A culture medium combination for Yunnan agarwood tissue culture and its application and a method for Yunnan agarwood tissue culture

Abstract

The invention belongs to the technical field of plant tissue culture, and specifically relates to a culture medium combination for Yunnan agarwood tissue culture and an application thereof and a Yunnan agarwood tissue culture method. The culture medium combination comprises an induction culture medium, a seedling-strengthening culture medium and a rooting culture medium. The plant growth regulator and other components in the culture medium provided by the invention are all cheap and readily available, easy to prepare, and simple to operate and highly efficient. The invention can cultivate a large number of healthy transplantable Yunnan agarwood tissue culture seedlings by using the screened culture medium composition through three stages of inducing callus tissue and clustered buds, strengthening seedlings and rooting, thereby simplifying the culture procedure and saving the culture cost, and a large number of transplantable Yunnan agarwood seedlings can be obtained in a relatively short time, which is of great significance for solving the problem that rare tree species are difficult to reproduce rapidly and in large quantities in a short period of time, meeting market demand and improving economic benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture medium combination for Yunnan agarwood tissue culture and an application thereof, and a Yunnan agarwood tissue culture method. Background Art

[0002] Aquilaria sinensis is a species of Thymelaeaceae ( Thymelaeaceae )Aquilaria Aquilaria The resinous wood produced by the plant after it is damaged is a traditional precious medicinal material and natural spice. There are about 15 species of Aquilaria in the world, and there are 2 species in China: Yunnan Aquilaria ( Aquilaria yunnanensis SC Huang) and Aquilaria sinensis ( Aquilaria sinensis (Lour.) Spreng.). The Yunnan Aquilaria tree is 3 to 8 meters tall, with a straight trunk and few branches. It has good ecological adaptability. Its bark can be used as a raw material for papermaking and artificial cotton. It can warm the middle and regulate qi, strengthen the spleen, and benefit the kidneys for medicinal purposes (Flora of China, 1999, 52(1): 290-292.). It is a valuable and precious tree species that can form agarwood with a strong and long-lasting fragrance, making it a top-quality agarwood.

[0003] Yunnan agarwood is mainly produced in tropical and subtropical areas such as Xishuangbanna and Lincang in Yunnan. With the gradual depletion of white wood fragrance resources, the development and utilization of Yunnan agarwood resources has gradually been valued (Su Juan et al. Comparative Anatomical Study on Wood Structure of Yunnan Agarwood and White Wood fragrance, Tropical Agricultural Science, 2016, 36 (4): 30-34.). At present, the breeding method of Yunnan agarwood is mainly seed seedling cultivation. The survival period of seeds is very short and cannot be stored for a long time. They need to be harvested and sown immediately, with low breeding efficiency, and seed seedling cultivation is prone to mutation, making it difficult to maintain excellent germplasm. Carrying out tissue culture of Yunnan agarwood is not affected by the external environment, can increase the proliferation rate of Yunnan agarwood, and keep the traits stable. It is also a fast and effective method to solve the contradiction between supply and demand of excellent and precious varieties, and is of great significance to the protection and preservation of the excellent germplasm resources. There is no report on the tissue culture and rapid propagation of Yunnan agarwood and related biotechnology in the prior art. Summary of the invention

[0004] The object of the present invention is to provide a culture medium combination for Yunnan agarwood tissue culture and its application and a method for Yunnan agarwood tissue culture. The culture medium combination and method of the present invention can simplify the culture procedure, save culture costs, and realize rapid and large-scale reproduction of Yunnan agarwood.

[0005] The present invention provides a culture medium combination for Yunnan agarwood tissue culture, including an induction culture medium, a seedling growth culture medium and a rooting culture medium;

[0006] The induction medium is based on MS medium and further includes the following components: 25.0-35.0 g / L sucrose, 0.05-0.2 mg / L IBA, 0.05-0.2 mg / L NAA, 2.5-3.5 mg / L 6-BA and 0.03-0.06 mg / L GA. 3 and 6.8-7.3 g / L agar;

[0007] The seedling medium is based on MS medium and further comprises the following components: 25.0-35.0 g / L sucrose, 1.5-2.5 mg / L NAA, 0.3-0.7 mg / L 6-BA, 0.5-1.5 g / L activated carbon and 6.8-8.0 g / L agar;

[0008] The rooting medium is based on 1 / 2MS medium and further comprises components with the following concentrations: 10.0-20.0 g / L of sucrose, 0.3-0.7 mg / L of NAA and 6.8-8.0 g / L of agar.

[0009] Preferably, the induction medium is based on MS medium and further comprises the following components: 30.0 g / L sucrose, 0.1 mg / L IBA, 0.1 mg / L NAA, 3.0 mg / L 6-BA and 0.05 mg / L GA. 3 and 7.0 g / L agar;

[0010] The seedling medium is based on MS medium and also includes the following components: 30.0 g / L sucrose, 2.0 mg / L NAA, 0.5 mg / L 6-BA, 1.0 g / L activated carbon and 7.0 g / L agar;

[0011] The rooting medium is based on 1 / 2MS medium and further comprises components of the following concentrations: 15.0 g / L sucrose, 0.5 mg / L NAA and 7.0 g / L agar.

[0012] The present invention also provides the application of the culture medium combination described in the above technical solution in the tissue culture of Yunnan agarwood.

[0013] The present invention also provides a method for culturing Yunnan agarwood tissue based on the culture medium combination described in the above technical solution, comprising the following steps:

[0014] inoculating the explants treated with the anti-browning agent onto an induction medium for induction culture to obtain clustered seedlings;

[0015] The clustered seedlings are inoculated onto a seedling culture medium for seedling culture, and then transferred onto a rooting culture medium for rooting culture to obtain Yunnan agarwood tissue culture seedlings;

[0016] The anti-browning agent includes PVP; the explant includes the young stem segment with axillary buds of Yunnan Aquilaria sinensis.

[0017] Preferably, the method for preparing the explant treated with the anti-browning agent comprises: soaking the explant in a 0.3wt.% to 0.6wt.% PVP solution for 10 to 20 minutes.

[0018] Preferably, after the explant is immersed in the PVP solution, the method further comprises: cutting off 2 mm stem segments at both ends of the explant in the 0.3 wt.% to 0.6 wt.% PVP solution.

[0019] Preferably, the rooting culture time is until the height of the clustered seedlings reaches 31-36 mm.

[0020] Preferably, the conditions for the induction culture, seedling culture and rooting culture are: temperature of 23-25°C, photoperiod of 16h light / 8h dark, and light intensity of 3000Lx.

[0021] Preferably, the induction culture time is 40 to 50 days; the seedling culture time is 35 to 45 days; and the rooting culture time is 40 to 50 days.

[0022] The present invention also provides application of the method described in the above technical solution in the cultivation of Yunnan agarwood.

[0023] Beneficial effects:

[0024] The present invention provides a culture medium combination for tissue culture of Yunnan agarwood, comprising a bud cluster induction culture medium, a seedling growth culture medium and a rooting culture medium; the plant growth regulators and other components in the culture medium combination provided by the present invention are cheap, readily available, easy to prepare, and simple to operate and highly efficient; the present invention can culture a large number of healthy transplantable Yunnan agarwood tissue culture seedlings through three stages of inducing callus tissue and bud clusters, strengthening seedlings and rooting by using the screened culture medium composition, thereby simplifying the culture procedure and saving the culture cost.

[0025] The present invention also provides a tissue culture method of Yunnan agarwood. The present invention uses young stem segments with axillary buds of Yunnan agarwood as explants, and uses PVP as an anti-browning agent to treat the explants, thereby overcoming the limitations of Yunnan agarwood explants that are easy to brown and callus tissue that is difficult to differentiate. The induction and differentiation of callus tissue and clustered buds are studied through multiple aspects such as explant type and size, inoculation method, and type and concentration combination of plant hormones, so as to obtain optimized conditions for tissue culture, thereby obtaining a large number of transplantable Yunnan agarwood seedlings in a relatively short period of time, which is of great significance for solving the problem that rare tree species are difficult to reproduce rapidly and in large quantities in a short period of time, meeting market demand, and improving economic benefits. 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 Schematic diagram of the browning of Yunnan Aquilaria sinensis stem segment explants cultured for 3 days in Test Example 2, wherein A is an explant treated with ultrapure water, and B is an explant treated with 0.6wt.% PVP solution as an anti-browning agent solution;

[0028] Figure 2 The differentiation of clustered buds of the Yunnan Aquilaria explants after induction culture on induction medium No. 1 in Example 1, wherein A is the clustered buds after 25 days of induction culture; B is the clustered buds after 45 days of induction culture;

[0029] Figure 3 The growth state of the clustered buds of Yunnan Aquilaria sinensis in Example 1 after 40 days of seedling cultivation on No. 1 seedling medium;

[0030] Figure 4 The growth state of the clustered buds of Yunnan Aquilaria sinensis in Example 1 after rooting and culture on rooting medium No. 1 for 45 days;

[0031] Figure 5 The differentiation of clustered buds of the stem segment explants with buds in comparative example 1 after induction culture on IV induction medium, wherein A is the stem segment callus after 14 days of induction culture, and B is the stem segment callus after 60 days of induction culture;

[0032] Figure 6 The growth of callus tissue of leaf explants after induction culture on induction medium No. 1 in comparative example 2, wherein A is the leaf callus tissue after 10 days of induction culture, and B is the leaf callus tissue after 60 days of induction culture;

[0033] Figure 7 The growth state of the clustered buds of Yunnan Aquilaria sinensis in Comparative Example 3 after being cultured on the No. III seedling medium for 40 days;

[0034] Figure 8 The growth state of the clustered buds of Yunnan Aquilaria in Comparative Example 4 after rooting and culture on rooting medium II for 45 days;

[0035] Fig. 9 The growth state of the clustered buds of Yunnan Aquilaria in Comparative Example 5 after rooting and culture on rooting medium III for 45 days;

[0036] Fig.10 This is the growth state of the clustered buds of Yunnan Aquilaria in Comparative Example 6 after 45 days of rooting culture on rooting medium IV. DETAILED DESCRIPTION

[0037] The present invention provides a culture medium combination for Yunnan agarwood tissue culture, including a cluster bud induction culture medium, a seedling growth culture medium and a rooting culture medium;

[0038] The induction medium is based on MS medium and further includes the following components: 25.0-35.0 g / L sucrose, 0.05-0.2 mg / L IBA, 0.05-0.2 mg / L NAA, 2.5-3.5 mg / L 6-BA and 0.03-0.06 mg / L GA. 3 and 6.8-7.3 g / L agar;

[0039] The seedling medium is based on MS medium and further comprises the following components: 25.0-35.0 g / L sucrose, 1.5-2.5 mg / L NAA, 0.3-0.7 mg / L 6-BA, 0.5-1.5 g / L activated carbon and 6.8-8.0 g / L agar;

[0040] The rooting medium is based on 1 / 2MS medium and further comprises components with the following concentrations: 10.0-20.0 g / L of sucrose, 0.3-0.7 mg / L of NAA and 6.8-8.0 g / L of agar.

[0041] In the present invention, the induction medium is based on MS medium and also includes the following components: 25.0-35.0 g / L sucrose, 0.05-0.2 mg / L IBA, 0.05-0.2 mg / L NAA, 2.5-3.5 mg / L 6-BA and 0.03-0.06 mg / L GA. 3 and 6.8-7.3 g / L agar. It is further preferred that the MS medium is used as the basic medium, and only contains the following components at the following concentrations: 25.0-35.0 g / L sucrose, 0.05-0.2 mg / L IBA, 0.05-0.2 mg / L NAA, 2.5-3.5 mg / L 6-BA and 0.03-0.06 mg / L GA3 and 6.8-7.3 g / L agar. In the present invention, the induction medium is based on MS medium, preferably further comprising 25.0-35.0 g / L sucrose, more preferably 30.0 g / L sucrose; preferably further comprising 0.05-0.2 mg / L IBA, more preferably 0.1 mg / L IBA; preferably further comprising 0.05-0.2 mg / L NAA, more preferably 0.1 mg / L NAA; preferably further comprising 2.5-3.5 mg / L 6-BA, more preferably 3.0 mg / L 6-BA; preferably further comprising 0.03-0.06 mg / L GA 3 , more preferably 0.05 mg / L GA 3 ; Preferably, it also includes 6.8-7.3 g / L agar, and more preferably 7.0 g / L agar. As a preferred embodiment of the present invention, the induction medium uses MS medium as the basal medium and also includes the following components: 30.0 g / L sucrose, 0.1 mg / L IBA, 0.1 mg / L NAA, 3.0 mg / L 6-BA and 0.05 mg / L GA 3 and 7.0 g / L agar.

[0042] The seedling medium is based on MS medium, and preferably further comprises the following components: 25.0-35.0 g / L sucrose, 1.5-2.5 mg / L NAA, 0.3-0.7 mg / L 6-BA, 0.5-1.5 g / L activated carbon, and 6.8-8.0 g / L agar. It is further preferred that the MS medium is used as the base medium, and only contains the following components: 25.0-35.0 g / L sucrose, 1.5-2.5 mg / L NAA, 0.3-0.7 mg / L 6-BA, 0.5-1.5 g / L activated carbon, and 6.8-8.0 g / L agar. In the present invention, the seedling culture medium is based on MS culture medium, preferably further comprising 25.0-35.0 g / L sucrose, more preferably 30.0 g / L sucrose; preferably further comprising 1.5-2.5 mg / L NAA, more preferably 2.0 mg / L NAA; preferably further comprising 0.3-0.7 mg / L 6-BA, more preferably 0.5 mg / L 6-BA; preferably further comprising 0.5-1.5 g / L activated carbon, more preferably 1.0 g / L activated carbon; preferably further comprising 6.8-8.0 g / L agar, more preferably 7.0 g / L agar. As a preferred embodiment of the present invention, the seedling culture medium is based on MS culture medium, and further comprising the following concentrations of components: 30.0 g / L sucrose, 2.0 mg / L NAA, 0.5 mg / L 6-BA, 1.0 g / L activated carbon and 7.0 g / L agar.

[0043] The rooting medium is based on 1 / 2MS medium, and preferably also includes the following concentrations of components: 10.0-20.0 g / L sucrose, 0.3-0.7 mg / L NAA and 6.8-8.0 g / L agar. It is further preferred that the 1 / 2MS medium is the base medium, and only contains the following concentrations of components: 15.0 g / L sucrose, 0.5 mg / L NAA and 7.0 g / L agar. In the present invention, the rooting medium is based on 1 / 2MS medium, and preferably also includes 10.0-20.0 g / L sucrose, and more preferably 15.0 g / L sucrose; preferably also includes 0.3-0.7 mg / L NAA, and more preferably 0.5 mg / L NAA; preferably also includes 6.8-8.0 g / L agar, and more preferably 7.0 g / L agar. As a preferred embodiment of the present invention, the rooting medium uses 1 / 2MS medium as the basic medium and also includes the following components: 15.0g / L sucrose, 0.5mg / L NAA and 7.0g / L agar. The plant growth regulators and other components in the medium provided by the present invention are cheap and easy to obtain, easy to prepare, and simple to operate and highly efficient; the present invention can cultivate a large number of healthy transplantable Yunnan agarwood tissue culture seedlings through the three stages of inducing callus tissue and clustered buds, strong seedlings and rooting by using the screened medium composition, simplifying the cultivation procedure and saving the cultivation cost.

[0044] The present invention also provides the application of the culture medium combination described in the above technical solution in the tissue culture of Yunnan agarwood.

[0045] The present invention also provides a method for culturing Yunnan agarwood tissue based on the culture medium combination described in the above technical solution, comprising the following steps:

[0046] The explant treated with the anti-browning agent is inoculated on an induction medium for induction culture to obtain clustered seedlings; the clustered seedlings are inoculated on a seedling medium for seedling culture, and then transferred to a rooting medium for rooting culture to obtain a Yunnan agarwood tissue culture seedling; the anti-browning agent includes PVP; the explant includes a young stem segment with axillary buds of Yunnan agarwood. The present invention uses the young stem segment with axillary buds of Yunnan agarwood as an explant, which overcomes the limitation that the callus tissue of the Yunnan agarwood explant is not easy to differentiate.

[0047] In the present invention, the explant is preferably an explant obtained in spring; the method for preparing the explant treated with an anti-browning agent preferably includes: soaking the explant in a 0.3wt.%~0.6wt.% PVP solution for 10~20min. The present invention soaks the explant in a 0.3wt.%~0.6wt.% PVP solution, more preferably in a 0.6wt.% PVP solution; the soaking time is 10~20min, more preferably 15min; the Yunnan agarwood tissue culture seedling is preferably a transplantable Yunnan agarwood sterile seedling. The present invention can effectively improve the problem of easy browning of Yunnan agarwood explants by reasonably selecting the type of anti-browning agent and adjusting the method of treating the explant with the anti-browning agent.

[0048] After the explant is immersed in the PVP solution, the present invention preferably further comprises sterilizing the explant, and after sterilization, the stem segments of 2 mm at both ends of the explant are respectively cut off in a 0.3wt.% to 0.6wt.% PVP solution; more preferably, after sterilization, the stem segments of 2 mm at both ends of the explant are respectively cut off in a 0.6wt.% PVP solution; after the stem segments at both ends of the explant are cut off, the length of the explant is preferably 1.0 to 1.5 cm; the sterilization preferably comprises the following steps:

[0049] Soak in 75% v / v ethanol and rinse three times with sterile water, 30 seconds each time;

[0050] Then use 0.1wt.% HgCl 2 Soak the explants and wash them 5 times with sterile water, 1 min each time;

[0051] The soaking time in 75% ethanol is preferably 15 to 25 seconds, more preferably 20 seconds; the soaking time in 0.1 wt.% HgCl 2 The soaking time of the explant is preferably 7 to 10 minutes, more preferably 8 minutes; the present invention preferably uses 0.1wt.% HgCl 2 The explant is gently shaken during immersion. The present invention cuts off 2 mm stem segments at both ends of the explant, thereby removing the stem segment portion that is in direct contact with the disinfectant and effectively reducing the browning rate of the explant.

[0052] In the present invention, the rooting culture time is preferably until the height of the clustered seedlings is 31-36 mm.

[0053] In the present invention, the conditions for the cluster bud induction culture, cluster bud induction culture and rooting culture are preferably: temperature 23~25°C, photoperiod 16h light / 8h dark, light intensity 3000Lx; the time of the induction culture is preferably 40~50d, more preferably 45d; the time of the seedling culture is preferably 35~45d, more preferably 40d; the time of the rooting culture is preferably 40~50d, more preferably 45d.

[0054] The present invention also provides application of the method described in the above technical solution in the cultivation of Yunnan agarwood.

[0055] In order to further illustrate the present invention, a culture medium combination for Yunnan agarwood tissue culture and its application and a method for Yunnan agarwood tissue culture provided by the present invention are described in detail below in combination with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present invention.

[0056] The English abbreviations in the present invention are as follows:

[0057] NAA: 1-naphthaleneacetic acid;

[0058] IBA: 3-Indole butyric acid;

[0059] 6-BA: 6-Benzylamino purine;

[0060] GA 3 :Gibberellic acid, gibberellin;

[0061] PVP: Polyvinyl pyrrolidone.

[0062] In the present embodiment, MS medium was purchased from PhytoTechnology Laboratories; sucrose, IBA, NAA, 6-BA and GA 3 purchased from BBI Life Sciences Co., Ltd., and agar was purchased from Sangon (Sangon Biotechnology (Shanghai) Co., Ltd.).

[0063] Test Example 1

[0064] Samples were collected in different seasons to obtain Yunnan Aquilaria sinensis stem segment explants. The spring sampling time was March 2022, the summer sampling time was July 2022, the autumn sampling time was October 2022, and the winter sampling time was January 2023.

[0065] The specific steps for obtaining explants in each season are:

[0066] After cutting the stem explants of about 1.5-2.0 cm in length, soak them in ultrapure water for 15 min, and then rinse them with ultrapure running water for 5 min;

[0067] In an ultra-clean workbench, soak the rinsed explants in 75% v / v ethanol for 20 seconds, and then rinse them three times with sterile water, each time for 30 seconds;

[0068] With 0.1wt.% HgCl 2 Soak the explants, gently shaking them, and wash them five times with sterile water after soaking for 8 minutes, each time for 1 minute.

[0069] Use sterile filter paper to gently dry the surface water of the explants, inoculate them on MS medium for culture, inoculate 11 bottles in total, inoculate 2 explants in each bottle, and repeat the test 3 times. After 3 days of culture, the browning rate of the explants was counted.

[0070] The browning rate of explants after sampling in each season is shown in Table 1.

[0071] Table 1 Effects of different sampling seasons on explant browning

[0072]

[0073] As shown in Table 1, the browning rate was the lowest when the Yunnan Aquilaria sinensis stem explants were taken in spring.

[0074] Test Example 2

[0075] By comparing the browning control experiment with different anti-browning agents, we found a treatment method that effectively inhibits the browning of Yunnan Aquilaria sinensis stem segment explants. The specific method is as follows:

[0076] The Yunnan Aquilaria sinensis stem segment explants were divided into 9 treatment groups. The anti-browning agents used in each treatment group and their solution concentrations are shown in Table 2.

[0077] In spring, each group cut a 1.5-2.0 cm long Yunnan Aquilaria sinensis stem explant and quickly soaked it in an anti-browning agent solution. After soaking for 15 min, it was rinsed with ultrapure water for 5 min. Then, it was soaked in 75% v / v ethanol for 20 s in an ultra-clean bench, rinsed three times with sterile water for 30 s each time, and then rinsed with 0.1 wt.% HgCl 2 The explants were soaked, gently shaken during soaking, and washed 5 times with sterile water after 8 minutes, each time for about 1 minute, and then the surface moisture of the explants was gently absorbed with sterile filter paper, and inoculated on MS medium for culture. After 3 days of culture, the browning rate of the explants in each treatment group was counted. Each treatment group was inoculated with 11 bottles, and each bottle was inoculated with 2 explants, and the test was repeated 3 times.

[0078] Table 2 Effects of pretreatment with anti-browning agents on browning of explants

[0079]

[0080] The browning of explants was compared among the groups. The results are shown in Table 2 and Figure 1 As shown, the results show that the anti-browning effect of pre-treating the explants with a 0.6wt.% PVP solution is the best, so in the subsequent examples, the explants are treated with a 0.6wt.% PVP solution as an anti-browning agent.

[0081] Example 1

[0082] Step 1, obtaining sterile stem segment explants:

[0083] (1) Newly sprouted, well-grown, and non-lignified shoots of Yunnan agarwood in the spring of the same year were selected as experimental materials, and stem segments with axillary buds on the shoots were cut as explants.

[0084] (2) After cutting the stem explants with a length of 1.5-2.0 cm, they were quickly immersed in a 0.6 wt.% PVP solution for 15 min, and then rinsed with ultrapure water for 5 min;

[0085] (3) In a clean bench, soak the rinsed explants in 75% v / v ethanol for 20 seconds, and then rinse them three times with sterile ultrapure water, each time for 30 seconds;

[0086] (4) Using 0.1wt.% HgCl 2 The explants were immersed in the solution with gentle shaking for 8 min and then rinsed with sterile ultrapure water 5 times, each wash for 1 min;

[0087] (5) Cut off 2 mm stem segments from both ends of the explant and place them in a filter-sterilized 0.6 wt.% PVP solution. Then, use sterile filter paper to gently absorb the moisture on the surface of the explant to obtain sterile explants.

[0088] Step 2, cluster shoot induction:

[0089] In the clean bench, pick up the sterile stem segment and gently place it flat on the No. 1 induction medium. Inoculate one explant in each culture bottle, inoculate 10 bottles each time, and repeat the experiment three times. After 30 days of culture, change the induction medium (transfer the explant to the same No. 1 cluster bud induction medium for continued culture). After 25 days and 45 days of culture on the induction medium, observe and count the cluster bud differentiation, such as Figure 2 shown.

[0090] Depend on Figure 2It can be seen that after about 25 days of culture on the induction medium, the stem segments swelled and callus tissue and clustered buds appeared. A small part of them formed a large amount of callus tissue, which then differentiated into clustered buds. According to statistics, after 45 days of induction culture, an average of 16 clustered buds were produced (16.40±3.34).

[0091] Formula of induction medium No. I: MS medium (4.4 g / L) + sucrose (30.0 g / L) + IBA (0.1 mg / L) + NAA (0.1 mg / L) + 6-BA (3.0 mg / L) + GA 3 (0.05 mg / L) + agar (7.0 g / L), pH 5.8±0.05.

[0092] Step 3, cultivation of strong seedlings of clustered buds:

[0093] After 45 days of induction culture, in a clean bench, cut the clustered buds with a height of 21-25 mm and transfer them to the No. 1 seedling medium. Inoculate one clustered bud seedling per bottle, inoculate 10 bottles each time, and repeat the experiment three times. After 30 days of culture, replace the seedling medium (transfer the clustered bud seedlings to the same No. 1 seedling medium for continued culture).

[0094] Formula of No. I seedling culture medium: MS medium (4.4g / L) + sucrose (30.0g / L) + NAA (2mg / L) + 6-BA (0.5mg / L) + activated carbon (1.0g / L) + agar (7.0g / L), pH 5.8±0.05.

[0095] The plant height was recorded at the beginning of seedling cultivation and on the 40th day. The growth status of clustered seedlings after 40 days of cultivation was as follows: Figure 3 As shown; according to statistics, the plant height difference after 40 days of cultivation of strong seedlings was 10.64±1.59mm.

[0096] Step 4, rooting culture:

[0097] After 40 days of seedling cultivation, the Yunnan Aquilaria sinensis clustered seedlings with a plant height of 31-36 mm were transferred to the No. 1 rooting medium for cultivation. One clustered seedling was transferred to each culture bottle, and 10 bottles were inoculated each time. The experiment was repeated three times in total. After 30 days of cultivation, the rooting medium was changed (the clustered seedlings were transferred to the same No. 1 rooting medium for continued cultivation). After 45 days of rooting culture, the number of roots was counted. The growth of the clustered seedlings after 45 days of rooting culture was as follows: Figure 4 As shown, the average number of roots is about 22 (21.57±3.30).

[0098] Rooting medium formula No. I: 1 / 2MS medium (2.2 g / L) + sucrose (15.0 g / L) + NAA (0.5 mg / L) + agar (7.0 g / L), pH 5.8±0.05.

[0099] The conditions for induction culture, seedling culture and rooting culture are: temperature of 24℃±1℃, photoperiod of 16h light / 8h dark, and light intensity of 3000Lx.

[0100] Example 2

[0101] Step 1, obtaining sterile stem segment explants: same as Example 1.

[0102] Step 2, cluster shoot induction:

[0103] In the clean bench, pick up the sterile stem segment and gently place it flat on the No. II induction medium. Inoculate one explant in each culture bottle, inoculate 10 bottles each time, and repeat the experiment three times. After 30 days of culture, change the induction medium (transfer the explant to the same No. II cluster bud induction medium for continued culture).

[0104] Formula of cluster bud induction medium II: MS medium (4.4g / L) + sucrose (30.0g / L) + IBA (0.05mg / L) + NAA (0.05mg / L) + 6-BA (2.5mg / L) + GA 3 (0.05 mg / L) + agar (7.0 g / L), pH 5.8±0.05.

[0105] The differentiation of cluster buds was observed and counted. The results showed that after 30 days of culture on cluster bud induction medium II, the explants expanded and callus tissue and cluster bud points appeared. A small part of them formed a large amount of callus tissue, which would then differentiate into cluster buds. According to statistics, after 45 days of induction culture, an average of about 11 cluster buds were produced (10.87±1.78).

[0106] Step 3, cultivation of strong seedlings of clustered buds: same as in Example 1.

[0107] Step 4, rooting culture: same as in Example 1.

[0108] The conditions for induction culture, seedling culture and rooting culture are: temperature of 24℃±1℃, photoperiod of 16h light / 8h dark, and light intensity of 3000Lx.

[0109] Example 3

[0110] Step 1, obtaining sterile stem segment explants: same as Example 1.

[0111] Step 2, cluster shoot induction:

[0112] In the clean bench, pick up the sterile stem segment and gently place it flat on the No. III induction medium. Inoculate one explant in each culture bottle, inoculate 10 bottles each time, and repeat the experiment three times. After 30 days of culture, change the induction medium (transfer the explant to the same No. II cluster bud induction medium for continued culture).

[0113] Formula of cluster bud induction medium III: MS medium (4.4g / L) + sucrose (30.0g / L) + IBA (0.2mg / L) + NAA (0.2mg / L) + 6-BA (3.5mg / L) + GA 3 (0.05 mg / L) + agar (7.0 g / L), pH 5.8±0.05.

[0114] The differentiation of cluster buds was observed and counted. The results showed that after 25 days of culture on cluster bud induction medium II, the explants expanded and callus tissue and cluster bud points appeared. A small part of them formed a large amount of callus tissue, which would then differentiate into cluster buds. According to statistics, after 45 days of induction culture, an average of about 15 cluster buds were produced (14.83±2.52).

[0115] Step 3, cultivation of strong seedlings of clustered buds: same as in Example 1.

[0116] Step 4, rooting culture: same as in Example 1.

[0117] The conditions for induction culture, seedling culture and rooting culture are: temperature of 24℃±1℃, photoperiod of 16h light / 8h dark, and light intensity of 3000Lx.

[0118] Example 4

[0119] Step 1, obtaining sterile stem segment explants: same as Example 1.

[0120] Step 2, cluster shoot induction: same as in Example 1.

[0121] Step 3, cultivation of strong seedlings of clustered buds:

[0122] After 45 days of induction culture, in the clean bench, cut the clustered buds with a height of 21-25 mm and transfer them to the No. II seedling medium. Inoculate one seedling per bottle, and inoculate 10 bottles each time. Repeat the experiment three times. After 30 days of culture, replace the seedling medium (transfer the clustered seedlings to the same No. II seedling medium for continued culture). The formula of No. II seedling medium: MS medium (4.4g / L) + sucrose (30.0g / L) + NAA (2.5mg / L) + 6-BA (0.3mg / L) + activated carbon (1.0g / L) + agar (7.0g / L), pH 5.8±0.05.

[0123] The plant heights were recorded at the beginning of seedling cultivation and 40 days later. According to statistics, the difference in plant height after 40 days of seedling cultivation was 9.16±1.21 mm.

[0124] Step 4, rooting culture: same as in Example 1.

[0125] The conditions for induction culture, seedling culture and rooting culture are: temperature of 24℃±1℃, photoperiod of 16h light / 8h dark, and light intensity of 3000Lx.

[0126] Comparative Example 1

[0127] Step 1, obtaining sterile stem segment explants: same as Example 1.

[0128] Step 2, cluster shoot induction:

[0129] In the clean bench, pick up the sterile stem segment and gently place it flat on the IV induction medium. Inoculate one explant in each culture bottle, inoculate 10 bottles each time, and repeat the experiment three times. After 30 days of culture, replace the induction medium (transfer the explant to the same IV cluster bud induction medium for continued culture). The conditions for induction culture are: temperature 24℃±1℃, photoperiod 16h light / 8h dark, light intensity 3000Lx.

[0130] The differentiation of cluster buds was observed. After about 7 days of culture on the induction medium, the stem segments began to swell and callus appeared, and then a large amount of callus was formed. This may be because the increase in the dosage of plant growth regulators accelerated the formation of callus. However, the growth of callus after 14 days and 60 days of induction culture was as follows Figure 5 As shown by Figure 5 It can be seen that after 60 days of co-cultivation at this stage, no clustered buds were differentiated and the callus tissue gradually turned brown.

[0131] Formula of cluster shoot induction medium No. IV: MS medium (4.4 g / L) + sucrose (30.0 g / L) + IBA (0.5 mg / L) + NAA (0.5 mg / L) + 6-BA (5.0 mg / L) + agar (7.0 g / L), pH 5.8±0.05.

[0132] Comparative Example 2

[0133] Step 1, obtaining sterile leaf explants:

[0134] (1) Select the newly sprouted young leaves of Yunnan Aquilaria sinensis in the spring of the current year as explants.

[0135] (2) After cutting the young leaf explants, quickly soak them in a 0.6wt.% PVP solution for 15 min, and then rinse them with ultrapure water for 5 min;

[0136] (3) In a clean bench, soak the rinsed explants in 75% v / v ethanol for 15 seconds, and then rinse them three times with sterile ultrapure water, each time for 30 seconds.

[0137] (4) Using 0.1wt.% HgCl 2 The explants were immersed in the solution with gentle shaking for 6 min and then rinsed with sterile ultrapure water 5 times, each wash for 30 s;

[0138] (5) Use sterile filter paper to gently absorb the moisture on the leaf surface. Use a blade to remove the base, tip, and edge tissue of the leaf in a filtered and sterilized 0.6 wt.% PVP solution to obtain a rectangular leaf explant with a length of about 15 mm and a width of about 10 mm.

[0139] Step 2, cluster shoot induction:

[0140] In the clean bench, place the sterile leaf explants flat (with the adaxial surface of the leaf facing up) on the No. 1 induction medium. Inoculate one leaf explant in each culture bottle, inoculate 10 bottles each time, and repeat the experiment three times. After 30 days of culture, replace the induction medium (transfer the explants to the same No. 1 cluster bud induction medium for continued culture). The conditions for induction culture are: temperature 24℃±1℃, photoperiod 16h light / 8h dark, light intensity 3000Lx.

[0141] Formula of induction medium No. I: MS medium (4.4 g / L) + sucrose (30.0 g / L) + IBA (0.1 mg / L) + NAA (0.1 mg / L) + 6-BA (3.0 mg / L) + GA 3 (0.05 mg / L) + agar (7.0 g / L), pH 5.8±0.05.

[0142] The differentiation of clustered buds was observed. After about 10 days of culture on the induction medium, the leaf explants began to swell and form callus tissue, and then a large amount of callus tissue was formed. The growth of leaf callus tissue after 10 days and 60 days of induction culture was as follows: Figure 6 As shown by Figure 6 It can be seen that no clustered buds were differentiated after 60 days of culture.

[0143] Comparative Example 3

[0144] Step 1, obtaining sterile stem segment explants: same as Example 1.

[0145] Step 2, cluster shoot induction: same as in Example 1.

[0146] Step 3, cultivation of strong seedlings of clustered buds:

[0147] After 45 days of induction culture, in the clean bench, cut the clustered buds with a height of about 21-25 mm and transfer them to the No. III seedling medium. Inoculate one bud in each bottle, and inoculate 10 bottles each time. Repeat the experiment three times. After 30 days of culture, replace the seedling medium (transfer the clustered buds to the same No. III seedling medium for continued culture).

[0148] Formula of No. III seedling culture medium: MS medium (4.4g / L) + sucrose (30.0g / L) + NAA (1mg / L) + 6-BA (1mg / L) + agar (7.0g / L), pH 5.8±0.05.

[0149] The conditions for induction culture and seedling culture were: temperature of 24°C ± 1°C, photoperiod of 16h light / 8h dark, and light intensity of 3000Lx.

[0150] After about 10 days of cultivation, the tips of the seedlings began to turn brown, the leaves fell off and even died. The plant height was recorded at the beginning of the seedling cultivation and 40 days later. The growth status of the clustered seedlings at 40 days of seedling cultivation was as follows: Figure 7 As shown, according to statistics, the plant height difference after 40 days of cultivation of strong seedlings is about 5.92±1.09mm.

[0151] Comparative Example 4

[0152] Step 1, obtaining sterile stem segment explants: same as Example 1.

[0153] Step 2, cluster shoot induction: same as in Example 1.

[0154] Step 3, cultivation of strong seedlings of clustered buds: same as in Example 1.

[0155] Step 4, rooting culture:

[0156] After 40 days of seedling cultivation, the clustered buds of Yunnan Aquilaria sinensis with a plant height of about 31-36 mm were transferred to the rooting medium No. II for cultivation. One plant was transferred to each culture bottle, and 10 bottles were inoculated each time. The test was repeated three times. After 30 days of cultivation, the rooting medium was replaced (the clustered buds were transferred to the same rooting medium No. II for continued cultivation). The conditions for induction culture, seedling culture and rooting culture were: temperature 24℃±1℃, photoperiod 16h light / 8h dark, and light intensity 3000Lx.

[0157] The growth status of the clustered seedlings after 45 days of rooting culture is as follows Figure 8 As shown, according to statistics, the average number of roots at this time is 5 (5.33±1.03).

[0158] Rooting medium formula No. II: 1 / 2MS medium (2.2 g / L) + sucrose (15.0 g / L) + NAA (0.2 mg / L) + agar (7.0 g / L), pH 5.8±0.05.

[0159] Comparative Example 5

[0160] Step 1, obtaining sterile stem segment explants: same as Example 1.

[0161] Step 2, cluster shoot induction: same as in Example 1.

[0162] Step 3, cultivation of strong seedlings of clustered buds: same as in Example 1.

[0163] Step 4, rooting culture:

[0164] After 40 days of seedling cultivation, the clustered buds of Yunnan Aquilaria sinensis with a plant height of about 31-36 mm were transferred to the rooting medium No. III for cultivation. One plant was transferred to each culture bottle, and 10 bottles were inoculated each time. The test was repeated three times in total. After 30 days of cultivation, the rooting medium was replaced (the clustered buds were transferred to the same rooting medium No. III for continued cultivation). The conditions for induction culture, seedling culture and rooting culture were: temperature 24℃±1℃, photoperiod 16h light / 8h dark, and light intensity 3000Lx.

[0165] The growth status of the clustered seedlings after 45 days of rooting culture is as follows Fig. 9 As shown, according to statistics, the average number of roots at this time is 10 (9.50±1.81).

[0166] Rooting medium formula No. III: 1 / 2MS medium (2.2g / L) + sucrose (15.0g / L) + NAA (0.5mg / L) + IBA (0.5mg / L) + agar (7.0g / L), pH 5.8±0.05.

[0167] Comparative Example 6

[0168] Step 1, obtaining sterile stem segment explants: same as Example 1.

[0169] Step 2, cluster shoot induction: same as in Example 1.

[0170] Step 3, cultivation of strong seedlings of clustered buds: same as in Example 1.

[0171] Step 4, rooting culture:

[0172] After 40 days of seedling cultivation, the clustered buds of Yunnan Aquilaria sinensis with a plant height of about 31-36 mm were transferred to the IV rooting medium for cultivation. One plant was transferred to each culture bottle, and 10 bottles were inoculated each time. The experiment was repeated three times. After 30 days of cultivation, the rooting medium was replaced (the clustered buds were transferred to the same IV rooting medium for continued cultivation). The conditions for induction culture, seedling culture and rooting culture were: temperature 24℃±1℃, photoperiod 16h light / 8h dark, and light intensity 3000Lx.

[0173] The growth status of the clustered seedlings after 45 days of rooting culture is as follows Fig.10 As shown, according to statistics, the average number of roots at this time is 0 (0.37±0.06).

[0174] Rooting medium formula No. IV: 1 / 2MS medium (2.2 g / L) + sucrose (15.0 g / L) + agar (7.0 g / L), pH 5.8±0.05.

[0175] It can be seen from the above embodiments that the present invention uses young stem segments with axillary buds of Yunnan agarwood as explants, and uses PVP as an anti-browning agent to treat the explants, thereby overcoming the limitations that Yunnan agarwood explants are easy to brown and callus tissue is not easy to differentiate; by using the screened culture medium composition, a large number of healthy and transplantable Yunnan agarwood tissue culture seedlings can be cultivated through three stages of inducing callus tissue and clustered buds, strengthening seedlings and rooting, thereby simplifying the culture procedure and saving the culture cost.

[0176] 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 culture medium combination for Yunnan agarwood tissue culture, characterized in that: Including induction medium, seedling medium and rooting medium; The induction medium is based on MS medium and also includes the following components: 25.0-35.0 g / L sucrose, 0.05-0.2 mg / L IBA, 0.05-0.2 mg / L NAA, 2.5-3.5 mg / L 6-BA, 0.05 mg / L GA3 and 6.8-7.3 g / L agar; The seedling medium is based on MS medium and further comprises the following components: 25.0-35.0 g / L sucrose, 1.5-2.5 mg / L NAA, 0.3-0.7 mg / L 6-BA, 0.5-1.5 g / L activated carbon and 6.8-8.0 g / L agar; The rooting medium is based on 1 / 2MS medium and further comprises components with the following concentrations: 10.0-20.0 g / L of sucrose, 0.3-0.7 mg / L of NAA and 6.8-8.0 g / L of agar.

2. The culture medium combination according to claim 1, characterized in that: The induction medium is based on MS medium and also includes the following components: 30.0 g / L sucrose, 0.1 mg / L IBA, 0.1 mg / L NAA, 3.0 mg / L 6-BA, 0.05 mg / L GA3 and 7.0 g / L agar; The seedling medium is based on MS medium and also includes the following components: 30.0 g / L sucrose, 2.0 mg / L NAA, 0.5 mg / L 6-BA, 1.0 g / L activated carbon and 7.0 g / L agar; The rooting medium uses 1 / 2MS medium as the basic medium and also includes the following components in the following concentrations: 15.0 g / L sucrose, 0.5 mg / L NAA and 7.0 g / L agar.

3. A method for culturing Yunnan agarwood tissue based on the culture medium combination according to claim 1 or 2, characterized in that: The following steps are involved: inoculating the explants treated with the anti-browning agent onto an induction medium for induction culture to obtain clustered sprouts; The clustered seedlings are inoculated onto a seedling culture medium for seedling culture, and then transferred onto a rooting culture medium for rooting culture to obtain Yunnan agarwood tissue culture seedlings; The anti-browning agent includes PVP; the explant includes the young stem segment with axillary buds of Yunnan Aquilaria sinensis.

4. The method according to claim 3, characterized in that The method for preparing the explant treated with the anti-browning agent comprises: soaking the explant in a 0.3wt.% to 0.6wt.% PVP solution for 10 to 20 minutes.

5. The method according to claim 4, characterized in that After the explant is immersed in the PVP solution, the method further comprises: cutting off 2 mm stem segments at both ends of the explant in the 0.3wt.% to 0.6wt.% PVP solution.

6. The method according to claim 3, characterized in that: The rooting culture time is until the height of the clustered seedlings reaches 31-36 mm.

7. The method according to claim 3, characterized in that The conditions for the induction culture, seedling culture and rooting culture are: temperature of 23-25° C., photoperiod of 16 h light / 8 h dark, and light intensity of 3000 Lx.

8. The method according to claim 3, characterized in that The time of the induction culture is 40 to 50 days; the time of the seedling culture is 35 to 45 days; and the time of the rooting culture is 40 to 50 days.

9. Application of the method according to any one of claims 3 to 8 in the cultivation of Yunnan agarwood.

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