A method for tissue culture seedlings of superior Schima superba family

Abstract

The invention belongs to the technical field of Schima superba tissue culture seedlings, and is specifically related to a tissue culture seedling method of a Schima superba excellent family. A combination of primary culture, subculture, root culture, domestication and seedling hardening methods is adopted, wherein two culture media are used for alternating culture in subculture, one for promoting budding, and the other for making the seedlings more robust and the leaves more stretched, and the alternating culture makes the Schima superba seedlings maintain the high quality of the subculture seedlings while maintaining a high value-added coefficient, which is conducive to subsequent root culture, and camphor essential oil / hydrosol or Melaleuca alternifolia essential oil / hydrosol is added to the culture medium, which is conducive to antibacterial disinfection, prevents the culture medium from getting moldy and the explant from browning, and in the domestication and hardening of the Schima superba tissue culture seedlings, firstly, the roots are stained with yellow heart soil slurry, and the root system will be more capable of retaining water after being wrapped with a layer of mud, and the yellow heart soil and perlite in the cultivation medium ensure the water retention and air permeability of the matrix, so that the transplant survival rate is greatly improved, and finally high-quality tissue culture seedlings are obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of Schima superba tissue culture seedlings, and particularly relates to a method for tissue culture seedlings of a superior Schima superba family. Background Art

[0002] Generally speaking, the offspring of sexual reproduction cannot completely maintain the excellent traits consistent with the mother plant; among asexual reproduction methods, tissue culture has higher reproduction efficiency than cuttings and grafting, and the seedlings are more uniform. Secondly, the success or failure of tissue culture is closely related to the genetic background of the material. For the excellent culture of plant tissues, the existing reports are not suitable for the cultivation of excellent Schima superba families. The roots of Schima superba tissue culture seedlings are slender and soft. Even after hardening, their lignification degree is still very low, resulting in a low survival rate of transplantation after the bottle is taken out. Therefore, it is necessary to develop more targeted tissue culture technology.

[0003] Patent CN201610265515.3 discloses a breeding method for a new fast-growing and cold-resistant variety of Schima superba, and CN201210121927.1 discloses an efficient in vitro propagation method for 20-year-old Schima superba trees. Although both are Schima superba, the specific varieties / strains are different, and the requirements for culture medium and environmental conditions for tissue culture are different. The former is for the breeding of cold-resistant varieties and does not adopt transplanting technology. In the technical scheme of the latter, its transplanting technology is not suitable for the excellent Schima superba strain cultivated by the Guangdong Academy of Forestry. The bottle seedlings with weaker root systems will be damaged due to long-term exposure of the roots after being taken out of the bottle. Therefore, it is still necessary to explore the tissue culture of the excellent Schima superba strain of this application. Summary of the invention

[0004] In view of the above problems, the object of the present invention is to provide a method for tissue culture seedlings of superior Schima superba families.

[0005] The technical contents of the present invention are as follows:

[0006] The present invention provides a method for tissue culture seedlings of a superior family of Schima superba, comprising the following steps:

[0007] 1) Primary culture: Take young branches of Schima superba, clean, disinfect and sterilize them, cut the branches into stem segments, and insert the stem segments obliquely into the primary culture medium;

[0008] The primary culture medium comprises the components of DCR+6-BA 2.0-4.0 mg / L+NAA 0.1-0.25 mg / L+plant essential oil 1-20 μL / L+sucrose 20-30 g / L+carrageenan 5-10 g / L;

[0009] 2) Subculture: After the stem segments of the primary culture grow new shoots, cut off the new shoots and transfer them to GD medium for culture. After that, clustered shoots are obtained and transferred to 1 / 2DCR medium. After that, it is decided whether to transfer to GD or 1 / 2DCR medium again according to the growth of the seedlings (if the proliferation coefficient is not enough and more clustered shoots are needed, they can be transferred to GD again; if the seedlings are not strong enough and the leaves are not stretched enough, they can be transferred to 1 / 2DCR again);

[0010] The GD culture medium comprises the following components: GD+6-BA 0.5-1.5 mg / L+IAA 0.1-0.5 mg / L+plant essential oil 1-20 μL / L+sucrose 20-40 g / L+carrageenan 5-10 g / L;

[0011] The 1 / 2DCR culture medium comprises the following components: 1 / 2DCR+6-BA 1.0-3.0 mg / L+NAA 0.02-0.1 mg / L+plant essential oil 1-20 μL / L+sucrose 20-40 g / L+carrageenan 5-10 g / L;

[0012] 3) Rooting culture: Take the seedlings that are larger than 2 cm and have strong growth obtained through subculture and transfer them to the rooting medium;

[0013] The rooting medium comprises the components of WPM+IBA 0.2-2.0 mg / L+IAA 0.2-0.5 mg / L+plant essential oil 50 μL / L-2 mL / L sucrose 20-40 g / L+carrageenan 10-15 g / L;

[0014] 4) Acclimation: Place the rooting medium at a temperature of 26-28°C and a light intensity of 2000-3000 lux for 15-20 days, then move it to a greenhouse with a shade of 70-75% and a daytime light intensity of 2000-3000 lux for 30 days. When the seedlings are more than 3 cm tall, they can be transplanted.

[0015] 5) Hardening of seedlings: Before transplanting, the roots of each seedling are smeared with mud, and then the seedlings are transplanted to hardening medium A, and covered with a cover with vents, the vents are closed, and the hardening medium is placed at a temperature of 26-28°C and a light of 2000-3000 lux for cultivation. After the seedlings grow new leaves, the vents are opened. After they grow to 10 cm, they are transplanted to hardening medium B, the vents are opened, and they are placed at a temperature of 26-28°C and a light of 2000-3000 lux for cultivation. After new leaves grow, the vent cover is removed, and after cultivation for 10-15 days, they are moved to a place with a shade degree of 70-75% and a daytime light of 2000-3000 lux for cultivation;

[0016] The plant essential oils include camphor tree essential oil (obtained by steam distillation from branches and leaves of camphor tree, a plant of the Lauraceae family), cinnamomum burmannii essential oil (obtained by steam distillation from branches and leaves of cinnamomum burmannii, a plant of the Lauraceae family), or melaleuca alternifolia essential oil (obtained by steam distillation from branches and leaves of melaleuca alternifolia, a plant of the Myrtaceae family);

[0017] The types of the camphor tree essential oil include more than one of linalool type, borneol type, 1,8-cineole type, camphor type, caryophyllene type, nerolidol type, and phellandrene type; the types of the cinnamomea ursinensis essential oil include more than one of borneol type, cinnamaldehyde type, eucalyptol type, caryophyllene oxide type, cymene type, and phellandrene type; the types of the Melaleuca alternifolia essential oil / include more than one of terpinene-4-ol type and citral type.

[0018] The camphor tree essential oil, burmanii cinnamomea essential oil and melaleuca alternifolia essential oil are extracted by steam distillation, and the upper oil phase obtained by extraction is the essential oil.

[0019] The mud comprises a mixture formed by stirring loess soil (the volume ratio of loess soil to water is 1:3-10);

[0020] The seedling hardening media A and B respectively comprise components of loess soil, perlite and peat soil, and the volume ratio of the components is (1-2): (1-2): (3-4);

[0021] The seedling hardening medium A comprises a crushed material of the residue of branches and leaves after the essential oil of Cinnamomum camphora / Cinnamomum burmanii / Telaleuca alternifolia is extracted, coconut bran, peat soil and perlite components, and the volume ratio of the components is (1-2): (2-4): (2-4): (1-2);

[0022] The seedling hardening matrix B comprises crushed branch and leaf residues after essential oils of camphor tree / Cinnamomum burmanii / Melaleuca alternifolia are extracted, loess soil, peat soil and perlite components, and the volume ratio of the components is (1-2): (2-4): (2-4): (1-2).

[0023] The beneficial effects of the present invention are as follows:

[0024] The tissue culture seedling method of the superior family of Schima superba adopts a combination of primary culture, subculture, rooting culture, domestication and seedling hardening methods, wherein two culture media are used for alternating culture in subculture, one for promoting budding and the other for making the seedlings thicker and the leaves more stretched, and the alternating culture enables the Schima superba seedlings to maintain a high value-added coefficient while maintaining the high quality of the subculture seedlings, which is beneficial to the subsequent rooting culture, and the addition of camphor essential oil or Melaleuca alternifolia essential oil in the culture medium is beneficial to antibacterial disinfection, and prevents the culture medium from getting moldy and the explant from browning (the contamination rate of the tissue culture seedlings with an appropriate amount of essential oil added can be controlled within 3%). Generally speaking, the roots of the obtained Schima superba rooted seedlings are slender, soft and have a very low degree of lignification. If they are immediately transplanted to an environment with unstable temperature and humidity (such as an ordinary greenhouse), they will die of water loss because the water absorption efficiency of the lower root system cannot keep up with the transpiration of the upper branches and leaves, so the survival rate of the ordinary transplanting method is very low. In the domestication and hardening of Schima superba tissue culture seedlings by the present invention, firstly, the roots are coated with yellow heart soil slurry, and the root system will be wrapped with a layer of mud to have a stronger ability to retain water. Secondly, combined with the designed culture cover, it is ensured that the unstable seedlings live in an environment with relatively constant humidity, and the culture room with temperature controlled by air conditioning provides a stable temperature for the survival of the seedlings. Thirdly, by closing the vents of the culture cover → opening the vents → uncovering the culture cover and the culture room → greenhouse, the adaptability of the seedlings is enhanced step by step, the survival rate of transplantation is greatly improved, and finally high-quality tissue culture seedlings are obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram showing the effect of subculture in Example 1;

[0026] Figure 2 This is the rooting effect diagram of Example 1;

[0027] Figure 3 This is a diagram showing the effect of the rooted seedlings after domestication in Example 1;

[0028] Figure 4 This is a diagram showing the effect of the tissue culture seedlings being taken out of the bottle in Example 1;

[0029] Figure 5 This is the effect diagram of subculture of Comparative Example 1;

[0030] Figure 6 This is the rooting culture effect diagram of Comparative Example 2;

[0031] Figure 7 This is the rooting culture effect diagram of Comparative Example 2;

[0032] Figure 8 The figure is a comparison diagram of the transplanting seedling effects of Example 1 and Comparative Example 3;

[0033] Fig. 9The seedling raising effect comparison diagram of Example 1, Comparative Example 4 and Comparative Example 5;

[0034] Fig.10 It is a strain isolated and cultured from Schima superba contaminated tissue culture seedlings;

[0035] Fig.11 This is a diagram showing the antibacterial experimental effects of camphor tree essential oil, burmannia glutinosa essential oil and Melaleuca alternifolia essential oil. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0037] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0038] Extraction of Cinnamomum camphora / Cinnamomum burmanii / Melaleuca alternifolia essential oil: extraction is carried out using the steam distillation method in Appendix XD of the Pharmacopoeia of the People's Republic of China (2015 edition) (the only difference is the extraction materials, which are the leaves of Cinnamomum camphora / Cinnamomum burmanii / Melaleuca alternifolia respectively).

[0039] Example

[0040] A method for tissue culture seedlings of superior Schima superba family

[0041] 1) Primary culture: Take young branches of Schima superba, remove leaves, wash with ultrasonic cleaner for 30 min, wash with sterile water 6 times, then soak in Tween 80 for 5 min, wash with sterile water 6 times, soak in 0.1% HgCl2 for 5 min, wash with sterile water 6 times, after disinfection and sterilization, cut the branches into 1-2 cm stem segments, each with 1-2 axillary buds, and obliquely insert into primary culture medium, with one stem segment inserted into each culture bottle;

[0042] 2) Subculture: After the stem segment of primary culture grows new shoots, the entire stem segment is removed from the primary culture medium, the new shoots are cut off, and transferred to GD culture medium for culture. After 25 days, clustered shoots are obtained, which are transferred to 1 / 2DCR culture medium. After 25 days, tissue culture seedlings are obtained. Figure 1 As shown, the sprouts are strong and the leaves are stretched;

[0043] Afterwards, decide whether to transfer to GD or 1 / 2DCR medium again according to the growth of the seedlings (if the proliferation coefficient is not enough and more clustered buds are needed, transfer to GD; if the seedlings are not strong enough and the leaves are not stretched enough, transfer to 1 / 2DCR);

[0044] 3) Rooting culture: Take the seedlings that are more than 2 cm and grow robustly after subculture and transfer them to the rooting medium. After 25 days, the rooted seedlings Figure 2 As shown, the rooting rate is high, reaching more than 95%, and the root system is well developed;

[0045] 4) Acclimation: The rooting medium was placed at a temperature of 26-28°C and a light intensity of 2000-3000 lux for 15-20 days, and then moved to a greenhouse with a shade of 70-75% and a daytime light intensity of 2000-3000 lux for 30 days. The tissue culture seedlings obtained were Figure 3 , Figure 4 As shown, the root system is well developed and the plant is strong. When the seedling height exceeds 3cm, it can be transplanted.

[0046] 5) Hardening of seedlings: Before transplanting, the roots of each seedling were smeared with mud formed by stirring yellow heart soil, and then the seedlings were transplanted into the hardening medium A (composed of the crushed residues of branches and leaves after the essential oils of Cinnamomum camphora / Cinnamomum burmanii / Telium alternifolium were extracted, coconut bran, peat soil and perlite in a volume ratio of 1:2:2:1, and the culture container was a 72-hole tray). After transplanting, water was immediately applied and covered with a cover with ventilation holes. The ventilation holes were closed and the hardening medium was placed at a temperature of 26-28°C and a light of 2000-3000 lux. After the seedlings grew new leaves, the ventilation holes were opened and grown to 10c m, transplant to hardening medium B (comprises the component of 1:2:2:1 volume ratio, namely, the residue crushed material of branches and leaves after camphor tree / Cinnamomum burmanii / Telium alternifolium essential oil extraction, yellow heart soil, peat soil and perlite, and the culture container is a 15-hole plug tray), water immediately after transplanting, open the vent, be placed in a temperature of 26-28 ℃, and cultivate at the place of 2000-3000 lux of illumination, after growing new leaves, remove the ventilation cover, cultivate 10-15 d later, move to a greenhouse with 70-75% shade and 2000-3000 lux of daylight, and carry out normal water and fertilizer management;

[0047] The seedling hardening medium A is composed of pulverized residues of camphor tree / Cinnamomum burmanii / Melaleuca alternifolia essential oils, coconut bran, peat soil and perlite components, and the volume ratio of the components is (1-2): (2-4): (2-4): (1-2), and the preferred ratio is 1:2:2:1;

[0048] The seedling hardening medium B is composed of the components of the residues of camphor tree / Cinnamomum burmanii / Melaleuca alternifolia after essential oil extraction, loess soil, peat soil and perlite, and the volume ratio of the components is (1-2): (2-4): (2-4): (1-2), and the preferred ratio is 1:2:2:1;

[0049] Table 1: Components of the culture medium in Examples and Comparative Examples

[0050]

[0051]

[0052] Comparative Example 1

[0053] Compared with Example 1, in Comparative Example 1, only GD medium was used for culture in subculture, and the state after 25 days was as follows Figure 5 As shown, the seedlings are not strong enough, the leaf tips are slightly curled, and the leaves are not stretched out.

[0054] Comparative Example 2

[0055] Compared with Example 1, in the rooting culture of Comparative Example 2, the composition of the rooting medium is the rooting medium 1 / 4MS+IBA1.00mg / L+IAA0.40mg / L described in the paper "Tissue Culture Research of Superior Families of Schima superba" (Zhou Lihua, Lan Yanqun, He Boxiang, et al. Guangdong Forestry Science and Technology, 2015, 31(2):6.), and the rooting effect after 25 days of rooting culture is as follows: Figure 6 , Figure 7 As shown, Figure 6 The seedlings used for rooting were subcultured seedlings cultured only on GD medium. Figure 7 The seedlings used for rooting were cultured alternately in GD and 1 / 2DCR medium. It can be seen that no matter how the subculture seedlings grew, they could not obtain a developed and strong root system in rooting medium B.

[0056] Comparative Example 3

[0057] Compared with Example 1, in the seedling hardening of Comparative Example 3, the roots of the seedlings were not stained with loess mud. The tissue culture seedlings of Comparative Example 3 (without loess mud) and Example 1 (with loess mud) were compared on the day of transplanting, 20 days after transplanting, and 60 days after transplanting. The results are as follows: Figure 8 As shown, the tissue culture seedlings of Comparative Example 3 grew weakly, with a survival rate of about 70% after 60 days of transplantation, while the tissue culture seedlings of Example 1 grew well, the plants were strong, and the survival rate of more than 90% after 60 days of transplantation was achieved.

[0058] Comparative Example 4

[0059] Compared with Example 1, in the tissue seedling cultivation method of Comparative Example 4, no plant essential oil is added to the culture medium used.

[0060] Comparative Example 5

[0061] Compared with Example 1, in the tissue seedling raising method of Comparative Example 4, the content of plant essential oil added to the culture medium used is excessive.

[0062] The growth of the tissue culture seedlings of Example 1, Comparative Example 4 and Comparative Example 5 was compared:

[0063] like Fig. 9 As shown, relative to the robust and sterile tissue culture seedlings in Example 1, the contamination rate of the tissue culture seedlings in Comparative Example 4 (no essential oil added) was higher (about 10%-15%), and the tissue culture seedlings in Comparative Example 5 (excessive plant essential oil added) showed a "burned seedling" state and ultimately failed to survive.

[0064] The antibacterial effect of the plant essential oil used in the present invention on the culture of Schima superba:

[0065] After separating the bacteria from the infected seedlings and diseased seedlings, they were cultured. Fig.10 As shown in the figure, three main strains were found. The three strains were tested for antibacterial activity against different pathogens using camphor tree essential oil, burmannia glutinosa essential oil and Melaleuca alternifolia essential oil. The results are shown in the figure. Fig.11 As shown, the inhibition rate of camphor tree essential oil on different pathogens is 25.67% to 63.56%, the inhibition rate of burdock osmanthus essential oil on different pathogens is 26.15% to 71.23%, and the inhibition rate of Melaleuca alternifolia essential oil on different pathogens is 35.67% to 83.56%.

[0066] Comparative Example 6

[0067] Compared with Example 1, in the seedling hardening of Comparative Example 7, the seedling hardening matrix A includes components composed of coconut bran, peat soil and perlite, and the composition volume ratio is 2:2:1, and the seedling hardening matrix B includes components composed of loess soil, peat soil and perlite, and the composition volume ratio is 2:2:1, and other conditions remain unchanged.

[0068] Comparative Example 7

[0069] Compared with Example 1, in the seedling hardening of Comparative Example 7, the seedling hardening matrix A includes components composed of perlite and peat soil, and the composition volume ratio is 2:4, and the seedling hardening matrix B includes components composed of perlite and peat soil, and the composition volume ratio is 3:4, and other conditions remain unchanged.

[0070] The growth of the bottle seedlings obtained by different hardening substrates of Example 1, Comparative Example 6 and Comparative Example 7 was recorded, and the results are shown in the following table:

[0071] Table 2 Survival rate of tissue culture seedlings

[0072] Example 1 Comparative Example 6 Comparative Example 7 Survival rate of seedlings out of bottles using hardening medium A ≥90％ 60～70％ 50～65％ The survival rate of seedlings in hardening medium B ≥95％ 70～80％ 60～70％

[0073] As can be seen from Table 2, compared with Comparative Example 6, Example 1 uses the crushed branches and leaves residues after the essential oil of Camphor tree / Cinnamomum burmanii / Alternaria alternifolia is extracted to increase the fertility of the matrix while also reducing the incidence of pests and diseases, which is beneficial to improve the survival rate of the bottle seedlings to 90% or more; compared with Comparative Example 7, the seedling hardening matrix A in Comparative Example 6 uses coconut bran, and the root lignification degree of the bottle seedlings is improved after a period of domestication in the seedling hardening matrix A, and they are more robust, while the seedling hardening B uses the components of yellow heart soil plus light matrix, the yellow heart soil enhances the fertility and pH value regulation ability of the matrix, and the light matrix ensures the water retention and air permeability of the matrix. It can be seen from the results of Example 1 that the crushed branches and leaves residues after the essential oil of Camphor tree / Cinnamomum burmanii / Alternaria alternifolia is extracted to increase the fertility of the matrix while also reducing the incidence of soil-borne pests and diseases, and the survival rate of the seedlings after transplanting can reach more than 95%.

Claims

1. A method for tissue culture seedlings of superior Schima superba families, characterized in that: The steps include: 1) Primary culture: Take young branches of Schima superba, clean, disinfect and sterilize them, cut the branches into stem segments, and insert the stem segments obliquely into the primary culture medium; The primary culture medium is composed of DCR+6-BA 2.0~4.0 mg / L+NAA 0.1~0.25 mg / L+plant essential oil 1~20 μL / L+sucrose 20~30 g / L+carrageenan 5~10 g / L; The plant essential oil includes camphor tree essential oil, burmannia glutinosa essential oil or Melaleuca alternifolia essential oil; 2) Subculture: After the stem segments grown from the primary culture have new shoots, cut off the new shoots and transfer them to GD medium for culture. After that, clustered shoots are obtained and transferred to 1 / 2DCR medium. After that, it is decided whether to transfer to GD or 1 / 2DCR medium again according to the growth of the shoots. The GD culture medium is composed of GD+6-BA 0.5-1.5 mg / L+IAA 0.1-0.5 mg / L+plant essential oil 1-20 μL / L+sucrose 20-40 g / L+carrageenan 5-10 g / L; The 1 / 2DCR culture medium is composed of 1 / 2DCR+6-BA 1.0~3.0 mg / L+NAA 0.02~0.1 mg / L+plant essential oil 1~20 μL / L+sucrose 20~40 g / L+carrageenan 5~10 g / L; The plant essential oil includes camphor tree essential oil, burmannia glutinosa essential oil or Melaleuca alternifolia essential oil; 3) Rooting culture: Take the seedlings that are larger than 2 cm and have strong growth obtained through subculture and transfer them to the rooting medium; The rooting medium is composed of WPM+IBA 0.2~2.0 mg / L+IAA 0.2~0.5 mg / L+plant essential oil 50 μL / L ~2mL / L+sucrose 20~40 g / L+carrageenan 10~15 g / L; The plant essential oil includes camphor tree essential oil, burmannia glutinosa essential oil or Melaleuca alternifolia essential oil; 4) Acclimation: Place the rooting medium at a temperature of 26-28°C and a light intensity of 2000-3000 lux for 15-20 days, then move it to a greenhouse with a shade of 70-75% and a daytime light intensity of 2000-3000 lux for 30 days. When the seedlings are more than 3 cm tall, they can be transplanted. 5) Hardening of seedlings: Before transplanting, apply mud to the roots of each seedling, then transplant the seedlings to hardening medium A, cover them with a cover with air holes, close the air holes, and place the hardening medium at a temperature of 26-28°C and a light of 2000-3000 lux. After the seedlings grow new leaves, open the air holes. After they grow to 10 cm, transplant them to hardening medium B, open the air holes, and place them at a temperature of 26-28°C and a light of 2000-3000 lux. After new leaves grow, remove the air cover. After 10-15 days of cultivation, move them to a place with a shade of 70-75% and a daytime light of 2000-3000 lux for cultivation. The mud is formed by stirring loess soil, and the volume ratio of loess soil to water is 1:3-10; The seedling hardening medium A is composed of pulverized residues of camphor tree / Cinnamomum burmanii / Melaleuca alternifolia essential oils, coconut bran, peat soil and perlite components, and the volume ratio of the components is (1-2): (2-4): (2-4): (1-2); The seedling hardening medium B is composed of crushed branch and leaf residues after essential oil extraction from camphor tree / Cinnamomum burmanii / Melaleuca alternifolia, loess soil, peat soil and perlite components, and the volume ratio of the components is (1-2): (2-4): (2-4): (1-2).

2. The method for tissue culture seedlings of Schima superba superior family according to claim 1, characterized in that: The types of the camphor tree essential oil include more than one of linalool type, borneol type, 1,8-cineole type, camphor type, caryophyllene type, nerolidol type, and phellandrene type; the types of the cinnamomea ursinensis essential oil include more than one of borneol type, cinnamaldehyde type, eucalyptol type, caryophyllene oxide type, cymene type, and phellandrene type; the types of the Melaleuca alternifolia essential oil / include more than one of terpinene-4-ol type and citral type.

3. The method for tissue culture seedlings of Schima superba superior family according to claim 2, characterized in that: The camphor tree essential oil, burmanii cinnamomea essential oil and melaleuca alternifolia essential oil are extracted by steam distillation, and the upper oil phase obtained by extraction is the essential oil.

4. The method for tissue culture seedlings of Schima superba superior family according to claim 1, characterized in that: The seedling hardening medium A comprises crushed branch and leaf residues after essential oil extraction from camphor tree / Cinnamomum burmanii / Melaleuca alternifolia, coconut bran, peat soil and perlite components, and the volume ratio of the components is 1:2:2:

1.

5. The method for tissue culture seedlings of Schima superba superior family according to claim 1, characterized in that: The seedling hardening medium B comprises crushed branch and leaf residues after essential oils of Cinnamomum camphora / Cinnamomum burmanii / Melaleuca alternifolia are extracted, loess soil, peat soil and perlite components, and the volume ratio of the components is 1:2:2:1.

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