Regeneration breeding method of hydrangea plants

Abstract

The invention discloses a regeneration breeding method of hydrangea plants, and belongs to the technical field of breeding of hydrangea tissue culture seedlings. Comprising the following steps: (1) taking leaves with petioles at the upper parts of hydrangea tissue culture seedlings as explants, putting the explants on an induction and proliferation culture medium, and performing induction culture to form cluster buds; (2) segmenting the cluster buds, and transferring the cluster buds to an adventitious bud culture medium for elongation growth; and (3) transferring the subcultured tissue culture seedlings into a rooting culture medium, and carrying out rooting culture to complete regenerated plants. According to the method, the induction culture medium and the regeneration culture medium are combined, so that the induction regeneration culture time is shortened (60 days), the cost is saved, and the pollution rate is reduced. The method disclosed by the invention is wide in applicable variety range, is far larger than a previously reported hydrangea induction regeneration culture method, has higher induction regeneration rate in a plurality of varieties such as' endless summer ', self-fertile strain' M1084 'and' Shenzhi ', and can be applied to actual production.

Description

Technical Field

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

[0002] Hydrangea macrophylla, also known as hydrangea or hydrangea, is a plant of the genus Hydrangea in the family Hydrangeaceae. Hydrangea has a variety of species and rich colors. It not only has high ornamental and ecological value, but also has certain medicinal value. Its flowers, leaves and roots can be used as medicine (Feng Weisheng et al. 2011). With the development of society, it has received widespread attention from the flower market. It is often used in garden greening, potted plants, fresh cut flowers and dried flowers, and is also widely used in gardens and green spaces. At present, in commercial production, the reproduction of hydrangea mainly relies on conventional breeding methods such as cuttings. Traditional breeding methods are not only limited by the growing season and breeding materials, but also have many consumables and low breeding coefficients, which cannot meet the large-scale production of seedlings (Zeng Yi 2018). Therefore, it is becoming increasingly important to find a fast and convenient tissue culture method for asexual reproduction of plants.

[0003] Tissue culture technology is a modern biotechnology that has gradually developed in recent years. Its advantages are fast reproduction speed, large coefficient, short culture cycle, low cost, and the individual can well maintain the characteristics of the mother, effectively making up for the shortcomings of traditional asexual reproduction. Therefore, this technology has good application prospects and has received widespread attention in the cultivation of various plants. However, there is currently a lack of an efficient tissue culture propagation technology for hydrangea.

[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 Hydrangea grandiflora 'Endless Summer' variety as experimental materials. After 20 days of dark treatment and 40 days of light treatment, the callus induction rate could reach more than 98%; then they were transferred to the 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 best medium for inducing callus was MS+6-BA 1.0-2.0 mg / L+NAA0.50 mg / L, of which petioles had the best culture effect, with a callus rate of up to 87.5% after 28 days, while leaves had the worst induction effect, with an induction rate of only 47.1%; the medium suitable for bud proliferation was MS+6-BA 1.0 mg / L+NAA 0.5 mg / L, with 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 MS+2,4-D 0.5 mg / L+KT0.5 mg / L or MS+2,4-D 0.15 mg / L solid medium was 100% (Ruffoni B 2013), and the highest regeneration rate was 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 still problems such as a long budding cycle (85-112 days), a small number of varieties applicable to the regeneration breeding method, and complex steps in the regeneration system. Summary of the invention

[0005] The purpose of the present invention is to provide a method for regenerating and breeding hydrangea plants to solve the problems existing in the above-mentioned prior art. The present invention studies the effects of explant type, plant growth regulator, basal culture medium, culture environment, type of coagulant and other additives on regeneration, in order to establish an efficient and stable hydrangea tissue culture regeneration system, and lay a theoretical foundation for promoting the breeding and commercial production of excellent 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, placing the explants on an induction and proliferation medium, and inducing the formation of clustered buds;

[0009] (2) cutting the clustered buds and transferring them to adventitious bud 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 effect of hydrangea by about 50%.

[0013] 2. The present invention combines the induction medium and the regeneration medium, simplifies the regeneration system steps, shortens the time of induction regeneration culture (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 the culture medium.

[0014] 3. The method of the present invention is applicable to a wide range of varieties, which is much greater than 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

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

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

[0018] Figure 3The effect of plant growth regulator concentration on plant regeneration. Among them, A is the effect of 6-BA on the regeneration rate of adventitious buds of 'Bailmer' and 'M0184', B is the effect of 6-BA on the average number of regenerated buds of 'Bailmer' and 'M0184', C is the effect of IBA on the regeneration rate of adventitious buds of 'Bailmer' and 'M0184', and D is the effect of IBA on the average number of regenerated buds of '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 The effect of basal culture medium on plant regeneration. A is the effect of different basal culture medium on the regeneration rate of adventitious shoots of 'Bailmer' and 'M0184', and B is the effect of different basal culture medium on the average number of regenerated shoots of 'Bailmer' and 'M0184'.

[0021] Figure 6 The influence of culture conditions on plant regeneration. A is the influence of different culture conditions on the regeneration rate of adventitious shoots of 'Bailmer' and 'M0184', and B is the influence 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

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

[0027] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[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, placing the explants on an induction and proliferation medium, and inducing the formation of clustered buds;

[0030] (2) cutting the clustered buds and transferring them to adventitious bud 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 20 days of dark culture, and then transferred 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 varieties of Hydrangea truncatum include 'Endless Summer', 'M1084' and 'Temple of the Sun'.

[0040] The present invention provides a method for regeneration and efficient breeding of hydrangea explants, including the effects of explant types, plant growth regulators, basal culture medium, culture environment, types of coagulants and other additives on regeneration, in order to establish a hydrangea leaf explant regeneration system, provide a reference for tissue culture of more excellent plant resources of the genus Hydrangea, lay a foundation for establishing a stable genetic regeneration and transformation system for hydrangea, and provide technical support for the breeding and commercial production of its excellent varieties.

[0041] Example 1

[0042] 1. A method for optimizing the 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 the regeneration ability of different explants. The study of three different explants, petioles, leaves and leaves with petioles, of two genotypes of plants 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-BA2.25 mg / L+IBA0.1 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 the regeneration of Hydrangea chinensis

[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 the regeneration of Hydrangea chinensis

[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 can be seen from 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 maximum average number of regenerated buds was 1.28, which was significantly higher than 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, it can be seen that only under IBA treatment, petiole leaves can be regenerated, and the regeneration rate is 55.56%, indicating that IBA is the most suitable auxin for the regeneration of petiole 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 rates of the two genotypes showed a trend of first increasing and then decreasing. -1 When the concentration of 6-BA was 0 mg·L, the adventitious bud regeneration rate of the two genotypes was significantly higher than that of the other concentrations, and the average number of regenerated buds was also significantly higher than that of the treatment with 0 mg·L -1 and 2mg·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 1.5mg·L -1 +IBA 0.1mg·L -1 It is the optimal hormone treatment combination for regeneration of petiolate leaves.

[0069] 3.5 Effect of coagulant type on plant regeneration

[0070] Coagulant is one of the essential components of solid culture medium. In the medium with carrageenan as coagulant, the adventitious bud regeneration rate of the two genotypes and the average number of regenerated buds of 'Bailmer' were significantly higher than those of other treatments. For 'M0184', agar made its average number of regenerated buds the highest, followed by carrageenan and plant gel ( Figure 4 ).

[0071] On the whole, 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 the regeneration rate of adventitious buds among the three different basal media, but the average number of regenerated buds in 1 / 2MS medium was significantly higher than that in other treatments. For 'M0184', although the regeneration rate of adventitious buds in 1 / 2MS medium was not the highest, its average number of regenerated buds was the highest.

[0074] Therefore, the combined results showed that 1 / 2MS medium was a more suitable basal medium for regeneration of petiole 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 effect of petiolate leaves. In both genotypes, the regeneration rate and average number of adventitious buds in the treatments 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 in the treatments 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 varieties, '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 out 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 cultured for rooting.

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

[0086] Unless otherwise specified, 30 g / L sucrose and 7.4 g / L carrageenan were added to the culture medium, and the pH of the culture medium was adjusted to 5.8 before high-pressure sterilization. The sterilization procedure was 121°C for 20 min. Except for special treatments, all culture conditions were culture temperature 25±2°C, light / dark time of the culture was 16h / 8h, light source was fluorescent lamp, and light intensity was 1000lx.

[0087] The above-mentioned optimal culture conditions of the present invention are used for asexual reproduction of multiple varieties such as 'Endless Summer', the self-fertile 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), saves costs, and reduces the contamination rate caused by changing the culture medium. Each variety has 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 clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope 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, placing the explants on an induction and proliferation medium, and inducing the formation of clustered buds; (2) cutting the clustered buds and transferring them to adventitious bud 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, characterized in that: The upper leaves with petioles are specifically the second and third pairs of leaves from the tissue culture seedlings.

3. The regeneration breeding method according to claim 1, characterized in that: The components of the induction and proliferation culture 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.

4. The regeneration and breeding method according to claim 1, characterized in that: The induction culture conditions are: firstly carry out 20 days of dark culture, and then transfer to light for light culture for 40 days.

5. The regeneration and breeding method according to claim 1, characterized in that: 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.

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

7. The regeneration and breeding method according to claim 4, characterized in that: The conditions of the light culture are light / dark time of 16h / 8h.

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

9. The method according to claim 1, characterized in that: The cultivars of hydrangea include 'Endless Summer', 'M1084' and 'Temple of the Sun'.

Images