A tissue culture rapid propagation method of dictamnus dasycarpus turcz

Abstract

The application provides a tissue culture rapid propagation method of Dictamnus dasycarpus Turcz, which directly induces the cluster buds by taking the seeds of the Dictamnus dasycarpus Turcz as explants, the explants can sprout on the culture medium for 7-10 days, and a large number of cluster buds can be generated for 20-25 days, so that the growth cycle and seedling time are greatly shortened. The existing tissue culture method mainly includes the callus induction, sprout induction, bud proliferation and rooting induction stages. The application directly induces the cluster buds without the callus induction and bud proliferation stages, so that the process is simplified, and the regeneration efficiency and propagation coefficient are improved. The traditional seed propagation can only be carried out in April-May or October-November every year, in addition, the seeds of the Dictamnus dasycarpus Turcz need to be stored in low temperature sand for several months to sprout, and the tissue culture rapid propagation seeds adopting the application do not need low temperature treatment, and can realize the year-round production in the room.

Description

Technical Field

[0001] This invention belongs to the field of plant regeneration technology through tissue culture, specifically relating to a rapid propagation method for Dictamnus dasycarpus through tissue culture. Background Technology

[0002] Dictamnus dasycarpus Turcz. is a perennial herb belonging to the genus Dictamnus in the family Dictamnusaceae, and it has high medicinal value. The dried root bark of Dictamnus dasycarpus is called Dictamnus root bark, and it is mainly used to treat skin itching, eczema, impetigo, and rheumatism, and is a commonly used traditional Chinese medicine in clinical practice. In addition, the main chemical components of Dictamnus dasycarpus include alkaloids, limonenes, flavonoids, and their glycosides, which can not only treat various diseases but also be used as a pollution-free pesticide. Currently, the Dictamnus root bark supplied in the market is mainly from wild Dictamnus dasycarpus. However, because the recovery period of wild Dictamnus dasycarpus is long, relying solely on wild resources would cause serious damage to wild Dictamnus dasycarpus resources and would be insufficient to meet current demand.

[0003] Currently, the common propagation methods for Dictamnus dasycarpus are sowing and division. However, because Dictamnus dasycarpus seeds undergo physiological after-ripening, requiring several months of low-temperature sand stratification to break dormancy, and because division has a low propagation coefficient and limited quantity, it is rarely used in production. Plant tissue culture can achieve industrialized production of seedlings using small amounts of material in a short time, thus meeting the demand for Dictamnus dasycarpus. Therefore, research on rapid propagation technology of Dictamnus dasycarpus through tissue culture is of great significance for the preservation of Dictamnus dasycarpus germplasm resources, the protection of wild resources, and meeting the needs of traditional Chinese medicine.

[0004] Currently, in vitro culture of Dictamnus dasycarpus mainly uses young stem segments and buds as explants for rapid propagation, with seeds rarely used. The number of young stem segments and buds as explants is extremely limited, far fewer than that of seeds. Using seeds as explants involves callus induction and differentiation, adventitious bud proliferation, and rooting stages, making it impossible to directly induce clustered buds without going through the callus formation stage. This results in a long culture cycle and a low proliferation coefficient. Furthermore, because the process of callus redifferentiation into buds is lengthy, its genetic material may be altered, which is detrimental to the preservation of Dictamnus dasycarpus germplasm resources. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a rapid tissue culture propagation method for Dictamnus dasycarpus, directly inducing shoot clusters using Dictamnus dasycarpus seeds as explants. The induction rate of this method reaches over 93%, the proliferation coefficient reaches over 7.82 times, and the shortest time from seed to shoot cluster formation is only 20-25 days, enabling the rapid acquisition of genetically stable Dictamnus dasycarpus seedlings.

[0006] The rapid propagation method of Dictamnus dasycarpus by tissue culture provided by the present invention uses Dictamnus dasycarpus seeds as explants, removes the seed coat, and sterilizes them with a disinfectant; after sterilization, the bottom part of the seeds is cut off and placed on a budding induction medium to induce clustered buds; the induced buds are inoculated in a rooting medium and cultured until roots are formed.

[0007] As one embodiment, the disinfection steps for Dictamnus dasycarpus seeds are as follows: first, disinfect with 75% ethanol for 30 seconds, then rinse with sterile water, and then disinfect with 2% sodium hypochlorite for 18 minutes, followed by rinsing with sterile water.

[0008] The germination medium is MS medium supplemented with 3% sucrose (g / mL), 0.7% agar (g / mL), 1.5 mg / L 6-BA and 0.5 mg / L NAA.

[0009] Furthermore, the explants are cultured on a budding induction medium for 20-25 days until clustered buds are produced.

[0010] The rooting medium is a 1 / 2 MS medium supplemented with 3% sucrose (g / mL), 0.7% agar (g / mL), and 0.4 mg / L IBA.

[0011] Furthermore, the adventitious buds were cultured on a budding induction culture medium for 30 days to obtain rooted tissue culture seedlings of Dictamnus dasycarpus.

[0012] Furthermore, the culture conditions for inducing and rooting the clustered shoots are: 24±1℃, 16 / 8h photoperiod, and light intensity of 2000lx.

[0013] This invention uses Dictamnus dasycarpus seeds as explants to directly induce shoot clusters. Explants germinate in 7-10 days on the culture medium, and produce a large number of shoot clusters in 20-25 days, significantly shortening the growth cycle and seedling time (as short as 4-5 weeks). Existing tissue culture methods mainly involve callus induction, shoot induction, shoot proliferation, and rooting induction stages. This invention directly induces shoot clusters without callus induction and shoot proliferation stages, simplifying the process and improving regeneration efficiency and propagation coefficient. Traditional seed propagation can only be carried out in April-May or October-November each year. Furthermore, Dictamnus dasycarpus seeds require several months of low-temperature sand stratification to germinate. The tissue culture rapid propagation method of this invention does not require low-temperature treatment and can achieve year-round indoor production. In addition, traditional seed propagation is slow, has a low propagation coefficient, and produces limited quantities. The tissue culture rapid propagation method of this invention can obtain a large number of offspring in a short period with stable traits. Attached Figure Description

[0014] Figure 1 This is a photograph of the clustered shoots induced by Dictamnus dasycarpus seeds in an embodiment of the present invention.

[0015] Figure 2 This is a photograph of the early stage of root induction in tissue culture seedlings of Dictamnus dasycarpus in an embodiment of the present invention.

[0016] Figure 3 This is a photograph of the rooting condition after 30 days of cultivation in the rooting medium in an embodiment of the present invention. Detailed Implementation

[0017] The abbreviations used in this invention are explained below:

[0018] Table 1: List of English Abbreviations

[0019]

[0020]

[0021] The present invention will be further explained and described below with reference to figures and embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may use the following technical content to make changes or modifications to obtain equivalent embodiments.

[0022] Example 1

[0023] Select plump, white seeds, peel off the black seed coat, wash them with running water, and place them in a clean conical flask on a laminar flow hood. Sterilize with 75% ethanol for 30 seconds, gently shaking the flask during sterilization. Rinse twice with sterile water, then sterilize in 2% sodium hypochlorite solution for 18 minutes, gently shaking the flask during sterilization. Rinse 5-6 times with sterile water and place on sterile filter paper to absorb surface moisture.

[0024] Cut off the base of the sterilized Dictamnus dasycarpus seeds, leaving the tip, and inoculate them into the following shoot induction medium (MS + 3% sucrose + 0.7% agar + 1.5 mg / L 6-BA + 0.5 mg / L NAA, pH = 5.8) and culture for 20-25 days. The culture conditions are 24±1℃, 16 / 8h photoperiod, and 2000 lx light intensity.

[0025] The induced 2-3 cm clustered buds were cut off from the base and divided into individual seedlings. They were then transferred to rooting medium (1 / 2 MS + 3% sucrose + 0.7% agar + 0.4 mg / L IBA, pH = 5.8) and cultured at 24±1℃, 16 / 8h photoperiod, and 2000 lx light intensity. Rooted Dictamnus dasycarpus tissue culture seedlings were obtained after 30 days and could then be domesticated and transplanted.

[0026] Comparative Example 1: Conventional Tissue Culture Method for Dictamnus dasycarpus

[0027] Remove the seed coat of Dictamnus dasycarpus, select mature and plump seeds, rinse with 75% ethanol for 1 minute, then disinfect in 1% mercuric chloride solution for 8 minutes, rinse 5 times with sterile water, and place on sterile filter paper to absorb surface moisture to complete the disinfection of Dictamnus dasycarpus seeds.

[0028] Sterile Dictamnus dasycarpus seeds are inoculated into callus induction medium and cultured for about 10 days to form yellow-green callus tissue with a callus emergence rate of up to 80%. The formula of the callus induction medium is: MS + 0.5-4.0 mg / L 6-BA + 0.2-2.0 mg / L NAA, pH = 5.8.

[0029] Cut the callus tissue into 0.5cm pieces. 2 Small pieces of tissue, when inoculated onto callus differentiation medium and cultured for 20 days, can form adventitious shoots with a differentiation rate of over 79%. The differentiation medium formula is: MS + 0.2-2.0 mg / L KT + 0.5-4.0 mg / L NAA, pH = 5.8.

[0030] Using the differentiated adventitious buds as experimental material, they were cut into 1-2 cm sections, each with 3-4 cm axillary buds. After inoculating them onto the proliferation medium and culturing for 20 days, vigorous adventitious buds were obtained with a proliferation coefficient of 4.75. The proliferation medium formula was: MS + 0.5-4.0 mg / L 6-BA + 2.0 mg / L NAA, pH = 5.8.

[0031] Cut vigorous adventitious buds into sections of about 3 cm and inoculate them onto adventitious bud rooting medium to induce rooting. A large number of roots can grow in 30 days. The rooting medium formula is: 1 / 2 MS + 0.2-1.0 mg / L NAA + 0.5-1.5 mg / L IAA, pH=5.8.

[0032] Comparing the method of this invention with the method described in the comparative example, firstly, the method of this invention simplifies the tissue culture process, mainly including two stages: shoot induction and rooting culture, while the comparative example's culture procedure includes four stages: callus induction, shoot induction, proliferation induction, and rooting induction. Secondly, the method of this invention can achieve rapid shoot emergence by directly cutting off the bottom part of the seed and placing it on the shoot induction medium, which can obtain a large number of shoots in as little as 20 days. In contrast, the comparative example requires inducing callus from the seed, cutting the callus into pieces for differentiation induction, and then undergoing proliferation induction to obtain shoots, a process that takes about 50 days. In comparison, Example 1 greatly accelerates shoot emergence and shortens the seedling time.

[0033] Comparative Example 2: Effects of different hormone ratios on the induction of clustered shoots

[0034] The base of sterilized Dictamnus dasycarpus seeds was cut off, leaving the tip. These seeds were then inoculated into the following shoot induction medium (MS + 3% sucrose + 0.7% agar + 0-1.5 mg / L 6-BA + 0-1 mg / L NAA, pH = 5.8). Continuous observation and recording were performed for one month, with timely removal of contaminated explants. The medium was replaced with fresh medium approximately every 15 days. After culturing at 24±1℃, a 16 / 8h photoperiod, and a light intensity of 2000 lx for 30 days, the germination rate and adventitious shoot proliferation coefficient were calculated. Each treatment was replicated three times.

[0035] Adventitious shoot induction rate = number of shoot-producing explants / number of inoculated explants × 100%;

[0036] Proliferation coefficient = the sum of adventitious buds induced by each explant / the total number of explants.

[0037] Table 2: Effect of the composition of the bud induction medium on the induction of adventitious buds of Dictamnus dasycarpus.

[0038]

[0039]

[0040] Note: Different lowercase letters in the table represent differences at the 0.05 level, and the same applies below.

[0041] The experimental results of inducing adventitious buds in Dictamnus dasycarpus are shown in Table 2. After culturing on the budding medium for 20-25 days, a large number of vigorous clustered buds were formed, such as... Figure 2 As shown in Table 1, different culture medium components have significant effects on the germination rate and proliferation coefficient of Dictamnus dasycarpus. Among them, the MS medium + 3% sucrose + 0.7% agar + 1.5 mg / L 6-BA + 0.5 mg / L NAA showed the best effect, with an adventitious bud induction rate of over 93% and an adventitious bud proliferation coefficient of 7.82.

[0042] Example 3: Effects of different hormone ratios on rooting induction

[0043] When the bud clusters reached 2-3 cm in length, they were cut off from the base and separated into individual seedlings. These seedlings were then transferred to rooting medium (1 / 2 MS + 3% sucrose + 0.7% agar + 0.2-0.6 mg / L IBA and 1 / 2 MS + 3% sucrose + 0.7% agar + 0.2-0.6 mg / L IAA, pH = 5.8) and cultured at 24±1℃, 16 / 8h photoperiod, and 2000 lx light intensity. After 30 days, the rooting rate and number of roots were recorded. Each treatment was replicated three times.

[0044] Table 3: Screening data for Dictamnus dasycarpus rooting medium

[0045]

[0046]

[0047] In the rooting induction of *Dictamnus dasycarpus*, rooting media containing different concentrations of IBA and IAA were designed. Table 3 shows a significant difference in rooting effects between *Dictamnus dasycarpus* tissue culture seedlings on media containing different concentrations of IAA and IBA. When IAA was added to the media as the rooting medium, the rooting rate of *Dictamnus dasycarpus* tissue culture seedlings was the highest at 58.02%, while the rooting rate of *Dictamnus dasycarpus* in media containing IBA reached as high as 83.78%. *Dictamnus dasycarpus* rooted on media containing different concentrations of IBA, with relatively ideal rooting effects of 80.37%, 83.78%, and 79.32%, respectively. Furthermore, in terms of the number of roots, the rooting effect of *Dictamnus dasycarpus* on media containing different concentrations of IAA was worse than that on media containing IBA. The highest number of roots was observed in media containing 0.4 mg / L IBA, reaching 7.36 or more. Therefore, the optimal rooting medium for *Dictamnus dasycarpus* was determined to be 1 / 2 MS + 3% sucrose + 0.7% agar + 0.4 mg / L IBA.

[0048] In summary, traditional seed propagation is slow, has a low propagation coefficient, and produces a limited number of offspring. In contrast, the tissue culture rapid propagation of this invention can produce a large number of offspring in a short period of time, and the offspring have stable traits.

Claims

1. A method for rapid propagation of Dictamnus dasycarpus through tissue culture, characterized in that, The method involves using Dictamnus dasycarpus seeds as explants, removing the seed coat, and sterilizing them with a disinfectant; after sterilization, the bottom part of the seeds is cut off, and the seeds are placed on a budding induction medium to induce shoot clusters; the induced shoots are inoculated into a rooting medium and cultured until roots are formed. The budding induction medium is MS medium supplemented with 3% sucrose, 0.7% agar, 1.5 mg / L 6-BA and 0.5 mg / L NAA; The rooting medium is a 1 / 2 MS medium supplemented with 3% sucrose, 0.7% agar, and 0.4 mg / L IBA.

2. The method as described in claim 1, characterized in that, The disinfection steps for Dictamnus dasycarpus seeds are as follows: first, disinfect with 75% ethanol for 30 seconds, then rinse with sterile water, and then disinfect with 2% sodium hypochlorite for 18 minutes. After disinfection, rinse with sterile water as well.

3. The method as described in claim 1, characterized in that, The explants were cultured on a budding induction medium for 20-25 days until they produced clustered buds.

4. The method as described in claim 1, characterized in that, The induced buds were inoculated into rooting medium and cultured for 30 days to obtain rooted tissue culture seedlings of Dictamnus dasycarpus.

5. The method as described in claim 1, characterized in that, The culture conditions for inducing and rooting shoots are as follows: 24±1℃, 16 / 8 h photoperiod, and light intensity of 2000 lx.

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