Tissue culture method of quercus acutissima

Abstract

The invention provides a tissue culture method of quercus acutissima, and belongs to the technical field of plant tissue culture. The method comprises the following steps: sterilizing stems with buds of quercus acutissima, inoculating the stems into a germination culture medium, and culturing to obtain germinated seedlings; cutting the germinated seedlings into stem segments with axillary buds, and inoculating the stem segments into a rapid propagation culture medium for subculture multiplication culture; transferring the tissue culture seedlings obtained by subculture into a rooting culture medium for rooting culture until normal growth quercus acutissima tissue culture seedlings are obtained; by improving an explant disinfection method, a WPM culture medium and a 1 / 2 WPM culture medium, the problems of high disinfection contamination rate and low proliferation efficiency are solved, the quality of quercus acutissima tissue culture seedlings is improved, and the propagation speed is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cultivation, relates to asexual reproduction technology of Quercus acutissima, and specifically relates to a tissue culture method of Quercus acutissima. Background Art

[0002] Quercus variabilis Bl. is a deciduous tree of the genus Quercus (Quercus L.) of the Fagaceae Dumort. family. It can reach a height of 15 to 25 meters and is a widely distributed pioneer tree species in my country. Its wood is hard, and its leaves, bark, and fruits can all be used as medicine. It is also a unique economic tree species in my country. It has a strong root system with a wide range of extension. It is resistant to drought and barrenness, has strong adaptability and vitality, can play a role in maintaining water and soil and reducing soil erosion, and also plays an important role in ecological construction.

[0003] Tissue culture is a technology that separates biological tissues or cells from organisms and cultivates them under artificially controlled environmental conditions through aseptic operations. Specifically, tissue culture is a technology that separates plant tissues with differentiation ability from plants, cultivates them on artificially prepared culture media under aseptic conditions, keeps them alive and continues to grow and develop, and then transplants them to greenhouses or farmlands to breed a large number of seedlings. This method has the advantages of short breeding cycle, fast speed, high production efficiency, and is not restricted by seasons and climate. The development of tissue culture technology for Quercus acutissima is a quick and effective way to solve the problems of Quercus acutissima such as difficulty in harvesting fruits and mixed varieties. It can play an important role in the fields of rapid propagation of Quercus acutissima seedlings, breeding of improved varieties, rapid propagation of seedlings, and preservation of germplasm resources.

[0004] Although there are reports on the successful planting of oak seedlings using tissue culture methods, there are still difficulties in tissue culture, such as low rapid propagation coefficient of oak, difficulty in rooting, and easy death of stem tips in tissue culture seedlings. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a tissue culture method of Quercus acutissima.

[0006] To solve the above problems, the present invention provides the following technical solutions:

[0007] A tissue culture method of Quercus acutissima, comprising the following steps:

[0008] After sterilizing the stem segments of Quercus acutissima with buds, the stem segments are inoculated into a germination medium to obtain germination seedlings. The germination medium comprises a modified WPM medium, 6-BA and NAA.

[0009] Cutting the germinated seedlings into stem segments with axillary buds, inoculating the segments into a rapid propagation medium for subculture and proliferation culture, and obtaining tissue culture seedlings, wherein the formula of the rapid propagation medium includes a modified WPM medium, 6-BA, NAA, calcium nitrate, sucrose and agar;

[0010] The tissue culture seedlings obtained by subculture 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 improved 1 / 2WPM medium, NAA, IBA, calcium nitrate, sucrose and agar.

[0011] In some embodiments of the present invention, the formula of the germination medium is modified WPM medium + 6-BA 1.2 mg / L + NAA 0.02 mg / L.

[0012] In some embodiments of the present invention, the formula of the rapid propagation medium is: improved WPM medium + 6-BA 1.2-1.8 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.

[0013] In some embodiments 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 0-0.02 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.

[0014] In some embodiments 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.

[0015] In some embodiments of the present invention, the formula of the rooting medium is: 1 / 2WPM medium + NAA 2.5-3.5 mg / L + IBA 0.5-1.5 mg / L + calcium nitrate 300-400 mg / L + sucrose 20 g / L + agar 6 g / L, and the pH value is adjusted to 5.8-6.0.

[0016] In some embodiments of the present invention, the formula of the rooting medium is: 1 / 2WPM medium + NAA 2.5 mg / L + IBA 1 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.

[0017] In some embodiments of the present invention, the conditions of the rapid propagation medium and the rooting medium are: temperature 25° C., light exposure time 16 h, and light intensity 5000 lux.

[0018] In some embodiments of the present invention, the oak stem segments with buds are collected from wild oak, and the specific method of disinfection is: disinfection with 3% NaClO solution for 3 minutes.

[0019] In some embodiments of the present invention, the rapid propagation medium and the rooting medium are sterilized by high temperature and high pressure.

[0020] In some embodiments of the present invention, the rapid propagation medium and the rooting medium are sterilized at 121° C. for 18 min.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The invention provides a method for tissue culture of Quercus acutissima, comprising explant disinfection, tissue culture rapid propagation and a rooting method. The method for disinfecting the explant, a WPM culture medium and a 1 / 2WPM culture medium are improved, so as to overcome the problems of high bacterial infection rate and low proliferation coefficient of Quercus acutissima after disinfection, and improve the rooting rate of tissue culture seedlings, thereby improving the quality of the Quercus acutissima tissue culture seedlings as a whole and increasing the propagation speed. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a growth comparison chart of the infected germination and normal germination during the germination process of the explant after disinfection;

[0024] Figure 2 This is the effect of different disinfection methods on the growth of oak stem segments after disinfection;

[0025] Figure 3 This is a graph showing the effect of the concentration of each component on plant growth in the screening of rapid propagation medium;

[0026] Figure 4 These are the leaf morphologies of two fast-propagating plants;

[0027] Figure 5 This is a graph showing the effect of the concentration of each component on plant growth in the optimization of rapid propagation medium;

[0028] Figure 6 This is a diagram showing the effect of each component concentration on plant rooting in the rooting experiment;

[0029] Figure 7 This is a comparison chart of the results of various treatments in the rooting experiment. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of this application.

[0031] The "range" disclosed in this application is defined in the form of a lower limit and an upper limit, and 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 arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range.

[0032] 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 + sodium ferric ethylenediaminetetraacetate (Fe NaEDTA) 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 (Pyridoxine HCl) 0.5mg / L.

[0033] The formula of the modified 1 / 2WPM medium used in the following examples is: calcium chloride (CaCl 2 )36.25mg / L+ potassium dihydrogen phosphate (KH2 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 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+Thiamin HCl (Thia mine HCl) 1mg / L+Pyridoxine HCl (Pyridoxine HCl) 0.5mg / L.

[0034] Example 1: Disinfection of Quercus acutissima explants

[0035] Wild oak stem segments with buds were collected as explants, and first disinfected. The steps of the disinfection method of wild oak stem segments with buds were as follows: the wild oak stem segments with buds were trimmed to 2.5-3 cm, washed with detergent to remove surface stains, rinsed under running water for 24 hours, treated with 75% ethanol for 30 seconds, rinsed with sterile water 3 times, and then treated with 1% and 5% sodium hypochlorite (NaClO) solution for 3 minutes and 5 minutes respectively (as shown in Table 1), rinsed with sterile water 3 times, and inoculated into a modified WPM medium supplemented with 1.2 mg / L 6-benzyladenine (6-BA) and 0.02 mg / L naphthaleneacetic acid (NAA) for sterile culture. The inoculated explants were germinated under the culture conditions of 25°C during the day, 18°C ​​at night, 16 hours of light, and 5000 lux of light intensity. The contamination and survival of the explants were counted at 30 days.

[0036] Table 1 Experimental design of stem segment disinfection

[0037]

[0038] 4 days after the stems were disinfected, contamination began to appear, mainly due to mold and bacteria contamination at the contact point between the stems and the culture medium ( Figure 1 (A) After 10 days, the axillary buds grow significantly, and the base swells to form callus tissue. After 30 days, the leaves are obviously expanded. Figure 1 When the concentration of NaClO is the same, prolonging the disinfection time can reduce the contamination rate and achieve a better disinfection effect, but the mortality rate will increase accordingly; the change in survival rate is related to the NaClO treatment time. When the concentration is low (1%), the survival rate increases when the disinfection time is extended ( Figure 2 B1, B2), when the concentration is higher (3%), the survival rate decreases with prolonged disinfection time ( Figure 2 B3 and B4). When the treatment time is the same, increasing the NaClO concentration can reduce the contamination rate, and the mortality rate increases accordingly. The survival rate increases with the increase of NaClO concentration when the treatment time is 3 minutes, and decreases with the increase of NaClO concentration when the treatment time is 5 minutes ( Figure 2 In summary, although extending the disinfection time and increasing the NaClO concentration can reduce the contamination rate, the mortality rate is also increasing, indicating that the damage to the explants is more serious. The survival rate does not increase linearly with these two factors. When the disinfection time is too long or the NaClO concentration is too high, the survival rate decreases instead. This experiment found that using 3% NaClO for disinfection for 3 minutes, the survival rate of the stem segments is the highest and the contamination rate is the lowest, which is a suitable disinfection method for oak stem segments.

[0039] Example 2: Rapid propagation culture medium screening

[0040] The stem segments with axillary buds that germinated after disinfection were trimmed to 1-2 cm and inoculated into the culture medium. The composition of the culture medium was: modified WPM medium, 20 g / L sucrose, 6 g / L agar, 1.2 mg / L, 1.5 mg / L, 1.8 mg / L 6-BA, 0 mg / L, 0.02 mg / L, 0.04 mg / L NAA, 500 mg / L, 600 mg / L, 700 mg / L calcium nitrate (Table 2). The culture conditions were an ambient temperature of 25 ° C, a light duration of 16 h, and a light intensity of 5000 lux. Each treatment was repeated in 3 groups, with 20-30 stem segments in each group. After 30 days, the proliferation coefficient (total number of buds / number of inoculated stem segments) and average height (cm) (total height of strong buds / total number of strong buds) of Quercus acutissima tissue culture seedlings under different treatment conditions were counted.

[0041] Table 2 Orthogonal experimental design of rapid propagation medium

[0042]

[0043] Table 3 Analysis of variance for rapid propagation culture medium screening

[0044]

[0045] Note: P < 0.05 was significant, P < 0.01 was extremely significant.

[0046] The experimental results are as follows Figure 2 The average heights of the nine treatment groups were not much different, with the highest being 1.4 cm and the shortest being 1.14 cm. The proliferation coefficients varied significantly, with the highest being 12.08 in group C6 and the lowest being 4.32 in group C7. The proliferation coefficients of groups C1 to C6 were all greater than 8, and the proliferation coefficients of groups C7 to C9 were lower. The results of variance analysis were performed on the test results, as shown in Table 3. The effect of 6-BA on the proliferation coefficient of Quercus acutissima reached a significant level. Figure 3 Analysis shows that the proliferation coefficient is higher when the 6-BA concentration is 1.2mg / L-1.5mg / L. From the growth state, the proliferation of Quercus acutissima seedlings shows different growth states under different culture conditions. When the amount of 6-BA added is high, the plant's buds and lateral buds grow vigorously, while the lateral buds are sparse. Figure 4 As shown, plants with high proliferation coefficients usually have more buds and smaller leaves ( Figure 4 A), while those with a small proliferation coefficient tend to have large leaf width ( Figure 4 B), but when the 6-BA concentration reaches 1.8 mg / L, vitrification occurs. The proliferation coefficient is negatively correlated with the calcium nitrate concentration, but appropriate addition of calcium nitrate can promote plant growth ( Figure 3 ).

[0047] Example 3: Rapid propagation culture medium optimization

[0048] The stem segments with axillary buds that germinated after disinfection were trimmed to 1-2 cm and inoculated into the culture medium. The composition of the culture medium was: modified WPM medium, 20 g / L sucrose, 6 g / L agar, 1.0 mg / L, 1.2 mg / L, 1.5 mg / L 6-BA, 0 mg / L, 0.01 mg / L, 0.02 mg / L NAA, 150 mg / L, 250 mg / L, 350 mg / L calcium nitrate (Table 4), and the culture conditions were an ambient temperature of 25 ° C, a light duration of 16 h, and a light intensity of 5000 lux. 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 (height>0.5 cm) (total number of robust buds / inoculated number) and average height (cm) of Quercus acutissima tissue culture seedlings under different treatment conditions were counted.

[0049] Table 4 Rapid propagation culture medium optimization experimental design

[0050]

[0051] Table 5 Analysis of variance for optimization of rapid propagation medium

[0052]

[0053] Note: P < 0.05 was significant, P < 0.01 was extremely significant.

[0054] The results of variance analysis are shown in Table 5. The effect of calcium nitrate on the average height of Quercus acutissima seedlings reached a significant level, while the effects of 6-BA, NAA, and calcium nitrate on the proliferation coefficient and the average number of robust buds did not reach a significant level. Figure 5 It can be seen that after optimization, the D5 treatment group has the highest proliferation coefficient, which can reach 13.9. The average height is as low as 1.38cm and as high as 1.69cm, which is significantly improved compared with before optimization. The various indicators of the D3 treatment are roughly similar to those of the D5 treatment group, but the D5 treatment group has the highest proliferation coefficient, and the average height is as high as 1.61cm, second only to the 1.69cm of D7, which has a clear advantage. The D9 proliferation coefficient is 12.1, but the average number of robust buds is small, only 3.7, indicating that although the D9 treatment group has a large number of buds, most of them are small buds below 0.5cm, and there are fewer buds above 0.5cm, which makes it difficult to provide robust inoculation materials for subsequent rapid propagation or rooting.

[0055] In summary, the optimized rapid propagation medium composition 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.

[0056] Example 4: Rooting culture

[0057] Using the improved 1 / 2WPM medium as the basic medium, a three-factor three-level orthogonal experimental design (as shown in Table 6) was adopted, and 20 g / L agar and 6 g / L sucrose were added to test the effects of different concentrations of NAA, 6-BA, and calcium nitrate on the rooting of Quercus variabilis tissue culture seedlings. The survey indicators are rooting rate (%) = (number of rooted explants / total number of inoculated explants) × 100%; shoot tip mortality (%) = (number of stem tips of inoculated explants / total number of inoculated explants) × 100%; average number of roots (roots) = total number of roots of all rooted explants / total number of inoculated explants.

[0058] Table 6 Orthogonal table for rooting experiment

[0059]

[0060] Table 7 Analysis of variance

[0061]

[0062] Note: P < 0.05 was significant, P < 0.01 was extremely significant.

[0063] The results of variance analysis are shown in Table 7. The effect of IBA on rooting rate reached a significant level, and the effects of IBA and NAA on shoot tip mortality both reached a significant level, indicating that IBA and NAA are the main factors affecting the rooting rate and survival rate of Quercus acutissima seedlings. Shoot tip mortality is a common phenomenon in the rooting process of Quercus acutissima and is the main factor causing the death of rooted Quercus acutissima seedlings. Figure 6 As shown, the Z2 treatment group had the highest rooting rate among the 9 treatments, which could reach 89.35%, and the stem tip mortality rate was the lowest, only 26%, and the average number of roots was the highest at 3.07. The Z5 treatment group was second, with a rooting rate of 81.16%. The treatment groups Z7 and Z8 had the lowest rooting rates of 58.1% and 60.5%, and the stem tip mortality rates were the highest, at 81.16% and 76.61%, respectively. In terms of the average number of roots, the Z9 treatment group had the lowest number of roots, only 1.97 ( Figure 7 (Z9).

[0064] In summary, in the Z2 treatment group, when 2.5 mg / L NAA, 1 mg / L IBA, and 350 mg calcium nitrate were added to the culture medium, the rooting rate was the highest and the shoot tip mortality was the lowest. At this time, the conditions were most suitable for the rooting culture of Quercus acutissima seedlings. The roots of the Quercus acutissima seedlings were numerous and strong, and the leaves were green and not withered ( Figure 7 (Z2).

Claims

1. A method for tissue culture of Quercus acutissima, characterized in that: The following steps are involved: After the budded stem segments of Quercus acutissima are sterilized, they are inoculated into a germination medium to obtain germination seedlings. The germination medium comprises a modified WPM medium, 6-BA and NAA. Cutting the germinated seedlings into stem segments with axillary buds, inoculating the segments into a rapid propagation medium for subculture and proliferation culture, and obtaining tissue culture seedlings, wherein the formula of the rapid propagation medium includes a modified WPM medium, 6-BA, NAA, calcium nitrate, sucrose and agar; The tissue culture seedlings obtained by subculture 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 improved 1 / 2WPM medium, NAA, IBA, calcium nitrate, sucrose and agar.

2. The tissue culture method of Quercus acutissima according to claim 1, characterized in that: The formula of the germination medium is improved WPM medium+6-BA1.2mg / L+NAA0.02mg / L.

3. The tissue 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 1.2-1.8 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.

4. The tissue culture method of Quercus acutissima according to claim 3, 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 0-0.02 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.

5. The tissue culture method of Quercus acutissima according to claim 4, characterized in that: The formula of the rapid propagation culture medium is: improved WPM culture 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.

6. The tissue culture method of Quercus acutissima according to claim 1, characterized in that: The formula of the rooting culture medium is: 1 / 2WPM culture medium + NAA 2.5-3.5 mg / L + IBA 0.5-1.5 mg / L + calcium nitrate 300-400 mg / L + sucrose 20 g / L + agar 6 g / L, and the pH value is adjusted to 5.8-6.

0.

7. The tissue culture method of Quercus acutissima according to claim 6, characterized in that: The formula of the rooting medium is: 1 / 2WPM medium + NAA 2.5 mg / L + IBA 1 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.

8. The tissue culture method of Quercus acutissima according to claim 1, characterized in that: The conditions of the rapid propagation medium and the rooting medium are: temperature 25° C., light exposure time 16 h, and light intensity 5000 lux.

9. The tissue culture method of Quercus acutissima according to claim 1, characterized in that: The oak stem segments with buds are collected from wild oak, and the specific method of disinfection is: disinfection with 3% NaClO solution for 3 minutes.

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