Plant protoplast semi-fusion breeding method

Abstract

The invention relates to the technical field of plant cell engineering, and particularly discloses a plant protoplast semi-fusion breeding method which comprises the following three stages of seedling material selection, preparation, fusion, induction and cultivation. A large number of fused plant seedlings are obtained through a series of operations and used for large-scale planting. The method comprises the following steps: selecting excellent varieties of a plant A and a plant B, respectively preparing the plant A and the plant B into protoplasts in a preparation process, culturing the protoplasts into corresponding calluses, and performing half-cell fusion on a fusion part to obtain mosaic calluses. Then, the callus of the plant A to be used as the root is induced to locally induce adventitious roots, the callus of the plant B to locally induce adventitious buds, and finally, breeding is carried out to obtain the super seedlings. According to the method, the grafting principle is combined, the degree and conditions of cell fusion are optimized, the breeding cost is reduced, meanwhile, it is ensured that the fusion body can stably express the excellent characters of parents at different parts, and agricultural large-scale production is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant cell engineering, and particularly relates to a method for semi-fusion breeding of plant protoplasts. Background Art

[0002] In the field of modern agricultural biotechnology, breeding new plant varieties through cell fusion technology is a potential method. This method can create plants with new genetic characteristics by merging cells of different plants, but in practical applications, especially when breeding specific plant varieties, many challenges still exist.

[0003] For example, the cell fusion of tomatoes and potatoes, although achievable under laboratory conditions, the resulting seedlings cannot exhibit the expected parental traits, that is, growing both potatoes and tomatoes. This may be due to physiological and genetic barriers during the cell fusion process, resulting in the inability of the fused plants to normally express the genetic characteristics of both parents. Currently, plant grafting technology is a traditional means of plant propagation and genetic improvement in the fields of agriculture and horticulture. Through grafting, the genetic characteristics of two different plants can be combined to create new varieties with specific traits. For example, grafting tomatoes and potatoes, two plants from different families, can combine the fruit production ability of tomatoes and the root characteristics of potatoes to achieve a crop with dual economic value. Although grafting technology has been quite mature in some plant species, the low grafting survival rate is a common problem.

[0004] Under laboratory conditions, the grafting survival rate of tomatoes and potatoes is only 16.7%, which far from meets the requirements of commercial production. Secondly, existing grafting technologies often require a large amount of manpower and material resources, resulting in high costs and making large-scale production difficult to achieve.

[0005] Due to the differences in fields, no method has been found to reduce the breeding cost while ensuring that the fusants can stably express the excellent traits of both parents in different parts, opening up a new and efficient way for genetic improvement in agricultural production. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for semi-fusion breeding of plant protoplasts to solve the problems raised in the above background art.

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] A method for semi-fusion breeding of plant protoplasts, comprising the following steps:

[0009] S1. Selection and preparation:

[0010] Select plants A and B with excellent genetic characteristics as parental materials, plant A with excellent genetic characteristics as the root source and plant B as the bud source, and prepare a protoplast medium, which includes but is not limited to MS medium, B5 medium, KM8P medium, NT medium, and Km8T medium;

[0011] S2. High-efficiency protoplast preparation:

[0012] Prepare the tissues of plants A and B into corresponding protoplasts respectively; plants A and B include but are not limited to tomatoes, peppers, potatoes, cucumbers, cauliflower, and eggplants;

[0013] S3. Cultivation of mosaic callus:

[0014] Cultivate the protoplasts in layers to form corresponding mosaic callus; adopt the semi-cell fusion technology, fuse some of the protoplasts from plants A and B, and introduce electric stimulation as an auxiliary means to promote the formation of mosaic callus;

[0015] S4. Inductive differentiation:

[0016] Perform local treatment on the mosaic callus, and use hormones to respectively induce adventitious roots with the characteristics of plant A and adventitious buds with the characteristics of plant B to achieve specific differentiation of roots and stems; the hormones include but are not limited to 6-benzyladenine, kinetin, and zeatin;

[0017] S5. Seedling cultivation:

[0018] Cultivate the mosaic callus to obtain artificial seeds.

[0019] Preferably, the protoplast preparation in S2 adopts the enzymatic hydrolysis method, which includes the following steps:

[0020] (a) Use a mixed enzyme solution of cellulase and pectinase for enzymatic hydrolysis of the cell wall, where the final concentration of cellulase is 1.5%, the final concentration of pectinase is 0.1%, or use Driselase at a final concentration of 2%;

[0021] (b) The formula of the enzymatic hydrolysis buffer is: 100 mM KCl, 20 mM MgCl 2 , 20 mM CaCl 2 , 0.1% (w / v) bovine serum albumin (BSA), 80 mM 2-(N-morpholino)ethanesulfonic acid (MES), 0.6 M mannitol, and adjust the pH to 5.5 with 0.1 M Tris HCl;

[0022] (c) The enzymatic hydrolysis process is carried out at 28 °C, and the time is adjusted according to the type and tenderness of the plant tissue, usually 10 - 300 minutes;

[0023] (d) The tissue fluid after enzymatic hydrolysis is filtered through a nylon mesh, and the protoplasts are washed twice with an isotonic washing buffer / protoplast buffer. The buffer formulation is: 100 mM KCl, 20 mM MgCl2, 0.1% (w / v) BSA, 0.4 M mannitol;

[0024] (e) The debris of the washed protoplasts is removed by centrifugation. Centrifugation is carried out at a centrifugal force of 50 - 100 g for 1 - 2 minutes. Finally, the protoplasts are resuspended in mannitol at an appropriate concentration to complete the purification process.

[0025] Preferably, the cultivation of the S3 mosaic callus specifically includes the following steps:

[0026] The protoplasts A and B in S2 are filtered respectively. First, the protoplast A is spread flat in a culture dish, and then the protoplast B is slowly and evenly spread flat on the plane of the protoplast A. The mixed protoplasts in this culture dish are subjected to electric stimulation, and then placed in the corresponding MC medium for cultivation to obtain A - B mosaic callus.

[0027] Preferably, the S3 semi - cell fusion technology is electrofusion technology. For an electroporation cuvette with a size of 0.2 cm, the voltage range is 80 - 160 V, the capacitance is also 800 - 1000 μF, and the pulse length is 10 - 20 milliseconds.

[0028] Preferably, step S5 includes using specific plant growth regulators. The plant growth regulators include but are not limited to indoleacetic acid, indolebutyric acid, and naphthaleneacetic acid; the plant growth regulators are used for local treatment of the mosaic callus to obtain adventitious roots and adventitious buds.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] The present invention proposes an innovative genetic improvement method applicable to a variety of plant species. This method ingeniously combines the grafting principle with modern cell engineering technology. It not only optimizes the process and conditions of cell fusion, designs the subsequent culture environment, and ensures that some heterologous plant cells and tissues can be retained during the cultivation process, realizes the precise integration of the genetic materials of both parents, and then cultivates chimeric plants. During the growth process of these plants, the excellent traits of both parents can be stably expressed, and the expected genetic characteristics can be presented in the roots, stems, and leaves.

[0031] Compared with traditional breeding methods, the present invention not only significantly reduces the breeding cost, but also greatly improves the efficiency and accuracy of genetic improvement, providing high-quality germplasm resources for subsequent agricultural production. It has opened up a new and efficient way of genetic improvement with low cost. By cultivating chimeric plants (including potential super crops), it is conducive to promoting the sustainable development of agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the basic process of the fusion breeding method of the present invention;

[0033] Figure 2 It is a schematic diagram of a finished product case of the fusion breeding method of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] Example 1:

[0036] Please refer to Figures 1 to 2 as shown in

[0037] A method for semi-fusion breeding of plant protoplasts includes the following steps:

[0038] S1. Selection and preparation:

[0039] Select plants A and B with excellent genetic characteristics as parental materials, and prepare the culture medium;

[0040] S2. Protoplast preparation: Prepare the corresponding protoplasts from the tissues of plants A and B respectively;

[0041] S3. Cultivation of mosaic callus: Stack-culture the protoplasts into the corresponding mosaic callus; adopt the semi-cell fusion technology to fuse some of the protoplasts from plants A and B, and give a stimulus of simulated injury to promote the formation of mosaic callus;

[0042] S4. Inductive differentiation: Locally induce adventitious roots in the callus of plant A as the root; locally induce adventitious buds in the callus of plant B as the bud;

[0043] S5. Seedling cultivation: Cultivate the mosaic callus to obtain artificial seeds.

[0044] Among them, in the above-mentioned semi-fusion breeding method of plant protoplasts, the above-mentioned processes are all operated in a sterile laminar flow hood.

[0045] Specifically, the materials in this example are tomato plant cell A and potato plant cell B.

[0046] First, the preparation of the culture medium: According to the different plant species, explant types and culture stages, select an appropriate culture medium formula.

[0047] Preferably, MS medium is used and adjusted if necessary. The preparation process includes steps such as melting agar, adding sucrose and mother liquor, adjusting the pH, making up the volume, and aliquoting. The aliquoted culture medium is autoclaved and the sterilized culture medium is placed in the inoculation room.

[0048] Establishment and expansion of sterile cultures: Explants (involving plants A and B) such as seeds, shoot tips, leaves, inflorescences, etc. are disinfected to ensure aseptic operation and prevent cross-contamination. Then these tissues are enzymatically digested to obtain a protoplast suspension for subculture. A method for enzymatically digesting the cell wall of a plant is involved, which is used to prepare a single-cell suspension or protoplasts. After enzymatic digestion and washing, subculture is carried out in the corresponding nutrient solution to obtain a large number of protoplasts.

[0049] Specifically, tomato plant cell A and potato plant cell B are treated with cellulase and pectinase to produce protoplasts:

[0050] A mixed enzyme solution of cellulase and pectinase is used for enzymatic digestion of the cell wall, where the final concentration of cellulase is 1.5% and the final concentration of pectinase is 0.1%, or Driselase is used at a final concentration of 2%.

[0051] The formula of the enzymatic digestion buffer is: 100 mM KCl, 20 mM MgCl 2 , 20 mM CaCl 2 , 0.1% (w / v) bovine serum albumin (BSA), 80 mM 2-(N-morpholino)ethanesulfonic acid (MES), 0.6 M mannitol, and the pH is adjusted to 5.5 with 0.1 M Tris HCl.

[0052] The enzymatic digestion process is carried out at 28°C, and the time is adjusted according to the type and tenderness of the plant tissue, usually ranging from dozens of minutes to several hours. The enzymatically digested tissue fluid is filtered through a nylon mesh, and the protoplasts are washed twice with an isotonic washing buffer / protoplast buffer, and the buffer formula is: 100 mM KCl, 20 mM MgCl2, 0.1% (w / v) BSA, 0.4 M mannitol.

[0053] The washed protoplasts are centrifuged to remove debris at a centrifugal force of 100 g for 2 minutes, and finally the protoplasts are resuspended in mannitol at an appropriate concentration to complete the purification process.

[0054] Protoplasts A of tomato plant cells and protoplasts B of potato plant cells are obtained, and the protoplasts A and B are filtered separately.

[0055] First, spread the protoplasts A evenly in a culture dish to form a uniform bottom layer. Then, slowly spread the protoplasts B evenly on top of the plane of the protoplasts A, thus forming a mixed layer of A and B in the same culture dish. The culture dish containing the mixed protoplasts is subjected to electrical stimulation, which can promote the fusion of protoplasts or activate cell processes. Then, the culture dish is placed in a specific MC medium for culture. After a period of culture, A-B mosaic callus can be obtained. This kind of callus is formed after the fusion or interaction of the two types of protoplasts A and B, and has a unique cell structure and genetic characteristics.

[0056] Example 2:

[0057] Material selection: Select healthy, disease-free, and genetically excellent pepper and potato plants as parents. Ensure that the pepper variety has excellent fruit quality or stress resistance, and the potato variety is selected for high yield, strong disease resistance, or specific underground characteristics (such as tuber shape and size).

[0058] Culture medium preparation: According to the plant species and experimental requirements, prepare MS medium as the basic medium and adjust the components as needed to adapt to the preparation, fusion, and subsequent culture of protoplasts. Consider adding appropriate plant hormones, growth regulators, and antibiotics to inhibit bacterial contamination and promote cell division.

[0059] High-efficiency protoplast preparation

[0060] Tissue selection: Select young leaves or shoot tips from pepper plants and young tuber cortex or apical buds from potato plants as materials for protoplast preparation.

[0061] Enzymatic digestion treatment: Use a mixture of cellulase and pectinase to enzymatically digest the selected tissues under appropriate temperature and pH conditions to remove the cell walls and obtain free protoplasts.

[0062] Purification and counting: Purify the protoplasts by centrifugation, filtration, etc., and use a hemocytometer for counting to ensure sufficient cell quantity and quality for subsequent experiments.

[0063] Cultivation of mosaic callus

[0064] Protoplast fusion: Using the semi - cell fusion technique, mix the protoplasts of peppers and potatoes in proportion. Use polyethylene glycol (PEG) as the fusogen and supplement with appropriate electro - stimulation parameters to promote cell fusion.

[0065] Cultivation conditions: Evenly spread the fused protoplasts on the MS medium, place them in the dark, and control the appropriate temperature and humidity to promote the formation of callus.

[0066] Induction of differentiation

[0067] Hormone treatment: After the formation of the mosaic callus, transfer it to a medium containing 6 - benzyladenine (BA), etc., and perform local treatment to induce the differentiation of adventitious buds with pepper characteristics and adventitious roots with potato characteristics.

[0068] Observation of differentiation: Regularly observe and record the differentiation of the callus, and adjust the hormone ratio to optimize the differentiation efficiency.

[0069] Seedling cultivation and transplantation

[0070] Artificial seed production: Carefully take out the mosaic plants with pepper buds and potato roots differentiated from the medium, wash them clean, and wrap them in artificial seed shells containing nutrient soil and moisture - retaining materials to form artificial seeds.

[0071] Germination and transplantation: Place the artificial seeds under suitable germination conditions. After the seedlings grow a certain number of leaves and roots, transplant them to the greenhouse or field for further growth observation and trait evaluation.

[0072] After obtaining the A - B mosaic callus, induce the formation of intermediate propagules such as buds and embryoids on the medium. By adjusting the composition of the medium and cultivation conditions, it is possible to stimulate the cells in the callus to differentiate and proliferate, thus forming the required intermediate propagules.

[0073] Example 3:

[0074] Material selection and preparation: Eggplant: Select eggplant varieties with excellent fruit quality, strong disease resistance, and vigorous growth potential. Potato: Select potato varieties with high yield, good tuber shape, and strong disease resistance.

[0075] Medium preparation:

[0076] Prepare the MS medium as the basic medium for the preparation, fusion, and subsequent cultivation of protoplasts.

[0077] Ensure the sterility of the medium and add appropriate antibiotics to inhibit bacterial contamination.

[0078] High - efficiency protoplast preparation

[0079] Tissue selection and treatment: Select young leaves or shoot tips from eggplant plants and young tuber cortex or apical buds from potato plants. Wash the selected tissues thoroughly with sterile water to remove surface attachments.

[0080] Enzymolysis and purification: Use a mixture of cellulase and pectinase to enzymatically treat the tissues at an appropriate temperature (such as 25 - 28°C) and pH value (such as 5.5 - 6.0) to remove the cell walls and obtain free protoplasts. Purify the protoplasts by methods such as centrifugation and filtration to remove undissociated cell debris and enzyme solution. Count the protoplasts using a hemocytometer to ensure a sufficient number of cells for subsequent experiments.

[0081] Cultivation of mosaic callus

[0082] Protoplast fusion: Mix the protoplasts of eggplant and potato in a certain proportion and use polyethylene glycol (PEG) as a fusogen to promote cell fusion. Introduce electric stimulation as an auxiliary means and set appropriate electric stimulation parameters to improve the fusion efficiency.

[0083] Evenly coat the fused protoplasts on MS medium and culture them in the dark or under weak light. Regularly observe the formation of callus, and record the fusion efficiency and the growth status of callus.

[0084] Hormone treatment: After the formation of mosaic callus, transfer it to a medium containing kinetin (KT). According to the characteristics of eggplant buds and potato roots, adjust the hormone ratio to induce adventitious buds with eggplant characteristics and adventitious roots with potato characteristics respectively.

[0085] Regularly observe and record the differentiation of callus, and screen out the plants with obvious characteristics of eggplant buds and potato roots. Further culture the screened plants to promote their growth and development.

[0086] Seedling cultivation and transplantation: Transfer the screened mosaic plants to a medium containing appropriate auxin and cytokinin to promote the further growth of their roots and stems and leaves. When the seedlings grow a certain number of leaves and roots, gradually reduce the hormone concentration in the medium to promote their independent growth.

[0087] Among them, the formation of intermediate propagules can be achieved through cutting and subculture. Cutting means dividing the intermediate propagules into multiple small parts, each part containing active cells and tissues. Subculture is to transfer these cut parts to a new medium for continuous culture so that they can continue to grow and proliferate. Through multiple subcultures, a large number of intermediate propagules can be obtained, providing sufficient materials for subsequent plant propagation and cultivation.

[0088] In summary, through somatic hybridization and tissue culture techniques, the genetic materials of plants A and B with excellent genetic characteristics are fused together, and super crop seedlings or artificial seeds with the excellent characteristics of both sides are cultivated to create chimeric plants that can stably express the excellent traits of both parents. The cultivation method is particularly applicable to plants that are difficult to improve by traditional breeding, opening up a new way for agricultural production and having broad application prospects and important agricultural value.

[0089] It should be understood that in the development of any actual implementation, such as in any engineering or design project, a large number of specific implementation decisions can be made. Such development efforts may be complex and time-consuming, but for those of ordinary skill in the art who benefit from this disclosure, without excessive experimentation, the development efforts will be a routine task of design, manufacture, and production.

[0090] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A plant protoplast semi-fusion breeding method, characterized in that: The following steps are involved: S1. Material selection and preparation: Selecting plant A and plant B with excellent genetic characteristics as parent materials, plant A with excellent genetic characteristics as a root source and plant B as a shoot source, preparing a protoplast culture medium, the culture medium includes but is not limited to MS culture medium, B5 culture medium, KM8P culture medium, NT culture medium and Km8T culture medium; S2. Efficient protoplast preparation: The tissues of plant A and plant B are respectively prepared into corresponding protoplasts; the plant A and plant B include but are not limited to tomato, pepper, potato, cucumber, cauliflower and eggplant; S3. Mosaic callus cultivation: The protoplasts are stacked and cultivated into corresponding mosaic callus tissues; a semi-cell fusion technique is used to fuse part of the protoplasts from plant A and plant B, and electric stimulation is introduced as an auxiliary means to promote the formation of mosaic callus tissues; S4. Inducing differentiation: The mosaic callus is locally treated, and adventitious roots with characteristics of plant A and adventitious buds with characteristics of plant B are induced using hormones to achieve specific differentiation of roots and stems; the hormones include but are not limited to 6-benzyladenine, kinetin and zeatin; S5. Seedling cultivation: The mosaic callus is cultivated to obtain artificial seeds.

2. A plant protoplast semi-fusion breeding method according to claim 1, characterized in that: The protoplast preparation in S2 adopts an enzymatic hydrolysis method, comprising the following steps: (a) using a mixed enzyme solution of cellulase and pectinase to enzymatically hydrolyze the cell wall, wherein the final concentration of cellulase is 1.5%, and the final concentration of pectinase is 0.1%, or using Driselase at a final concentration of 2%; (b) The enzymatic buffer was formulated as follows: 100 mM KCl, 20 mM MgCl2, 20 mM CaCl2, 0.1% (w / v) bovine serum albumin (BSA), 80 mM 2-(N-morpholino)ethanesulfonic acid (MES), 0.6 M mannitol, and the pH was adjusted to 5.5 using 0.1 M Tris HCl; (c) The enzymatic hydrolysis process is carried out at 28° C., and the time is adjusted according to the type and tenderness of the plant tissue, usually 10-300 minutes; (d) The tissue fluid after enzymatic hydrolysis was filtered through a nylon mesh, and the protoplasts were washed twice with an isotonic washing buffer / protoplast buffer, wherein the buffer formula is: 100 mM KCl, 20 mM MgCl2, 0.1% (w / v) BSA, 0.4 M mannitol; (e) The washed protoplasts are centrifuged to remove debris at 50-100 g for 1-2 minutes, and the protoplasts are resuspended in an appropriate concentration of mannitol to complete the purification process.

3. A plant protoplast semi-fusion breeding method according to claim 1, characterized in that: The S3 mosaic callus cultivation specifically comprises the following steps: The protoplasts A and B described in S2 are filtered separately, and protoplast A is first spread flat on a culture dish, and then protoplast B is slowly and evenly spread on the plane of protoplast A. The mixed protoplasts in the culture dish are electrically stimulated, and then placed in a corresponding MC culture medium to obtain AB mosaic callus.

4. A plant protoplast semi-fusion breeding method according to claim 1, characterized in that: The semi-cell fusion technology described in S3 is an electric fusion technology, wherein for a 0.2 cm electric rotating cup, the voltage range is 80-160 V, the capacitance is also 800-1000 μF, and the pulse length is 10-20 milliseconds.

5. A plant protoplast semi-fusion breeding method according to claim 1, characterized in that: Step S5 includes using a specific plant growth regulator, which includes but is not limited to indoleacetic acid, indolebutyric acid and naphthylacetic acid; the plant growth regulator is used to locally treat the mosaic callus to obtain adventitious roots and adventitious buds.

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