Culture medium for rapid propagation of sweet potato test-tube seedlings and preparation method thereof

Abstract

This invention discloses a culture medium for the rapid propagation of sweet potato test-tube seedlings, comprising the following components: MS + 0.2-0.4 mg / L 6-BA + 0.05-0.15 mg / L NAA + 0.1-0.3 mg / L IAA + 0.02-0.04 mg / L LPP333 + 0.7-0.9 g / L KNO3 + 0.21-0.23 g / L MgSO4 + 0.43-0.45 g / L CaCl2. The preparation method specifically includes the following steps: (1) preparation of plant hormones and salts; (2) melting agar; (3) adding mother liquor; (4) adjusting pH; (5) dispensing; and (6) autoclaving. This invention, by optimizing the culture medium components, shortens the growth time of sweet potato test-tube seedlings, increases the propagation coefficient of sweet potato test-tube seedlings, and reduces the production cost of tissue culture, thus achieving the goal of cost reduction and efficiency improvement in the sweet potato virus-free seedling propagation technology system.

Description

Technical Field

[0001] This invention relates to the field of rapid propagation technology, and more specifically to a culture medium for the rapid propagation of sweet potato test-tube seedlings and its preparation method. Background Technology

[0002] Sweet potato cultivation has a long history. As grain production has increased and the food problem has been gradually solved, the planting area of ​​sweet potatoes has gradually decreased, and it has been transformed from a major food crop to an economic crop.

[0003] Sweet potatoes are considered "the most ideal food of the 21st century" by experts because they are produced and stored without pollution or harmful substances and are rich in nutrients. Sweet potatoes are rich in nutrients needed by the human body. According to the *Complete Encyclopedia of Traditional Chinese Medicine*, sweet potatoes are alkaline foods containing protein, fat, carbohydrates, crude fiber, calcium, phosphorus, iron, potassium, sodium, magnesium, chlorine, carotene, vitamin B1, vitamin B2, niacin, and vitamin C, as well as tomatine and malic acid. Furthermore, sweet potatoes have excellent medicinal and health benefits. They contain the essential amino acid lysine, which provides the body with a large amount of mucoprotein, offering special protection. They also maintain the elasticity of cardiovascular walls, preventing arteriosclerosis, and reduce subcutaneous fat, avoiding excessive obesity. They can also prevent the atrophy of connective tissue in the liver and kidneys, preventing collagen diseases. Simultaneously, they keep the digestive tract, respiratory tract, joint cavities, and serous cavities lubricated. Sweet potatoes are high in starch and fiber, which can prevent constipation and intestinal diseases, and help prevent the formation of cholesterol in the blood. They are said to have the effects of "tonifying the middle and harmonizing the blood, replenishing qi and promoting body fluids, widening the intestines and stomach, and relieving constipation." Li Shizhen wrote in the Compendium of Materia Medica that sweet potatoes "replenish deficiency and fatigue, replenish qi and strength, strengthen the spleen and stomach, and strengthen kidney yin," and that long-term consumption "makes people live longer and have fewer diseases."

[0004] In recent years, sweet potato virus diseases have severely damaged sweet potato production, becoming one of the main reasons for reduced yield, quality, and seed quality degeneration. Sweet potato is an asexually propagated crop; due to long-term asexual reproduction, viruses can be transmitted through tubers and seedlings, leading to increased virus accumulation and severe degradation of yield and seed quality. Currently, there are no effective chemical control methods for sweet potato viruses. Therefore, virus-free seedling propagation technology remains the most effective measure for controlling sweet potato virus diseases.

[0005] Traditional sweet potato virus-free seedling propagation technology has certain shortcomings: First, it takes 3-4 years from virus-free seedlings to production seed, which is a long cycle; second, during the growth and propagation of virus-free seedlings, sweet potatoes are susceptible to viral infection, and the infection rate increases with each generation, resulting in a decline in seed potato quality; third, the propagation process requires more manpower and resources, leading to higher economic costs.

[0006] Therefore, how to shorten the growth time of sweet potato test-tube seedlings is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a culture medium for rapid propagation of sweet potato test-tube seedlings and a method for preparing the same, so as to overcome the shortcomings of the prior art.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A culture medium for rapid propagation of sweet potato test-tube seedlings comprises the following components: MS + 0.2-0.4 mg / L 6-BA (6-benzylaminopurine) + 0.05-0.15 mg / L NAA (naphthaleneacetic acid) + 0.1-0.3 mg / L IAA (indoleacetic acid) + 0.02-0.04 mg / L PP333 (paclobutrazol) + 0.7-0.9 g / L KNO3 (potassium nitrate) + 0.21-0.23 g / L MgSO4 (magnesium sulfate) + 0.43-0.45 g / L CaCl2 (calcium chloride);

[0010] The preferred concentration is: MS + 0.3 mg / L 6-BA + 0.1 mg / L NAA + 0.2 mg / L IAA + 0.03 mg / L P333 + 0.8 g / L KNO3 + 0.22 g / L MgSO4 + 0.44 g / L CaCl2.

[0011] In this invention:

[0012] MS medium has a high concentration of inorganic salts, ensuring sufficient mineral nutrition for tissue growth and accelerating callus growth. Due to the high ion concentration in its formula, slight variations in some components during preparation, storage, and sterilization will not affect the ion balance. The quantity and proportion of inorganic nutrients in MS medium are suitable, sufficient to meet the nutritional and physiological needs of plant cells. Compared to the basic components of other culture media, MS medium has a high content of nitrates, potassium, and ammonium, which is its significant characteristic.

[0013] 6-BA, or 6-benzylaminopurine, also known as 6-phenylmethyladenine, primarily functions to promote bud formation.

[0014] NAA, or 1-naphthaleneacetic acid, has the molecular formula C2. 12 H 10 O2 is a plant growth regulator with auxin-like activity. It is absorbed by roots, stems, and leaves, and its main function is to induce the formation of adventitious roots.

[0015] IAA, or auxin, is chemically indoleacetic acid and has the functions of regulating stem growth rate, inhibiting lateral buds, and promoting rooting.

[0016] PP333, or paclobutrazol, is a triazole plant growth regulator that inhibits the synthesis of endogenous gibberellins. It has the effects of delaying plant growth, inhibiting stem elongation, shortening internodes, promoting plant tillering, promoting flower bud differentiation, increasing plant stress resistance, and increasing yield.

[0017] KNO3 provides nitrogen and potassium ions.

[0018] MgSO4 provides magnesium ions.

[0019] CaCl2 provides calcium ions.

[0020] A method for preparing a culture medium for rapid propagation of sweet potato test-tube seedlings specifically includes the following steps:

[0021] (1) Preparation of plant hormones and salts

[0022] Prepare 6-BA, NAA, IAA, PP333, KNO3, MgSO4 and CaCl2 according to the concentrations of the above-mentioned culture medium;

[0023] (2) Melt the agar

[0024] Place the agar in a stainless steel pot, add water to half the volume of the required culture medium, heat to melt the agar, and obtain the agar solution for later use.

[0025] (3) Add mother liquor

[0026] Add the prepared stock solutions of 6-BA, NAA, IAA, PP333, KNO3, MgSO4 and CaCl2, various stock solutions of MS medium, and sucrose to the agar solution, continue heating and stirring, and after the sucrose dissolves, add water to the required volume of medium and stir well.

[0027] (4) Adjust pH value

[0028] Measure the pH value of the culture medium and adjust the pH value of the culture medium to 5.8-6.0;

[0029] (5) Packaging

[0030] The culture medium is dispensed and sealed with high-temperature and high-pressure resistant material;

[0031] (6) High-pressure sterilization

[0032] Place the culture medium in an autoclave, thoroughly purge the air from the autoclave with saturated steam, and autoclave. Once the pressure in the autoclave has dropped to normal, transfer the culture medium to a clean room.

[0033] Furthermore, in step (1) above, the preparation of 6-BA is as follows: Weigh 1.0 mg / mL of 6-BA, dissolve it with 1 mol / L NaOH, then dilute it to 0.2-0.4 mg / L with distilled water, and store it in a refrigerator at 4℃ for later use.

[0034] Furthermore, in step (1) above, the preparation of NAA is as follows: weigh 0.1 mg / mL of NAA, dissolve it in 95% alcohol, then dilute it to 0.05-0.15 mg / L with distilled water, and store it in a refrigerator at 4°C for later use.

[0035] Furthermore, in step (1) above, the preparation of IAA is as follows: Weigh 0.1 mg / mL of IAA, dissolve it in 95% alcohol, then dilute it to 0.1-0.3 mg / L with distilled water, and store it in a refrigerator at 4°C for later use.

[0036] Furthermore, in step (1) above, the preparation of PP333 is as follows: weigh 0.1 mg / mL of PP333, dissolve it fully in distilled water, then make up to 0.02-0.04 mg / L with distilled water, and store it in a refrigerator at 4℃ for later use.

[0037] Furthermore, in step (4) above, the measuring tool is a pH meter or precision pH test paper; the solution for adjusting the pH value is a 0.1 mol / L HCl solution or a 0.1 mol / L NaOH solution.

[0038] Furthermore, in step (5) above, the containers for dispensing are test tubes, Erlenmeyer flasks, or culture flasks (do not allow culture medium to adhere to the mouth of the culture flask).

[0039] Furthermore, in step (6) above, the temperature for autoclaving is 121°C and the time is 20-40 min (the larger the volume of the object to be sterilized, the longer the sterilization time is required).

[0040] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] This invention shortens the growth time of sweet potato test-tube seedlings, increases the propagation coefficient of sweet potato test-tube seedlings, and reduces the production cost of tissue culture by optimizing the culture medium composition, thus enabling the sweet potato virus-free seedling propagation technology system to achieve the goal of cost reduction and efficiency improvement. Attached Figure Description

[0042] Figure 1 This is a photograph of the overall growth of the test-tube seedlings after 20 days of culture in culture medium A, Comparative Example 1.

[0043] Figure 2 This is a photograph of the stem and leaf growth of test-tube seedlings after 20 days of culture in culture medium A, Comparative Example 1.

[0044] Figure 3 This is a photograph of the root growth of test-tube seedlings after 20 days of culture in culture medium A, Comparative Example 1.

[0045] Figure 4 This is a photograph of the overall growth of the test-tube seedlings after 20 days of culture in culture medium B, Comparative Example 2.

[0046] Figure 5 This is a photograph of the stem and leaf growth of test-tube seedlings after 20 days of culture in culture medium B, Comparative Example 2.

[0047] Figure 6 This is a photograph of the root growth of test-tube seedlings after 20 days of culture in culture medium B, Comparative Example 2.

[0048] Figure 7 This is a photograph of the overall growth of test-tube seedlings after 20 days of culture in culture medium C (Comparative Example 3).

[0049] Figure 8 This is a photograph of the stem and leaf growth of test-tube seedlings after 20 days of culture in culture medium C (Comparative Example 3).

[0050] Figure 9 This is a photograph of the root growth of test-tube seedlings after 20 days of culture in culture medium C (Comparative Example 3).

[0051] Figure 10 This is a photograph of the overall growth of the test-tube seedlings after 20 days of culture in culture medium D, Comparative Example 4.

[0052] Figure 11 This is a photograph of the stem and leaf growth of test-tube seedlings after 20 days of culture in culture medium D, as shown in Comparative Example 4.

[0053] Figure 12 This is a photograph of the root growth of test-tube seedlings after 20 days of culture in culture medium D (Comparative Example 4).

[0054] Figure 13 This is a photograph of the overall growth of the test-tube seedlings after 20 days of culture in the culture medium of Example 1.

[0055] Figure 14 This is a photograph of the stem and leaf growth of test-tube seedlings after 20 days of culture in the culture medium of Example 1.

[0056] Figure 15 This is a photograph of the root growth of test-tube seedlings after 20 days of culture in the culture medium of Example 1. Detailed Implementation

[0057] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] In the following examples and comparative examples, the composition and amount of MS culture medium are shown in Table 1.

[0059] Table 1. Components and dosage of MS culture medium

[0060]

[0061] Example 1

[0062] The culture medium for rapid propagation of sweet potato test-tube seedlings includes the following components: MS + 0.3 mg / L 6-BA + 0.1 mg / L NAA + 0.2 mg / L IAA + 0.03 mg / L PP333 + 0.8 g / L KNO3 + 0.22 g / L MgSO4 + 0.44 g / L CaCl2;

[0063] The method for preparing the culture medium for rapid propagation of sweet potato test-tube seedlings described above specifically includes the following steps:

[0064] (1) Preparation of plant hormones and salts

[0065] Prepare 6-BA, NAA, IAA, PP333, KNO3, MgSO4 and CaCl2 according to the concentrations of the above-mentioned culture medium;

[0066] The specific preparation of 6-BA is as follows: Weigh 1.0 mg / mL of 6-BA, dissolve it in 1 mol / L NaOH, then dilute it to 0.3 mg / L with distilled water, and store it in a refrigerator at 4℃ for later use.

[0067] The specific preparation method for NAA is as follows: Weigh 0.1 mg / mL of NAA, dissolve it in 95% alcohol, then dilute it to 0.1 mg / L with distilled water, and store it in a refrigerator at 4°C until use.

[0068] The specific preparation method for IAA is as follows: Weigh 0.1 mg / mL of IAA, dissolve it in 95% alcohol, then dilute it to 0.2 mg / L with distilled water, and store it in a refrigerator at 4°C until use.

[0069] The specific preparation method for PP333 is as follows: Weigh 0.1 mg / mL of PP333, dissolve it thoroughly in distilled water, then make up the volume with distilled water to 0.03 mg / L, and store it in a refrigerator at 4℃ until use.

[0070] (2) Melt the agar

[0071] Place the agar in a stainless steel pot, add water to half the volume of the required culture medium, heat to melt the agar, and obtain the agar solution for later use.

[0072] (3) Add mother liquor

[0073] Add the prepared stock solutions of 6-BA, NAA, IAA, PP333, KNO3, MgSO4 and CaCl2, various stock solutions of MS medium, and sucrose to the agar solution, continue heating and stirring, and after the sucrose dissolves, add water to the required volume of medium and stir well.

[0074] (4) Adjust pH value

[0075] The pH of the culture medium was measured using a pH meter, and 0.1 mol / L HCl solution was added to adjust the pH of the culture medium to 6.0.

[0076] (5) Packaging

[0077] The culture medium was dispensed into test tubes and sealed with high-temperature and high-pressure resistant material;

[0078] (6) High-pressure sterilization

[0079] Place the culture medium in an autoclave, thoroughly purge the air from the autoclave with saturated steam, heat to 121°C, and autoclave for 30 minutes. After the pressure in the autoclave drops to normal, transfer the culture medium to a clean room.

[0080] Comparative Example 1

[0081] Culture medium A differs from that in Example 1 only in that it includes the following component: MS.

[0082] Comparative Example 2

[0083] Culture medium B differs from that in Example 1 only in that it includes the following components: MS + 0.1 mg / L 6-BA + 0.05 mg / L NAA.

[0084] Comparative Example 3

[0085] Culture medium C differs from that in Example 1 only in that it includes the following components: MS + 0.3 mg / L 6-BA + 0.1 mg / L NAA + 0.2 mg / L IAA + 0.03 mg / L P333.

[0086] Comparative Example 4

[0087] Culture medium D differs from that in Example 1 only in that it includes the following components: MS + 0.8 g / L KNO3 + 0.22 g / L MgSO4 + 0.44 g / L CaCl2.

[0088] Performance testing

[0089] 1. Sample processing

[0090] The culture medium AD prepared in Comparative Examples 1-4 and the culture medium for rapid propagation of sweet potato test-tube seedlings prepared in Example 1.

[0091] 2. Inoculation method

[0092] Select test-tube seedlings of Fushu 604 with the same growth cycle, thick main stems, and uniform growth. Cut them into single stem segments with one axillary bud, about 1.5 cm in length. Inoculate 4 stem segments per bottle, 5 bottles per treatment, and repeat 3 times.

[0093] 3. Cultivation conditions

[0094] Temperature 27±1℃, light intensity 2000-3000lx, light cycle 16h / d.

[0095] 4. Measurement Items and Methods

[0096] Twenty days after inoculation, the rooting time, average fresh weight per plant, average number of leaves per plant, average plant height, and root development of sweet potato test-tube seedlings in each treatment were investigated.

[0097] Thirty days after inoculation, sweet potato test-tube seedlings were inoculated in single stem segments, and the proliferation coefficient was calculated.

[0098] 5. Test Results

[0099] As shown in Table 1 and Figure 1-15 As shown.

[0100] Table 1. Growth of sweet potato in vitro seedlings under each treatment 20 days after inoculation and proliferation coefficient 30 days after inoculation.

[0101]

[0102] From Table 1 and Figure 1-15 It can be seen that, compared with Comparative Examples 1-4, the culture medium used in Example 1 for rapid propagation of sweet potato test-tube seedlings significantly shortened the rooting time of sweet potato test-tube seedlings and significantly improved the growth of stems, leaves and roots of sweet potato test-tube seedlings.

[0103] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A culture medium for the rapid propagation of sweet potato test-tube seedlings, characterized in that, It is prepared from the following components: MS + 0.2-0.4 mg / L 6-BA + 0.05-0.15 mg / L NAA + 0.1-0.3 mg / L L IAA + 0.02-0.04 mg / L LPP333 + 0.7-0.9 g / L KNO3 + 0.21-0.23 g / L MgSO4 + 0.43-0.45 g / L CaCl2; The explants inoculated into the culture medium are single stem segments with one axillary bud.

2. The culture medium for rapid propagation of sweet potato test-tube seedlings according to claim 1, characterized in that, It is made from the following components: MS + 0.3 mg / L 6-BA + 0.1 mg / L NAA + 0.2 mg / L L1AA + 0.03 mg / L LPP333 + 0.8 g / L KNO3 + 0.22 g / L MgSO4 + 0.44 g / L CaCl2.

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