Method for saving cost of production of in vitro culture seedling of lycoris radiata and application thereof

Abstract

The application discloses a method for saving production cost of lycoris radiata tissue culture seedlings and application thereof, and relates to the technical field of plant tissue culture, and comprises the following steps: taking lycoris radiata bulbs, and cutting the bulbs into four pieces in a cross shape; and culturing and inducing small bulbs to proliferate in a proliferation culture medium containing rice powder under greenhouse conditions; wherein, the composition of the proliferation culture medium comprises conventional MS components and special additives, namely rice powder. The method has the beneficial effect that rice powder is used to replace coconut juice as an exogenous additive for lycoris radiata tissue culture production, the proliferation coefficient is improved, and the production cost is remarkably saved, thereby providing technical support for industrialized production of lycoris radiata seedlings.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for saving costs in cultivating Lycoris radiata tissue culture seedlings and its application. Background Technology

[0002] *Lycoris radiata* Herb. is a perennial bulbous herbaceous plant belonging to the Amaryllidaceae family. Due to its unique flower shape, robust flower stalk, vibrant flower color, and elegant leaf shape, it is known in the West as the "Chinese tulip." Endemic to East Asia, *Lycoris radiata* blooms during the hot summer and autumn seasons when few flowers bloom, making it widely used in flower borders, forest edges, and as ground cover. It is also an excellent cut flower. Its underground bulbs are rich in lycorine, galantamine, and other chemical substances, giving it high medicinal value. *Lycoris radiata* has very high economic value; however, current techniques for propagation through seed, natural bulb division, and artificial bulb division cannot meet the requirements for large-scale seedling production, severely restricting the development of the industry. Rapid bulb propagation of *Lycoris radiata* is currently the biggest obstacle to the industrialization of the plant. While there has been considerable research on propagation of *Lycoris radiata* seedlings through tissue culture, the long growth cycle and high cost make it difficult to meet production needs.

[0003] Previous studies have shown that adding coconut juice can significantly improve the proliferation coefficient of Lycoris radiata bulblets. However, the cost of using coconut juice is high. Therefore, finding a substitute for coconut juice to reduce the cost of tissue culture production is of great significance for the tissue culture production of Lycoris radiata.

[0004] Therefore, establishing a low-cost and efficient tissue culture method for Lycoris radiata is of great significance to the industrialization of Lycoris radiata. Summary of the Invention

[0005] This invention aims to provide a method and its application for saving the production cost of Lycoris radiata tissue culture seedlings. It can obtain proliferating seedlings with good bulb proliferation coefficient and normal morphology at low cost compared with coconut juice culture, providing technical support for the industrialized production of high-quality Lycoris radiata seedlings.

[0006] In a first aspect, the present invention provides a method for saving the production cost of Lycoris radiata tissue culture seedlings, comprising the following steps:

[0007] First-generation aseptic proliferating bulbs of Lycoris radiata were placed in a proliferation medium and cultured under greenhouse conditions;

[0008] The proliferation medium includes MS medium, 1-10 g / L rice flour, 5.0 mg / L 6-BA, 3 mg / L IBA, 1 g / L AC, 30 g / L sucrose, and 7 g / L carrageenan.

[0009] Optionally, the size of the bulb is 1.0-1.5 cm.

[0010] Optionally, the preparation of the tissue culture-proliferated bulbs of Lycoris radiata includes the following steps:

[0011] Take the unsprouted bulbs of red stone garlic, remove the outer skin, and rinse with running water for 3-4 hours;

[0012] Under aseptic conditions, soak the bulbs in 75% ethanol solution for 30-40 seconds, then add 0.1% HgCl2 for 10-15 minutes for disinfection. After disinfection, wash the bulbs 5 times with sterile water.

[0013] After washing, the bulbs are cut into bulb pieces and cultured in primary culture medium for 30-40 days to obtain primary sterile proliferating bulbs.

[0014] Optionally, the primary culture medium includes MS medium, 1 g / L AC, 5.0 mg / L 6-BA, 3.0 mg / L IBA, 30 g / L sucrose, and 7 g / L carrageenan.

[0015] Optionally, the room temperature condition is 25°C, and the light-induced period is 30-40 days.

[0016] Secondly, the present invention provides an application of the method described above in the production of Lycoris radiata tissue culture seedlings.

[0017] The beneficial effects of this invention include:

[0018] (1) The rice flour provided by this invention can replace coconut juice as the main component of the culture medium to carry out small bulb proliferation culture of Lycoris radiata. After 30-40 days, proliferating seedlings with good proliferation coefficient and normal morphology are obtained.

[0019] (2) The method provided by the present invention uses rice flour to reduce the cost of culture medium with coconut juice as the main component. Each liter of culture medium requires 50 mL of coconut juice. Each coconut costs 5 yuan and contains 250 mL of coconut juice, so the cost of coconut juice is about 1 yuan. However, each liter of culture medium only requires 1-10 g of rice flour, so the cost is about 0.004 yuan. The method of the present invention greatly reduces the production cost of Lycoris radiata tissue culture seedlings and plays an important role in promoting the industrialization of Lycoris radiata tissue culture. Attached Figure Description

[0020] Figure 1 The results show the effects of different culture media on the proliferation of small bulbs of Lycoris radiata. Detailed Implementation

[0021] The present invention will be further described in conjunction with the accompanying drawings and through the following embodiments.

[0022] In a first aspect, embodiments of the present invention provide a method for saving production costs of Lycoris radiata tissue culture seedlings, comprising the following steps:

[0023] Red spider lily bulbs were cultured in a proliferation medium under greenhouse conditions;

[0024] The proliferation medium includes MS medium, 1-10 g / L rice flour, 5.0 mg / L 6-BA, 3 mg / L IBA, 1 g / L AC, 30 g / L sucrose, and 7 g / L carrageenan.

[0025] Specifically, the size of the bulb is 1.0-1.5cm.

[0026] In some embodiments, the preparation of the tissue culture-proliferated bulbs of Lycoris radiata includes the following steps:

[0027] Take the unsprouted bulbs of red stone garlic, remove the outer skin, and rinse with running water for 3-4 hours;

[0028] Under aseptic conditions, soak the bulbs in 75% ethanol solution for 30-40 seconds, then add 0.1% HgCl2 for 10-15 minutes for disinfection. After disinfection, wash the bulbs 5 times with sterile water.

[0029] After washing, the bulbs are cut into four bulb pieces with basal discs and cultured in primary culture medium for 30-40 days to obtain primary sterile proliferating bulbs.

[0030] Specifically, the primary culture medium includes MS medium, 1 g / L AC, 5.0 mg / L 6-BA, 3.0 mg / L IBA, 30 g / L sucrose, and 7 g / L carrageenan.

[0031] Specifically, the room temperature condition is 25°C, and the light induction period is 30-40 days.

[0032] Secondly, embodiments of the present invention provide an application of the method in the production of Lycoris radiata tissue culture seedlings.

[0033] The reagents, culture media, other culture media additives, rice, coconut, soluble starch, etc. used in the embodiments of this invention are all commercially available.

[0034] Example 1

[0035] Embodiment 1 of the present invention provides a method for saving the production cost of Lycoris radiata tissue culture seedlings, comprising the following steps:

[0036] S1, Primary culture of Lycoris radiata bulbs

[0037] Take unsprouted bulbs of wild Lycoris radiata, remove the outer skin, and rinse with running water for 3 hours;

[0038] Under aseptic conditions, soak the bulbs in 75% ethanol for 40 seconds, then add 0.1% HgCl2 for 10-15 minutes for disinfection.

[0039] After disinfection, wash five times with sterile water, cut into four bulb pieces and culture in primary culture medium for 30-40 days to obtain primary sterile proliferating bulbs of Lycoris radiata.

[0040] The primary culture medium consisted of MS medium, 1 g / L LAc, 5.0 mg / L, 6-BA, 3.0 mg / L IBA, 30 g / L sucrose, and 7 g / L carrageenan.

[0041] S2, Propagation culture of Lycoris radiata bulbs

[0042] The primary sterile proliferating bulbs obtained in step S1 were divided into four bulb pieces, placed in a proliferation medium, and induced to proliferate under a light intensity of 3000 lx and a temperature of 25°C for 30-40 days.

[0043] The proliferation medium consisted of MS medium, 5.0 mg / L 6-BA, 3.0 mg / L IBA, and 1 g / L rice flour.

[0044] The method for preparing rice flour is as follows: take raw rice grains, grind them into powder, and pass them through an 80-mesh sieve to obtain fine and uniform rice flour. After weighing, add it to the culture medium.

[0045] Example 2

[0046] Example 2 of this invention provides a method for saving production costs of Lycoris radiata tissue culture seedlings. The difference from Example 1 is that the amount of rice flour added is 5g / L, while other conditions and steps are the same as in Example 1.

[0047] Example 3

[0048] Example 2 of this invention provides a method for saving production costs of Lycoris radiata tissue culture seedlings. The difference from Example 1 is that the amount of rice flour added is 10g / L, while other conditions and steps are the same as in Example 1.

[0049] Comparative Example 1

[0050] Comparative Example 1 of this invention provides a method for producing Lycoris radiata tissue culture seedlings, which differs from Example 1 in that rice flour is not added, while other conditions and steps are consistent with Example 1.

[0051] Comparative Example 2

[0052] Comparative Example 2 of this invention provides a method for producing Lycoris radiata tissue culture seedlings. The difference from Example 1 is that rice flour is replaced with 50 mL / L coconut juice, while other conditions and steps are the same as in Example 1.

[0053] Comparative Example 3

[0054] Comparative Example 3 of this invention provides a method for producing Lycoris radiata tissue culture seedlings. The difference from Example 1 is that rice flour is replaced with 100 mL / L coconut juice, while other conditions and steps are the same as in Example 1.

[0055] Comparative Example 4

[0056] Comparative Example 4 of this invention provides a method for producing Lycoris radiata tissue culture seedlings. The difference from Example 1 is that rice flour is replaced with 200 mL / L coconut juice, while other conditions and steps are the same as in Example 1.

[0057] Comparative Example 5

[0058] Comparative Example 5 of this invention provides a method for producing Lycoris radiata tissue culture seedlings. The difference from Example 1 is that rice flour is replaced with soluble starch, and the amount added is 1g / L. Other conditions and steps are the same as in Example 1.

[0059] Comparative Example 6

[0060] Comparative Example 6 of this invention provides a method for producing tissue culture seedlings of Lycoris radiata. The difference from Example 1 is that rice flour is replaced with soluble starch, and the amount added is 5g / L. Other conditions and steps are the same as in Example 1.

[0061] Comparative Example 7

[0062] Comparative Example 7 of this invention provides a method for producing Lycoris radiata tissue culture seedlings. The difference from Example 1 is that rice flour is replaced with soluble starch, and the amount added is 10g / L. Other conditions and steps are the same as in Example 1.

[0063] Property testing

[0064] (1) Analysis of the bulbs after proliferation culture of Lycoris radiata in the examples and comparative examples.

[0065] Each example and comparative example had 3 replicates, with 10 explants per replicate. The total number of proliferating bulbs and the bulb proliferation coefficient were statistically analyzed 30-40 days after inoculation into the proliferation medium. The calculation formula is as follows:

[0066] Proliferation coefficient = Total number of proliferating bulbs / Total number of inoculated bulb tissue blocks; Proliferation results are as follows: Figure 1 As shown in Table 1, the calculation results are as follows.

[0067] (2) Cost calculation for the proliferation culture of Lycoris radiata bulbs in the examples and comparative examples.

[0068] Cost accounting is based on 250mL of coconut juice per coconut, with each coconut costing 5 yuan and coconut juice costing 20 yuan / L; soluble starch costs 120 yuan / kg, and rice flour only requires 1-10g per liter of culture medium, with an average cost of approximately 0.004 yuan. The standard cost accounting for conventional MS culture medium is shown in Table 2, and the costs in each example and comparative example are shown in Table 3.

[0069] Table 1. Efficiency and Cost Calculation of Small Bulb Propagation Culture of Lycoris radiata

[0070]

[0071] Table 2. Cost Accounting Table for the Propagation and Culture of Small Bulbs of Lycoris radiata using MS

[0072]

[0073] Table 3. Cost of each culture medium in the examples and comparative examples.

[0074]

[0075] Referring to Tables 1 and 3, compared with the comparative examples, the culture medium with 1 g / L rice flour added in Example 1 showed the best culture effect and the highest proliferation coefficient, reaching 4.2, which was not significantly different from the proliferation coefficient of 4.13 of the culture medium with added coconut juice. Among the culture media with added soluble starch, Comparative Example 6 was the best, with a proliferation coefficient of 2.37. The rice flour provided by the present invention can achieve the same effect as coconut juice, or even slightly better than coconut juice culture medium, while reducing the cost of coconut juice. Soluble starch has no obvious effect on the proliferation of Lycoris radiata, and its proliferation coefficient is not significantly different from the control group in Comparative Example 1 that did not add rice flour or coconut juice.

[0076] See Figure 1 The small bulb tissue culture seedlings induced by the culture medium with added rice flour grew healthily, with normal bulb morphology and bright green leaves, making it very easy for them to root later. This shows that the Red Stone Garlic tissue culture seedlings prepared by the proliferation culture medium with added rice flour provided by this invention are effective seedlings and can be applied to large-scale production.

[0077] Referring to Table 3, further analysis of production costs shows that using rice flour increases the multiplication coefficient while saving 73.53% in production costs compared to using coconut milk.

[0078] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A method for saving production costs of Lycoris radiata tissue culture seedlings, characterized in that, Includes the following steps: The first generation of aseptic proliferating bulbs of Lycoris radiata were divided into four pieces, placed in a proliferation medium, and induced under light at 25°C for 30-40 days. The proliferation medium is MS medium + 1-5 g / L rice flour + 5.0 mg / L 6-BA + 3 mg / L IBA + 1 g / L AC + 30 g / L sucrose + 7 g / L carrageenan; the rice flour is obtained by crushing raw rice grains and passing them through an 80-mesh sieve.

2. The method according to claim 1, characterized in that, The size of the bulb is 1.0-1.5 cm.

3. The method according to claim 1, characterized in that, The preparation of the primary aseptic proliferating bulbs of Lycoris radiata includes the following steps: Take unsprouted bulbs of Lycoris radiata, remove the outer skin, and rinse with running water for 3-4 hours; Under aseptic conditions, soak the bulbs in 75% ethanol solution for 30-40 seconds, then add 0.1% HgCl2 for 10-15 minutes for disinfection. After disinfection, wash the bulbs 5 times with sterile water. After washing, the bulbs are cut into bulb pieces with basal discs and cultured in primary culture medium for 30-40 days to obtain the primary sterile proliferating bulbs.

4. The method according to claim 3, characterized in that, The primary culture medium consisted of MS medium + 1 g / L AC + 5.0 mg / L 6-BA + 3.0 mg / L IBA + 30 g / L sucrose + 7 g / L carrageenan.

5. The application of the method as described in claim 1 in the production of Lycoris radiata tissue culture seedlings.

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