Culture medium optimization method for improving plant tissue culture regeneration efficiency

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a culture medium optimization method for improving plant tissue culture regeneration efficiency, which comprises the following steps: S1, culture medium optimization; s2, selecting and treating an explant; s3, domesticating, transplanting and optimizing; by comprehensively optimizing all stages of plant tissue culture, the defects of a traditional culture medium in the aspects of wide applicability, regeneration efficiency, rooting ability, survival rate and the like are overcome, a new solution is provided for large-scale commercial application of a plant tissue culture technology, and the method is suitable for laboratory research and also suitable for large-scale commercial application of plant tissue culture. And the method is also suitable for large-scale plant propagation, breeding and genetic improvement, and promotes a plant tissue culture technology to develop towards a more efficient and economic direction.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a culture medium optimization method for improving the regeneration efficiency of plant tissue culture. Background Art

[0002] Plant tissue culture is an important asexual reproduction technology that achieves rapid plant reproduction and genetic improvement through somatic cell regeneration. It is widely used in agricultural breeding, genetic engineering, germplasm resource protection and other fields. However, the successful establishment of a plant regeneration system depends on the optimization of the culture medium formula, especially the selection of explants, bud induction and rooting culture, which directly affects the regeneration efficiency and the final survival rate.

[0003] In the prior art, Murashige and Skoog (MS) medium is a basic medium widely used in plant tissue culture. By adding appropriate amounts of plant hormones (such as 6-BA, IAA, GA3, etc.), it can promote bud induction and elongation of plant explants. However, although MS medium has shown good effects in many plant species, its regeneration efficiency for different explants and plant varieties varies greatly, especially in some economic crops and genetically modified plants, the regeneration efficiency is unstable. In addition, the performance of traditional culture media is weak in the rooting stage, and the root system is not fully developed, resulting in a low survival rate of regenerated plants, limiting its potential in large-scale commercial applications.

[0004] For example, the study by Zhang et al. (2018) explored the effects of adding different hormone combinations to MS medium on bud induction of tomato explants. The results showed that different hormone ratios significantly affected the regeneration efficiency of explants, and different explants had different sensitivities to hormone concentrations. Similarly, the study by Liu et al. (2020) focused on optimizing the tissue culture system of grapes. The study found that adding appropriate amounts of hydrolyzed casein and glutamine can promote the induction and elongation of grape buds. However, the applicability of these formulas is usually limited to certain specific plants and cannot be widely used in other plant varieties. In addition, although these studies have made some progress in bud induction, there is still much room for improvement in rooting culture.

[0005] In terms of patent technology, patent CN102345678A describes an optimized MS culture medium, which improves the bud induction rate of certain plant explants by adding specific concentrations of hormones and organic nutrients. However, this patent solution still has problems with low rooting rate and low survival rate of regenerated plants, and cannot meet the needs of large-scale production.

[0006] The main problems of the existing technology include: (1) poor adaptability of the culture medium: the existing culture medium formula has a good effect on specific plant varieties, but generally lacks adaptability and versatility and cannot be widely used in the rapid regeneration of different plant varieties; (2) low regeneration efficiency: in the bud induction and rooting stages, the existing culture medium often shows low induction rate and rooting rate, especially in commercial applications, it is difficult to meet the efficiency requirements of large-scale production; (3) the survival rate of regenerated seedlings is not high: after acclimatization and transplanting, the survival rate of regenerated seedlings is unstable, which affects the promotion and application of regenerated plants.

[0007] Therefore, in order to address the problems of poor adaptability of the above-mentioned culture medium, low regeneration efficiency, unstable rooting rate and low survival rate of regenerated seedlings, which limit the promotion of plant tissue culture technology in large-scale commercial applications, a culture medium optimization method for improving the regeneration efficiency of plant tissue culture can be designed. Summary of the invention

[0008] In order to overcome the problems of poor culture medium adaptability, low regeneration efficiency, unstable rooting rate and low survival rate of regenerated seedlings, the promotion of plant tissue culture technology in large-scale commercial applications is limited.

[0009] The technical solution of the present invention is: a culture medium optimization method for improving the regeneration efficiency of plant tissue culture, comprising the following steps:

[0010] S1: Culture medium optimization

[0011] Adjust the nutrients, hormone combinations and organic additives for efficient bud induction and rooting culture of different plant explants, and regulate the concentration of growth hormones at the critical stage of plant somatic cell culture;

[0012] S2: Explant selection and processing

[0013] Select suitable explants. According to the characteristics of different plant varieties, choose the most suitable explant type, and perform disinfection and cutting pretreatment to ensure efficient induction during the cultivation process;

[0014] S3: Acclimation and Transplanting Optimization

[0015] During the rooting stage of the regenerated seedlings, use optimized rooting medium to enhance the development ability of the root system, and adopt reasonable acclimatization and transplanting methods to ensure that the regenerated seedlings can survive smoothly when transplanted to the greenhouse or soil.

[0016] Preferably, the organic additives include but are not limited to hydrolyzed casein, proline, and glutamine.

[0017] Preferably, the growth hormone includes but is not limited to cytokinins such as 6-BA and IBA.

[0018] Preferably, the explant is an epicotyl, a hypocotyl or a cotyledon node.

[0019] Beneficial effects of the present invention:

[0020] 1. By comprehensively optimizing each stage of plant tissue culture, the shortcomings of traditional culture media in terms of wide applicability, regeneration efficiency, rooting ability and survival rate have been overcome, providing a new solution for the large-scale commercial application of plant tissue culture technology. It is not only suitable for laboratory research, but also for large-scale plant reproduction, breeding and genetic improvement, promoting the development of plant tissue culture technology in a more efficient and economical direction;

[0021] 2. By optimizing the nutrient components and hormone ratios in the culture medium, the success rate of bud induction of different plant explants is effectively improved. Experiments have shown that the efficiency of inducing buds in the bud induction culture medium is significantly higher than that of the prior art, especially the induction rate of cotyledon nodes reaches 86.67%, which greatly improves the success rate of plant regeneration. This efficient bud induction ability enables the present invention to meet the tissue culture needs of various plant varieties;

[0022] 3. By optimizing the hormones and nutrients in the rooting medium, the rooting efficiency and root development quality of the regenerated seedlings are effectively improved. The rooting rate is between 13.15% and 29.21%, which is significantly improved compared with the existing technology. The root system is more robust, which enhances the adaptability of the regenerated seedlings after transplanting, reduces the problems of poor root development and rooting difficulties, and ensures the stability of the plant regeneration process;

[0023] 4. By improving the acclimatization and transplanting technology, the regenerated seedlings have a high survival rate after being transplanted into the greenhouse or soil. The root system of the regenerated seedlings maintains good water absorption capacity during the transplanting process, and the roots are healthy and developed. The survival rate reaches 71.67% to 88.14%. This high survival rate greatly reduces the loss rate of regenerated seedlings and reduces waste in plant reproduction and production;

[0024] 5. The optimized culture medium is not only suitable for specific plant varieties, but also has a wide adaptability and can meet the regeneration requirements of a variety of plant explants. Whether it is explants of different varieties or explants of different parts of the same plant, the culture medium of the present invention can ensure a high regeneration efficiency. This wide adaptability effectively solves the problem of the limitations of the culture medium in the prior art, so that the present invention can be extended to more plant species;

[0025] 6. By optimizing the composition of the culture medium, the growth rate and regeneration efficiency of the explants are improved, thereby shortening the regeneration cycle. The shortening of the regeneration cycle not only improves the production efficiency, but also reduces the resource consumption and time cost required in the breeding process. In addition, the realization of high rooting rate and survival rate enables the present invention to show significant cost advantages in large-scale commercial production, and is suitable for industrial applications in plant breeding, seedling propagation and genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 What is shown are the bud induction rate, rooting rate and survival rate of different explants of different species in the present invention;

[0027] Figure 2 Shown is a flow chart of plant tissue culture regeneration of the present invention. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0029] See also Figure 1-Figure 2 The present invention provides an embodiment: a culture medium optimization method for improving the regeneration efficiency of plant tissue culture, comprising the following steps:

[0030] S1: Culture medium optimization

[0031] Adjust the nutrients, hormone combinations and organic additives for efficient bud induction and rooting culture of different plant explants, and regulate the concentration of growth hormones at the critical stage of plant somatic cell culture;

[0032] S2: Explant selection and processing

[0033] Select suitable explants. According to the characteristics of different plant varieties, choose the most suitable explant type, and perform disinfection and cutting pretreatment to ensure efficient induction during the cultivation process;

[0034] S3: Acclimation and Transplanting Optimization

[0035] During the rooting stage of the regenerated seedlings, use optimized rooting medium to enhance the development ability of the root system, and adopt reasonable acclimatization and transplanting methods to ensure that the regenerated seedlings can survive smoothly when transplanted to the greenhouse or soil.

[0036] Preferably, the organic additives include but are not limited to hydrolyzed casein, proline, and glutamine.

[0037] Preferably, the growth hormone includes but is not limited to cytokinins such as 6-BA and IBA.

[0038] Preferably, the explant is an epicotyl, a hypocotyl or a cotyledon node.

[0039] Example

[0040] (1) Preparation of sterile seedlings, the steps are as follows:

[0041] Step 1: Seed disinfection

[0042] Select suitable plant seeds, first sterilize them, place the seeds in a sealed dryer, and use NaClO solution for vapor phase sterilization for 10 to 12 hours to ensure that the microorganisms on the seed surface are completely removed;

[0043] Step 2: Germination and cultivation

[0044] The sterilized seeds are inoculated into the germination medium and cultured for 5 to 7 days until the seeds germinate into sterile seedlings. The specific formula of the germination medium is:

[0045]

[0046] Culture conditions: 16 h light / 8 h dark, light intensity of 3000 Lux, temperature of 25 °C.

[0047] (2) Explant preparation and bud induction, the steps are as follows:

[0048] Step 1: Explant selection and processing

[0049] Epicotyls, hypocotyls and cotyledon nodes were cut from sterile seedlings as explants. Each explant was repeated in 3 groups, with 30 explants in each epicotyl and hypocotyl group and 60 explants in each cotyledon node group. The explants were properly cut to ensure that each explant had a uniform length, so as to facilitate subsequent induction and growth.

[0050] Step 2: Bud induction

[0051] The explants were inoculated into bud induction medium and after 2 weeks of culture, they were transferred into fresh bud induction medium for a second round of screening. The optimized formula of the bud induction medium was:

[0052]

[0053] The culture medium formula of the present invention significantly improves the bud induction rate of explants, with the highest induction rate of the cotyledon node reaching 86.67%. Meanwhile, the bud induction rates of the epicotyl and hypocotyl are also significantly improved compared with the prior art.

[0054] (3) Bud elongation and rooting culture, the steps are as follows:

[0055] Step 1: Bud elongation culture

[0056] After the induction of clustered buds, the explants were transferred to the bud elongation medium for culture. After 2 weeks, the medium was replaced with fresh medium. The optimized formula of the bud elongation medium was:

[0057]

[0058] Through the regulation of this formula, the growth of clustered buds is accelerated, and the plants grow well, providing an ideal foundation for subsequent rooting culture;

[0059] Step 2: Rooting Culture

[0060] When the buds grow to 2-3 cm, dip the bud base in 2 mg / mL IBA solution for 30 minutes and then transfer to the optimized rooting medium for rooting culture. The specific formula of the rooting medium is:

[0061]

[0062]

[0063] The culture medium can significantly improve the development efficiency of the root system, with the rooting rate ranging from 13.15% to 29.21%.

[0064] (4) Domestication and transplanting, the steps are as follows:

[0065] Step 1: Acclimation process

[0066] When the roots of the regenerated seedlings grow to 10-20 cm, transfer the regenerated seedlings to sterile water for acclimatization. The acclimatization process lasts for 5-days, and the caps of the tissue culture bottles are gradually opened to allow the plants to adapt to the external environment.

[0067] Step 2: Transplanting

[0068] After acclimation, the regenerated seedlings were taken out of the sterile water, the roots were washed and transplanted into a matrix containing vermiculite and nutrient soil in the greenhouse. Statistical results showed that the survival rate of the regenerated seedlings was between 71.67% and 88.14%, which was significantly higher than the traditional culture system.

[0069] Table 1 is an analysis table of the bud induction rate, rooting rate and survival rate of different explants of different plants.

[0070]

[0071] Table 1

[0072] Table 1 and Appendix Figure 1The epicotyls, hypocotyls and cotyledon nodes of the two species in the present invention are used as explants. After culturing on a bud induction medium for 4 weeks, the results show that the bud induction efficiency of the cotyledon node is the highest (86.67% and 82.78%, respectively), followed by the epicotyl and the lowest is the hypocotyl. There are significant differences in the bud induction rates of different explants, but there are no significant differences between different varieties of the same explants. In addition, the rooting culture results show that the rooting rate is between 13.15% and 29.21%, and there is no significant difference in the rooting rate under each treatment. The survival rate of the regenerated seedlings after acclimatization and transplantation is between 71.67% and 88.14%, and there is no significant difference in the survival rate between different species and explants. These results show that the present invention improves the bud induction rate and the survival rate of the regenerated seedlings by optimizing the culture medium formula and explant selection, but there is still room for further optimization of the rooting conditions.

[0073] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of those skilled in the art without departing from the spirit of the present invention.

Claims

1. A method for optimizing a culture medium for improving the regeneration efficiency of plant tissue culture, characterized in that: The steps include: S1: Culture medium optimization Adjust the nutrients, hormone combinations and organic additives for efficient bud induction and rooting culture of different plant explants, and regulate the concentration of growth hormones at the critical stage of plant somatic cell culture; S2: Explant selection and processing Select suitable explants. According to the characteristics of different plant varieties, choose the most suitable explant type, and perform disinfection and cutting pretreatment to ensure efficient induction during the cultivation process; S3: Acclimation and Transplanting Optimization During the rooting stage of the regenerated seedlings, use optimized rooting medium to enhance the development ability of the root system, and adopt reasonable acclimatization and transplanting methods to ensure that the regenerated seedlings can survive smoothly when transplanted to the greenhouse or soil.

2. The method for optimizing the culture medium for improving the regeneration efficiency of plant tissue culture according to claim 1, characterized in that: Organic additives include but are not limited to hydrolyzed casein, proline, and glutamine.

3. The method for optimizing the culture medium for improving the regeneration efficiency of plant tissue culture according to claim 1, characterized in that: Growth hormones include, but are not limited to, cytokinins such as 6-BA and IBA.

4. The method for optimizing the culture medium for improving the regeneration efficiency of plant tissue culture according to claim 1, characterized in that: The explant is an epicotyl, hypocotyl or cotyledon node.

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