of low propagation efficiency, can provide a large number of high-quality seedlings in a short period, and provides scientific basis and technical guarantee for development and utilization of the crocodile flower on a large-scale production in the future.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the state of rapid tissue culture and growth of the stem segments of alligator sinensis, in which FIGS. A-C show the induction of the stem segments of alligator sinensis in the culture medium MS +6-BA 1.0mg/L + NAA0.1mg/L at 10d,20d,30d, respectively; FIGS. D-F are graphs showing induction of crocodile flower at 10d,20d, and 30d in culture medium MS + CPPU0.1mg/L + KT0.2mg/L + NAA0.05mg/L, respectively; FIG. G shows the induction of crocodile flower in MS +6-BA3.0mg/L + NAA0.1mg/L medium for 30 days; FIG. H shows the rooting induction and growth conditions of crocodile flower shoot cultured in 1/2MS + IBA0.2+2% sucrose for 25 d; FIG. I shows the rooting induction and growth conditions of crocodile flower stem section cultured in MS +6-BA0.1mg/L + KT0.05mg/L + NAA0.05mg/L +3% sucrose for 25 days.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

1.1 test materials

The test material is taken from excellent wild plants collected from Wuzhishan mountain of Hainan province, the height of the collected plants is 72cm, the plants are dug together with roots, the plants are potted and maintained after the plants are cut horizontally at the position 15cm above the root neck, when new branches germinate and grow to 15-20cm, robust branches without diseases and insect pests on the upper parts are selected, leaves and tender tips are removed, and the branches are cut into small segments (7-8 cm). Washing with tap water, soaking in washing powder for 3-5 min, and washing with running water for 40min. Placing the cleaned stem segment of the crocodile mouth on a super-clean workbench, washing with sterile water for 2 times, absorbing surface moisture by sterile filter paper, sterilizing with 75% alcohol for 35-40s, washing with sterile water for 2 times, absorbing moisture by sterile filter paper, sterilizing with 2% sodium hypochlorite solution for 20-25min, washing with sterile water for 3 times, and absorbing moisture by sterile filter paper for later use.

1.2 culture conditions

The culture medium used in the test is MS and B5 culture medium, pH5.8, and is autoclaved at 121 deg.C for 15min; the culture temperature is 26 +/-1 ℃, the illumination time is 13.5h/d, and the illumination intensity is 2500lx.

1.3 test methods

1.3.1 Alligator flower Stem segment axillary bud Induction

The sterilized new shoots of the crocodile mouth flowers are cut into small sections of 1.5-2cm, each section is provided with a pair of axillary buds, the small sections are respectively inoculated on 4 culture media of different combined growth regulators (table 1), each bottle is inoculated with one branch, each group is inoculated with 20 stem sections, and the induction result is observed after 30 days (see figures 1A-C).

1.3.2 Cluster bud Induction and subculture

The lateral branches (about 3.5-4.5 cm) induced by axillary buds of crocodile mouth flowers are taken as materials, cut into segments or top tips with the length of 0.6-1.5cm and at least one stem node, and respectively inoculated into culture media (table 2) containing different growth regulator combinations, 40 seeds are inoculated into each group, 10 seeds are inoculated into each bottle, 4 bottles in total, and the process is repeated for 3 times. After 30 days, the induction and growth of multiple shoots were counted for each group (see FIGS. 1D-G). And (3) performing subculture after 30d, dividing the induced cluster buds into 2-3 buds per cluster, inoculating the buds to a subculture medium, culturing 10 clusters per bottle, and continuing to culture for 30d.

1.3.3 rooting culture

The cluster buds subcultured for 30d were divided, and sprouts (top shoots and stem segments) with a height of 1cm to 1.5cm were inoculated onto media of different minimal media and different combinations of growth regulators (table 3), 10 per bottle, 40 plants per group, and repeated 3 times. After 25 days, the rooting and growth were counted (see FIGS. 1H and I).

1.3.4 hardening off and transplanting

And (4) culturing 25d rooting bottle seedlings in a rooting culture medium, under the condition of semi-shading natural light in a greenhouse, unscrewing a bottle cap, and hardening the seedlings for 3d. And (4) after hardening off the seedlings, taking out the seedlings by using tweezers, and cleaning the culture medium. After cleaning, the seeds are transplanted to a disinfected sand bed. Keeping 50% shade, 85% humidity, 20-30 deg.C. After 10 days, the shading net is removed in the morning and evening, and 1500 times of the Huabao No. 2 fertilizer is sprayed and fertilized every 7 days.

2 results and analysis

2.1 crocodile flower test Material Sterilization

The newly born twigs of the crocodile flower are disinfected by a 2% sodium hypochlorite solution for 20-25min, the pollution rate is 23% after 30d, and is 6% compared with the pollution rate of a 0.1% mercury-mercuric chloride solution disinfected for 15min, the pollution rate is higher, but good aseptic materials of the crocodile flower can be obtained by two disinfection modes, and an aseptic propagation system is established. In consideration of toxicity and harmfulness of mercuric chloride, the test uses 2% sodium hypochlorite solution for disinfection for 20-25min.

2.2 Alligator flower Stem segment axillary bud Induction

Taking tender stem segments of alligator crocodile flowers as explants, and growing axillary buds on one side after 15 days in MS culture medium added with growth regulators with different concentrations. As shown in Table 1, the culture medium No. 3, namely MS +6-BA0.5mg/L + NAA0.05mg/L, is most beneficial to the induction and the elongation of axillary buds of stem segments, and has robust growth and the induction rate of 90 percent.

TABLE 1 Effect of different combination media on the Induction of axillary buds of young stems of Alligator

2.3 Cluster bud Induction and subculture

As can be seen from Table 2, both the B5 medium and the MS medium induced lateral buds or multiple buds in selection of the basal medium, and the MS medium was superior to the B5 medium in terms of overall growth and multiple bud development. On the growth regulator, the 6-BA + NAA combination and the 6-BA + KT + NAA combination can well induce lateral buds, but cluster buds are difficult to generate, firstly, only 1-2 lateral buds rapidly extend along with the growth of the lateral buds, and the phenomena of inhibiting the generation and growth of basal lateral buds appear; secondly, as the concentration of 6-BA increases (above 2.0 mg/L), the callus at the base part also grows larger, and beyond 30 days, the callus is browned and soft. The CPPU-containing culture medium has a remarkable effect on promoting the emergence of flower bud clusters of the alligator, and the lateral buds of the base can be observed to grow at 10 days, and cluster bud points appear at the bases of the newly grown lateral buds; at 20d dense clumpy buds appeared under the new lateral buds, but different combinations showed differences in the growth of clumpy buds. In a comprehensive view, MS, CPPU0.1mg/L, KT0.2mg/L and NAA0.05mg/L can well induce the cluster buds, the induction rate is 100%, the generation of the cluster buds and the elongation of the cluster buds are considered, no or few calluses are generated, and the formula can be used as an optimal combination formula for cluster bud induction and subculture. The proliferation rate averaged 5.02 per month by subculture.

TABLE 2 Effect of different combination media on Alligator floral bud Induction

2.4 rooting culture

Cutting the subcultured cluster buds, inoculating stem segments and terminal buds with the height of 1-1.5cm on rooting culture media with different combinations, and observing that white radicles grow on the stem segments at the lower parts of the crocodile flowers after 5 days. After 20 days, the rooting rate is 100%, the average root length exceeds 3.5cm, and the longest root length is 6cm. As can be seen from Table 3, the crocodile mouth peanut roots are relatively fast, and comprehensive analysis shows that the optimal rooting culture medium for the top shoots is 1/2MS + IBA0.2mg/L +2% of sucrose, and the optimal rooting culture medium for the stem segments is MS +6-BA0.1mg/L + KT0.05mg/L + NAA0.05mg/L +3% of sucrose. The 1/2MS culture medium added with the NAA and IBA mixed solution and the N6+ IBA culture medium have yellow upper leaves in the growth process and have poor effect.

TABLE 3 Effect of different combination media on Alligator peanut root induction

2.5 hardening off and transplanting

Taking out the rooted seedlings after seedling hardening for 3d, cleaning, and pruning old roots and overlength roots. Soaking in 1000 times of chlorothalonil or carbendazim solution for 10min, and planting in sterilized seedling hardening sand bed. The sand bed is made of common river sand, and larger particles need to be sieved. After 20 days, the survival rate of the rooted seedlings is 100 percent. After survival, the air humidity is gradually reduced until the air humidity is level with the outside, and the illumination is increased to the natural illumination intensity. After 40 days, the stem leaves grow rapidly, the average height is more than 8cm, the plants are robust, and then the transplanting can be carried out. The crocodile mouth flower has high seedling hardening survival rate on the premise of ensuring certain temperature and humidity. The river sand is used as a seedling hardening and transplanting substrate, is low in price and can be recycled, so that the production cost is reduced; meanwhile, the damage to the alligator flower root system is small during transplanting, and the planting and transplanting are facilitated.

Discussion 3

The crocodile mouth flower has strong erectness and few side branches, and has obvious top end advantages. In the cluster bud induction stage, it is difficult to induce new lateral buds at the base after the lateral buds have grown. In plant tissue culture, cytokinin can effectively break the apical dominance and is widely applied to the induction of lateral buds and cluster buds; the balance between the generation quantity of buds and the high growth speed can be well achieved by combining different ratios of cytokinin to auxin. In the aspect of growth regulator selection, in previous researches, the alligator flower bud cluster bud induction mainly adopts a combination of 6-BA and NAA (Bihua Chen (2015) and Wangqiang (2018)), and 1-2 lateral buds can be induced by low-concentration 6-BA; along with the increase of the concentration of 6-BA, when a plurality of lateral buds and even clump buds are induced, the base parts generate callus and grow and expand rapidly, simultaneously along with the prolonging of the period, 1-2 new buds in the clump buds grow rapidly, and other lateral buds are inhibited to grow slowly or even stop growing, so that effective clump buds cannot be formed, and the purposes of proliferation subculture and rapid propagation cannot be achieved.

CPPU (forchlorfenuron) is a phenylurea plant growth regulator with cytokinin activity, and can promote cell division, differentiation and expansion, promote organ formation and chlorophyll synthesis, break apical dominance, promote lateral bud growth, improve photosynthetic efficiency and prevent plant aging. CPPU biological activity is 10-100 times higher than 6-BA, and activity for inducing cell division and promoting organogenesis is far higher than that of general purine cytokinin. It is common in agricultural production for parthenocarpy and for the production of increased yields in root tubers and fruits. The invention shows that CPPU plays a key role in inducing the bud formation of the crocodile blossom buds, and can well break the apical dominance to generate the lateral buds. CPPU with the use concentration of 0.05mg/L-0.1mg/L can effectively induce the bud of the crocodile flower bud, and the problem of uneven growth in the callus and culture period in the induction process of the bud of the crocodile flower bud is effectively solved by combining low-concentration NAA. As CPPU concentration increased, more multiple shoots were produced by the alligator flower stem section, but grew slowly; as the NAA concentration increases, the cluster buds decrease, and the phenomenon that the 1-2 branches grow rapidly and other lateral buds are inhibited from growing occurs.

The crocodile peanut root is relatively easy, and in the past research, the crocodile peanut root only adopts a culture medium containing auxin. Practical experiments show that in a culture medium only containing auxin, a stem section with a terminal bud has inconsistent performance with a stem section without the terminal bud, the stem section with the terminal bud can rapidly take root and grow, lateral buds of the stem section without the terminal bud slowly germinate and rapidly grow only after the lateral buds grow, and the period is obviously longer than that of the stem section with the terminal bud. According to the invention, the low-concentration 6-BA and KT are combined with IBA, so that the stem section without the terminal bud can be quickly induced to generate the lateral bud, and the rooting length is high. The medium containing a low concentration of mitogen can significantly shorten the growth cycle and provide good rooting compared to the medium using only auxin.

The invention needs 85 days from the explant to the rooting seedling obtaining, and the rooting process can root in the fastest 15 days, so the rooting time can be shortened to 75 days. The invention can continuously transfer and repeat after obtaining the cluster buds, simultaneously carry out rooting and proliferation (can be used for rooting and can also be used for continuously transferring and proliferating after obtaining the cluster buds), each period is only 30d, the period is greatly shortened, and the proliferation multiple is obviously improved, thereby meeting the requirement of providing a large number of seedlings in a short period in production and having important significance. Meanwhile, the invention simultaneously utilizes the stem section without terminal bud to take root, fully utilizes the material and improves the utilization rate.

In the actual production, the shortening of the period means the reduction of the production cost and the improvement of the production efficiency, which has important significance on the tissue culture production of alligator flowers.

4 conclusion

The research shows that in the tissue culture of the crocodile flower, CPPU plays a key role in inducing the cluster buds at the stem section of the crocodile flower, and the MS culture medium combined with 0.1mg/L CPPU, 0.2mg/L KT and 0.05mg/LNAA is the optimal combination for inducing the cluster buds at the stem section of the crocodile flower; optimal rooting culture medium 1/2MS + IBA0.2mg/L +2% sucrose for stem segments with terminal buds, optimal rooting culture medium MS +6-BA0.1mg/L + KT0.05mg/L + NAA0.05mg/L +3% sucrose for stem segments without terminal buds. The research effectively establishes a new crocodile flower tissue culture rapid propagation system, particularly successfully induces and subcultures cluster buds, and provides effective technical support for large-scale production of crocodile flowers from explants to bottle seedlings with roots at the fastest speed of 85 days through proliferation subculture in each month, wherein the proliferation multiple is more than 5 times, and the research has important practical significance for large-scale production and preservation of seedlings in the north of Yangtze river.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. The tissue culture and rapid propagation method of alligator sinensis is characterized by comprising the following steps:

(2) Axillary bud growth induction: cutting the disinfected twig into stem sections, inoculating the stem sections into an induction culture medium, and inducing the growth of axillary buds; the induction culture medium is as follows: MS +6-BA0.5mg/L + NAA0.05mg/L;

(3) Cluster bud induction and subculture: cutting lateral branches obtained by axillary bud growth into small segments, inoculating the small segments into a differentiation culture medium, inducing the growth of cluster buds, and inoculating the obtained cluster buds into the differentiation culture medium for subculture; the differentiation medium is as follows: MS + CPPU0.1mg/L + KT0.2mg/L + NAA0.05mg/L;

(4) Rooting culture: before inoculating into rooting culture medium, firstly, cutting subcultured cluster buds into stem segments or top tips with the height of 1-1.5 cm; the rooting culture medium for stem segment inoculation is as follows: MS +6-BA0.1mg/L + KT0.05mg/L + NAA0.05mg/L +3% sucrose, and the rooting culture medium for apical tip inoculation is as follows: 1/2MS + IBA0.2mg/L +2% sucrose;

(5) Hardening and transplanting seedlings: and (4) hardening the obtained rooted seedlings for 3d, transplanting to a sand bed, and culturing.

2. The tissue culture and rapid propagation method of alligator sinensis as claimed in claim 1, wherein in step (1), the shoots are cut into 7-8cm stem segments before being cleaned.

3. The tissue culture and rapid propagation method of alligator sinensis as claimed in claim 1, wherein the cleaning in step (1) is specifically washing under tap water, then soaking for 3-5 minutes with washing powder, and then washing for 40min with running water.

4. The tissue culture and rapid propagation method for crocodile flowers according to claim 1, wherein the disinfection treatment in the step (1) comprises the steps of washing the cleaned explants with sterile water for 2 times, absorbing water, disinfecting with 75% alcohol by volume for 35-40s, washing with sterile water for 2 times, absorbing water, disinfecting with 2% sodium hypochlorite solution by mass for 20-25min, and finally washing with sterile water for 3 times, and absorbing water.

5. The tissue culture and rapid propagation method of alligator sinensis as claimed in claim 1, wherein the stem section in step (2) is a 1.5-2cm long section with axillary buds.

6. The tissue culture and rapid propagation method of alligator sinensis as claimed in claim 1, wherein in step (3), before the obtained lateral branches are inoculated into the culture medium, the lateral branches are cut into stem segments or terminal tips with the height of 0.6-1.5cm and at least one stem node.

7. The tissue culture and rapid propagation method of alligator sinensis as claimed in claim 1, wherein the culture conditions in step (5) are 50% shade, 85% humidity and 20-30 ℃.

8. The tissue culture and rapid propagation method of alligator sinensis as claimed in claim 1, wherein the tissue culture conditions in steps (1) to (4) are temperature

The illumination time is 13.5h/d, and the illumination intensity is 2500lx.