Tissue culture system construction method for inducing regenerated seedlings by using triticale mature embryos

Abstract

The invention discloses a tissue culture system construction method for inducing regenerated seedlings by using triticale mature embryos, and belongs to the technical field of agricultural biology. Aiming at the problems of low efficiency, poor effect and the like of the existing triticale mature embryo regeneration system, the method comprises the stages of callus induction, differentiation and rooting. In the induction stage, on the basis of an MS culture medium, picloram, dicamba, 2, 4-dichlorphenoxyacetic acid, trehalose and zinc sulfate heptahydrate with specific concentrations are added, hormone combination is optimized through a Dicamba (+ trehalose + zinc sulfate) ternary synergistic formula and an orthogonal test, and a universal culture system is formed. In the differentiation stage, a 1 / 2MS culture medium is combined with a specific hormone combination to improve the differentiation rate, and in the rooting stage, an optimized 1 / 2MS + NAA formula is used for improving root development. The method effectively solves the problems of strong genotype dependence, high pollution rate, long differentiation period and the like of a triticale regeneration system, and establishes an efficient and stable technical platform for triticale molecular breeding.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and specifically to a method for constructing a tissue culture system for inducing regenerated seedlings using mature embryos of triticale. Background Art

[0002] Triticale, a polyploid intergeneric hybrid of wheat and rye, combines the excellent characteristics of both parents, such as high yield potential, strong stress tolerance, and wide adaptability, making it an important food and feed crop. However, its genetic improvement, particularly the introduction of desirable traits through gene editing or transgenic techniques, relies heavily on efficient in vitro regeneration systems. Currently, genetic transformation and regeneration technologies for triticale, particularly those based on mature embryos, face significant bottlenecks, hindering the advancement of its molecular breeding.

[0003] Existing regeneration systems for mature triticale embryos exhibit significant variability in their response to different genotypes. This high genotype dependence makes it difficult to directly apply existing regeneration systems to popular varieties with superior agronomic traits (such as high-yield, disease-resistant, and stress-tolerant varieties). Culture medium components and hormone ratios often need to be re-optimized for each specific genotype, which consumes considerable time and resources. Regeneration efficiency is low and unstable, primarily manifested in the difficulty of callus induction, the complex and inefficient differentiation process, and the high dependence of the differentiation stage on specific hormone combinations. The final differentiation cycle typically lasts several weeks, making efficiency difficult to guarantee. Callus induction rates among different triticale lines on MS, B5, and N6 media can vary by more than three-fold, indicating that the compatibility of triticale genotypes with callus induction media still needs to be optimized.

[0004] These issues directly limit the application of Agrobacterium-mediated genetic transformation techniques for improving triticale varieties. Traditional regeneration systems are often effective only on a few "model" genotypes, failing to meet the demand for efficient genetic improvement of widely disseminated varieties.

[0005] Therefore, constructing a mature triticale embryo regeneration system that is highly universal, efficient, stable, and easy to operate has become a major technical challenge that the industry urgently needs to solve. Summary of the Invention

[0006] In order to solve the problem of low efficiency and poor regeneration effect of the existing triticale mature embryo regeneration system, the present invention provides a method for constructing a tissue culture system for inducing regenerated seedlings using triticale mature embryos to solve the above problems.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for constructing a tissue culture system for inducing regenerated seedlings using mature triticale embryos, comprising callus induction, differentiation, and rooting stages of triticale seeds; the environmental conditions for the callus induction stage are a temperature of 26±2°C, a humidity of 60% to 70%, and dark culture;

[0009] The components of the callus induction medium are as follows: MS medium as the basis, sucrose as the carbon source, agar as the solidifying agent, and 1-3 mg / L of cypermethrin, 2-6 mg / L of dicamba, 1-3 mg / L of 2,4-dichlorophenoxyacetic acid, 8-12 g / L of trehalose, and 40-60 μmol / L of zinc sulfate heptahydrate are added to each liter of the induction medium.

[0010] Preferably, before the callus induction of triticale seeds, the triticale seeds are pretreated, and the specific steps are as follows:

[0011] (1) Select healthy and plump seeds and wash them;

[0012] (2) Soak the cleaned seeds in 75% ethanol for 5 minutes;

[0013] (3) Soak the seeds in 0.1% mercuric chloride for 15 minutes, then wash them with sterile water for more than three times;

[0014] (4) Soak the seeds in 4°C sterile water for 15-18 hours;

[0015] (5) Secondary disinfection with 10% sodium hypochlorite for 5 minutes, final rinse with sterile water until neutral; place on sterile filter paper to dry.

[0016] Preferably, 2 mg / L of chlorpyrifos, 4 mg / L of dicamba, and 2 mg / L of 2,4-dichlorophenoxyacetic acid are added per liter of induction culture medium.

[0017] Preferably, 2 mg / L of chlorpyrifos, 6 mg / L of dicamba, and 1 mg / L of 2,4-dichlorophenoxyacetic acid are added to each liter of induction culture medium.

[0018] Preferably, the callus differentiation medium used in the triticale callus differentiation process has the following composition: 1 / 2MS medium; 0.3g / L CH; 1.5mg / L KT; 0.2mg / L NAA; 2.5g / L phytagel; 15g / L sucrose, pH = 5.8.

[0019] Preferably, the environmental conditions during the differentiation of triticale callus are: temperature of 26±2°C, humidity of 60% to 70%, photoperiod of 16 hours of light and 8 hours of darkness, and light intensity of 1000-2500 lux.

[0020] Preferably, the rooting medium used in the rooting tissue differentiation process of triticale is formulated as follows: 1 / 2MS medium; 0.2mg / LNAA; 30g / L sucrose; 8g / L agar; PH = 5.8.

[0021] Preferably, the environmental conditions during the rooting process of the rooting medium are: 23–25°C (day) / 18–20°C (night); light intensity is 1000-1500 Lux; and air humidity is 70–80%.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention innovatively utilizes a ternary synergistic formula of "Dicamba (+ trehalose + zinc sulfate)" in the callus induction medium, forming a chain of action during the callus induction phase: activation of drought-resistance genes, enhanced osmotic protection, and antioxidant maintenance. Dicamba specifically induces the expression of the DREB2A transcription factor, while trehalose enhances the induction efficiency of Dicamba's drought-resistance genes through the osmotic signaling pathway (BIK1-SnRK2.4), significantly improving the cell's water retention capacity. Zinc sulfate plays a dual role: first, it reduces contamination risk by inhibiting mold metabolic genes (such as Chs3), and second, it prolongs the protective effect of drought-resistance genes by stabilizing antioxidant enzymes such as SOD.

[0024] 2. In the present invention, the hormone combination of the culture medium in the induction stage was optimized by orthogonal experiment; two optimal hormone combination schemes were obtained (B5 scheme: 2 mg / L of piclopirox + 4 mg / L of dicamba + 2 mg / L of 2,4-D; B6 scheme: 2 mg / L of piclopirox + 6 mg / L of dicamba + 1 mg / L of 2,4-D), and a universal culture system was successfully constructed across genotypes such as octoploid (Zhongli No. 6), hexaploid (CM-Z2), winter (Beilian No. 1), and spring (New Triticale No. 1).

[0025] 3. In this invention, the culture medium for triticale callus differentiation was optimized, using 1 / 2 MS medium instead of traditional high-salt or complex additives (such as L-Glu / L-Pro in the M3 group). This medium, combined with hydrolyzed casein (0.3 g / L) to provide essential amino acids, and supplemented with a hormone combination of KT (1.5 mg / L) and NAA (0.2 mg / L). This synergistic effect overcomes the differentiation bottleneck of traditional MS medium and significantly improves the differentiation rate.

[0026] 4. In the present invention, the culture medium for the rooting stage of triticale callus is optimized and selected, and the optimized 1 / 2MS+NAA (0.2 mg / L) formula significantly improves the quality of root development (comprehensive improvement in total length, surface area and volume) compared with the traditional N6 culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present application 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 only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0028] Figure 1 This is a comparison chart of triticale callus induction effects;

[0029] Figure 2 This is a comparison chart of differentiation rates during the differentiation stage;

[0030] Figure 3 This is a comparison chart of root system development during the rooting stage;

[0031] Figure 4 It is a schematic diagram of the entire process of tissue culture system. DETAILED DESCRIPTION

[0032] In order to make the purpose, features, and advantages of this application more obvious and easy to understand, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the embodiments described below are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0033] Example

[0034] 1. Selection of triticale varieties:

[0035] To construct an efficient mature embryo regeneration system and verify its genotypic universality, the following five triticale varieties were selected for experiments, covering different ploidy, ecotypes, and agronomic characteristics:

[0036] Variety 1: Middle Rye No. 6 (octoploid AABBDDRR)

[0037] Source: Variety bred by the Crop Research Institute of the Chinese Academy of Agricultural Sciences.

[0038] Variety 2: CM-Z2 (hexaploid AABBRR)

[0039] Source: CIMMYT Stress-Resistant Germplasm Bank

[0040] Variety 3: New Triticale No. 1 (Spring Hexaploid)

[0041] Source: Bred by Shihezi University, Xinjiang approved variety

[0042] Variety 4: Beilian No. 1 (winter hexaploid)

[0043] Source: Cold-resistant varieties from Heilongjiang Academy of Agricultural Sciences

[0044] Variety 5: Gannong No. 2 (hexaploid for feed)

[0045] Source: Breeding by Gansu Agricultural University

[0046] The above-mentioned wheat varieties come from three categories: octoploid / hexaploid, winter / spring, and grain / feed, which can make the tissue culture system obtained from the research more universal.

[0047] 2. Triticale Callus Induction

[0048] 2.1 The pretreatment steps of triticale are as follows:

[0049] Select seeds: Select healthy, plump seeds from the five varieties listed above, and discard damaged, moldy, or insect-infested seeds. In the following processing steps, process the different varieties of triticale separately to avoid mixing.

[0050] Initial sterile water wash: Wash the seeds three times with sterile water to remove physical contaminants such as dust, soil, and organic debris from the seed surface. These impurities can interfere with the effective contact between the subsequent disinfectant and microorganisms and may form a protective layer around the microorganisms.

[0051] Ethanol surface disinfection: Soak the seeds in 75% ethanol for 5 minutes, ensuring they are completely immersed in the ethanol. Discard the waste liquid after immersion. Ethanol dissolves lipids, destroying the cell membranes of most bacteria and fungi, quickly killing most surface microorganisms. As a first step in chemical disinfection, it quickly reduces the microbial population, reducing the burden on subsequent, more potent but slower-penetrating disinfectants (mercuric chloride), improving their effectiveness.

[0052] Mercuric chloride deep disinfection: Mercuric chloride is a highly toxic, broad-spectrum fungicide (particularly effective against bacterial endospores and some stubborn fungi), with exceptional penetrating power and bactericidal efficacy. The 15-minute treatment is designed to kill stubborn microorganisms (especially endospores) that are difficult for ethanol to kill and that lurk in the crevices of the seed coat or even in the superficial layers of the endosperm. This procedure is high-risk and should be performed in a fume hood while wearing protective equipment such as a respirator, rubber gloves, and goggles. Soak in 0.1% mercuric chloride for 15 minutes. Afterwards, wash with sterile water at least three times, shaking for 30 seconds each time, to completely remove any mercury residue.

[0053] Low-temperature soaking activation: soak the triticale in sterile water, and then place it in a 4°C refrigerator to soak for 15-18 hours. The seeds should be 3 cm below the liquid surface during soaking. At the same time, the soaking activation process can remove residual trace disinfectants or substances that seep from the inside of the seeds.

[0054] Secondary disinfection with sodium hypochlorite: Soak in 10% sodium hypochlorite for 5 minutes. Sodium hypochlorite releases available chlorine, making it a strong oxidizing disinfectant effective against mold spores, some bacteria (especially Gram-negative bacteria), and viruses. It kills microorganisms that may have been secondary contaminated during the previous rinse step and compensates for the ineffectiveness of mercuric chloride against some fungal spores.

[0055] Final rinse: Rinse the triticale seeds with sterile water until there is no chlorine smell (neutral according to pH paper).

[0056] Drying with sterile filter paper: Spread a single layer of seeds and place them in a clean bench for ventilation and drying (avoid direct sunlight).

[0057] 2.2 Preparation of culture medium

[0058] MS medium was used as the basal medium for cultivating triticale, and different concentration combinations of 2.4-D, Picloram and Dicamba were added to the MS medium. Orthogonal experiments were used to set treatment combinations of different concentrations of 2.4-D, Dicamba and Picloram. Then, the induction medium was modified, and sucrose and agar were added to each induction medium. The amount of sucrose added to each liter of the induction medium was 30 g, and the amount of agar added to each liter of the induction medium was 8 g.

[0059] Among them, 1,2,4-dichlorophenoxyacetic acid (2,4-D) can stimulate explant dedifferentiation and promote callus formation, making it a key initiating factor in tissue culture. Picloram can effectively induce embryonic callus in difficult-to-regenerate crops, increasing regeneration rates. Dicamba specifically induces the DREB2A transcription factor, synthesizing osmoprotectants and enhancing cellular water retention.

[0060] The environmental humidity in conventional plant tissue culture is generally set at 70%~80%; however, the germination activity of mold spores is significantly improved when the humidity is ≥65%, and the reproduction rate increases exponentially when the relative humidity is >70%, which makes it easy for mold to grow during the cultivation of callus tissue.

[0061] In order to reduce the environmental humidity requirement during the primary callus cultivation process, 8-12 g of trehalose was added to each liter of the induction medium, and the added amount in this embodiment was 10 g / L; each liter of the induction medium contained 40-60 μmol / L of ZnSO4·7H2O, and the added amount in this embodiment was 50 μmol / L.

[0062] To adapt triticale to lower humidity environments, Dicamba, trehalose, and zinc sulfate form a synergistic regulatory network. Dicamba, as a core component, triggers the expression of drought-resistance genes such as DREB2A in triticale by activating the auxin signaling pathway, a key molecular basis for triticale's adaptation to low humidity. Trehalose enhances Dicamba's induction of drought-resistance genes through the osmotic signaling pathway (BIK1-SnRK2.4), significantly improving cellular water retention. Zinc sulfate plays a dual role: first, it reduces contamination risk by inhibiting mold metabolism genes (such as Chs3), and second, it prolongs the protective effect of drought-resistance genes by stabilizing antioxidant enzymes such as SOD. These three ingredients work together to form a complete chain of action: Dicamba activates drought-resistance genes, trehalose strengthens signaling, and zinc ions maintain protection. This allows the plant to maintain efficient growth induction and excellent stress tolerance at 60-70% humidity.

[0063] The specific modification scheme is as follows:

[0064] Petri dish grouping:

[0065] Prepare 18 sterile culture dishes and name them B1a, B1b; B2a, B2b, B3a, B3b; B4a, B4b; B5a, B5b; B6a, B6b; B7a, B7b; B8a, B8b; B9a, B9b.

[0066] Pour the induction medium corresponding to its number into each dish; for example, B1a and B1b should be filled with B1 induction medium, and so on. Each dish should be filled with 30ml of induction medium. Once prepared, set aside.

[0067] 2.3 The two-stage callus induction steps under the orthogonal optimized hormone combination are as follows:

[0068] The conditions in the tissue culture room where the culture dishes are located during the entire culture stage are: temperature 26±2°C, humidity 60%-70%; and the culture dishes are protected from light during the culture process to ensure that the entire culture process is dark culture.

[0069] The specific cultivation process is as follows:

[0070] Primary callus induction stage (days 0-7):

[0071] Take nine culture dishes, B1a, B2a...B9a, and inoculate triticale seeds into each of the nine culture dishes. Inoculate 20 seeds per dish (four seeds of each triticale variety) evenly to reduce edge effects.

[0072] When inoculating sterilized seeds in a culture dish, gently press the radicle downward into the surface of the culture medium (depth ≤ 1 mm) to avoid complete burial; primary callus tissue is obtained after 7 days of cultivation.

[0073] Subculture stage (Day 8-14): Take nine culture dishes, B1b, B2b...B9b, to obtain primary callus tissue, which is then transferred to the corresponding induction medium for subculture for 7 days (the induction medium used in the subculture and primary callus induction stages corresponds one to one, for example, the primary callus tissue obtained by culture B1a is continued to be cultured with B1b).

[0074] Investigate the data such as the size and induction rate of callus tissue. Figure 1 Shown are the development of seeds of various varieties on the surface of the culture medium after 14 days of induction culture of triticale.

[0075] Repeat the experiment

[0076] The above research steps of the mature embryo regeneration system were repeated three times to investigate the size of the formed callus tissue, the induction rate and other data; and the corresponding data were obtained.

[0077] 2.4 Data Processing

[0078] The data of the study on the mature embryo regeneration system and three repeated experiments are shown in the following table:

[0079]

[0080] From the calculation in the table above, it can be seen that the callus induction rates of B5 and B6 callus media are the highest, exceeding 98%.

[0081] 3. Triticale Callus Differentiation

[0082] The callus obtained in the above steps was transferred to callus differentiation medium after 14 days of culture and continued to grow for 30 days. The differentiation rate and green spot rate of the callus were investigated. Differentiation culture was performed as shown in the table below. Different culture media and different plant growth regulators were selected for screening.

[0083]

[0084] Tissue culture room conditions: temperature is 26±2℃, humidity is 60%~70%, photoperiod is 16h light and 8h dark, and light intensity is 1000-2500lux.

[0085] like Figure 2 The figure shows the development of triticale callus on M1, M2, and M3 media. As can be seen from the figure, M2 differentiation medium has the highest green spot rate and differentiation rate.

[0086] 4. Rooting of triticale callus

[0087] Regenerated seedlings with consistent growth and good condition were selected from the differentiation medium and inoculated into the rooting medium listed in the table below. After 20 days, the plant height and stem diameter of the regenerated seedlings were measured. Root system parameters such as total root length, average root diameter, root surface area, and root volume were analyzed using a root analysis system (Regent Instruments, Canada) and an auxiliary scanner (Eposon, Japan) to select the optimal rooting medium. Environmental conditions during rooting in the rooting medium were: 23–25°C (daytime) / 18–20°C (nighttime), 1000–1500 Lux of light, and 70–80% humidity.

[0088] rooting medium

[0089]

[0090] like Figure 3 As shown in the figure, the culture effect of R1 rooting medium was significantly better than that of R2, so R1 was selected as the rooting medium.

[0091] It will be apparent to those skilled in the art that the present application is not limited to the details of the exemplary embodiments described above, and that the present application can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the present application is defined by the appended claims, not the foregoing description, and all variations within the meaning and scope of the appended claims are intended to be included herein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0092] As described above, the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for constructing a tissue culture system for inducing regenerated seedlings from mature triticale embryos, comprising the stages of callus induction, differentiation, and rooting of triticale seeds; characterized in that: The environmental conditions during the callus induction stage were a temperature of 26 ± 2°C, a humidity of 60% to 70%, and dark culture; The components of the callus induction medium are as follows: MS medium as the basis, sucrose as the carbon source, agar as the solidifying agent, and 1-3 mg / L of cypermethrin, 2-6 mg / L of dicamba, 1-3 mg / L of 2,4-dichlorophenoxyacetic acid, 8-12 g / L of trehalose, and 40-60 μmol / L of zinc sulfate heptahydrate are added to each liter of the induction medium.

2. The method for constructing a tissue culture system for inducing regeneration of seedlings using mature embryos of triticale according to claim 1, characterized in that: Before callus induction of triticale seeds, the triticale seeds were pretreated, and the specific steps were as follows: (1) Select healthy and plump seeds and wash them; (2) Soak the cleaned seeds in 75% ethanol for 5 minutes; (3) Soak the seeds in 0.1% mercuric chloride for 15 minutes, then wash them with sterile water for more than three times; (4) Soak the seeds in 4°C sterile water for 15-18 hours; (5) Secondary disinfection with 10% sodium hypochlorite for 5 minutes, and final rinse with sterile water until neutral; place on sterile filter paper to dry.

3. The method for constructing a tissue culture system for inducing regenerated seedlings using mature embryos of triticale according to claim 1, characterized in that: Add 2 mg / L of cypermethrin, 4 mg / L of dicamba, and 2 mg / L of 2,4-dichlorophenoxyacetic acid per liter of induction culture medium.

4. The method for constructing a tissue culture system for inducing regenerated seedlings using mature embryos of triticale according to claim 1, characterized in that: Add 2 mg / L of cypermethrin, 6 mg / L of dicamba, and 1 mg / L of 2,4-dichlorophenoxyacetic acid per liter of induction culture medium.

5. The method for constructing a tissue culture system for inducing regenerated seedlings using mature embryos of triticale according to claim 1, characterized in that: The callus differentiation medium used in the triticale callus differentiation process has the following composition: 1 / 2MS medium; 0.3 g / L CH; 1.5 mg / L KT; 0.2 mg / L NAA; 2.5 g / L phytagel; 15 g / L sucrose, pH=5.

8.

6. The method for constructing a tissue culture system for inducing regeneration of seedlings using mature embryos of triticale according to claim 5, characterized in that: The environmental conditions during the differentiation of triticale callus were as follows: temperature of 26±2°C, humidity of 60% to 70%, photoperiod of 16 h light and 8 h dark, and light intensity of 1000-2500 lux.

7. The method for constructing a tissue culture system for inducing regeneration of seedlings using mature embryos of triticale according to claim 1, characterized in that: The rooting medium used in the root tissue differentiation process of triticale is as follows: 1 / 2MS medium; 0.2mg / L NAA; 30g / L sucrose; 8g / L agar, pH=5.

8.

8. The method for constructing a tissue culture system for inducing regenerated seedlings using mature embryos of triticale according to claim 7, characterized in that: The environmental conditions during the rooting process of the rooting medium are: 23–25°C (day) / 18–20°C (night); light intensity is 1000-1500 Lux; air humidity is 70–80%.

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