A method for regenerating hosta plant plants using leaves as explants
By using leaves as explants and inducing and proliferating hydrangea plants with specific culture media, the problem of propagation being limited by season and materials has been solved, and year-round propagation and genetic transformation have been supported.
Patent Information
- Application Number
- CN202410503266.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-25
- Publication Date
- 2026-02-27
- Estimated Expiration
- 2044-04-25
AI Technical Summary
Existing technologies make it difficult to establish a universal regeneration system for hydrangea paniculata, resulting in limited propagation due to the constraints of growing season and materials, a long propagation cycle, and difficulty in meeting large-scale market demands.
Leaves were used as explants. Callus was induced using an induction medium, then inoculated into a somatic embryo proliferation medium for proliferation, and finally formed into plants in a somatic embryo germination medium. A specific culture medium was used to promote the occurrence and proliferation of somatic embryos.
It enables year-round propagation of panicle hydrangeas, provides a good recipient for genetic transformation, simplifies the propagation process, and improves propagation efficiency and flexibility.
Smart Images
Figure CN118216428B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of plant tissue culture technology, and more particularly, to a method for regenerating Hydrangea paniculata plants using leaves as explants. BACKGROUND
[0002] Hydrangea paniculata Sieb. is a deciduous shrub or small tree of the genus Hydrangea in the family Hydrangeaceae, which is native to China. It has a large plant size, high flower yield, long flowering period, strong adaptability, and high ornamental value. It has been widely used in gardens and landscaping in Europe, the United States, and Japan. In addition, the whole plant of Hydrangea paniculata contains mucilage, which can be used as a paste, and has the effects of clearing heat, resisting malaria, and preventing diabetic nephropathy. After the peak flowering period in autumn, the color of Hydrangea paniculata flowers gradually changes from green to bronze, and the spent branches remain on the tree for a long time. It can be used for pot culture and planted in gardens, parks, and green spaces. It is an important ornamental plant in gardens, and the demand for seedlings is large. However, the propagation of Hydrangea paniculata mainly relies on cutting, layering, and division, which is limited by the growing season and propagation material, and is also time-consuming, labor-intensive, and low in propagation efficiency. Therefore, it is necessary to develop a method for propagating Hydrangea paniculata that is not limited by environmental conditions and seasons.
[0003] Plant somatic embryogenesis is a process in which somatic cells of a plant develop into new individuals under in vitro conditions through a similar developmental pathway to zygotic embryos. The establishment of a somatic embryogenesis pathway regeneration system can help to accelerate the propagation of Hydrangea paniculata test-tube seedlings, produce and store artificial seeds, and use embryonic cells as the best recipients for genetic transformation of Hydrangea paniculata.
[0004] According to the applicant's search, different Hydrangea paniculata varieties, explants, and experimental methods have been applied to the study of Hydrangea paniculata regeneration systems. The results show that it is difficult to find a universal method for establishing a Hydrangea paniculata regeneration system due to genotype limitations. Currently, there is no report on the somatic embryogenesis of Hydrangea paniculata 'Shihui Deng'.
[0005] To solve the above problems, a technical solution is provided. SUMMARY
[0006] To overcome the above-mentioned defects of the prior art, embodiments of the present application provide a method for regenerating Hydrangea paniculata plants using leaves as explants to solve the problems raised in the background art.
[0007] To achieve the above-mentioned objects, the present application provides the following technical solutions:
[0008] A method for regenerating plants of Hydrangea paniculata by using leaves as explants, comprising the following steps:
[0009] Step S1: using leaves of Hydrangea paniculata 'Limelight' tissue culture seedlings as explants, culturing the explants in an induction medium to induce callus, and inducing somatic embryos from the callus;
[0010] It should be noted that the source of the explants is not limited by season, and the tissue culture of Hydrangea paniculata 'Limelight' can be carried out throughout the year;
[0011] Step S2: inoculating the somatic embryos in step S1 into a somatic embryo proliferation medium for proliferation culture to obtain secondary somatic embryos;
[0012] Step S3: inoculating the secondary somatic embryos obtained in step S2 into a somatic embryo germination medium to form plants.
[0013] In a preferred embodiment, in step S1, the induction medium is composed of: basic medium 1 / 2MS or MS, 2,4-dichlorophenoxyacetic acid 4.0-5.0 mg / L, kinetin 0.5 mg / L, acid hydrolysis casein 500 mg / L, glucose 45 g / L, and plant gel 3.0 g / L.
[0014] In a preferred embodiment, in step S2, the somatic embryo proliferation medium is composed of: basic medium MS, 2,4-dichlorophenoxyacetic acid 0-2.0 mg / L, kinetin 0.5 mg / L, acid hydrolysis casein 0-500 mg / L, glucose 45 g / L, and agar powder 7.5 g / L.
[0015] In a preferred embodiment, in step S3, the somatic embryo germination medium is composed of: basic medium 1 / 2MS, 6-benzyladenine 0-1.5 mg / L, naphthalene acetic acid 0.2 mg / L, sucrose 30 g / L, and agar powder 7.5 g / L.
[0016] In a preferred embodiment, in step S2, the somatic embryos are inoculated into a somatic embryo subculture medium for long-term subculture and maintaining the differentiation ability.
[0017] The method for regenerating plants of Hydrangea paniculata by using leaves as explants has the following technical effects and advantages:
[0018] 1. The method uses leaves of Hydrangea paniculata 'Limelight' tissue culture seedlings as explants, induces somatic embryo occurrence, and maintains the characteristics of continuous proliferation and seedling formation of the somatic embryos, which provides good recipients for Agrobacterium-mediated genetic transformation of Hydrangea paniculata and lays a certain technical foundation for preparing new transgenic Hydrangea paniculata varieties.
[0019] 2、The application has the advantages of wide source of explants and convenient material taking, and the obtained somatic embryo is a good receptor of Agrobacterium-mediated genetic transformation of Spirea triloba, and can be used for genetic transformation research of Spirea triloba. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 The photo is for callus induction of Spirea triloba 'Limestone Lamp'.
[0021] Figure 2 The photo is for somatic embryo of Spirea triloba 'Limestone Lamp' induced by callus induction.
[0022] Figure 3 The photo is for proliferation of somatic embryo of Spirea triloba 'Limestone Lamp'.
[0023] Figure 4 The photo is for germination embryo of somatic embryo of Spirea triloba 'Limestone Lamp'.
[0024] Figure 5 The photo is for plant formed after germination of somatic embryo of Spirea triloba 'Limestone Lamp'. DETAILED DESCRIPTION
[0025] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.
[0026] Embodiment 1
[0027] The following operations are sequentially performed:
[0028] 1, Source of test materials and treatment:
[0029] The test materials in the application are selected from laboratory-preserved healthy and uniform Spirea triloba 'Limestone Lamp' sterile seedlings, the middle and upper leaves are cut off, the main leaf veins are scratched, and the leaf back is inoculated on the induction medium with the leaf back facing down.
[0030] 2, Design of culture medium:
[0031] Table 1 Composition and amount of various culture media
[0032]
[0033] Note: The MS basic medium is prepared according to Murashige T. and F. Skoog, 1962. The 1 / 2MS basic medium is the MS basic medium with half of the macroelements, half of the microelements and half of the Fe salts.
[0034] The somatic embryo subculture medium in Table 1 is used as a general subculture medium and also as a long-term subculture medium for somatic embryos.
[0035] The codes of the components in the medium are as follows: 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (6-BA), naphthalene acetic acid (NAA), kinetin (KT), plant gel (GEL), and agar powder (Agar) can be purchased commercially.
[0036] 3. Culture conditions:
[0037] The culture temperature in the culture room is 24±2°C, the light intensity is 1000-1500 lx, and the light cycle is 14 h. The dark culture temperature is 24±2°C.
[0038] 4. Inoculation and culture:
[0039] The upper leaves of the aseptic seedlings of Hydrangea paniculata 'Limelight' were inoculated on the induction medium (see Table 1) on a clean bench, 9 explants were inoculated in each culture dish, and the culture was performed in the dark. After 8 weeks, callus was produced at the edges of the leaves (see Fig. 2), and then somatic embryos were produced (see Fig. 3). Some explants turned brown and died, and somatic embryos were not induced. Figure 1 Figure 2 The somatic embryos obtained by induction were inoculated on the somatic embryo proliferation medium (see Table 1), and the culture was performed under light. These somatic embryos could proliferate rapidly on the somatic embryo proliferation medium (see Fig. 4). The somatic embryos obtained by induction were inoculated on the somatic embryo subculture medium (see Table 1), and the somatic embryos could be stored for a long time. The secondary somatic embryos were inoculated on the somatic embryo germination medium (see Table 1), and the somatic embryos could germinate into seedlings (see Fig. 5).
[0040] The somatic embryos obtained by induction were inoculated on the somatic embryo proliferation medium (see Table 1), and the culture was performed under light. These somatic embryos could proliferate rapidly on the somatic embryo proliferation medium (see Fig. 4). The somatic embryos obtained by induction were inoculated on the somatic embryo subculture medium (see Table 1), and the somatic embryos could be stored for a long time. The secondary somatic embryos were inoculated on the somatic embryo germination medium (see Table 1), and the somatic embryos could germinate into seedlings (see Fig. 5). Figure 3 Figure 5
[0041] Example 2
[0042] The following operations were performed in sequence:
[0043] The aseptic seedling leaves of Hydrangea paniculata 'Limelight' were used as explants for callus induction. When the 2,4-D concentration was 4.0 mg / L on the 1 / 2MS basic medium, the somatic embryo induction rate was the highest, which was 87.21%. The experimental results are shown in Table 2.
[0044] Table 2 Effects of different basic medium and 2,4-D concentration on callus induction of S. involucrata
[0045] Basal medium 2,4-D (mg / L) Callus induction rate (%) MS 1.0 54.81±1.47c MS 2.0 68.75±3.84b 1 / 2MS 1.0 72.58±1.58b 1 / 2MS 2.0 87.21±0.47a
[0046] Data are shown as mean ± standard error, and different letters indicate significant difference at P < 0.05 level.
[0047] This example is used to verify the effects of different concentrations of 2,4-D on callus induction of S. involucrata.
[0048] Example 3
[0049] The following operations were performed in sequence:
[0050] The sterile seedling leaves of S. involucrata 'Lime Light' were used as explants to induce somatic embryogenesis. The highest somatic embryogenesis induction rate was 16.87% when the 2,4-D concentration was 4.0 mg / L on 1 / 2MS basic medium. The experimental results are shown in Table 3:
[0051] Table 3 Effects of different basic medium and 2,4-D concentration on somatic embryogenesis of S. involucrata
[0052] Basal medium 2,4-D (mg / L) Somatic embryo induction rate (%) 1 / 2MS 4.0 16.87±0.72a 1 / 2MS 5.0 8.79±1.15b MS 4.0 10.57±0.82b MS 5.0 5.31±0.67c
[0053] Data are shown as mean ± standard error, and different letters indicate significant difference at P < 0.05 level.
[0054] This example is used to verify the effects of different concentrations of 2,4-D on somatic embryogenesis of S. involucrata.
[0055] Example 4
[0056] The following operations were performed in sequence:
[0057] In the somatic embryogenesis proliferation test, there were significant differences in the effects of different media on the proliferation of S. involucrata somatic embryos. The MS medium containing 2,4-D 2.0 mg / L, KT 0.5 mg / L, acid hydrolysis casein 500 mg / L, sucrose 45 g / L, and Agar 7.5 g / L was more conducive to the occurrence and growth and development of secondary somatic embryos of S. involucrata 'Lime Light'.
[0058] Table 4 Effects of different media on the growth and development state of S. involucrata somatic embryos
[0059]
[0060] Note: waterlogging 1, 2, 3 represent no waterlogging, light waterlogging, and serious waterlogging, respectively, and granularity 1, 2 represent obvious granularity and non-obvious granularity, respectively. The data represent mean ± standard error, and different letters represent significant differences at the P < 0.05 level.
[0061] This example is used to verify the effects of different media on the proliferation of cell embryos of S. paniculatum.
[0062] Example 5
[0063] The following operations are performed in sequence:
[0064] In the somatic embryo germination test, there were significant differences in the effects of different media on the germination of somatic embryos of S. paniculatum. The somatic embryo germination rate of S. paniculatum ‘Shihui Deng’ was the highest, reaching 26.38%, on the 1 / 2MS medium containing 6-BA 1.5 mg / L, NAA 0.2 mg / L, sucrose 30 g / L, and agar 7.5 g / L.
[0065] Table 5 Effects of different germination media on the germination of somatic embryos of S. paniculatum
[0066]
[0067]
[0068] Note: The data represent mean ± standard error, and different letters represent significant differences at the P < 0.05 level. Somatic embryo germination rate (%) = number of germinated somatic embryos / total number of inoculated somatic embryos × 100%.
[0069] This example is used to verify the effects of different media on the germination of somatic embryos of S. paniculatum.
[0070] The above merely describes the specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.
[0071] Finally, the above merely describes the preferred embodiments of the present application and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims
1. A method for regenerating plants of Hydrangea paniculata using leaves as explants, characterized in that, The method comprises the following steps: Step S1: taking leaf of tissue culture seedling of Hydrangea paniculata 'Limed Lamp' as explant, and culturing the explant in induction medium to induce callus, and inducing somatic embryo from the callus; Step S2: inoculating the somatic embryo in step S1 into somatic embryo proliferation medium to carry out proliferation culture, and obtaining secondary somatic embryo; Step S3: inoculating the secondary somatic embryo obtained in step S2 into somatic embryo germination medium to form plantlet; In step S1, the induction medium comprises the following components: basic medium 1 / 2MS or MS, 2,4-dichlorophenoxyacetic acid 4.0-5.0 mg / L, Kinetin 0.5 mg / L, acid hydrolysis casein 500 mg / L, glucose 45 g / L, and plant gel 3.0 g / L; In step S2, the somatic embryo proliferation medium comprises the following components: basic medium MS, 2,4-dichlorophenoxyacetic acid 0-2.0 mg / L, Kinetin 0.5 mg / L, acid hydrolysis casein 0-500 mg / L, glucose 45 g / L, and agar powder 7.5 g / L; In step S3, the somatic embryo germination medium comprises the following components: basic medium 1 / 2MS, 6-benzyladenine 0.1-1.5 mg / L, naphthalene acetic acid 0.2 mg / L, sucrose 30 g / L, and agar powder 7.5 g / L.
2. The method for regenerating plantlets of Hydrangea paniculata using leaves as explants according to claim 1, characterized in that: In step S2, the somatic embryo is inoculated into somatic embryo subculture medium to carry out long-term subculture and keep differentiation ability.