Rapid propagation method for pleione bulbocodioides seedlings

Abstract

The invention relates to the technical field of pleione bulbocodioides planting, in particular to a pleione bulbocodioides seedling rapid propagation method. The pleione bulbocodioides seedling rapid propagation method comprises the following steps: sowing pleione bulbocodioides seeds in a seed germination culture medium, and culturing to obtain pleione bulbocodioides rootless seedlings, wherein the germination culture medium contains a seed germination agent for promoting the germination of the pleione bulbocodioides seeds; the pleione bulbocodioides rootless seedlings are transferred to a large corm culture medium to be cultured into pleione bulbocodioides rooted seedlings with pleione bulbocodioides corms larger than 5-9 mm, and the large corm culture medium contains a corm growth agent for promoting pleione bulbocodioides corm development. Therefore, the problem of long cultivation period of the bulbocodioides bulbocodioides bulb is solved.

Description

Technical Field

[0001] The invention relates to the technical field of Orchidaceae planting, in particular to a method for rapid propagation of Orchidaceae seedlings. Background Art

[0002] The dried pseudobulb of the orchid, commonly known as "ice balls," is a semi-epiphytic perennial herb in the Orchidaceae family. It often grows on decaying tree trunks, humus-rich forest soil, and moss-covered rocks. It is commonly found in shaded, moist areas at altitudes between 1,100 and 3,500 meters. The dried pseudobulbs of the orchid, commonly known as "ice balls," are collectively known as "mountain cilia" along with the cilia edodes. They are included in various editions of the Chinese Pharmacopoeia and are primarily used for clearing heat and detoxification, relieving cough and expectoration, relieving inflammation and pain, and treating traumatic bleeding and tumors. While promising medicinal applications, they are expensive and currently endangered. They are currently on the verge of extinction and are listed as Class II rare and endangered plants in my country.

[0003] A mature capsule of the genus Solanum contains thousands of dust-like seeds. These seeds have incomplete embryonic development, degenerate seed coat cytoplasm and organelles, and lack the nutrients provided by the endosperm. This results in a high mortality rate during natural germination. Given the large number of seeds in the orchid family, symbiotic germination of Solanum seeds with symbiotic fungi can promote germination and seedling formation, but this is difficult to implement in practice. Solanum interacts with multiple fungi simultaneously, making it difficult to determine the ecological function of each fungus. Furthermore, the germination-promoting effect only persists until the protocorm stage, and its role in natural environments is uncertain. Propagation of Solanum using tissue culture has been reported, but in practice, it still faces the challenge of balancing the interplay between plant growth regulator levels to promote plant growth and pollution rates and mortality rates. Therefore, natural division is generally used in production, with each plant producing one to three new corms per year. This slow growth and reproduction rate has severely limited the development of the Trillium industry. Currently, the market for Orchidaceae rooted seedlings is in disarray, with varying quality. This creates a significant market gap and leads to a shortage of supply. The existing tissue culture and rapid propagation technology for Orchidaceae rooted seedlings in actual production has a long growth cycle, high cost, and unsatisfactory quality of rooted seedlings. The lack of efficient and mature tissue culture and rapid propagation technology for rooted seedlings has severely restricted the development of this industry. The present invention aims to provide an efficient and rapid propagation production technology for Orchidaceae rooted seedlings, thereby providing a technical research foundation for the development of the Chinese medicinal material Psoralea corylifolia industry. Summary of the Invention

[0004] In order to solve the problems of long cultivation period and poor seedling quality of Orchidaceae, the present invention provides a method for rapid propagation of Orchidaceae seedlings, comprising the following steps:

[0005] Sowing the seeds of the Orchidaceae on a seed germination medium to obtain strong and robust rootless seedlings of the Orchidaceae, wherein the germination medium contains a seed germination agent that promotes the germination of the Orchidaceae seeds;

[0006] The rootless seedlings of the Orchidaceae are transferred to a large bulb culture medium for cultivation to become rooted seedlings of the Orchidaceae with bulbs larger than 5-9 mm. The large bulb culture medium contains a bulb growth agent that promotes the development of the Orchidaceae bulbs.

[0007] By adding a seed germination agent, the plant height of the rootless seedlings of the garlic orchid is greater than 26 mm, the germination rate is greater than 88%, the number of leaves reaches 2, and the stem thickness of the rootless seedlings of the garlic orchid reaches more than 1.21 mm, so that when the rootless seedlings of the garlic orchid are cultured in a subsequent large bulb culture medium, the rootless seedlings of the garlic orchid can obtain large bulbs in a shorter culture cycle, thereby shortening the time for cultivating large bulbs of the garlic orchid.

[0008] Preferably, the large bulb culture medium is used to culture the rooted seedlings of Orchidaceae with bulbs larger than 9 mm.

[0009] Preferably, 50-100 of the Cymbidium orchid seeds are sown in each of the seed germination culture mediums, and 10-15 of the germinated seedlings are transferred into each of the large bulb culture mediums.

[0010] By choosing the right number of seeds to be sown, there is enough space and nutrition for the seeds to grow into rootless seedlings. By choosing the right number of rootless seedlings to be sown, there is enough space and nutrition for the rootless seedlings to be cultivated.

[0011] According to one embodiment of the present invention, the seed germination agent includes 1-2 mg / L peptone, 0.25-1.5 mg / L naphthaleneacetic acid, 0-1 mg / L activated carbon and 0-1 mg / L Huabao No. 1, wherein the activated carbon and / or Huabao No. 1 is greater than 0 mg / L.

[0012] According to one embodiment of the present invention, the seed germination agent includes 2 mg / L peptone, 0.5-1 mg / L naphthaleneacetic acid, 0.25-0.75 mg / L activated carbon and 0.125-0.25 mg / L Huabao No. 1.

[0013] According to one embodiment of the present invention, the seed germination agent includes 2 mg / L peptone, 0.5 mg / L naphthaleneacetic acid, 0.5 g / L activated carbon and 0.125 g / L Huabao No. 1.

[0014] According to one embodiment of the present invention, the bulb growth agent includes 0.5-2 mg / L peptone, 0.5-1.5 mg / L naphthaleneacetic acid, 0.5-1.5 g / L activated carbon and 25-150 mL / L organic additives.

[0015] According to one embodiment of the present invention, the bulb growth agent includes 0.75-1.5 mg / L peptone, 0.5-1 g / L activated carbon, 0.5-1 mg / L naphthaleneacetic acid and 75-100 mL / L organic additives.

[0016] According to one embodiment of the present invention, the bulb growth agent includes 1.0 g / L peptone, 0.5 g / L activated carbon, 0.5 mg / L naphthaleneacetic acid and 75 ml / L organic additives.

[0017] According to one embodiment of the present invention, the organic additive includes coconut milk, mashed bananas or mashed potatoes.

[0018] According to one embodiment of the present invention, before culturing the rootless seedlings of the Orchidaceae seeds, the Orchidaceae seeds are sterilized in a sodium hypochlorite solution for 4-12 minutes.

[0019] According to one embodiment of the present invention, the method for rapid propagation of Orchidaceae seedlings also includes pretreatment, after disinfecting the surface of the fresh and mature capsule of Orchidaceae, soaking the fresh and mature capsule of Orchidaceae in ethanol solution and mercuric chloride solution in sequence, rinsing the fresh and mature capsule of Orchidaceae with sterile water to obtain sterile fresh and mature capsule of Orchidaceae, and obtaining the Orchidaceae seeds from the sterile fresh and mature capsule of Orchidaceae.

[0020] According to one embodiment of the present invention, the basic substances in the seed germination medium and the large bulb culture medium include 1 / 2MS, 20-30 g / L sucrose and 5-7 g / L agar, and the pH of the seed germination medium and the large bulb culture medium is 5.8-6.0.

[0021] The present invention has the following beneficial effects:

[0022] 1. The best cultivation method for the germination of Orchidaceae seeds is to pretreat the seeds with 2% sodium hypochlorite for 8 minutes, and then culture them in 1 / 2MS + 2g / L peptone + 0.5mg / L naphthaleneacetic acid + 0.5g / L activated carbon + 0.125g / L Huabao No. 1. Sodium hypochlorite pretreatment can make the seeds germinate earlier and shorten the growth cycle. Adding appropriate amounts of activated carbon and Huabao No. 1 can obtain relatively sturdy Orchidaceae rootless seedlings in 3 months, which are more suitable for the induction and cultivation of large bulbs in the next step.

[0023] 2. The best effect was achieved when subcultured large corms were cultured in 1 / 2MS + 0.5g / L activated carbon + 0.5mg / L naphthaleneacetic acid + 75ml / L coconut juice + 1.0g / L peptone for 3 months. The maximum corm diameter reached 7.59mm and the plant height was 58.60mm. Plants with corms larger than 5mm accounted for 91% of the total. The larger the corm of the tissue culture seedling, the higher the transplant survival rate, which effectively improved the quality of the seedlings.

[0024] 3. The present invention adopts a two-stage tissue culture method of sterile sowing stage and seedling growth stage. It only takes 6 months from sowing to obtaining high-quality rooted seedlings with a high survival rate, which simplifies the production process and shortens the growth cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shown is a diagram showing the actual cultivation of the orchid of the present invention. DETAILED DESCRIPTION

[0026] The present disclosure will now be discussed with reference to several exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the present disclosure, rather than to imply any limitation on the scope of the present disclosure.

[0027] As used herein, the term "including" and its variations are to be interpreted as open-ended terms meaning "including but not limited to". The term "based on" is to be interpreted as "based at least in part on". The terms "one embodiment" and "an embodiment" are to be interpreted as "at least one embodiment". The term "another embodiment" is to be interpreted as "at least one other embodiment". In addition, the terms "first", "second", etc. are mainly used to distinguish different devices, elements or components (the specific types and structures may be the same or different), and are not used to indicate or imply the relative importance and quantity of the indicated devices, elements or components. Unless otherwise specified, "plurality" means two or more.

[0028] 1. Materials and Methods

[0029] 1.1 Materials

[0030] The fresh and mature capsules of the perennial Orchidaceae planted in the Chinese medicinal material resource garden of Guizhou Academy of Agricultural Sciences were selected as materials.

[0031] 1.2 Explant disinfection

[0032] Pick fresh mature capsules of Orchidaceae, wipe them three times with cotton soaked in 75% ethanol, soak them in 75% ethanol for 30 seconds, wash them three times with sterile water, soak them in 0.1% mercuric chloride for 15 minutes, rinse them three times with sterile water, place them on inoculation paper, blow dry and set aside.

[0033] 1.3 Culture conditions

[0034] 1.3.1 Use 1 / 2MS (the macronutrients in MS basal medium are halved, while other components remain unchanged) as the basal medium, add 30 g / L sucrose and 7 g / L agar to the medium, and set the pH to 5.8. Prepare the culture medium by sterilizing at 121°C for 20 min and maintaining a photoperiod of 12 h / d. The culture period is determined based on the experimental purpose.

[0035] 1.3.2 The environmental conditions for cultivating rootless seedlings in the seed germination medium of Orchidaceae were as follows: photoperiod of 12 h / d, light intensity of 40-45 μmol / (m2 / s), and temperature of 25±2℃.

[0036] 1.3.3 The environmental conditions for cultivating rooted seedlings in large corm culture medium are a photoperiod of 12 h / d, a light intensity of 40-45 μmol / (m2 / s), and a temperature of 25±2℃.

[0037] 1.4 Data Statistics and Analysis

[0038] The culture cycle for sterile sowing and large corm culture is 2-3 months, and the initial germination time is counted when 30% of the seeds have germinated. At the end of the culture cycle, seeds are examined under an optical microscope (germination is counted as breaking through the seed coat), and the germination rate is calculated. After the culture is completed, the corm size and fresh weight are measured using a vernier caliper and an electronic analytical balance, respectively. The data are analyzed using SPSS17 software.

[0039] 1.5 Methods

[0040] 1.5.1 Sterile seeding

[0041] Cut the sterilized and ready-to-use capsules of the garlic orchid to obtain the exposed dust-like garlic orchid seeds, and evenly sow 50-100 seeds on a solid culture medium with a diameter of 63 mm, a bottom diameter of 65 mm, a height of 95 mm, and a volume of about 66.33 cm 3 The seeds of Orchidaceae were cultured on the corresponding seed germination medium until the bulblets of Orchidaceae were larger than 1 mm. 30 bottles were treated in each experiment and repeated 3 times. 3 bottles were randomly selected from each treatment to calculate the starting germination time, germination rate, plant height and other indicators.

[0042] 1.5.1.1 Effect of naphthaleneacetic acid content on aseptic germination of Cymbidium truncatum

[0043] The germination medium for the seeds of Cymbidium uniflorum was 1 / 2MS+2g / L peptone+(0, 0.25, 0.5, 0.75, 1.0, 1.5) mg / L naphthaleneacetic acid. The culture period was 2 months. The effects of different naphthaleneacetic acid contents on the aseptic germination of Cymbidium uniflorum seeds are shown in Table 1.

[0044] Table 1 Effect of naphthaleneacetic acid content on aseptic germination of Cymbidium truncatum

[0045] Naphthaleneacetic acid (mg / L) Germination start time (d) Germination rate (%) Plant height(mm) 0.00 27.67±1.45d 39.67±0.88c 5.33±0.30d 0.25 19.00±0.58c 41.33±0.88c 7.85±0.15c 0.50 16.33±0.33b 60.00±1.15a 10.44±0.69a 0.75 15.33±0.33a 51.67±2.03b 9.15±0.50b 1.00 13.67±0.33a 50.67±0.67b 8.53±0.10bc 1.50 19.00±0.58c 36.00±1.00d 7.60±0.07c

[0046] Note: Different lowercase letters indicate significant differences in the same indicator (P<0.05).

[0047] As shown in Table 1, in culture media with the same content of naphthaleneacetic acid, the germination rate, initial germination time and plant height of the seeds of Orchidaceae were significantly different, all showing a normal curve distribution trend. Adding a certain concentration of naphthaleneacetic acid can promote seed germination. Among them, the culture medium containing 1.0 mg / L naphthaleneacetic acid had the fastest seed germination speed, starting to germinate about 14 days. The germination rate was the highest at 60% when 0.5 mg / L naphthaleneacetic acid was added, and the plant height was 10.44 mm.

[0048] 1.5.1.2 Effect of Sodium Hypochlorite Treatment Time on Seed Germination of Cymbidium spp.

[0049] Before sowing the seeds of the Orchidaceae, the seeds were soaked in 2% sodium hypochlorite solution for 0 min, 4 min, 6 min, 8 min, 10 min or 12 min, washed three times with clean water, filtered and spread out on filter paper, placed on a clean bench to dry the water, and sown in a culture medium of 1 / 2MS+2 g / L peptone+0.5 mg / L naphthaleneacetic acid and cultured for 2 months. The effect of the soaking time of the Orchidaceae seeds in 2% sodium hypochlorite solution on the sterile germination of the Orchidaceae seeds is shown in Table 2.

[0050] Table 2 Effect of sodium hypochlorite treatment time on seed germination of Cymbidium truncatum

[0051]

[0052]

[0053] Note: Different lowercase letters indicate significant differences in the same indicator (P<0.05).

[0054] As shown in Table 2, treatment time of 10 minutes or more increased seed mortality, and treatment for 8 minutes had the best effect. In fact, the germination time was about 9 days, the germination rate increased to 86.67%, and the plant height was 12.49 mm.

[0055] 1.5.1.3 Effect of activated carbon content on seed germination of Cymbidium spp.

[0056] The germination medium for the seeds of Cymbidium dulcis was 1 / 2MS + 2 g / L peptone + 0.5 mg / L naphthaleneacetic acid + activated carbon (0, 0.125, 0.25, 0.5, 0.75, 1.0) g / L, and the culture was carried out for 2 months. The effects of different activated carbon contents on the germination of Cymbidium dulcis seeds are shown in Table 3.

[0057] Table 3 Effect of activated carbon content on germination of Orchidaceae seeds

[0058] Activated carbon (g / L) Germination rate (%) Initial germination time (d) Plant height(mm) 0 85.33±0.33b 9.00±0.00a 12.38±0.09f 0.125 85.00±0.58b 8.67±0.33a 12.71±0.09ef 0.250 86.67±0.67ab 8.33±0.33a 13.62±0.13c 0.500 88.33±0.88a 8.33±0.33a 15.99±0.10a 0.750 85.00±0.58b 10.33±0.33b 15.18±0.16b 1.000 80.33±0.88c 13.00±0.58c 12.85±0.12d

[0059] Note: Different lowercase letters indicate significant differences in the same indicator (P<0.05).

[0060] As shown in Table 3, compared with no activated carbon addition, the addition of 0.25-0.5 g / L activated carbon can promote germination and seedling growth. When the activated carbon concentration is higher than 0.5 g / L, the initial germination time is prolonged, and when it is higher than 1.0 g / L, it no longer has a promoting effect on the seedling height. Activated carbon 0.5 g / L has the best effect, with the germination rate increased to 88.33%, the highest plant height is 15.99 mm, and the initial germination time is no different from that without activated carbon addition.

[0061] 1.5.1.4 Effect of Huabao No. 1 Content on Seed Germination of Cymbidium dulcis

[0062] The germination medium for Orchidaceae seeds was 1 / 2MS + 2g / L peptone + 0.5mg / L naphthaleneacetic acid + 0.5g / L activated carbon + Huabao No. 1 (0, 0.125, 0.25, 0.5, 0.75, 1.0) g / L. The culture was carried out for 3 months, and statistics were collected at 2 months and 3 months respectively. The effects of different contents of Huabao No. 1 on the germination of Orchidaceae seeds are shown in Table 4.

[0063] Table 4 Effects of Huabao No. 1 content on seed germination of Orchidaceae

[0064]

[0065] Note: Different lowercase letters indicate significant differences in the same indicator (P<0.05).

[0066] As shown in Table 4, the effect of adding Huabao No. 1 below 0.5 g / L on the germination rate and the initial germination time was not significant. When the concentration exceeded this, the initial germination was delayed and the germination rate decreased. In terms of plant height, at 2 months, low concentrations of Huabao No. 1 below 0.25 g / L promoted plant height. There was no difference between 0.5 g / L Huabao No. 1 and the control. Concentrations exceeding this concentration had an inhibitory effect on plant height. At 3 months, adding Huabao No. 1 within 0.5 g / L promoted plant height. Adding a certain amount of Huabao No. 1 promoted the growth of leaves and stem diameter. Overall, adding 0.125 g / L Huabao No. 1 had the best effect. The highest plant heights reached 20.17 mm and 26.53 mm at 2 and 3 months, respectively. At 3 months, the stem diameter and number of leaves were better than the control, with 2 leaves and a stem diameter of 1.21 mm. The best culture method for seed germination of Orchidaceae is to pretreat Orchidaceae seeds with 2% sodium hypochlorite for 8 minutes, and 1 / 2MS + 2g / L peptone + 0.5mg / L naphthaleneacetic acid + 0.5g / L activated carbon + 0.125g / L Huabao No. 1 is the best seed germination medium. The results of sterile sowing culture in the best seed germination medium are shown in Figure 1 .

[0067] 1.5.2 Large corm culture

[0068] The rootless seedlings with a height of more than 2.5 cm were transferred to a large corm culture medium with a diameter of 63 mm, a bottom diameter of 65 mm, a height of 95 mm, and a solid culture medium volume of about 66.33 cm. 3 , each treatment had 15 bottles, 8 seedlings in each bottle, and the experiment was repeated 3 times. After the cultivation period, the plant height, leaf length, leaf width, bulb diameter, and bulb fresh weight were examined, and the bulbs were cultivated to be larger than 5-9 mm.

[0069] 1.5.2.1 Effects of coconut juice and banana puree content on the growth of Orchidaceae seedlings

[0070] The culture medium for large corms was 1 / 2MS+0.5g / L activated carbon+0.5mg / L naphthaleneacetic acid+organic additives, and the organic additives were (50, 75, 100, 125, 150, 200)ml / L coconut juice or (25, 50, 75, 100, 125, 150)mg / L banana puree. The culture was continued for 2 months. The effects of different contents of coconut juice and banana puree on the growth of the seedlings of Orchidaceae are shown in Table 5. Coconut juice was obtained directly from coconut fruit, and banana puree was banana puree made from fruit bananas.

[0071] Table 5 Effects of coconut juice and banana puree content on the growth of Orchidaceae seedlings

[0072] additives Blade length (mm) Plant height(mm) Bulb diameter (mm) Fresh weight of bulb (g) CK 20.01±0.31cd 26.05±0.06cd 3.20±0.23ab 0.030±0.008cd Coconut juice 50ml / L 22.28±1.33abc 29.50±0.78abc 3.34±0.30a 0.044±0.002abc Coconut juice 75ml / L 24.53±2.38ab 30.67±2.21ab 3.84±0.28a 0.054±0.002a Coconut juice 100ml / L 25.47±1.08a 31.34±0.79a 3.78±0.29ab 0.039±0.009abc Coconut juice 125ml / L 20.37±0.32cd 26.41±1.74bc 3.11±0.45ab 0.038±0.005bcd Coconut juice 150ml / L 19.78±1.04cd 25.89±1.44cd 2.88±0.20ab 0.033±0.007bcd Banana puree 25g / L 21.26±1.49bc 27.03±0.95abc 3.26±0.14abc 0.032±0.004cd Banana puree 50g / L 23.21±0.72abc 29.18±2.57abc 3.21±0.39ab 0.048±0.002ab Banana puree 75g / L 23.36±1.04abc 29.39±0.47abc 3.54±0.33abc 0.046±0.002abc Banana puree 100g / L 19.94±0.71cd 25.79±0.82cd 2.81±0.09c 0.023±0.002d 125g / L banana puree 17.18±0.66d 21.94±0.52d 2.72±0.04c 0.022±0.002d

[0073] Note: Different lowercase letters indicate significant differences in the same indicator (P<0.05).

[0074] As shown in Table 5, the CK group did not add coconut juice or banana puree. Compared with the CK group, the addition of 50-100 ml / L coconut juice promoted the growth of leaves, plant height and bulbs of Orchidaceae seedlings. Adding 75 ml / L coconut juice had the best effect, with the best plant height and leaves, and the bulb diameter was 3.78 mm; adding less than 100 g / L banana puree was beneficial to the growth of Orchidaceae seedlings, among which adding 50 g / L banana puree had the best effect, and the bulb diameter was 3.21 mm. Overall, adding coconut juice was better than adding banana puree, with 75 ml / L coconut juice having the best effect, and 50 g / L banana puree second.

[0075] 1.5.2.2 Effects of Huabao No. 1, Huabao No. 4, and Peptone Content on the Growth of Cymbidium dulcis Seedlings

[0076] The culture medium for large corms consisted of 1 / 2 MS, 0.5 g / L activated carbon, 0.5 mg / L naphthaleneacetic acid, 75 ml / L coconut water, and organic additives. The organic additives included Huabao No. 1 (0.065, 0.125, 0.25, 0.5, 0.75, 1) g / L, Huabao No. 4 (0.065, 0.125, 0.25, 0.5, 0.75, 1) g / L, or peptone (0.25 / 0.5 / 0.75, 1.0, 1.5, 2.0) g / L. The growth of Orchidaceae seedlings cultured with different concentrations of Huabao No. 1, Huabao No. 4, and peptone for two months is shown in Table 6. The growth of Orchidaceae seedlings cultured with different concentrations of Huabao No. 1, Huabao No. 4, and peptone for three months is shown in Table 6.

[0077] Table 6 Growth of 2-month cultured Orchidaceae seedlings with Huabao No. 1, Huabao No. 4, and peptone content

[0078] Additives (g / L) Blade width (mm) Plant height(mm) Bulb diameter (mm) Fresh weight of bulb (g) CK 1.36±0.16d 31.56±0.23cd 3.11±0.08i 0.06±0.00f 0.125 Huabao No. 1 1.55±0.08d 37.02±0.35ab 3.71±0.05g 0.08±0.00e 0.25 Huabao No. 1 1.68±0.12d 36.67±1.71b 3.79±0.04g 0.08±0.00e 0.50 Huabao No. 1 1.74±0.11cd 33.51±1.13c 3.43±0.02h 0.09±0.02e 0.75 Huabao No. 1 5.28±0.75a 29.77±0.79de 4.70±0.08e 0.14±0.01d 1.00 Huabao No. 1 3.75±0.33abc 24.98±0.75g 4.83±0.11e 0.09±0.01e 0.065 Huabao No. 4 5.71±0.92a 30.46±1.12de 5.25±0.13d 0.17±0.00bc 0.125 Huabao No. 4 5.59±0.72a 29.58±0.34de 6.02±0.11a 0.19±0.00ab 0.25 Huabao No. 4 4.45±0.72ab 28.03±1.22ef 5.27±0.08d 0.14±0.01d 0.50 Huabao No. 4 3.81±0.57abc 26.26±0.24fg 4.40±0.07f 0.08±0.00ef 0.75 Huabao No. 4 2.92±0.33bcd 24.25±0.17g 4.23±0.03f 0.08±0.00ef 0.5 peptone 4.95±0.30ab 37.36±0.84ab 5.28±0.16d 0.15±0.00cd 0.75 peptone 4.91±0.75ab 37.57±0.87ab 5.51±0.11cd 0.17±0.01bc 1.0 Peptone 4.65±1.34ab 39.59±0.55a 6.00±0.00a 0.20±0.01a 1.5 Peptone 4.68±0.56ab 36.40±0.56b 5.83±0.10ab 0.19±0.01a 2.0 Peptone 5.14±1.11ab 31.81±1.08cd 5.63±0.08bc 0.17±0.01b

[0079] Note: Different lowercase letters indicate significant differences in the same indicator (P<0.05).

[0080] Table 6 shows that the CK group, which did not include Huabao No. 1, Huabao No. 4, or peptone, showed superior growth compared to the CK group with Huabao No. 4. The CK group showed the worst growth. The best results were achieved with 0.75 g / L Huabao No. 1 and 0.065-0.125 g / L Huabao No. 4. Regarding plant height, peptone was superior to both Huabao No. 1 and the CK group. However, adding Huabao No. 4 had an inhibitory effect on plant height, resulting in the worst effect. Peptone at 1.0 g / L had the best effect on plant height. Regarding bulb diameter and fresh weight, peptone was superior to Huabao No. 4, Huabao No. 1, and the CK group. Peptone at 1.0 g / L showed the best results, achieving a leaf width of 4.95 mm, a plant height of 39.59 mm, a bulb diameter of 6 mm, and a fresh weight of 0.2 g. Overall, the best results were achieved with 1.0 g / L peptone added for two months.

[0081] Table 7 Growth of Huabao No. 1, Huabao No. 4 and Peptone Content Cultured Orchidaceae Seedlings for 3 Months

[0082]

[0083]

[0084] Note: Different lowercase letters indicate significant differences in the same indicator (P<0.05). Large-bulb plants refer to plants with pseudobulb diameters greater than 5 mm.

[0085] Table 7 shows that the CK group (which lacked Huabao No. 1, Huabao No. 4, or peptone) showed significant differences in the growth of Orchidaceae grown on large corm medium after three months compared to the CK group. In terms of plant height, peptone outperformed Huabao No. 4, Huabao No. 1, and the CK group, with 1.0 g / L peptone being the most effective. In terms of corm diameter, 0.065-0.5 g / L Huabao No. 4 and 0.5-1.0 g / L peptone showed the best results, with no significant difference, both outperforming Huabao No. 1 and the CK group. In terms of the proportion of Orchidaceae plants with corms 5 mm or larger, the peptone group had the highest proportion, reaching over 90% and a rooting rate of 84%. Furthermore, considering the rooting rate, 1.0 g / L peptone achieved the best results after three months of culture, achieving a large corm proportion of 91%, a corm diameter of 7.59 mm, a plant height of 58.60 mm, and a rooting rate of 67%. 1 / 2MS+0.5g / L activated carbon+0.5mg / L naphthaleneacetic acid+75ml / L coconut juice+1.0g / L peptone is the best medium for large corm culture. The best medium for large corm culture is shown in Figure 1 , Figure 1 Figure A is a plant of Orchidaceae; BD are the growth conditions of Orchidaceae after aseptic sowing and culture on the optimal seed germination medium at 45 days, 60 days, and 90 days; EF are the growth conditions of Orchidaceae after large corm culture on the optimal large corm medium.

[0086] 1.5.3 Transplanting

[0087] The above-mentioned rooted seedlings of the Orchidaceae obtained by large corm culture with a plant height of more than 3.5 cm and a corm diameter of more than 5 mm were moved out of the tissue culture bottle of the Orchidaceae tissue culture room, placed for one week, and then the bottle cap was opened and placed for another week. After the rooted seedlings with roots were taken out and cleaned, the rooted Orchidaceae seedlings were transplanted into a matrix with a ratio of humus soil: pine bark: sawn wood = 1:1:1 for cultivation. After 2 months, the survival rate was more than 93.75%.

[0088] The growth of seedlings obtained during the germination stage of the seeds of the garlic orchid largely determines the formation of large bulbs. The formation of large bulbs is related to the culture cycle and growth conditions. Plants with strong growth and many leaves will have large bulbs earlier in the large bulb formation stage. Therefore, the cultivation goal during the seed germination stage is to obtain strong, leafy garlic orchid seedlings (rootless garlic orchid seedlings). Peptone is a complex nutrient source containing a rich nitrogen source and a small amount of carbon source. It can serve as a nutrient required for cell growth, promote plant growth and metabolism, and provide sufficient nutrients for the garlic orchid during the germination stage. Adding activated carbon to the culture medium improves the permeability of the culture medium, increases some trace elements, or absorbs inhibitory substances produced during seed germination. Huabao No. 1 culture medium is suitable for the growth of new stems and leaves. Adding an appropriate amount of Huabao No. 1 during seed germination can promote faster and stronger seedling growth and promote the growth of large corms. Sodium hypochlorite is a strong base and weak acid and is commonly used to disinfect explants in tissue culture. The seeds of the genus Echinops are small and have thick seed coats. Pre-treating them with 2% sodium hypochlorite, whose corrosive properties can somewhat wear away the seed coat and promote seed germination. Organic additives generally promote seed germination and protocorm development, but some can inhibit it. Organic additives promote corm growth more than seed germination, but they can inhibit germination to some extent. Coconut water, as an organic additive, contains amino acids, vitamins, carbohydrates, plant growth regulators, and various inorganic ions such as phosphorus, magnesium, potassium, and sodium.

[0089] Plants have different growth characteristics at different stages of growth, and require different nutrients. In the first stage, the cultivation goal is to have a high seed germination rate, fast germination, tall plant height, and many large leaves, which are manifested in the growth of sturdy seedlings. Such rootless seedlings are more likely to produce larger bulbs in the next stage of growth. In the seed germination stage, it is necessary to consider how to make the culture medium nutritious without affecting the germination rate. Excessive nutrients or excessive concentrations of certain hormones will affect the germination rate and the growth of seedlings. The cultivation goal of the second stage is to increase plant height, large bulbs, and a high rooting rate. It is necessary to consider how to improve the culture medium and subculture it based on the plant growth regulators in the first stage and the nutrients required for bulb formation. The two stages have different cultivation goals and growth characteristics. By selecting appropriate culture media according to different stages of the cultivation of the garlic orchid, the preset needs can be met.

[0090] Based on the above, it can be seen that pre-treating seeds with 2% sodium hypochlorite during the sterile sowing stage can promote early seed germination and increase the germination rate. Adding a certain amount of activated carbon is beneficial to the growth of D. uniflora, with 0.5g / L activated carbon having the best effect. Low concentrations of Huabao can promote the growth of seedlings, with 0.125g / L Huabao No. 1 having the best effect. The best culture method for D. uniflora seed germination is to pre-treat D. uniflora seeds with 2% sodium hypochlorite for 8 minutes, and then culture them in 1 / 2MS + 2g / L peptone + 0.5mg / L naphthaleneacetic acid + 0.5g / L activated carbon + 0.125g / L Huabao No. 1 for 3 months to obtain relatively sturdy D. uniflora rootless seedlings, which are more suitable for the induction and cultivation of large corms in the next step.

[0091] During the seedling growth stage, adding a certain concentration of coconut juice was better than banana puree. On this basis, adding a certain concentration of Huabao No. 1, Huabao No. 4 and peptone showed significant differences. Huabao No. 1 promoted the growth of stems and leaves, and Huabao No. 4 had a more obvious promoting effect on leaf width, plant height and bulb growth. Peptone had a significant promoting effect on leaf width, plant height, bulb and root growth. The best effect was achieved by subculturing large bulbs in 1 / 2MS+0.5g / L activated carbon+0.5mg / L naphthaleneacetic acid+75ml / L coconut juice+1.0g / L peptone for 3 months. The maximum bulb diameter reached 7.59mm and the plant height was 58.60mm. Plants with bulbs larger than 5mm accounted for 91% of the total, and the rooting rate was 67%.

[0092] The purpose of cultivating and growing sturdy rootless seedlings in the first stage is that the sturdy plants have stronger photosynthesis and grow faster, which is more conducive to the cultivation of large bulbs in the second stage. Bulbs are modified stems of orchids that store nutrients and water to adapt to special habitats. When tissue culture seedlings are transferred from a better tissue culture environment to a natural environment, the growth environment is relatively poor. At this time, the nutrients and water stored in the modified pseudobulbs are needed to help alleviate and adapt. Plants with larger bulbs have a higher transplant survival rate. Orchid plants with pseudobulbs exceeding 5mm have a high transplant survival rate. Therefore, the second stage cultivation target is larger bulbs with a diameter greater than 5mm.

[0093] The present invention adopts a two-stage tissue culture method of a sterile sowing stage and a strong seedling growth stage, and only takes 6 months from sowing to obtaining high-quality rooted seedlings with a high survival rate, thereby simplifying the production procedure and shortening the growth cycle. The method of the present invention is suitable for industrial production and lays a foundation for the cultivation of Solanum sempervirens.

[0094] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present disclosure, and that in actual applications, various changes may be made thereto in form and detail without departing from the scope of the present disclosure.

Claims

1. A method for rapid propagation of Orchidaceae seedlings, characterized in that: The steps include: Sowing the seeds of the Orchidaceae on a seed germination medium and cultivating them to obtain Orchidaceae rootless seedlings, wherein the germination medium contains a seed germination agent that promotes the germination of the Orchidaceae seeds; The rootless seedlings of the Orchidaceae are transferred to a large bulb culture medium for cultivation to become rooted seedlings of the Orchidaceae with bulbs larger than 5-9 mm. The large bulb culture medium contains a bulb growth agent.

2. A method for rapid propagation of Orchidaceae seedlings according to claim 1, characterized in that, The seed germination agent includes 1-2 mg / L peptone, 0.25-1.5 mg / L naphthaleneacetic acid, 0-1 mg / L activated carbon and 0-1 mg / L Huabao No. 1, wherein the activated carbon and / or Huabao No. 1 are greater than 0 mg / L.

3. A method for rapid propagation of Orchidaceae seedlings according to claim 2, characterized in that, The seed germination agent includes 2 mg / L peptone, 0.5-1 mg / L naphthaleneacetic acid, 0.25-0.75 mg / L activated carbon and 0.125-0.25 mg / L Huabao No.

1.

4. A method for rapid propagation of Orchidaceae seedlings according to claim 3, characterized in that, The seed germination agent includes 2 mg / L peptone, 0.5 mg / L naphthaleneacetic acid, 0.5 g / L activated carbon and 0.125 g / L Huabao No.

1.

5. A method for rapid propagation of Orchidaceae seedlings according to claim 1, characterized in that, The bulb growth agent comprises 0.5-2 mg / L peptone, 0.5-1.5 mg / L naphthaleneacetic acid, 0.5-1.5 g / L activated carbon and 25-150 mL / L organic additives.

6. A method for rapid propagation of Orchidaceae seedlings according to claim 5, characterized in that, The bulb growth agent comprises 0.75-1.5 mg / L peptone, 0.5-1 g / L activated carbon, 0.5-1 mg / L naphthaleneacetic acid and 75-100 mL / L organic additive.

7. A method for rapid propagation of Orchidaceae seedlings according to claim 6, characterized in that, The bulb growth agent includes 1.0 g / L peptone, 0.5 g / L activated carbon, 0.5 mg / L naphthaleneacetic acid and 75 ml / L organic additives.

8. A method for rapid propagation of Orchidaceae seedlings according to any one of claims 5 to 7, characterized in that: The organic additives include coconut water, mashed bananas or mashed potatoes.

9. A method for rapid propagation of Orchidaceae seedlings according to claim 1, characterized in that, The bulb growth agent includes 0.065-1 mg / L of Huabao, 0.5-1.5 mg / L of naphthaleneacetic acid, 0.5-1.5 g / L of activated carbon and 25-150 mL / L of organic additives; The flower treasure is flower treasure No. 1 or flower treasure No.

4.

10. A method for rapid propagation of Orchidaceae seedlings according to claim 1, characterized in that: Before the rootless seedlings of the Orchidaceae seeds are cultured, the Orchidaceae seeds are sterilized in a sodium hypochlorite solution for 4-12 minutes.

11. A method for rapid propagation of Orchidaceae seedlings according to claim 1, characterized in that, The basic substances in the seed germination medium and the large bulb culture medium include 1 / 2MS, 20-30g / L sucrose and 5-7g / L agar, and the pH of the seed germination medium and the large bulb culture medium is 5.8-6.0.

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