Application of amphotericin B in inhibiting endophyte pollution of stem tissue culture of ancient cinnamomum camphora

Abstract

The invention relates to the technical field of plant tissue culture, in particular to application of amphotericin B in inhibition of endophyte pollution of ancient cinnamomum camphora stem tissue culture. It is found that amphotericin B can effectively inhibit endophyte pollution in the ancient cinnamomum camphora stem tissue culture process, meanwhile, it is avoided that high-concentration antibiotics generate a toxic effect on stem induction, the stem induction endophyte pollution rate in the tissue culture process is effectively reduced and can be reduced to 6%, the stem induction rate can still reach 78%, and the tissue culture cost is reduced. The bottleneck problem of endophyte pollution in the ancient cinnamomum camphora stem tissue culture process is solved, and technical support is provided for efficient propagation and preservation of precious germplasm resources of ancient cinnamomum camphora.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to application of amphotericin B in inhibiting endophytic bacteria contamination in tissue culture of ancient camphor tree stem segments. Background Art

[0002] Ancient trees have rich scientific, cultural, landscape and economic values, and the preservation of ancient tree germplasm resources is particularly important. Camphor (Cinnamomum camphora) is an ancient tree species native to China, with functions such as spices, medicine, timber, chemical industry, ornamental and ecological protection. As an important garden ornamental tree species, camphor has become a characteristic landscape in many places. It contains important genetic information and is the basis for scientific research and application. Ancient camphor (camphor trees over 100 years old) are hundreds or even thousands of years old, and their physiological age is already in the aging period of growth and development. Many of them have aged or even died, and precious germplasm and gene resources are gradually lost. However, the current protection methods for ancient camphor are basically in-situ protection such as prohibiting felling, destruction and migration, and there is a lack of effective methods and technologies for preserving germplasm resources. Asexual reproduction and rejuvenation are widely used in forestry scientific research because they can maintain the characteristics of the parents. It is the main way to preserve and utilize ancient tree germplasm resources. Cuttings are an important method of asexual propagation, but cuttings require a large number of explants. The weak growth and few branches of ancient camphor trees are particularly limited in their propagation by cuttings. Tissue culture has the advantages of fast propagation, no seasonal influence on material collection, high propagation coefficient, and rapid acquisition of a large number of asexual seedlings. Compared with cuttings, tissue culture requires less explants. For the same explant branches, the number of asexual seedlings obtained by tissue culture is 2 to 3 times that of cuttings. Therefore, in the conservation of ancient camphor germplasm resources, tissue culture is the most suitable and important asexual propagation method.

[0003] Due to the hundreds and thousands of years of life history and the influence of various biological and abiotic stresses and other external environments, the ancient camphor has formed a large number of microorganisms such as bacteria and fungi in long-term symbiosis with its various tissues, collectively referred to as endophytes. The presence of endophytes can cause excessive growth of endophytes in the explants during tissue culture, contaminating the explants, leading to failure of explant induced culture, and thus making it impossible to obtain healthy and sterile tissue culture seedlings. At present, there is no substantial solution to the problem of endophyte contamination of ancient camphor tissue culture explants. Therefore, a method that can inhibit the growth and contamination of endophytes during explant tissue culture is urgently needed for the preservation of ancient camphor germplasm resources. Summary of the invention

[0004] In order to solve the above problems, the present invention provides the use of amphotericin B in inhibiting endophyte contamination in tissue culture of ancient camphor stem segments. The present invention finds that amphotericin B at an appropriate concentration can effectively inhibit endophyte contamination in the tissue culture process of ancient camphor stem segments, and reduce the contamination rate of tissue culture stem segments without affecting the induction rate of the stem segments, thereby achieving efficient asexual breeding of ancient camphor, and providing technical support for the efficient expansion and preservation of valuable germplasm resources of ancient camphor.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The invention provides application of amphotericin B in inhibiting endophyte contamination in tissue culture of ancient camphor tree stem segments. The concentration of the amphotericin B is 100-200 mg / L.

[0007] Preferably, the concentration of amphotericin B is 200 mg / L.

[0008] The invention provides an induction culture medium for ancient camphor trees. The induction culture medium uses an MS culture medium as a basic culture medium and further comprises components with the following concentrations: 1.2-1.5 mg / L of 6-benzylaminoadenine, 0.03-0.06 mg / L of naphthaleneacetic acid and 100-200 mg / L of amphotericin B.

[0009] Preferably, the concentration of amphotericin B in the induction medium is 200 mg / L.

[0010] The present invention provides the use of the induction culture medium described in the above technical solution in inhibiting endophytic bacteria contamination in tissue culture of ancient camphor tree stem segments.

[0011] The present invention provides a tissue culture method for ancient camphor stem segments, comprising the following steps:

[0012] Inoculating the disinfected ancient camphor stem segments into the induction culture medium described in the above technical solution for induction culture, and inducing the axillary buds to grow into small buds;

[0013] cutting off the small buds and inoculating them into a proliferation culture medium for proliferation culture to obtain clustered buds;

[0014] The clustered buds are divided into individual plants and inoculated into a rooting medium for rooting culture to obtain rooted seedlings;

[0015] The rooted seedlings are hardened and transplanted into a cultivation medium to obtain ancient camphor asexual plants.

[0016] Preferably, the proliferation medium uses MS medium as the basic medium and further includes the following components at the following concentrations: 1.8-2.0 mg / L 6-benzylaminoadenine and 0.1-0.2 mg / L naphthaleneacetic acid;

[0017] The rooting medium uses 1 / 2MS medium as the basic medium and also includes the following components in concentrations: 0.15-0.25 mg / L of naphthaleneacetic acid and 1 g / L of activated carbon;

[0018] The cultivation matrix comprises the following components in parts by volume: 1 part of nutrient soil, 1 part of vermiculite and 1 part of perlite.

[0019] Preferably, the induction culture conditions include: light intensity of 1500-1800 Lux; photoperiod of 12 hours of light and 12 hours of darkness; temperature of 24-26° C.;

[0020] The conditions for the proliferation culture include: light intensity of 1500-1800 Lux; photoperiod of 12 hours of light and 12 hours of darkness; temperature of 24-26° C.;

[0021] The rooting culture conditions include: light intensity of 1500-1800 Lux; photoperiod of 12 hours of light and 12 hours of darkness; and temperature of 24-26°C.

[0022] Preferably, the disinfection method comprises: soaking the ancient camphor stem segment in a carbendazim solution, transferring it to 75% alcohol by volume for washing, and finally transferring it to 0.1% mercuric chloride solution for washing, to obtain the disinfected ancient camphor stem segment after washing.

[0023] Preferably, the length of the ancient camphor tree stem segment is 3 to 4 cm, and each ancient camphor tree stem segment has 2 to 3 axillary buds.

[0024] Beneficial effects:

[0025] The present invention provides an application of amphotericin B in inhibiting endophyte contamination in tissue culture of ancient camphor stem segments. The present invention finds that amphotericin B at an appropriate concentration can effectively inhibit endophyte contamination in the tissue culture process of ancient camphor stem segments, while avoiding the toxic effect of high concentrations of antibiotics on the induction of stem segments, effectively reducing the endophyte contamination rate induced by the stem segments in the tissue culture process, which can be reduced to 6%, and the induction rate of the stem segments can still reach 78%, solving the bottleneck problem of endophyte contamination in the tissue culture process of ancient camphor stem segments, and providing technical support for the efficient propagation and preservation of valuable germplasm resources of ancient camphor. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 The results of endophyte contamination of ancient camphor stem segments in tissue culture in the induction medium in Example 1; A, no amphotericin B was added to the medium; B, 200 mg / L amphotericin B was added to the medium;

[0028] Figure 2 The clustered buds formed by the aseptic buds of ancient camphor in the proliferation medium in Example 1;

[0029] Figure 3 The present invention relates to tissue culture rooted seedlings formed by the aseptic clustered buds of ancient camphor in Example 1 in a rooting medium. DETAILED DESCRIPTION

[0030] The invention provides application of amphotericin B in inhibiting endophyte contamination in tissue culture of ancient camphor tree stem segments. The concentration of the amphotericin B is 100-200 mg / L.

[0031] As an embodiment, the concentration of amphotericin B is 200 mg / L.

[0032] The invention can effectively inhibit the growth of endophytes in the ancient camphor stem segments in the induction culture medium by adding amphotericin B of appropriate concentration into the ancient camphor stem segment induction culture medium, and the contamination rate of endophytes is as low as 6% to 12%. Meanwhile, the toxic effect of high concentration of antibiotics on the induction and differentiation of the ancient camphor stem segments is avoided, and the induction rate of the stem segments can reach 72% to 78%, thereby solving the bottleneck problem of endophyte contamination in the tissue culture of the ancient camphor stem segments and providing technical support for the efficient propagation of the asexual lines and the preservation of germplasm resources of the ancient camphor.

[0033] Based on the above advantages, the present invention provides an induction culture medium for ancient camphor, which uses MS culture medium as a basic culture medium and also includes components with the following concentrations: 1.2-1.5 mg / L of 6-benzylaminoadenine, 0.03-0.06 mg / L of naphthaleneacetic acid, and 100-200 mg / L of amphotericin B.

[0034] As an embodiment, the concentration of 6-benzylaminoadenine in the induction medium is 1.5 mg / L.

[0035] As an embodiment, the concentration of naphthylacetic acid in the induction medium is 0.04 mg / L.

[0036] As an embodiment, the concentration of amphotericin B in the induction medium is 200 mg / L.

[0037] The induction culture medium provided by the invention can effectively inhibit the growth of endophytes in the stem segments in the induction culture medium, and the contamination rate of endophytes is as low as 6% to 12%. Meanwhile, the toxic effects of high concentrations of antibiotics on the induction and differentiation of ancient camphor stem segments are avoided, and the induction rate of the stem segments can reach 72% to 78%.

[0038] Based on the above advantages, the present invention provides the use of the induction culture medium described in the above technical solution in inhibiting endophytic contamination of ancient camphor stem segment tissue culture.

[0039] The present invention provides a tissue culture method for ancient camphor stem segments, comprising the following steps:

[0040] Inoculating the disinfected ancient camphor stem segments into the induction culture medium described in the above technical solution for induction culture, and inducing the axillary buds to grow into small buds;

[0041] cutting off the small buds and inoculating them into a proliferation culture medium for proliferation culture to obtain clustered buds;

[0042] The clustered buds are divided into individual plants and inoculated into a rooting medium for rooting culture to obtain tissue culture rooted seedlings;

[0043] The rooted seedlings are hardened and then transplanted into a substrate to obtain ancient camphor asexual plants.

[0044] As an implementation mode, the length of the ancient camphor tree stem segment is 3 to 4 cm, and each ancient camphor tree stem segment has 2 to 3 axillary buds.

[0045] As an embodiment, the present invention soaks the ancient camphor stem segment in a carbendazim solution and then transfers it to a mercuric chloride solution for soaking, and washes to obtain the disinfected ancient camphor stem segment. As an embodiment, the carbendazim is a commercially available carbendazim with a content of 50%, the carbendazim solution is a solution prepared by diluting the carbendazim 100 times, and the ancient camphor stem segment is soaked in the carbendazim solution for 2 to 3 hours. As another embodiment, the ancient camphor stem segment is soaked in the carbendazim solution and then rinsed with sterile water 3 to 5 times and then transferred to an alcohol solution for washing. As an embodiment, the alcohol concentration is 75%, fully washed for 45 to 60 seconds, then washed with sterile water 3 to 5 times, and finally the surface moisture of the ancient camphor stem segment is dried with sterile filter paper, and then transferred to a mercuric chloride solution for washing. As an embodiment, the concentration of the mercuric chloride solution is 0.1% (1g / L), and the ancient camphor stem segment is soaked in the mercuric chloride solution for 8 to 10 minutes. The present invention can kill microorganisms on the surface of the ancient camphor tree stem segment through disinfection treatment, thereby reducing the contamination rate of subsequent tissue culture.

[0046] After obtaining the disinfected ancient camphor stem segment, the present invention inoculates the disinfected ancient camphor stem segment into the induction culture medium described in the above technical solution for induction culture, and the axillary buds are induced to grow into small buds. As an embodiment, the conditions of the induction culture include: light intensity of 1500-1800 Lux; photoperiod of 12 hours of light and 12 hours of darkness; temperature of 24-26°C.

[0047] After obtaining the small buds, the present invention cuts off the small buds and inoculates them into a proliferation culture medium for proliferation culture to obtain clustered buds. As one embodiment, the proliferation culture medium uses MS culture medium as the basic culture medium, and also includes components of the following concentrations: 1.8-2.0 mg / L of 6-benzylaminoadenine and 0.1-0.2 mg / L of naphthaleneacetic acid. As another embodiment, the proliferation culture medium uses MS culture medium as the basic culture medium, and also includes components of the following concentrations: 1.8 mg / L of 6-benzylaminoadenine and 0.15 mg / L of naphthaleneacetic acid. As one embodiment, the conditions of the proliferation culture include: light intensity of 1500-1800 Lux; photoperiod of 12 h light and 12 h dark; temperature of 24-26°C.

[0048] After obtaining the clustered buds, the present invention divides the clustered buds into individual plants and inoculates them into a rooting medium for rooting culture to obtain rooted seedlings. As an embodiment, the rooting medium uses 1 / 2MS medium as the basic medium, and also includes components with the following concentrations: 0.15-0.25 mg / L of naphthaleneacetic acid and 1 g / L of activated carbon. As another embodiment, the rooting medium uses 1 / 2MS medium as the basic medium, and also includes components with the following concentrations: 0.2 mg / L of naphthaleneacetic acid and 1 g / L of activated carbon. As an embodiment, the conditions for the rooting culture include: light intensity of 1500-1800 Lux; photoperiod of 12 h light and 12 h dark; temperature of 24-26°C.

[0049] After obtaining the rooted seedlings, the present invention transplants the rooted seedlings into a cultivation medium after hardening to obtain ancient camphor plants. As an embodiment, the cultivation medium includes the following components by volume: 1 part of nutrient soil, 1 part of vermiculite and 1 part of perlite. As an embodiment, the cultivation medium is mixed with 50% carbendazim at a ratio of 2g / kg of the cultivation medium, and sealed for 7 days to sterilize it.

[0050] To further illustrate the present invention, the application of amphotericin B provided by the present invention in inhibiting endophyte contamination in tissue culture of ancient camphor stem segments, the induction culture medium of ancient camphor and the tissue culture method of ancient camphor are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] Unless otherwise specified, the drugs, reagents and materials used in the following examples can be obtained from commercial sources.

[0053] Example 1

[0054] 1. The origin of ancient camphor trees and the collection of their branches

[0055] The ancient camphor tree material used in the present invention comes from an ancient camphor tree with a tree age of 2000 in Jingkou Township, Nanchang City, Jiangxi Province (28°63′69″N, 116°27′02″E). Semi-lignified branches of the current year are collected in July and brought back to the laboratory for processing. The leaves of the branches are cut off and the branches are cut into stem segments with 2-3 axillary buds, uniform thickness and hardness, and a length of 3-4 cm.

[0056] 2. Treatment and washing of ancient camphor stems

[0057] Rinse the cut ancient camphor tree stem segments with clean water for 2 to 3 times to wash away the dirt and impurities on the surface of the stem segments, then soak them in a carbendazim solution (50% carbendazim diluted 100 times) for 2 hours, and rinse them with clean water again for 3 to 5 times to wash away the residual carbendazim on the surface of the stem segments.

[0058] 3. Disinfection of stem surface

[0059] In the ultra-sterile workbench, the stem segments were divided into sterile conical bottles, and 75% alcohol was added to fully wash for 45-60 seconds, and then washed with sterile water for 3-5 times, and finally the surface moisture of the ancient camphor stem segments was absorbed with sterile filter paper. A 0.1% mercuric chloride solution prepared with sterile water was added, and the stem segments were soaked for 8-10 minutes according to the tenderness of the ancient camphor stem segments. During the soaking process, the stem segments were constantly stirred with a glass rod to make the stem segments fully contact with the mercuric chloride solution to achieve a full sterilization effect. The soaked mercuric chloride solution was poured into the waste liquid bottle, and the stem segments were washed with sterile water for 3-5 times. Finally, the surface moisture of the ancient camphor stem segments was absorbed with sterile filter paper and inoculated into the induction medium.

[0060] 4. Inoculate into induction medium

[0061] The induction medium used MS medium as the basic medium, and the hormone concentration of 6-benzylaminoadenine (6-BA) was added at 1.5 mg / L, and the hormone concentration of naphthaleneacetic acid (NAA) was added at 0.04 mg / L. In order to determine the inhibition and effect of different types and concentrations of antibiotics on endophytes in the ancient camphor stem segments in the induction medium, the induction medium without antibiotics was used as the blank control (CK), and three different final concentrations of antibiotics were added to the induction medium: streptomycin sulfate (50 mg / L, 100 mg / L, 200 mg / L and 400 mg / L), ampicillin sodium (50 mg / L, 100 mg / L, 200 mg / L and 400 mg / L) and amphotericin B (50 mg / L, 100 mg / L, 200 mg / L and 400 mg / L). After adding antibiotics for 20 days, the contamination of the ancient camphor stem segments and the induction of the stem segments were observed, and the contamination rate and induction rate of the stem segments were counted. The induction medium of the inoculated stem segment was cultured in a tissue culture room, the light intensity was set to 1500-1800 Lux, the photoperiod was set to 12 h / 12 ​​h (light / dark), and the culture temperature was 24-26°C.

[0062] 5. Inoculate into proliferation medium

[0063] After 20 days of induction in the induction medium, the stem segments swelled at the base and the axillary buds differentiated into small buds. When the small buds germinated to 2-3 cm, the new leaf area of ​​the small buds reached 2-3 cm. 2 , cut the uninfected buds from the base and inoculate them into the proliferation medium. The proliferation medium uses MS as the basic medium, adds 6-benzylaminoadenine (6-BA) hormone concentration of 1.8 mg / L, and naphthaleneacetic acid (NAA) hormone concentration of 0.15 mg / L. The proliferation medium of the inoculated stem segment is placed in the tissue culture room for culture, the light intensity is set to 1500-1800 Lux, the photoperiod is set to 12h / 12h (light / dark), and the culture temperature is 24-26°C.

[0064] 6. Inoculate into rooting medium

[0065] After culturing in the proliferation medium for 25 days, the base of the buds will continue to differentiate, swell and proliferate to form clustered buds ( Figure 2 ), when the buds grow to 3-5 cm, and the new leaves of the buds grow to 3-5 cm 2 , in the super-bacteria workbench, the clustered buds were divided into individual plants and inoculated into the rooting medium for further cultivation. The rooting medium used 1 / 2MS as the basic medium, with 0.2mg / L of naphthaleneacetic acid (NAA) hormone and 1g / L of activated carbon. The rooting medium of the inoculated stem segments was placed in the tissue culture room for cultivation, with the light intensity set to 1500-1800Lux, the photoperiod set to 12h / 12h (light / dark), and the culture temperature set to 24-26℃.

[0066] 7. Hardening and transplanting

[0067] Continue to culture in the rooting medium for 25 to 30 days until the root system of the rooted seedlings grows to more than 2 cm and the height reaches more than 5 cm ( Figure 3 ). Open the bottle cap of the rooted seedlings with good growth, place them at room temperature for 5-7 days, wash off the base culture medium of the rooted seedlings with new leaves and no pollution, and transplant them into a matrix of nutrient soil: vermiculite: perlite = 1:1:1 (volume ratio), stir and mix the matrix with 50% carbendazim at a ratio of 2g / kg, seal it for one week to sterilize it, cover it with a light-transmitting film after transplanting, remove the film after 20-25 days, and manage light and water under normal conditions.

[0068] The contamination rate and induction rate of stem segments with different types and concentrations of antibiotics added to the induction medium were statistically analyzed. The results are shown in Tables 1 and Figure 1 .

[0069] Table 1 Contamination rate and induction rate of stem segments with different types and concentrations of antibiotics added to the induction medium

[0070]

[0071] The results show that:

[0072] 1. In the absence of any antibiotics in the induction medium, white hyphae began to appear at the base of the stem segments after 10-15 days of tissue culture. Figure 1 A), and quickly spread to the entire culture medium within 3 to 5 days. The endophyte contamination was serious, with the endophyte contamination rate reaching 66%, and the stem segment tissue culture induction rate was reduced to 36%.

[0073] 2. After adding different concentrations of streptomycin sulfate to the induction medium (50 mg / L, 100 mg / L, 200 mg / L and 400 mg / L), the endophyte contamination rate of stem segment tissue culture gradually decreased from 28% to 10%. However, when the concentration reached 400 mg / L, the antibiotics had a toxic effect on the stem segments, and the tissue culture induction rate decreased to 42%.

[0074] 3. When different concentrations of ampicillin sodium (50 mg / L, 100 mg / L, 200 mg / L and 400 mg / L) were added to the induction medium, the endophyte contamination rate of the stem segment tissue culture gradually decreased from 36% to 20%. However, when the concentration reached 200 mg / L, the antibiotics had a toxic effect on the stem segments, and the tissue culture induction rate was as low as 48%.

[0075] 4. When different concentrations of amphotericin B (50 mg / L, 100 mg / L, 200 mg / L and 400 mg / L) were added to the induction medium, the endophyte contamination rate of stem segment tissue culture gradually decreased from 22% to 4%. When the concentration of amphotericin B was 200 mg / L ( Figure 1 In B), there is no endophyte contamination and the tissue culture induction rate can reach 78%.

[0076] In summary, when amphotericin B was added to the induction medium of ancient camphor tree at a concentration of 200 mg / L, the endophyte contamination rate was reduced to 6%, and the tissue culture induction rate still reached 78%. Therefore, compared with streptomycin sulfate and ampicillin sodium, amphotericin B is the most suitable antibiotic to inhibit the endophyte contamination of ancient camphor tree stem segments in tissue culture.

[0077] 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. Application of amphotericin B in inhibiting endophyte contamination in tissue culture of ancient camphor tree stem segments, wherein the concentration of amphotericin B is 100-200 mg / L.

2. The use according to claim 1, characterized in that: The concentration of amphotericin B is 200 mg / L.

3. An induction culture medium for ancient camphor, characterized in that: The induction culture medium uses MS culture medium as the basic culture medium and further comprises the following components in concentrations: 1.2-1.5 mg / L of 6-benzylaminoadenine, 0.03-0.06 mg / L of naphthaleneacetic acid and 100-200 mg / L of amphotericin B.

4. The induction medium according to claim 3, characterized in that The concentration of amphotericin B in the induction medium is 200 mg / L.

5. Use of the induction culture medium according to claim 3 or 4 in inhibiting endophyte contamination in tissue culture of ancient camphor tree stem segments.

6. A tissue culture method for ancient camphor stem segments, characterized in that: The following steps are involved: Inoculating the disinfected ancient camphor stem segment into the induction culture medium described in claim 3 or 4 for induction culture, and inducing the axillary buds to grow into small buds; cutting off the small buds and inoculating them into a proliferation culture medium for proliferation culture to obtain clustered buds; The clustered buds are divided into individual plants and inoculated into a rooting medium for rooting culture to obtain rooted seedlings; The rooted seedlings are hardened and then transplanted into a cultivation medium to obtain ancient camphor asexual plants.

7. The tissue culture method according to claim 6, characterized in that The proliferation culture medium uses MS culture medium as the basic culture medium, and also includes the following components in concentrations: 1.8-2.0 mg / L of 6-benzylaminoadenine and 0.1-0.2 mg / L of naphthaleneacetic acid; The rooting medium uses 1 / 2MS medium as the basic medium and also includes the following components in concentrations: 0.15-0.25 mg / L of naphthaleneacetic acid and 1 g / L of activated carbon; The cultivation matrix comprises the following components in parts by volume: 1 part of nutrient soil, 1 part of vermiculite and 1 part of perlite.

8. The tissue culture method according to claim 6, characterized in that The induction culture conditions include: light intensity of 1500-1800 Lux; photoperiod of 12 hours of light and 12 hours of darkness; temperature of 24-26° C.; The conditions for the proliferation culture include: light intensity of 1500-1800 Lux; photoperiod of 12 hours of light and 12 hours of darkness; temperature of 24-26° C.; The rooting culture conditions include: light intensity of 1500-1800 Lux; photoperiod of 12 hours of light and 12 hours of darkness; and temperature of 24-26°C.

9. The tissue culture method according to claim 6, characterized in that The disinfection method comprises: soaking the ancient camphor stem segment in a carbendazim solution, transferring it to an alcohol solution with a volume concentration of 75% for washing, and finally transferring it to a 0.1% mercuric chloride solution for washing, and obtaining the disinfected ancient camphor stem segment after washing.

10. The tissue culture method according to claim 6 or 9, characterized in that: The length of the ancient camphor stem segment is 3 to 4 cm, and each ancient camphor stem segment has 2 to 3 axillary buds.

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