Cymbidium goeringii Rhizome Tissue Culture Medium Containing Animal-Derived Additives and Tissue Culture Method

Abstract

The present invention belongs to the field of plant cell engineering, and specifically relates to a spring orchid rhizome tissue culture medium and a tissue culture method containing animal-derived additives and anti-browning agents. The spring orchid rhizome tissue culture medium includes a proliferation culture medium, and the spring orchid tissue culture medium containing an anti-browning agent includes a differentiation culture medium. The proliferation culture medium is a basic proliferation culture medium added with animal-derived additives; the animal-derived additives include clams, preserved egg yolks, shrimps and squids. The differentiation culture medium is a basic differentiation culture medium added with an anti-browning agent; the anti-browning agent includes dithiothreitol, cysteine, glutathione, polyvinyl pyrrolidone, citric acid and succinic acid. This technical solution overcomes the technical problems of low reproduction coefficient, high browning rate and long culture cycle of the existing spring orchid tissue culture system. This solution can not only fill the gap in the study of factors affecting the growth of spring orchids, but also directly guide tissue culture and rapid propagation, provide a reference for factory seedling cultivation, and has important theoretical significance and practical value.

Description

Technical Field

[0001] The invention belongs to the field of plant cell engineering, and in particular relates to a cymbidium rhizome tissue culture medium containing animal-source additives and a tissue culture method. Background Art

[0002] Cymbidium is one of the five ornamental genera of the Orchidaceae family. As of December 20, 2023, the World Plant Online (https: / / powo.science.kew.org) records a total of 105 native species of Cymbidium plants worldwide, and the China Biological Species List (http: / / www.sp2000.org.cn / ) records 58 native species distributed in China. Terrestrial orchid species of the genus Cymbidium, such as C. goeringii, C. sinense, C. faberi, C. ensifolium, C. kanran, C. sinense and C. tortisepalum, are called Chinese orchids, or Chinese orchids for short (Xu Wan, Research Progress on Resources and Breeding of Cymbidium Plants, Acta Horticulturae Sinica, 2022). Chunlan is one of the most abundant and widely distributed species among my country's national orchids (Chen Xinqi, Complete Book of National Orchids and Their Varieties, Beijing: China Forestry Publishing House, 2011). It is named for its flowering in spring (January-March). After flowering, it has a delicate fragrance and is one of China's traditional famous flowers. Chunlan is loved by Chinese people for its elegant color, rich fragrance, elegant leaves and long-standing national orchid culture. It has a long history of cultivation and has both aesthetic, cultural and economic values. Therefore, the market demand continues to increase. In recent years, due to problems such as over-exploitation and habitat destruction, the wild resources of Chunlan have decreased sharply, and there has been a phenomenon of supply exceeding demand.

[0003] To protect the wild resources of Cymbidium goeringii and meet the unprecedented market demand, it is particularly important to conduct research on the tissue culture and rapid propagation system of Cymbidium goeringii. Like other Orchidaceae plants, Cymbidium goeringii seeds lack endosperm and are difficult to germinate under natural conditions. It is commonly propagated by division, which takes a long time and has low propagation efficiency, making it difficult to meet market demand. Using tissue culture technology for rapid propagation can quickly obtain excellent varieties with good quality and fast growth cycle, and solve the development bottlenecks such as propagation speed and new variety cultivation. Therefore, to protect the wild resources of Cymbidium goeringii and meet the unprecedented market demand, the research on the tissue culture and rapid propagation system is undoubtedly an important means. Rhizome is a unique proliferation stage of Cymbidium goeringii and other national orchids, and it is also the key process restricting its tissue culture and rapid propagation. Subculture and proliferation through rhizome is the key to realizing the rapid propagation of national orchids. During the tissue culture process of Cymbidium, rhizome can be induced from explants, and seeds and shoot tips are the best explants for inducing rhizome (Bhadra S K, In vitro germination and micropropagation of Geodorum densiforum (Lam.) Schitr, an endangered orchid species. Plant Tissue Culture, 2003; Shimasaki K, Rhizome induction and plantlet regeneration of Cymbidium goeringii from flower bud cultures iv itro. Plant Cell Tssue and organ Culture, 1991). However, the Cymbidium goeringii rhizome has a slow proliferation speed, low proliferation efficiency, few seedlings formed by differentiation, and the rhizome is prone to browning or death during the differentiation process. At the same time, the characteristics of long tissue culture cycle and high production cost of Cymbidium goeringii severely limit the development process of its rapid propagation technology. Therefore, in the commercial production of Cymbidium plant seedlings, it is necessary to find effective methods to improve the existing tissue culture system. To increase the propagation coefficient, reduce the browning rate and shorten the culture cycle, exploring new natural additives and anti-browning agents is an inevitable way to optimize the in vitro rapid propagation system. Summary of the Invention

[0004] The purpose of the present invention is to provide a Cymbidium goeringii rhizome tissue culture medium containing animal-derived additives and a Cymbidium goeringii rhizome tissue culture medium containing an anti-browning agent, which overcomes the technical problems of low propagation coefficient, high browning rate and long culture cycle in the existing Cymbidium goeringii tissue culture system.

[0005] To solve the above technical problems, the technical solution of the present invention is as follows:

[0006] The spring orchid rhizome tissue culture medium containing animal-derived additives includes a proliferation culture medium; the proliferation culture medium is a basic proliferation culture medium added with animal-derived additives; the raw materials of the animal-derived additives include at least one of clam meat, preserved egg yolk, shrimp and squid.

[0007] The above technical scheme is adopted, and the technical principle and beneficial effects are as follows: rotten animal additives are rich in putrescine, which is also called diaminobutane. It belongs to aliphatic straight-chain polyamine biogenic amines, which play an important role in regulating plant growth and development, delaying aging and improving stress resistance. In the relevant literature reported so far, putrescine is often used to treat and induce post-harvest cold resistance of fruits such as cucumbers, tomatoes, sweet oranges, and apricots, and significantly maintain the quality of the fruit. However, in current plant tissue culture experiments, the use of putrescine to enhance the proliferation effect of plants (such as rhizomes of orchids) is still relatively rare. In addition, putrescine has a certain toxic effect on organisms, which limits its application and promotion (Zhou Lingyu et al., Research Progress on Detection Technology of Biogenic Amines in Aquatic Products, Meat Research, 2022).

[0008] Since putrescine is commonly found in aquatic products and livestock and poultry foods, the present technical solution directly adds a certain amount of rotten animal-derived additives (animal tissues that have been sealed and placed, etc.) to the culture medium to improve the efficiency of tissue culture and rapid propagation, which not only avoids the problem of drug toxicity, but also plays the role of a circular economy. Additives derived from rotten animals have not been used in plant tissue culture and rapid propagation, and are new additives to be developed. In this study, the inventors found that adding rotten squid to the culture medium can significantly promote the proliferation of rhizomes. Compared with other rotten animal-derived additives, rotten squid has a better technical effect on the proliferation of rhizomes. In addition, rotten animal-derived additives such as squid are safe and non-toxic, easy to obtain, and adding a small amount can significantly promote the proliferation and growth of explants, which is significantly higher than the control treatment without adding rotten animal-derived additives.

[0009] Furthermore, the basic proliferation culture medium is a 1 / 2MS culture medium containing 6-BA, NAA, sucrose, agar (Ag), and activated carbon (AC).

[0010] Furthermore, the animal-derived additive is prepared by the following method: the raw material is sealed and placed, and then homogenized to obtain the additive.

[0011] Furthermore, calculated on a solid matter basis, when the raw material of the animal-source additive is clam meat, the amount of the animal-source additive added is 0.5-10.0 g / L; when the raw material of the animal-source additive is preserved egg yolk, the amount of the animal-source additive added is 0.1-5.0 g / L; when the raw material of the animal-source additive is shrimp, the amount of the animal-source additive added is 0.2-5.0 g / L; when the raw material of the animal-source additive is squid, the amount of the animal-source additive added is 0.05-1.0 g / L.

[0012] Among them, 0.1 g / L and 0.5 g / L of preserved egg yolks both promoted proliferation. 1.0 g / L of razor clam meat and 0.5 g / L of shrimps had a very significant promoting effect on the proliferation of rhizomes.

[0013] Furthermore, the raw material of the animal-derived additive is squid; the addition amount of the animal-derived additive is 0.1 g / L. Through the screening study of four kinds of rotten animal-derived additives, it was found that squid had a significant promoting effect on the proliferation of Cymbidium goeringii rhizomes. Squid can be added to the proliferation medium of Cymbidium goeringii rhizomes to promote tissue proliferation and accelerate the cultivation process of Cymbidium goeringii.

[0014] Furthermore, the Cymbidium goeringii rhizome tissue culture medium containing the animal-derived additive further includes a differentiation medium; the differentiation medium is a basic differentiation medium added with an anti-browning agent; the anti-browning agent includes at least one of dithiothreitol (DTT), cysteine, glutathione (GSH), polyvinylpyrrolidone (PVP), citric acid (CA), and succinic acid.

[0015] Furthermore, the basic differentiation medium is a 1 / 2MS medium containing 6-BA, NAA, sucrose, and agar.

[0016] Furthermore, the concentration of polyvinylpyrrolidone in the differentiation medium is 1.0 - 5.0 g / L, the concentrations of CA and DTT in the differentiation medium are 0.25 - 3.0 g / L respectively, the concentrations of succinic acid and cysteine in the differentiation medium are 0.05 - 0.25 g / L, and the concentration of GSH in the differentiation medium is 0.1 - 0.5 g / L.

[0017] Furthermore, the anti-browning agent is succinic acid, and its working concentration is 0.05 - 0.25 g / L.

[0018] Through the screening and research of six anti-browning agents, it was found that succinic acid has a significant promoting effect on the differentiation of Cymbidium goeringii rhizomes. Succinic acid can be added to the differentiation medium of Cymbidium goeringii rhizomes to promote tissue differentiation and accelerate the cultivation process of Cymbidium goeringii. In plant tissue culture research, two anti-browning agents, CA and PVP, are often added to reduce the browning rate. Fang Zhongming et al. found that 1.0 g / L CA can control the browning of the medium in the culture of Cymbidium nanulum rhizomes and promote the differentiation of rhizomes (Fang Zhongming et al., Research on the mechanism of citric acid inhibiting the differentiation and browning of Cymbidium nanulum rhizomes, Journal of South China Normal University (Natural Science Edition), 2012). Chen et al. found that the combination of anti-browning agents AC and PVP has a good promoting effect on the rooting of rootless seedlings of Paeonia ostii ‘Feng Dan’ embryos, but there are few reports on the effect of their combination on the rooting and seedling formation of rootless seedlings of Orchidaceae plants (Chen X, The Effect of Anti-browning Agent Activated Carbon and Polyvinyl Pyrrolidone on the Rooting of Embryo Seedlings of “Feng Dan” and Its Transcriptome Analysis. Frontiers in Plant Science, 2022). In the experiment with Cymbidium goeringii flower stalks and sepals as explants, different ratios of anti-browning agents PVP, CA, and AC and different dark culture times were used as treatments, and it was found that adding 5.0 g / L PVP and dark culture for 10 days at the initial stage of explant inoculation were the most effective in reducing explant browning (Wang Baoning, Research on the control of browning during the tissue culture of wild Cymbidium goeringii in the Qinling Mountains, Northern Horticulture, 2011). In the research on anti-browning of Orchidaceae plants, Li Can (2011) found that PVP, ascorbic acid (VC), CA, and 8-hydroxyquinoline sulfate (8-HQS) all have significant inhibitory effects on the browning of Cymbidium tortisepalum flower stalk segments (Li Can, Research on the in vitro culture of Cymbidium tortisepalum flower organs, Chengdu: Sichuan Agricultural University, 2011). Liu Zhenhua et al. (2005) found that adding 0.2 g / L GSH to the tissue culture of aseptic seedlings of Phalaenopsis R4 in Orchidaceae plants can not only better inhibit browning but also promote growth and differentiation, and the use of GSH has not been reported in the anti-browning research of Cymbidium goeringii (Liu Zhenhua, Research on the control of browning in the tissue culture of Phalaenopsis, Acta Horticulturae Sinica, 2005). Cysteine and DTT have not been used in the anti-browning research of Orchidaceae plants. Liu Yue et al. (2017) found that 0.1 g / L cysteine can effectively control the browning of explants of adult stem segments of grapefruit (Liu Yue, Disinfection and anti-browning of adult stem segment explants of grapefruit, Northern Horticulture, 2017).When the concentration of DTT added to the sterile seedlings of Paulownia elongata was greater than 0.2 g / L, the browning rate was 0 (Yang Xiaojuan, Effects of exogenous substances and culture conditions on in vitro plant regeneration of Paulownia elongata leaves, Zhengzhou: Henan Agricultural University, 2005). However, succinic acid, the optimal anti-browning agent in this technical solution, has not been used in anti-browning experiments in plant tissue culture.

[0019] Except for the anti-browning agents AC, CA and PVP, which are commonly used in plant tissue culture, the anti-browning agents DTT, GSH, cysteine and succinic acid are rarely used in tissue culture, and the latter four anti-browning agents have not been used in the study of anti-browning of spring orchids. Although AC is the most widely used anti-browning agent, it is not suitable for the differentiation stage, and CA and PVP also have certain limitations. Lv Zongyou et al. found that CA can increase the callus induction rate but cannot prevent browning in the induction of Sudan grass (Sorghum sudanense) spikelets (Lv Zongyou, Study on the effect of different anti-browning measures on Sudan grass callus induction and anti-browning, Journal of Grassland, 2011). PVP is less effective than VC and CA in inhibiting the browning of Miscanthus sacchariflorus explants and callus (Peng Sijia, Effects of anti-browning agents on browning of Miscanthus explants and callus growth, Grassland and Turf, 2015). All growth stages of spring orchids are prone to browning, which hinders growth, especially the differentiation stage. Therefore, it is of great significance to explore the research on the anti-browning effect of the above anti-browning agents on the differentiation stage during the tissue culture of spring orchids. The inventors found in this study that PVP, CA and succinic acid have good anti-browning effects in the differentiation culture of spring orchids, and cysteine, DTT, succinic acid and GSH effectively promote the differentiation of spring orchid rhizomes (differentiation bud number and average bud height). It can be seen that succinic acid can reduce the browning problem in rhizome differentiation and promote the growth of rhizome differentiation (average bud number and average bud height). Adding succinic acid to the culture medium can significantly promote the differentiation of rhizomes. Compared with other anti-browning agents, succinic acid has a better technical effect on the differentiation of rhizomes. In the differentiation culture after proliferation culture, succinic acid can significantly prevent the browning of rhizomes, and at the same time significantly promote the differentiation efficiency of rhizomes, including the number of differentiation buds and bud height.

[0020] The technical solution also provides a method for culturing rhizome tissue of Cymbidium orchid, comprising the following steps performed in sequence:

[0021] S1 seed germination culture steps: inducing the culture of spring orchid capsules to obtain spring orchid rhizomes;

[0022] S2 proliferation culture step: using a proliferation culture medium to culture the rhizomes of Cymbidium orchid to obtain proliferation culture tissue; the proliferation culture medium is a basic proliferation culture medium supplemented with animal-derived additives; the raw materials of the animal-derived additives include at least one of flower clams, preserved egg yolks, shrimps and squids;

[0023] S3 differentiation culture step: Cultivate the rhizomes separated from the proliferated culture tissue with a differentiation medium to obtain rootless seedlings; the differentiation medium is a basic differentiation medium added with an anti-browning agent, and the anti-browning agent for differentiation promotion includes at least one of DTT, cysteine, GSH, PVP, CA, and succinic acid;

[0024] S4 rooting culture step: Induce the culture of rootless seedlings to obtain small seedlings with roots.

[0025] Adopting the above technical solution, the rhizomes of Cymbidium goeringii are now subjected to proliferative culture to expand the biomass and accumulate a material basis for subsequent differentiation culture. Then, through differentiation culture, the explant cells are differentiated into corresponding tissues and young buds grow out for subsequent rooting culture of rootless seedlings. In this solution, the explant is selected as the rhizome of Cymbidium goeringii, which is easy to inoculate and not easily contaminated, has a fast proliferation and differentiation speed, a high proliferation rate, a low browning rate of the explant and the culture medium, and can grow into relatively large seedlings without frequent subculture inoculation.

[0026] In the above process, in the S2 stage, a proliferation medium is used, and its basic proliferation medium is a 1 / 2MS medium added with basic proliferation components, and the basic proliferation components include 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), sucrose, Ag, and AC; in the S3 stage, a differentiation medium is used, and its basic differentiation medium is a 1 / 2MS medium added with basic differentiation components, and the basic differentiation components include 6-BA, NAA, Ag, and sucrose. In the S1 stage, a basic seed germination medium is used, which is a 1 / 2MS medium added with basic germination components, and the basic germination components include 6-BA, NAA, Ag, AC, peptone, and sucrose. In the S4 stage, a basic rootless seedling rooting medium is used, which is a 1 / 2MS medium added with basic rootless seedling rooting components, and the basic rootless seedling rooting components include indolebutyric acid (IBA), NAA, Ag, AC, PVP, coconut water (CW), and sucrose. The 1 / 2MS medium containing 6-BA, NAA, sucrose, Ag, and AC can provide a suitable environment and nutrients for the proliferation of the rhizomes of Cymbidium goeringii; the 1 / 2MS medium containing 6-BA, NAA, Ag, and sucrose can provide a suitable environment and nutrients for the differentiation of the rhizomes of Cymbidium goeringii; the 1 / 2MS medium containing 6-BA, NAA, Ag, AC, peptone, and sucrose can provide a suitable environment and nutrients for the germination of Cymbidium goeringii seeds; the 1 / 2MS medium containing IBA, NAA, Ag, AC, PVP, CW, and sucrose can provide a suitable environment and nutrients for the rooting of rootless seedlings of Cymbidium goeringii.

[0027] In addition, in the S1 seed germination culture step, the spring orchid seeds are cultured using seed germination medium for 120 days; in the S2 proliferation culture step, the spring orchid rhizomes are cultured using proliferation medium for 90 days; in the S3 differentiation culture step, the spring orchid rhizomes are cultured using differentiation medium for proliferation culture for 90 days; in the S4 rooting culture step, the spring orchid rootless seedlings are cultured using rooting medium for 90 days.

[0028] The above-mentioned seed germination culture, proliferation culture, differentiation culture and rooting culture time can ensure that the spring orchid seeds, rhizomes and rootless seedlings grow fully, creating conditions for subsequent growth. The flower clams, preserved egg yolks, shrimps and squids added to the proliferation culture medium used in the method of the present invention can promote the division of rhizome cells, thereby achieving rapid proliferation. PVP, CA and succinic acid added to the differentiation culture medium used can prevent the browning problem that occurs during the differentiation of rhizomes and promote the differentiation of rhizomes. DTT, cysteine, GSH and succinic acid effectively promote the differentiation of rhizomes into buds, but DTT, GSH and succinic acid easily cause the rhizomes to brown. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 These are typical photos of the results of the rhizome proliferation experiment of Cymbidium orchid (Example 3 and Experimental Example 1, scale 1m).

[0030] Figure 2 These are typical photos of the results of the rhizome differentiation experiment of Cymbidium orchid (Example 4 and Experimental Example 1).

[0031] Figure 3 Statistical graph of the experimental results of the effects of four animal-derived additives on the proliferation of Cymbidium orchid (Example 3 and Experimental Example 1).

[0032] Figure 4 Statistical graph of experimental results of the effects of six anti-browning agents on the anti-browning effect in rhizome differentiation of Cymbidium orchid (Example 4 and Experimental Example 1).

[0033] Figure 5 Statistical graph of experimental results of the effects of six anti-browning agents on the average number of buds in rhizome differentiation of Cymbidium orchid (Example 4 and Experimental Example 1).

[0034] Figure 6 Statistical graph of experimental results of the effects of six anti-browning agents on the average bud height (cm) during rhizome differentiation of Cymbidium orchid (Example 4 and Experimental Example 1). DETAILED DESCRIPTION

[0035] The invention will be introduced in detail below with specific examples. In the following specific embodiments, the reagents used are as follows: 1 / 2MS powder (Shanghai Yika Biotechnology Co., Ltd.); 6-BA, NAA, IBA (Beijing Kangbeisi Technology Co., Ltd.); sucrose, Ag (Shanghai Yika Biotechnology Co., Ltd.); AC (Beijing Kangbeisi Technology Co., Ltd.); clam meat, preserved egg yolk, shrimps and squids; various anti-browning agents (Plant Tissue Culture Biochemical Reagents Co., Ltd.). The Cymbidium goeringii capsules used in the present invention are from the Cymbidium germplasm resource nursery of Guizhou University.

[0036] Example 1: Preparation of culture media

[0037] Prepare seed germination medium, proliferation medium, differentiation medium and rooting medium, which are used for Cymbidium goeringii seed germination experiment, Cymbidium goeringii rhizome proliferation experiment, Cymbidium goeringii rhizome differentiation experiment and Cymbidium goeringii rootless seedling rooting experiment respectively.

[0038] The basic seed germination medium is 1 / 2MS medium (pH 5.6) containing basic germination components, and the germination promoting components and their contents are respectively: 0.5 mg / L 6-BA, 0.5 mg / L NAA, 30.0 g / L sucrose, 7.5 g / L Ag, 1.0 g / L AC, 2 g / L peptone.

[0039] The basic proliferation medium is 1 / 2MS medium (pH 5.6) containing basic proliferation components, and the proliferation promoting components and their contents are respectively: 0.5 mg / L 6-BA, 1.0 mg / L NAA, 30.0 g / L sucrose, 7.5 g / L Ag, 1.0 g / L AC.

[0040] The basic differentiation medium is 1 / 2MS medium (pH 5.6) containing basic differentiation components, and the growth promoting components and their contents are respectively: 1.0 mg / L 6-BA, 0.1 mg / L NAA, 30.0 g / L sucrose, 7.5 g / L Ag.

[0041] The basic rooting medium is 1 / 2MS medium (pH 5.6) containing basic rooting components, and the rooting promoting components and their contents are respectively: 1.0 mg / L IBA (indolebutyric acid), 0.5 mg / L NAA, 20.0 g / L sucrose, 7.5 g / L Ag, 2.0 g / L AC, 2.0 g / L PVP, 100 mL / L CW.

[0042] An animal-derived additive or an anti-browning agent is added to the above-mentioned basic proliferation medium and basic differentiation medium. The animal-derived additive is prepared by the following method: The animal-derived components of clam meat, preserved egg yolk, shrimp, and squid are respectively controlled within the temperature range of 25-30°C and sealed for 3 days (in the actual operation process, the sealing time can be 2-4 days, and relatively ideal effects can be achieved). The medium without the animal-derived additive is used as a control. Four concentrations are set for each treatment; in the differentiation medium, four anti-browning agents, namely DTT, cysteine, GSH, and succinic acid, must be filter-sterilized, and two anti-browning agents, PVP and CA, are sterilized using an autoclave. Five concentrations are set for each anti-browning agent, and the stock solution concentration is configured to be 0.1 g / mL. The medium without the anti-browning agent is used as a control.

[0043] The 1 / 2MS medium is a conventional medium in the prior art, and the conventional preparation method and formula are as follows (commercial 1 / 2MS medium powder can also be purchased and dissolved in water to prepare the 1 / 2MS medium):

[0044] Preparation of the 1 / 2MS medium stock solution:

[0045] 20× Macronutrients (1L): 19 g KNO3, 16.5 g NH4NO3, 4.4 g CaCl2·2H2O, 3.7 g MgSO4·7H2O, 1.7 g KH2PO4;

[0046] 200× Micronutrients (1L): 4.46 g MnSO4·4H2O, 1.72 g ZnSO4·7H2O, 0.166 g KI, 1.24 g H3BO3, 0.05 g Na2MoO4·2H2O, 0.005 g CuSO4·5H2O, 0.005 g CoCl2·6H2O.

[0047] 200× Vitamins and Amino Acids (1L): 20 g inositol, 0.1 g niacin (VB3), 0.1 g pyridoxine hydrochloride (VB6), 0.02 g thiamine hydrochloride (VB1), 0.4 g glycine.

[0048] 200× Iron Salt (1L): 5.56 g FeSO4·7H2O, 7.46 g Na2EDTA·2H2O, first dissolve them separately in 200 mL of distilled water, heat and stir, and make up to 1L.

[0049] Using the above-prepared stock solution, according to the dilution ratio, prepare the basic 1 / 2MS culture solution: 1× macronutrients, 1× micronutrients, 1× vitamins and amino acids, 1× iron salt, adjust the pH to 5.6 after volume adjustment to obtain the 1 / 2MS medium (1× liquid medium, which needs to be sterilized by high temperature and high pressure).

[0050] On the basis of 1 / 2 MS medium, other components such as plant growth regulators were added to obtain a basic proliferation medium and a basic differentiation medium respectively. A rotting animal-derived additive to be tested and an anti-browning agent were added to the basic proliferation medium and the basic differentiation medium respectively to obtain a proliferation medium to be tested and a differentiation medium to be tested for subsequent experiments.

[0051] Example 2: Cymbidium goeringii seed germination experiment

[0052] Prepare 3 Cymbidium goeringii capsules pollinated for 130 days. After disinfection, they were evenly sown on the medium. Disinfection process: soak in washing powder for 30 min, then rinse with running water and place in a laminar flow hood. First soak in 75% alcohol for 30 s on the laminar flow hood, rinse once with sterile water, then soak in 1% sodium hypochlorite solution for 20 min, and then rinse with sterile water 5 times. Place on sterile paper for use. A total of 30 bottles were sown; at 25 °C, with a light intensity of 2000 Lx and a photoperiod of 12 h / d; the germination rate was counted after 120 days.

[0053] Preparation of the seed germination medium to be tested (taking 1 L as an example): Heat about 300 mL of distilled water. When small bubbles appear, add 7.5 g of Ag, stir evenly, and after complete dissolution, add 30.0 g of sucrose, 2.47 g of 1 / 2 MS medium powder, 1.0 g of AC and 2.0 g / L of peptone, and stir until completely dissolved. Add 0.5 mL of 1.0 mg / mL 6-BA stock solution and 0.5 mL of 1.0 mg / mL NAA stock solution of plant growth regulators. Make the solution up to 1 L, adjust the pH to 5.6, and divide it into orchid bottles respectively, with a filling volume of 100 mL / bottle, a total of 10 bottles. The medium was sterilized using an autoclave. After taking it out of the pot, shake the medium well and let it cool for later use.

[0054] Example 3: Cymbidium goeringii rhizome proliferation experiment

[0055] Prepare aseptic and well-growing rhizomes (the rhizomes were induced from seeds, which is a conventional method in the prior art, and can be referred to in Example 2), with a diameter of about 2 mm, cut into 1 cm lengths for standby. Transfer 1 cm-length Cymbidium goeringii rhizomes into the proliferation medium, 10 in each bottle, and evenly inoculate the rhizome materials on the surface of the medium (the proliferation medium to be tested). At 25 °C, with a light intensity of 2000 Lx and a photoperiod of 12 h / d. After 90 days, take out the rhizomes in the bottles and count the number of newly added rhizomes, and record the number of rhizomes of the newly added rhizomes on each medium.

[0056] Preparation of the proliferation culture medium to be tested (taking 1L as an example): Heat about 300mL of distilled water. When small bubbles appear, add 7.5g of agar, stir evenly, and after complete dissolution, add 30.0g of sucrose, 2.47g of 1 / 2MS culture medium powder and 1.0g of activated carbon, stir until completely dissolved, add 0.5mL of 1.0mg / mL 6-BA stock solution of plant growth regulator and 1.0mL of 1.0mg / mL NAA stock solution, and add animal-derived additives of various concentrations to each treatment. The required animal-derived additives are all daily ingredients, and are sealed at 25-30℃ for 3 days before the experiment (in actual operation, the sealed storage time can be 2-4 days, which can achieve relatively ideal results). Then process each material: Use shrimps, remove the heads and shells, and remove the shrimp threads to get the shrimps, wash them with tap water, and cut them into pieces of about 0.5cm in volume in advance. 3 Remove the shell and internal organs of the clams and use the meat, wash with tap water and cut into pieces of about 0.5cm in size in advance. 3 Use small squids about 6-8cm long, remove the internal organs, wash with tap water and cut into pieces about 0.5cm in size in advance. 3Small pieces; For the egg yolk of preserved eggs, Songhua preserved eggs are selected as raw materials. After the preserved eggs are peeled, the egg white part is removed, and the egg yolk is left and cut into thin slices about 1 mm thick in advance. The addition amounts of different concentrations of animal-derived additives are calculated based on the mass of solid substances before juicing. For example, to prepare 1 L of a medium with a squid concentration of 0.05 g / L, 0.05 g of small squid pieces should be added, poured into a beaker, made up to 1000 mL with distilled water, then poured into a juicer and juiced using the fruit and vegetable cold drink mode. For convenience of use and accurate dosage, a squid stock solution can be prepared. Taking the preparation of 1 L of each of the media with squid concentrations of 0.05 g / L, 0.1 g / L, 0.5 g / L, and 1.0 g / L of animal-derived additives as an example, calculate the total mass of small squid pieces required for the experiment as 0.05 g + 0.1 g + 0.5 g + 1.0 g = 1.65 g, make up to 1 L, and juice to obtain a mixture which is the squid stock solution (a suspension of squid tissue, without filtration treatment). After mixing evenly, calculate the addition amount of the animal-derived additive according to (mass of each treatment / total mass of the additive) × 1000 mL. Calculate the addition amount of the squid stock solution in 1 L of the medium with a squid concentration of 0.05 g / L as (0.05 g / 1.65 g) × 1000 mL, that is, 30.3 mL; calculate the addition amount of the squid stock solution in 1 L of the medium with a squid concentration of 0.1 g / L as (0.1 g / 1.65 g) × 1000 mL, that is, 60.6 mL; calculate the addition amount of the squid stock solution in 1 L of the medium with a squid concentration of 0.5 g / L as (0.5 g / 1.65 g) × 1000 mL, that is, 303.0 mL; calculate the addition amount of the squid stock solution in 1 L of the medium with a squid concentration of 1.0 g / L as (1.0 g / 1.65 g) × 1000 mL, that is, 606.1 mL. Make up the medium to 1 L and adjust the pH to 5.6. In this way, in the prepared proliferation medium, the squid concentrations are 0.05 g / L, 0.1 g / L, 0.5 g / L, and 1.0 g / L respectively. Each is sub-packed into orchid bottles, with a filling amount of 100 mL / bottle, a total of 10 bottles. After autoclaving at high temperature and high pressure, shake the medium well and set aside after cooling.

[0057] Example 4: Cymbidium goeringii rhizome differentiation experiment

[0058] Transfer Cymbidium goeringii rhizomes with a length of 1 cm into the differentiation medium, 10 per bottle, and culture in a culture room. 25 °C, light 2000 Lx, 12 h / d. After about 15 d, the rhizomes start to differentiate, and after about 90 d, the small buds grow to 2 - 4 cm. Record the number and length of the newly added buds on the medium, and count the browning situation of the materials and the medium.

[0059] Preparation of differentiation medium to be tested (taking 1L as an example): Take about 300mL of distilled water and heat it. When small bubbles appear, add 7.5g of agar, stir evenly, and after dissolving completely, add 30.0g of sucrose and 2.47g of 1 / 2MS medium powder, stir until completely dissolved, and add 1.0mL of 1.0mg / mL 6-BA mother solution and 0.1mL of 1.0mg / mL NAA mother solution of plant growth regulator. Make the solution volume to 1L, adjust the pH to 5.6, and dispense it into orchid bottles, with a filling volume of 100mL / bottle, for a total of 10 bottles. PVP and CA are directly added to the culture medium in the early stage, sterilized in a high-pressure sterilizer, shake the culture medium evenly after taking it out of the pot, cool it and set it aside, and filter and sterilize the other four anti-browning agents. Place the prepared four anti-browning agents on a clean bench, filter and sterilize them with a 0.22μm water filter membrane and set them aside. When the culture medium is prepared and cooled to 50-60°C, add different volumes of filtered anti-browning agent mother solution to the culture medium in a clean bench, wherein the concentration of the anti-browning agent mother solution is 0.1 g / mL, and mix well for later use.

[0060] Example 5: Rooting experiment of spring orchid rootless seedlings

[0061] Select 1.5-2.5cm spring orchid tissue culture rootless seedlings with relatively consistent growth status and transfer them to rooting medium. 5 experimental materials per bottle, cultured in the culture room. 25℃, light 2000Lx, 12h / d. After 90 days, the rooting rate, average root length and average number of roots of each treatment were counted. Rooting rate = number of rooted seedlings / number of inoculated seedlings × 100%, average root length = total root length / number of rooted seedlings, average number of roots = total number of roots / number of rooted seedlings.

[0062] Preparation of the rooting medium to be tested (taking 1L as an example): Heat about 300mL of distilled water. When small bubbles appear, add 7.5g of agar, stir evenly, and after complete dissolution, add 20.0g of sucrose, 2.47g of 1 / 2MS medium powder, 2.0gAC, 2.0g / L PVP and 100mL / L CW, stir until completely dissolved, and add 1.0mL of 1.0mg / mL IBA mother solution and 0.5mg / mL NAA mother solution of plant growth regulator. Make the solution dilute to 1L, adjust the pH to 5.6, and dispense into orchid bottles, with a filling volume of 100mL / bottle, for a total of 10 bottles. Sterilize the culture medium in a high pressure sterilizer, shake the culture medium evenly after taking it out of the pot, and cool it for use.

[0063] Experimental Example 1: Screening of proliferation medium and differentiation medium

[0064] An animal-derived additive was added to the basal proliferation medium to obtain the proliferation medium to be tested, and Cymbidium goeringii rhizomes were proliferated and cultured according to the method in Example 2. After the culture was completed, the proliferation multiple was calculated. Proliferation multiple = number of new lateral branches on the inoculated rhizomes / number of inoculated rhizomes (4 replicates were set for each group of experiments).

[0065] An anti-browning agent was added to the basal differentiation medium to obtain the differentiation medium to be tested, and Cymbidium goeringii rhizomes were differentiated and cultured according to the method in Example 3. After the culture was completed, the browning situation of the rhizomes was observed, and the browning rate, average number of buds, and average bud height were calculated. The browning rate, average number of buds, and average bud height were statistically analyzed after 90 d. Browning rate = number of browned rhizomes / number of inoculations × 100%. Only buds with a length > 0.2 cm were counted for the average number of buds and average bud height (5 replicates were set for each group of experiments).

[0066] Different proliferation media to be tested and differentiation media to be tested were prepared, and Cymbidium goeringii rhizome proliferation experiments and Cymbidium goeringii rhizome differentiation experiments were carried out according to the methods in Example 3 and Example 4. The results of rhizome proliferation culture and differentiation culture are as Figure 1-6 shown. Figures 3-6 The results were analyzed for significant differences using Duncan's test, and different lowercase letters indicate significant differences at the 0.05 level.

[0067] Among them, Figure 1 are the experimental results when 4 animal-derived additives were added in the Cymbidium goeringii proliferation experiment, with no addition of rotten animal-derived additive as the control (basal proliferation medium). A-I respectively represent: A: control (basal proliferation medium), which is the control when the concentration of the added animal-derived additive is 0 g / L; B: squid 0.1 g / L; C: shrimp 0.5 g / L; D: preserved egg yolk 0.5 g / L; E: razor clam meat 1.0 g / L; F: razor clam meat 0.5 g / L.

[0068] For the Cymbidium goeringii rhizome proliferation culture experiment, referring to Figure 3 , adding animal-derived additives to the basal proliferation medium can significantly increase the proliferation multiple of rhizomes at a certain concentration. When adding squid and preserved egg yolk, all concentrations have a promoting effect on the proliferation of Cymbidium goeringii, and squid has a more obvious promoting effect on the proliferation of rhizomes: when the additive is squid, the highest number of new rhizomes in a single rhizome reaches 22, and the average proliferation multiple is above 10. Low concentrations of shrimp have a promoting effect on rhizome proliferation, but high concentrations of shrimp have an obvious inhibitory effect on rhizome proliferation, and the inhibitory effect becomes more obvious with the increase in concentration. Razor clam meat has a promoting effect on rhizome proliferation, but the effect is not obvious.

[0069] Combined with Figure 1 and Figure 3 shown, 0.1 g / L squid ( Figure 1B) and 0.5g / L shrimp ( Figure 1 C) can significantly promote the proliferation of rhizomes. When the concentration of 0.1g / L squid is 15, the proliferation of the rhizomes is only 7.5 in the control group. Low concentrations of preserved egg yolk have a better promoting effect on the proliferation of Chunlan rhizomes ( Figure 1 D). Clam meat has no obvious effect on the proliferation of spring orchid rhizomes. Even low and high concentrations have an inhibitory effect on the growth of spring orchid rhizomes. At 0.5g / L, it has a significant inhibitory effect on the proliferation of spring orchid rhizomes ( Figure 1 F). In summary, 0.1g / L squid can increase the proliferation multiple to 15; shrimp can only increase the proliferation multiple to about 10 when used in a larger amount (0.5g / L); preserved egg yolk can only increase the proliferation multiple to about 9; and the proliferation effect of clam meat on the rhizomes of spring orchids is even inhibited. This shows that although animal-derived additives all contain putrescine (putrescine is commonly found in aquatic and livestock products), the components of putrescine-containing additives from different animal sources are different, which ultimately leads to differences in the growth-promoting effects of additives on the rhizomes of spring orchids. Some even inhibit the proliferation of spring orchid rhizomes due to their toxic effects on plants. As an animal-derived additive for spring orchids, squid has a very significant growth-promoting effect compared to other substances, which was not expected by the inventor before the experiment. In addition, the concentration setting of squid additives to achieve growth-promoting effects is very critical. At a concentration of 0.1g / L, it significantly promotes the proliferation of spring orchid rhizomes. However, at other concentrations, it is basically difficult to show a promoting effect.

[0070] Figure 2 These are photos of rhizome growth in the differentiation experiment of Cymbidium orchid. When the added concentration is 0 g / L, it is the control (basic differentiation medium). AK represent: A: control (basic differentiation medium), when the anti-browning added concentration is 0 g / L; B: polyvinyl pyrrolidone (PVP) 5.0 g / L; C: dithiothreitol (DTT) 0.25 g / L; D: cysteine 0.25 g / L; E: glutathione (GSH) 0.1 g / L; F: succinic acid 0.15 g / L; G: succinic acid 0.25 g / L; H: cysteine 0.25 g / L; I: cysteine 0.25 g / L; J: dithiothreitol (DTT) 0.5 g / L; K: cysteine 0.25 g / L; L: glutathione (GSH) 0.2 g / L.

[0071] For the rhizome differentiation culture experiment of spring orchid, refer to Figure 2 , Figure 4 , Figure 5 , Figure 6, all six anti-browning agents can increase the number of differentiated buds of Cymbidium goeringii rhizomes and promote the differentiation of Cymbidium goeringii. PVP, CA, and succinic acid have obvious anti-browning effects during the differentiation process of Cymbidium goeringii rhizomes, and DTT and succinic acid promote the growth of the height of Cymbidium goeringii buds. The anti-browning effects of DTT, cysteine, and GSH are relatively poor, and the browning phenomenon becomes more serious with the increase in concentration. Low-concentration cysteine promotes the growth of the average bud height of Cymbidium goeringii, and high-concentration (0.25 g / L) cysteine can significantly promote the differentiation of rhizomes, with the number of differentiated buds being 11.5 (2.5 times that of the control). Succinic acid has more obvious effects on promoting the differentiation of rhizomes, anti-browning, and the growth of the average bud height, and the average number of buds in all treatments is higher than that of the control.

[0072] As Figure 2 shown, 5.0 g / L PVP ( Figure 2 B), 0.25 g / L DTT ( Figure 2 C), 0.25 g / L cysteine ( Figure 2 D), 0.1 g / L GSH ( Figure 2 E), and 0.15 g / L succinic acid ( Figure 2 F) can significantly promote the increase in the average number of buds of rhizomes and promote differentiation. 0.25 g / L succinic acid promotes the differentiation of Cymbidium goeringii rhizomes and simultaneously significantly promotes the growth of the average bud height of Cymbidium goeringii, with a relatively obvious anti-browning effect ( Figure 2 G). 0.05 g / L cysteine also promotes the differentiation of Cymbidium goeringii rhizomes and simultaneously significantly promotes the growth of the average bud height of Cymbidium goeringii, but the rhizomes are prone to browning ( Figure 2 H), and the browning becomes more serious with the increase in concentration (2I). 0.25 g / L succinic acid can promote the differentiation of Cymbidium goeringii rhizomes and simultaneously significantly promote the growth of the average bud height of Cymbidium goeringii, with a relatively obvious anti-browning effect ( Figure 2 G). At the same time, during the differentiation process of Cymbidium goeringii, excessive use of DTT will cause the growth of rhizomes to be restricted and will cause the browning and even albino of rhizomes. The rhizomes are severely browned at 0.5 g / L DTT ( Figure 2 J), and when its concentration increases to 1.0 g / L, albino phenomena begin to occur in the rhizomes ( Figure 2 K). 0.2 g / L GSH promotes the proliferation of rhizomes, and the rhizomes are severely browned ( Figure 2 L), and with the increase in concentration, the proliferation effect is better and the browning phenomenon is more obvious.

[0073] According to Figure 4The experimental results show that succinic acid has the most significant browning inhibition effect, and can significantly inhibit the browning of Cymbidium goeringii rhizomes at a low concentration of 0.05 g / L. Moreover, the concentration range of the anti-browning effect of succinic acid is relatively wide, and it can play a role in the range of 0.05 - 0.25 g / L. In actual use, the concentration of succinic acid can be adjusted according to other actual needs, and it will not overly affect the anti-browning effect of succinic acid. That is, the concentration requirements for achieving the anti-browning effect of succinic acid are relatively flexible, which is also the advantage of using succinic acid as an additive. Combining Figures 4-6 with the experimental results, 0.25 g / L of succinic acid simultaneously has significant effects of anti-browning and promoting the differentiation of Cymbidium goeringii rhizomes (the number of differentiated buds and bud height). However, when using other anti-browning agents at their corresponding test concentrations, none of them can achieve the triple effects of significantly inhibiting browning, significantly promoting the number of differentiated buds of Cymbidium goeringii rhizomes, and significantly promoting the bud height of rhizomes.

[0074] The above are only examples of the present invention. Specific technical solutions and / or common knowledge such as characteristics well known in the art are not described in detail herein. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several deformations and improvements can still be made, and these should also be regarded as the protection scope of the present invention, and none of these will affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. Cymbidium goeringii rhizome tissue culture medium containing animal-derived additives, characterized in that: It includes a proliferation medium; the proliferation medium is a basic proliferation medium added with an animal-derived additive; The raw material of the animal-derived additive is squid or shrimp; When the raw material of the animal-derived additive is squid, its addition amount is 0.1 g / L; When the raw material of the animal-derived additive is shrimp, its addition amount is 0.5 g / L; The processing method of the animal-derived additive is: placing the raw material at 25 - 30 °C for 2 - 4 days under sealed conditions to obtain it.

2. The Cymbidium goeringii rhizome tissue culture medium containing animal-derived additives according to claim 1, wherein: The basic proliferation medium is a 1 / 2 MS medium containing 6-BA, NAA, sucrose, agar, and activated carbon.

3. The Cymbidium goeringii rhizome tissue culture medium containing animal-derived additives according to claim 1 or 2, characterized in that: It also includes a differentiation medium; the differentiation medium is a basic differentiation medium added with an anti-browning agent; the anti-browning agent includes at least one of dithiothreitol, cysteine, glutathione, polyvinylpyrrolidone, citric acid, and succinic acid.

4. The Cymbidium goeringii rhizome tissue culture medium containing animal-derived additives according to claim 3, characterized in that: The basic differentiation medium is a 1 / 2 MS medium containing 6-BA, NAA, sucrose, and agar.

5. The Cymbidium goeringii rhizome tissue culture medium containing animal-derived additives according to claim 4, characterized in that: The concentration of polyvinylpyrrolidone in the differentiation medium is 1.0 - 5.0 g / L, the concentrations of citric acid and dithiothreitol in the differentiation medium are 0.25 - 3.0 g / L respectively, the concentrations of succinic acid and cysteine in the differentiation medium are 0.05 - 0.25 g / L, and the concentration of glutathione in the differentiation medium is 0.1 - 0.5 g / L.

6. The Cymbidium goeringii rhizome tissue culture medium containing animal-derived additives according to claim 5, characterized in that: The anti-browning agent is succinic acid, and its working concentration is 0.05 - 0.25 g / L.

7. A method for tissue culture of Cymbidium goeringii rhizome, characterized in that: It includes the following steps carried out in sequence: S1 Seed germination and culture step: Inducing the culture of Cymbidium goeringii capsules to obtain Cymbidium goeringii rhizomes; S2 Proliferation culture step: Using the proliferation medium to culture Cymbidium goeringii rhizomes to obtain proliferation culture tissues; the proliferation medium is a basic proliferation medium added with an animal-derived additive; the raw material of the animal-derived additive is squid or shrimp; When the raw material of the animal-derived additive is squid, its addition amount is 0.1 g / L; When the raw material of the animal-derived additive is shrimp, its addition amount is 0.5 g / L; The processing method of the animal-derived additive is: placing the raw material at 25 - 30 °C for 2 - 4 days under sealed conditions to obtain it; S3 Differentiation culture step: Using the differentiation medium to culture the rhizomes separated from the proliferation culture tissues to obtain rootless seedlings; the differentiation medium is a basic differentiation medium added with an anti-browning agent, and the anti-browning agent for differentiation promotion includes at least one of dithiothreitol, cysteine, glutathione, polyvinylpyrrolidone, citric acid, and succinic acid; S4 Rooting culture step: Inducing the culture of rootless seedlings to obtain small seedlings with roots.

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