A tissue culture method for an oak tree

Abstract

The present invention discloses a tissue culture method for oak trees, which comprises the following steps: Cluster bud differentiation culture: Using the aseptic seedlings of Quercus alba as materials, cutting off the redundant new leaves, retaining the aseptic buds of 1.5-2 cm per plant and inoculating them onto the cluster bud differentiation medium for differentiation culture; Rooting culture: Obtaining the young and tender buds of aseptic seedlings through step (1), cutting off the redundant new leaves, retaining 1.5-2 cm per plant, and inoculating them onto the rooting medium for rooting culture. The cluster bud differentiation medium can rapidly breed a large number of cluster seedlings, and the multiplication coefficient can reach 7.13; The rooting medium can rapidly induce rooting, with a rooting rate of 93%, fast rooting speed and a large number of root numbers; At the same time, an appropriate concentration of Cef is added to the medium, which can further inhibit the activity of endophytic bacteria and reduce the influence of endophytic bacteria on the growth of Quercus alba seedlings, thereby increasing the multiplication coefficient and rooting rate.

Description

Technical Field

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

[0002] Oak (Quercus palustris Münchh.), also known as oak or oak, is a large deciduous tree of the genus Quercus (Quercus L.) of the Fagaceae family. It usually refers to plants of the genus Quercus, not a specific species. Native to Europe, Anatolia and the Caucasus, it has the characteristics of tall tree body, luxuriant branches and leaves, beautiful tree shape, cold and drought resistance, and strong adaptability. Oak wood is solid and has beautiful texture. It can be used in the construction industry, as well as in the manufacture of oak barrels and precious furniture, and has good economic value; its leaves are beautiful in shape, turn golden in autumn and last for a long time, making it an excellent ornamental tree species and landscaping tree species; its seeds have a high starch content and can be used for food and winemaking, and have a very broad development prospect.

[0003] Tissue culture technology has been widely used in asexual reproduction of more than 1,500 plant species. The study of tissue culture of Fagaceae plants originated in the 1950s and became popular in the 1980s. So far, studies on Quercus, Castanopsis, Castanea and Lithospermum have been reported. Tissue culture has become one of the important means of rapid propagation of Fagaceae plants. Oak has important application prospects in my country, but its fruiting quantity is obvious, flowering and fruiting are late, vegetative propagation is difficult, it is difficult to establish a seed garden and cannot obtain pure lines through continuous backcrossing, seed germination is difficult and growth is slow, and seeds have poor uniformity of germination under natural conditions, which makes it difficult to produce oak seedlings on a large scale, and it is even more difficult to cultivate selected high-quality seedlings, which seriously restricts the development of my country's oak industry. Therefore, there is an urgent need for an oak tissue culture method with a short breeding cycle, simple operation, high reproduction coefficient and high rooting rate. Summary of the invention

[0004] Based on the above problems, the present invention provides a tissue culture method for oak trees, which has high propagation coefficient and high rooting rate.

[0005] The present invention is achieved by adopting the following technical solutions:

[0006] A method for tissue culture of oak trees comprises the following steps:

[0007] ⑴ Cluster bud differentiation culture: Use oak sterile seedlings as materials, cut off the extra new leaves of the sterile seedlings, keep the sterile buds of 1.5-2 cm per plant, and inoculate them into cluster bud differentiation medium for differentiation culture to obtain young buds of sterile seedlings;

[0008] ⑵ Rooting culture: Obtain the young shoots of the aseptic seedlings through step ⑴, cut off the redundant newly grown leaves, retain 1.5 - 2 cm per single plant, and transfer them onto the rooting medium for rooting culture.

[0009] Furthermore, the cluster bud differentiation medium in step (1) is the WPM basic medium supplemented with 0.2 - 0.3 mg / L 6 - BA and 50 - 100 mg / L Cef.

[0010] Furthermore, the cluster bud differentiation medium is supplemented with 29 - 31 g / L of sucrose, 5.8 - 6.2 g / L of agar, and the pH is 5.7 - 5.9.

[0011] The rooting medium in step (2) is the 1 / 2MS basic medium supplemented with 0.1 - 0.5 mg / L of naphthaleneacetic acid (NAA) and 100 mg / L of cefotaxime sodium (Cef).

[0012] The rooting medium is supplemented with 29 - 31 g / L of sucrose, 5.8 - 6.2 g / L of agar, and the pH is 5.7 - 5.9.

[0013] The conditions for the cluster bud differentiation culture in step ⑴ and the rooting culture in step ⑵ are both: culture temperature (25 ± 2)°C, light duration 16 - 17 h / d.

[0014] The culture of the Quercus aseptic seedlings includes the following steps:

[0015] (1) Seed pretreatment: Remove the seed coat of the Quercus alba seeds to obtain the zygotic embryos with cotyledons;

[0016] (2) Disinfection and sterilization of the seeds: Rinse the zygotic embryos with running water, rinse with alcohol, rinse with sterile water, soak in sodium hypochlorite solution, rinse with sterile water again, and soak in plant tissue culture antibacterial agent solution;

[0017] (3) Seed germination culture: Inoculate the sterilized zygotic embryos into the seed germination medium to promote the germination of roots, shoots and stems to obtain Quercus alba aseptic seedlings.

[0018] Preferably, the disinfection and sterilization steps of the seeds in step ⑵ are: Rinse the zygotic embryos with running water for 4 h, rinse with 75% (v / v) alcohol for 90 s, rinse with sterile water once, soak in 10% (w / v) sodium hypochlorite solution for 15 min, rinse with sterile water three times, and soak in plant tissue culture antibacterial agent (PPM) solution for 24 h.

[0019] Preferably, the seed germination medium in step ⑶ is the WPM basic medium supplemented with 0.002 mg / L TDZ and 100 mg / L Cef.

[0020] Further, the oak seeds in step (1) are selected from the American white oak plants of excellent individual plants with good growth, no pests and diseases, and strong stress resistance.

[0021] The advantages and beneficial effects of the present invention are as follows:

[0022] In the cluster bud differentiation medium of the present invention, an appropriate concentration of 6-benzylaminopurine (6-BA) and sodium cephalosporin are added to the WPM basic medium. The WPM basic medium, 6-BA, and sodium cephalosporin act together to have a better effect of promoting cell differentiation, can improve the differentiation of buds, inhibit the reproduction of endophytes, and reduce the negative impact of endophytes on bud differentiation. Under their combined action, the cluster bud differentiation medium can rapidly breed a large number of cluster seedlings, and the multiplication coefficient can reach 7.13. The rooting medium is prepared by adding naphthaleneacetic acid (NAA) and sodium cephalosporin to the 1 / 2MS basic medium. The 1 / 2MS medium contains a small amount of nutrients. The 1 / 2MS medium, naphthaleneacetic acid (NAA), and sodium cephalosporin act together to not only reduce the negative impact of endophytes on rooting but also accelerate the growth of plant roots to adapt to the environment. Under the combined action of the three, rooting can be rapidly induced, and the rooting rate reaches 93%. Moreover, the rooting is fast and the number of rootings is large. The seed germination medium is the WPM basic medium added with 0.002 mg / L TDZ and 100 mg / L Cef. After adding thidiazuron (TDZ) and Cef to the WPM basic medium, the activity of endophytes can be inhibited, the influence of endophytes on the growth of oak seedlings can be reduced, thereby increasing the multiplication coefficient and rooting rate. The cultivation method of the oak sterile seedlings of the present invention is to germinate and culture the zygotic embryos with the seed coats removed from the oak on the seed germination medium, which can make the zygotic embryos germinate multiple times with a high emergence rate; then combined with soaking the zygotic embryos in a 5% PPM solution for 24 h, rapid and efficient sterilization and long-lasting antibacterial effects can be achieved. The present invention has established an oak tissue culture method with a short breeding cycle, simple operation, high propagation coefficient, and high rooting rate, which can obtain a large number of seedlings in a short time and provides technical support for the rapid development of the oak industry. Description of the Drawings

[0023] Figure 1 It is a flow chart of a tissue culture method for an oak tree of the present invention;

[0024] Figure 2 It is a state diagram of the zygotic embryo of the oak tree with the outer shell removed in Example 1;

[0025] Figure 3 It is a state diagram of the sterilized zygotic embryo of the oak tree in Example 1.

[0026] Figure 4 It is a germination state diagram of the zygotic embryo of the oak tree in Example 1.

[0027] Figure 5It is a diagram of the differentiation state of adventitious buds of oak trees;

[0028] Figure 6 It is a diagram of the root growth state of oak trees. Specific implementation manners

[0029] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments, and do not limit the present invention. In the present invention, the materials and reagents involved are all commercially available products, or can be obtained by conventional technical means in the art.

[0030] The oak trees described in the embodiments and experimental examples of this application are Quercus alba, and the Latin name is: Quercus alba L.

[0031] The composition of the WPM basal medium described in the embodiments and experimental examples of this application is: K2SO4 990 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4 170 mg / L, NH4NO3 400 mg / L, MnSO4·4H2O 22.5 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 36.2 mg / L, CuSO4·5H2O 0.25 mg / L, Na2MoO4·2H2O 0.25 mg / L, Ca(NO3)2·4H2O 556 mg / L, FeSO4·7H2O 27.8 mg / L, Na2EDTA 37.3 mg / L, inositol 100 mg / L, glycine 2.0 mg / L, VB1 1.0 mg / L, VB6 0.5 mg / L, VB5 0.5 mg / L.

[0032] The composition of the 1 / 2MS basal medium is as follows: KNO3 950 mg / L, NH4NO3 825 mg / L, CaCl2·2H2O 220 mg / L, MgSO4·7H2O 185 mg / L, KH2PO4 85 mg / L, MnSO4·4H2O 11.15 mg / L, ZnSO4·7H2O 4.3 mg / L, H3BO3 43.1 mg / L, KI 0.415 mg / L, Na2MoO4·2H2O 0.125 mg / L, CuSO4·5H2O 0.0125 mg / L, CoCl·6H2O 0.0125 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, inositol 100 mg / L, glycine 2 mg / L, nicotinic acid 0.5 mg / L, VB6 0.5 mg / L, VB1 0.1 mg / L.

[0033] In the examples and experimental examples of this application, the brand of the plant tissue culture antibacterial agent (PPM) is PCT lnc., the number is PPM-100mL, and the product name is PPM. TM (Plant Preservative Mixture) Plant tissue culture antibacterial agent.

[0034] In the examples and experimental examples of this application, the aseptic seedlings of Quercus can be the commercially available aseptic seedlings of Quercus alba, or the aseptic seedlings of Quercus alba cultivated by oneself. In the examples of this application, the aseptic seedlings of Quercus alba cultivated by oneself are used.

[0035] Example 1

[0036] As Figure 1 shown, this example provides a tissue culture method for Quercus, including the following steps:

[0037] (1) Seed pretreatment: As Figure 2 shown, select the seeds of Quercus alba plants with good growth, no pests and diseases, and strong stress resistance from excellent individual plants, remove the seed coats of the Quercus alba seeds to obtain zygotic embryos with cotyledons;

[0038] (2) Disinfection and sterilization of seeds: Rinse the zygotic embryos with running water for 4 h, rinse them with 75% (v / v) alcohol for 90 s, rinse once with sterile water, soak them in 10% (w / v) sodium hypochlorite solution for 15 min, rinse three times with sterile water, and soak them in 5% (w / v) PPM solution for 24 h. The sterilized zygotic embryos are as Figure 3 shown.

[0039] ⑶ Seed germination culture: Inoculate the sterilized zygotic embryos into the seed germination medium to promote the germination of roots, shoots and stems, and obtain aseptic seedlings of Quercus alba. The culture conditions are as follows: the culture temperature is (25±2)°C, and the light duration is 16 h / d; the composition of the seed germination medium is: WPM + 0.002 mg / L TDZ + 100 mg / L Cef + 30 g / L sucrose + 6 g / L agar, and the pH is 5.8. The germinated zygotic embryos are as Figure 4 shown.

[0040] ⑷ Cluster bud differentiation culture: Divide the new shoots of aseptic seedlings of Quercus alba induced in the seed germination medium into single aseptic seedlings, cut off the redundant new leaves, and retain the aseptic buds of 1.5 cm per single plant and inoculate them onto the cluster bud differentiation medium for differentiation culture to obtain young and tender buds of aseptic seedlings; the culture conditions are as follows: the culture temperature is (25±2)°C, and the light duration is 16 h / d; the composition of the cluster bud differentiation medium is: WPM + 0.3 mg / L 6-BA + 100 mg / L Cef + 30 g / L sucrose + 6 g / L agar, and the pH is 5.8.

[0041] ⑸ Rooting culture: Obtain the young and tender buds of aseptic seedlings through step ⑷, cut off the redundant new leaves, retain 1.5 cm per single plant, and inoculate them onto the rooting medium for rooting culture. The culture conditions are as follows: the culture temperature is (25±2)°C, and the light duration is 16 h / d. The composition of the rooting medium is: 1 / 2MS + 0.5 mg / L NAA + 100 mg / L Cef + 30 g / L sucrose + 6 g / L agar, and the pH is 5.8.

[0042] After 30 days, count the rooting rate, rooting time, length of the longest root, average number of roots, and average root length. The results are shown in Table 5.

[0043] Example 2

[0044] This example provides a tissue culture method for oak trees, including the following steps:

[0045] (1) Seed pretreatment: Select the seeds of Quercus alba plants with good growth, no diseases and pests, and strong stress resistance from excellent single plants, remove the seed coats of Quercus alba seeds to obtain zygotic embryos with cotyledons;

[0046] ⑵ Seed disinfection and sterilization: Rinse the zygotic embryos with running water for 4 h, rinse them with 75% alcohol by volume for 90 s, rinse once with sterile water, soak them in 10% sodium hypochlorite solution by mass for 15 min, rinse three times with sterile water, and soak them in 5% PPM solution by mass for 24 h.

[0047] ⑶ Seed germination culture: The sterilized zygotic embryos were inoculated into the seed germination medium to promote the germination of roots, shoots and stems, and sterile seedlings of Quercus alba were obtained. The culture conditions were: culture temperature (25±2)°C, light duration 16 h / d; the seed germination medium in step ⑶ had the following composition: WPM + 0.002 mg / L TDZ + 100 mg / L Cef + 30 g / L sucrose + 6 g / L agar, with a pH of 5.8.

[0048] ⑷ Cluster bud differentiation culture: The new shoots of the sterile seedlings induced in the seed germination medium were divided into single sterile seedlings, the redundant new leaves were removed, and the 1.5 cm sterile buds of each single plant were inoculated onto the cluster bud differentiation medium for differentiation culture. The culture conditions were: culture temperature (25±2)°C, light duration 16 h / d; the cluster bud differentiation medium had the following composition: WPM + 0.2 mg / L 6-BA + 50 mg / L Cef + 30 g / L sucrose + 6 g / L agar, with a pH of 5.8.

[0049] ⑸ Rooting culture: The young tender buds of the sterile seedlings obtained from ⑷ were used, the redundant new leaves were removed, and the remaining 1.5 cm was inoculated onto the rooting medium for rooting culture. The culture conditions were: culture temperature (25±2)°C, light duration 16 h / d. The rooting medium in ⑸ had the following composition: 1 / 2MS + 0.2 mg / L NAA + 100 mg / L Cef + 30 g / L sucrose + 6 g / L agar, with a pH of 5.8.

[0050] After 30 days, the rooting rate, rooting time, length of the longest root, average number of roots, and average root length were counted, and the results are shown in Table 5.

[0051] Experimental Example 1

[0052] Experiment on the Effect of Different Growth Regulators and Their Concentrations on Cluster Bud Differentiation

[0053] Five concentration gradients of 6-BA were set at 0.1, 0.2, 0.3, 0.4, and 0.5 mg / L, and five concentration gradients of TDZ were set at 0.002, 0.004, 0.01, 0.02, and 0.05 mg / L. They were added to the WPM medium, and a cluster bud differentiation experiment was carried out on the sterile oak seedlings obtained in Example 1. The new shoots of the sterile oak seedlings were divided into single sterile seedlings, the redundant new leaves were removed, and the 1.5 cm sterile buds of each single plant were inoculated onto the WPM medium, which also contained 30 g / L of sucrose, 6 g / L of agar, with a pH of 5.8. The temperature for cluster bud differentiation culture was (25±2)°C, and the light duration was 16 h / d. The explant was a 1.5 cm single sterile bud. There were five groups for each treatment, and each group had three single buds. After 30 days, the differentiation of adventitious buds was counted. The results are shown in Table 1.

[0054] Table 1. Effects of Different Plant Growth Regulators and Different Concentrations on the Differentiation of Cluster Buds of Oak

[0055]

[0056] (Note: Different lowercase letters indicate significant differences (P < 0.05))

[0057] The results showed that 6-BA had a better effect on inducing the differentiation of cluster buds and a faster differentiation time. Among the many experimental groups, 0.3 mg / L 6-BA had the best effect on inducing the differentiation of cluster buds, with a proliferation coefficient of 7.08, showing significant differences from other treatments.

[0058] Experimental Example 2

[0059] Experiment on the Effects of Different Antibiotics and Different Concentrations on the Differentiation of Cluster Buds

[0060] Five concentration gradients of cefotaxime sodium (Cef) were set at 0, 10, 50, 100, and 200 mg / L, five concentration gradients of plant tissue culture antibacterial agent (PPM) were set at 0.005, 0.01, 0.02, 0.05, and 0.1%, and five concentration gradients of ticarcillin / clavulanate potassium (Tim) were set at 0, 10, 50, 100, and 300 mg / L. They were added to the WPM + 0.3 mg / L 6-BA medium to conduct an experiment on the differentiation of cluster buds of the sterile oak seedlings obtained in Example 1. The new shoots of the sterile oak seedlings were divided into single-plant sterile seedlings, the excess new leaves were cut off, and the 1.5 cm sterile buds of each single plant were retained and inoculated onto the WPM + 0.3 mg / L 6-BA medium for differentiation culture. The medium also contained 30 g / L of sucrose, 6 g / L of agar, with a pH of 5.8. The temperature for the differentiation culture of cluster buds was (25 ± 2) °C, and the light time was 16 h / d. The explants were 1.5 cm single-plant sterile buds. There were five groups for each treatment, with three single-plant buds in each group. After 30 days, the differentiation of adventitious buds was counted. The results are shown in Table 2.

[0061] Table 2. Effects of Different Antibiotics and Different Concentrations on the Differentiation of Cluster Buds of Oak

[0062]

[0063] (Note: Different lowercase letters indicate significant differences (P < 0.05))

[0064] From the experimental data in Table 2, it can be seen that the effect of adding PPM was better than that of Tim, but the effect of adding Cef was better. Moreover, when 100 mg / L Cef was added, the proliferation coefficient was the highest, reaching 7.13, showing significant differences from other treatments.

[0065] As can be seen from Table 1 and Table 2, when the culture medium formula is WPM + 0.3 mg / L 6-BA + 100 mg / L Cef + 30 g / L sucrose + 6 g / L agar, the induction effect on the adventitious bud differentiation of Quercus alba is the best, the induction coefficient is the highest, and a large number of adventitious buds can be obtained in a short time. The effect of adventitious bud differentiation is as Figure 5 shown.

[0066] Experimental Example 3

[0067] Experiment on the effect of different basic culture media on the rooting of oak trees:

[0068] Add 0.3 mg / L IBA and 0.1 mg / L NAA to four basic culture media of 1 / 4 MS, 1 / 2 MS, MS, and WPM. At the same time, it also contains 30 g / L sucrose, 6 g / L agar, the pH is 5.8, the temperature for rooting culture is (25±2) °C, and the light time is 16 h / d. The explants are the sterile seedling young shoots obtained in Example 1. Cut off the redundant new leaves, retain 1.5 cm per single plant. There are five groups for each treatment, three single plant buds in each group. After 30 days, count the rooting rate, the length of the longest root, the average number of roots, and the average root length. The results are shown in Table 3.

[0069] Table 3 shows the effect of different basic culture media on the rooting of oak trees, as shown in Table 3:

[0070]

[0071] As can be seen from Table 3, the rooting rates on the four culture media are 73.3%, 80.0%, 20.0%, and 60.0% respectively. The MS culture medium is the least suitable for the rooting of oak trees, and the 1 / 2 MS culture medium is the most suitable for the rooting of oak trees, with a rooting rate of 80.0% and the longest root reaching 9.4 cm.

[0072] Experimental Example 4

[0073] Experiment on the effect of different plant growth regulators and different concentrations on the rooting of oak trees:

[0074] Set five concentration gradients of IBA at 0.2, 0.4, 0.8, 1.0, and 1.6 mg / L and five concentration gradients of NAA at 0.05, 0.1, 0.2, 0.5, and 1.0 mg / L, and add them to the 1 / 2MS medium to root the young tender buds of the aseptic seedlings obtained in Example 1. Cut off the excess new leaves of the young tender buds of the aseptic seedlings, retain 1.5 cm per plant, and also contain 30 g / L of sucrose and 6 g / L of agar, with a pH of 5.8. The temperature for root culture is (25±2)°C, and the light time is 16 h / d. The explant is the 1.5 cm single aseptic bud grown in step (4). There are five groups for each treatment, three single aseptic buds in each group. After 30 days, count the rooting rate, rooting time, length of the longest root, average number of roots, and average root length. The results are shown in Table 4.

[0075] Table 4 shows the effects of different plant growth regulators and different concentrations on the rooting of oak trees, as shown in Table 4:

[0076]

[0077] As can be seen from Table 4, when 0.5 mg / L of NAA is selected, the rooting rate is the highest, reaching 93%, and the rooting time is the fastest, which can induce the rooting of young tender buds of oak trees in a short time. The root growth schematic diagram is as Figure 6 shown.

[0078] Comparative Example 1

[0079] This comparative example provides a tissue culture method for oak trees. The difference between this comparative example and Example 1 is only that in step (4), 1 / 2MS is used instead of WPM in the cluster bud differentiation medium, and in step (5) during the root culture process, the excess new leaves of the germinated aseptic seedlings are cut off, and the 1 cm aseptic bud is retained and inoculated onto the root culture medium for root culture; the rest is the same as in Example 1.

[0080] After 30 days, count the rooting rate, rooting time, length of the longest root, average number of roots, and average root length. The results are shown in Table 5.

[0081] Table 5

[0082]

[0083] As can be seen from Table 5, using the method of the present invention with the aseptic seedlings of Quercus alba as the material, cutting off the excess new leaves, retaining the 1.5 - 2 cm single aseptic bud per plant and inoculating it onto the specially prepared cluster bud differentiation medium of the present invention for differentiation culture to obtain young tender buds of aseptic seedlings; through step Obtain aseptic seedlings of young shoots, cut off the redundant newly grown leaves, retain 1.5 - 2 cm for each single plant, inoculate them onto the special rooting medium of the present invention, and conduct rooting culture. It has a good proliferation effect on inducing clustered buds and an effect on inducing the rooting of buds. The rooted seedlings induced have strong roots and grow well.

[0084] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as a limitation to the present invention. The protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art in this technical field, without departing from the spirit and scope of the present invention, several improvements and refinements can also be made, and these improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A tissue culture method for an oak tree, characterized in that , including the following steps: The oak tree is Quercus alba, ⑴ Cluster bud differentiation culture: Using the aseptic seedlings of Quercus alba as materials, cut off the redundant new leaves, and inoculate the aseptic buds with a single plant of 1.5 - 2 cm into the cluster bud differentiation medium for differentiation culture to obtain young tender buds of aseptic seedlings; ⑵ Rooting culture: Through the young tender buds of aseptic seedlings obtained in step ⑴, cut off the redundant new leaves, retain a single plant of 1.5 - 2 cm, and inoculate it into the rooting medium for rooting culture; The cluster bud differentiation medium in step (1) is the WPM basal medium added with 0.2 - 0.3 mg / L 6-benzylaminopurine, 50 - 100 mg / L sodium thiosporin, 29 - 31 g / L sucrose, 5.8 - 6.2 g / L agar, and the pH is 5.7 - 5.9; The rooting medium in step (2) is the 1 / 2MS basal medium added with 0.1 - 0.5 mg / L naphthylacetic acid and 100 mg / L sodium thiosporin, 29 - 31 g / L sucrose, 5.8 - 6.2 g / L agar, and the pH is 5.7 - 5.

9.

2. The tissue culture method of an oak tree according to claim 1, characterized in that, The conditions for the cluster bud differentiation culture in step ⑴ and the rooting culture in step ⑵ are both: culture temperature (25 ± 2) °C, light time 16 - 17 h / d.

3. The tissue culture method of an oak tree according to claim 1, characterized in that , the culture of the aseptic seedlings of Quercus alba includes the following steps: (1) Seed pretreatment: Remove the seed coat of the Quercus alba seeds to obtain the zygotic embryo with cotyledons; (2) Disinfection and sterilization of seeds: Rinse the zygotic embryo with running water, rinse it with alcohol, rinse it with sterile water, soak it in sodium hypochlorite solution, rinse it with sterile water again, and soak it in the plant tissue culture antibacterial agent solution; (3) Seed germination culture: Inoculate the sterilized zygotic embryo into the seed germination medium to promote the germination of roots, buds and stems to obtain aseptic seedlings of oak trees.

4. The tissue culture method of an oak tree according to claim 3, characterized in that , the disinfection and sterilization of the seeds in step ⑵ includes the following steps: Rinse the zygotic embryo with running water for 4 h, rinse it with 75% (v / v) alcohol for 90 s, rinse it with sterile water once, soak it in 10% (w / v) sodium hypochlorite solution for 15 min, rinse it with sterile water three times, and soak it in the plant tissue culture antibacterial agent solution for 24 h.

5. The tissue culture method of an oak tree according to claim 4, wherein: The seed germination medium in step ⑶ is the WPM basal medium added with 0.002 mg / L thidiazuron and 100 mg / L sodium thiosporin.

6. The tissue culture method of an oak tree according to claim 3, characterized in that: The Quercus alba seeds in step ⑴ are selected from the Quercus alba plants of excellent single plants with good growth, no diseases and pests, and strong stress resistance.

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