Culture medium combination for cultivating malus hallings and application thereof in rapid propagation of malus hallings

Abstract

The present application relates to the field of tissue culture, in particular to a culture medium combination for culturing Hibiscus syriacus and application thereof in rapid propagation of Hibiscus syriacus. The culture medium combination for culturing Hibiscus syriacus comprises a differentiation medium, an adventitious bud elongation subculture medium and a strong root culture medium, wherein the differentiation medium comprises differentiation medium I and differentiation medium II, the present application uses part of the differentiation medium in different stages, can fully meet the application required by growth of callus in different stages, improves callus differentiation rate and average bud number, and improves propagation efficiency; the adventitious bud elongation subculture medium is optimized from the perspective of different basic culture media and different growth hormones, finally guarantees to obtain the best adventitious bud elongation subculture medium, increases effective bud number, guarantees survival rate, finally selects a specific rooting culture medium, and increases rooting rate. It can be seen that the culture medium combination can improve propagation rate of Hibiscus syriacus and guarantee survival rate of Hibiscus syriacus.

Description

Technical Field

[0001] The present invention relates to the field of tissue culture, and particularly to a culture medium combination for culturing Hibiscus syriacus and its application in rapid propagation of Hibiscus syriacus. Background Art

[0002] Hibiscus syriacus, also known as cotton tree and China rose, is a shrub or deciduous small tree belonging to the genus Hibiscus in the family Malvaceae. Its small branches are densely covered with yellow stellate villi. The leaves are rhomboid to triangular-ovate, 3-10 cm long and 2-4 cm wide, with 3 lobes of different depths or without lobes, the apex is obtuse, the base is cuneate, the margin has irregular serrations, and the lower surface is slightly hairy or nearly hairless along the veins. The flowers are solitary in the axils of the terminal leaves of the branches. The calyx is campanulate, 14-20 mm long, densely covered with stellate short villi, with 5 lobes, triangular; the flower colors are pure white, light pink, light purple, purplish red, etc., the flower shape is bell-shaped, and there are single-petal, double-petal, and multi-petal types; the outside is sparsely covered with cilia and stellate long pubescence. The capsule is ovoid, about 12 mm in diameter, densely covered with yellow stellate villi; the seeds are kidney-shaped, with yellowish-white long pubescence on the back, and the flowering period is from July to October. Hibiscus syriacus has a cultivation history of more than 3,000 years in China, and has the characteristics of rich varieties, large and colorful flowers, and strong adaptability. It is drought-tolerant, salt-tolerant, and barren-tolerant, has low requirements for the soil, and can be used as an excellent summer flowering tree species.

[0003] Hibiscus syriacus is a common shrub flower species in gardens. It is native to the central provinces of China and is cultivated everywhere. Hibiscus syriacus is one of the most commonly used ornamental plants in landscaping seedlings. It can be used as a hedge-style green hedge, and can be planted alone or in clusters. The seeds of Hibiscus syriacus are used as medicine, called "Chaotianzi". Hibiscus syriacus is the national flower of South Korea and Malaysia, and also has the alias of desert rose in North America.

[0004] The breeding of Hibiscus syriacus is mainly divided into sexual reproduction and asexual reproduction. Among them, sexual reproduction is mainly used for the cultivation of new varieties of Hibiscus syriacus, but there are problems such as low natural germination rate of seeds. Asexual reproduction mainly includes methods such as dividing plants, cutting, layering, and tissue culture. Among them, cutting is the most common, but the propagation coefficient is low and the rooting time is long, resulting in the production quantity not meeting the market requirements. At present, there are endless studies on the tissue culture of Hibiscus syriacus, but the effects are not satisfactory.

[0005] Patent application number 201711200887.9 discloses a method for hibiscus tissue culture regeneration: using current-year lignified branches of hibiscus as explants, initial seedlings are obtained induction medium formulated as 1 / 2 modified MS medium + 6-8 mg / L 6-BA + 2-3 mg / L IBA + 15 mg / L LVC + 15 mg / L LVB2 + 50 mg / L gibberellin + 3.6 g / L agar powder + 20 g / L sucrose; buds differentiate in proliferation medium formulated as 1 / 2 modified MS medium + 3-5 mg / L 6-BA + 1.5-2 mg / L NAA + 0.1-0.5 mg / L ZT + 10 mL / L L-cysteine ​​+ 50 mg / L gibberellin + 3.6 g / L agar powder + 30 g / L sucrose; and buds differentiate in proliferation medium formulated as 1 / 2 modified MS medium + 1.5-2 mg / L 6-BA + 0.5-1 mg / L L... Hibiscus was rooted in a rooting medium consisting of NAA + 10 mg / L LVC + 20 mg / L VB2 + 30 mg / L gibberellin + 10 ml / L L-cysteine ​​+ 3.6 g / L agar powder + 30 g / L sucrose. While this method is effective for cultivating hibiscus, the addition of large amounts of hormones to the medium makes its preparation cumbersome and increases costs.

[0006] Therefore, it is essential to break through the bottleneck in hibiscus propagation and develop a simple and effective hibiscus propagation technique. Summary of the Invention

[0007] The purpose of this invention is to provide a culture medium composition for cultivating hibiscus and its application in the rapid propagation of hibiscus, thereby solving the problems existing in the prior art. The culture medium composition provided by this invention enables rapid propagation of hibiscus, and its components are simple, resulting in good propagation effects.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] This invention provides a culture medium combination for cultivating hibiscus, the culture medium combination comprising a differentiation medium, an adventitious shoot elongation subculture medium, and a root strengthening medium;

[0010] The differentiation medium includes differentiation medium I and differentiation medium II; differentiation medium I uses MS medium as the basal medium and further includes 1.0 mg / L 6-benzylaminopurine, 0.5 mg / L IAA and 30.0 g / L sucrose; differentiation medium II uses MS medium as the basal medium and further includes 1.0 mg / L 6-benzylaminopurine, 0.2 mg / L NAA and 30.0 g / L sucrose.

[0011] The adventitious shoot elongation subculture medium is based on DKW medium and also includes ZT 2.0 mg / L, IBA 0.2 mg / L and sucrose 15.0 g / L;

[0012] The rooting medium is based on 1 / 2 MS medium and also includes IBA 0.1 mg / L and sucrose 15.0 g / L.

[0013] This invention provides the application of the above-described culture medium combination in the rapid propagation of Hibiscus syriacus.

[0014] This invention provides a method for rapid propagation of Hibiscus syriacus, comprising the following steps:

[0015] Hypocotyls of hibiscus seedlings were used as explants for callus culture to obtain callus tissue.

[0016] The callus tissue was subjected to differentiation culture, adventitious shoot elongation subculture, and root strengthening culture in sequence to obtain the hibiscus.

[0017] Preferably, the differentiation culture includes a first differentiation culture and a second differentiation culture; the first differentiation culture uses MS medium as the basic medium and further includes 1.0 mg / L of 6-benzylaminopurine, 0.5 mg / L of IAA and 30.0 g / L of sucrose;

[0018] The second differentiation was carried out using MS medium as the basic medium, which also included 1.0 mg / L of 6-benzylaminopurine, 0.2 mg / L of NAA and 30.0 g / L of sucrose.

[0019] Preferably, the temperature of the first differentiation culture is 25°C, the light intensity is 1800 lx, the time is 14 days, and the light duration is 16 h.

[0020] The second differentiation culture was conducted at a temperature of 25°C, a light intensity of 1800 lx, for 10 days, with a light duration of 16 hours.

[0021] Preferably, the culture medium used for the adventitious shoot elongation subculture is based on DKW medium, and also includes ZT 2.0 mg / L, IBA 0.2 mg / L and sucrose 15.0 g / L.

[0022] Preferably, the temperature for the adventitious bud elongation subculture is 25°C, the light intensity is 1800 lx, the time is 30 days, and the light duration is 16 h.

[0023] Preferably, the root-strengthening culture medium is based on 1 / 2 MS medium and also includes 0.1 mg / L IBA and 15.0 g / L sucrose.

[0024] Preferably, the temperature for root cultivation is 25°C, the light intensity is 1800 lx, and the light duration is 16 h.

[0025] Preferably, the induction culture is based on MS medium, which also includes 0.5 mg / L 2,4-D, 0.1 mg / L kinetin (KT), and 30.0 g / L sucrose.

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

[0027] This invention provides a culture medium combination for cultivating Hibiscus syriacus, which includes a differentiation medium, an adventitious shoot elongation subculture medium, and a root-strengthening medium. The differentiation medium includes differentiation medium I and differentiation medium II. This invention uses partial differentiation medium at different stages, which can fully meet the needs of callus growth at different stages, improve the callus differentiation rate and average number of shoots, and improve propagation efficiency. 6-BA at an appropriate concentration can promote cell division and elongation, promote cell differentiation, and promote the synthesis and transport of substances. According to literature reports, the addition of 6-BA to Hibiscus syriacus in the Malvaceae family leads to accelerated cell division of axillary bud meristem cells in its young shoots, producing a large number of shoots. The formation of explants induced by 6-BA and NAA / IAA from light green to dark green callus is more conducive to the differentiation of adventitious shoots. The adventitious shoot elongation subculture medium is optimized from the perspective of different basal media and different growth hormones, ultimately ensuring the acquisition of the best adventitious shoot elongation subculture medium, increasing the number of effective shoot nodes, ensuring the survival rate, and finally selecting a specific rooting medium to increase the rooting rate. Therefore, the culture medium combination provided by the present invention can improve the hibiscus reproduction rate and ensure the hibiscus survival rate.

[0028] The present invention also provides a rapid propagation method based on the above-mentioned culture medium combination for cultivating hibiscus. This method is simple and has the advantages of high propagation rate and high survival rate. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1This diagram illustrates the process of inducing callus differentiation from aseptic hibiscus seedlings; where a represents callus induction from hibiscus hypocotyls; b represents callus induction from hypocotyls; c represents adventitious bud differentiation from callus on differentiation medium I; df represents the state of adventitious buds under an electron microscope; g represents the growth of adventitious buds on differentiation medium II; and hi represents the rooting of hibiscus seedlings.

[0031] Figure 2 Figure 1 shows the growth of hibiscus adventitious buds on some adventitious bud elongation subculture media; where a represents the growth of hibiscus adventitious buds on medium 1; b represents the growth of hibiscus adventitious buds on medium 2; c represents the growth of hibiscus adventitious buds on medium 3; d represents the growth of hibiscus adventitious buds on medium 4; and e represents the growth of hibiscus adventitious buds on medium 5.

[0032] Figure 3 Figure (a) shows the elongation and subculturing of hibiscus adventitious buds on the optimal culture medium (based on DKW medium, which also contains ZT 2.0 mg / L, IBA 0.2 mg / L and sucrose 15.0 g / L) and figure (b) shows the rooting of hibiscus. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] Example 1: Screening of Differentiation Culture Media

[0039] (1) Aseptic germination of seeds

[0040] The germination medium was based on 1 / 2 MS medium and also contained 15 g / L of sucrose.

[0041] Select mature and plump hibiscus seeds, soak them in concentrated sulfuric acid for 5 minutes, and then rinse them with running water for 2 hours. In a clean bench, disinfect them with 75% ethanol + Triton-80 for 10 minutes, rinse them three times with sterile water, then soak them in 0.1% HgCl2 for 10 minutes, rinse them six times with sterile water, and blot them dry with filter paper before inoculating them into germination medium and culturing them until the hibiscus seeds germinate and produce two cotyledons.

[0042] (2) Callus induction culture

[0043] The induction medium was based on MS medium, and also contained 0.5 mg / L 2,4-D, 0.1 mg / L kinetin (KT) and 30.0 g / L sucrose.

[0044] The hypocotyls of hibiscus seedlings with two cotyledons were cut into 0.5cm segments and placed in induction medium for dark culture for 14 days, then transferred to light culture. The temperature in the tissue culture room was 25℃, the light intensity was about 1800lx, and the light-dark cycle was 16h / 8h (16h light time and 8h dark time). The callus induction rate was 100%.

[0045] (3) Callus differentiation and culture

[0046] Dense, yellowish-white callus tissue was inoculated onto different differentiation medium I and cultured under a lamp at 25℃ and a light intensity of about 1800 lx. After 14 days, the callus tissue turned dark green and adventitious shoots were obtained. The adventitious shoots were then transferred to different differentiation medium II and cultured for another 10 days under the same light conditions until the callus differentiated into seedlings. The differentiation rate and the average number of shoots per callus were investigated. The components and concentrations of different differentiation mediums and the investigation results are shown in Table 1.

[0047] Table 1. Specific components and survey results of different differentiation media.

[0048]

[0049] The data in Table 1 show that the basal culture medium also has a significant impact on the callus differentiation ability of Hibiscus syriacus, and there are also requirements for the concentration of hormones. Table 1 indicates that treatment 1 was the most suitable for callus differentiation culture among the four culture media, achieving a differentiation rate of over 80% and an average of four shoots per callus.

[0050] Example 2: Screening of culture medium for adventitious shoot elongation subculture

[0051] The process by which callus is induced from the hypocotyl of a hibiscus sterile seedling to differentiate into a plant is as follows: Figure 1 As shown, specifically:

[0052] (1) Aseptic germination of seeds

[0053] The germination medium was based on 1 / 2 MS medium and also contained 15 g / L of sucrose.

[0054] Select mature and plump hibiscus seeds, soak them in concentrated sulfuric acid for 5 minutes, and then rinse them with running water for 2 hours. In a clean bench, disinfect them with 75% ethanol + Triton-80 for 10 minutes, rinse them three times with sterile water, then soak them in 0.1% HgCl2 for 10 minutes, rinse them six times with sterile water, and blot them dry with filter paper before inoculating them into germination medium and culturing them until the hibiscus seeds germinate and produce two cotyledons.

[0055] (2) Callus induction culture

[0056] The induction medium was based on MS medium, and also contained 0.5 mg / L 2,4-D, 0.1 mg / L kinetin (KT) and 30 g / L sucrose.

[0057] The hypocotyls of hibiscus seedlings with two cotyledons were cut into 0.5cm segments for induction culture. Figure 1 (a) The tissue was placed in induction medium and cultured in the dark for 14 days, then transferred to light culture. The temperature of the tissue culture room was 25℃, the light intensity was about 1800 lx, and the light-dark cycle was 16h / 8h (16h light time and 8h dark time) to obtain callus tissue. Figure 2 In b), the callus induction rate was 100%.

[0058] (3) Callus differentiation and culture

[0059] Differentiation medium I used MS medium as the basal medium and also contained 1.0 mg / L 6-benzylaminopurine, 0.5 mg / L IAA and 30.0 g / L sucrose, with a pH of 5.8; differentiation medium II used MS medium as the basal medium and also contained 1.0 mg / L 6-benzylaminopurine, 0.2 mg / L NAA and 30.0 g / L sucrose, with a pH of 5.8.

[0060] Dense, yellowish-white callus tissue was inoculated onto differentiation medium I and cultured under a lamp at 25℃ and a light intensity of approximately 1800 lx. After 14 days, the callus tissue turned dark green, and adventitious shoots were obtained. Figure 1 In section C), the adventitious buds were observed using an electron microscope, and the state diagram is shown below. Figure 1 As shown in df; then the adventitious shoots were transferred to differentiation medium II and cultured for another 10 days under the same light conditions until callus differentiated into seedlings. Figure 1 The differentiation rate reached 80.16%, and the average number of shoots from a single callus was 4. When the seedlings grew to 2-3 cm, they were transferred to the adventitious shoot elongation subculture medium.

[0061] (4) Screening of culture medium for adventitious shoot elongation subculture

[0062] Seedlings differentiated on differentiation medium II were cut and transferred to nine different adventitious bud elongation subculture media for screening. The culture conditions were: 25℃ in the tissue culture room, light intensity of approximately 1800 lx, and a light / dark cycle of 16h / 8h (16h light time, 8h dark time). After 30 days of culture, subcultured seedlings were obtained, and plant height and the number of effective bud nodes were recorded. The specific components and results of the nine different adventitious bud elongation subculture media are shown in Table 2. Meanwhile, the growth of Hibiscus syriacus on some media is also shown in Table 2. Figure 2 As shown.

[0063] Table 29 shows the specific components and survey results of different adventitious shoot elongation subculture media.

[0064]

[0065] From Table 2 and Figure 2 It was found that treatment 3 (based on DKW medium, also containing ZT 2.0 mg / L, IBA 0.2 mg / L, and sucrose 1.5 wt.%) was the most suitable for adventitious shoot elongation and subculture among the nine culture media, with an average plant height of 6.8 cm and 6 effective bud nodes. The subculture diagram of hibiscus elongation obtained from treatment 3 is shown below. Figure 3 As shown in 'a'.

[0066] The tissue culture room temperature was then set to 25℃, the light intensity to approximately 1800 lx, and the light-dark cycle to 16h / 8h (16h light, 8h dark). Simultaneously, the healthy, yellowish-green callus tissue at the base of the subcultured seedlings was transferred to a new differentiation medium (MS medium as the basal medium, also containing 0.5 mg / L 6-BA, 0.1 mg / L NAA, and 1.5 wt.% sucrose) to re-differentiate adventitious shoots. Because the callus differentiates multiple times and accumulates a certain amount of hormones, the concentrations of 6-BA and NAA in the new differentiation medium were halved. Under this formulation, the callus tissue could cyclically differentiate into adventitious shoots, significantly saving costs and improving the differentiation rate. When the seedlings reached 4-5 cm in height, rooting culture was initiated.

[0067] (5) Rooting culture

[0068] The rooting medium was based on 1 / 2 MS medium, and also contained 0.1 mg / L IBA and 1.5 wt.% sucrose.

[0069] Hibiscus seedlings 4-5cm tall were grafted onto rooting medium. The culture conditions were: 25℃ in the tissue culture room, light intensity of approximately 1800 lx, and a light / dark cycle of 16h / 8h (16h light, 8h dark). After approximately 20 days, 4-6 new roots would have emerged. Figure 3 b) Figure 1 The figure in 'hi' shows the results after 30 days of growth on the rooting medium.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A composite culture medium for cultivating hibiscus, characterized in that, The combined culture medium includes callus differentiation medium, adventitious shoot elongation subculture medium, and rooting medium. The callus differentiation culture medium includes differentiation culture medium I and differentiation culture medium II; differentiation culture medium I is MS basal medium + 6-benzylaminopurine 1.0 mg / L + IAA 0.5 mg / L + sucrose 30.0 g / L; differentiation culture medium II is MS basal medium + 6-benzylaminopurine 1.0 mg / L + NAA 0.2 mg / L + sucrose 30.0 g / L. The adventitious shoot elongation subculture medium was DKW basal medium + ZT 2.0 mg / L + IBA 0.2 mg / L + sucrose 15.0 g / L; The rooting medium consisted of 1 / 2 MS basal medium + IBA 0.1 mg / L + sucrose 15.0 g / L.

2. The application of the combined culture medium according to claim 1 in the rapid propagation of Hibiscus syriacus.

3. A method for rapid propagation of Hibiscus syriacus, characterized in that, Includes the following steps: Using the hypocotyl of hibiscus seedlings as explants, callus induction culture was performed to obtain callus tissue; The callus tissue was successively subjected to differentiation culture, adventitious bud elongation subculture culture and rooting culture to obtain hibiscus regenerated plants; The differentiation culture consists of a first differentiation culture and a second differentiation culture; the culture medium used in the first differentiation culture is the differentiation culture medium I described in claim 1; The culture medium used for the second differentiation is the differentiation culture medium II described in claim 1; The culture medium used for the adventitious shoot elongation subculture is the adventitious shoot elongation subculture medium described in claim 1; The rooting culture medium used is the rooting medium described in claim 1.

4. The method according to claim 3, characterized in that, The first differentiation culture was conducted at a temperature of 25°C, a light intensity of 1800 lx, for 14 days, with a light duration of 16 hours. The second differentiation culture was conducted at a temperature of 25°C, a light intensity of 1800 lx, for 10 days, with a light duration of 16 hours.

5. The method according to claim 3, characterized in that, The adventitious shoot elongation subculture was conducted at a temperature of 25℃, a light intensity of 1800 lx, for 30 days, with a light duration of 16 h.

6. The method according to claim 3, characterized in that, The rooting culture was conducted at a temperature of 25°C, a light intensity of 1800 lx, and a light duration of 16 h.

7. The method according to claim 3, characterized in that, The induction culture was performed using the following culture medium: MS basal medium + 2,4-D 0.5 mg / L + kinetin 0.1 mg / L + sucrose 30.0 g / L.

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