Rapid propagation rooting culture medium for peony seed tissue culture and culture method

Abstract

The invention discloses a rapid propagation rooting culture medium for peony seed tissue culture and a culture method, and belongs to the technical field of plant tissue culture. The rapid propagation rooting culture medium takes 1 / 2 MS as a basic culture medium, and further comprises the following components: ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulfate, monopotassium phosphate, a carbon source, kinetin and a curing agent. By adding kinetin into the culture medium, dependence of peony seed tissue culture seedlings on low-temperature treatment is broken, low-temperature pretreatment necessary in a traditional method is avoided, the peony seed tissue culture process is greatly simplified, the culture period is shortened, the rooting rate and the root strengthening rate are increased, the rooting rate is stabilized to be 95% or above, and the rooting rate is stabilized to be more than 95%. Technical support is provided for establishment of a new peony seed tissue culture and rapid propagation technology, and a material foundation is laid for promotion of peony seed tissue culture and rapid propagation factorization.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a rapid rooting culture medium and culture method for peony seed tissue culture. Background Technology

[0002] As an important ornamental and medicinal plant, peony tissue culture propagation technology is key to overcoming traditional propagation bottlenecks. However, existing technologies face severe challenges in peony tissue culture, especially in the in vitro rooting stage of seeds or varieties that are difficult to root, becoming a major obstacle to industrialization. These challenges are mainly manifested in the following ways: ① Low rooting efficiency: The rooting induction rate of conventional formulas for peony seedlings is usually less than 40%, and is extremely unstable (Zhang Qian et al., 2012). ② Long cycle: From inoculation to the formation of transplantable roots, it usually takes 60 to 90 days or even longer, resulting in high production costs (Liu Huichao et al., 2010). ③ Dependence on low-temperature treatment: Most effective methods require several weeks of vernalization treatment at low temperatures (around 4℃), which is cumbersome, increases equipment and energy costs, and is not easy to standardize (Cheng Fangyun et al., 2008). ④ Poor root quality and transplant survival rate: The induced roots are often weak and few in number, accompanied by callus tissue (Zhang Shaowei et al., 2017), resulting in extremely low survival rates during the transplant acclimatization stage. Therefore, developing a peony tissue culture rooting technology that can eliminate the need for low-temperature pretreatment, significantly shorten the rooting cycle, and achieve an extremely high rooting rate is of urgent practical significance for promoting the industrialization of peony. Summary of the Invention

[0003] The purpose of this invention is to provide a rapid rooting culture medium and method for peony seed tissue culture, thereby solving the problems existing in the prior art. This invention, by adding kinetin to the culture medium, breaks the dependence of peony seed tissue culture seedlings on low-temperature treatment, significantly shortening the rooting cycle and improving the rooting rate.

[0004] To achieve the above objectives, the present invention provides the following solution: This invention provides a rapid propagation and rooting culture medium for peony seed tissue culture. The rapid propagation and rooting culture medium is based on 1 / 2 MS medium and also includes the following components: basic medium, ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulfate, potassium dihydrogen phosphate, carbon source, kinetin, and curing agent.

[0005] Furthermore, based on the final concentration, the rapid propagation and rooting medium, with 1 / 2 MS as the basal medium, also includes the following components: ammonium nitrate 800-900 mg / L, potassium nitrate 900-1000 mg / L, calcium chloride 150-170 mg / L, magnesium sulfate 90-95 mg / L, potassium dihydrogen phosphate 80-90 mg / L, carbon source 25-40 g / L, kinetin 0.2-1.5 mg / L, and curing agent 3-5 g / L.

[0006] Optionally, the carbon source includes sucrose.

[0007] Optionally, the curing agent includes plant gel.

[0008] Furthermore, the pH value is 5.6-5.8.

[0009] The present invention also provides a method for rooting peony tissue culture seedlings, comprising the steps of inoculating peony seed embryos onto the above-mentioned rapid propagation and rooting culture medium, and performing tissue culture and inducing rooting.

[0010] Furthermore, the temperature for tissue culture is 22±1℃.

[0011] Furthermore, the optical density of the tissue culture is 50-55 μmol / (m²). 2 •s), the light cycle is 16 h of light / 8 h of darkness.

[0012] The present invention discloses the following technical effects: 1. Simplified process and reduced cost: The rapid propagation and rooting medium provided by this invention completely eliminates the low-temperature vernalization treatment process that lasts for several weeks, simplifies the operation process, significantly reduces equipment investment and energy consumption, and makes it easier to carry out large-scale and standardized production.

[0013] 2. The rooting cycle is greatly shortened: The complete rooting cycle of peony seed tissue culture seedlings is stably shortened from the traditional 60-90 days to about 50 days, and the production efficiency is increased by about 30%-50%.

[0014] 3. Breakthrough improvement in rooting rate: Using the culture method provided by this invention, the rooting rate of peony seed tissue culture seedlings can be stably maintained at over 95%, and can reach 100% in the best embodiment, solving the core problem of low and unstable rooting rate.

[0015] 4. Excellent root quality: The induced roots are robust and well-developed, without any deformities such as callus formation, laying a solid foundation for a high survival rate after transplanting. 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 1This is a schematic diagram of the rooting culture method for peony tissue culture seedlings of the present invention; wherein, A is a mature peony fruit; B is a mature seed pod; C is a complete seed; D is a complete embryo; E is embryo germination 7 days after inoculation; F is bud morphology 35 days after inoculation; Figure 2 Morphology of peony tissue culture seedlings 50 days after inoculation; Figure 3 The germination status of peony embryos in other hormone treatment groups is shown; where A is treatment 1; B is treatment 2; C is treatment 3; and D is treatment 4. 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] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0024] Example 1 This example provides a rapid propagation and rooting medium for tissue culture of peony seeds. Its formula contains a basal medium, macronutrients, a carbon source, a solidifying agent, and a unique plant growth regulator. This plant growth regulator can effectively bypass the physiological dependence of peony rooting on low-temperature signals and omit the low-temperature treatment step in the tissue culture process of peony seeds.

[0025] The specific composition of this rapid propagation and rooting medium is as follows: Basal medium: 1 / 2 MS; Macronutrients: ammonium nitrate (NH4NO3) 800 - 900 mg / L, potassium nitrate (KNO3) 900 - 1000 mg / L, calcium chloride (CaCl2) 150 - 170 mg / L, magnesium sulfate (MgSO4) 90 - 95 mg / L, and potassium dihydrogen phosphate (KH2PO4) 80 - 90 mg / L; Carbon source: sucrose 25 - 40 g / L; Plant growth regulator: KT (Chinese full name: Kinetin; English full name: Kinetin) 0.2 - 1.5 mg / L; Solidifying agent: phytagel 3 - 5 g / L; pH value: 5.6 - 5.8.

[0026] Among them, kinetin, as a cytokinin, can effectively promote cell division and differentiation and participate in regulating the hormone balance and signal transduction pathways in plants. In this invention, kinetin activates the gene expression related to rooting (such as auxin response factors, genes related to root development, etc.) by mimicking or substituting the endogenous hormone response induced by low temperature, thus bypassing the necessary low-temperature vernalization signal in the traditional method. In addition, kinetin can cooperate with endogenous auxin at low concentrations to promote the formation and elongation of root primordia, avoid callus formation, and directly induce the development of healthy roots.

[0027] Example 2 This example provides a method for culturing peony tissue culture seedlings to root, and the specific steps are as follows: (1) Explant preparation: Select healthy and mature peony seeds at the S4 stage with strong growth as the rooting explants (see Figure 1 A - C).

[0028] (2) Pretreatment: Select peony seeds with uniform maturity, plump and undamaged kernels, soak them in tap water at room temperature for 24 h to soften the seed coat, and then remove them and blot dry with sterile filter paper. Use tweezers to carefully peel off the seed coat along the ventral suture, strictly avoiding mechanical damage to the endosperm, and quickly carry out subsequent experiments after obtaining the complete endosperm.

[0029] (3) The above-mentioned intact endosperm is subjected to aseptic treatment in sequence: surface disinfection with 75% ethanol for 1 min, then disinfection in 2-5% sodium hypochlorite solution for 18 min (replace with fresh 2-5% sodium hypochlorite solution once during the disinfection process); after disinfection, rinse with sterile water 3-5 times to completely remove residual disinfectant on the surface, and finally dry the surface moisture of the endosperm with sterile filter paper for later use.

[0030] (4) Inoculation: Under aseptic conditions, remove the endosperm with sterile forceps and scissors to obtain the complete embryo (see...). Figure 1 (D), inoculated onto the rapid rooting medium.

[0031] Rapid propagation and rooting medium: 1 / 2 MS, ammonium nitrate 825 mg / L, potassium nitrate 950 mg / L, calcium chloride 166.1 mg / L, magnesium sulfate 90.35 mg / L, potassium dihydrogen phosphate 85 mg / L, sucrose 30 g / L, kinetin 0.5 mg / L and plant gel 4 g / L, pH=5.6.

[0032] (5) Culture conditions: The culture was placed in a tissue culture room at a temperature of (22±1)℃, and the daylight lamp density was 54 μmol / (m²). 2 •s), 16 h light / 8 h dark. No low-temperature treatment is required at any stage of the entire culture process.

[0033] Following the steps described above, a total of 100 embryos were inoculated. Within 50 days post-inoculation, all embryos germinated and rooted, achieving a 100% rooting rate. Germination of the peony embryos was clearly observed 7 days after inoculation (see...). Figure 1 (E); 35 days after inoculation, it can be clearly observed that the peony embryos have sprouted and rooted (see E). Figure 1 (F); 50 days after inoculation, it can be clearly observed that the peony seed embryos are developing normally, the root length can reach 2-3cm, and the root system is robust and well-developed (see F). Figure 2 ).

[0034] Example 3 In the process of developing a rapid propagation and rooting medium for peony seed tissue culture, this invention also experimented with various plant growth regulators, such as using auxin alone, using cytokinin alone, and combining auxin and cytokinin, to explore the effects of different types of hormones on peony seed germination and growth. The results showed significant differences in seed germination among different hormone treatments. Only the kinetin treatment group (0.2-1.5 mg / L) showed rooting, with robust root development and excellent overall growth; the other treatment groups did not show rooting. In the higher concentration of cytokinin treatment group, root development was completely inhibited. Even with other auxins (such as IAA), only the epicotyl and cotyledon development was observed, with no signs of rooting. The following are some of the treatment groups using auxin (IAA) and cytokinin; specific treatment methods are shown in Table 1. Various hormones were added to the rapid propagation and rooting medium of Example 1 (replacing kinetin) at the concentrations recorded in Table 1, and peony tissue culture seedlings were rooted according to the method of Example 2. The germination rate and rooting rate of each group 50 days after inoculation are shown in Table 1, and the growth status is shown in [Table 1]. Figure 3 .

[0035] Table 1. Germination results of different hormone treatments In summary, this invention has found that kinetin can activate the expression of genes related to rooting (such as auxin response factors and root development-related genes) by simulating or replacing the endogenous hormone response induced by low temperature. This breaks the dependence of peony seeds on low temperature treatment, bypasses the necessary low temperature pretreatment in traditional methods, greatly simplifies the process of peony seed tissue culture, shortens the culture cycle, and improves the rooting rate.

[0036] 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 rapid rooting culture medium for peony seed tissue culture, characterized in that, The rapid propagation and rooting medium is based on 1 / 2MS and also includes the following components: basal medium, ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulfate, potassium dihydrogen phosphate, carbon source, kinetin, and curing agent.

2. The rapid propagation and rooting culture medium according to claim 1, characterized in that, Based on the final concentration, the rapid propagation and rooting medium, with 1 / 2 MS as the basal medium, also includes the following components: ammonium nitrate 800-900 mg / L, potassium nitrate 900-1000 mg / L, calcium chloride 150-170 mg / L, magnesium sulfate 90-95 mg / L, potassium dihydrogen phosphate 80-90 mg / L, carbon source 25-40 g / L, kinetin 0.2-1.5 mg / L, and curing agent 3-5 g / L.

3. The rapid propagation and rooting culture medium according to claim 1 or 2, characterized in that, The carbon source includes sucrose.

4. The rapid propagation and rooting culture medium according to claim 1 or 2, characterized in that, The curing agent includes plant gel.

5. The rapid propagation and rooting culture medium according to claim 1 or 2, characterized in that, The pH value is 5.6-5.

8.

6. A method for rooting peony tissue culture seedlings, characterized in that, The method includes the steps of inoculating peony seed embryos onto the rapid propagation and rooting medium described in any one of claims 1-5, and performing tissue culture and inducing rooting.

7. The method for rooting peony tissue culture seedlings according to claim 6, characterized in that, The temperature for tissue culture was 22±1℃.

8. The method for rooting peony tissue culture seedlings according to claim 6, characterized in that, The optical density of the tissue culture was 50-55 μmol / (m²). 2 •s), the light cycle is 16 h of light / 8 h of darkness.

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