A method for rapid propagation of camphor

Abstract

The present invention relates to the technical field of camphor tree propagation, and in particular to a method for rapid propagation of camphor tree. The present invention uses the stem segments of sterile seedlings as explants, and directly induces rooting by inoculating them into a suitable rooting medium without callus induction and proliferation, thereby greatly shortening the cultivation period. After rooting induction, the rooting rate can reach 90%; then, the seedlings are cultured in a suitable seedling culture medium. After the seedling culture, the leaves of the tissue culture seedlings are larger and more buds are sprouted, and they can be directly hardened and transplanted. The method provided by the present invention can realize seedling cultivation throughout the year. Furthermore, the sterile seedlings after seedling culture can be cut into micro-stem segments for further breeding, which become the source of materials for in vitro rapid breeding, greatly reducing the amount of seeds used for breeding under natural conditions, and can cyclically and rapidly cultivate a large number of high-quality tissue culture seedlings.

Description

Technical Field

[0001] The invention relates to the technical field of camphor tree propagation, in particular to a method for rapid propagation of camphor tree. Background Art

[0002] Cinnamomum longipaniculatum (Gamble) N. Chao ex HWLi is an evergreen tree of the Lauraceae family, Cinnamomum genus. Its trunk and branches contain aromatic oil. The main components of the oil are linalool, linalool and camphor, and the oil content is higher than that of other Lauraceae species. It has the characteristics of fast growth, strong sprouting, many leaves, beautiful tree shape, majestic posture, flexible wood and dense texture. It has a lifespan of up to a thousand years and is also the preferred tree species for large-scale afforestation and greening of surrounding areas.

[0003] At present, the main methods of propagation of camphor tree seedlings are sowing and cuttings. Seed propagation has high requirements on seed collection period and handling methods. Improper handling can easily cause problems such as seed mildew, water loss and oiling, resulting in a lower germination rate. Cutting propagation requires inducing the healing and rooting of the incision. The comprehensive effect of cuttings is easily affected by the cutting time, the age of the mother tree, and the cultivation conditions, resulting in an unstable cutting survival rate.

[0004] Overall, the traditional breeding technology of Cinnamomum camphora has major problems such as low breeding efficiency, poor stability, and seasonal restrictions, making it impossible to produce high-quality seedlings on a large scale and in a stable manner. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for rapid propagation of camphor. The method provided by the present invention overcomes the problems of serious pollution, easy browning and difficulty in inducing rooting of stem segments collected in the wild. At the same time, the direct rooting method is adopted, the callus induction and proliferation steps are eliminated, the cultivation cycle is greatly shortened, and a large number of high-quality camphor seedlings can be cultivated cyclically and quickly, providing technical support for the breeding of camphor seedlings.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for rapid propagation of camphor trees, comprising the following steps:

[0008] The explants are inoculated into a rooting medium for rooting culture to obtain primary tissue culture seedlings; the explants include stem segments of sterile camphor seedlings; the rooting medium is based on WPM medium and also includes 25-35 g / L sugar, 3-4 g / L agar, 0.1-0.5 mg / L indolebutyric acid and 0.5-2.0 mg / L naphthaleneacetic acid;

[0009] The primary tissue culture seedlings are inoculated into a seedling culture medium for seedling culture to obtain tissue culture seedlings; the seedling culture medium uses 2WPM culture medium as a basic culture medium and also includes 25-35 g / L sugar, 3-4 g / L agar and 0.1-0.5 mg / L indolebutyric acid.

[0010] Preferably, the pH value of the rooting medium is 5.6-6.0; the pH value of the seedling growth medium is 5.6-6.0.

[0011] Preferably, the stem segments include stem segments with nodes.

[0012] Preferably, the length of the stem segment is 4 to 6 mm.

[0013] Preferably, the rooting culture temperature is 24° C., the light intensity is 2000-3000 Lux, the lighting time is 16 h / d, and the culture time is 14-21 d.

[0014] Preferably, the temperature for culturing the strong seedlings is 24°C, the light intensity is 2000-3000 Lux, the lighting time is 16 h / d, and the culturing time is 21-28 d.

[0015] Preferably, the method for culturing the sterile camphor tree seedlings comprises: inoculating camphor tree embryos into MS solid culture medium, and culturing aseptically to obtain the sterile camphor tree seedlings; the MS solid culture medium further contains 25-35 g / L of sugar.

[0016] Preferably, the conditions for the aseptic culture include: transferring to light culture after 24 hours of dark culture; the light intensity of the light culture is 2000-3000 Lux, the light time is 16 h / d, and the light culture time is 15 days.

[0017] Preferably, the seedling cultivation further includes seedling hardening and transplanting in sequence to obtain Cinnamomum camphora seedlings; the light intensity of the seedling hardening is 5000-10000 Lux, the ambient temperature is 20-32° C., and the time is 7-14 days.

[0018] Preferably, the stem segments of the sterile Cinnamomum elata seedlings are replaced with the stem segments of the tissue culture seedlings.

[0019] Beneficial effects:

[0020] The invention provides a method for rapid propagation of camphor, comprising the following steps: inoculating an explant into a rooting medium, performing rooting culture, and obtaining a primary tissue culture seedling; the explant comprises a stem segment of a sterile camphor seedling; the rooting medium uses a WPM medium as a basic medium and further comprises 25-35 g / L of sugar, 3-4 g / L of agar, 0.1-0.5 mg / L of indoleacetic acid (IBA) and 0.5-2.0 mg / L of naphthaleneacetic acid (NAA); inoculating the primary tissue culture seedling into a seedling-strengthening medium for seedling-strengthening culture, and obtaining a tissue culture seedling; the seedling-strengthening medium uses a 2WPM medium as a basic medium and further comprises 25-35 g / L of sugar, 3-4 g / L of agar and 0.1-0.5 mg / L of indolebutyric acid. The present invention uses the stem segments of sterile seedlings as explants, and directly induces rooting by inoculating them into a suitable rooting medium, without callus induction and proliferation, greatly shortening the cultivation period. After rooting induction, the rooting rate can reach 90%; and then the seedlings are cultured in a suitable seedling culture medium. After the seedling culture, the tissue culture seedlings have larger leaves and more buds, and can be directly hardened and transplanted. The method provided by the present invention can achieve seedling cultivation throughout the year.

[0021] Furthermore, after the sterile seedlings have been cultured for strong seedlings, microstem segments can be cut for further breeding, becoming a source of material for in vitro rapid propagation, greatly reducing the amount of seeds used for breeding under natural conditions, and allowing for the recycling and rapid cultivation of a large number of high-quality tissue culture seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 This is a schematic diagram of the various stages of rapid reproduction of Cinnamomum camphora. DETAILED DESCRIPTION

[0024] The present invention provides a method for rapid propagation of camphor trees, comprising the following steps:

[0025] The explants are inoculated into a rooting medium for rooting culture to obtain primary tissue culture seedlings; the explants include stem segments of sterile Cinnamomum camphora seedlings; the rooting medium is based on WPM medium and also includes 25-35 g / L sugar, 3-4 g / L agar, 0.1-0.5 mg / L IBA and 0.5-2.0 mg / L NAA;

[0026] The primary tissue culture seedlings are inoculated into a seedling culture medium for seedling culture to obtain tissue culture seedlings; the seedling culture medium uses 2WPM culture medium as a basic culture medium and further includes 25-35 g / L sugar, 3-4 g / L agar and 0.1-0.5 mg / L IBA.

[0027] The invention inoculates the explant into a rooting culture medium for rooting culture to obtain a primary tissue culture seedling; the explant comprises a stem segment of a sterile seedling of Cinnamomum oleifera.

[0028] In the present invention, the method for culturing the aseptic seedlings of camphor trees preferably includes: inoculating camphor tree embryos into MS solid culture medium, aseptically culturing, and obtaining the aseptic seedlings of camphor trees. The method for preparing camphor tree embryos preferably includes: selecting camphor tree fruits that mature in the current year, removing the pulp and disinfecting, peeling off the seed shell, and obtaining the camphor tree embryos; the MS solid culture medium preferably also contains 25 to 35 g / L of sugar, more preferably 30 g / L; the pH value of the MS solid culture medium is preferably 5.6 to 6.0, more preferably 5.8; the conditions for aseptic culture preferably include: transferring to light culture after 24 hours of dark culture; the light intensity of the light culture is preferably 2000 to 3000 Lux, more preferably 2500 Lux, the light time is preferably 16 h / d, and the light culture time is preferably 15 d.

[0029] In the present invention, the stem segment preferably includes a stem segment with nodes; the length of the stem segment is preferably 4 to 6 mm, more preferably 5 mm.

[0030] The invention adopts seed embryos for aseptic culture to obtain aseptic seedlings, which grow neatly and well with a germination rate of 90%, and can obtain high-quality micro stem segment (4-6 mm) explants, thus solving the problems of easy browning, high contamination rate and difficulty in inducing rooting of mature stem segments collected in the wild.

[0031] In the present invention, the rooting medium uses WPM medium as the basic medium and also includes 25-35 g / L of sugar, 3-4 g / L of agar, 0.1-0.5 mg / L of IBA and 0.5-2.0 mg / L of NAA; the concentration of sugar in the rooting medium is preferably 30 g / L, and the sugar preferably includes sucrose; the concentration of agar in the rooting medium is preferably 3.5 g / L; the concentration of IBA in the rooting medium is preferably 0.2-0.4 mg / L, more preferably 0.3 mg / L; the concentration of NAA in the rooting medium is preferably 1-1.5 mg / L, more preferably 1 mg / L; the pH value of the rooting medium is preferably 5.6-6.0, more preferably 5.8.

[0032] In the present invention, the temperature of the rooting culture is preferably 24°C; the light intensity of the rooting culture is preferably 2000-3000 Lux, more preferably 2500 Lux; the illumination time of the rooting culture is preferably 16 h / d; the time of the rooting culture is preferably 14-21 d, further preferably 15-20 d, more preferably 17-18 d.

[0033] The invention directly induces rooting by inoculating the explant into a suitable rooting medium without callus induction and proliferation, thus greatly shortening the cultivation period. After the rooting induction, the rooting rate can reach 90%.

[0034] After obtaining the primary tissue culture seedlings, the present invention inoculates the tissue culture seedlings into a seedling-strengthening culture medium, and performs seedling-strengthening culture to obtain tissue culture seedlings.

[0035] The seedling-strengthening culture medium of the present invention uses 2WPM culture medium as the basic culture medium, and further includes 25-35 g / L of sugar, 3-4 g / L of agar and 0.1-0.5 mg / L of IBA; the concentration of IBA in the seedling-strengthening culture medium is preferably 0.1-0.3 mg / L, more preferably 0.1 mg / L; the concentration of sugar in the seedling-strengthening culture medium is preferably 30 g / L, and the sugar preferably includes sucrose; the concentration of agar in the seedling-strengthening culture medium is preferably 3.5 g / L; the pH value of the seedling-strengthening culture medium is preferably 5.6-6.0, more preferably 5.8.

[0036] In the present invention, the temperature for culturing the strong seedlings is preferably 24°C; the light intensity for culturing the strong seedlings is preferably 2000-3000Lux, more preferably 2500Lux; the light time for culturing the strong seedlings is preferably 16h / d; the time for culturing the strong seedlings is preferably 21-28d, further preferably 22-27d, more preferably 24-25d.

[0037] After being cultured in a suitable seedling culture medium, the primary tissue culture seedlings have larger leaves and more sprouts, and can be directly hardened and transplanted.

[0038] After the strong seedlings are cultured, the present invention preferably sequentially hardens and transplants the tissue culture seedlings after the strong seedlings to obtain Cinnamomum camphora seedlings.

[0039] The present invention can also use the tissue culture seedlings obtained by strong seedling culture as the sterile seedlings described in the above technical solution, preferably cut off part of the stem segment as an explant, and continue to induce rooting culture and strong seedling culture according to the method described in the above technical solution. The tissue culture seedlings after strong seedling culture of the present invention can become the source of materials for in vitro rapid propagation, so that production can be circulated throughout the year.

[0040] In the present invention, the seedling height of the tissue culture seedlings after seedling cultivation is ≥3.5cm, and the seedling hardening is preferably carried out when the root length is 2-3cm; the light intensity of the seedling hardening is preferably 5000-10000Lux, more preferably 6000-9000Lux, and more preferably 7500Lux; the environmental temperature of the seedling hardening is preferably 20-32°C, more preferably 23-30°C, and more preferably 25-28°C; the seedling hardening time is preferably 7-14d, and more preferably, the seedling hardening is stopped when the seedling height of the tissue culture seedlings is 4.5-6.5cm.

[0041] After the hardening is completed, the present invention transplants the hardened tissue culture seedlings; the transplanting method preferably comprises: transplanting the hardened tissue culture seedlings into a cultivation substrate, shading and culturing for 7 to 10 days, and then removing the shading facility cultivation; the cultivation substrate preferably comprises the following components by volume: 3 parts of peat soil and 7 parts of perlite; the environmental humidity at the initial stage of transplanting is preferably 80% to 95%; the shading cultivation time is preferably 8 to 9 days; the shading facility cultivation time is preferably 20 to 30 days, further preferably 22 to 28 days, and more preferably 25 days; when the shading facility cultivation is removed, it is preferred to apply a compound fertilizer once; the mass ratio of nitrogen, phosphorus and potassium in the compound fertilizer is preferably 15:15:15, and the total nutrients are preferably ≥45%; the compound fertilizer application method is preferably spraying a 1% compound fertilizer solution.

[0042] When the height of the tissue culture seedlings after transplanting is preferably ≥40 cm, the camphor tree seedlings are obtained; the obtained camphor tree seedlings can be planted in the field.

[0043] In order to further illustrate the present invention, a method for rapid propagation of Cinnamomum camphora provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] A method for rapid propagation of camphor trees, comprising the following steps:

[0046] Select the mature camphor fruits of the year, remove the pulp and rinse them with detergent once, then rinse with clean water for 2 hours, disinfect them with 75% alcohol for 30 seconds on a clean bench, then disinfect them with 8 times sodium hypochlorite solution for 8 minutes, and rinse them with sterile water for 5 times; then use sterile absorbent paper to dry the surface moisture, peel off the seed shell and inoculate them into MS culture medium, which only contains 30g / L sucrose and 3.5g / L agar, and the pH value of the MS culture medium is 5.8; first culture in the dark for 24 hours, and then culture for 15 days under the environment of light intensity of 1500Lux, temperature of 24℃, and light time of 16h / d, to obtain sterile seedlings ( Figure 1 A in

[0047] Cut the node stem segment of the sterile seedling, the length of the node stem segment is 5mm; inoculate the cut node stem segment into the rooting medium ( Figure 1 B), cultured for 14 days at a temperature of 24°C, a light intensity of 2500 Lux, and a light duration of 16 h / d, to obtain primary tissue culture seedlings ( Figure 1 C); the rooting medium is based on WPM medium, and also contains sucrose 30g / L, agar 3.5g / L, IBA 0.3mg / L and NAA1mg / L;

[0048] The tissue culture seedlings were inoculated into a seedling medium and cultured for 21 days at a temperature of 24° C., a light intensity of 2500 Lux, and a light duration of 16 h / d. Figure 1 D); the seedling medium is based on 2WPM medium, and also contains 30g / L sucrose, 3.5g / L agar and 0.1mg / L IBA;

[0049] When the height of the tissue culture seedlings after strong seedling cultivation reaches more than 3.5 cm and the root length is 2.5 cm, the tissue culture seedlings after strong seedling cultivation are moved from the culture room to the seedling hardening greenhouse for seedling hardening, covered with a sunshade net, and the light intensity is controlled at 7000 Lux and the ambient temperature is 25°C to avoid direct sunlight and high temperature burns to the tissue culture seedlings;

[0050] When the rooted seedlings obtained by hardening grow to 5cm, they can be transplanted into nutrition bags (brand: Huinong nutrition pot, specifications: diameter 12cm, height 10cm). In the early stage of transplanting, the environmental humidity is maintained at 80% to 95%, and the sunshade net is used to cover and cultivate for 8 days. After that, the sunshade net is untied and cultivated for 25 days, and 1wt.% compound fertilizer solution (brand: Stanley, total nutrients of compound fertilizer ≥45%, N-P2O5-K2O ratio is 15-15-15) is sprayed. When the seedling height reaches 40cm, it can be planted in the field.

[0051] Example 2

[0052] A method similar to Example 1, the only difference being that the concentration of IBA in the seedling-strengthening medium is 0.3 mg / L.

[0053] Comparative Example 1

[0054] A method similar to Example 1, the only difference being that the concentration of IBA in the rooting medium is 0.1 mg / L.

[0055] Comparative Example 2

[0056] A method similar to Example 1, except that the basic culture medium of the rooting culture medium is 2WPM culture medium, and the concentration of IBA in the rooting culture medium is 0.1 mg / L.

[0057] Comparative Example 3

[0058] A method similar to Example 1, the only difference being that the basic culture medium of the rooting culture medium is 2WPM culture medium.

[0059] Comparative Example 4

[0060] A method similar to Example 1, except that the basic culture medium of the seedling-strengthening culture medium is WPM culture medium.

[0061] Comparative Example 5

[0062] A method similar to Example 1, except that the basic culture medium of the seedling-strengthening culture medium is WPM culture medium, and the concentration of IBA in the seedling-strengthening culture medium is 0.3 mg / L.

[0063] Comparative application example 1

[0064] The effects of different rooting culture media on root induction and different seedling cultivation culture media on seedling cultivation in Example 1, Example 2 and Comparative Examples 1 to 5 were determined as follows: 10 experimental materials were treated in each case and repeated 3 groups; the root number, root length and rooting rate of the rooting culture media were observed 21 days after inoculation of the rooting culture media, and the seedling height, leaf number and leaf length of the seedling cultivation media were observed 28 days after inoculation of the seedling cultivation culture media. The determination indicators and results are shown in Tables 1 and 2.

[0065] Table 1 Effects of different rooting media on root induction

[0066]

[0067]

[0068] Table 2 Effects of different seedling culture media on seedling cultivation

[0069] Group deal with Basic medium IBA(mg / L) Seedling height (cm) Number of leaves Leaf length(cm) Comparative Example 4 S1 WPM 0.1 1.2 3.4 1.2 Comparative Example 5 S2 WPM 0.3 2.3 3.1 1.5 Example 1 S3 2WPM 0.1 3.7 5.2 2.6 Example 2 S4 2WPM 0.3 3.2 4.8 2.1

[0070] As shown in Table 1, under the WPM basic culture medium, when the IBA concentration is 0.3 mg / L and the NAA concentration is 1 mg / L, the rooting rate can reach up to 90%, the average number of roots can reach 2.1 roots, and the average root length is 4.99 cm.

[0071] As shown in Table 2, under the 2WPM basic culture medium, when the IBA concentration is 0.1-0.3 mg / L, the seedling strengthening effect is best, the average seedling height reaches 3.2-3.7 cm, the average number of leaves can reach 4.8-5.2, and the average leaf length is 2.1-2.6 cm.

[0072] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for rapid propagation of camphor trees, characterized in that: The following steps are involved: Inoculating the explant into a rooting medium, performing rooting culture, and obtaining primary tissue culture seedlings; the explant comprises a stem segment with nodes of a sterile seedling of Cinnamomum oleifera; the rooting medium is based on WPM medium and further contains 25-35 g / L sugar, 3-4 g / L agar, 0.3 mg / L indolebutyric acid, and 1 mg / L naphthaleneacetic acid; The primary tissue culture seedlings are inoculated into a seedling medium for seedling culture to obtain tissue culture seedlings; the seedling medium is based on 2WPM medium and further contains 25-35 g / L sugar, 3-4 g / L agar and 0.1 mg / L indolebutyric acid; The method for culturing aseptic seedlings of Cinnamomum camphora comprises: inoculating Cinnamomum camphora embryos into MS solid culture medium, and aseptically culturing to obtain the aseptic seedlings of Cinnamomum camphora; the MS solid culture medium further contains 25-35 g / L of sugar.

2. The method according to claim 1, characterized in that The pH value of the rooting medium is 5.6-6.0; the pH value of the seedling-strengthening medium is 5.6-6.

0.

3. The method according to claim 1, characterized in that The length of the stem segment is 4-6 mm.

4. The method according to claim 1, characterized in that The rooting culture temperature is 24° C., the light intensity is 2000-3000 Lux, the light time is 16 h / d, and the culture time is 14-21 d.

5. The method according to claim 1, characterized in that The temperature for culturing the strong seedlings is 24° C., the light intensity is 2000-3000 Lux, the light time is 16 h / d, and the culturing time is 21-28 d.

6. The method according to claim 1, characterized in that The sterile culture conditions include: dark culture for 24 hours followed by light culture; the light intensity of the light culture is 2000-3000 Lux, the light time is 16 hours per day, and the light culture time is 15 days.

7. The method according to claim 1, characterized in that The method further comprises seedling hardening and transplanting in sequence after the seedling cultivation to obtain camphor tree seedlings; the light intensity of the seedling hardening is 5000-10000 Lux, the ambient temperature is 20-32° C., and the time is 7-14 days.

8. The method according to claim 1, characterized in that The tissue culture seedlings obtained from the strong seedling culture were used as sterile seedlings, and some stem segments were cut off as explants to continue the root induction culture and strong seedling culture.

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