Potato explant tissue culture one-step seedling culture medium and potato tissue culture method

Abstract

According to the potato explant tissue culture one-step seedling culture medium and the potato tissue culture method, an MS culture medium is improved, ammonium nitrate is replaced with ammonium sulfate, ammonium chloride and ammonium dihydrogen phosphate, the purchase mode of ammonium chloride, ammonium sulfate and ammonium dihydrogen phosphate is simple, and the safety coefficient is higher; as ammonium chloride is used in MS, calcium chloride is replaced by calcium nitrate tetrahydrate, so that the use of chloride ions can be reduced while the content of calcium element is ensured. According to the application, the casein polypeptide with a specific concentration is added, so that the phenomena of weak growth of root systems, stem leaves and plants of the potato tissue culture seedlings and yellowing of the plants can be solved, and the plant height and diameter thickness of the potato tissue culture seedlings can also be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a one-step seedling culture medium for potato explant tissue culture and a potato tissue culture method. Background Art

[0002] Potato (scientific name: Solanum tuberosum L.), botanically classified as Solanum, Solanaceae, annual herb. Also known as potato, sweet potato, yam egg, etc. The tuber is edible and is an important crop used as food, vegetable, feed and industrial raw material. Potato tubers are rich in nutrients, 76-85% of which is water and the dry matter content is 15-24%. In the dry matter, starch and sugar account for 13-22%, and protein accounts for 1.6-2.1%. Protein has high nutritional value, is easy to digest and absorb, equivalent to the protein in eggs, and contains 8 essential amino acids for the human body, especially lysine and leucine.

[0003] The cultivation conditions of potatoes are poor, but they are resistant to adversity, have high nutritional value, and are highly adaptable to the external environment. Therefore, potato cultivation technology is constantly developing, and existing research mainly uses tissue culture and genetic transformation to carry out potato genetic breeding.

[0004] Plant tissue culture is also called in vitro culture in a broad sense, which refers to the technology of separating required tissues, organs or cells, protoplasts, etc. from the plant body, and cultivating them under sterile conditions on a culture medium containing various nutrients and plant hormones to obtain regenerated complete plants or produce other products with economic value through aseptic operation. In a narrow sense, it refers to tissue culture, which refers to the use of various parts of plant tissues, such as cambium, parenchyma, mesophyll tissue, endosperm, etc. to cultivate regenerated plants. It also refers to the culture of callus tissue produced from various organs during the culture process, and the callus tissue is then redifferentiated to form regenerated plants. Plant tissue culture can be divided into embryo culture, organ culture, tissue culture, cell culture and protoplast culture. The basic process of plant tissue culture includes surface sterilization, callus induction, detoxification and bud differentiation, bud subculture, rooting culture, seedling hardening and transplanting.

[0005] At present, there have been relevant studies on potato tissue culture. For example, Chinese patent application No. 202210151270.7 discloses a culture medium for potato tissue culture and its application, which is composed of 0.5-1.0 mg CCPU, 0.1-0.2 mg KT, 15-20 mg PAA, 35-40 g sucrose and 5-6 g agar per liter of MS culture medium; the invention reasonably regulates the ratio of the types and dosages of various plant hormones, and the prepared culture medium can shorten the culture time by about 15 days compared with the conventional one-step seedling culture medium, reducing the production cost and improving the economic value of potato tissue culture and rapid propagation. In addition, the culture medium is suitable for a variety of potato varieties, providing a new and effective way for their tissue culture and rapid propagation and variety improvement. However, the culture medium is based on MS culture medium.

[0006] MS medium is a commonly used medium in the rapid propagation of potato tissue culture seedlings, and ammonium nitrate is one of the main components of MS. Since ammonium nitrate is a raw material for industrial explosives and is prone to explosion under specific conditions such as high temperature, its production and sales are strictly controlled, making it very difficult to purchase. At present, in the tissue culture of plants such as apples, strawberries, grapes, cilantro, and Sophora flavescens, it has been found that ammonium sulfate, urea, ammonium chloride, and ammonium bicarbonate can be used to replace ammonium nitrate. Ammonium sulfate has stable chemical properties and can hydrolyze to produce NH4 + 、SO4 2- , which can provide nitrogen nutrition for adventitious buds without adding new elements to the mother solution of macronutrients, is a good substitute for ammonium sulfate. However, there are large differences in the adaptability and suitable concentration of ammonium sulfate among different plants. For example, plants such as Flame Nandina domestica and Sophora flavescens are not suitable for growing in a culture medium where ammonium sulfate replaces ammonium nitrate. At the same time, studies have also shown that high concentrations of NH4 + It has an inhibitory effect on plant roots. There have been studies on the use of potassium chloride and calcium nitrate to replace ammonium nitrate in potatoes, but there is still a question of how to improve the MS medium and replace ammonium nitrate, which can shorten the time for callus formation and differentiation into buds, and increase the induction rate and differentiation rate. There are few related reports. Summary of the invention

[0007] In the prior art, MS culture medium is commonly used as a culture medium in the rapid propagation production of potato tissue culture seedlings. In the MS culture medium, ammonium nitrate is one of the main components. There are some problems with the use of ammonium nitrate. Since ammonium nitrate is a raw material for industrial manufacturing of explosives, it is easy to explode under specific environments such as high temperature. Its production and sales are strictly controlled and it is difficult to purchase. In view of these problems, the present invention provides a one-step seedling culture medium for potato explant tissue culture and a method for potato tissue culture. By improving the MS culture medium and replacing ammonium nitrate, the plant height and stem reduction of potato tissue culture seedlings can be effectively promoted.

[0008] The purpose of the present invention is achieved through the following technical solutions: A one-step seedling culture medium for potato explant tissue culture, the formula of which is as follows: The composition and concentration of macroelements are: potassium nitrate 2400 mg / L, ammonium sulfate 240 mg / L, ammonium chloride 350 mg / L, diammonium phosphate 250 mg / L, potassium dihydrogen phosphate 120 mg / L, magnesium sulfate heptahydrate 370 mg / L, calcium nitrate tetrahydrate 707 mg / L; The composition and concentration of organic compounds were: inositol 200 mg / L, niacin 2 mg / L, pyridoxine hydrochloride 2 mg / L, thiamine hydrochloride 0.5 mg / L, and glycine 2 mg / L; Casein polypeptide 350-450 mg / L + zeatin (ZT) 3.0-5.0 mg / L + indolebutyric acid (IBA) 0.1-0.2 mg / L + sucrose 30 g / L + agar 5-8 g / L, pH 5.5-6.0; The composition and concentration of trace elements and iron salts are the same as those of MS medium.

[0009] The present invention also relates to a method for potato tissue culture, comprising the following steps: 1) Explant selection: Select sterile test tube potato seedlings of 20-30 days old and cut 0.5cm-1cm stem segments without axillary buds as explants under sterile conditions; 2) Tissue culture: The explants from the previous step are inoculated into the above-mentioned one-step seedling medium for potato explant tissue culture, and cultured at 25-27°C, a light intensity of 4000-4500 Lux, and a light duration of 14 h / d until regenerated plants are formed. The potato tissue culture seedlings have a plant height of 16.2-16.7 cm and a stem diameter of 2.18-2.32 mm.

[0010] In the present invention: The potato sterile test tube seedlings described in step 1) are selected from Mian Zi Yu No. 1 or Mian Yu No. 1.

[0011] The adjustments for a large number of elements are as follows: Compound Molecular weight MS(mg / L) Modified MS (mg / L) Ammonium Nitrate 80.05 1650 - Potassium Nitrate 101.10 1900 2400 Ammonium sulfate 132.14 - 240 Ammonium chloride 53.49 - 350 Ammonium dihydrogen phosphate 115.03 - 250 Potassium dihydrogen phosphate 136.09 170 120 Magnesium sulfate heptahydrate 246.47 370 370 Calcium chloride 110.99 332.25 - Calcium nitrate tetrahydrate 236.15 - 707 Compared with the prior art, the present invention has the following advantages: 1. Ammonium nitrate is a raw material for industrial explosives and is prone to explosion under specific conditions such as high temperature. Its production and sales are strictly controlled and difficult to purchase. The one-step seedling culture medium for potato explant tissue culture described in the present invention replaces ammonium nitrate with ammonium sulfate, ammonium chloride and diammonium phosphate by improving the MS culture medium. The procurement method of ammonium chloride, ammonium sulfate and diammonium phosphate is simple and has a higher safety factor. Since ammonium chloride has been used in MS, the present invention uses calcium nitrate tetrahydrate instead of calcium chloride to reduce the use of chloride ions while ensuring the calcium content.

[0012] 2. The present invention has found that ammonium nitrate is replaced by ammonium sulfate, ammonium chloride and diammonium phosphate. Although the procurement method of ammonium chloride, ammonium sulfate and diammonium phosphate is simple and the safety factor is higher, the potato tissue culture seedlings after treatment have the phenomenon of weak root system and stem and leaf growth and yellowing of plants, which is not suitable for potato propagation. Therefore, the present invention adds casein polypeptide to the one-step seedling medium of potato explant tissue culture. Casein polypeptide is a small molecule protein polypeptide extracted and hydrolyzed from bovine milk casein. It can complete the whole process of activation, repair, regeneration and activity enhancement for human immune cells (such as lymphocyte-specific antibodies, T cells, B cells, etc.), stimulate the initiative of the autoimmune system, and fundamentally restore and strengthen the autoimmune system. By adding a specific concentration of casein polypeptide, the present application can solve the problem of weak root system and stem and leaf growth and yellowing of potato tissue culture seedlings, and can also improve the plant height and diameter of potato tissue culture seedlings.

[0013] 3. The one-step seedling medium for tissue culture of potato explants provided by the present invention uses MS medium as the basic medium, which can meet the inorganic nutrients and part of the organic nutrients required for tissue culture. The casein polypeptide added thereto can more effectively promote the plant height and stem thickness of potato tissue culture seedlings than conventional synthetic auxins such as NAA and 2,4-D. The present invention has found through research that the addition of casein polypeptides to the one-step seedling medium for tissue culture of potato explants is not necessarily the more the better. An addition amount of casein polypeptide of 350-450 mg / L is more appropriate, and a satisfactory plant height and stem thickness can be obtained. With the increase of the addition amount of casein polypeptide, the plant height of potato tissue culture seedlings is reduced, and the stem thickness does not increase. Therefore, the present invention controls the addition amount of casein polypeptide to 350-450 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a planting site photo of tissue culture seedlings obtained by the one-step seedling culture medium and tissue culture method for potato explant tissue culture according to an embodiment of the present invention (3 months after planting); Figure 2This is a planting site photo of tissue culture seedlings obtained by the one-step seedling culture medium and tissue culture method for potato explant tissue culture according to an embodiment of the present invention (4 months after planting); Figure 3 This is a planting site photo of tissue culture seedlings obtained by the one-step seedling culture medium and tissue culture method for potato explant tissue culture according to an embodiment of the present invention (5 months after planting). DETAILED DESCRIPTION

[0015] The specific implementation modes of the present invention are further described below in conjunction with examples. Example

[0016] A one-step seedling culture medium for potato explant tissue culture, the formula of which is as follows: The composition and concentration of macroelements are: potassium nitrate 2400 mg / L, ammonium sulfate 240 mg / L, ammonium chloride 350 mg / L, diammonium phosphate 250 mg / L, potassium dihydrogen phosphate 120 mg / L, magnesium sulfate heptahydrate 370 mg / L, calcium nitrate tetrahydrate 707 mg / L; The composition and concentration of organic compounds were: inositol 200 mg / L, niacin 2 mg / L, pyridoxine hydrochloride 2 mg / L, thiamine hydrochloride 0.5 mg / L, and glycine 2 mg / L; Casein polypeptide 350-450 mg / L+ZT 3.0-5.0 mg / L+IBA 0.1-0.2 mg / L+sucrose 30g / L+agar 5-8 g / L, pH 5.5-6.0; The composition and concentration of trace elements and iron salts are the same as those of MS medium.

[0017] The above-mentioned method for potato tissue culture comprises the following steps: 1) Explant selection: Select sterile test tube potato seedlings of 20-30 days old and cut 0.5cm-1cm stem segments without axillary buds as explants under sterile conditions; 2) Tissue culture: The explants from the previous step are inoculated into the above-mentioned one-step seedling medium for potato explant tissue culture, and cultured at 25-27°C, a light intensity of 4000-4500 Lux, and a light duration of 14 h / d until regenerated plants are formed. The potato tissue culture seedlings have a plant height of 16.2-16.7 cm and a stem diameter of 2.18-2.32 mm.

[0018] Table 1: Culture medium Plant height (cm) Stem diameter (mm) Example 1 The composition and concentration of macroelements are: potassium nitrate 2400 mg / L, ammonium sulfate 240 mg / L, ammonium chloride 350 mg / L, ammonium dihydrogen phosphate 250 mg / L, potassium dihydrogen phosphate 120 mg / L, magnesium sulfate heptahydrate 370 mg / L, calcium nitrate tetrahydrate 707 mg / L; the composition and concentration of organic compounds are: inositol 200 mg / L, nicotinic acid 2 mg / L, pyridoxine hydrochloride 2 mg / L, thiamine hydrochloride 0.5 mg / L, glycine 2 mg / L; casein polypeptide 400 mg / L + ZT 4.0 mg / L + IBA 0.2 mg / L + sucrose 30 g / L + agar 5-8 g / L, pH 5.5-6.0; the composition and concentration of trace elements and iron salts are the same as MS medium 16.5 2.24 Example 2 Casein polypeptide 350 mg / L, other ingredients are the same as in Example 1 16.2 2.20 Example 3 Casein polypeptide 450 mg / L, other ingredients are the same as in Example 1 16.7 2.32 Example 4 ZT 3.0 mg / L, other contents are the same as in Example 1 16.4 2.18 Example 5 IBA 0.1 mg / L, other contents are the same as in Example 1 16.3 2.20 Comparative Example 1 Casein polypeptide 0 mg / L, other contents are the same as in Example 1 14.5 2.06 Comparative Example 2 Casein polypeptide 150 mg / L, other contents are the same as in Example 1 14.6 2.12 Comparative Example 3 Casein polypeptide 250 mg / L, other ingredients are the same as in Example 1 14.9 2.13 Comparative Example 4 Casein polypeptide 300 mg / L, other ingredients are the same as in Example 1 15.3 2.15 Comparative Example 5 Casein polypeptide 500 mg / L, other ingredients are the same as in Example 1 15.8 2.17 Comparative Example 6 Casein polypeptide 550 mg / L, other contents are the same as in Example 1 15.3 2.10 Control group (MS) 14.8 1.92 Results and conclusions: 1. By comparing Examples 1-5 with the control group (MS), the potato tissue culture seedlings using the culture medium of Examples 1-5 had a plant height of 16.2-16.7 cm and a stem diameter of 2.18-2.32 mm, while the potato tissue culture seedlings using the MS culture medium had a plant height of 14.8 cm and a stem diameter of 1.92 mm, indicating that using MS as a one-step seedling culture medium for potato explant tissue culture has the phenomenon that the potato tissue culture seedlings have weak plant height and stem and leaf growth, and are not suitable for potato propagation.

[0019] 2. By comparing Examples 1-5 with Comparative Example 1, the potato tissue culture seedlings using the culture medium of Examples 1-5 had a plant height of 16.2-16.7 cm and a stem thickness of 2.18-2.32 mm, while the potato tissue culture seedlings of Comparative Example 1 had a plant height of 14.2 cm and a stem thickness of 2.06 mm, indicating that no casein polypeptide was added. The plant height and stem thickness of the potato tissue culture seedlings of Comparative Example 1 were significantly different from those of Examples 1-5, indicating that the potato explant tissue culture one-step seedling culture medium of the present invention added casein polypeptide, which can effectively improve the plant height and stem thickness of potato tissue culture seedlings.

[0020] 3. By comparing the comparative example 1 with the control group, the potato tissue culture seedlings of the comparative example 1 had a plant height of 14.2 cm and a stem thickness of 2.06 mm, while the potato tissue culture seedlings of the control group using MS medium had a plant height of 14.8 cm and a stem thickness of 1.92 mm, indicating that without adding casein polypeptides, only the ammonium nitrate was replaced by ammonium sulfate, ammonium chloride and diammonium phosphate in the macroelements, and calcium chloride was replaced by calcium nitrate tetrahydrate, which had little effect on the plant height and stem thickness of the potato tissue culture seedlings.

[0021] 4. By comparing Examples 1-5 with Comparative Examples 1-5, it is shown that adding casein polypeptides to the one-step seedling culture medium for potato explant tissue culture is not necessarily better. In Examples 1-5, the addition amount of casein polypeptides of 350-450 mg / L is more appropriate, and satisfactory plant height and stem thickness can be obtained. With the increase of the addition amount of casein polypeptides, the plant height of potato tissue culture seedlings is reduced, and the stem thickness does not increase. Therefore, the present invention controls the addition amount of casein polypeptides to 350-450 mg / L.

[0022] 5. In the one-step seedling medium for tissue culture of potato explants described in the present invention, the improved MS increases the dosage of potassium nitrate to replace the use of potassium dihydrogen phosphate. Since ammonium dihydrogen phosphate has been used in MS, the use of potassium nitrate instead of dipotassium hydrogen phosphate can reduce the concentration of phosphate ions and the probability of phosphate ions forming precipitation with calcium ions, which is beneficial to the induction of callus tissue.

[0023] Figure 1It is a planting site picture of the potato explant tissue culture one-step seedling medium and tissue culture method obtained in the embodiment (a growth photo after 3 months of planting); Figure 2 It is a planting site picture of the tissue culture seedlings obtained by the one-step seedling culture medium and tissue culture method of the potato explant tissue culture of the embodiment (a growth photo of about 4 months of planting, autumn planting potatoes in mid-to-late September, and winter planting in mid-to-late December); Figure 3 This is a planting site photo of the tissue culture seedlings obtained by the one-step seedling culture medium and tissue culture method of potato explants in the embodiment (a photo taken after harvesting about 5 months after planting). The above description is only a preferred embodiment of the present invention. It should be pointed out that it is possible for a person skilled in the art to make several improvements and changes without departing from the creative concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A one-step seedling culture medium for potato explant tissue culture, characterized in that: The recipe is as follows: The composition and concentration of macroelements are: potassium nitrate 2400mg / L, ammonium sulfate 240mg / L, ammonium chloride 350mg / L, diammonium phosphate 250mg / L, potassium dihydrogen phosphate 120mg / L, magnesium sulfate heptahydrate 370mg / L, calcium nitrate tetrahydrate 707mg / L; The composition and concentration of organic compounds are: inositol 200 mg / L, niacin 2 mg / L, pyridoxine hydrochloride 2 mg / L, thiamine hydrochloride 0.5 mg / L, glycine 2 mg / L; Casein polypeptide 350-450mg / L+ZT 3.0-5.0mg / L+IBA 0.1-0.2mg / L+sucrose 30g / L+agar 5-8g / L, pH 5.5-6.0; The composition and concentration of trace elements and iron salts are the same as those of MS medium.

2. A method for potato tissue culture, characterized in that: The following steps are involved: 1) Explant selection: Select sterile test tube potato seedlings of 20-30 days old, and cut 0.5 cm-1 cm stem segments without axillary buds as explants under sterile conditions; 2) Tissue culture: inoculating the explants from the previous step into a one-step seedling culture medium for potato explant tissue culture, and culturing at 25-27° C., a light intensity of 4000-4500 Lux, and a light duration of 14 h / d until regenerated plants are formed, wherein the potato tissue culture seedlings have a plant height of 16.2-16.7 cm and a stem diameter of 2.18-2.32 mm; The one-step seedling culture medium for potato explant tissue culture has the following formula: The composition and concentration of macroelements are: potassium nitrate 2400mg / L, ammonium sulfate 240mg / L, ammonium chloride 350mg / L, diammonium phosphate 250mg / L, potassium dihydrogen phosphate 120mg / L, magnesium sulfate heptahydrate 370mg / L, calcium nitrate tetrahydrate 707mg / L; The composition and concentration of organic compounds are: inositol 200 mg / L, niacin 2 mg / L, pyridoxine hydrochloride 2 mg / L, thiamine hydrochloride 0.5 mg / L, glycine 2 mg / L; Casein polypeptide 350-450mg / L+ZT 3.0-5.0mg / L+IBA 0.1-0.2mg / L+sucrose 30g / L+agar 5-8g / L, pH 5.5-6.0; the composition and concentration of trace elements and iron salts are the same as those of MS culture medium.

3. A method for potato tissue culture according to claim 2, characterized in that: The sterile potato seedlings in step 1) are selected from Mian Zi Yu No. 1 or Mian Yu No. 1.

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