A method for obtaining Dendrobium officinale seedlings

Abstract

The present invention relates to a method for obtaining Dendrobium officinale seedlings, and belongs to the technical field of artificial planting of Dendrobium officinale. The main technical scheme adopted is: the method for obtaining Dendrobium officinale seedlings comprises the following steps: using Dendrobium officinale waste stubble as raw material; making the Dendrobium officinale waste stubble into cuttings; inserting the cuttings into a culture matrix, placing them in a light culture box for light culture treatment, and obtaining Dendrobium officinale seedlings. The present invention uses Dendrobium officinale waste stubble as raw material for the first time, and cooperates with the set light culture treatment conditions to successfully obtain Dendrobium officinale seedlings. Compared with the prior art, the method for obtaining Dendrobium officinale seedlings of the present invention has the advantages of low cost, simple operation, short time, and suitability for industrialization.

Description

Technical Field

[0001] The invention relates to the technical field of artificial planting of Dendrobium officinale, in particular to a method for obtaining Dendrobium officinale seedlings. Background Art

[0002] Dendrobium is a traditional precious Chinese medicinal material made from the stems of the Dendrobium plant of the Orchidaceae family. It was first recorded in the "Shennong Bencao Jing". Dendrobium candidum is the most precious and expensive of the Dendrobium varieties, and enjoys the reputation of "life-saving magic grass". Among the "Nine Great Magic Grasses of China" listed in the "Daoist Canon" of the Tang Dynasty, Dendrobium candidum is listed first. Dendrobium candidum is regarded as the "top grade of medicine" and has many functions such as nourishing yin and promoting body fluid, strengthening the spleen and appetite, protecting the liver and promoting gallbladder, relieving fatigue, nourishing the skin, anti-oxidation, anti-tumor, lowering blood sugar, and enhancing immunity. It has extremely high medicinal and economic value.

[0003] Dendrobium officinale Kimura et Migo is a perennial epiphytic herb of the genus Dendrobium in the orchid family. It likes to grow in a semi-shady and semi-sunny environment. It is mainly distributed in southern and southwestern China. It grows in valleys or forest rocks or trees at high altitudes. It has strict requirements on the environment, and its seeds are small and have no endosperm. It is extremely difficult to germinate in the natural environment. In addition, the low reproductive capacity of wild Dendrobium officinale and the indiscriminate collection and digging of artificial plants have caused wild Dendrobium to be on the verge of extinction. As early as the 1980s, wild Dendrobium officinale had been listed as a rare and endangered plant under key protection by the state, and its collection and sale were prohibited. Therefore, artificial cultivation of Dendrobium officinale has become the only way to meet market demand. The biggest cost of artificial cultivation is the cost of seedlings. Therefore, reducing the cost of seedling cultivation is the key to developing the Dendrobium officinale industry.

[0004] In recent years, due to the emergence of Dendrobium officinale tissue culture technology and intensive artificial cultivation technology, great progress has been made in obtaining Dendrobium officinale seedlings. At present, the research on Dendrobium officinale tissue culture technology mainly includes: sterile sowing, protocorm induction, proliferation and differentiation, seedling rooting, explant induction of organs and protocorms, cell culture, in vitro flowering, artificial seed and germplasm resource preservation, tetraploid induction, etc.

[0005] Domestic scholars have conducted a lot of research on the selection of explants for Dendrobium officinale. Most of them use explants such as seeds, stem segments, root tips, adventitious buds, young leaves, and sterile seedling stem segments of Dendrobium officinale to obtain protocorms, and obtain regenerated seedlings or complete plants through proliferation, differentiation, and rooting culture. However, in the tissue culture method using Dendrobium officinale seeds as materials, due to the shortage of seed resources, it is difficult to produce Dendrobium officinale seedlings in factories. The proliferation rate of protocorms as explants is relatively low, and the cost of large-scale production is relatively high. When stem tips are used as explants, most of them are attached to stem segments with buds, and the survival rate of stem tips alone as explants is low. When leaves are used as explants, the degree of differentiation of leaves is relatively high, and the ability to be cultivated into complete plants is relatively low, and it is not suitable for factory production.

[0006] In summary, the existing methods for obtaining Dendrobium officinale seedlings have at least the problems of high cost, long time, and complicated operation, and are not suitable for factory production. Summary of the invention

[0007] In view of this, the present invention provides a method for obtaining Dendrobium officinale seedlings, the main purpose of which is to provide a method for obtaining Dendrobium officinale seedlings with low cost, short time and simple operation, so as to be suitable for factory production.

[0008] In order to achieve the above object, the present invention mainly provides the following technical solutions:

[0009] On the one hand, an embodiment of the present invention provides a method for obtaining a Dendrobium officinale seedling, wherein the method for obtaining a Dendrobium officinale seedling comprises the following steps:

[0010] The method comprises the following steps: using the waste stubble of Dendrobium officinale as raw material; preparing the waste stubble of Dendrobium officinale into cutting strips;

[0011] Light culture step: inserting the cuttings into a culture matrix, placing them in a light culture box for light culture treatment, and obtaining Dendrobium officinale seedlings.

[0012] Preferably, in the light culturing step: the light culturing treatment includes: first culturing under the first set conditions for 15-40 days, and then culturing under the second set conditions for 25-35 days; wherein,

[0013] The first setting conditions include: light intensity of 20000-40000LX, preferably 30000LX; temperature of 20-35°C, preferably 28°C; humidity of 50-80%, preferably 60-75%;

[0014] The second setting conditions include: alternating light treatment for 10-14 hours and dark treatment for 10-14 hours; wherein, during the light treatment: humidity is 50-80%, preferably 60-75%; light intensity is 3000-10000LX, preferably 5000LX; temperature is 20-35°C, preferably 30°C; wherein the temperature of the dark treatment is 20-35°C, preferably 25°C.

[0015] Preferably, the culture is carried out under the first set conditions for 25-35 days, preferably 29-31 days; preferably, the culture is carried out under the second set conditions for 29-31 days.

[0016] Preferably, the step of preparing cuttings comprises: removing the fibrous roots on the waste stubble of Dendrobium officinale, leaving the stem and the roots connected to the stem base to obtain cuttings; and disinfecting the cuttings.

[0017] Preferably, the length of the cuttings is 3-5 cm.

[0018] Preferably, the step of disinfecting the cuttings comprises: soaking the cuttings in lime water for disinfection.

[0019] Preferably, the cuttings are immersed in lime water for 8-15 hours; and / or the lime water is a solution prepared from slaked lime and water with a mass concentration of 1-5%, preferably 2%.

[0020] Preferably, in the light culturing step: the culture matrix includes one or two of pine bark and vermiculite.

[0021] Preferably, the thickness of the culture matrix is ​​1.5-2 cm.

[0022] Preferably, in the light culture step: the root end of the cutting is inserted into the culture matrix; preferably, the length of the cutting inserted into the culture matrix is ​​0.8-1.2 cm.

[0023] Preferably, the cutting density of the cuttings on the culture matrix is ​​4500-5200 plants per square meter.

[0024] Compared with the prior art, the method for obtaining Dendrobium officinale seedlings of the present invention has at least the following beneficial effects:

[0025] The embodiment of the present invention proposes a method for obtaining Dendrobium officinale seedlings, which uses the discarded tissue (i.e., the waste stubble of Dendrobium officinale) after the fresh strips of Dendrobium officinale are harvested as raw materials, and adopts the set light culture conditions to successfully obtain Dendrobium officinale seedlings. Here, (1) the raw materials used in the embodiment of the present invention are usually directly discarded by the planting base. Therefore, the method of the embodiment of the present invention not only realizes the recycling and full utilization of agricultural residues, but also has low cost. (2) The method of the embodiment of the present invention only needs to disinfect the waste stubble of Dendrobium officinale with slaked lime, cut it into a common culture matrix, and carry out light culture treatment. In contrast, the conventional tissue culture seedlings in the prior art need to be proliferated and differentiated, and the seedlings need to be rooted in a sterile environment. In contrast, the method of the embodiment of the present invention only needs to be operated in a conventional environment, and ordinary personnel can complete the operation. Therefore, the method of the embodiment of the present invention has the advantage of being easy to operate. (3) The method of the embodiment of the present invention takes 60 days (about 60 days) to obtain strong seedlings with an average plant height of 7 cm, an average total root length of 10 cm, and a root number of 4.26. To obtain a Dendrobium officinale seedling of the same size, it takes 180 days to use a hormone-free tissue culture method and 75 days to use a hormone tissue culture method; the methods of the prior art are much longer than the time used by the method of the embodiment of the present invention. For example, in the method of using seeds for tissue culture, it usually takes 1-2 years from seed germination to transplanting into seedlings. (4) The method of the embodiment of the present invention only uses waste stubble of Dendrobium officinale, plus common materials such as slaked lime and pine bark, and then treats it with light. The conventional tissue culture method of Dendrobium officinale in the prior art usually requires processes such as protocorm induction, proliferation and differentiation, and rooting of seedlings, which require professionals to use professional equipment and reagents in a special tissue culture environment, and require a lot of manpower and material resources. In summary, the method for obtaining Dendrobium officinale seedlings proposed in the embodiment of the present invention is a method with low cost, short time, and simple operation, which is suitable for factory production.

[0026] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a real picture of the waste stubble of Dendrobium officinale;

[0028] Figure 2 This is a physical picture of the cuttings;

[0029] Figure 3 This is a photo of the seedlings germinating after the cuttings were inserted into the culture matrix (vermiculite) and cultured under light for 20 days in Example 1;

[0030] Figure 4This is a photo of the germination of seedlings after the cuttings were inserted into the culture medium (pine bark) and cultured under light for 20 days in Example 1;

[0031] Figure 5 yes Figure 4 A partial enlarged physical picture;

[0032] Figure 6 This is a photo of the germination of seedlings after the cuttings were inserted into the culture medium (pine bark and vermiculite) and then cultured under light for 40 days in Example 1;

[0033] Figure 7 yes Figure 6 A partial enlarged physical picture;

[0034] Figure 8 This is a physical picture of the seedlings germinating after the cuttings were inserted into the culture matrix (vermiculite) and cultured under light for 60 days in Example 1;

[0035] Fig. 9 yes Figure 8 A partial enlarged physical picture;

[0036] Fig.10 This is a photo of the germination of seedlings after the cuttings were implanted in a culture medium (pine bark) and cultured under light for 60 days in Example 1;

[0037] Fig.11 yes Fig.10 A partial enlarged physical picture;

[0038] Fig.12 It is the change of growth-related hormone content in cuttings under strong light treatment;

[0039] Fig.13 It is the change of hormone content related to aging of cuttings under strong light treatment. DETAILED DESCRIPTION

[0040] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention application are described in detail below in conjunction with the accompanying drawings and preferred embodiments. In the following description, different "one embodiment" or "embodiment" does not necessarily refer to the same embodiment. In addition, specific features, structures, or characteristics in one or more embodiments may be combined in any suitable form.

[0041] The embodiment of the present invention provides a method for obtaining Dendrobium officinale seedlings, which mainly proposes for the first time to use Dendrobium officinale waste stubble as raw material, select a set culture medium and corresponding light culture conditions to culture Dendrobium officinale seedlings.

[0042] Among them, the "waste stubble of Dendrobium candidum" in the embodiment of the present invention is described as follows: Dendrobium candidum begins to sprout in spring, and the growth period is from May to October. After October, the stems enter the mature stage, the new stems thicken but the leaves no longer increase, and the leaves fall off naturally around November, entering the low-temperature dormancy period. After the leaves fall off, the leaf sheaths are tightly wrapped and white, which is called white strips by herbal farmers. The harvesting time of artificially cultivated Dendrobium candidum is preferably from November to March of the following year. When harvesting white strips, it cannot be cut from the base, and at least two thicker stem nodes should be retained to provide sufficient nutrients and water for the germination of new buds next year. Although Dendrobium candidum is a perennial plant, usually after 2-3 times of harvesting, that is, when it is planted for about 4 years, the germination rate of the old stems is severely reduced, and even new buds are basically not sent out. At this time, the growers will pull out Dendrobium candidum by the roots. The pulled-out Dendrobium officinale is the material used in this experiment, which is defined as the waste stubble of Dendrobium officinale (this part of the material is usually directly discarded by the planting base). It should be noted that the explants used in the prior art, except for seeds, are all healthy plant tissues; while the materials used in the embodiments of the present invention are agricultural waste.

[0043] The culture medium selected in the embodiment of the present invention is mainly one or two of pine bark and vermiculite.

[0044] The light culture conditions selected in the embodiment of the present invention refer to: first culturing under the first set conditions for 15-40 days (preferably 15-35 days, further preferably 29-31 days), and then culturing under the second set conditions for 25-35 days (preferably 29-31 days); wherein the first set conditions include: light intensity of 20000-40000LX, preferably 30000LX; temperature of 20-35°C, preferably 28°C; humidity of 50-80%, preferably 60-75%; the second set conditions include: alternating light treatment for 10-14h and dark treatment for 10-14h; wherein, in the light treatment: humidity is 50-80%, preferably 60-75%; light intensity is 3000-10000LX, preferably 5000LX; temperature is 20-35°C, preferably 30°C; wherein the temperature of the dark treatment is 20-35°C, preferably 25°C. This study is different from the existing technology in that it uses extreme light treatment, specifically a method of obtaining seedlings by utilizing the extreme response of Dendrobium officinale to stress.

[0045] The present invention is further described below by way of examples and comparative examples:

[0046] Example 1

[0047] Example 1 provides a method for obtaining Dendrobium officinale seedlings, which is as follows:

[0048] 1. Selected raw materials (plant materials)

[0049] Dendrobium officinale waste. Among them, the actual picture of Dendrobium officinale waste is as follows Figure 1 shown.

[0050] 2. Main equipment and reagents

[0051] Light incubator (GXZ-310D, Ningbo Southeast Instrument Co., Ltd.), professional root analysis system (WinRHIZOTM, 2013e, Regent Instruments, Canada), ultra-high performance liquid chromatography-mass spectrometry (SHIMADZU, Japan), chromatographically pure methanol, chromatographically pure acetonitrile, chromatographically pure plant hormone standards, slaked lime, etc.

[0052] 3. Experimental Methods

[0053] 1) Processing of plant materials

[0054] Take the waste stubble of Dendrobium officinale and remove the fibrous root system (generally, the fibrous roots of the waste stubble of Dendrobium officinale have rotted, and the rotten roots have no biological function and may also cause pollution). Figure 2 Take slaked lime, add clean water to make lime water with a mass fraction of 2%, soak for 8-15 hours, put it in a tray to dry, and use it as cuttings for later use.

[0055] 2) Cutting method and culture conditions

[0056] Take some pine bark and vermiculite, soak them in clean water, and spread them flat on a plastic tray (length, width and height 48×33.5×3cm) as a culture matrix with a thickness of 1.5-2cm.

[0057] Take cuttings and insert the bottom end (root end) about 0.8-1.2 cm into the culture matrix. The cutting density is 4500-5200 plants per square meter.

[0058] Place the trays randomly in the light incubator, and first set the incubator parameters as follows: light intensity of 30000LX, temperature of 28℃, humidity of 60-75%, 24h strong light treatment for 30 days. Then adjust the incubator parameters as follows: alternating light treatment of 12h and dark treatment of 12h until light culture for 30 days. Among them, the light treatment conditions: humidity of 60-75%, light intensity of 5000LX, temperature of 30℃. The temperature of dark treatment is 25℃.

[0059] The entire light culture process described above lasts for 60 days.

[0060] The germination rates of seedlings were counted on the 20th and 45th days of culture, and the endogenous hormone content of cuttings was measured on 0h, 24h, 11th and 21st days of culture. The plant height and root length of newly germinated seedlings were measured on the 60th day after culture.

[0061] Among them, the germination percentage (GP)

[0062] The length of the young shoots ≥ 2 mm is used as the standard for seedling germination. The calculation formula for the seedling germination rate is as follows:

[0063]

[0064] In the above formula, SNG is the number of germinated plants, and SN0 is the number of cuttings in a single tray.

[0065] 4. Extraction and determination of seedling hormones

[0066] (1) Hormone extraction steps

[0067] a. Take a fresh sample, add liquid nitrogen, and grind it into powder using a grinder (50 Hz, 1 minute).

[0068] b. Weigh 100 mg (±1 mg) of fresh sample powder, add 1 mL of extract (wherein the extract includes methanol, water, and formic acid; wherein the volume ratio of methanol, water, and formic acid is 75:23:2), and oscillate at room temperature for 15 min.

[0069] c. After centrifugation (centrifugation parameters are set as follows: 12000 rpm, 4°C, 10 minutes), take the supernatant and put it into a new centrifuge tube.

[0070] d. Add 0.5 mL of the extract to the residue, repeat shaking and centrifugation, and combine the supernatants.

[0071] e. Concentrate to dryness in a concentrator at 4°C, and add 200 μL of a reconstitution solution (the reconstitution solution includes methanol and water; wherein the volume ratio of methanol to water is 4:1) for reconstitution.

[0072] f. After centrifugation (rotation speed 12000 rpm, 10 minutes), the supernatant was aspirated, the sample was filtered with a microporous filter membrane (0.22 μm pore size), and stored in a sample injection bottle for LC-MS / MS analysis.

[0073] (2) Chromatographic mass spectrometry acquisition conditions

[0074] The data acquisition instrument system mainly includes:

[0075] Ultra Performance Liquid Chromatography (UPLC) (Shim-pack UFLC SHIMADZU CBM30A);

[0076] Tandem mass spectrometry (MS / MS) (Applied Biosystems 4500QTRAP).

[0077] The liquid phase conditions mainly include:

[0078] 1) Chromatographic column: Waters ACQUITY HSS T3 C18 1.8μm, 2.1mm×100mm;

[0079] 2) Mobile phase: Phase A is ultrapure water (containing 0.04% acetic acid), and phase B is acetonitrile (containing 0.04% acetic acid);

[0080] 3) Elution gradient: 0 min A / B 95:5 (V / V), 10.0 min A / B 5:95, 11.0 min A / B 5:95, 11.1 min A / B 95:5, 14 min A / B 95:5.

[0081] 4) Flow rate: 0.40 mL / min, column temperature 40 °C, injection volume 5 μm.

[0082] Among them, the mass spectrometry conditions are shown in Table 1:

[0083] Table 1 (Mass spectrometry detection parameters)

[0084] Mass spectrometry conditions parameter Electrospray ionization (ESI) temperature 550℃ Ion source voltage 5500V / -4500V Curtain gas (CUR) 35psi Collision-induced ionization (CAD) 2 / -2 Declustering potential (DP) optimization Collision energy (CE) optimization

[0085] 5. Measurement of seedling roots and stems

[0086] The WinRHIZOTM root system analysis system was used to measure the root and stem lengths of seedlings. The specific system parameter settings of WinRHIZOTM are as follows:

[0087] Mode: Professional mode;

[0088] Document type: Film (with film area guide);

[0089] Film type: positive film;

[0090] Image type: 8-bit grayscale;

[0091] Resolution: 600dpi;

[0092] Manuscript size: 203×254mm;

[0093] Target size: original;

[0094] Adjuster: Unsharp Mask.

[0095] 6. Experimental Results

[0096] 1) In Example 1, the cuttings were inserted into the culture medium (vermiculite, pine bark), and the seedlings sprouted after 20 days, 40 days, and 60 days of light culture. Figure 3-Figure 11 shown.

[0097] from Figure 3-Figure 11 It can be seen that the embodiment of the present invention successfully obtained Dendrobium officinale seedlings.

[0098] 2) Seedling germination rate after 20 and 45 days of light culture.

[0099] Table 2 shows the seedling germination rate 20 days and 45 days after cutting under two culture substrates.

[0100] Table 2

[0101]

[0102] Note: The data in Table 2 are mean ± standard error, and different letters indicate significant differences (p<0.05).

[0103] Here, the cuttings of the present embodiment were treated with 24h strong light (30000LX) for 15-18 days, and the seedling buds can be clearly observed. The germination rates of the seedlings under the two culture substrates were counted on the 20th and 40th days after cutting, and the results are shown in Table 2. Under the same culture conditions, the seedling germination rate of the pine bark matrix group was significantly higher than that of the vermiculite matrix group. On the 20th and 40th days after cutting, the germination rates of the pine bark group were 14.82% and 9.55% higher than those of the vermiculite group, respectively.

[0104] 3) Root and stem indicators after 40 and 60 days of cultivation

[0105] Table 3 shows the growth index of seedlings 40 days after cutting under two culture substrates

[0106] Table 3

[0107]

[0108] Note: The data in Table 3 are mean ± standard error, and different letters indicate significant differences (p<0.05).

[0109] Table 4. Growth index of seedlings 60 days after cuttings in two culture substrates

[0110] Table 4

[0111]

[0112] Note: The data in Table 4 are mean ± standard error, and different letters indicate significant differences (p<0.05).

[0113] As shown in Tables 3 and 4, the two culture substrates had little effect on the stem height of seedlings 40 and 60 days after cutting; however, the root growth in the pine bark substrate group was significantly higher than that in the vermiculite substrate group. On the 40th and 60th days after cutting, the root length in the pine bark substrate group was 58.2% and 63.89% higher than that in the vermiculite substrate group, respectively.

[0114] Judging from the seedling germination rate and rhizome growth indicators, on the 45th day after cutting, the seedling germination rate of the pine bark matrix group could reach 89.59%; on the 60th day, the seedling height (stem length) of the pine bark matrix group could reach an average of 7.47 cm.

[0115] 4) Changes in endogenous hormones in cuttings under strong light treatment

[0116] At present, the research on the effect of light treatment on Dendrobium officinale mainly focuses on two aspects: one is to use the second-generation transcriptome sequencing technology to study the changes in gene regulation of Dendrobium officinale under light treatment; the other is to study the optimal light conditions for Dendrobium officinale seedlings (especially test tube seedlings). Here, the existing technologies are extracted as follows:

[0117] Wang Xueni et al. found in the study "The effect of light treatment on the reproduction and growth of Dendrobium officinale" that the protocorms of Dendrobium officinale were used as the test material, and the culture seedlings were obtained by inducing the proliferation and differentiation of the protocorms. The growth of Dendrobium officinale seedlings was best when the light time was 10h / d, the light intensity was 2500Lx, and the red-blue light quality ratio was 1:1. (Wang Xueni, Lin Wei, Wang Jingjing, Li Jing, Fan Zhengyang, Ye Xiaojie, He Bizhu. Effect of light treatment on the reproduction and growth of Dendrobium officinale [J]. Anhui Agricultural Sciences, 2020, 48(17): 49-52+76.)

[0118] Chen Zhi et al. conducted a study on the effects of different color temperature LED white light on the growth of Dendrobium officinale tissue culture seedlings and believed that 3000K LED white light can completely replace fluorescent lamps for the cultivation of Dendrobium officinale tissue culture seedlings, and the appropriate illumination time is 12 hours. (Chen Zhi, Wang Yiting, Lv Yongping, Mou Haojie. Effects of different color temperature LED white light on the growth of Dendrobium officinale tissue culture seedlings [J]. Zhejiang Agricultural Sciences, 2018, 59(11): 2013-2016. DOI: 10.16178 / j.issn.0528-9017.20181119.)

[0119] Lin Xiaoping's research results show that keeping the CO2 concentration (350±50mg / L) unchanged, increasing the light intensity has no obvious promoting effect on the growth of test tube seedlings induced by protocorms of Dendrobium officinale, and sugar-free culture is not as good as sugar culture; keeping the light intensity at 3500Lx unchanged and increasing the CO2 concentration has a greater promoting effect on the photosynthesis of test tube seedlings (Lin Xiaoping. Preliminary study on sugar-free tissue culture technology of Dendrobium officinale [J]. Fujian Tropical Crops Science and Technology, 2018, 43(02): 6-10.)

[0120] Huangfu Sensen found in the study of "The effect of LED light on the growth characteristics of Dendrobium officinale" that the growth characteristics of Dendrobium officinale in adult stage were significantly different from those in adult stage (red light: blue light = 24:96 μmol·m -2 ·s -1 8 / 16h) light environment has the best comprehensive index. -2 ·s -1 10 / 14h) light environment has the best comprehensive indicators (Huangfu Sensen. Research on the growth characteristics of Dendrobium officinale under LED lighting [D]. Fujian Agriculture and Forestry University, 2018. DOI: 10.27018 / d.cnki.gfjnu.2018.000006.)

[0121] Mai Xiaoyan et al. studied the effects of different culture media and light intensity on the test tube seedlings of Dendrobium officinale. The results showed that the rooting rate of the test tube seedlings of Dendrobium officinale under 5000Lx strong light was higher than that under 500Lx weak light. The authors believe that only under appropriate culture media and certain strong light conditions can the test tube seedlings of Dendrobium officinale be cultivated into strong seedlings with well-developed root systems and strong stems and leaves, thereby improving the survival rate of transplantation. The appropriate light intensity range for cultivating the test tube seedlings of Dendrobium officinale needs further study (Mai Xiaoyan, Zheng Ping, Liu Rongwei, Wang Tao, Zhou Rong. Effects of different culture media and light intensity on the test tube seedlings of Dendrobium officinale [J]. Guangdong Agricultural Sciences, 1997(03):26-27. DOI:10.16768 / j.issn.1004-874x.1997.03.011.).

[0122] Chou Minxia et al. conducted a study on the effect of light intensity on the growth and metabolism of Dendrobium officinale and believed that the cultivation conditions for the tillering period of Dendrobium officinale were 12 h / d of light duration, 80% relative humidity, and 320 μmol·m -2 ·s -1 It is appropriate (Chou Minxia, ​​Zhu Liquan, Zhang Yujin, Zhang Ming, Bie Zhilong, Chen Shijiang, Li Quansen. Effects of light intensity on growth and metabolism of Dendrobium officinale [J]. Acta Horticulturae Sinica, 2000(05):380-382.).

[0123] Gao Libin et al. found in the study of rooting technology of Dendrobium officinale tissue culture seedlings that the rooting medium formula has the greatest impact on the rooting rate of Dendrobium officinale tissue culture seedlings, followed by light intensity, but the effect of light time on the rooting rate is not significant. The best laboratory environmental conditions for rooting of Dendrobium officinale tissue culture seedlings are light intensity of 2000Lx, culture room temperature controlled at (20±2)℃, and the best rooting medium is 1 / 2MS+NAA 0.1mg / L+banana juice 100mg / L (Gao Libin, Cao Zhengyu, Gu Yunli, Zhou Yahui. Research on rooting technology of Dendrobium officinale tissue culture seedlings [J]. Shanghai Agricultural Science and Technology, 2017(04):32+46.).

[0124] Long Xiangyou et al. found in their study on "Suitable light duration for the growth of Dendrobium officinale test tube seedlings" that the suitable light duration for the proliferation stage of Dendrobium officinale test tube seedlings is 10 h / d, and the suitable light duration for the rooting stage is 12 h / d. (Long Xiangyou, Zhu Hong, Sun Changsheng, Liu Xuelan, Li Lixia. Suitable light duration for the growth of Dendrobium officinale test tube seedlings[J]. Guizhou Agricultural Sciences, 2015, 43(04): 173-175.)

[0125] Different from the above studies, the present invention is a method of obtaining seedlings by using extreme light treatment and the extreme response of Dendrobium officinale to stress. By measuring the changes in the endogenous hormone content of Dendrobium officinale under extreme light treatment conditions, the internal mechanism of the present invention can be preliminarily explored.

[0126] like Fig.12 and Fig.13 As shown, under the all-weather uninterrupted strong light treatment, the endogenous hormones of the cutting strips change significantly with the extension of the treatment time. Under the 24h treatment, growth-related hormones decrease slightly, and mature aging-related hormones, especially ethylene, rise sharply, indicating that the cutting strips are in a stress response state under strong light stress. And under the continuous strong light treatment, after 11 days of treatment, growth-related hormones rise, and mature aging-related hormones decline, indicating that the cutting strips of Dendrobium candidum do not die under long-term strong light stress, but have produced resistance, and mobilize endogenous hormones to resist stress. Under 21 days of strong light treatment, the measured 4 kinds of endogenous hormone levels are consistent with the control group, reflecting that the cutting strips have adapted to the strong light stress, and the observed result is that new buds sprout on the cutting strips. This also fully illustrates that the folk saying about Dendrobium candidum is also called "unkillable", and Dendrobium candidum has the ability to adapt to extreme conditions, which also provides possibility for the success of the present invention.

[0127] Comparative Example 1

[0128] Comparative Example 1 is a method of the prior art, see: Cai Xuanmei. Research on tissue culture and rapid propagation system of Dendrobium officinale in Fujian [J]. Fujian Tropical Crops Science and Technology, 2020, 45(04): 1-4. The main steps are as follows:

[0129] 1 Materials and Methods

[0130] 1.1 Materials

[0131] The test materials were taken from the mature capsules of wild Dendrobium officinale in Liancheng, Fujian.

[0132] 1.2 Culture conditions

[0133] The culture conditions were 23±2℃, 1500Lx light, and 12h·d. -1 , ambient humidity 60-80%.

[0134] 1.3 Methods

[0135] 1.3.1 Obtaining protocorms

[0136] Select healthy, disease-free and insect-free capsules, rinse them with running water, soak them with detergent for 5 minutes, brush them gently with a brush, and then rinse them with tap water. Soak them in 75% alcohol for 30 seconds on a clean bench, disinfect them with 0.1% mercuric chloride for 10 minutes, rinse them with sterile water 3-4 times, cut one end of the disinfected capsule with a scalpel under sterile conditions, and gently shake the capsule to inoculate the seeds on the induction medium.

[0137] 1.3.2 Seed germination culture

[0138] MS medium, sucrose 3%, agar 0.65%, pH 5.8-6.0. Cultivation for 100 days.

[0139] 1.3.3 Protocorm proliferation culture

[0140] The protocorms cultured for 100 days were cut and transferred to organic supplement medium (6-BA2.2+NAA0.2+2,4-D0.1+banana extract 50+potato extract 50) and cultured for 120 days.

[0141] 1.3.4 Seedling strengthening and rooting culture

[0142] After differentiation, seedlings 2-3 cm high and with 4-5 leaves were cut and inoculated into seedling and rooting medium (6-BA0.5+NAA0.5+IBA0.1+banana extract 100) and cultured (the literature did not specify how long the culture was carried out), obtaining seedlings with an average root number of 4.63 and a plant height of 4.14 cm.

[0143] Comparative Example 2

[0144] Comparative Example 2 is a method of the prior art, see: Xia Jiucheng, Zheng Yi, Dai Qinchuan. Research on tissue culture technology of Dendrobium officinale [J]. Heilongjiang Agricultural Science, 2022(02): 69-74.

[0145] 1 Materials and methods

[0146] 1.1 Materials

[0147] The noded stem segments of Dendrobium officinale were used as explants, and the experimental materials were plants with soil from Huoshan County, Anhui Province.

[0148] 1.2 Methods

[0149] The light culture conditions were as follows: culture temperature (25±1)℃, light intensity 1600Lx, and light duration 12h·d -1 .

[0150] 1.2.1 Explant sterilization

[0151] Select robust plants of Huoshan Dendrobium officinale, cut off the internode stem segments with sterile scissors as explants, rinse under running water for 30-40 minutes, air-dry, and remove 35-45% of the water in the fresh strips. Before inoculation, wipe your hands with alcohol cotton in the clean bench, then spray with 75% alcohol to reduce dust, and wipe the work surface. Use a sterilized scalpel to trim the explants into 0.5-0.8cm internode stem segments, remove the leaves, and then disinfect the explants. Soak in 75% alcohol for 30 seconds, then divide the explants into 3 groups, soak them in 0.1% HgCl2 for 10 minutes, and then inoculate them on ordinary MS culture medium (0.5mg·L - 1 NAA and 2.0mg·L -1 6-BA), with one explant inoculated per bottle.

[0152] 1.2.2 Explant culture

[0153] The culture medium was 1 / 2MS medium, and the cells were subcultured once after 35 days of culture. The total culture period was 75 days, with two subcultures.

[0154] 1.2.3 Induction of axillary buds

[0155] The culture medium for inducing axillary buds from the noded stems of Dendrobium officinale is 1 / 2MS + 10 g·L -1 Sucrose + 0.6 mg·L -1 NAA+1.5mg·L -1 6-BA, cultured for 75 days.

[0156] 1.2.4 Proliferation of buds

[0157] The culture medium is 1 / 2MS+0.6mg·L -1 NAA+1.5mg·L -1 6-BA, cultured for 75 days.

[0158] 1.2.5 Rooting culture of tissue culture seedlings

[0159] The formula of the rooting medium for the noded stem segments of Dendrobium officinale is 1 / 2MS + 0.9 mg·L -1 NAA+0.5mg·L - 1 IBA, cultured for 45 days.

[0160] Here, compared with Comparative Example 1 and Comparative Example 2, the method for obtaining Dendrobium officinale seedlings proposed in the embodiment of the present invention has at least the following advantages:

[0161] 1. Reuse of plant residues; the cuttings used in the method of the embodiment of the present invention are discarded tissues after the fresh Dendrobium officinale strips are harvested. In actual large-scale production, this part of the material is usually directly discarded by the planting base.

[0162] The raw material of Comparative Example 1 is the mature capsule of wild Dendrobium officinale, and the raw material of Comparative Example 2 is the stem segment with nodes of Dendrobium officinale from Huoshan. Obviously, the raw materials of Comparative Examples 1 and 2 are relatively expensive and costly.

[0163] 2. Easy to operate: The method of the embodiment of the present invention only requires disinfecting the residue of Dendrobium with slaked lime, cutting it onto a common culture matrix, and subjecting it to 24-hour full light treatment for a period of time according to the method of the embodiment of the present invention. Conventional tissue culture seedlings need to be proliferated and differentiated, and the seedlings need to be rooted in a sterile environment. In contrast, the method of the embodiment of the present invention can be completed by ordinary personnel in a conventional environment.

[0164] Comparative Example 1 and Comparative Example 2 are both operated under sterile conditions, and require the completion of operations such as medium preparation, sterilization, inoculation, primary culture, subculture, rooting culture, and test tube seedling transplanting and domestication, which is too cumbersome.

[0165] 3. Short time: The method of the embodiment of the present invention takes 60 days to obtain robust seedlings with an average plant height of 7 cm, an average total root length of 10 cm, and a root meristem number of 4.26. For the same size of Dendrobium officinale seedlings, it takes 180 days to grow them by the hormone-free tissue culture method and 75 days by the hormone tissue culture method; the methods of the comparative examples are much longer than the time taken by the method of the present invention. For example, in the method of tissue culture using seeds, it usually takes 1-2 years from seed germination to transplantation into seedlings.

[0166] 4. Low cost: This method only uses waste stubble of Dendrobium officinale, common materials such as slaked lime and pine bark, and then treats it with light. The conventional tissue culture method of Dendrobium officinale usually requires processes such as protocorm induction, proliferation and differentiation, and rooting of seedlings. It requires professionals to use professional equipment and reagents in a special tissue culture environment, which consumes a lot of manpower and material resources.

[0167] In summary, the method for obtaining Dendrobium officinale seedlings proposed in the embodiment of the present invention is a method with low cost, short time and simple operation, which is suitable for factory production.

[0168] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. A method for obtaining Dendrobium officinale seedlings, characterized in that: The method for obtaining Dendrobium officinale seedlings comprises the following steps: The step of preparing cutting strips: using waste stubble of Dendrobium officinale as raw material; making the waste stubble of Dendrobium officinale into cutting strips; wherein the step of preparing cutting strips comprises: removing the fibrous roots on the waste stubble of Dendrobium officinale, leaving the stem and the roots connected to the stem base, and obtaining the cutting strips; disinfecting the cutting strips; wherein the waste stubble of Dendrobium officinale is the discarded tissue after the fresh strips of Dendrobium officinale are harvested; Light culture step: inserting the cuttings into a culture matrix, placing them in a light culture box for light culture treatment, and obtaining Dendrobium officinale seedlings; Wherein, in the light culturing step: the light culturing treatment includes: first culturing under the first set conditions for 15-40 days, and then culturing under the second set conditions for 25-35 days; wherein, The first setting conditions include: light intensity of 20000-40000LX, temperature of 20-35°C, and humidity of 50-80%; The second setting conditions include: alternating light treatment for 10-14 hours and dark treatment for 10-14 hours; wherein, during the light treatment: humidity is 50-80%, light intensity is 3000-10000LX, and temperature is 20-35°C; wherein, the temperature of the dark treatment is 20-35°C.

2. The method for obtaining Dendrobium officinale seedlings according to claim 1, characterized in that: In the first setting condition: the light intensity is 30000LX.

3. The method for obtaining Dendrobium officinale seedlings according to claim 1, characterized in that: In the first setting condition: the temperature is 28°C.

4. The method for obtaining Dendrobium officinale seedlings according to claim 1, characterized in that: In the first setting condition: the humidity is 60-75%.

5. The method for obtaining Dendrobium officinale seedlings according to claim 1, characterized in that: In the illumination treatment of the second setting condition: the humidity is 60-75%.

6. The method for obtaining Dendrobium officinale seedlings according to claim 1, characterized in that: In the illumination treatment under the second setting condition: the illumination intensity is 5000LX.

7. The method for obtaining Dendrobium officinale seedlings according to claim 1, characterized in that: In the light treatment of the second setting condition: the temperature is 30°C.

8. The method for obtaining Dendrobium officinale seedlings according to claim 1, characterized in that: In the dark process of the second setting condition, the temperature of the dark process is 25°C.

9. The method for obtaining Dendrobium officinale seedlings according to claim 1, characterized in that: The culture was carried out under the first set condition for 25-35 days.

10. The method for obtaining Dendrobium officinale seedlings according to claim 9, characterized in that: The culture was carried out under the first set condition for 29-31 days.

11. The method for obtaining Dendrobium officinale seedlings according to claim 1, characterized in that: The cells were cultured under the second set condition for 29-31 days.

12. The method for obtaining Dendrobium officinale seedlings according to claim 1, characterized in that: The length of the cuttings is 3-5 cm.

13. The method for obtaining Dendrobium officinale seedlings according to claim 1, characterized in that: The step of disinfecting the cuttings comprises: soaking the cuttings in lime water for disinfection.

14. The method for obtaining Dendrobium officinale seedlings according to claim 13, characterized in that: The cuttings are immersed in lime water for 8-15 hours.

15. The method for obtaining Dendrobium officinale seedlings according to claim 13, characterized in that: The lime water is a solution prepared from slaked lime and water with a mass concentration of 1-5%.

16. The method for obtaining Dendrobium officinale seedlings according to claim 13, characterized in that: The lime water is a solution with a mass concentration of 2% prepared from slaked lime and water.

17. The method for obtaining Dendrobium officinale seedlings according to any one of claims 1 to 16, characterized in that: In the light culturing step: the culture matrix includes one or two of pine bark and vermiculite.

18. The method for obtaining Dendrobium officinale seedlings according to claim 17, characterized in that: The thickness of the culture matrix is ​​1.5-2 cm.

19. The method for obtaining Dendrobium officinale seedlings according to any one of claims 1 to 16, characterized in that: In the light culture step: the root end of the cutting is inserted into the culture matrix.

20. The method for obtaining Dendrobium officinale seedlings according to claim 19, characterized in that: The length of the cuttings inserted into the culture matrix is ​​0.8-1.2 cm.

21. The method for obtaining Dendrobium officinale seedlings according to any one of claims 1 to 16, characterized in that: The cutting density of the cuttings on the culture matrix is ​​4500-5200 plants per square meter.

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