A method for tissue culture and rapid propagation of prunus mume

Abstract

The application discloses a method for tissue culture and rapid propagation of Prunus mume var. insititia, and belongs to the technical field of plant tissue culture and rapid propagation. The method comprises the following steps: taking a stem segment with nodes of aseptic seedling of Prunus mume var. insititia, inoculating the stem segment into an axillary bud germination culture medium WPM+6-BA 1-4 mg / L to induce axillary bud or adventitious bud germination and proliferation; inoculating the adventitious bud into an adventitious root induction culture medium WPM+IBA 0.5-1 mg / L for adventitious root induction after subculture or elongation growth; and transplanting the rooted tissue culture seedling into a substrate. The method realizes rapid propagation of Prunus mume var. insititia through a tissue culture and rapid propagation system, is simple and efficient, and can be widely applied to the fields of Prunus mume var. insititia germplasm resource preservation and excellent strain cultivation.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture and rapid propagation, in particular to a method for rapid propagation of Prunus mume by tissue culture. Background Art

[0002] The genus Cinnamomum sempervirens is an evergreen tree of the Lauraceae family, with an attractive shape, straight trunk, and dense foliage. It is an excellent species for gardens, roadside trees, and roadside gardens. The genus Cinnamomum sempervirens is also a medicinal plant, rich in borneol (also known as natural borneol), the active ingredient of which is widely used in the formulation of various famous and high-quality Chinese patent medicines, such as Suhexiang Pills, Angong Niuhuang Jiedu Pills, Liushen Pills, Tankejing, and Shuangliaohoufengsan. Due to the extremely high medicinal value of the genus Cinnamomum sempervirens, market demand is increasing, while wild resources are decreasing. Therefore, vigorously developing the artificial cultivation of the genus Cinnamomum sempervirens to increase its production has great social benefits. Plant tissue culture propagation technology has the advantages of maintaining the excellent traits of the original variety, a high propagation coefficient, and a short propagation cycle. Plant tissue culture technology can rapidly propagate a large number of seedlings, shortening the propagation cycle, improving propagation efficiency, and enabling large-scale seedling production, which is conducive to the sustainable development of the plant industry and improving economic and social benefits. Therefore, the development of an efficient tissue culture rapid propagation method for the genus Cinnamomum sempervirens is highly desirable. Summary of the Invention

[0003] The invention provides a method for tissue culture and rapid propagation of Prunus mume. The method comprises the following steps: taking a noded stem segment of a sterile Prunus mume seedling, inoculating the segment into an axillary bud germination medium containing WPM and 1-4 mg / L of 6-BA, and inducing germination and proliferation of axillary buds or adventitious buds; performing subculture or elongation growth on the adventitious buds, and then inoculating the segment into an adventitious root induction medium containing WPM and 0.5-1 mg / L of IBA to induce adventitious roots; and transplanting the rooted tissue culture seedlings to a substrate.

[0004] Preferably, the axillary bud germination medium is WPM+6-BA2-4 mg / L.

[0005] Preferably, the axillary bud germination medium is WPM+6-BA2 mg / L.

[0006] Preferably, the adventitious root induction medium is WPM+IBA0.5 mg / L.

[0007] Preferably, the matrix has a volume ratio of vermiculite:sand=1:1, vermiculite:peat soil=1:1, peat soil:sand=1:1, perlite:peat soil=1:1 or vermiculite:perlite=1:1.

[0008] Preferably, the noded stem segment of the sterile seedling is 0.5-1.0 cm, containing 1-2 nodes and 1-2 leaves.

[0009] Preferably, the adventitious buds are cut into 1.0-1.2cm single buds containing more than 2 nodes and 2-4 leaf blades before inoculation in the adventitious root induction medium.

[0010] Preferably, the elongation growth medium is WPM+6-BA 1.0mg / L.

[0011] Preferably, the subculture is performed by cutting or stolonization.

[0012] Preferably, the method for preparing the sterile seedlings of Prunus serrulata is as follows: young branches of Prunus serrulata are taken, 0.5-1.0cm stem segments with nodes are cut, and after sterilization, the stem segments are inoculated in WPM medium added with 6-BA 1mg / L and NAA 0.1mg / L to induce axillary bud germination and obtain sterile seedlings.

[0013] Advantages of the present application:

[0014] The present application provides an efficient tissue culture and rapid propagation method for Prunus serrulata, which solves the problems in the prior art. The method comprises the following steps: sterilization of explants to obtain sterile seedlings; induction of axillary buds and adventitious buds; induction of adventitious roots; and transplanting of tissue culture seedlings. Through the axillary bud germination medium, the plant adventitious root induction formula and the transplanting substrate of the present application, Prunus serrulata can be rapidly and massively propagated through tissue culture and rapid propagation system. The method of the present application is simple and efficient, and can be widely applied in the fields of Prunus serrulata germplasm resource preservation and excellent strain cultivation BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the axillary bud germination status of Prunus serrulata stem segments under the treatment of different plant growth regulators, the scale is 1cm, and the unit of plant growth regulator is mg / L.

[0016] Figure 2 is the effect of different plant growth regulators on the induction of adventitious roots of Prunus serrulata tissue culture seedlings, CK is the group without addition of plant growth regulators. The scale is 1cm, and the unit of plant growth regulator is mg / L.

[0017] Figure 3 is the growth status of Prunus serrulata plants after transplanting in different substrate ratios for 30d, the scale is 5cm. DETAILED DESCRIPTION

[0018] The technical route of the present application is as follows:

[0019] (1) Sterilization of explants to obtain sterile seedlings

[0020] Select healthy young branch of Mei slice tree, cut into 0.5-1.0cm around with nodal stem segment, soak in 75% volume percent alcohol for 1min, wash with sterile water three times, then soak in 0.1% mass fraction mercuric chloride for 8-15min, wash with sterile water six times. The sterilized material is dried on sterile filter paper, inoculated in WPM medium added with 6-BA 1mg / L and NAA 0.1mg / L, the axillary bud is induced to germinate, and the aseptic seedling is obtained. The material in the medium is observed and recorded regularly, and subculture is carried out by cutting or branching as needed, and the subculture medium of aseptic seedling is WPM added with 6-BA 1mg / L and NAA 0.1mg / L.

[0021] The WPM medium contains sucrose 20g / L, agar powder 5g / L, and PH 5.8-6.0. The light cycle is 12 / 12h (light / dark), the light intensity is 1000-1500lx, and the temperature is 25±2℃. The same below.

[0022] (2) Axillary bud and adventitious bud induction

[0023] The aseptic seedling obtained in (1) is used as the material, which is cut into 0.5-1.0cm stem segment (containing 1-2 nodes and 1-2 leaves), inoculated in WPM medium containing different concentrations of plant growth regulators (6-BA, KT, ZT, TDZ, BR; 0.0-4.0mg / L), and the axillary bud or adventitious bud is induced to germinate and proliferate. A large number of tissue culture seedlings can be continuously obtained by subculture on the optimal axillary bud or adventitious bud germination and proliferation medium. The material in the medium is observed and recorded regularly, and subculture is carried out by cutting or branching as needed, and the bud subculture medium is the optimal axillary bud or adventitious bud germination and proliferation medium screened.

[0024] (3) Adventitious root induction

[0025] The adventitious bud obtained in (2) is used as the material, which is inoculated in WPM medium added with 6-BA 1.0mg / L for elongation growth, then the cluster bud is cut into 1.0cm single bud (containing more than 2 nodes and 2-4 leaves), inoculated in WPM medium containing different concentrations of plant growth regulators (NAA, IBA, IAA; 0.0-2.0mg / L), and the adventitious root is induced to occur, and the optimal adventitious root induction medium is selected.

[0026] (4) Acclimatization and transplanting

[0027] The rooted tissue culture seedling obtained in (3) is used as the material, which is transplanted in the substrate of vermiculite:sand=1:1, vermiculite:peat soil=1:1, peat soil:sand=1:1, perlite:peat soil=1:1, vermiculite:perlite=1:1, and the optimal transplanting substrate is selected for acclimatization and transplanting.

[0028] The present application takes Prunus serrulata as material, disinfects the explant, obtains sterile seedlings, and then uses the preferred optimal axillary bud or adventitious bud germination proliferation, adventitious root induction and transplanting substrate formula to realize the rapid propagation of Prunus serrulata.

[0029] The following examples are further illustrations of the application and are not intended to limit the same.

[0030] Example 1:

[0031] This example takes Prunus serrulata as an example to illustrate the specific application of the present application.

[0032] 1. Select healthy Prunus serrulata young branches, cut into 0.5-1.0 cm long stem segments, soak in 75% volume percentage alcohol for 1 min, wash with sterile water three times, then soak in 0.1% mass fraction mercury chloride for 15 min, wash with sterile water six times. Dry the disinfected material on sterile filter paper, inoculate in WPM medium added with 6-BA 1 mg / L and NAA 0.1 mg / L, induce axillary bud germination, and culture for 30 d to obtain sterile seedlings.

[0033] 2. Take the sterile seedlings obtained in step 1 as material, cut into 0.5-1.0 cm long stem segments (containing 1-2 nodes and 1-2 leaves), inoculate in WPM medium containing different plant growth regulators, and culture for 60 d for axillary bud induction. The optimal axillary bud germination medium is WPM+6-BA 2.0 mg / L, the axillary bud induction rate is 87.78±7.70%, and the average number of buds is 8.27±0.23 (Table 1 and Figure 1 );

[0034] 3. Take the adventitious buds obtained in step 2 in WPM+6-BA 2.0 mg / L as material, culture in WPM medium added with 6-BA 1.0 mg / L for 30 d for elongation growth, cut the cluster buds into 1.0 cm long single buds (containing more than 2 nodes and 2-4 leaves), inoculate in WPM medium containing different plant growth regulators, and culture for 30 d for adventitious root induction. The optimal adventitious root induction medium is WPM+IBA 0.5 mg / L, the adventitious root induction rate is 80.00±5.77%, the average number of roots is 3.13±0.23, and the average root length is 5.31±1.06 cm (Table 2 and Figure 2 );

[0035] 4. The rooted plantlets obtained in step 3 were transplanted into different substrates, and the survival rate was calculated after 30 days. The survival rate of the plantlets was close to 100% in the substrates of vermiculite: sand = 1:1, vermiculite: peat soil = 1:1, peat soil: sand = 1:1, perlite: peat soil = 1:1, and vermiculite: perlite = 1:1 (Table 3 and Figure 3 ).

[0036] The WPM medium in steps 1-4 contained sucrose 20 g / L, agar powder 5 g / L, and the pH was 5.8. The culture conditions were as follows: light cycle 12 / 12 h (light / dark), light intensity 1000-1500 lx, and temperature 25±2℃.

[0037] Table 1 Effects of different plant growth regulators on the germination of Prunus mume axillary buds

[0038]

[0039]

[0040] Note: Different letters represent significant differences between the treatment groups under the Duncan's new multiple range test, p < 0.05. The same below.

[0041] Table 2 Effects of different plant growth regulators on the adventitious root formation of Prunus mume plantlets

[0042]

[0043] Table 3 Effects of different substrates on the survival rate of Prunus mume plantlets after transplantation

[0044]

Claims

1. A method for tissue culture rapid propagation of Prunus domestica L. trees, characterized by, The method comprises the following steps: taking the stem section with nodes of the sterile seedling of Prunus serrulata, inoculating in the axillary bud germination medium WPM+6-BA 2-4 mg / L to induce the axillary bud or adventitious bud germination and proliferation; inoculating the adventitious bud after subculture or elongation growth in the adventitious root induction medium WPM+IBA 0.5-1 mg / L to induce the adventitious root; transplanting the rooted tissue culture seedling to the substrate, wherein the elongation growth medium is WPM+6-BA 1.0 mg / L, and the substrate is vermiculite:sand=1:1, vermiculite:peat soil=1:1, peat soil:sand=1:1, perlite:peat soil=1:1 or vermiculite:perlite=1:

1.

2. The method of claim 1, wherein, The axillary bud germination medium is WPM+6-BA 2 mg / L.

3. The method of claim 1, wherein, The adventitious root induction medium is WPM+IBA 0.5 mg / L.

4. The method of claim 1, wherein, The stem section with nodes of the sterile seedling is 0.5-1.0 cm long, contains 1-2 nodes and 1-2 leaf blades.

5. The method of claim 1, wherein, The adventitious bud is cut into a single bud of 1.0-1.2 cm long before inoculation in the adventitious root induction medium, and the single bud contains more than 2 nodes and 2-4 leaf blades.

6. The method of claim 1, wherein, The subculture is performed by cutting or branching.

7. The method of claim 1, wherein, The preparation method of the sterile seedling of Prunus serrulata is as follows: taking the young branch of Prunus serrulata, cutting the stem section with nodes of 0.5-1.0 cm long, inoculating in the WPM medium added with 6-BA 1 mg / L and NAA 0.1 mg / L after sterilization, inducing the axillary bud germination and obtaining the sterile seedling.

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