Grape solid-liquid double-layer culture method

Abstract

The invention discloses a grape solid-liquid double-layer culture method, and relates to the technical field of agricultural science, and the method comprises the following steps: obtaining a plurality of plump Chardonnay seeds; the solid substrate is placed in a seedling raising pot, the Chardonnay seeds are planted in the solid substrate of the seedling raising pot, the solid substrate comprises fine soil, vermiculite and perlite, and the proportion of the fine soil to the vermiculite to the perlite is 6: 1: 1; adding a culture solution into a liquid culture medium, and putting the seedling culture bowl into the liquid culture medium; the liquid level of the culture solution abuts against the bowl bottom of the seedling raising bowl, a plurality of seedling raising holes are formed in the bottom of the seedling raising bowl, and rhizomes obtained after sprouting and rooting of Chardonnay seeds planted in the seedling raising bowl extend into the culture solution through the seedling raising holes to absorb nutrition and moisture of the culture solution. According to the method, a solid-liquid co-culture mode is adopted, the problem of independent water culture and soil culture is avoided, the advantages of the water culture and the soil culture are combined, watering and fertilization are not needed, manpower and material resource investment is remarkably reduced, the overall cost is reduced, and the method can be used for genetic transformation screening.

Description

Technical Field

[0001] The present application relates to the field of agricultural science and technology, and in particular to a solid-liquid double-layer culture method for grapes. Background Art

[0002] Chardonnay, also known as Sherdonnay, Chardonnay, and Chardonnay, is a Eurasian variety, a medium-early maturing wine grape variety, and is currently the most popular white grape variety in the world. It is suitable for all types of climates, is cold-resistant, has high and stable yields, is easy to cultivate, and the plants sprout early, have strong growth potential, and have semi-erect branches, which are suitable for medium and long tip pruning; it has good adaptability to terroir and is more suitable for calcareous soils with medium fertility, and the best soil is calcareous soil with marl. Therefore, it is planted in major production areas around the world; in northern my country, Chardonnay is mainly distributed in Shandong, Hebei, Shanxi, Xinjiang and Inner Mongolia. With the continuous development of production, people have put forward higher requirements for grape varieties.

[0003] The cultivation methods of Chardonnay seeds are divided into soil cultivation and soilless cultivation. Soilless cultivation includes aseptic cultivation of plant tissue culture. Soil cultivation mainly depends on the type and ratio of the substrate, while soilless cultivation mainly depends on the type and ratio of the nutrient solution. When the related art uses a single soil cultivation or soilless cultivation method to cultivate Chardonnay seeds, the germination rate of Chardonnay seeds is relatively low, the seedlings are weak, and the fruiting cycle is long. In addition, when using soil cultivation to plant Chardonnay seeds, it is impossible to perform genetic transformation screening on Chardonnay seeds, which is not conducive to the development of industrialization. Summary of the invention

[0004] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a grape solid-liquid double-layer culture method, which adopts a solid-liquid co-culture method, which not only avoids the problems of separate hydroponics and soil culture, but also combines the advantages of the two, does not require watering and fertilization, significantly reduces the input of manpower and material resources, thereby reducing the overall cost, and can be used for genetic transformation screening.

[0005] The present application provides a grape solid-liquid double-layer culture method, comprising:

[0006] Get several full-grained Chardonnay seeds;

[0007] Placing a solid matrix in a seedling pot, and planting the Chardonnay seeds in the solid matrix of the seedling pot, wherein the solid matrix comprises fine soil, vermiculite and perlite, and the ratio of the fine soil, the vermiculite and the perlite is 6:1:1;

[0008] A culture solution is added to a liquid culture medium, and the seedling pot is placed in the liquid culture medium, wherein the liquid surface of the culture solution is in contact with the bottom of the seedling pot, and a plurality of seedling holes are provided at the bottom of the seedling pot. After germination and rooting, the rhizomes of the Chardonnay seeds planted in the seedling pot extend into the culture solution through the seedling holes to absorb nutrients and water from the culture solution.

[0009] According to some embodiments of the present application, the culture medium is 1 / 2MS culture medium.

[0010] According to some embodiments of the present application, when the culture solution is a screening solution, after obtaining a plurality of Chardonnay seeds with full grains, the method includes:

[0011] By using Agrobacterium transformation technology, a null vector carrying an auxiliary selection marker gene is transferred into each of the Chardonnay seeds to obtain a plurality of transgenic Chardonnay seeds;

[0012] Prepare 50 mg / ml kanamycin screening solution;

[0013] The 50 mg / ml kanamycin screening solution was diluted with distilled water to obtain 30 mg / L kanamycin screening solution, 50 mg / L kanamycin screening solution, and 70 mg / L kanamycin screening solution.

[0014] According to some embodiments of the present application, four seedling raising pots are provided, and the four seedling raising pots are respectively a first seedling raising pot, a second seedling raising pot, a third seedling raising pot and a fourth seedling raising pot, and the grape solid-liquid double-layer culture method further includes:

[0015] Solid matrix is ​​added to the first seedling raising pot, the second seedling raising pot, the third seedling raising pot and the fourth seedling raising pot respectively to obtain the first seedling raising pot, the second seedling raising pot, the third seedling raising pot and the fourth seedling raising pot containing solid matrix.

[0016] According to some embodiments of the present application, four liquid culture media are provided, and the four liquid culture media correspond to the first seedling raising pot, the second seedling raising pot, the third seedling raising pot, and the fourth seedling raising pot, respectively. The four liquid culture media are the first liquid culture medium, the second liquid culture medium, the third liquid culture medium, and the fourth liquid culture medium, respectively. The grape solid-liquid double-layer culture method further includes:

[0017] preparing 0 mg / L kanamycin screening solution and adding the 0 mg / L kanamycin screening solution to the first liquid culture medium;

[0018] The 30 mg / L kanamycin screening solution was added to the second liquid culture medium, the 50 mg / L kanamycin screening solution was added to the third liquid culture medium, and the 70 mg / L kanamycin screening solution was added to the fourth liquid culture medium.

[0019] According to some embodiments of the present application, the grape solid-liquid double-layer culture method further comprises:

[0020] Planting a plurality of the transgenic Chardonnay seeds in the first seedling raising pot, the second seedling raising pot, the third seedling raising pot and the fourth seedling raising pot containing the solid matrix respectively;

[0021] The first seedling raising pot is placed in the first liquid culture medium, the second seedling raising pot is placed in the second liquid culture medium, the third seedling raising pot is placed in the third liquid culture medium, and the fourth seedling raising pot is placed in the fourth liquid culture medium. The bottoms of the first seedling raising pot, the second seedling raising pot, the third seedling raising pot and the fourth seedling raising pot are all provided with a plurality of seedling raising holes.

[0022] According to some embodiments of the present application, the seedling raising pot is a six-hole seedling raising pot, and six seedling raising holes are opened at the bottom of the six-hole seedling raising pot.

[0023] The beneficial effects of the present invention are as follows: a seedling pot is placed in a liquid culture medium, and the liquid surface of the culture solution is flush with the bottom of the seedling pot; a plurality of seedling holes are provided at the bottom of the seedling pot; the rhizomes of the Chardonnay seeds planted in the seedling pot, after germination and rooting, extend into the culture solution through the seedling holes to absorb nutrients and water from the culture solution; the solid-liquid co-cultivation method combining soil cultivation with soilless cultivation is adopted, which not only avoids the problems of separate hydroponics and soil cultivation, but also combines the advantages of the two, does not require watering and fertilizing, significantly reduces the input of manpower and material resources, and thus reduces the overall cost; secondly, the cultivation method has high repeatability and universality, is simple to operate, is easy to repeat, and can be used for genetic transformation screening of soil-cultured transgenic plants.

[0024] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0026] Figure 1 A schematic diagram of the process of the solid-liquid double-layer culture method of grapes provided in the embodiments of the present application;

[0027] Figure 2 A schematic diagram of the cultivation of Chardonnay grape seeds provided in an embodiment of the present application;

[0028] Figure 3 A schematic diagram of the process of the solid-liquid double-layer culture method of grapes provided in the embodiments of the present application;

[0029] Figure 4 A schematic diagram of the screening of transgenic grape seedlings after planting using different concentrations of kan provided in the examples of the present application. DETAILED DESCRIPTION

[0030] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0031] In the description of the present application, it should be understood that descriptions involving orientation, such as up, down, front, back, left, right, etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0032] In the description of this application, if there is a description of first or second, it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0033] In the description of this application, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in this application based on the specific content of the technical solution.

[0034] Chardonnay, also known as Sherdonnay, Chardonnay, and Chardonnay, is a Eurasian variety, a medium-early maturing wine grape variety, and is currently the most popular white grape variety in the world. It is suitable for all types of climates, is cold-resistant, has high and stable yields, is easy to cultivate, and the plants sprout early, have strong growth potential, and have semi-erect branches, which are suitable for medium and long tip pruning; it has good adaptability to terroir and is more suitable for calcareous soils with medium fertility, and the best soil is calcareous soil with marl. Therefore, it is planted in major production areas around the world; in northern my country, Chardonnay is mainly distributed in Shandong, Hebei, Shanxi, Xinjiang and Inner Mongolia. With the continuous development of production, people have put forward higher requirements for grape varieties.

[0035] The cultivation methods of Chardonnay seeds are divided into soil cultivation and soilless cultivation. Soilless cultivation includes aseptic cultivation of plant tissue culture. Soil cultivation mainly depends on the type and ratio of the substrate, while soilless cultivation mainly depends on the type and ratio of the nutrient solution. When the related art uses a single soil cultivation or soilless cultivation method to cultivate Chardonnay seeds, the germination rate of Chardonnay seeds is relatively low, the seedlings are weak, and the fruiting cycle is long. In addition, when using soil cultivation to plant Chardonnay seeds, it is impossible to perform genetic transformation screening on Chardonnay seeds, which is not conducive to the development of industrialization.

[0036] In order to solve the above problems, the present application proposes a solid-liquid double-layer culture method for grapes. The embodiments of the present application are further described below in conjunction with the accompanying drawings.

[0037] Embodiment 1:

[0038] Reference Figure 1 to Figure 2 , Figure 1 A grape solid-liquid double-layer culture method provided by an embodiment of the first aspect of the present invention is shown, and the method comprises the following steps:

[0039] Step S110, obtaining a plurality of Chardonnay seeds with full grains.

[0040] Step S120, placing a solid matrix in a seedling raising pot, and planting Chardonnay seeds in the solid matrix of the seedling raising pot.

[0041] In this step, the solid matrix includes fine soil, vermiculite and perlite, and the ratio of the fine soil, vermiculite and perlite is 6:1:1.

[0042] It should be noted that peat in the solid matrix is ​​the main source of organic matter and can provide nutrients needed for plant growth; lightweight materials such as perlite and vermiculite can increase soil aeration and water permeability, which is beneficial to the growth of plant roots.

[0043] Step S130, adding culture solution into the liquid culture medium and placing the seedling pot into the liquid culture medium.

[0044] In this step, the liquid surface of the culture solution abuts against the bottom of the seedling pot, and a plurality of seedling holes are provided at the bottom of the seedling pot. After the Chardonnay seeds planted in the seedling pot germinate and take root, the rhizomes extend into the culture solution through the seedling holes to absorb nutrients and water from the culture solution.

[0045] In a possible implementation, the culture medium is 1 / 2MS culture medium.

[0046] It should be noted that MS medium was designed by Murashige and Skoog for tobacco cell culture in 1962. It is characterized by high concentrations of inorganic salts and ions, a relatively stable ion balance solution, high nitrate content, and appropriate quantity and proportion of nutrients to meet the nutritional and physiological needs of plant cells. Therefore, it has a wide range of applications and is used as the basic medium for most plant tissue culture rapid propagation. 1 / 2MS medium is a medium in which the trace elements are reduced to 1 / 2 of the original.

[0047] Specifically, refer to Figure 2 , select full-grained Chardonnay seeds and plant them in the solid matrix of the seedling pot. Put 1 / 2MS culture medium nutrient solution in the cup below the seedling pot. The surface of the nutrient solution should be level with the bottom of the seedling pot. When the Chardonnay seeds germinate and take root, there is insufficient water in the solid matrix, and the roots will extend into the nutrient solution below through the small holes at the bottom of the seedling pot, and then the plant will be supplied with nutrition and water through the nutrient solution.

[0048] In the present application, a seedling pot is placed in a liquid culture medium, and the liquid surface of the culture solution is level with the bottom of the seedling pot. A plurality of seedling holes are provided at the bottom of the seedling pot. The rhizomes of the Chardonnay seeds planted in the seedling pot, after germination and rooting, extend into the culture solution through the seedling holes to absorb nutrients and water from the culture solution. The solid-liquid co-cultivation method combining soil cultivation and soilless cultivation is adopted, which not only avoids the problems of separate hydroponics and soil cultivation, but also combines the advantages of the two. No watering or fertilizing is required, which significantly reduces the input of manpower and material resources, thereby reducing the overall cost.

[0049] Embodiment 2:

[0050] Reference Figure 3 to Figure 4 , Figure 3 The solid-liquid double-layer culture method for grapes provided by the second embodiment of the present invention is shown. When the culture liquid is a screening liquid, the method comprises the following steps:

[0051] Step S210, obtaining a plurality of Chardonnay seeds with full grains.

[0052] Step S211, using Agrobacterium transformation technology to transfer the empty vector carrying the auxiliary selection marker gene into each Chardonnay seed to obtain a plurality of transgenic Chardonnay seeds.

[0053] In this step, GFP (Green Fluorescence Protein) green fluorescent protein gene is used as an auxiliary screening marker gene. In some embodiments, the empty vector with the auxiliary screening marker gene is pCAMBIA2300 vector, referred to as PC2300 vector.

[0054] Step S212, preparing 50 mg / ml kanamycin screening solution.

[0055] Step S213, diluting the 50 mg / ml kanamycin screening solution with distilled water to obtain 30 mg / L kanamycin screening solution, 50 mg / L kanamycin screening solution, and 70 mg / L kanamycin screening solution.

[0056] It should be noted that kanamycin (Kanamycin, abbreviated as Kan) is used to screen the transformed plants.

[0057] Step S220, adding solid matrix into the first seedling raising pot, the second seedling raising pot, the third seedling raising pot and the fourth seedling raising pot respectively to obtain the first seedling raising pot, the second seedling raising pot, the third seedling raising pot and the fourth seedling raising pot containing solid matrix.

[0058] In this step, four seedling raising pots are provided, and the four seedling raising pots are respectively a first seedling raising pot, a second seedling raising pot, a third seedling raising pot and a fourth seedling raising pot.

[0059] Step S221 , planting a plurality of transgenic Chardonnay seeds in a first seedling raising pot, a second seedling raising pot, a third seedling raising pot and a fourth seedling raising pot containing a solid matrix respectively.

[0060] In this step, the solid matrix includes fine soil, vermiculite and perlite, and the ratio of the fine soil, vermiculite and perlite is 6:1:1.

[0061] Step S230, preparing 0 mg / L kanamycin screening solution and adding 0 mg / L kanamycin screening solution into the first liquid culture medium.

[0062] Step S240, adding 30 mg / L kanamycin screening solution to the second liquid culture medium, adding 50 mg / L kanamycin screening solution to the third liquid culture medium, and adding 70 mg / L kanamycin screening solution to the fourth liquid culture medium.

[0063] In this step, four liquid culture media are provided, and the four liquid culture media correspond to the first seedling raising pot, the second seedling raising pot, the third seedling raising pot and the fourth seedling raising pot respectively. The four liquid culture media are the first liquid culture medium, the second liquid culture medium, the third liquid culture medium and the fourth liquid culture medium respectively.

[0064] Step S250, placing the first seedling raising pot in the first liquid culture medium, placing the second seedling raising pot in the second liquid culture medium, placing the third seedling raising pot in the third liquid culture medium, and placing the fourth seedling raising pot in the fourth liquid culture medium.

[0065] In this step, a plurality of seedling raising holes are provided at the bottom of the first seedling raising pot, the second seedling raising pot, the third seedling raising pot and the fourth seedling raising pot.

[0066] It should be noted that the 0 mg / L kanamycin screening solution was distilled water. Using distilled water as the control group, transgenic Chardonnay seeds were planted in the matrix of a six-hole seedling pot. 0 mg / L, 30 mg / L, 50 mg / L, and 70 mg / L of kan screening solution were added to the bottom of the seedling pot, and the plant growth was observed after 35 days. Figure 4 As shown, the higher the kan concentration, the better the screening effect and high concentration of kan inhibits plant growth.

[0067] In a possible implementation, the seedling raising pot is a six-hole seedling raising pot, and six seedling raising holes are opened at the bottom of the six-hole seedling raising pot.

[0068] In the present application, the screening of transgenic plants can be achieved through steps S210 to S250; secondly, the cultivation method has high repeatability and universality, is simple to operate, and is easy to repeat, and can be used for genetic transformation screening of soil-cultured transgenic plants.

[0069] In other embodiments, the grape solid-liquid double-layer culture method can not only be used to screen transgenic plants, but also can be used to determine the gradient of some plant stresses such as salt stress, ABA, heavy metals and other stress substances that can be configured in liquids.

[0070] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0071] The above is a preferred embodiment of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications are also considered to be within the scope of protection of the present application.

[0072] Although the embodiments of the present application have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the claims and their equivalents.

Claims

1. A grape solid-liquid double-layer culture method, characterized in that: include: Get several full-grained Chardonnay seeds; Placing a solid matrix in a seedling pot, and planting the Chardonnay seeds in the solid matrix of the seedling pot, wherein the solid matrix comprises fine soil, vermiculite and perlite, and the ratio of the fine soil, the vermiculite and the perlite is 6:1:1; A culture solution is added to a liquid culture medium, and the seedling pot is placed in the liquid culture medium, wherein the liquid surface of the culture solution is in contact with the bottom of the seedling pot, and a plurality of seedling holes are provided at the bottom of the seedling pot. After germination and rooting, the rhizomes of the Chardonnay seeds planted in the seedling pot extend into the culture solution through the seedling holes to absorb nutrients and water from the culture solution.

2. The grape solid-liquid double-layer culture method according to claim 1, characterized in that: The culture medium is 1 / 2MS culture medium.

3. The grape solid-liquid double-layer culture method according to claim 1, characterized in that: In the case where the culture solution is a screening solution, after obtaining a plurality of Chardonnay seeds with full grains, the method comprises: By using Agrobacterium transformation technology, a null vector carrying an auxiliary selection marker gene is transferred into each of the Chardonnay seeds to obtain a plurality of transgenic Chardonnay seeds; Prepare 50 mg / ml kanamycin screening solution; The 50 mg / ml kanamycin screening solution was diluted with distilled water to obtain 30 mg / L kanamycin screening solution, 50 mg / L kanamycin screening solution, and 70 mg / L kanamycin screening solution.

4. The grape solid-liquid double-layer culture method according to claim 3, characterized in that: There are four seedling raising pots, which are respectively a first seedling raising pot, a second seedling raising pot, a third seedling raising pot and a fourth seedling raising pot. The grape solid-liquid double-layer culture method also includes: Solid matrix is ​​added to the first seedling raising pot, the second seedling raising pot, the third seedling raising pot and the fourth seedling raising pot respectively to obtain the first seedling raising pot, the second seedling raising pot, the third seedling raising pot and the fourth seedling raising pot containing solid matrix.

5. The grape solid-liquid double-layer culture method according to claim 4, characterized in that: The four liquid culture media are provided, and the four liquid culture media correspond to the first seedling raising pot, the second seedling raising pot, the third seedling raising pot, and the fourth seedling raising pot, respectively. The four liquid culture media are the first liquid culture medium, the second liquid culture medium, the third liquid culture medium, and the fourth liquid culture medium. The grape solid-liquid double-layer culture method also includes: preparing 0 mg / L kanamycin screening solution and adding the 0 mg / L kanamycin screening solution to the first liquid culture medium; The 30 mg / L kanamycin screening solution was added to the second liquid culture medium, the 50 mg / L kanamycin screening solution was added to the third liquid culture medium, and the 70 mg / L kanamycin screening solution was added to the fourth liquid culture medium.

6. The grape solid-liquid double-layer culture method according to claim 5, characterized in that: The grape solid-liquid double-layer culture method also includes: Planting a plurality of the transgenic Chardonnay seeds in the first seedling raising pot, the second seedling raising pot, the third seedling raising pot and the fourth seedling raising pot containing the solid matrix respectively; The first seedling raising pot is placed in the first liquid culture medium, the second seedling raising pot is placed in the second liquid culture medium, the third seedling raising pot is placed in the third liquid culture medium, and the fourth seedling raising pot is placed in the fourth liquid culture medium. The bottoms of the first seedling raising pot, the second seedling raising pot, the third seedling raising pot and the fourth seedling raising pot are all provided with a plurality of seedling raising holes.

7. The grape solid-liquid double-layer culture method according to claim 1, characterized in that: The seedling raising pot is a six-hole seedling raising pot, and six seedling raising holes are opened at the bottom of the six-hole seedling raising pot.

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