Tissue culture method of aquilaria agallocha seedlings

Abstract

The invention discloses a tissue culture method of aquilaria agallocha seedlings, and belongs to the technical field of plant tissue culture. The tissue culture method comprises the following steps: (1) inoculating a disinfected explant into an adventitious bud induction culture medium, and carrying out induction culture to obtain an adventitious bud; (2) inoculating the adventitious buds into a proliferation culture medium, and carrying out proliferation culture to obtain proliferation seedlings; (3) inoculating the proliferation seedlings into a rooting culture medium, and performing rooting culture to obtain test-tube plantlets; and (4) hardening the test-tube plantlet, and transplanting to obtain the agilawood seedling. According to the method, the agilawood explants are induced to form the adventitious buds, the proliferation seedlings and the rooting test-tube plantlets by adopting the specific culture medium under the specific culture conditions, the adventitious bud induction rate is high, the proliferation speed and the rooting rate are high, the survival rate of the test-tube plantlets is high, the loss of the agilawood explants can be reduced, the agilawood high-quality seedling breeding process is accelerated, and the survival rate of the test-tube plantlets is increased. The economic benefits of the agilawood are improved.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a tissue culture method for agarwood seedlings. Background Art

[0002] Agarwood is naturally distributed in tropical forests of Vietnam, Thailand, Malaysia, India and other countries. It was introduced and planted in Yunnan (Xishuangbanna), Guangdong, Guangxi, Fujian and other places in China. It is one of the main tree species of traditional precious agarwood and agarwood oil. Because agarwood is rare and precious, the market price is very expensive.

[0003] The propagation methods of agarwood include seed propagation, cutting propagation and grafting propagation. However, the seeds of agarwood should be selected from excellent mother trees over 8 years old. The seeds mature in June and July, and the fruits are harvested and placed in a ventilated place to dry in the shade. They cannot be exposed to the sun. The seeds are sown as soon as they are picked. If they need to be stored for standby, they should be stored in wet sand for ≤7 days, otherwise the germination rate will decrease; and the seedlings propagated by seeds cannot maintain their excellent fragrance characteristics. Therefore, it is difficult to obtain a large number of high-quality agarwood seedlings in a short time by seed propagation. Cutting propagation requires the selection of healthy, pest-free branches with branches and buds, which causes great damage to the mother plant; and because the growth rate of agarwood seedlings is very slow, it requires long-term cultivation and care, and the success rate of cuttings of agarwood seedlings is relatively low, about 30%. Grafting propagation requires certain grafting skills and experience, and has certain requirements for rootstock varieties. Therefore, it is difficult to achieve industrial breeding and propagation.

[0004] Plant tissue culture, also known as in vitro culture, refers to the technology of separating the required tissues, organs or cells, protoplasts, etc. from the plant body, inoculating them on a culture medium containing various nutrients and plant hormones under sterile conditions to obtain regenerated complete plants or produce other products with economic value. Plant tissue culture technology is not limited by the growing season, has a short culture cycle, can be reproduced in large quantities, and is easy to preserve the excellent properties of the plant. It can be used to save endangered plants that are difficult to reproduce naturally. Therefore, the use of plant tissue culture technology to obtain high-quality seedlings of agarwood is conducive to the preservation and rapid reproduction of the excellent traits of agarwood, and has excellent application prospects. Summary of the invention

[0005] The purpose of the present invention is to provide a tissue culture method for agarwood seedlings to solve the problems existing in the above-mentioned prior art. The present invention uses a specific culture medium to induce agarwood explants to form adventitious buds, proliferated seedlings and rooted test tube seedlings under specific culture conditions. The adventitious bud induction rate is high, the proliferation rate is fast, the rooting rate is high, and the survival rate of the test tube seedlings is high. The loss of agarwood explants can be reduced, the breeding process of high-quality agarwood seedlings can be accelerated, and the economic benefits of agarwood can be improved.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a tissue culture method for agarwood seedlings, comprising the following steps:

[0008] (1) inoculating the sterilized explants into an adventitious bud induction medium, inducing culture, and obtaining adventitious buds;

[0009] (2) inoculating the adventitious buds into a proliferation medium, culturing them for proliferation, and obtaining proliferated seedlings;

[0010] (3) inoculating the proliferated seedlings into a rooting medium, performing rooting culture, and obtaining test tube seedlings;

[0011] (4) after hardening the test tube seedlings, transplanting them to obtain the agarwood seedlings;

[0012] The adventitious bud induction medium is: MS+6-BA (0.3-0.6) mg / L+TDZ (0.1-0.2) mg / L+sucrose (12-18) g / L+agar (5-10) g / L;

[0013] The proliferation medium is: 1 / 2MS+6-BA (0.05-0.2) mg / L+IBA (0.1-0.3) mg / L+agar (5-10) g / L;

[0014] The rooting medium is: 1 / 2MS+NAA (0.01-0.1) mg / L+Vitamin A (0.5-1.5) mg / L+Agar (5-10) g / L;

[0015] The induction culture conditions are dark culture first and then light culture.

[0016] Furthermore, the adventitious bud induction medium is: MS+6-BA0.5mg / L+TDZ 0.1mg / L+sucrose 15g / L+agar 5g / L;

[0017] The proliferation medium is: 1 / 2MS+6-BA 0.1mg / L+IBA 0.2mg / L+agar 5g / L;

[0018] The rooting medium is: 1 / 2MS+NAA 0.05mg / L+Vitamin A 1.0mg / L+Agar 5g / L.

[0019] Furthermore, the temperature during the dark culture is 24±1°C and the time is 3-6 days.

[0020] Furthermore, the temperature during the light culture is 24±1°C, the light intensity is 1500 lx, the light conditions are 8h light / 16h dark, and the total light culture time is 60-85 days. Furthermore, during the induction culture, a new adventitious bud induction medium is transferred every 20-25 days of culture.

[0021] Furthermore, the conditions for the proliferation culture are: the culture temperature is 24±1°C, the light intensity is 1500lx, and the illumination time is 10h light / 14h dark.

[0022] Furthermore, the rooting culture conditions are: culture temperature is 24±1°C, light intensity is 2500lx, and lighting time is 12h light / 12h dark.

[0023] Furthermore, the light intensity during the hardening of the seedlings is 5000-8000lx.

[0024] Furthermore, the proliferation culture time is 50-60 days. Furthermore, during the proliferation culture, a new proliferation culture medium is transferred every 25-30 days.

[0025] Furthermore, the rooting culture time is 25-30 days.

[0026] Furthermore, the time for hardening the seedlings is 8-15 days.

[0027] The present invention discloses the following technical effects:

[0028] The invention optimizes the types and concentrations of components in the adventitious bud induction medium, the proliferation medium and the rooting medium in the tissue culture process of agarwood, as well as the conditions for adventitious bud induction culture, thereby greatly improving the adventitious bud induction rate, the proliferation multiple and the rooting rate of agarwood, and significantly improving the survival rate of agarwood test tube seedlings, and is expected to provide new experience and technology for the rapid propagation of high-quality agarwood varieties and the retention of excellent properties, reduce the introduction and breeding costs of agarwood, and improve the planting benefits of agarwood. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] In the present invention, 6-BA is 6-benzyladenine, which is a cytokinin whose main function is to promote cell division. In plant tissue culture, it is often used to induce callus formation and cell division, thereby promoting plant growth and development. In some embodiments of the present invention, the concentration of 6-BA in the adventitious bud induction medium may be 0.3-0.6 mg / L, specifically selected from 0.3 mg / L, 0.4 mg / L, 0.45 mg / L, 0.5 mg / L, 0.55 mg / L, 0.6 mg / L; in some specific embodiments of the present invention, 0.5 mg / L of 6-BA is used as an example. In some embodiments of the present invention, the concentration of 6-BA in the proliferation medium may be 0.05-0.2 mg / L, specifically selected from 0.05 mg / L, 0.1 mg / L, 0.15 mg / L, 0.2 mg / L; in some specific embodiments of the present invention, 0.1 mg / L of 6-BA is used as an example.

[0035] In the present invention, TDZ is thidiazuron, which is a urea plant growth regulator with strong cytokinin activity (CTK), plays an important role in plant tissue culture, can induce a series of reactions from callus formation to somatic embryogenesis of explants, and has the special function of dual action of auxin and cytokinin. In some embodiments of the present invention, the concentration of TDZ in the adventitious bud induction medium can be 0.1-0.2 mg / L, specifically selected from 0.1 mg / L, 0.12 mg / L, 0.15 mg / L, 0.18 mg / L, 0.2 mg / L; in some specific embodiments of the present invention, the concentration of TDZ in the adventitious bud induction medium can be selected from 0.1 mg / L and 0.2 mg / L.

[0036] In the present invention, IBA is indolebutyric acid, which is a plant hormone with multiple physiological effects, mainly including promoting cell division and growth, inducing the formation of adventitious roots, increasing fruit setting, preventing fruit drop, changing the ratio of male and female flowers, etc. In some embodiments of the present invention, the concentration of IBA in the proliferation medium can be 0.1-0.3 mg / L, specifically selected from 0.1 mg / L, 0.15 mg / L, 0.2 mg / L, 0.25 mg / L, 0.3 mg / L; in some specific embodiments of the present invention, the concentration of IBA in the proliferation medium can be selected from 0.1 mg / L, 0.2 mg / L, 0.3 mg / L.

[0037] In the present invention, NAA is naphthylacetic acid, which is a synthetic plant growth regulator, having multiple functions such as promoting plant rooting, germination, and improving transplant survival rate. During plant growth, NAA can promote cell division and expansion, induce the formation of adventitious roots, increase fruit setting, prevent fruit drop, change the ratio of male and female flowers, and promote plant metabolism and photosynthesis, thereby accelerating growth and development and enhancing resistance. In some embodiments of the present invention, the concentration of NAA in the rooting medium can be 0.01-0.1 mg / L, specifically selected from 0.01 mg / L, 0.02 mg / L, 0.03 mg / L, 0.04 mg / L, 0.05 mg / L, 0.06 mg / L, 0.07 mg / L, 0.08 mg / L, 0.09 mg / L, 0.1 mg / L; in some specific embodiments of the present invention, 0.05 mg / L of NAA is used as an example.

[0038] In the present invention, vitamin A is a fat-soluble vitamin, a chemical precursor in plants, which can be converted into an active form by enzyme catalytic reactions in plants. Vitamin A can promote the growth and development of plants, increase the green pigment content of leaves, improve photosynthesis efficiency, enhance the stress resistance of plants, and enable plants to better adapt to environmental changes. In some embodiments of the present invention, the concentration of vitamin A in the rooting medium can be 0.5-1.5 mg / L, specifically selected from 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L; in some specific embodiments of the present invention, the concentration of vitamin A in the rooting medium can be selected from 0.5 mg / L, 1.0 mg / L, 1.5 mg / L.

[0039] In the present invention, adventitious bud induction culture is divided into the first, second and third rounds of culture. The first round of culture is dark culture first, and then light culture. Dark culture can promote the dedifferentiation of plant explants into callus tissue, which is convenient for the subsequent differentiation of adventitious buds. In some embodiments of the present invention, the dark culture time can be 3-6d, specifically 3, 4, 5, and 6d; in some specific embodiments of the present invention, 5d is used as an example. In some embodiments of the present invention, the light culture time can be 20-25d, specifically 20, 21, 22, 23, 24, and 25d; in some specific embodiments of the present invention, 20d is used as an example. The second round of culture is light culture. In some embodiments of the present invention, the light culture time can be 20-25d, specifically 20, 21, 22, 23, 24, and 25d; in some specific embodiments of the present invention, 25d is used as an example. The third round of culture is light culture. In some embodiments of the present invention, the time of light culture may be 20-25 days, specifically 20, 21, 22, 23, 24, 25 days; in some specific embodiments of the present invention, 25 days is taken as an example.

[0040] In the present invention, the proliferation culture is divided into a first round and a second round of culture. In some embodiments of the present invention, the time of the first round of culture may be 25-30 days, specifically 25, 26, 27, 28, 29, 30 days; in some specific embodiments of the present invention, 25 days is taken as an example. In some embodiments of the present invention, the time of the second round of culture may be 25-30 days, specifically 25, 26, 27, 28, 29, 30 days; in some specific embodiments of the present invention, 25 days is taken as an example.

[0041] In the present invention, the explant of Aquilaria sinensis is young and tender tissue. In some embodiments of the present invention, the young and tender tissue can be selected from young branches, young leaves, young buds, young and tender stem segments, etc.; in some specific embodiments of the present invention, the young and tender tissue is selected from young leaves, young buds and young and tender stem segments.

[0042] In the present invention, the agarwood is a common cultivated variety on the market. In some embodiments of the present invention, the variety of agarwood can be selected from grapevine, golden silk oil, watermelon leaf, purple chess, oil leaf, tiger stripe agarwood, etc.; in some specific embodiments of the present invention, golden silk oil agarwood is taken as an example.

[0043] Unless otherwise specified, the reagents or materials used in the following examples of the present invention can be purchased through conventional channels.

[0044] Example 1

[0045] 1. Explant disinfection: Collect the tender leaves of Aquilaria sinensis, rinse them with running water for 5 minutes to remove dirt and other impurities attached to the surface, soak the rinsed leaves in 75% alcohol for 1 minute, then transfer the leaves to 0.1% mercuric chloride for disinfection for 10 minutes, and finally rinse them with sterile water for 5 times. Place the disinfected leaves on a sterile workbench and trim them into 1cm×1cm explant tissue blocks.

[0046] 2. Adventitious bud induction culture: inoculate the explant tissue block into the adventitious bud induction medium and carry out the first round of culture at 24±1℃; then transfer it to a new adventitious bud induction medium and carry out the second round of culture at 24±1℃. After culturing for 25 days, transfer it to a new adventitious bud induction medium and carry out the third round of culture at 24±1℃. After culturing for 25 days, adventitious buds are obtained.

[0047] In order to study the influencing factors of adventitious bud induction rate, the present invention screens the induction conditions and the adventitious bud induction medium formula.

[0048] The induction conditions were set as follows: A1: the first round of culture was dark culture for 5 days, then light culture for 20 days, with a light intensity of 1500lx, 8h light / 16h dark; the second and third rounds of culture were both at a light intensity of 1500lx, 8h light / 16h dark, and cultured for 25 days; A2: the first, second, and third rounds of culture were all at a light intensity of 1500lx, 8h light / 16h dark, and cultured for 25 days; the transfer culture conditions remained unchanged.

[0049] The formula of adventitious bud induction medium was set as: B1: MS + 6-BA 0.5 mg / L + sucrose 15 g / L + agar 5 g / L; B2: MS + 6-BA 0.5 mg / L + TDZ 0.1 mg / L + sucrose 15 g / L + agar 5 g / L; B3: MS + 6-BA 0.5 mg / L + TDZ 0.2 mg / L + sucrose 15 g / L + agar 5 g / L; B4: MS + 6-BA 0.5 mg / L + TDZ 0.3 mg / L + sucrose 15 g / L + agar 5 g / L.

[0050] Adventitious bud induction culture was carried out according to the above conditions. A total of 100 explant tissue blocks were inoculated, and after three rounds of induction culture, the adventitious bud induction rate (number of adventitious buds / number of explant tissue blocks) was calculated. The results are shown in Table 1.

[0051] Table 1 Induction rate of adventitious buds under different conditions (%)

[0052] Adventitious bud induction rate B1 B2 B3 B4 A1 41％ 74％ 69％ 60％ A2 45％ 62％ 57％ 64％

[0053] As can be seen from Table 1, the agarwood explant tissue blocks were first cultured in the dark and then in the light, and induced using the medium of MS+6-BA0.5mg / L+TDZ(0.1-0.2)mg / L+sucrose 15g / L+agar 5g / L, and the induction rate of adventitious buds was high.

[0054] 3. Proliferation culture: Transfer the adventitious buds to the proliferation culture medium and carry out the first round of proliferation culture at 24±1℃, light intensity of 1500lx, and lighting time of 10h light / 14h dark. After 25 days of culture, transfer them to new proliferation culture medium and carry out the second round of proliferation culture under the same conditions. After 25 days of culture, proliferated seedlings are obtained.

[0055] In order to study the factors affecting the proliferation speed of the proliferating seedlings, the invention screens the formula of the proliferating culture medium.

[0056] The proliferation medium formula was set as: C1: 1 / 2MS+6-BA 0.1mg / L+agar 5g / L; C2: 1 / 2MS+IBA0.2mg / L+agar 5g / L; C3: 1 / 2MS+6-BA 0.1mg / L+IBA 0.1mg / L+agar 5g / L; C4: 1 / 2MS+6-BA0.1mg / L+IBA 0.2mg / L+agar 5g / L; C5: 1 / 2MS+6-BA0.1mg / L+IBA 0.3mg / L+agar 5g / L.

[0057] Proliferation culture was carried out according to the above-mentioned proliferation medium formula. A total of 100 adventitious buds were inoculated. After 25 days of culture, the number of proliferated buds was counted and the proliferation multiple (number of proliferated buds / number of adventitious buds inoculated) was calculated. The results are shown in Table 2.

[0058] Table 2 Proliferation results of different proliferation medium

[0059]

[0060] As can be seen from Table 2, when the culture medium of 1 / 2MS+6-BA 0.1mg / L+IBA (0.1-0.3)mg / L+agar 5g / L is used for proliferation culture, the proliferation rate of adventitious buds is faster, and the proliferation rate is fastest when the IBA concentration is 0.2mg / L.

[0061] 4. Rooting culture: Transfer the proliferated seedlings to the rooting medium and carry out rooting culture at 24±1℃, light intensity of 2500lx, and lighting time of 12h light / 12h dark. After 25 days of culture, rooted test tube seedlings are obtained.

[0062] In order to study the factors affecting the rooting speed of the proliferated seedlings, the present invention screens the formula of the rooting medium.

[0063] The rooting medium formula was set as: D1: 1 / 2MS+NAA0.05mg / L+agar 5g / L; D2: 1 / 2MS+Vitamin A1.0mg / L+Agar 5g / L; D3: 1 / 2MS+NAA 0.05mg / L+Vitamin A 0.5mg / L+Agar 5g / L; D4: 1 / 2MS+NAA0.05mg / L+Vitamin A1.0mg / L+Agar 5g / L; D5: 1 / 2MS+NAA 0.05mg / L+Vitamin A1.5mg / L+Agar 5g / L; D6: 1 / 2MS+NAA0.05mg / L+Vitamin A 2.0mg / L+Agar 5g / L.

[0064] Rooting culture was carried out according to the above rooting medium formula. A total of 100 single buds of the proliferation seedlings with a height range of 1.8-2.0 cm were inoculated. After 25 days of culture, the rooting rate (number of roots / number of proliferation seedlings inoculated) was counted, and the proportion of test tube seedlings with more than 10 roots was counted. The results are shown in Table 3.

[0065] Table 3 Rooting results of different rooting media

[0066]

[0067]

[0068] It can be seen from Table 3 that the use of 1 / 2MS + NAA 0.05 mg / L + vitamin A (0.5-1.5) mg / L + agar 5 g / L for rooting culture can improve the rooting rate and rooting potential of the proliferated seedlings.

[0069] 5. Hardening and transplanting: Place 50 rooted test tube seedlings under natural light, use a sunshade to control the light intensity to about 6000lx for 10 days. After hardening, take out the test tube seedlings, wash off the culture medium on the roots, transplant them to sandy loam, and cultivate them for 2 months according to conventional field management methods. The survival rate of the seedlings is 90%.

[0070] Example 2

[0071] 1. Disinfection of explants: Collect young stem segments of agarwood, rinse the stem segments with running water for 5 minutes to remove dirt and other impurities attached to the surface, soak the rinsed stem segments in 75% alcohol for 1 minute, then transfer the stem segments to 0.1% mercuric chloride for disinfection for 10 minutes, and finally rinse with sterile water for 4 times. Place the disinfected stem segments on a sterile workbench and trim them into 1cm×1cm explant tissue blocks.

[0072] 2. Adventitious bud induction culture: Inoculate the explant tissue block in the adventitious bud induction medium for the first round of culture: 24±1℃ dark culture for 5 days, then adjust the light intensity to 1500lx, 8h light / 16h dark, culture for 20 days; then transfer the tissue block to a new adventitious bud induction medium for the second round of culture: 24±1℃, light intensity of 1500lx, 8h light / 16h dark, culture for 25 days; transfer to a new adventitious bud induction medium again for the third round of culture: 24±1℃, light intensity of 1500lx, 8h light / 16h dark, culture for 25 days, and obtain adventitious buds. The adventitious bud induction rate is 75%.

[0073] The adventitious bud induction medium is: MS+6-BA 0.5mg / L+TDZ 0.1mg / L+sucrose 15g / L+agar 5g / L.

[0074] 3. Proliferation culture: The adventitious buds were transferred to the proliferation culture medium for the first round of proliferation culture: 24±1℃, light intensity 1500lx, lighting time 10h light / 14h dark, cultured for 25 days; then transferred to a new proliferation culture medium, and the second round of proliferation culture was carried out under the same conditions. After 25 days of culture, the proliferated seedlings were obtained. The proliferation multiple was 3.47.

[0075] The proliferation medium is: 1 / 2MS+6-BA 0.1mg / L+IBA0.2mg / L+agar 5g / L.

[0076] 4. Rooting culture: Transfer the proliferated seedlings to the rooting medium and carry out rooting culture at 24±1℃, light intensity of 2500lx, and light time of 12h light / 12h dark. After 25 days of culture, rooted test tube seedlings were obtained. The rooting rate was 90%.

[0077] The rooting medium is: 1 / 2MS+NAA 0.05mg / L+Vitamin A 1.0mg / L+Agar 5g / L.

[0078] 5. Hardening and transplanting: Place the rooted test tube seedlings under natural light, and use a sunshade to control the light intensity to about 6000lx for 10 days. After hardening, take out the test tube seedlings, wash off the culture medium on the roots, transplant them to sandy loam, and cultivate them for 2 months according to conventional field management methods. The survival rate of the seedlings is 88%.

[0079] Example 3

[0080] 1. Disinfection of explants: Collect the tender shoots of agarwood, rinse them with running water for 5 minutes to remove dirt and other impurities attached to the surface, soak the rinsed tender shoots in 75% alcohol for 1 minute, then transfer the tender shoots to 0.1% mercuric chloride for disinfection for 10 minutes, and finally rinse them with sterile water for 5 times. Place the disinfected tender shoots on a sterile workbench and trim them into 0.5cm×1cm explant tissue blocks.

[0081] 2. Adventitious bud induction culture: Inoculate the explant tissue block in the adventitious bud induction medium for the first round of culture: 24±1℃ dark culture for 5 days, then adjust the light intensity to 1500lx, 8h light / 16h dark, culture for 20 days; then transfer the tissue block to a new adventitious bud induction medium for the second round of culture: 24±1℃, light intensity of 1500lx, 8h light / 16h dark, culture for 25 days; transfer to a new adventitious bud induction medium again for the third round of culture: 24±1℃, light intensity of 1500lx, 8h light / 16h dark, culture for 25 days, and obtain adventitious buds. The adventitious bud induction rate is 80%.

[0082] The adventitious bud induction medium is: MS+6-BA 0.5mg / L+TDZ 0.1mg / L+sucrose 15g / L+agar 5g / L.

[0083] 3. Proliferation culture: The adventitious buds were transferred to the proliferation culture medium for the first round of proliferation culture: 24±1℃, light intensity 1500lx, lighting time 10h light / 14h dark, cultured for 25 days; then transferred to a new proliferation culture medium, and the second round of proliferation culture was carried out under the same conditions. After 25 days of culture, the proliferated seedlings were obtained. The proliferation multiple was 3.56.

[0084] The proliferation medium is: 1 / 2MS+6-BA 0.1mg / L+IBA0.2mg / L+agar 5g / L.

[0085] 4. Rooting culture: Transfer the proliferated seedlings to the rooting medium and carry out rooting culture at 24±1℃, light intensity of 2500lx, and light time of 12h light / 12h dark. After 25 days of culture, rooted test tube seedlings were obtained. The rooting rate was 90%.

[0086] The rooting medium is: 1 / 2MS+NAA 0.05mg / L+Vitamin A 1.0mg / L+Agar 5g / L.

[0087] 5. Hardening and transplanting: Place the rooted test tube seedlings under natural light, and use a sunshade to control the light intensity to about 6000lx for 10 days. After hardening, take out the test tube seedlings, wash off the culture medium on the roots, transplant them to sandy loam, and cultivate them for 2 months according to conventional field management methods. The survival rate of the seedlings is 93%.

[0088] in conclusion:

[0089] Agarwood is an extremely precious wood, and is often made into hand strings, pendants and other cultural and artistic ornaments. In recent years, the antique market has risen, and the market value of agarwood has been rising, and the problem of the scarcity of high-quality agarwood seedlings has followed. At present, common plant breeding and propagation methods are not applicable to agarwood, and the tissue culture method of agarwood is rare. The common adventitious bud induction rate, slow proliferation rate, and low survival rate in its tissue culture process are not clearly solved. The present invention has found that 6-BA and TDZ have a greater impact on the adventitious bud induction rate of agarwood explants. By adjusting the concentration of 6-BA and TDZ, combined with the dark culture in the early stage of induction, the explant cells are dedifferentiated, and callus with strong differentiation ability is generated, which improves the induction rate of adventitious buds on the one hand, and the activity of cells is strong on the other hand, which lays a material foundation for the next step of cell proliferation. The present invention also found that IBA, as a plant growth regulating hormone, has a greater influence on the proliferation rate of adventitious buds of agarwood. By optimizing the concentration of IBA in the proliferation medium, the proliferation rate of adventitious buds is increased, and the time for agarwood seedlings to leave the factory is shortened. The present invention also increases the rooting rate of agarwood by adding NAA and vitamin A to the rooting medium. NAA is a plant growth regulator that can promote cell division, and vitamin A is a fat-soluble vitamin that has the effect of promoting plant growth and development. The present invention found that the combined use of NAA and vitamin A can increase the rooting rate to 92%, and at the same time increase the number of roots of the proliferated seedlings, that is, increase the rate of strong seedlings of the proliferated seedlings, so that the proliferated seedlings are not prone to yellowing, falling off, death, etc. during subsequent seedling hardening, and are easier to survive after transplanting. The tissue culture method of the present invention can increase the induction rate of adventitious buds to 74%, the proliferation rate to 3.52, the rooting rate to 92%, and the survival rate to 93%, and can be used to cultivate high-quality agarwood seedlings in large quantities.

[0090] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for tissue culture of Aquilaria sinensis seedlings, characterized in that: The steps include: (1) inoculating the sterilized explants into an adventitious bud induction medium, inducing culture, and obtaining adventitious buds; (2) inoculating the adventitious buds into a proliferation medium, culturing them for proliferation, and obtaining proliferated seedlings; (3) inoculating the proliferated seedlings into a rooting medium, performing rooting culture, and obtaining test tube seedlings; (4) after hardening the test tube seedlings, transplanting them to obtain the agarwood seedlings; The adventitious bud induction medium is: MS+6-BA (0.3-0.6) mg / L+TDZ (0.1-0.2) mg / L+sucrose (12-18) g / L+agar (5-10) g / L; The proliferation medium is: 1 / 2MS+6-BA (0.05-0.2) mg / L+IBA (0.1-0.3) mg / L+agar (5-10) g / L; The rooting medium is: 1 / 2MS+NAA (0.01-0.1) mg / L+Vitamin A (0.5-1.5) mg / L+Agar (5-10) g / L; The induction culture conditions are dark culture first and then light culture.

2. The tissue culture method according to claim 1, characterized in that The adventitious bud induction medium is: MS+6-BA 0.5mg / L+TDZ 0.1mg / L+sucrose 15g / L+agar 5g / L; The proliferation medium is: 1 / 2MS+6-BA 0.1mg / L+IBA 0.2mg / L+agar 5g / L; The rooting medium is: 1 / 2MS+NAA 0.05mg / L+Vitamin A 1.0mg / L+Agar 5g / L.

3. The tissue culture method according to claim 1, characterized in that The temperature during the dark culture is 24±1° C. and the time is 3-6 days.

4. The tissue culture method according to claim 1, characterized in that The temperature during the light culture is 24±1° C., the light intensity is 1500 lx, the light conditions are 8 h light / 16 h dark, and the total time of the light culture is 60-85 days.

5. The tissue culture method according to claim 1, characterized in that The conditions for the proliferation culture are: the culture temperature is 24±1° C., the light intensity is 1500 lx, and the illumination time is 10 h light / 14 h dark.

6. The tissue culture method according to claim 1, characterized in that The rooting culture conditions are as follows: the culture temperature is 24±1° C., the light intensity is 2500 lx, and the lighting time is 12 h light / 12 h dark.

7. The tissue culture method according to claim 1, characterized in that The light intensity during the hardening of the seedlings is 5000-8000lx.

8. The tissue culture method according to claim 1, characterized in that The proliferation culture time is 50-60 days.

9. The tissue culture method according to claim 1, characterized in that: The rooting culture time is 25-30 days.

10. The tissue culture method according to claim 1, characterized in that: The time for hardening the seedlings is 8-15 days.

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