A culture medium and its application in reducing browning of Kandelia candel leaves in tissue culture

Abstract

The present invention provides a culture medium and its application in reducing the browning of Kandelia candel leaves in tissue culture, belonging to the field of bio-agricultural technology. The present invention solves the problem that Kandelia candel leaves in tissue culture are prone to browning, which limits the induction of leaf callus, and provides a new way for tissue culture propagation of Kandelia candel, overcoming the shortcomings of traditional cutting or sowing propagation that are affected by seasons, have low propagation coefficients, and cause great damage to endangered mangrove plant mother plants, and is helpful to promote the research on Kandelia candel leaf callus induction seedling technology. Mangrove plants are rich in secondary metabolites such as tannins, which makes explants prone to browning, and there are few reports on tissue culture research. The present invention provides a reference for the development of mangrove plant tissue culture technology, is conducive to promoting the application of mangrove plant tissue culture in actual production, and is of great significance to the protection of mangrove plant germplasm resources.

Description

Technical Field

[0001] The invention relates to the technical field of biological agriculture, and in particular to a culture medium and application thereof in reducing browning of Kandelia candel leaves in tissue culture. Background Art

[0002] Mangroves occupy a vital position in the ecosystem and have extremely high ecological value and certain ornamental value. In terms of coastal protection, mangroves can effectively resist wind and wave erosion, slow down the flow of seawater, and protect the ecological stability of the coastal zone and land safety; in terms of biodiversity maintenance, they provide a place for many marine organisms to live, forage and reproduce, and are a key shelter for many rare species.

[0003] In the field of reproduction, although the cutting or sowing propagation technology of common mangrove plants such as Avicennia marina and Bruguiera gymnorrhiza has achieved a certain degree of maturity, there are still significant defects. Seasonal factors are particularly restrictive on their reproduction. The success rate of reproduction outside of a specific season is greatly reduced, making it difficult to ensure the efficiency of reproduction. The reproduction coefficient is generally low, which makes it difficult to meet the demand for the number of seedlings for large-scale planting or ecological restoration. At the same time, for endangered mangrove plants with low seed germination rates, cutting propagation is not only difficult to operate, but also causes serious damage to the mother plant, which may affect the growth and survival of the mother plant, and then threaten the safety of its germplasm resources, which is not conducive to the protection of endangered species and population recovery.

[0004] In terms of tissue culture research, although there are relevant reports on major mangrove plants such as Aglaonema and Kandelia ovata, due to their woody characteristics and rich in secondary metabolites such as tannins, the explants are very likely to brown during the culture process. Browning will seriously hinder the normal growth and development of the explants, making it difficult to smoothly advance tissue culture. This is also one of the key reasons for the relative scarcity of tissue culture research on mangrove plants. This situation greatly limits the application of tissue culture technology in the actual production and protection of mangrove plants. Innovative solutions are urgently needed to break through these bottlenecks and achieve the goal of efficient reproduction of mangrove plants and sustainable protection of germplasm resources. Summary of the invention

[0005] The purpose of the present invention is to provide a culture medium and its application in reducing the browning of Kandelia candel leaves in tissue culture, so as to provide an effective means for promoting the research on the callus seedling formation technology of Kandelia candel leaves.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a Kandelia candel tissue culture medium, which contains kinetin, 2.4-D, oxadiazole, citric acid, ascorbic acid and additives;

[0008] The additive is banana juice or boiled concentrated liquid of Kandelia candel hypocotyl root system.

[0009] Preferably, in the Kandelia candel tissue culture medium, the added amount of the kinetin is 0.3-0.5 mg / L.

[0010] Preferably, in the Kandelia candel tissue culture medium, the added amount of 2.4-D is 0.2-0.4 mg / L.

[0011] Preferably, in the Kandelia candel tissue culture medium, the added amount of oxadiazole is 0.8-1.2 mg / L.

[0012] Preferably, in the Kandelia candel tissue culture medium, the added amount of citric acid is 0.03-0.08 g / L.

[0013] Preferably, in the Kandelia candel tissue culture medium, the added amount of ascorbic acid is 0.03-0.08 g / L.

[0014] Preferably, in the Kandelia candel tissue culture medium, the banana juice is added at a concentration of 1% to 3%;

[0015] The added concentration of the boiled Kandelia candel hypocotyl root concentrate is 10 mL / L.

[0016] Preferably, the Kandelia candel tissue culture medium uses MS medium as the basal medium.

[0017] The present invention also provides application of the Kandelia candel tissue culture medium in reducing browning of Kandelia candel leaves in tissue culture.

[0018] The present invention also provides a method for tissue culture of Kandelia candel, comprising the following steps:

[0019] After the Kandelia candel hypocotyl germinates, cut off the terminal bud and the connecting hypocotyl 1-2 cm to obtain the hypocotyl incision;

[0020] New branches and leaves grown from the hypocotyl incision are taken as explants, inoculated with the above-mentioned Kandelia candel tissue culture medium, and then tissue culture is performed.

[0021] Beneficial effects of the present invention:

[0022] The invention solves the problem that the tissue culture of Kandelia candel leaves easily turns brown, which restricts the induction of leaf callus, provides a new way for the tissue culture reproduction of Kandelia candel, overcomes the shortcomings of traditional cutting or sowing reproduction that are affected by seasons, have a low reproduction coefficient, and cause great damage to the mother plants of endangered mangrove plants, and is helpful to promote the research on the technology of inducing callus and seedling formation of Kandelia candel leaves.

[0023] Mangrove plants are rich in secondary metabolites such as tannins, which makes explants prone to browning, and there are few reports on tissue culture research. The present invention provides a reference for the development of mangrove plant tissue culture technology, is conducive to promoting the application of mangrove plant tissue culture in actual production, and is of great significance to the protection of mangrove plant germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of embryonic budding of Kandelia candel hypocotyl;

[0025] Figure 2 This is the result of tissue culture of Kandelia candel of formula 1;

[0026] Figure 3 This is the result of tissue culture of Kandelia candel of recipe 2;

[0027] Figure 4 This is the result of tissue culture of Kandelia candel of formula 3;

[0028] Figure 5 This is the result of tissue culture of Kandelia candel of formula 4;

[0029] Figure 6 This is the result of tissue culture of formula 5 Kandelia candel;

[0030] Figure 7 This is the result of tissue culture of Kandelia candel of formula 6;

[0031] Figure 8 This is the result of tissue culture of formula 7 Kandelia candel;

[0032] Fig. 9 This is the result of tissue culture of Kandelia candel of recipe 8;

[0033] Fig.10 This is the result of tissue culture of Kandelia candel of formula 9;

[0034] Fig.11 This is the leaf tissue culture result of formula 9 for embryonic axis germination of Kandelia candel. DETAILED DESCRIPTION

[0035] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] Example

[0037] Preparation of culture medium for reducing browning of Kandelia candel leaves in tissue culture:

[0038] MS+kinetin 0.4 mg / L +2,4-dichlorophenoxyacetic acid 0.3 mg / L + oxathiapiprolin 1.0 mg / L + citric acid 0.05 g / L + ascorbic acid 0.05 g / L + 5% banana juice.

[0039] Effects of different formulations on browning of Kandelia candel leaves in tissue culture

[0040] Experimental materials: After the embryonic axis of Kandelia candel germinates, the terminal bud and the 2 cm connection to the embryonic axis are cut off, and new branches and leaves emerge from the cut of the embryonic axis.

[0041] Test period: 3 months.

[0042] Test recipe treatment:

[0043] Formula 1, MS + 6-benzylaminopurine 0.5 mg / L + kinetin 1.0 mg / L + naphthaleneacetic acid 0.2 mg / L + oxazolidinone 0.1 mg / L

[0044] Formula 2: MS + Thidiazuron 0.4 mg / L + Naphthaleneacetic acid 0.2 mg / L + Ethiconazole 0.1 mg / L

[0045] Formula 3: MS + Thidiazuron 0.4 mg / L + Naphthaleneacetic acid 0.5 mg / L + Ethiconazole 0.1 mg / L

[0046] Formula 4: MS + 6-benzylaminopurine 0.8 mg / L + naphthaleneacetic acid 0.4 mg / L + abscisic acid 0.1 mg / L

[0047] Formula 5: MS + 6-benzylaminopurine 0.8 mg / L + kinetin 1.0 mg / L + naphthaleneacetic acid 0.4 mg / L + 5% banana juice

[0048] Formula 6: MS + thidiazuron 0.4 mg / L + naphthaleneacetic acid 0.2 mg / L + oxathiapiprolin 0.1 mg / L + citric acid 0.05 g / L + ascorbic acid 0.05 g / L + 5% banana juice

[0049] Formula 7, MS + thidiazuron 0.4 mg / L + 2,4-dichlorophenoxyacetic acid 0.3 mg / L + oxathiapiprolin 1.0 mg / L + citric acid 0.05 g / L + ascorbic acid 0.05 g / L + 5% banana juice

[0050] Formula 8: MS + thidiazuron 0.4 mg / L + naphthaleneacetic acid 0.2 mg / L + oxathiapiprolin 0.5 mg / L + citric acid 0.05 g / L + ascorbic acid 0.05 g / L + 5% banana juice + Kandelia candel root pre-boiled liquid 10 mL / L

[0051] Formula 9, MS + kinetin 0.4 mg / L + 2,4-dichlorophenoxyacetic acid 0.3 mg / L + oxathiapiprolin 1.0 mg / L + citric acid 0.05 g / L + ascorbic acid 0.05 g / L + 5% banana juice

[0052] Thidiazuron TDZ: 96%, 5g / bottle, Mumu Biotechnology.

[0053] Naphthaleneacetic acid NAA: CP / Shanghai 10g.

[0054] 6-Benzylaminopurine 6-BA: 1g / bottle, Sinopharm.

[0055] S-3307: active ingredient content 5%, 50g / bag, Guoguang.

[0056] Citric acid: 500g / bottle, Xilong.

[0057] Ascorbic acid: 100 g / bottle, Chinese medicine.

[0058] Kinetin KT: 1 g / bottle, Mumu Biotech.

[0059] Abscisic acid ABA: 30mg / bottle, Mumu Biotechnology.

[0060] 2,4-Dichlorophenoxyacetic acid 2.4-D: 5g / bottle, Mumu Biotechnology.

[0061] Test steps:

[0062] Preparation process of Kandelia candel pre-boiled liquid: cut 50g Kandelia candel hypocotyl root system, add 1 liter of pure water, boil and concentrate to 100 ml as the original concentration, filter, cool and sterilize under high pressure to obtain the pre-boiled liquid.

[0063] Collect healthy and mature hypocotyls of Kandelia candel, rinse with tap water, and then soak and disinfect in 1% potassium permanganate solution for 20 minutes. After disinfection, insert the hypocotyl into peat soil to a depth of 1.5 to 2.5 cm, then compact the soil by hand and water it. After 30 days, the top of the hypocotyl begins to sprout. Use scissors to cut off 2 cm of the hypocotyl carrying the apical bud, and wait for the lateral buds to sprout from the hypocotyl cut (such as Figure 1 The leaves were then taken for tissue culture anti-browning study.

[0064] Take the leaves, wash them with clean water on a clean bench, disinfect them with 75% alcohol for 30 seconds, disinfect them with 0.1% mercuric chloride for 8 minutes, rinse them with sterile water three times, and then transfer them to the autoclaved culture medium for culture observation, 2 to 3 leaves / bottle. Culture on a light culture rack with a light intensity of 20 to 30 μmol / m 2 •s, photoperiod 10h / d. Temperature 20~24℃.

[0065] Test results and analysis

[0066] from Figure 2 to Figure 4 It can be seen that the leaves of Kandelia candel under formulations 1 to 3 began to turn brown on the 7th day of culture until all the leaves turned brown and died. When a certain concentration of abscisic acid was added to formulation 4, the leaves did not turn brown completely, indicating that abscisic acid has a certain effect on reducing the browning of Kandelia candel leaves in tissue culture. Figure 5 As shown in the figure, from the comparison between formula 1 and formula 5, it can be seen that when the oxadiazole 7 in formula 1 was replaced with banana juice, the leaves of Kandelia candel also began to turn brown on the 7th day of cultivation until the entire leaves turned brown and died. Figure 6Formula 6 is based on Formula 3 and adds a certain concentration of citric acid, ascorbic acid and 5% banana juice. Figure 7 It can be seen that all the leaves turned brown and died. In the combination of thidiazuron, naphthylacetic acid and oxadiazole, adding conventional anti-browning agents had no anti-browning effect. Formula 7 is based on Formula 6, but the naphthylacetic acid in Formula 6 is replaced with 2,4-dichlorophenoxyacetic acid. The browning phenomenon of the Kandelia candel leaves in tissue culture was still not alleviated. Figure 8 Formula 8 is based on Formula 6 and continues to add the root pre-boiled liquid of Kandelia candel. The leaves have browned, but some remain green. It can be seen that the root pre-boiled liquid of Kandelia candel has a certain anti-browning effect. Fig. 9 Formula 9 is a combination of kinetin, 2,4-dichlorophenoxyacetic acid and oxadiazole, with a certain concentration of citric acid, ascorbic acid and 5% banana juice added. This formula has a good anti-browning effect on the tissue culture of Kandelia candel leaves. The leaves remain green after 3.5 months of culture. Fig.10 After more than 3.5 months of cultivation, some leaves gradually turned brown, but the browning speed was slow.

[0067] In summary, if the pre-cooked liquid of Kandelia candel and banana are combined with appropriate hormones, they will have a certain effect on reducing the browning of Kandelia candel leaves in tissue culture (after the Kandelia candel hypocotyl germinates, the terminal bud and the 2 cm connection to the hypocotyl are cut off, and the new branch leaves at the hypocotyl incision are grown). A better culture medium for reducing the browning of Kandelia candel leaves in tissue culture is MS + kinetin 0.4 mg / L + 2,4-dichlorophenoxyacetic acid 0.3 mg / L + oxathiapiprolin 1.0 mg / L + citric acid 0.05 g / L + ascorbic acid 0.05 g / L + 5% banana juice. This formula also has a certain anti-browning effect on Kandelia candel leaf tissue culture after the hypocotyl germinates, but the effect is poor, the anti-browning effect time is short, and the leaves are not fresh. Fig.11 shown.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Kandelia candel tissue culture medium, characterized in that Contains kinetin, 2.4-D, oxathiapiprolin, citric acid, ascorbic acid and additives; The additive is banana juice or boiled concentrate of Kandelia candel hypocotyl roots; The added amount of the kinetin is 0.3-0.5 mg / L; the added amount of the 2.4-D is 0.2-0.4 mg / L; the added amount of the oxadiazole is 0.8-1.2 mg / L; the added amount of the citric acid is 0.03-0.08 g / L; the added amount of the ascorbic acid is 0.03-0.08 g / L; and the added concentration of the boiled concentrate of the Kandelia candel hypocotyl root system is 10 mL / L.

2. The Kandelia candel tissue culture medium according to claim 1, characterized in that The Kandelia candel tissue culture medium uses MS culture medium as the basal culture medium.

3. Use of the Kandelia candel tissue culture medium according to claim 1 or 2 in reducing the browning of Kandelia candel leaves in tissue culture.

4. A method for tissue culture of Kandelia candel, characterized in that: The following steps are involved: After the Kandelia candel hypocotyl germinates, cut off the terminal bud and the connecting hypocotyl 1-2 cm to obtain the hypocotyl incision; New branches and leaves grown from the hypocotyl incision are taken as explants, inoculated into the Kandelia candel tissue culture medium according to claim 1 or 2, and tissue culture is performed.

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