Tissue culture and propagation method of East China raspberry and its application

Abstract

The invention relates to the technical field of plant tissue culture, and specifically to a method for tissue culture and propagation of East China raspberry and its application; the steps include: selecting explants for cleaning and disinfection, inoculating the disinfected explants in a primary generation cluster bud induction medium for culture, cutting the obtained cluster buds into single buds and inoculating them in a secondary proliferation medium for culture, and cutting the obtained cluster buds into single buds and inoculating them in a rooting medium. The method of the invention reduces the contamination rate to below 5%, increases the cluster bud induction rate from about 10% of a conventional plant growth regulator combination to more than 70%, stabilizes the proliferation coefficient to more than 4, increases the rooting rate to more than 99%, and can also make the seedlings more robust, which is more conducive to the survival of domestication.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and propagation method of East China raspberry and its application. Background Art

[0002] Rubus chingii Hu is a plant of the Rosaceae family. Its fruit is used as medicine, which has the effects of nourishing the kidney, consolidating essence and reducing urine, nourishing the liver and improving eyesight. It is often used for spermatorrhea, nocturnal emission, frequent urination, impotence, premature ejaculation, and blurred vision.

[0003] East China raspberry seedlings are generally propagated by root suckers and cuttings. Division propagation: A large number of root suckers are produced every spring and autumn. When they grow to 1-1.3m, they are topped and the soil is loosened for trench fertilization to promote the development of adventitious roots. Division can be carried out at the end of the season or early spring. Each mother plant can be divided into 2-10 seedlings each year. Cutting propagation: Before germination in spring, cut the lignified branches with buds, cut them into about 10cm, and insert them into a well-drained nursery bed covered with mulch. The survival rate is about 40%.

[0004] CN105918126A discloses a method for rapid propagation of Raspberry palmata virus-free seedlings in vitro; CN117322331A discloses a method for propagating high-quality seedlings of Raspberry palmata through tissue culture; Wang Liping et al. Establishment of a high-quality Raspberry palmata rapid propagation system. Zhejiang Agricultural Science. 2013, (Issue 8), pp. 967-970. However, no method for rapid tissue propagation of Raspberry palmata in East China has been reported so far. Summary of the invention

[0005] The invention aims to provide a tissue culture and propagation method of East China raspberry and its application, so as to realize the rapid propagation of East China raspberry.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for tissue culture and propagation of East China raspberry, comprising the following steps:

[0007] (1) Selection and cleaning of explants: Select young, unlignified stem segments 15 to 25 cm from the end, remove the leaves, cut into 2 to 3 cm long segments with buds, rinse with tap water for 30 to 40 minutes, then rinse with saturated soap solution for 10 to 15 minutes, rinse with clean water until no obvious soap solution remains, add 2 to 3 drops of Tween-80, shake for 10 to 15 minutes, and finally rinse with tap water for 50 to 70 minutes;

[0008] (2) Disinfection of explants: Transfer the cleaned explants to a clean bench and disinfect them with 75% alcohol, saturated bleach solution, 75% alcohol, and 0.08% mercuric chloride in sequence;

[0009] (3) Primary bud induction: Use sterile paper to absorb the surface moisture of the sterilized explants, cut off the wounds at both ends, inoculate them into the primary bud induction medium, and culture them in an environment with a light intensity of 3000-4000 lx, a single day of light for 10 hours, and a temperature of 25±2℃ for 40-50 days;

[0010] The formula of the primary generation clustered bud induction medium is: WPM medium + NAA 0.02-0.05 mg / L + CPPU 0.10-0.15 mg / L + TDZ 2.0-3.0 mg / L;

[0011] (4) Subculture proliferation culture: After primary induction culture, the clustered buds are cut into single buds, inoculated into subculture proliferation medium, and cultured in an environment with a light intensity of 3000-4000 lx, a single-day light intensity of 10 h, and a temperature of 25±2°C for 25-35 days;

[0012] The formula of the secondary proliferation medium is: improved MS medium + IBA 0.1-0.2 mg / L + TDZ 1.0-2.0 mg / L + KT 5.0-6.0 mg / L;

[0013] (5) Rooting culture: The clustered buds obtained through subculture proliferation culture are cut into single buds, inoculated into rooting medium, and cultured in a dark environment at a temperature of 28±2℃ for 5 to 7 days, and then cultured in a dark environment at a light intensity of 3000 to 4000 lx, a single day of light for 10 hours, and a temperature of 25±2℃ for 30 to 35 days;

[0014] The formula of the rooting medium is: 1 / 4 improved MS medium + IBA 0.4-0.6 mg / L + coconut juice 50-70 ml / L + AC 0.2-0.4 g / L;

[0015] The formula of the improved MS culture medium is as follows: the amount of magnesium sulfate is adjusted to 0.50 g / L, the amount of ferrous sulfate is adjusted to 0.064 g / L, the amount of EDTA-disodium is adjusted to 0.087 g / L, the amount of calcium chloride dihydrate is adjusted to 0.33 g / L, the pH is adjusted to 5.4, and the other ingredients remain unchanged.

[0016] Further: In step (2), the disinfection is specifically as follows: 75% alcohol disinfection for 50-60 seconds, rinsing with sterile water 3-4 times, saturated bleach solution disinfection for 15-20 minutes, rinsing with sterile water 4-5 times, 75% alcohol disinfection for 5-10 seconds, rinsing with sterile water 2-3 times, 0.08% mercuric chloride solution disinfection for 8-9 minutes, rinsing with sterile water 6-7 times.

[0017] Further: In step (3), the formula of the primary cluster bud induction medium is: WPM medium + NAA 0.03 mg / L + CPPU 0.12 mg / L + TDZ 2.5 mg / L.

[0018] Further: In step (4), the formula of the subculture proliferation medium is: modified MS medium + IBA 0.15 mg / L + TDZ 1.5 mg / L + KT 5.5 mg / L.

[0019] Further: In step (5), the formula of the rooting medium is: 1 / 4 modified MS medium + IBA 0.5 mg / L + coconut juice 60 ml / L + AC 0.3 g / L.

[0020] The beneficial technical effects of the present invention are:

[0021] 1. The disinfection method adopted by the present invention adopts an organic combination of 75% alcohol, saturated bleach solution and 0.08% mercuric chloride solution, which can reduce the contamination rate to the greatest extent without damaging the explants, so that the contamination rate in the process of establishing the sterile system is reduced to less than 5%, and the success rate of East China raspberry in the process of establishing the sterile system is as high as more than 90%.

[0022] 2. During the primary induction of Raspberry, most of the leaves unfold but clustered buds occur. The present invention found in the study of primary clustered bud induction that Raspberry is extremely sensitive to CPPU. When the concentration is too low, clustered buds cannot be induced. When the concentration is too high, it is very easy to cause browning or even necrosis of the explants, and clustered buds cannot be induced. During the primary clustered bud induction, the present invention uses an organic combination of NAA, CPPU and TDZ with appropriate concentrations to increase the clustered bud induction rate from about 10% of the conventional plant growth regulator combination to more than 70%.

[0023] 3. NAA has a significant inhibitory effect on rooting of Raspberry. When NAA is used in the subculture medium, no matter how high the concentration is, it will reduce the rooting rate during rooting culture and prolong the rooting time. Therefore, in the subculture proliferation culture, IBA is used instead of NAA. On the one hand, it can achieve the proliferation effect, and on the other hand, it is beneficial to the rooting during rooting culture. At the same time, Raspberry is too sensitive to CPPU, which leads to the accumulation of CPPU during subculture proliferation culture, making the proliferation coefficient unstable and causing poisoning. In addition, the cost of using CPPU is high, which is ultimately not conducive to factory production. The present invention uses a combination of IBA, TDZ and KT during subculture proliferation culture to stabilize the proliferation coefficient to above 4.0.

[0024] 4. The present invention creatively adds coconut juice to the rooting medium, and simultaneously adopts high temperature, lightless, dark cultivation for 5 to 7 days combined with a conventional cultivation environment for 30 to 35 days. On the one hand, the rooting time can be shortened from 55 to 65 days without adding coconut juice to 35 to 40 days, and on the other hand, the rooting rate can be increased to more than 99%, and at the same time, the seedlings can be made thicker and more conducive to domestication and survival.

[0025] 5. During the proliferation culture and rooting culture process, when the basic culture medium commonly used in tissue culture, such as WPM, MS, N6, B5, etc., most of the East China raspberries will have unhealthy conditions such as leaf shrinkage and leaf yellowing. When the most classic MS is used as the basic culture medium, the symptoms are improved but cannot be eliminated. The present invention creatively changes the concentration of iron salts, calcium salts and magnesium salts, and lowers the pH at the same time, which can completely eliminate the occurrence of unhealthy conditions such as leaf shrinkage and leaf yellowing, and reduce the loss of commercial seedlings during factory production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0027] Figure 1 This is a diagram of the primary culture results of Example 1 of the present invention.

[0028] Figure 2 It is a diagram of the successive culture results of Example 1 of the present invention.

[0029] Figure 3 This is a diagram showing the rooting culture results of Example 1 of the present invention. DETAILED DESCRIPTION

[0030] 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 ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] A method for tissue culture and propagation of East China raspberry, comprising the following steps:

[0033] (1) Selection and cleaning of explants: Select a 20 cm non-lignified young stem segment, remove the leaves, cut into 2.5 cm long segments with buds, rinse with tap water for 35 min, then rinse with saturated soap solution for 13 min, rinse with clean water until no obvious soap solution remains, add 3 drops of Tween-80, shake for 13 min, and finally rinse with tap water for 60 min.

[0034] (2) Disinfection of explants: Transfer the cleaned explants to a clean bench and disinfect them with 75% alcohol for 55 seconds and rinse with sterile water four times, disinfect them with saturated bleach solution for 17 minutes and rinse with sterile water four times, disinfect them with 75% alcohol for 8 seconds and rinse with sterile water twice, and disinfect them with 0.08% mercuric chloride solution for 8 minutes and rinse with sterile water six times.

[0035] (3) Primary bud induction: Use sterile paper to absorb the surface moisture of the sterilized explants, cut off the wounds at both ends, inoculate them into primary bud induction medium (formula: WPM medium + NAA 0.03mg / L + CPPU 0.12mg / L + TDZ2.5mg / L), and culture them in an environment with a light intensity of 3500lx, a single day of light for 10h, and a temperature of 25±2℃ for 45 days. The culture results are as follows: Figure 1 As shown, the explants developed new leaves.

[0036] (4) Subculture proliferation culture: After primary induction culture, the clustered buds were cut into single buds, inoculated into subculture proliferation medium (formula: modified MS medium + IBA 0.15mg / L + TDZ 1.5mg / L + KT 5.5mg / L), and cultured in an environment with a light intensity of 3500lx, a single day of light for 10h, and a temperature of 25±2℃ for 30 days. The culture results are as follows: Figure 2 shown.

[0037] (5) Rooting culture: The clustered buds obtained by subculture proliferation culture were cut into single buds and inoculated into rooting medium (formula: 1 / 4 modified MS medium + IBA 0.5 mg / L + coconut juice 60 ml / L + AC 0.3 g / L), and cultured in a dark environment at a temperature of 28±2℃ for 6 days. Then, cultured in a dark environment at a light intensity of 3500 lx, a single day of light for 10 hours, and a temperature of 25±2℃ for 30 days. The culture results are as follows: Figure 3 As shown, the explants grew into tissue culture plantlets with roots and leaves.

[0038] Among them, the formula of the improved MS culture medium is: the amount of magnesium sulfate is adjusted to 0.50 g / L, the amount of ferrous sulfate is adjusted to 0.064 g / L, the amount of EDTA-disodium is adjusted to 0.087 g / L, the amount of calcium chloride dihydrate is adjusted to 0.33 g / L, the pH is adjusted to 5.4, and the other ingredients remain unchanged.

[0039] Example 2

[0040] A method for tissue culture and propagation of East China raspberry, comprising the following steps:

[0041] (1) Selection and cleaning of explants: Select a 15 cm non-lignified young stem segment, remove the leaves, cut into 2 cm long segments with buds, rinse with tap water for 30 min, then rinse with saturated soap solution for 10 min, rinse with clean water until no obvious soap solution remains, add 2 drops of Tween-80, shake for 10 min, and finally rinse with tap water for 50 min.

[0042] (2) Disinfection of explants: Transfer the cleaned explants to a clean bench and disinfect them with 75% alcohol for 50 seconds, rinse with sterile water three times, disinfect with saturated bleach solution for 15 minutes, rinse with sterile water four times, disinfect with 75% alcohol for 5 seconds, rinse with sterile water twice, and disinfect with 0.08% mercuric chloride solution for 8 minutes, rinse with sterile water six times.

[0043] (3) Primary cluster bud induction: Use sterile paper to absorb the surface moisture of the disinfected explants, cut off the wounds at both ends, inoculate them into primary cluster bud induction medium (formula: WPM medium + NAA 0.02mg / L + CPPU 0.10mg / L + TDZ2.0mg / L), and place them in an environment with a light intensity of 3000lx, a single day of light for 10h, and a temperature of 25±2℃ for 40 days.

[0044] (4) Subculture proliferation culture: The clustered buds obtained through primary induction culture were cut into single buds, inoculated into subculture proliferation medium (formula: modified MS medium + IBA 0.1 mg / L + TDZ 1.0 mg / L + KT 5.0 mg / L), and cultured in an environment with a light intensity of 3000 lx, a single day of light for 10 h, and a temperature of 25±2℃ for 25 days.

[0045] (5) Rooting culture: The clustered buds obtained through subculture proliferation culture were cut into single buds, inoculated into rooting medium (formula: 1 / 4 modified MS medium + IBA 0.4 mg / L + coconut juice 50 ml / L + AC 0.2 g / L), and cultured in a completely dark environment at a temperature of 28±2°C for 5 days. Thereafter, cultured in a completely dark environment at a light intensity of 3000 lx, a single day of light for 10 h, and a temperature of 25±2°C for 30 days.

[0046] Among them, the formula of the improved MS culture medium is: the amount of magnesium sulfate is adjusted to 0.50 g / L, the amount of ferrous sulfate is adjusted to 0.064 g / L, the amount of EDTA-disodium is adjusted to 0.087 g / L, the amount of calcium chloride dihydrate is adjusted to 0.33 g / L, the pH is adjusted to 5.4, and the other ingredients remain unchanged.

[0047] Example 3

[0048] A method for tissue culture and propagation of East China raspberry, comprising the following steps:

[0049] (1) Selection and cleaning of explants: Select a 25 cm non-lignified young stem segment, remove the leaves, cut into 3 cm long segments with buds, rinse with tap water for 40 min, then rinse with saturated soap solution for 15 min, rinse with clean water until no obvious soap solution remains, add 3 drops of Tween-80, shake for 15 min, and finally rinse with tap water for 70 min.

[0050] (2) Disinfection of explants: Transfer the cleaned explants to a clean bench and disinfect them with 75% alcohol for 60 seconds and rinse with sterile water four times, disinfect them with saturated bleach solution for 20 minutes and rinse with sterile water five times, disinfect them with 75% alcohol for 10 seconds and rinse with sterile water three times, and disinfect them with 0.08% mercuric chloride solution for 9 minutes and rinse with sterile water seven times.

[0051] (3) Primary cluster bud induction: Use sterile paper to absorb the surface moisture of the disinfected explants, cut off the wounds at both ends, inoculate them into primary cluster bud induction medium (formula: WPM medium + NAA 0.05mg / L + CPPU 0.15mg / L + TDZ3.0mg / L), and place them in an environment with a light intensity of 4000lx, a single day of light for 10h, and a temperature of 25±2℃ for 50 days.

[0052] (4) Subculture proliferation culture: The clustered buds obtained through primary induction culture were cut into single buds, inoculated into subculture proliferation medium (formula: modified MS medium + IBA 0.2 mg / L + TDZ 2.0 mg / L + KT 6.0 mg / L), and cultured in an environment with a light intensity of 4000 lx, a single day of light for 10 h, and a temperature of 25±2℃ for 35 days.

[0053] (5) Rooting culture: The clustered buds obtained through subculture proliferation culture were cut into single buds and inoculated into rooting medium (formula: 1 / 4 modified MS medium + IBA 0.6 mg / L + coconut juice 70 ml / L + AC 0.4 g / L), and cultured in a completely dark environment at a temperature of 28±2°C for 7 days. Thereafter, cultured in a completely dark environment at a light intensity of 4000 lx, a single day of light for 10 h, and a temperature of 25±2°C for 35 days.

[0054] The formula of the improved MS culture medium is as follows: the amount of magnesium sulfate is adjusted to 0.50 g / L, the amount of ferrous sulfate is adjusted to 0.064 g / L, the amount of EDTA-disodium is adjusted to 0.087 g / L, the amount of calcium chloride dihydrate is adjusted to 0.33 g / L, the pH is adjusted to 5.4, and the other ingredients remain unchanged.

[0055] Comparative Example 1

[0056] The steps (1) of selecting and washing the rubus idaeus were performed according to Example 1, and the subsequent steps were performed according to Yu Zhenming, Ying Yuxin, Chen Junyu, et al. A method for propagating high-quality seedlings of rubus idaeus through tissue culture: 202311285489[P][2024-07-03].

[0057] Comparative Example 2

[0058] The steps (1) of Example 1, i.e., selection and washing of Raspberries of East China, were followed, and the best method disclosed in Wang Liping, Chen Zhen, Jiang Jingyong, et al., Establishment of a rapid propagation system for high-quality Raspberries of Palmate Leaf [J]. Zhejiang Agricultural Sciences, 2013(8):4. DOI:10.3969 / j.issn.0528-9017.2013.08.019 was followed.

[0059] Comparative Example 3

[0060] The steps (1) of Example 1, i.e., selection and washing of Rubus idaeus, were followed, and then the best method disclosed by Xie Congshou, Chen Yonghe, Ying Xueyang, Zheng Pinghan, Yang Fuliang, Rao Baorong, Xiong Xinqin, Hu Jianzhu, and Chen Qihui. Rapid propagation technology of Rubus idaeus through tissue culture [J]. Fujian Agricultural Science and Technology, 2020, No.362(10):36-40. was followed.

[0061] During the cultivation process, statistics were collected on the disinfection mortality rate, disinfection contamination rate, primary generation induction rate, value-added coefficient and rooting rate in the above Examples 1-3 and Comparative Examples 1-3, and the results are shown in the following table.

[0062] Table 1 Statistical results

[0063] Disinfection mortality rate (%) Disinfection contamination rate (%) Primary induction rate (%) Value-added coefficient Rooting rate (%) Example 1 6.3% 3.8% 77.8% 4.8 100% Example 2 7.2% 4.1% 76.5% 4.2 99.1% Example 3 6.1% 4.7% 76.3% 4.6 99.8% Comparative Example 1 24.7% 13.2% 11.3% (large amount of callus, only a few clustered buds) 2.1 (still a lot of healing) 94.2% Comparative Example 2 2.3% 36.7% 48.9% 3.7 68.9% Comparative Example 3 10.9% 24.3% 36.0% 3.1 72.6%

[0064] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for tissue culture and propagation of raspberry in East China, characterized in that the steps include: (1) Selection and cleaning of explants: Select young, unlignified stem segments 15 to 25 cm from the end, remove the leaves, cut into 2 to 3 cm long segments with buds, rinse with tap water for 30 to 40 minutes, then rinse with saturated soap solution for 10 to 15 minutes, rinse with clean water until no obvious soap solution remains, add 2 to 3 drops of Tween-80, shake for 10 to 15 minutes, and finally rinse with tap water for 50 to 70 minutes; (2) Disinfection of explants: Transfer the cleaned explants to a clean bench and disinfect them with 75% alcohol, saturated bleach solution, 75% alcohol, and 0.08% mercuric chloride in sequence; (3) Primary bud induction: Use sterile paper to absorb the surface moisture of the sterilized explants, cut off the wounds at both ends, inoculate them into the primary bud induction medium, and culture them in an environment with a light intensity of 3000-4000 lx, a single day of light for 10 hours, and a temperature of 25±2℃ for 40-50 days; The formula of the primary generation cluster bud induction medium is: WPM medium + NAA 0.02-0.05 mg / L + CPPU 0.10-0.15 mg / L + TDZ 2.0-3.0 mg / L; (4) Subculture proliferation culture: The clustered buds obtained by primary induction culture are cut into single buds, inoculated into subculture proliferation medium, and cultured in an environment with a light intensity of 3000-4000 lx, a single-day light intensity of 10 h, and a temperature of 25±2°C for 25-35 days; The formula of the secondary proliferation medium is: improved MS medium + IBA 0.1-0.2 mg / L + TDZ 1.0-2.0 mg / L + KT 5.0-6.0 mg / L; (5) Rooting culture: The clustered buds obtained through subculture proliferation culture are cut into single buds, inoculated into rooting medium, and cultured in a dark environment at a temperature of 28±2℃ for 5 to 7 days, and then cultured in an environment with a light intensity of 3000 to 4000 lx, a single day of light for 10 hours, and a temperature of 25±2℃ for 30 to 35 days; The formula of the rooting medium is: 1 / 4 improved MS medium + IBA 0.4-0.6 mg / L + coconut juice 50-70 ml / L + AC 0.2-0.4 g / L; The formula of the improved MS culture medium is as follows: the amount of magnesium sulfate is adjusted to 0.50 g / L, the amount of ferrous sulfate is adjusted to 0.064 g / L, the amount of EDTA-disodium is adjusted to 0.087 g / L, the amount of calcium chloride dihydrate is adjusted to 0.33 g / L, the pH is adjusted to 5.4, and the other ingredients remain unchanged.

2. The method according to claim 1, characterized in that: In step (2), the disinfection is specifically as follows: disinfection with 75% alcohol for 50-60 seconds, rinsing with sterile water for 3-4 times, disinfection with saturated bleach solution for 15-20 minutes, rinsing with sterile water for 4-5 times, disinfection with 75% alcohol for 5-10 seconds, rinsing with sterile water for 2-3 times, disinfection with 0.08% mercuric chloride solution for 8-9 minutes, rinsing with sterile water for 6-7 times.

3. The method according to claim 1, characterized in that: In step (3), the formula of the primary cluster bud induction medium is: WPM medium + NAA 0.03 mg / L + CPPU 0.12 mg / L + TDZ 2.5 mg / L.

4. The method according to claim 1, characterized in that: In step (4), the formula of the secondary proliferation medium is: modified MS medium + IBA 0.15 mg / L + TDZ 1.5 mg / L + KT 5.5 mg / L.

5. The method according to claim 1, characterized in that: In step (5), the formula of the rooting medium is: 1 / 4 modified MS medium + IBA 0.5 mg / L + coconut juice 60 ml / L + AC 0.3 g / L.

6. Use of the method according to any one of claims 1 to 5 in tissue culture and propagation of Rubus idaeus.

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