A method for preserving germplasm resources of in vitro roots of herbaceous plants

Abstract

A method for preserving germplasm resources of in vitro roots of herbaceous plants, comprising the following steps: Selecting well-growing root materials to cut and obtain explants, placing the explants in subculture medium I for the first subculture. The subculture medium I is based on the conventional B5 medium, adding indolebutyric acid (IBA), sucrose and agar thereto, and adjusting the pH of the medium to 5.8 - 6.0; placing the cultured roots in subculture medium II for the second subculture. It is applicable to the preservation of germplasm resources of in vitro roots of herbaceous plants. It effectively extends the subculture cycle and reduces the number of subcultures. In one preservation cycle, the in vitro root materials can be stably preserved for more than 1 year, effectively and stably preserving the excellent traits of the germplasm resources of herbaceous plant roots, and the recovery rate of the root germplasm resources reaches over 95%.

Description

Technical Field

[0001] The present invention relates to the technical field of germplasm resource preservation, and particularly to a method for preserving herbaceous plant in vitro root germplasm resources. Background Art

[0002] Plant germplasm resources are an important basis for crop improvement, new variety breeding, and biodiversity conservation. The protection of plant germplasm resources is an important task for protecting biodiversity and supporting sustainable agricultural development. Currently, the main protection strategies include in-situ conservation and ex-situ conservation methods. Among them, conventional ex-situ conservation such as seed conservation has problems such as greater difficulty in long-term preservation, more space requirements, and easy occurrence of genetic drift. To solve the above problems, in vitro conservation technology in ex-situ conservation has emerged. In vitro conservation is to use plant biotechnology means to aseptically culture plant materials and add growth inhibitory conditions during the culture process to delay or stop their growth, thereby reducing the time of subculture to achieve long-term preservation of plant germplasm resources. This method can overcome the limitations of in-situ conservation and provide a more flexible and controllable preservation plan.

[0003] Among the materials for in vitro conservation, it is necessary to subculture frequently to maintain activity. For example, in Patent CN 104145818 A, after the second subculture, multiple steps of induced differentiation subculture and other operations are required to complete the preservation of germplasm resources, and the preservation time is extended by repeatedly subculturing frequently to maintain the activity of Gerbera jamesonii. Compared with materials of other plant parts, root materials can still maintain a relatively high survival rate and physiological functions under in vitro conditions. However, its strong regeneration ability also brings corresponding problems. The higher polyphenol oxidase content in root materials leads to a faster browning rate than materials of other parts. During this frequent subculture process, root materials are easily affected by factors such as environmental changes, physiological imbalance, and microbial infection, which limits their growth and recovery ability. This is an unavoidable problem in conventional preservation techniques. Therefore, cryopreservation technology has become a relatively ideal means for preserving root materials at present, but this method has high requirements for equipment, is expensive, and occupies a large space; some root materials with higher water content are not suitable for cryopreservation, and the cryoprotectants used during the preservation process may be toxic to cells and cannot be completely avoided. Therefore, a new method for preserving plant in vitro roots is needed, which can effectively preserve root materials at room temperature, avoid the limitations of cryopreservation, and improve the preservation success rate and recovery activity. This method will bring a breakthrough to the technology for preserving plant root materials and has important application value. Summary of the Invention

[0004] Aiming at the problems existing in the preservation of root germplasm resources at present, the purpose of the present invention is to provide a method for preserving in vitro root germplasm resources of herbaceous plants. This method is applicable to the in vitro roots of herbaceous plants, extends the subculture cycle, reduces the number of subcultures, and improves the preservation success rate and recovery activity.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A method for preserving in vitro root germplasm resources of herbaceous plants, characterized by comprising the following steps:

[0007] (1) Selection and treatment of explants

[0008] Select root materials with good growth, many branches and no contamination, select different cutting lengths according to the different materials, and select root materials of 1 - 2.5 cm in size to obtain the explants required for subsequent culture;

[0009] (2) First subculture

[0010] Place the explants in subculture medium I for the first subculture. The subculture medium I is based on the conventional B5 medium, and indolebutyric acid (IBA), sucrose and agar are added thereto, and the pH of the medium is adjusted to 5.8 - 6.0;

[0011] (3) Second subculture

[0012] Place the roots obtained by culturing in step (2) in subculture medium II for the second subculture. The subculture medium II is based on the B5 modified medium, sucrose and agar are added thereto, and the pH of the medium is adjusted to 5.8 - 6.0.

[0013] Further, in the subculture medium I, the concentration of IBA is 3.80 - 4.10 mg / L, the sucrose concentration is 30000 mg / L, and the agar concentration is 6500 mg / L.

[0014] Further, the first subculture is dark culture, the culture temperature is 22 - 25 °C, and the culture time is 21 - 24 days until the root tissue grows strong and branches appear.

[0015] Further, in the subculture medium II, the B5 modified medium is a culture solution in which the concentration of macronutrients in the conventional B5 medium is reduced to 1 / 2 of the original, the concentration of micronutrients is reduced to 3 / 4 of the original, the inositol concentration is 80 mg / L, the concentration of thiamine hydrochloride is 8 mg / L, and the proportions of the remaining components remain unchanged. The sucrose concentration is 20000 mg / L, and the agar concentration is 6500 mg / L.

[0016] Further, the second subculture is a dark culture, the culture temperature is 22-25 °C, and the culture is carried out for more than 1 year.

[0017] Most specifically, a method for preserving germplasm resources of in vitro roots of herbaceous plants is characterized by comprising the following steps:

[0018] (1) Selection and treatment of explants

[0019] Select root materials with good growth, a large number of branches and no contamination. According to the different materials, select different cutting lengths, and select root materials of 1-2.5 cm in size to obtain the explants required for subsequent culture;

[0020] (2) First subculture

[0021] Place the explants in subculture medium I for the first subculture. The culture temperature is 22-25 °C, and the culture time is 21-24 days. The subculture medium I is based on the conventional B5 medium, and indolebutyric acid (IBA), sucrose and agar are added thereto. The concentration of IBA is 3.80-4.10 mg / L, the sucrose concentration is 30000 mg / L, and the agar concentration is 6500 mg / L. Adjust the pH of the medium to 5.8-6.0;

[0022] (3) Second subculture

[0023] Place the roots obtained from the culture in step (2) in subculture medium II for the second subculture. The culture is a dark culture, the culture temperature is 22-25 °C, and the culture is carried out for more than 1 year; the subculture medium II is based on the B5 modified medium, and sucrose and agar are added thereto. The B5 modified medium is a culture solution in which the concentration of macronutrients in the conventional B5 medium is reduced to 1 / 2 of the original, the concentration of micronutrients is reduced to 3 / 4 of the original, the concentration of inositol is 80 mg / L, and the concentration of thiamine hydrochloride is 8 mg / L, and the proportions of the remaining components remain unchanged. The sucrose concentration is 20000 mg / L, the agar concentration is 6500 mg / L, and the pH of the medium is adjusted to 5.8-6.0.

[0024] Further, when the root materials preserved by the above method are resuscitated, the resuscitation medium used is based on the conventional B5 medium, and IBA, sucrose and agar are added thereto. The concentration of IBA is 3.80-4.10 mg / L, the sucrose concentration is 30000 mg / L, and the agar concentration is 6500 mg / L. Adjust the pH of the resuscitation medium to 5.8-6.0.

[0025] Further, the culture temperature for resuscitation culture is 22-25 °C, and the culture time is 21-24 days.

[0026] The present invention has the following technical effects:

[0027] In the present invention, by using B5 medium and formulating medium I with IBA at a specific concentration as an additive, and using B5 modified medium as subculture medium II, two different subculture media are used for two-step subculture, which is suitable for the preservation of in vitro root germplasm resources of herbaceous plants. It effectively prolongs the subculture cycle and reduces the number of subcultures. During a preservation cycle, the in vitro root materials can be stably preserved for more than 1 year, effectively and stably preserving the excellent traits of the root germplasm resources of herbaceous plants, and the recovery rate of the root germplasm resources reaches more than 95%.

[0028] Since there is no need for frequent subculture operations and no steps such as induction culture, this method greatly reduces the maintenance cost and labor input for root germplasm resources and improves the preservation efficiency. The method of the present invention is a new, effective and cost-saving method for preserving in vitro root germplasm resources of plants. Specific Embodiments

[0029] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and should not be construed as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above content of the present invention.

[0030] The composition of the conventional B5 medium used in the present invention is as follows:

[0031]

[0032] Example 1

[0033] A method for preserving in vitro root germplasm resources of herbaceous plants, comprising the following steps:

[0034] (1) Selection and treatment of explants

[0035] Select ginseng root materials with good growth, many branches and no contamination, cut 1.5 cm-sized root tissues therefrom to obtain the explants required for subsequent culture;

[0036] (2) First subculture

[0037] Place the explants in subculture medium I for the first subculture. The culture temperature is 25 °C and the culture time is 24 days until the root materials grow strong and produce branches. The subculture medium I is based on the conventional B5 medium, and indolebutyric acid (IBA), sucrose and agar are added thereto. The concentration of IBA is 4.0 mg / L, the sucrose concentration is 30000 mg / L, and the agar concentration is 6500 mg / L. Adjust the pH of the medium to 5.8;

[0038] (3) Second subculture

[0039] Place the roots obtained from the culture in step (2) into the subculture medium II for the second subculture. The culture is carried out in the dark at a temperature of 25 °C for 1 year and 6 months. The subculture medium II is based on the B5 modified medium, and sucrose and agar are added thereto. The B5 modified medium is a culture solution in which the concentration of macronutrients in the conventional B5 medium is reduced to 1 / 2 of the original, the concentration of micronutrients is reduced to 3 / 4 of the original, the concentration of inositol is 80 mg / L, the concentration of thiamine hydrochloride is 8 mg / L, and the proportions of the remaining components remain unchanged. The sucrose concentration is 20000 mg / L, the agar concentration is 6500 mg / L, and the pH of the medium is adjusted to 5.8.

[0040] When the root material preserved by the above method is resuscitated, the resuscitation medium used is the subculture medium I, that is, based on the conventional B5 medium, IBA, sucrose and agar are added thereto. The IBA concentration is 4.0 mg / L, the sucrose concentration is 30000 mg / L, the agar concentration is 6500 mg / L, and the pH of the resuscitation medium is adjusted to 5.8. The culture temperature for resuscitation culture is 25 °C, and the culture time is 24 days.

[0041] Comparative Example 1

[0042] Compared with Example 1, the difference is that the conventional MS medium is used as the basal medium instead of the B5 medium during the formulation process, and the remaining formulations and steps are the same as those in Example 1.

[0043] Comparative Example 2

[0044] Compared with Example 1, during the second subculture process, the B5 modified medium in the medium II has the total element concentration reduced to 1 / 2 and 1 / 4 of the original conventional B5 medium.

[0045] Comparative Example 3

[0046] Compared with Example 1, the difference is that when resuscitating the material, IBA is not added to the resuscitation medium used and the IBA concentration is reduced to 1 / 2 of that in Example 1, and the remaining formulations and steps are the same as those in Example 1.

[0047] Comparative Example 4

[0048] Compared with Example 1, NAA and IAA, which also have the effect of promoting root growth, are used to replace IBA in Example 1 and adjusted to the optimal concentration, and the remaining steps remain unchanged as those in Example 1.

[0049] Comparative test:

[0050] Observe the preservation time of the root germplasm resources obtained in Example 1, Comparative Example 1 - Comparative Example 4 within a complete subculture cycle, and observe the growth rate, survival rate, and recovery rate of the root planting resources preserved and recovered in each scheme within the same preservation time. Among them, the best preservation cycle is during the second subculture process. The survival rate and recovery rate are tested once every month, and the parameter changes are observed. When a significant change in the survival rate or recovery rate leads to a large-scale death of the root material (specifically, the recovery rate data drops below 50%), it is no longer suitable to continue extending the preservation time at this time. Then the previous test time is the best preservation cycle of the root material. The specific results are shown in Table 1.

[0051] Among them, the growth rate refers to the growth state of the roots during the first-stage culture in the subculture process; the survival rate refers to the proportion of root materials that do not turn brown or die old during the long-term preservation of the inoculated roots in the second subculture; the recovery rate refers to the proportion of root materials that will continue to grow and maintain a similar activity to that before preservation after the root materials obtained after preservation are inoculated into a new recovery medium for a period of time.

[0052] Table 1:

[0053]

[0054] Due to the change of the culture medium, as can be seen from the above table, for the root materials subcultured and recovered in Comparative Example 1, Comparative Example 2, and Comparative Example 4, within a complete subculture cycle, the best preservation time of the root materials is relatively short. Under the best preservation cycle, the growth rate, survival rate, and recovery rate of the root materials all decrease to varying degrees. In Comparative Example 1 and Comparative Example 2, when the second subculture is preserved to the best preservation cycle, the survival rate and recovery rate of the root materials have slowly dropped to a relatively low level. At this time, if the preservation time of the second subculture is continued to be extended and the survival rate and recovery rate are tested again after 1 month, the recovery rate has dropped below 50%. If repeated subculture is used to extend the preservation cycle, due to various problems faced during the repeated subculture process, the growth, survival, and recovery of the root materials all show varying degrees of decline. Eventually, the survival rate and recovery rate of the obtained root materials will also be lower than the data at this time, which has no meaning for improving the preservation effect of the root materials. Therefore, we did not conduct subsequent tests. In Comparative Example 3, due to the change in the addition amount of IBA, it can be seen that the recovery rate of the root materials has decreased significantly. In Comparative Example 4, due to the different selection of hormones, the growth rate, survival rate, and recovery rate all show significant decreases during the subculture process.

[0055] In this example, the growth rate of subculture is 99.8%. After 1 complete subculture (the storage time is 18 months), the survival rate and resuscitation rate of the obtained roots can reach 98.1% and 95.7% respectively. On this basis, after repeating 1 complete subculture (at this time, the total storage period is extended to 36 months), the survival rate and resuscitation rate of the obtained roots can reach 94.9% and 94.0% respectively.

[0056] Example 2

[0057] A method for preserving germplasm resources of in vitro roots of herbaceous plants, comprising the following steps:

[0058] (1) Selection and treatment of explants

[0059] Select the roots of Astragalus membranaceus with good growth, many branches and no contamination, cut 1 cm-sized root tissues from them to obtain the explants required for subsequent culture;

[0060] (2) First subculture

[0061] Place the explants in Subculture Medium I for the first subculture. The culture temperature is 22 °C and the culture time is 21 days. The Subculture Medium I is based on the conventional B5 medium, and indolebutyric acid (IBA), sucrose and agar are added thereto. The concentration of IBA is 4.00 mg / L, the sucrose concentration is 30000 mg / L, the agar concentration is 6500 mg / L, and the pH of the medium is adjusted to 6.0;

[0062] (3) Second subculture

[0063] Place the root tissues obtained from the culture in step (2) in Subculture Medium II for the second subculture. The culture is in the dark, the culture temperature is 22 °C, and the culture is carried out for 1 year; the Subculture Medium II is based on the B5 modified medium, and sucrose and agar are added thereto. The B5 modified medium is a culture solution in which the concentration of macronutrients in the conventional B5 medium is reduced to 1 / 2 of the original, the concentration of trace elements is reduced to 3 / 4 of the original, the inositol concentration is 80 mg / L, the concentration of thiamine hydrochloride is 8 mg / L, and the proportions of the remaining components remain unchanged. The sucrose concentration is 20000 mg / L, the agar concentration is 6500 mg / L, and the pH of the medium is adjusted to 6.0.

[0064] When resuscitating the root materials preserved by the above method, the resuscitation medium used is based on the conventional B5 medium, and IBA, sucrose and agar are added thereto. The concentration of IBA is 4.0 mg / L, the sucrose concentration is 30000 mg / L, the agar concentration is 6500 mg / L, and the pH of the resuscitation medium is adjusted to 6.0. The resuscitation culture temperature is 22 °C and the culture time is 21 days.

[0065] On the basis of Example 2, according to the scheme changes in Comparative Example 1, Comparative Example 2, and Comparative Example 3 above, the adventitious root system materials of Astragalus membranaceus were subcultured and preserved and then revived respectively. The results are shown in Table 2.

[0066] Table 2:

[0067]

[0068] In the above control tests, the growth rate, survival rate, and revival rate were all the data measured when the optimal preservation period was used as the subculture period. The subculture period of the adventitious root system of Astragalus membranaceus is extremely short. During the in vitro preservation process, frequent subculturing to extend the preservation time of germplasm resources will lead to problems such as root system degradation or contamination. However, the preservation method of this Example 2 only integrates the first subculture + the second subculture into a complete subculture period through two subcultures, extending the subculture period to more than 1 year, effectively and stably preserving the excellent traits of the root system germplasm resources of Astragalus membranaceus. The survival rate and revival rate of the root system of Astragalus membranaceus reached 97.5% and 96.8%. After 1 subsequent subculture, the root system survival rate and revival rate could still reach 96.2% and 95.5%. The adventitious root system materials of Astragalus membranaceus after revival were vigorous and grew well, and could be quickly used for subsequent research needs.

[0069] Example 3

[0070] A method for preserving in vitro root system germplasm resources of herbaceous plants, comprising the following steps:

[0071] (1) Selection and treatment of explants

[0072] Select Gerbera jamesonii root system materials with good growth, many branches and no contamination, cut 2.5 cm-sized root system tissues from them to obtain the explants required for subsequent culture;

[0073] (2) First subculture

[0074] Place the explants in Subculture Medium I for the first subculture. The culture temperature is 24 °C, and the culture time is 22 days. The Subculture Medium I is based on the conventional B5 medium, and indolebutyric acid (IBA), sucrose, and agar are added thereto. The concentration of IBA is 3.80 mg / L, the sucrose concentration is 30000 mg / L, and the agar concentration is 6500 mg / L. Adjust the pH of the medium to 5.9;

[0075] (3) Second subculture

[0076] The roots obtained from the culture in step (2) were placed in the subculture medium II for the second subculture. The culture was carried out in the dark at a temperature of 24 °C for 1 year and 2 months. The subculture medium II was based on the B5 modified medium, to which sucrose and agar were added. The B5 modified medium was a culture solution in which the concentrations of macronutrients in the conventional B5 medium were reduced to 1 / 2 of the original, the concentrations of micronutrients were reduced to 3 / 4 of the original, the inositol concentration was 80 mg / L, the thiamine hydrochloride concentration was 8 mg / L, and the proportions of the remaining components remained unchanged. The sucrose concentration was 20000 mg / L, the agar concentration was 6500 mg / L, and the pH of the medium was adjusted to 5.9.

[0077] When the root system materials preserved by the above method were resuscitated, the resuscitation medium used was based on the conventional B5 medium, to which IBA, sucrose and agar were added. The IBA concentration was 3.80 mg / L, the sucrose concentration was 30000 mg / L, the agar concentration was 6500 mg / L, and the pH of the resuscitation medium was adjusted to 5.9. The resuscitation culture temperature was 24 °C and the culture time was 22 days.

[0078] On the basis of Example 2, according to the scheme changes in the above Comparative Example 1, Comparative Example 2 and Comparative Example 3, the in vitro root systems of Gerbera jamesonii were subcultured and preserved and resuscitated respectively. The results are shown in Table 3.

[0079] Table 3:

[0080]

[0081] In the above table, the growth rate, survival rate and resuscitation rate of each control test were measured when the best preservation period was used as the subculture period. In this example, through subculture with two different subculture media in sequence, an integrated preservation period of complete subculture was formed. The root system of Gerbera jamesonii was stably preserved for 1 year and 2 months. The survival rate after root resuscitation reached 97.2%, and the resuscitation rate reached 96.8%. After repeating the complete subculture once, the survival rate of the root system could still be maintained at 96.1%, and the resuscitation rate reached 95.3%.

[0082] The preservation method of the in vitro root planting resources of the present invention is applicable to the in vitro roots of herbaceous plants. We adopted the scheme of the present invention and conducted a large number of preservation and resuscitation tests on the in vitro roots of herbaceous plants, such as the root materials of herbaceous plants like ginseng, Astragalus membranaceus, Glycyrrhiza uralensis, Adonis amurensis, Atractylodes japonica, Bupleurum chinense, lavender, dahlia, honeysuckle, plantain, dandelion, etc. The preservation effect on the in vitro roots of each herbaceous plant is excellent, and both the root survival rate and the resuscitation rate are maintained above 95%. After 1 complete subculture cycle, the stable preservation time of the roots is more than 1 year. The method of the present invention is applicable to in vitro materials with preservation problems due to various reasons, such as poor preservation effect of the above-ground part materials, short preservation period, low resuscitation rate, high human and material resources required for preservation, etc. The root materials preserved by the method of the present invention do not need to be subcultured frequently, have a long preservation time, a high survival rate, and a simple resuscitation step, and can be quickly applied to various needs such as scientific research and commercial applications after resuscitation.

[0083] In the present invention, each ratio is calculated as the ratio of the number of effective root materials at each stage to the number of the initial total inoculated root materials.

Claims

1. A method for preserving in vitro root germplasm resources of herbaceous plants, characterized in that: The steps include: (1) Selection and processing of explants Select ginseng, Astragalus membranaceus or Gerbera jamesonii root materials with good growth, large number of branches and no bacteria, select different cutting lengths according to different materials, and select root materials with a size of 1 to 2.5 cm to obtain explants required for subsequent culture; (2) First subculture The explants are placed in a subculture medium I for the first subculture, wherein the subculture medium I is based on a conventional B5 medium, to which only indolebutyric acid (IBA), sucrose and agar are added, the pH of the medium is adjusted to 5.8-6.0, the concentration of IBA is 3.80-4.10 mg / L, the concentration of sucrose is 30000 mg / L, and the concentration of agar is 6500 mg / L, the first subculture is dark culture, the culture temperature is 22-25°C, and the culture time is 21-24 days until the root tissue grows strong and branches grow; (3) Second subculture Placing the root system obtained by culture in step (2) in subculture medium II for a second subculture, wherein the subculture medium II uses B5 modified medium as the basic medium, to which only sucrose and agar are added, and the pH of the medium is adjusted to 5.8-6.

0. In the subculture medium II, the B5 modified medium is a medium in which the concentration of macroelements in the conventional B5 medium is reduced to 1 / 2 of the original, the concentration of trace elements is reduced to 3 / 4 of the original, the concentration of inositol is 80 mg / L, the concentration of ammonium sulfate hydrochloride is 8 mg / L, and the proportion of other components remains unchanged, the concentration of sucrose is 20,000 mg / L, and the concentration of agar is 6,500 mg / L. The second subculture is dark culture at a culture temperature of 22-25°C, and the culture is carried out for more than 1 year; When the preserved root material is revived, the resuscitation medium used is a conventional B5 medium as the base medium, to which only IBA, sucrose and agar are added, the IBA concentration is 3.80-4.10 mg / L, the sucrose concentration is 30000 mg / L, the agar concentration is 6500 mg / L, and the pH of the resuscitation medium is adjusted to 5.8-6.0.

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