A method for tissue culture of polyploid poplar

Abstract

The application provides a method for tissue culture of polyploid poplar, and relates to the technical field of plant tissue culture. The tissue culture method comprises the following steps: after the external explant is disinfected, the differentiated bud material is obtained through differentiation culture; the differentiated bud material is inoculated into the proliferation culture medium containing peanut bud brine extract to obtain the multiple shoots through proliferation culture; and the multiple shoots are inoculated into the rooting culture medium containing peanut leaf ethanol extract to obtain the poplar rooting seedlings through rooting culture. The application overcomes the defects of the prior art, effectively improves the efficiency of the tissue culture of polyploid poplar by optimizing the culture medium formula, and comprehensively improves the economic effect of the tissue culture.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and specifically to a method for culturing polyploid aspen tissue. Background Technology

[0002] Polyploid aspen is a variant of aspen, characterized by rapid growth, strong resistance to adverse conditions, and excellent wood quality, making it valuable for forestry production and ecological restoration. The probability of naturally forming polyploid aspen is low; most polyploid aspen is formed through artificial hybridization or artificial induction (colchicine induction).

[0003] The natural occurrence rate of polyploid aspen is low, and some superior varieties (such as triploid hybrids) are difficult to propagate through seeds. Therefore, asexual reproduction is the main propagation method for polyploid aspen, which is mainly divided into cutting propagation and tissue culture. Compared with cutting propagation, tissue culture is not limited by season and has a high propagation coefficient (one explant can produce dozens of buds). At the same time, by optimizing the culture medium formula (such as adjusting the hormone ratio) and culture conditions (such as light and temperature), the propagation efficiency can be further improved and the unit cost reduced. Therefore, optimizing tissue culture methods to comprehensively improve the tissue culture efficiency of polyploid aspen is a major research direction at present. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for culturing polyploid aspen tissue, which effectively improves the efficiency of polyploid aspen tissue culture by optimizing the culture medium formulation.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for culturing polyploid aspen tissue, the method comprising the following steps:

[0007] S1. Disinfection of explants: Select young stem segments with axillary buds that sprouted in the current year from polyploid poplar as explants, disinfect them, and obtain sterile explants for later use.

[0008] S2. Differentiation culture of explants: The above-mentioned sterile explants were inoculated into differentiation medium and cultured to obtain differentiated shoot materials. The formula of the differentiation medium was: MS + BA 1.0 mg / L + NAA 0.1 mg / L + sucrose 30 g / L + agar 5.5 g / L.

[0009] S3. Proliferation Culture: The differentiated buds were inoculated into a proliferation culture medium to obtain clustered buds. The formulation of the proliferation culture medium was MS + 30 g / L sucrose + 5 g / L agar powder + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 5-8 mg / L peanut bud extract. The peanut bud extract was obtained by soaking peanut buds in 3% saline solution and then drying them.

[0010] S4. Rooting Culture: The above-mentioned clustered buds were inoculated into a rooting medium for rooting culture to obtain rooted poplar seedlings. The formula of the rooting medium is: 1 / 2 MS + NAA 0.1 mg / L + ABA 0.5 mg / L + sucrose 30 g / L + agar powder 5 g / L + peanut leaf extract 10-12 mg / L; the peanut leaf extract is obtained by drying peanut leaves, ultrasonically extracting them with 75% ethanol, and then freeze-drying them.

[0011] Preferably, the disinfection method in step S1 is to cut off the leaves of the tender stem segments with axillary buds, rinse with Tween 80 and then rinse with sterile water, then treat with 75% ethanol for 30 seconds, treat with 0.1% mercuric chloride for 8 minutes, and then rinse with sterile water.

[0012] Preferably, the differentiation culture time in step S2 is 35-40 days, the culture temperature is 25℃±2℃, the light intensity is 1500-5000 Lux, and the daily light duration is 12-16 hours.

[0013] Preferably, the preparation method of the peanut sprout extract includes the following steps:

[0014] S3-1. Grind peanut sprouts into a paste by adding 3-5 times their volume of 3% salt water to obtain a mixed solution for later use.

[0015] S3-2. Place the mixture in a water bath at 40-50℃ and stir for 30-40 minutes. Then press and filter to obtain filtrate and residue. Add 3-5 times the volume of 3% saline to the residue and stir for 30-40 minutes in a water bath at 40-50℃. Press and filter, combine the two filtrates and dry to obtain peanut sprout extract.

[0016] Preferably, the drying method in step S3-2 is vacuum concentration to 1 / 5 of the original volume, followed by hot air drying at 35-45°C.

[0017] Preferably, the proliferation culture time is 50-60 days, the culture temperature is 25℃±2℃, the light intensity is 1500-5000 Lux, and the daily light duration is 12-16 hours.

[0018] Preferably, the preparation method of the peanut leaf extract includes the following steps:

[0019] S4-1. Dry fresh peanut leaves at 40-50℃ to constant weight, then pulverize them and pass them through an 80-mesh sieve to obtain peanut leaf powder for later use.

[0020] S4-2. Add 10-12 times the volume of 75% ethanol to peanut leaf powder, reflux for 1.5-2 hours, and then freeze-dry to obtain peanut leaf extract.

[0021] Preferably, the reflux extraction temperature in step S4-2 is 70-80℃.

[0022] Preferably, the rooting culture time is 35-40 days, the culture temperature is 25℃±2℃, the light intensity is 1500-5000 Lux, and the daily light duration is 12-16 hours.

[0023] This invention provides a method for culturing polyploid aspen tissue, which has the following advantages compared with the prior art:

[0024] This invention adds a certain amount of plant hormones and peanut sprout extract to the proliferation medium, which effectively promotes the proliferation and growth of polyploid poplar and ensures the proliferation rate. At the same time, the subsequent rooting culture is carried out using a rooting medium containing NAA, ABA and peanut leaf extract, which can effectively promote the early rooting of polyploid poplar and improve the rooting efficiency, achieve good seedling growth, ensure the survival rate of subsequent bottle-grown seedlings, and achieve the purpose of good tissue culture propagation. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the growth of explants treated with different disinfection methods in MS basal medium in an embodiment of the present invention.

[0026] Figure 2 This is a schematic diagram showing the germination of explants cultured in different differentiation media for 40 days in an embodiment of the present invention;

[0027] Figure 3 This is a schematic diagram illustrating the proliferation of explants cultured in different proliferation media for 60 days in an embodiment of the present invention.

[0028] Figure 4 This is a schematic diagram showing the rooting of explants cultured in different rooting media for 35 days in an embodiment of the present invention;

[0029] Figure 5 This is a schematic diagram showing the growth of tissue culture seedlings obtained using D4 rooting medium 30 days after transplanting, as described in this embodiment of the invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1:

[0031] Preparation of tissue culture materials:

[0032] 1. Preparation of PT-A:

[0033] Fresh peanuts were dried at 50℃ to constant weight, then crushed and passed through an 80-mesh sieve. Then, 10 times the volume of 75% ethanol was added, and the mixture was refluxed at 75℃ for 2 hours. Finally, it was freeze-dried to obtain PT-A.

[0034] 2. Preparation of PT-B:

[0035] (1) Grind fresh peanuts into a paste by adding 5 times their volume of 3% salt water, and set aside the mixture.

[0036] (2) The mixture was stirred for 30 minutes in a water bath at 45°C, then pressed and filtered. The filter residue was added to 5 times the volume of 3% brine and stirred for 30 minutes in a water bath at 45°C. The mixture was then pressed and filtered. The two filtrates were combined and vacuum concentrated to 1 / 5 of the original volume. The mixture was then dried with hot air at 35-45°C to obtain PT-B.

[0037] 3. Preparation of SN:

[0038] Fresh soybeans were dried at 50°C to constant weight, then pulverized and passed through an 80-mesh sieve. Ten times the volume of 75% ethanol was added, and the mixture was refluxed at 75°C for 2 hours. The mixture was then freeze-dried to obtain SN.

[0039] 4. Preparation of PTS-A:

[0040] Fresh peanut sprouts were dried at 50℃ to constant weight, then crushed and passed through an 80-mesh sieve. Then, 10 times the volume of 75% ethanol was added, and the mixture was refluxed at 75℃ for 2 hours. Finally, it was freeze-dried to obtain PTS-A.

[0041] 5. Preparation of PTS-B:

[0042] (1) Grind fresh peanut sprouts into a paste by adding 5 times their volume of 3% salt water, and prepare a mixture for later use;

[0043] (2) The mixture was stirred for 30 minutes in a water bath at 45°C, then pressed and filtered. The filter residue was then added to 5 times the volume of 3% brine and stirred for 30 minutes in a water bath at 45°C. The mixture was then pressed and filtered, and the two filtrates were combined. The mixture was then concentrated under vacuum to 1 / 5 of its original volume and dried with hot air at 35-45°C to obtain PTS-B.

[0044] 6. Preparation of PTL-A:

[0045] Fresh peanut leaves were dried at 50℃ to constant weight, then pulverized and passed through an 80-mesh sieve. Ten times the volume of 75% ethanol was added, and the mixture was refluxed at 75℃ for 2 hours. The resulting product was then freeze-dried to obtain PTL-A.

[0046] 7. Preparation of PTL-B:

[0047] (1) Grind fresh peanut leaves into a paste by adding 5 times their volume of 3% salt water, and prepare the mixture for later use;

[0048] (2) The mixture was stirred for 30 minutes in a water bath at 45°C, then pressed and filtered. The filter residue was added to 5 times the volume of 3% brine and stirred for 30 minutes in a water bath at 45°C. The mixture was then pressed and filtered. The two filtrates were combined and vacuum concentrated to 1 / 5 of the original volume. The mixture was then dried with hot air at 35-45°C to obtain PTL-B. Example 2:

[0049] Tissue culture of polyploid aspen:

[0050] 1. Material Acquisition:

[0051] Triploid aspen plants found in wild aspen populations in Mangkang County, Changdu City, Tibet Autonomous Region, were used to collect 50-80cm long branches, which were then brought back indoors for water culture. Young stem segments with axillary buds sprouting in the current year from these branches were selected as explants.

[0052] 2. Disinfection verification:

[0053] (1) Remove the leaves, rinse the explants with Tween 80 for 4 min, and rinse thoroughly with sterile water. Then, surface disinfect with 75% ethanol for 30 s, rinse twice with sterile distilled water, and conduct experiments with different disinfection times: using 0.1% mercuric chloride as the disinfectant, four treatments were set up according to different disinfection times, namely 6 min, 8 min, and 10 min. 20 bottles were used for each treatment. After disinfection, the explants were inoculated into hormone-free MS basal medium (culture temperature 25℃±2℃, light intensity 3000 Lux, daily light duration 14h), and the contamination rate was counted after 30 days:

[0054] Contamination rate = (Number of contaminated cells / Number of inoculated cells) × 100%;

[0055] The specific results are shown in Table 1 below. Figure 1 As shown:

[0056] Table 1

[0057]

[0058] As shown in the table above, the A3 disinfection method is the most effective.

[0059] 3. Differentiation culture:

[0060] (1) Prepare the differentiation medium with the following formulation:

[0061] B1: MS + BA 0.5 mg / L + NAA 0.1 mg / L + sucrose 30 g / L + agar 5.5 g / L;

[0062] B2: MS + BA 1.0 mg / L + NAA 0.1 mg / L + sucrose 30 g / L + agar 5.5 g / L;

[0063] B3: MS + BA 2.0 mg / L + NAA 0.1 mg / L + sucrose 30 g / L + agar 5.5 g / L

[0064] (2) Explants were treated according to the disinfection method described in A3 above, with three treatments. The explants from each treatment were inoculated into differentiation media containing different hormone combinations (culture temperature 25℃±2℃, light intensity 3000 Lux, daily light duration 14h), with 20 bottles per treatment. Germination rate and shoot length were recorded after 40 days. Specific results are shown in Table 2 below. Figure 2 As shown:

[0065] Table 2

[0066]

[0067] As shown in the table above, the overall germination and growth were optimal when the differentiation medium was B2.

[0068] 4. Proliferation culture:

[0069] (1) Prepare the proliferation medium with the following formula:

[0070] C1: MS + sucrose 30g / L + agar powder 5g / L + 6-BA 0.5mg / L + NAA 0.1mg / L + PT-A 6mg / L;

[0071] C2: MS + sucrose 30g / L + agar powder 5g / L + 6-BA 0.5mg / L + NAA 0.1mg / L + SN 6mg / L;

[0072] C3: MS + sucrose 30g / L + agar powder 5g / L + 6-BA 0.5mg / L + NAA 0.1mg / L + PTS-A 6mg / L;

[0073] C4: MS + sucrose 30g / L + agar powder 5g / L + 6-BA 0.5mg / L + NAA 0.1mg / L + PTL-A 6mg / L;

[0074] C5: MS + sucrose 30g / L + agar powder 5g / L + 6-BA 0.5mg / L + NAA 0.1mg / L + PTS-B 6mg / L.

[0075] (2) Buds obtained from differentiation culture medium of the above-mentioned B2 formula were selected, and stem segments above the cotyledons were cut into 1.5-2.0 cm stem segments with buds. These segments were then transferred to different proliferation culture media. In the experiment, single buds were first inoculated into C1 and C2 culture media, 20 bottles of each culture medium, and cultured for 60 days (culture temperature was 25℃±2℃, light intensity was 3000 Lux, and daily light duration was 15h). Finally, the proliferation rate of C1 with PT-A (i.e., peanut ethanol extract) reached 72.4%, which was significantly higher than the proliferation rate of C2 with SN (soybean ethanol extract) at 60.3%. Based on this, peanut extract was selected as the exogenous additive for the culture medium, which had a better effect. On this basis, extracts from different parts of peanut were added to the culture medium as exogenous additives. Additives, such as culture media C3 (PTS-A, peanut sprout ethanol extract) and C4 (PTL-A, peanut leaf ethanol extract), were used to inoculate and culture stem segments with buds, with 20 bottles per culture medium. The highest growth rate, reaching 77.3%, was observed in C3 culture medium after 60 days of cultivation. Based on this, peanut sprout extracts prepared by different methods were used, such as PTS-B (peanut sprout saline extract) added to C5. When 20 bottles of stem segments with buds were inoculated and cultured for 60 days using the same method, the final growth rate reached 82.1%. This indicates that adding PTS-B to the culture medium in C5 as an exogenous additive significantly improved the growth rate. See Table 3 below for details. Figure 3 :

[0076] Table 3

[0077]

[0078] 5. Rooting culture:

[0079] (1) Prepare the rooting medium with the following formula:

[0080] D1: 1 / 2 MS + NAA 0.1 mg / L + ABA 0.5 mg / L + sucrose 30 g / L + agar powder 5 g / L + PT-A 11 mg / L;

[0081] D2: 1 / 2 MS + NAA 0.1 mg / L + ABA 0.5 mg / L + sucrose 30 g / L + agar powder 5 g / L + SN 11 mg / L;

[0082] D3: 1 / 2 MS + NAA 0.1 mg / L + ABA 0.5 mg / L + sucrose 30 g / L + agar powder 5 g / L + PTS-A 11 mg / L;

[0083] D4: 1 / 2 MS + NAA 0.1 mg / L + ABA 0.5 mg / L + sucrose 30 g / L + agar powder 5 g / L + PTL-A 11 mg / L;

[0084] D5: 1 / 2MS + NAA 0.1 mg / L + ABA 0.5 mg / L + sucrose 30 g / L + agar powder 5 g / L + PTL-B 11 mg / L.

[0085] (2) The propagated seedlings obtained by culture in the above-mentioned C5 formula propagation medium were cut at a height of 3-4 cm and inoculated into the rooting medium of D1 and D2 for 35 days (20 bottles of each medium, 8-12 seedlings per bottle, culture temperature of 25℃±2℃, light intensity of 3000 Lux, and daily light duration of 14h). Finally, the rooting of D1 was significantly better than that of D2. Based on this, the exogenous addition of PT-A (peanut ethanol extract) in D1 was significantly better than that in D2 (soybean ethanol extract). Exogenous addition effectively promotes the rooting of Aspen. Based on this, D3 and D4 media were prepared using ethanol extracts from different peanut parts as exogenous additives for rooting culture (20 bottles of each media). Ultimately, PTL-A (peanut leaf ethanol extract) showed the best overall rooting effect in D4 medium as the exogenous additive. Furthermore, D5 medium using PTL-B (peanut leaf saline extract) as the exogenous additive also showed worse rooting results than D4. Therefore, D4 ​​is the optimal rooting medium for Aspen. Specific results are shown in Table 4 below. Figure 4 As shown:

[0086] Table 4

[0087]

[0088] (5) Transplant survival experiment:

[0089] A mixture of perlite and loam of equal weight was prepared as the transplanting substrate. After sterilization, the substrate was placed in a transplanting frame. After acclimatizing the tissue culture seedlings obtained from the D4 rooting culture for 3 days, the rooting medium was removed, and the seedlings were transplanted into the substrate. They were then cultured at 22±2℃ and 65-70% humidity for 30 days, with a final survival rate of 97% (e.g., ...). Figure 5 As shown in the figure, this means that a high survival rate can be achieved.

[0090] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for tissue culture of polyploid aspen, characterized in that, The method includes the following steps: S1. Disinfection of explants: Select young stem segments with axillary buds that sprouted in the current year from polyploid poplar as explants, disinfect them, and obtain sterile explants for later use. S2. Differentiation culture of explants: The above-mentioned sterile explants were inoculated into differentiation medium and cultured to obtain differentiated shoot materials. The formula of the differentiation medium was: MS + BA 1.0 mg / L + NAA 0.1 mg / L + sucrose 30 g / L + agar 5.5 g / L. S3. Proliferation culture: The above-mentioned differentiated buds were inoculated into the proliferation culture medium to obtain clustered buds. The formulation of the proliferation culture medium was MS + sucrose 30g / L + agar powder 5g / L + 6-BA 0.5mg / L + NAA 0.1mg / L + peanut bud extract 5-8mg / L. The preparation method of the peanut sprout extract includes the following steps: S3-1. Grind peanut sprouts into a paste by adding 3-5 times their volume of 3% salt water to obtain a mixed solution for later use. S3-2. Place the mixture in a water bath at 40-50℃ and stir for 30-40 minutes. Then press and filter to obtain filtrate and residue. Add 3-5 times the volume of 3% saline to the residue and stir in a water bath at 40-50℃ for 30-40 minutes. Press and filter again. Combine the two filtrates and dry to obtain peanut sprout extract. The drying method is to concentrate it under vacuum to 1 / 5 of the original volume and then dry it with hot air at 35-45℃. S4. Rooting culture: The above-mentioned clustered buds are inoculated into the rooting medium for rooting culture to obtain rooted poplar seedlings. The formula of the rooting medium is: 1 / 2MS + NAA 0.1 mg / L + ABA 0.5 mg / L + sucrose 30 g / L + agar powder 5 g / L + peanut leaf extract 10-12 mg / L. The preparation method of the peanut leaf extract includes the following steps: S4-1. Dry fresh peanut leaves at 40-50℃ to constant weight, then pulverize them and pass them through an 80-mesh sieve to obtain peanut leaf powder for later use. S4-2. Add 10-12 times the volume of 75% ethanol to peanut leaf powder, reflux for 1.5-2 hours, and then freeze-dry to obtain peanut leaf extract; the reflux extraction temperature is 70-80℃.

2. The method for polyploid aspen tissue culture according to claim 1, characterized in that: The disinfection method in step S1 is to cut off the leaves of the tender stem segments with axillary buds, rinse with Tween 80 and then rinse with sterile water, then treat with 75% ethanol for 30 seconds, treat with 0.1% mercuric chloride for 8 minutes, and then rinse with sterile water.

3. The method for polyploid aspen tissue culture according to claim 1, characterized in that: In step S2, the differentiation culture time is 35-40 days, the culture temperature is 25℃±2℃, the light intensity is 1500-5000 Lux, and the daily light duration is 12-16 hours.

4. The method for polyploid aspen tissue culture according to claim 1, characterized in that: The proliferation culture time is 50-60 days, and the culture temperature is 25℃±2℃, the light intensity is 1500-5000 Lux, and the daily light time is 12-16 hours.

5. The method for polyploid aspen tissue culture according to claim 1, characterized in that: The rooting culture period is 35-40 days, and the culture temperature is 25℃±2℃, the light intensity is 1500-5000 Lux, and the daily light duration is 12-16 hours.

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