Tissue optimization culture method of quercus acutissima

Abstract

The invention belongs to the technical field of plant culture, and particularly relates to a tissue optimization culture method of quercus acutissima. The method comprises the following steps that S1, after german oak seeds with seed coats removed are disinfected, the german oak seeds are inoculated into a germination culture medium, germination seedlings are obtained through culture, and the formula of the germination culture medium comprises an improved WPM culture medium, 6-BA and NAA; s2, the germinated seedlings are cut into stem segments with axillary buds, the stem segments are inoculated into a rapid propagation culture medium to be subjected to subculture multiplication culture, tissue culture seedlings are obtained through culture, and the rapid propagation culture medium is prepared from an improved WPM culture medium, 6-BA, NAA, calcium nitrate, cane sugar and agar; s3, transferring the tissue culture seedlings obtained by the subculture multiplication culture into a rooting culture medium for rooting culture to obtain quercus acutissima tissue culture seedlings; the formula of the rooting culture medium comprises an improved 1 / 2WPM culture medium, NAA, IBA, calcium nitrate, sucrose and agar. According to the method, a seed disinfection method, spectrum conditions, culture medium components and contents are improved, so that the proliferation efficiency is further improved, the problems of tip drying and rooting difficulty in the proliferation culture process are reduced, the quality of quercus acutissima tissue culture seedlings is improved, and the proliferation speed is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cultivation, and in particular relates to a tissue optimization cultivation method of Quercus acutissima. Background Art

[0002] Quercus variabilis Bl. belongs to the genus Quercus (Quercus L.) of the Fagaceae Dumort. family. It is a deciduous tree that is widely distributed in my country and is commonly found in North China, East China, Central China, and Southwest China. The height of the Quercus variabilis tree can reach 15 to 25 meters. Its wood is hard and resistant to decay and is widely used in construction, furniture manufacturing, tool making and other fields. Its fruit can be used to extract starch, which can be used in food processing and feed production. As an important economic and ecological tree species, Quercus variabilis has strong adaptability and a wide range of uses, and contains high cultural and ecological value.

[0003] At present, sowing and seedling raising is the main method of breeding oak, and cutting and grafting techniques are also commonly used for asexual reproduction of oak. In the research on oak tissue culture, although certain achievements have been made in the construction of sterile culture system, callus induction, plant regeneration and other links, it still faces problems such as difficult to control pollution and low regeneration efficiency. In addition, during the tissue culture process, oak plants are prone to stem tip drying and death, and rooting difficulties, which further increases the difficulty of tissue culture of oak plants.

[0004] Therefore, there is an urgent need to optimize the tissue culture method of Quercus acutissima to improve its reproductive efficiency and quality, thereby meeting the market demand for high-quality Quercus acutissima seedlings and promoting the sustainable development of the Quercus acutissima industry. Summary of the invention

[0005] Technical issues

[0006] In view of the above problems existing in the prior art, the present invention aims to provide a tissue culture optimization method capable of increasing the propagation speed of Quercus acutissima and improving the quality of Quercus acutissima.

[0007] Technical Solution

[0008] The first aspect of the present invention provides a tissue optimization culture method for Quercus acutissima, comprising the following steps: S1. After removing the seed coat of Quercus acutissima, the seeds are sterilized and inoculated into a germination medium to obtain germinated seedlings, wherein the formula of the germination medium includes a modified WPM medium, 6-BA and NAA; S2. The germinated seedlings are cut into stem segments with axillary buds, and the segments are inoculated into a rapid propagation medium for subculture and proliferation culture to obtain tissue culture seedlings, wherein the formula of the rapid propagation medium includes a modified WPM medium, 6-BA, NAA, calcium nitrate, sucrose and agar; S3. The tissue culture seedlings obtained by the subculture and proliferation culture are transferred to a rooting medium for rooting culture to obtain Quercus acutissima tissue culture seedlings; the formula of the rooting medium includes a modified 1 / 2WPM medium, NAA, IBA, calcium nitrate, sucrose and agar; wherein, the step S2 selects a specific spectrum lamp to provide a light source.

[0009] In one embodiment of the present invention, the specific conditions for disinfection are: 75% ethanol treatment for 30 seconds and 5% NaClO treatment for 6 minutes.

[0010] In one embodiment of the present invention, the light source characteristics of the specific spectrum lamp are: the three primary colors of the spectrum R:G:B=82.3%:0.7%:17.0%, and the luminous flux is 200lm; or: the three primary colors of the spectrum R:G:B=55.0%:22.2%:22.8%, and the luminous flux is 950lm.

[0011] In one embodiment of the present invention, the culture conditions of the germination medium and the rooting medium are: temperature of 25° C., light duration of 16 h, and light intensity of 5000 lux.

[0012] In one embodiment of the present invention, the culture conditions of the rapid propagation culture medium are: temperature of 25° C. and light exposure time of 16 h.

[0013] In one embodiment of the present invention, the rapid propagation culture time is 30 days.

[0014] In one embodiment of the present invention, the rooting culture time is 30 to 40 days.

[0015] In one embodiment of the present invention, the formula of the germination medium is: modified WPM medium + 6-BA 1.2 mg / L + NAA 0.01 mg / L + calcium nitrate 350 mg / L + sucrose 20 g / L + agar 6 g / L.

[0016] In one embodiment of the present invention, the formula of the rapid propagation culture medium is: improved WPM culture medium + 6-BA 1.0-1.8 mg / L + NAA 0-0.04 mg / L + calcium nitrate 0-700 mg / L + sucrose 20 g / L + agar 6 g / L, and the pH value is adjusted to 5.8-6.0.

[0017] In one embodiment of the present invention, the formula of the rapid propagation culture medium is: improved WPM culture medium + 6-BA 1.0-1.4 mg / L + NAA 0.01-0.02 mg / L + calcium nitrate 150-350 mg / L + sucrose 20 g / L + agar 6 g / L, and the pH value is adjusted to 5.8-6.0.

[0018] In one embodiment of the present invention, the formula of the rapid propagation culture medium is: improved WPM medium + 6-BA 1.2 mg / L + NAA 0.01 mg / L + calcium nitrate 350 mg / L + sucrose 20 g / L + agar 6 g / L, and the pH value is adjusted to 5.8-6.0.

[0019] In one embodiment of the present invention, the formula of the rooting medium is: improved 1 / 2WPM medium + NAA 0.5-3 mg / L + IBA 0.5-3 mg / L + calcium nitrate 100-450 mg / L + sucrose 20 g / L + agar 6 g / L, and the pH value is adjusted to 5.8-6.0.

[0020] In one embodiment of the present invention, the formula of the rooting medium is: improved 1 / 2WPM medium + NAA 0.5 mg / L + IBA 0.5 mg / L + calcium nitrate 150 mg / L + sucrose 20 g / L + agar 6 g / L, and the pH value is adjusted to 5.8-6.0.

[0021] In one embodiment of the present invention, the formula of the improved WPM medium is: CaCl 2 72.5 mg / L+KH 2 PO 4 170mg / L+K 2 SO 4 990 mg / L+MgSO 4 180.54 mg / L+NH 4 NO 3 400 mg / L+CuSO 4 ·5H 2 O 0.25mg / L+FeNaEDTA36.7mg / L+H 3 BO 3 6.2 mg / L+MnSO 4 ·H 2 O 22.3mg / L+Na2 MoO 4 0.25 mg / L+ZnSO 4 7H 2 O8.6mg / L+glycine 2mg / L+inositol 100mg / L+nicotinic acid 0.5mg / L+thiamine hydrochloride 1mg / L+pyridoxine hydrochloride 0.5mg / L; the formula of the improved 1 / 2WPM medium is: CaCl 2 36.25 mg / L+KH 2 PO 4 85 mg / L+K 2 SO 4 495 mg / L+MgSO 4 90.27 mg / L+NH 4 NO 3 200 mg / L+CuSO 4 ·5H 2 O0.25mg / L+FeNaEDTA36.7 mg / L+H 3 BO 3 6.2mg / L+MnSO 4 ·H 2 O 22.3mg / L+Na 2 MoO 4 0.25mg / L+ZnSO 4 7H 2 O 8.6mg / L+glycine 2mg / L+inositol 100mg / L+niacin 0.5mg / L+thiamine hydrochloride 1mg / L+pyridoxine hydrochloride 0.5mg / L.

[0022] In one embodiment of the present invention, the rapid propagation medium and the rooting medium are sterilized by high temperature and high pressure.

[0023] In one embodiment of the present invention, the rapid propagation medium and the rooting medium are sterilized at 121° C. for 18 minutes.

[0024] Technical Effects

[0025] The invention provides a method for tissue culture of Quercus acutissima, comprising seed disinfection, tissue culture rapid propagation, light quality proliferation and rooting method. By improving the seed disinfection method, different light quality treatments, WPM culture medium and 1 / 2WPM culture medium, the problems of high infection rate during disinfection, dry tips and difficulty in rooting during proliferation culture are overcome, the propagation speed is increased, and the quality of Quercus acutissima tissue culture seedlings is improved.

[0026] Specifically, the seed disinfection method provided by the present invention improves the seed germination rate while further reducing the contamination rate and the browning rate, indicating that the disinfection method is suitable for seed germination; the spectral conditions provided by the present invention enable the proliferation coefficient to reach about 15, the strong bud rate to reach about 50%, and the average number of strong buds to reach more than 7, indicating that the spectral conditions are suitable for cultivating tissue culture seedlings of Quercus acutissima; the rooting culture medium provided by the present invention enables the rooting rate to reach about 85%, while reducing the shoot tip mortality rate to below 25%, indicating that the culture medium is suitable for rooting culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a comparison chart between the bacteria infection during seed disinfection and normal growth;

[0028] Figure 2 This is the effect of different disinfection methods on the disinfection results of Quercus acutissima seeds;

[0029] Figure 3 The growth status of Quercus acutissima tissue culture seedlings under full spectrum and white light conditions;

[0030] Figure 4 This is a diagram showing the effects of different spectra on plant growth;

[0031] Figure 5 This is a diagram showing the effect of basic culture medium on rooting culture;

[0032] Figure 6 This is a comparison chart of poor growth and well-developed root system in rooting culture;

[0033] Figure 7 This is the effect of NAA concentration on rooting culture;

[0034] Figure 8 This is a graph showing the effect of IBA concentration on rooting culture. DETAILED DESCRIPTION

[0035] To facilitate the technical solution of the application, the following first provides a general description and definition of the terms and expressions involved in the present invention.

[0036] The "range" disclosed in the present invention is defined in the form of a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values ​​and can be combined in any way, that is, any lower limit can be combined with any upper limit to form a range.

[0037] In each group of comparative experiments provided by the present invention, unless otherwise specified, other experimental conditions, materials, etc., except for the differences indicated in each group, are kept consistent to ensure comparability.

[0038] Unless otherwise specified, the reagents and instruments used in the embodiments of the present invention can be purchased from the market.

[0039] The formula of the modified WPM medium used in the following examples is: calcium chloride (CaCl 2 )72.5mg / L+ potassium dihydrogen phosphate (KH 2 PO 4 )170mg / L+Potassium sulfate (K 2 SO 4 )990mg / L+magnesium sulfate (MgSO 4 )180.54mg / L+ammonium nitrate (NH 4 NO 3 )400mg / L+Copper sulfate pentahydrate (CuSO 4 ·5H 2 O) 0.25mg / L + FeNaEDTA 36.7mg / L + Boric acid (H 3 BO 3 )6.2mg / L+Manganese sulfate monohydrate (MnSO 4 ·H 2 O)22.3mg / L+Sodium molybdate (Na 2 MoO 4 )0.25mg / L+ZnSO 4 7H 2 O)8.6mg / L+Glycine (Glycine) 2mg / L+Myo-Inositol (myo-Inositol) 100mg / L+Nicotinic acid (nicotinic acid) 0.5mg / L+Thiamine HCl (Thiamine HCl) 1mg / L+Pyridoxine HCl (PyridoxineHCl) 0.5mg / L.

[0040] The formula of the modified 1 / 2WPM medium used in the following examples is: calcium chloride (CaCl 2 )36.25mg / L+ potassium dihydrogen phosphate (KH 2 PO 4 )85mg / L+Potassium sulfate (K 2 SO 4 )495mg / L+magnesium sulfate (MgSO 4 )90.27mg / L+ammonium nitrate (NH 4 NO 3 )200mg / L+Copper sulfate pentahydrate (CuSO 4 ·5H 2 O) 0.25mg / L + FeNaEDTA 36.7mg / L + Boric acid (H 3 BO3 )6.2mg / L+Manganese sulfate monohydrate (Mn SO 4 ·H 2 O)22.3mg / L+Sodium molybdate (Na 2 MoO 4 )0.25mg / L+ZnSO 4 7H 2 O)8.6mg / L+Glycine (Glycine) 2mg / L+Myo-Inositol (myo-Inositol) 100mg / L+Nicotinic acid (nicotini c acid) 0.5mg / L+Thiamine HCl (Thiamine HCl) 1mg / L+Pyridoxine HCl (Pyridoxine HCl) 0.5mg / L.

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0042] Example 1: Quercus acutissima seed disinfection experiment

[0043] Wild Quercus acutissima seeds were collected for disinfection. The disinfection steps were as follows: remove the shell and seed coat of wild Quercus acutissima seeds, wash under running water for 24 hours after removing surface stains with detergent, treat with 75% ethanol for 30 seconds and 60 seconds, rinse with sterile water three times, treat with 5% NaClO solution for 5 minutes and 6 minutes respectively (as shown in Table 1), rinse with sterile water three times, and inoculate the disinfected seeds into the modified WPM medium supplemented with 1.2 mg / L 6-BA and 0.01 mg / L NAA for aseptic culture 1. The inoculated seeds were germinated under the culture conditions of 25°C, 16 hours of light time, and 5000 lux of light intensity, and the contamination and survival of the explants were counted at 30 days.

[0044] Table 1 Seed disinfection experimental design

[0045]

[0046] Two days after seed disinfection, some seeds were contaminated, mainly by fungi (such as Figure 1 After 30 days, the roots elongated, the apical buds grew significantly, and cluster buds sprouted (as shown in Figure 2A). Figure 1 For seeds treated with 75% ethanol for 30 seconds, the germination rate increased with the extension of the 5% NaClO treatment time, but the contamination rate decreased (as shown in Figure 2A). Figure 2In the case of 75% ethanol treatment for 60 seconds, the contamination rate was lower, but the germination rate dropped rapidly and the browning was aggravated (as shown in Figure 1). Figure 1 When the 5% NaClO treatment time was the same, the 75% ethanol treatment time mainly affected the germination rate and browning rate. When the 5% NaClO treatment time was 5 min, the two values ​​increased with the extension of the 75% ethanol treatment time (as shown in Figure 2). Figure 2 When treated with 5% NaClO for 6 min, the germination rate decreased significantly and the browning rate increased significantly (as shown in S1 and S3 groups in the figure); Figure 2 The results of the experiment showed that the seeds were damaged by the long-term treatment with 75% ethanol, which affected the germination of the seeds and decreased the germination rate. Therefore, the best disinfection treatment was 75% ethanol for 30 seconds and 5% NaClO for 6 minutes.

[0047] Example 2 Spectral type screening experiment

[0048] Use the stem segments with axillary buds that germinate after disinfection, trim them to 1-2 cm, and inoculate them into a rapid propagation medium. The composition of the medium is: improved WPM medium + 6-BA 1.2 mg / L + NAA 0.01 mg / L + calcium nitrate 350 mg / L + sucrose 20 g / L + agar 6 g / L, and the pH value is adjusted to 5.8-6.0. Select the spectrum lamps L1 (spectral primary colors R:G:B=45.5%:39.8%:14.7%, luminous flux 370 lm), L2 (spectral primary colors R:G:B=66.1%:0.7%:33.2%, luminous flux 200 lm), L3 (spectral primary colors R:G:B=82.3%:0.7%:17.0%, luminous flux 200 lm), L4 (spectral primary colors R:G:B=54.0%:26.3%:19 .8%, luminous flux 950lm), L5 (spectral primary colors R:G:B=67.4%:18.8%:13.8%, luminous flux 840lm), L6 (spectral primary colors R:G:B=55.0%:22.2%:22.8%, luminous flux 950lm), L7 (spectral primary colors R:G:B=45.5%:39.8%:14.7%, luminous flux 1800lm), L0 (white light control) were used for spectrum treatment experiments. The culture conditions were ambient temperature 25℃ and light duration 16h. Each treatment was repeated in 3 groups, with 20-30 stem segments in each group. After 30 days, the proliferation coefficient, average number of robust buds (pieces), strong bud rate and average height (cm) of Quercus acutissima tissue culture seedlings under different treatment conditions were counted.

[0049] Table 2 Spectral one-way ANOVA

[0050]

[0051] Note: P < 0.05 was significant, P < 0.01 was extremely significant.

[0052] like Figure 3 As shown, the growth status of plants under spectral treatment (such as Figure 3 A) and the control group under white light (as shown in Figure 3 As shown in B), the comparison between the two clearly shows that changing the light conditions can effectively improve the dryness of the top stem tip of the plant, and is also conducive to leaf expansion and proliferation. As shown in Table 2, the effect of the spectrum on the proliferation coefficient, the average number of strong buds, and the strong bud rate all reached an extremely significant level, and the effect on the average height reached a significant level, indicating that the selection of spectrum lamps plays an important role in the differentiation and proliferation of Quercus acutissima tissue culture seedlings and the elongation of buds. Figure 4 As shown in the figure, from the analysis of proliferation coefficient, the proliferation coefficients of L3 and L6 treatment groups were significantly higher than those of other treatment groups, reaching 14.65 and 15.33 respectively, followed by L6 and L7 treatment groups, with proliferation coefficients above 13. From the perspective of robust buds, the L3 treatment group had the highest average number of robust buds, and the strong bud rate of this group was also the highest, reaching 49%. In the L0 control group, the average number of robust buds was not high, but the strong bud rate was second only to the L3 treatment group, reaching 43%. Although the L6 treatment group had the highest proliferation coefficient, the strong bud rate was only 29%. From the perspective of average height, the L0 treatment group had the highest average height, reaching 1.67cm, followed by the L3 treatment group at 1.47cm.

[0053] In summary, the proliferation efficiency of Quercus acutissima tissue culture seedlings in the L6 treatment group was the highest. Although the proliferation coefficient of the L3 treatment group was slightly lower than that of the L6 treatment group, the strong bud rate was significantly higher than that of other treatment groups, which was conducive to the subsequent rooting culture. Therefore, both L6 and L3 type spectrum lamps are suitable for the cultivation of Quercus acutissima tissue culture seedlings.

[0054] Example 3: Rooting medium screening experiment

[0055] The tissue culture seedlings with a height of more than 1 cm were inoculated into the rooting medium, and the basic medium screening and the concentration gradient single factor experiment of NAA, IBA and calcium nitrate were carried out. The experimental design is shown in Table 3-Table 5. The amount of sucrose in all the culture media was 20g / L, and the amount of agar was 6g / L. The culture conditions were an ambient temperature of 25°C, a light exposure time of 16h, and a light intensity of 5000lux. Each treatment was repeated in 3 groups, with 20-30 stem segments in each group. After 30 days, the rooting rate (%) (number of rooted explants / total number of inoculated explants), the shoot tip mortality rate (%) (number of inoculated explant dry tips / total number of inoculated explants) and the average number of roots (bars) (total number of all rooted explants / total number of inoculated explants) were statistically analyzed.

[0056] Table 3 Basic culture medium screening single factor test

[0057]

[0058] Table 4 NAA concentration gradient single factor experiment

[0059]

[0060] Table 5 IBA concentration gradient single factor experiment

[0061]

[0062]

[0063] like Figure 5 As shown in the single factor experiment of basic culture medium screening, the highest rooting rate was in the G3 treatment group, reaching 95.24%, and the lowest shoot tip mortality rate was 19.31%; followed by the G4 treatment group, with a rooting rate of 84% and a shoot tip mortality rate of 54.85%, but the average number of roots reached 3.6; the G2 treatment group had the lowest rooting rate, only 2%, and a shoot tip mortality rate of up to 91.37%. Different culture media have a great influence on the growth state of tissue culture seedlings. The oak seedlings in MS culture medium showed wilting, yellowing leaves or death (such as Figure 6 In the modified WPM medium, the oak seedlings grew robustly and had a well-developed root system (as shown in A). Figure 6 (shown in B).

[0064] like Figure 7 As shown in the single factor experiment of NAA concentration gradient, the highest rooting rate was in the N4 treatment group, which could reach 91.27%. Its shoot tip mortality rate was basically the same as that of the N1 treatment group, which were 22.49% and 21.7% respectively. The average number of roots was the highest among the four treatment groups, which was 3.92. After adding NAA, the rooting rate increased with the increase of NAA concentration. The N2 treatment group had a poor effect on the rooting rate, while the N3 treatment group had a high shoot tip mortality rate. Overall, the N4 treatment group was a better treatment combination.

[0065] like Figure 8 As shown in the single factor experiment of IBA concentration gradient, with the increase of IBA concentration, the shoot tip mortality rate and the average number of roots also increased, indicating that IBA can effectively promote the increase of the average number of roots, but at the same time it brings the negative effect of shoot tip death. The highest shoot tip mortality rate was in the I4 treatment group, reaching 33.33%. The average number of roots in the I3 treatment group and the I4 treatment group were similar, 3.07 and 3.02 respectively, with the highest number in the I3 treatment group being 3.07. There was no obvious trend in the rooting rate, all above 70%. Under the I2 treatment group, the rooting rate reached a maximum of 84.8%, and the shoot tip mortality rate was relatively small, at 24.34%, which was suitable for rooting culture.

[0066] In summary, selecting improved 1 / 2WPM as the basic culture medium for rooting culture, with NAA concentration of 0-3 mg / L, IBA concentration of 0-3 mg / L, and calcium nitrate concentration of 150 mg / L is beneficial to the rooting of Quercus acutissima; among them, the best rooting effect was achieved when using improved 1 / 2WPM as the basic culture medium, NAA0.5 mg / L, IBA0.5 mg / L, and calcium nitrate 150 mg / L.

Claims

1. A tissue optimization culture method for Quercus acutissima, characterized in that: The following steps are involved: S1. After removing the seed coat of Quercus acutissima, the seeds are sterilized and inoculated into a germination medium to obtain germinated seedlings, wherein the germination medium comprises a modified WPM medium, 6-BA and NAA; S2. cutting the germinated seedlings into stem segments with axillary buds, inoculating them into a rapid propagation medium for subculture and proliferation culture, and cultivating tissue culture seedlings, wherein the formula of the rapid propagation medium includes a modified WPM medium, 6-BA, NAA, calcium nitrate, sucrose and agar; S3. The tissue culture seedlings obtained by subculture proliferation culture were transferred to a rooting medium for rooting culture to obtain Quercus acutissima tissue culture seedlings; the formula of the rooting medium includes a modified 1 / 2WPM medium, NAA, IBA, calcium nitrate, sucrose and agar; Wherein, the step S2 selects a lamp with a specific spectrum to provide a light source.

2. The tissue optimization culture method of Quercus acutissima according to claim 1, characterized in that: The specific conditions of the disinfection are: 75% ethanol treatment for 30 seconds and 5% NaClO treatment for 6 minutes.

3. The tissue optimization culture method of Quercus acutissima according to claim 1, characterized in that: The light source characteristics of the specific spectrum lamp are: the three primary colors of the spectrum R:G:B=82.3%:0.7%:17.0%, and the luminous flux is 200lm; Or: the three primary colors of the spectrum R:G:B=55.0%:22.2%:22.8%, luminous flux 950lm.

4. The tissue optimization culture method of Quercus acutissima according to claim 1, characterized in that: The culture conditions of the germination medium and the rooting medium are: temperature of 25° C., light duration of 16 h, and light intensity of 5000 lux.

5. The tissue optimization culture method of Quercus acutissima according to claim 3, characterized in that: The culture conditions of the rapid propagation culture medium are: temperature of 25° C. and light exposure time of 16 h.

6. The method for optimizing tissue culture of Quercus acutissima according to claim 1, characterized in that: The formula of the germination medium is: improved WPM medium+6-BA 1.2 mg / L+NAA 0.01 mg / L+calcium nitrate 350 mg / L+sucrose 20 g / L+agar 6 g / L.

7. The tissue optimization culture method of Quercus acutissima according to claim 1, characterized in that: The formula of the rapid propagation culture medium is: improved WPM culture medium + 6-BA 1.0-1.4 mg / L + NAA 0.01-0.02 mg / L + calcium nitrate 150-350 mg / L + sucrose 20 g / L + agar 6 g / L, and the pH value is adjusted to 5.8-6.

0.

8. The method for optimizing tissue culture of Quercus acutissima according to claim 1, characterized in that: The formula of the rooting medium is: improved 1 / 2WPM medium + NAA 0.5-3 mg / L + IBA 0.5-3 mg / L + calcium nitrate 100-450 mg / L + sucrose 20 g / L + agar 6 g / L, and the pH value is adjusted to 5.8-6.

0.

9. The method for optimizing tissue culture of Quercus acutissima according to claim 1, characterized in that: The formula of the rooting medium is: improved 1 / 2WPM medium + NAA 0.5 mg / L + IBA 0.5 mg / L + calcium nitrate 150 mg / L + sucrose 20 g / L + agar 6 g / L, and the pH value is adjusted to 5.8-6.

0.

10. The method for optimizing tissue culture of Quercus acutissima according to any one of claims 1 to 8, characterized in that: The formula of the improved WPM medium is: CaCl2 72.5 mg / L+KH2PO4 170 mg / L+K2SO4 990 mg / L+MgSO4 180.54 mg / L+NH4NO3 400 mg / L+CuSO4·5H2O 0.25 mg / L+FeNaEDTA 36.7 mg / L+H3BO 36.2 mg / L+MnSO4·H2O 22.3 mg / L+Na2MoO4 0.25 mg / L+ZnSO4·7H2O 8.6 mg / L+Glycine 2 mg / L+Inositol 100 mg / L+Nicotinic acid 0.5 mg / L+Thiamine hydrochloride 1 mg / L+Pyridoxine hydrochloride 0.5 mg / L; The formula of the improved 1 / 2WPM medium is: CaCl2 36.25 mg / L+KH2PO4 85 mg / L+K2SO4 495 mg / L+MgSO490.27mg / L+NH4NO3200 mg / L+CuSO4·5H2O 0.25mg / L+FeNaEDTA36.7 mg / L+H3BO36.2mg / L+MnSO4·H2O 22.3mg / L+Na2MoO40.25 mg / L+ZnSO4·7H2O 8.6mg / L+Glycine 2mg / L+Inositol 100mg / L+Nicotinic acid 0.5mg / L+Thiamine hydrochloride 1mg / L+Pyridoxine hydrochloride 0.5mg / L.

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