A regeneration and breeding method for hydrangea plants

Abstract

The present invention discloses a method for regenerating and breeding hydrangea plants, belonging to the technical field of breeding hydrangea tissue culture seedlings. The method comprises the following steps: (1) using the petiole-bearing leaf of the upper part of the hydrangea tissue culture seedling as an explant, placing the explant on an induction and proliferation culture medium, and inducing and culturing to form clustered buds; (2) cutting the clustered buds and transferring them to an adventitious bud culture medium for elongation growth; (3) transferring the tissue culture seedlings after subculture to a rooting culture medium for rooting culture to completely regenerate the plants. The present invention combines the induction culture medium and the regeneration culture medium, shortening the time for induction and regeneration culture (60 days), saving costs, and reducing the contamination rate. The method of the present invention is applicable to a wide range of varieties, far exceeding the previously reported method for induction and regeneration culture of hydrangea. It has a high induction and regeneration rate in multiple varieties such as 'Endless Summer', the self-bred strain 'M1084', and 'Temple of the Sun', and can be applied to actual production.

Description

Technical Field

[0001] The invention relates to the technical field of propagation of hydrangea tissue culture seedlings, in particular to a regeneration and propagation method of hydrangea plants. Background Art

[0002] Hydrangea macrophylla, also known as hydrangea or purple hydrangea, is a plant of the genus Hydrangea in the family Hydrangeaceae. Hydrangea boasts diverse species and rich colors, possessing not only high ornamental and ecological value but also medicinal properties, with its flowers, leaves, and roots all used as medicine (Feng Weisheng et al. 2011). With the development of society, it has gained widespread attention in the flower market and is commonly used in garden landscaping, potted plants, and for fresh cut and dried flower production, as well as in landscape gardens. Currently, commercial propagation of hydrangea relies primarily on conventional propagation methods such as cuttings. However, these traditional propagation methods are not only limited by the growing season and the availability of propagation materials, but also consume a large amount of materials and have a low propagation coefficient, making them unsuitable for large-scale seedling production (Zeng Yi 2018). Therefore, the development of rapid and convenient tissue culture methods for asexual plant propagation has become increasingly important.

[0003] Tissue culture is a modern biotechnology technique that has been gradually developing in recent years. Its advantages include rapid propagation, high coefficients, short culture cycles, and low costs. Furthermore, the individual plants retain the characteristics of the parent plant, effectively compensating for the shortcomings of traditional asexual propagation methods. Therefore, this technique has promising application prospects and has attracted widespread attention in the cultivation of various plants. However, an efficient tissue culture propagation technique for hydrangea is currently lacking.

[0004] Sun Xiaobo et al. (Sun Xiaobo, invention patent: Method for rapid propagation of Hydrangea grandiflora Endless Summer using tissue culture seedling leaves, 2018) used tissue culture seedling leaves of the Hydrangea 'Endless Summer' variety as experimental materials. After 20 days of dark treatment and 40 days of light treatment, the callus induction rate reached more than 98%; then they were transferred to a callus proliferation and adventitious bud differentiation medium with a formula of MS+6-BA2.25 mg / L+IBA 0.1 mg / L. After 40 days, the adventitious bud induction rate was 67.1-74.3%. Zhao Yingying et al. (2015) used the leaves of the red variety 'Adria' hydrangea as explants for regeneration. The callus induction medium was MS+6-BA2.5 mL+IAA0.2 mg / L, and the callus rate reached 100%; the bud induction differentiation medium was MS+6-BA 3.0 mg / L+NAA0.3 mg / L. After 15 days of dark culture, light culture was carried out. After 70 days of light culture, the bud induction rate could reach 80%. Fan Xiaofeng et al. (2009) used stem segments, petioles, and leaves of Hydrangea chinensis as explants and found that the optimal medium for inducing callus formation was MS + 1.0–2.0 mg / L 6-BA + 0.50 mg / L NAA. The best culture results were achieved with petioles, achieving a callus induction rate of 87.5% after 28 days, while leaves showed the worst induction rate, with only 47.1%. The medium most suitable for bud proliferation was MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA, achieving a bud induction rate of 75.0% after 84 days. Ruffoni et al. (2013) reported that the callus induction rate of leaves of Hydrangea chinensis 'Snow Queen' on solid medium containing either MS + 0.5 mg / L 2,4-D + 0.5 mg / L KT or MS + 0.15 mg / L 2,4-D was 100% (Ruffoni B 2013), with a maximum regeneration rate of 44%. In summary, the current hydrangea explant induction regeneration method has a high callus induction rate, but the bud regeneration rate is not ideal; in addition, there are problems such as a long budding cycle (85-112 days), a small number of varieties applicable to the regeneration breeding method, and complex regeneration system steps. Summary of the Invention

[0005] The present invention aims to provide a method for regenerating and propagating hydrangea plants to address the aforementioned problems of the prior art. The present invention investigates the effects of explant type, plant growth regulators, basal culture medium, culture environment, coagulant type, and other additives on regeneration, with the goal of establishing an efficient and stable hydrangea tissue culture regeneration system. This will lay a theoretical foundation for promoting the breeding and commercial production of superior hydrangea varieties.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a method for regenerating and breeding hydrangea plants, comprising the following steps:

[0008] (1) Using the leaves with petioles on the upper part of the hydrangea tissue culture seedlings as explants, the explants were placed on an induction and proliferation medium and induced to form clustered buds;

[0009] (2) cutting the clustered buds and transferring them to adventitious bud culture medium for elongation growth;

[0010] (3) The tissue culture seedlings after subculture are transferred to the rooting medium for rooting culture to completely regenerate the plants.

[0011] Based on the above technical solution, the present invention has the following technical effects:

[0012] 1. Compared with the prior art, the present invention is the first to discover that a new additive, adenine sulfate, promotes the regeneration of hydrangea by about 50%.

[0013] 2. The present invention combines the induction culture medium and the regeneration culture medium, simplifies the regeneration system steps, shortens the induction regeneration culture time (60 days), which is much shorter than the reported induction regeneration culture time (85-112 days), saves costs, and reduces the contamination rate caused by switching culture media.

[0014] 3. The method of the present invention is applicable to a wide range of varieties, far exceeding the previously reported method of induced regeneration culture of Hydrangea chinensis. It has a high induced regeneration rate in multiple varieties such as 'Endless Summer', the self-bred strain 'M1084' and 'Temple of the Sun', and can be applied to actual production. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 The effect of explant type on hydrangea regeneration, where A is the regeneration rate and B is the average number of regenerated buds.

[0017] Figure 2 The effect of the tenderness of the explant on the regeneration of Hydrangea chinensis. A is the regeneration rate and B is the average number of regenerated buds.

[0018] Figure 3Figure 2 shows the effects of plant growth regulator concentrations on plant regeneration. A shows the effect of 6-BA on the regeneration rate of adventitious shoots in 'Bailmer' and 'M0184'; B shows the effect of 6-BA on the average number of regenerated shoots in 'Bailmer' and 'M0184'; C shows the effect of IBA on the regeneration rate of adventitious shoots in 'Bailmer' and 'M0184'; and D shows the effect of IBA on the average number of regenerated shoots in 'Bailmer' and 'M0184'.

[0019] Figure 4 The effects of different coagulant types on plant regeneration. A is the effect of different coagulant types on the regeneration rate of adventitious buds of 'Bailmer' and 'M0184', and B is the effect of different coagulant types on the average number of regenerated buds of 'Bailmer' and 'M0184'.

[0020] Figure 5 Effects of basal culture media on plant regeneration. A shows the effect of different basal culture media on the regeneration rate of adventitious shoots of 'Bailmer' and 'M0184', and B shows the effect of different basal culture media on the average number of regenerated shoots of 'Bailmer' and 'M0184'.

[0021] Figure 6 Figure 2 is the effect of culture conditions on plant regeneration. A is the effect of different culture conditions on the regeneration rate of adventitious shoots of 'Bailmer' and 'M0184', and B is the effect of different culture conditions on the average number of regenerated shoots of 'Bailmer' and 'M0184'. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0028] The embodiment of the present invention provides a method for regenerating and breeding hydrangea plants, comprising the following steps:

[0029] (1) Using the leaves with petioles on the upper part of the hydrangea tissue culture seedlings as explants, the explants were placed on an induction and proliferation medium and induced to form clustered buds;

[0030] (2) cutting the clustered buds and transferring them to adventitious bud culture medium for elongation growth;

[0031] (3) The tissue culture seedlings after subculture are transferred to the rooting medium for rooting culture to completely regenerate the plants.

[0032] In some specific embodiments, the upper leaves with petioles are specifically the second and third pairs of leaves from the tissue culture seedlings.

[0033] In some specific embodiments, the components of the induction and proliferation medium are: 1 / 2MS+6-BA 1.5 mg / L+IBA 0.1 mg / L+sucrose 30 g / L+carrageenan 7.4 g / L+adenine sulfate 5 mg / L.

[0034] In some specific embodiments, the induction culture conditions are: first culturing in darkness for 20 days, and then transferring to light for light culture for 40 days.

[0035] In some specific embodiments, the components of the adventitious bud culture medium are: MS+0.5 mg / L 6-BA+30 g / L sucrose+7.4 g / L carrageenan.

[0036] In some specific embodiments, the rooting medium comprises: 1 / 2MS+NAA 0.05 mg / L+sucrose 20 g / L+carrageenan 7.4 g / L.

[0037] In some specific embodiments, the light culture condition is a light / dark period of 16h / 8h.

[0038] In some specific embodiments, the intensity of the light is 1000 lx.

[0039] In some specific embodiments, the hydrangea varieties include 'Endless Summer', 'M1084' and 'Temple of the Sun'.

[0040] The present invention provides a method for regenerating and efficiently propagating hydrangea explants, including the effects of explant type, plant growth regulators, basal culture medium, culture environment, coagulant type, and other additives on regeneration. The aim is to establish a hydrangea leaf explant regeneration system. This method provides a reference for tissue culture of more high-quality hydrangea plant resources, lays the foundation for establishing a stable genetic regeneration and transformation system for hydrangea, and provides technical support for the breeding and commercial production of high-quality hydrangea varieties.

[0041] Example 1

[0042] 1. An optimization method for regeneration and propagation of hydrangea plants, comprising the following steps:

[0043] (1) Selection of explants: Cut the leaves with petioles on the upper part of the tissue culture seedlings of 'Endless Summer' as explants.

[0044] (2) Callus induction and adventitious bud proliferation: Leaves with petioles were placed on induction and proliferation medium, cultured in the dark for 20 days and then in the light for 40 days to form clustered buds.

[0045] (3) Subculture of adventitious buds: The clustered buds were cut and transferred to the adventitious bud subculture medium for elongation growth for 30 days.

[0046] (4) Rooting culture of tissue culture seedlings: After 30 days of subculture, the tissue culture seedlings are transferred to the rooting medium. After 30 days, the seedlings develop into complete regenerated plants with roots, stems and leaves.

[0047] 2 Regarding step (1), this example experiment compared the regeneration ability of different explants.

[0048] The results showed that there were significant differences in regeneration capacity between different explants. The study of three different explants, petioles, leaves and leaves with petioles, found that the leaf explants with petioles had a higher regeneration efficiency than the other two explants, with a regeneration rate of 77.77% ( Figure 1 The regeneration ability of explants varies with the degree of tenderness. The leaves with petioles on the upper part (the second and third pairs of leaves from the top) have better regeneration efficiency than the leaves with petioles on the lower part (the second and third pairs of leaves from the bottom), with a regeneration rate of 66.67% and an average number of regenerated buds of 1.67 ( Figure 2 Therefore, the leaves with petioles on the upper part of 'Endless Summer' hydrangea with good growth condition and consistent growth were selected as explants.

[0049] 3. Regarding step (2), callus induction and adventitious bud proliferation.

[0050] 3.1 This example experiment compared different culture medium components. Based on the initial culture medium formula MS+6-BA 2.25 mg / L+IBA 0.1 mg / L (Sun Xiaobo et al.), the effects of adding different additives on the regeneration of Hydrangea chinensis are shown in Table 1.

[0051] Table 1 Effects of different additives on hydrangea regeneration

[0052]

[0053]

[0054] As can be seen from Table 1, by comparing the three additives, it was found that the regeneration rate and average number of regenerated buds of the adenine sulfate added at 5m / L (D6 group) reached the maximum, which were 66.67% and 2.71 respectively, which was about 50% higher than the regeneration rate of the CK control group (44.45%), and the callus state was good.

[0055] 3.2 The effects of different cytokinins on hydrangea regeneration are shown in Table 2.

[0056] Table 2 Effects of different cytokinins on hydrangea regeneration

[0057]

[0058]

[0059] Note: The values ​​in the table are mean ± standard error, “-” means no addition, and different letters after the values ​​in the same column represent the difference of the values ​​at the P < 0.05 level.

[0060] As shown in Table 2, under different cytokinin treatments, the addition of 2.25 mg / L 6-BA had the highest regeneration rate of leaves with petioles, reaching 55.56%, and the average maximum number of regenerated buds was 1.28, which was significantly higher than that of other treatments. 6-BA is the most suitable cytokinin for the regeneration of leaves with petioles.

[0061] 3.3 The effects of different auxins on hydrangea regeneration are shown in Table 3.

[0062] Table 3 Effects of different auxins on hydrangea regeneration

[0063]

[0064]

[0065] From Table 3, we can see that only under IBA treatment, petiolate leaves can be regenerated, and the regeneration rate is 55.56%, indicating that IBA is the most suitable auxin for the regeneration of petiolate leaves.

[0066] 3.46-BA and IBA were the most suitable cytokinin and auxin, respectively. Next, the effects of different concentrations of 6-BA and IBA on plant regeneration were studied.

[0067] With the increase of 6-BA and IBA concentrations, the adventitious bud regeneration rate of both genotypes showed a trend of first increasing and then decreasing. -1 The adventitious bud regeneration rate of the two genotypes was significantly higher than that of the other concentration treatments, and the average number of regenerated buds was also significantly higher than that of the treatment with 6-BA concentration of 0 mg·L -1 and 2 mg·L -1 Based on the addition of 6-BA to the culture medium at an optimal concentration of 1.5 mg·L -1 When the IBA concentration is 0.1 mg·L -1 When , the average number of regenerated shoots of the two genotypes was significantly higher than that of other concentration treatments, and the adventitious shoot regeneration rate was significantly higher than that of other concentration treatments of IBA ( Figure 3 ).

[0068] Therefore, 6-BA 1.5 mg·L -1 +IBA 0.1mg·L -1 It is the best hormone treatment combination for regeneration of leaves with petioles.

[0069] 3.5 Effect of coagulant type on plant regeneration

[0070] Coagulants are essential components of solid culture media. In media containing carrageenan as a coagulant, the adventitious bud regeneration rates of both genotypes and the average number of regenerated buds in 'Bailmer' were significantly higher than those in other treatments. For 'M0184', agar resulted in the highest average number of regenerated buds, followed by carrageenan and phytol ( Figure 4 ).

[0071] Overall, carrageenan is the most suitable culture medium coagulant.

[0072] 3.6 Effect of basal medium type on plant regeneration

[0073] The effects of different basal culture medium types on plant regeneration were studied ( Figure 5 For 'Bailmer', there was no significant difference in adventitious bud regeneration rate among the three different basal media, but the average number of regenerated buds in the 1 / 2MS medium was significantly higher than in the other treatments. For 'M0184', although the adventitious bud regeneration rate in the 1 / 2MS medium was not the highest, its average number of regenerated buds was the highest.

[0074] Therefore, the comprehensive results showed that 1 / 2MS medium was a more suitable basal medium for regeneration of petiolate leaves.

[0075] 3.7 The final composition of the induction and proliferation medium was determined to be 1 / 2MS + 6-BA 1.5 mg / L + IBA 0.1 mg / L + sucrose 30 g / L + carrageenan 7.4 g / L + adenine sulfate 5 mg / L.

[0076] 3.8 Effects of culture conditions on plant regeneration

[0077] Different light and dark treatment times had a significant effect on the regeneration of petiolate leaves. In both genotypes, the regeneration rate and average number of adventitious buds after 20 days of dark culture and then light culture were significantly higher than those in other treatments, while the regeneration rate and average number of adventitious buds after 60 days of dark culture were the lowest ( Figure 6 ). It can be seen that appropriate dark culture in the early stage is helpful to improve the regeneration ability of petiolate leaves.

[0078] Three hydrangea cultivars, 'Endless Summer', 'M1084' and 'Temple of the Sun', were used as materials and cultured according to the above-mentioned optimal conditions. The results are shown in Table 4.

[0079] Table 4 Parallel experiments on three hydrangea varieties

[0080]

[0081] As shown in Table 4, the callus rate of the three varieties reached 100%, the regeneration rate reached a maximum of 90.1% (Temple of the Sun), and the average number of regenerated buds reached a maximum of 4.8 (M0184).

[0082] 4. Regarding the subculture of adventitious buds in step (3).

[0083] The best adventitious bud culture medium screened was: MS+0.5mg / L 6-BA+30g / L sucrose+7.4g / L carrageenan.

[0084] 5. With respect to step (4), the tissue culture seedlings are rooted.

[0085] The best seedling and rooting culture medium screened out was: 1 / 2MS+NAA0.05 mg / L+sucrose 20 g / L+carrageenan 7.4 g / L.

[0086] Unless otherwise specified, the culture medium was supplemented with 30 g / L sucrose and 7.4 g / L carrageenan. The pH of the culture medium was adjusted to 5.8 before autoclaving. Sterilization was performed at 121°C for 20 min. Unless otherwise specified, all cultures were maintained at 25 ± 2°C, with a 16 h / 8 h light / dark cycle and a fluorescent light source at 1000 lx.

[0087] The above-mentioned optimal culture conditions of the present invention are used for asexual propagation of multiple varieties such as 'Endless Summer', the self-bred strain 'M1084' and 'Temple of the Sun', which simplifies the regeneration system steps and shortens the time of induced regeneration culture (60 days), which is much shorter than the reported induced regeneration culture time (85-112 days). It also saves costs and reduces the contamination rate caused by changing the culture medium. All varieties have a high induced regeneration rate and can be applied to actual production.

[0088] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A method for regenerating and breeding hydrangea plants, characterized in that: The following steps are involved: (1) Using the leaves with petioles on the upper part of the hydrangea tissue culture seedlings as explants, the explants were placed on an induction and proliferation medium to induce and culture the formation of clustered buds; the induction and proliferation medium comprises the following components: 1 / 2MS + 6-BA 1.5 mg / L + IBA 0.1 mg / L + sucrose 30 g / L + carrageenan 7.4 g / L + adenine sulfate 5 mg / L; (2) cutting the clustered buds and transferring them to adventitious bud culture medium for elongation growth; (3) The tissue culture seedlings after subculture are transferred to the rooting medium for rooting culture to completely regenerate the plants.

2. The regeneration and breeding method according to claim 1, wherein: The upper leaves with petioles are specifically the second and third pairs of leaves from the tissue culture seedling.

3. The regeneration and breeding method according to claim 1, wherein: The induction culture conditions are: first carry out dark culture for 20 days, then transfer to light and carry out light culture for 40 days.

4. The regeneration and breeding method according to claim 1, wherein: The adventitious bud culture medium comprises the following components: MS+0.5 mg / L 6-BA+30 g / L sucrose+7.4 g / L carrageenan.

5. The regeneration and breeding method according to claim 1, characterized in that: The rooting medium comprises the following components: 1 / 2MS+NAA 0.05 mg / L+sucrose 20 g / L+carrageenan 7.4 g / L.

6. The regeneration and breeding method according to claim 3, characterized in that: The light culture conditions are light / dark time of 16h / 8h.

7. The regeneration and breeding method according to claim 6, characterized in that: The intensity of the light is 1000 lx.

8. The method according to claim 1, characterized in that The hydrangea varieties include 'Endless Summer' and 'Temple of the Sun'.

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