Ailanthus altissima somatic embryogenesis and plant regeneration method

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly discloses a somatic embryogenesis and plant regeneration method for ailanthus altissima. The method comprises the following steps: (1) taking a leaf axis, a small leaf or a compound leaf of an ailanthus altissima aseptic seedling as an explant, inoculating the explant on an embryogenic callus induction culture medium, and culturing to form an embryogenic callus; (2) inoculating the embryogenic callus obtained in the step (1) to a somatic embryo induction culture medium for culturing; and (3) selecting embryogenic calluses with good growth vigor and somatic embryos on the surfaces, and subculturing the embryogenic calluses in a somatic embryo germination culture medium to obtain somatic culture seedlings. According to the invention, a somatic embryo regeneration system of Ailanthus altissima is established for the first time, and the problem that the embryonic callus and somatic embryo of Ailanthus altissima are difficult to induce is effectively solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant tissue culture, and particularly relates to a somatic embryogenesis and plant regeneration method of Ailanthus altissima var. erythrocarpa. BACKGROUND

[0002] Ailanthus altissima var. erythrocarpa belongs to Simaroubaceae and Ailanthus, and is a deciduous tree. Its bark is smooth, and its crown is oval. Its small leaves are oval and have a special smell. The most distinctive feature of Ailanthus altissima var. erythrocarpa is that it has bright red cone inflorescences in summer, and the fruit period lasts for 3 months, which has excellent ornamental effect. It is a large ornamental tree with great potential, and has broad application prospects in landscape and urban greening.

[0003] The propagation of Ailanthus altissima var. erythrocarpa mainly adopts methods such as seedling, root cutting, grafting and tissue culture. However, seedling leads to separation of offspring traits due to natural hybridization, and cannot stably maintain the excellent characteristics of the mother plant; root cutting needs to damage the mother tree to collect cuttings, and has strict requirements on environmental conditions, with a survival rate of only 30%-50%, which is difficult to scale up; grafting can maintain the traits of the mother plant, but the compatibility of the stock and scion is unstable, the operation is complex, the labor cost is high, the survival rate is low and unstable (60%-85%). In comparison, tissue culture technology has more advantages in trait maintenance, propagation efficiency and material collection.

[0004] At present, the plant tissue culture technology of Ailanthus altissima var. erythrocarpa mainly focuses on the organogenesis pathway, such as inducing callus and producing adventitious buds from axillary buds, terminal buds and leaves, or constructing a regeneration system through young stem segments. However, this pathway has obvious defects: first, single bud propagation through axillary buds and terminal buds has low propagation coefficient, which is difficult to meet the demand of large-scale production; second, the process of inducing callus and adventitious buds from leaves is complicated, and the callus induction rate is unstable and easily affected by factors such as physiological state of explants and medium composition. In addition, variations may occur during the differentiation of adventitious buds, affecting the quality of offspring plants. When young stem segments are used to establish a regeneration system, the collection of explants is limited by seasons, and the mother tree is damaged. At the same time, stem segment culture is easily contaminated by microorganisms, and strict control of environmental conditions is required during the culture process, which increases the operation difficulty and cost.

[0005] Another way of tissue culture technology is somatic embryo regeneration way, however, due to the complex somatic embryogenesis mechanism, it needs to accurately regulate the ratio and concentration of multiple hormones and suitable culture environment, and the large amount of picraline, alkaloids and terpenoids contained in A. paniculata itself can inhibit the cell division and dedifferentiation (the first step of forming callus) of in vitro tissues, leading to the browning and death of callus, and even if callus is formed, it is mostly "non-embryogenic callus" (loose structure, no embryogenic potential), which is difficult to further differentiate into somatic embryo, further increasing the difficulty of somatic embryo induction. SUMMARY

[0006] In view of the technical problems existing in the current A. paniculata propagation method, the purpose of the present application is to provide a high-efficiency and stable A. paniculata somatic embryogenesis and plant regeneration method to meet the demand for high-quality and large-scale seedling raising.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme: The somatic embryogenesis and plant regeneration method of A. paniculata comprises the following steps: (1) Taking the leaf axis, small leaf or compound leaf of A. paniculata sterile seedling as explant, inoculating it on embryogenic callus induction medium to form embryogenic callus; (2) Selecting fresh and lustrous callus from the embryogenic callus obtained in step (1) as somatic embryo induction material, removing the browned embryogenic callus, and inoculating it on somatic embryo induction medium for culture; (3) Selecting embryogenic callus with good growth and surface somatic embryo, and subculturing it on somatic embryo germination medium for culture to obtain plantlets.

[0008] Further, the A. paniculata sterile seedling in step (1) is obtained by tissue culture with A. paniculata stem segments as explants.

[0009] Further, the embryogenic callus induction medium in step (1) has the formula: MS+1.0 mg·L -1 2,4-D +0.8~2.5 mg·L -1 6-BA+0.1~0.3 mg·L -1 KT+0.01~0.05 mg·L -1 TDZ, pH value 6.5~6.8; preferably MS+1.0 mg·L -1 2,4-D +0.8 mg·L -1 6-BA+0.25 mg·L -1 KT+0.05 mg·L -1TDZ, pH 6.5-6.8.

[0010] Further, the somatic embryo induction medium formula in step (2) is MS+0.1 mg·L -1 IBA+0.8~2.0mg·L -1 6-BA+0~0.3 mg·L -1 KT, pH 6.5-6.8; preferably MS+0.1 mg·L -1 IBA+1.0~1.5mg·L -1 6-BA+0~0.3 mg·L -1 KT, pH 6.5-6.8; more preferably MS+0.1 mg·L -1 IBA+1.5mg·L -1 6-BA+0~0.15mg·L -1 KT, pH 6.5-6.8; more preferably MS+0.1 mg·L -1 IBA+1.5mg·L -1 6-BA+0.05 mg·L -1 KT, pH 6.5-6.8.

[0011] Further, the somatic embryo induction medium formula in step (2) is MS+0.1 mg·L -1 6-BA+0.2 mg·L -1 IBA, pH 6.5-6.8.

[0012] Further, the step (1) uses small leaves or compound leaves of sterile seedlings of A. paniculata as explants.

[0013] Further, the culture condition of the embryogenic callus induction medium in step (1) is 25±2℃ dark culture for 10d, and then placed under 16h light / 8h dark conditions for culture for 20d.

[0014] Further, the culture condition of each medium in steps (2)-(3) is 16h light / 8h dark for culture for 20d.

[0015] Further, when the petiole is inoculated as the explant in step (1), the petiole is cut into small segments of 1-2cm in length; when the small leaf or compound leaf is inoculated as the explant, the petiole of the small leaf or compound leaf is placed downward and the leaf blade is placed upward in the culture dish, and according to the size of the small leaf or compound leaf, 3-4 wounds are made along the edge of the leaf blade perpendicular to the main vein by using a scalpel blade.

[0016] The present application has the following beneficial effects: The application takes the small leaf, leaf axis or compound leaf of the sterile seedling of Ailanthus altissima 'Rubra' as an explant, and first establishes a somatic embryo regeneration system of Ailanthus altissima 'Rubra', effectively solving the problem of difficult induction of embryogenic callus and somatic embryos of Ailanthus altissima 'Rubra'.

[0017] The application provides an important way for the preservation of Ailanthus altissima 'Rubra' germplasm resources and the rapid propagation of excellent varieties, and lays a foundation for genetic transformation and biotechnological breeding of Ailanthus altissima 'Rubra'. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Fig. a is a tissue culture seedling of Ailanthus altissima 'Rubra', and Figs. b, c and d are different explant materials, wherein Fig. b is a leaf as an explant material, Fig. c is a leaf axis as an explant material, and Fig. d is a compound leaf as an explant material.

[0019] Figure 2 Figs. a and b are callus states of different explants, wherein Fig. a is an initial callus state, and Fig. b is a callus state.

[0020] Figure 3 Fig. a is a somatic embryo induction state, wherein Fig. a is a spherical embryo, Fig. b is a heart-shaped embryo, Fig. c is a torpedo embryo, and Fig. d is a cotyledon embryo. DETAILED DESCRIPTION

[0021] The application will be further described below in combination with specific examples.

[0022] The MS medium described in the application comprises a macro nutrient element, a micro nutrient element, an iron salt and an organic substance.

[0023] The components of the macro nutrient element and the corresponding concentrations thereof are as follows: potassium nitrate 1900 mg / L, ammonium nitrate 1650 mg / L, magnesium sulfate 180.7 mg / L, potassium dihydrogen phosphate 170 mg / L, and calcium chloride 332.2 mg / L. The components of the micro nutrient element and the corresponding concentrations thereof are as follows: manganese sulfate monohydrate 16.9 mg / L, zinc sulfate 8.6 mg / L, boric acid 6.2 mg / L, potassium iodide 0.83 mg / L, sodium molybdate dihydrate 0.25 mg / L, copper sulfate pentahydrate 0.025 mg / L, and cobalt chloride hexahydrate 0.025 mg / L; and zinc sulfate heptahydrate 8.6 mg / L. The component of the iron salt and the corresponding concentration thereof are as follows: EDTA iron 36.7 mg / L. The components of the organic substance and the corresponding concentrations thereof are as follows: glycine 2 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, vitamin B6 0.5 mg / L, and vitamin B1 0.1 mg / L. EMBODIMENT

[0024] 1. Obtaining of aseptic seedlings This example takes the healthy and pest-free 2-year-old Ailanthus altissima with buds stem segments as explants for tissue culture to obtain aseptic seedlings after hydroponics in the room.

[0025] 2. Treatment of explants The aseptic seedlings of Ailanthus altissima obtained by germination of the tender stem segments in step 1 are used as initial materials for embryogenic callus induction. Figure 1 a) The leaf axis, small leaves and compound leaves of the aseptic seedlings of Ailanthus altissima are taken as explants, and the leaf axis is cut into small segments of 1 cm long on the sterile clean bench. According to the size of the small leaves and compound leaves, 3-4 wounds are cut along the edge of the leaf blade perpendicular to the main vein, and the main vein is not cut off. The leaf blade is placed flat on the medium with the front face upward. Figure 1 b-c).

[0026] 3. Embryogenic callus induction culture and proliferation culture The treated Ailanthus altissima explants are inoculated on different formula treated embryogenic callus induction medium, 10 dishes for each treatment, 3 explants per dish, 3 replicates, culture conditions are 25±2℃ dark culture for 10d, then placed in 16h light / 8h dark conditions, after 20d culture, the callus induction rate and the state of callus of the explants are counted, and part of the treatment and the corresponding statistical results are shown in Table 1.

[0027] Callus induction rate (%) = number of callus induction explants / total number of inoculated explants × 100.

[0028] Except for the cases of browning necrosis or explant death, after 10d dark culture and 20d 16h light / 8h dark induction, the compound leaves and small leaves start to callus 5d after inoculation, thicken and curl; 10d after inoculation, they present an enlarged state; 20d after inoculation, callus tissue has been induced. Figure 2 ).

[0029] Table 1

[0030] 4. Somatic embryo induction culture Fresh and glossy callus tissue induced from the three types of explants is selected as somatic embryo induction material, and the browned callus tissue is removed and inoculated on different treated somatic embryo induction medium, cultured at 25±2℃, 16h light / 8h dark conditions, and the somatic embryo induction rate is counted after 20d, and part of the treatment and the corresponding statistical results are shown in Table 2.

[0031] Embryo induction rate (%) = number of calli with somatic embryogenesis / number of inoculated calli x 100.

[0032] By Figure 3 It can be seen that the surface of embryogenic calli is loose and granular, and globular embryos can be observed on the surface after about 20 days, and with the extension of time, heart-shaped embryos, torpedo embryos and cotyledon embryos gradually appear, and somatic embryos will continue to callus during the culture process.

[0033] Table 2

[0034] 5. Plant regeneration in somatic embryo culture The embryogenic calli with good growth and somatic embryos on the surface were selected and subcultured in somatic embryo germination medium to obtain somatic embryo seedlings, and the somatic embryo seedlings were placed in 16h light / 8h dark conditions, and after about 20 days of culture, somatic embryo seedlings appeared, the seedling rate was counted, and the average value was taken.

[0035] Seedling rate (%) = number of plants with stem and leaf development / number of inoculated calli with somatic embryogenesis x 100%.

[0036] The somatic embryo seedling differentiation medium was MS+0.6 mg·L -1 6-BA+0.2 mg·L -1 IBA, pH 6.5~6.8, and the seedling rate was more than 98%.

[0037] Although the specific embodiments of the present application are described above in combination with the drawings, it is not a limitation on the protection scope of the present application, and those skilled in the art should understand that various modifications or changes made by those skilled in the art on the basis of the technical solutions of the present application without creative labor are still within the protection scope of the present application.

Claims

1. A method for somatic embryogenesis and plant regeneration of Ailanthus altissima var. simensis, characterized by, The method comprises the following steps: (1) taking leaf axis, small leaf or compound leaf of the sterile seedling of Ailanthus altissima as explants, inoculating the explants on embryogenic callus induction medium to form embryogenic callus; (2) selecting fresh and glossy callus from the embryogenic callus obtained in step (1) as somatic embryo induction material, removing the browned embryogenic callus, and inoculating the somatic embryo induction material on somatic embryo induction medium to culture; (3) selecting the embryogenic callus with good growth and surface somatic embryo, and subculturing the embryogenic callus on somatic embryo germination medium to obtain plantlets.

2. The somatic embryogenesis and plant regeneration method of A. rubra according to claim 1, characterized in that, The embryo callus induction medium formula of step (1) is: MS+1.0 mg·L -1 2,4-D +0.8~2.5 mg·L -1 6-BA+0.1~0.3 mg·L -1 KT+0.01~0.05 mg·L -1 TDZ, pH value 6.5~6.

8.

3. The somatic embryogenesis and plant regeneration method of A. rubra according to claim 2, characterized in that, The embryo callus induction medium formula in step (1) is: MS+1.0 mg·L -1 2,4-D +0.8 mg·L -1 6-BA+0.25 mg·L -1 KT+0.05 mg·L -1 TDZ, pH value 6.5~6.

8.

4. The somatic embryogenesis and plant regeneration method of A. rubra according to claim 1, characterized in that, The somatic embryo induction medium formula of step (2) is MS+0.1 mg·L -1 IBA+0.8~2.0mg·L -1 6-BA+0~0.3mg·L -1 KT, pH value 6.5~6.

8.

5. The somatic embryogenesis and plant regeneration method of A. rubra according to claim 4, characterized in that, The somatic embryo induction medium formula of step (2) is MS+0.1 mg·L -1 IBA+1.0~1.5 mg·L -1 6-BA+0~0.3 mg·L -1 KT, pH value 6.5~6.

8.

6. The somatic embryogenesis and plant regeneration method of A. rubra according to claim 1, characterized in that, The body embryo germination medium formula of step (3) is: MS+0.6 mg·L -1 6-BA+0.2 mg·L -1 IBA, pH value 6.5~6.

8.

7. The somatic embryogenesis and plant regeneration method of A. rubra according to claim 1, wherein, In step (1), the small leaf or compound leaf of the sterile seedling of Ailanthus altissima is taken as the explants.

8. The somatic embryogenesis and plant regeneration method of Ailanthus altissima var. sinensis according to claim 1, characterized in that, In step (1), the culture condition of the embryogenic callus induction medium is 25±2 DEG C dark culture for 10 days, and then 16h light / 8h dark culture for 20 days.

9. The somatic embryogenesis and plant regeneration method of Ailanthus altissima var. sinensis according to claim 1, characterized in that, In steps (2) and (3), the culture condition of each medium is 16h light / 8h dark culture for 20 days.

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