Culture medium for inducing adventitious buds of tagetes erecta and application thereof

Abstract

This invention discloses a culture medium for inducing adventitious shoots in marigolds and its application, relating to the field of plant tissue culture technology. The culture medium consists of 1 / 2 MS + 0.5 mg / L indoleacetic acid + 3.5 mg / L 6-benzylaminopurine + 30 g / L sucrose + 6 g / L agar. This invention significantly improves the efficiency of adventitious shoot induction and the quality of tissue culture seedlings from marigold explants by optimizing the concentration of indoleacetic acid and 6-benzylaminopurine. Using the culture medium provided by this invention, the browning rate of explants can be effectively reduced, and the differentiation rate and survival rate of adventitious shoots can be increased. Robust adventitious shoots can be obtained after 25 days of culture. The resulting tissue culture seedlings have dark green, well-developed leaves without yellowing or vitrification, a well-developed root system with a strong taproot, dense lateral roots with abundant branching, and strong transplant adaptability. The culture medium of this invention has simple components, low cost, high stability, and good reproducibility, making it suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a culture medium for inducing adventitious shoots of marigolds and its application. Background Technology

[0002] marigold( Tagetes erecta L Marigold (Maburoides) is an annual herbaceous plant belonging to the genus Marigold in the family Asteraceae. It possesses ornamental, medicinal, and economic value, and is widely used in landscaping, potted plant production, pigment extraction, and ecological restoration. With the continuous expansion of market demand, traditional seed propagation methods are limited by factors such as season, climate, and pollination purity, resulting in problems such as seed-seed trait segregation, poor uniformity, long propagation cycles, and low propagation efficiency, making it difficult to meet the needs of large-scale, standardized production. Plant tissue culture technology can obtain a large number of genetically stable sterile seedlings in a short time, which is an important technical approach to achieve efficient and rapid propagation of marigolds and has become a core supporting technology for modern horticultural breeding and industrialized production.

[0003] Currently, several technical bottlenecks remain in the tissue culture process of marigolds: First, severe browning of explants, especially leaves and young stem segments, is prone to oxidative browning during cutting and culture, leading to tissue necrosis and induction failure; second, the efficiency of adventitious bud induction is unstable, with different types, concentrations, and ratios of hormones significantly affecting differentiation, and conventional hormone combinations often resulting in low bud differentiation rates, weak seedling growth, and frequent vitrification; third, poor rooting quality, with induced adventitious buds exhibiting problems such as thin roots, sparse lateral roots, and low transplant survival rates, hindering the industrial application of tissue culture seedlings.

[0004] In existing technologies, culture media used for inducing adventitious shoots in marigolds mostly employ single cytokinins or auxins, or use conventional concentration ratios, lacking highly efficient compound systems specifically for marigold explants. Meanwhile, large-scale production places higher demands on the versatility, stability, and low cost of culture media. Some existing culture media have complex compositions, high hormone costs, and cumbersome operating procedures, which are not conducive to the promotion of industrialized seedling production. Therefore, developing a marigold adventitious shoot induction culture medium with simple components, precise hormone ratios, high induction efficiency, low browning rate, and good rooting effect, and establishing a standardized and efficient tissue culture method, is of significant practical importance for solving the shortage of high-quality marigold seedlings and improving propagation efficiency and seedling quality. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium for inducing adventitious shoots in marigolds and its application, thereby solving the problems existing in the prior art. The culture medium provided by this invention can significantly improve the efficiency of adventitious shoot induction from marigold explants and the quality of tissue culture seedlings.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a culture medium for inducing adventitious shoots of marigolds, wherein the culture medium is 1 / 2 MS + 0.5 mg / L indoleacetic acid + 3.5 mg / L 6-benzylaminopurine + 30 g / L sucrose + 6 g / L agar.

[0007] The present invention also provides a method for preparing the above-mentioned culture medium for inducing adventitious shoots of marigolds, comprising the following steps: Dissolve 1 / 2 MS, sucrose, and agar in water, adjust the pH, and sterilize to obtain the basal culture medium. After the basal culture medium cools, add indoleacetic acid and 6-benzylaminopurine and mix well to obtain the culture medium for inducing marigold adventitious shoots.

[0008] Furthermore, the pH value is adjusted to 5.8–6.0.

[0009] The present invention also provides an application of the above-mentioned culture medium, wherein the application is to induce adventitious buds in explants during marigold tissue culture.

[0010] Furthermore, the explants are marigold leaves and / or stem segments containing axillary buds.

[0011] The present invention also provides a method for inducing adventitious shoots from explants in marigold tissue culture, comprising the step of inducing adventitious shoots from explants using the above-mentioned culture medium for inducing adventitious shoots in marigolds.

[0012] Furthermore, the explants are marigold leaves and / or stem segments containing axillary buds.

[0013] Furthermore, the culture conditions for the adventitious bud induction culture are: temperature 23-25℃, light intensity 2000-2500 lx, and photoperiod 12 hours / day.

[0014] The present invention discloses the following technical effects: This invention significantly improves the efficiency of adventitious bud induction and the quality of tissue culture seedlings from marigold explants by optimizing the concentration of IAA and 6-BA. Using the culture medium provided by this invention, the browning rate of explants can be effectively reduced, and the differentiation and survival rates of adventitious buds can be increased. Robust adventitious buds can be obtained after 25 days of culture. The resulting tissue culture seedlings have dark green, well-developed leaves without yellowing or vitrification, a well-developed root system with a strong taproot, dense lateral roots, and abundant branching, exhibiting strong transplant adaptability.

[0015] The culture medium of this invention has simple components, low cost, high stability, and good reproducibility, making it suitable for large-scale production. Compared with conventional techniques, the adventitious bud induction cycle is shortened, the effective seedling rate is significantly improved, and problems such as severe browning, unstable differentiation, and poor rooting are solved. It provides a stable and reliable technical solution for the efficient tissue culture and rapid propagation of marigolds, and can meet the industrial seedling needs of landscaping, pigment extraction, and other applications. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 An observation diagram of marigold explant culture; Figure 2 Comparison of the adventitious bud induction effect of marigold under different hormone ratios; where A and J are observation images of the adventitious bud induction effect of marigold in treatment groups 1-10 respectively. Figure 3 An observation of the rooting culture of marigold tissue culture seedlings under the optimal hormone ratio; Figure 4 This is an observation image of marigold tissue culture seedlings under the optimal hormone ratio. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1 1. Culture medium preparation: Preparation of basal culture medium: Based on 1 / 2 MS medium, add 30 g / L sucrose and 6 g / L agar, and stir thoroughly to ensure complete dissolution of all components. Adjust the pH of the medium to 5.8–6.0 with 1 mol / L NaOH solution or 1 mol / L HCl solution, mix thoroughly again, and sterilize at 121°C under autoclave for 20 min. After sterilization, remove and cool in a sterile environment for later use.

[0024] Preparation of adventitious bud induction medium: When the above basic medium is cooled to 50-60℃ (avoid high temperature destroying hormone activity), add hormones of different final concentrations as shown in Table 1. Under aseptic conditions, add and quickly and gently mix, then dispense into aseptic culture containers. After cooling and solidification, the adventitious bud induction medium is obtained. Each group is repeated three times.

[0025] Table 1 Hormone Ratio Table 2. Inducing adventitious buds in marigolds 2.1 Seed disinfection and acquisition of sterile seedlings Healthy, plump seeds of the marigold variety "Te-2" were selected and subjected to explant disinfection treatment: the seeds were successively disinfected by soaking in 5% sodium hypochlorite solution and then in 75% ethanol. After disinfection, the seeds were repeatedly rinsed with sterile water to obtain sterile seeds. These sterile seeds were inoculated into 1 / 2 MS basal medium and cultured under suitable conditions to obtain robust and uniformly growing marigold seedlings.

[0026] 2.2 Explant wound creation Select healthy, vigorous, and well-grown sterile seedlings obtained through the above culture. In a clean bench, cut tender, fresh, and undamaged healthy leaves. Using a sterile scalpel, gently make several tiny incisions on the leaf surface (avoiding excessive damage or breakage of the leaf tissue). Simultaneously, cut robust stem segments into small sections approximately 0.5 cm in length, ensuring each section contains 1-2 axillary buds to guarantee subsequent culture activity (e.g., ...). Figure 1(As shown).

[0027] 2.3 Adventitious bud induction culture The cut leaf explants and 0.5 cm stem segment explants were inoculated into a culture medium containing 10 different combinations of hormone concentrations and cultured under the following conditions: temperature 23-25℃, light intensity 2000-2500 lx, and photoperiod 12 hours / day.

[0028] After about 25 days, observe the germination of new shoots and record the growth status of explants under different hormone combinations.

[0029] After 25 days of cultivation, the germination rate and plant growth were statistically analyzed and compared under 10 treatments with different hormone concentrations. The results are shown below. Figure 2 See Table 2. The results showed that different hormone ratios had significant effects on the growth status of tissue culture seedlings. Treatment group 2 performed best, with dark green and well-developed roots and the lowest browning rate. Treatment groups 1 and 5 were next, with a certain proportion of browning or differentiation inhibition. Treatment groups 3, 4, and 6-10 performed poorly, with fewer surviving seedlings, severe tissue browning and necrosis, and some treatments had almost no induction effect.

[0030] Table 2 Comparison and analysis of growth status of each group Marigold shoots cultivated under hormone No. 2 treatment were transferred to basal culture medium for root induction culture. After a period of culture, white adventitious roots began to appear at the base of the marigold seedlings. The root system was robust and evenly distributed, with the main root length being approximately 4-5 cm and the total number of lateral roots per plant exceeding 20. The lateral root density was 5-6 roots / cm per unit length of the main root. The lateral roots were distributed along the entire length of the main root and showed a large number of secondary branches. The plants exhibited good overall growth, reaching a height of approximately 10-12 cm. The leaves were fully expanded and dark green, without obvious yellowing, browning, or vitrification. Figure 3 As the number of cultivation days increases, the number of roots continues to increase. Figure 4 The plants grew stably and showed good rooting effects, which provided a guarantee for subsequent domestication and transplanting.

[0031] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A culture medium for inducing adventitious shoots of marigolds, characterized in that, The culture medium consisted of 1 / 2 MS + 0.5 mg / L indoleacetic acid + 3.5 mg / L 6-benzylaminopurine + 30 g / L sucrose + 6 g / L agar.

2. A method for preparing a culture medium for inducing adventitious shoots of marigolds as described in claim 1, characterized in that, Includes the following steps: Dissolve 1 / 2 MS, sucrose, and agar in water, adjust the pH, and sterilize to obtain the basal culture medium. After the basal culture medium cools, add indoleacetic acid and 6-benzylaminopurine and mix well to obtain the culture medium for inducing marigold adventitious shoots.

3. The preparation method according to claim 2, characterized in that, The pH value is adjusted to 5.8–6.

0.

4. An application of the culture medium as described in claim 1, characterized in that, The application is to induce adventitious buds in explants during marigold tissue culture.

5. The application according to claim 4, characterized in that, The explants are marigold leaves and / or stem segments containing axillary buds.

6. A method for inducing adventitious shoots from explants in marigold tissue culture, characterized in that, The method includes the step of inducing adventitious shoots in explants using the culture medium for inducing adventitious shoots of marigolds as described in claim 1.

7. The method according to claim 6, characterized in that, The explants are marigold leaves and / or stem segments containing axillary buds.

8. The method according to claim 6, characterized in that, The culture conditions for the adventitious bud induction culture are: temperature 23-25℃, light intensity 2000-2500 lx, and photoperiod 12 hours / day.

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