CN112154917A - Method for effectively inhibiting cluster bud browning in tissue culture process of phyllostachys pubescens - Google Patents
Method for effectively inhibiting cluster bud browning in tissue culture process of phyllostachys pubescens Download PDFInfo
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Abstract
The invention discloses a method for effectively inhibiting cluster bud browning in a tissue culture process of phyllostachys pubescens, and belongs to the technical field of plant tissue culture. The method comprises the following steps: selecting a brocade bamboo explant, sterilizing, performing primary induction culture, performing secondary multiplication culture, performing anti-browning culture and the like. Aiming at the problem of serious cluster bud browning in the existing brocade bamboo tissue culture, the invention effectively inhibits the oxidation of phenolic substances by adding the anti-browning agent and adjusting the medium components and hormone proportion, avoids the occurrence of the cluster bud browning phenomenon of the brocade bamboo, can reduce the cluster bud browning rate of the brocade bamboo to be below 5 percent, solves the problems of high cluster bud browning rate and easy death in the brocade bamboo tissue culture process, shows better technical advantages, and also lays a good technical foundation for improving the rapid propagation efficiency of the brocade bamboo.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for effectively inhibiting cluster bud browning in a brocade bamboo tissue culture process.
Background
Phyllostachys pubescens (Hibanobambus longifolia f. shiroshima) also named as phyllostachys albopictus, belongs to the genus Phyllostachys and mixed bamboo species. The height of the bamboo is 1.5-2m, the diameter is 0.8-1.4cm, the bamboo is shorter in the first planting period, the leaves are needle-shaped to broad-needle-shaped, the length is 9-25cm, and the width is 1.3-5 cm. In late 4 months of the bamboo shoot period, new leaves are yellow and white, wider yellow and white stripes are formed among gradual greenness, the leaf color is bright, and the bamboo is listed as a rare color leaf ornamental bamboo seed in China (Shi army, easy coculture, Marliza, Wang sea waves, Yanglin. China color bamboo resource and protection and utilization thereof, Sichuan forestry science and technology 2016,27 (3): 42-48).
The bamboo plant has luxuriant branches and leaves and evergreen seasons, and has great significance in conserving water sources, maintaining water and soil, preventing wind and fixing sand, adjusting climate, purifying air, reducing noise and the like. Meanwhile, the bamboo is not withered in winter, has unique shape, elegant style and high ornamental value, and is an important garden ornamental plant. In recent years, global attention to the quality of the living environment, rapid progress of urbanization and continuous improvement of living standard of people are focused, and the ecological and environmental landscape functions of bamboo plants are increasingly paid attention by people. With the increasing attention on ornamental bamboos, the traditional propagation methods such as bamboo removal, bamboo stump, penis burying, stalk burying, node burying and the like have the defects of more consumed mother bamboos, inconvenient seedling transportation, high labor intensity, low propagation coefficient and the like, and are difficult to meet the requirements of current commercial production. Aiming at the phenomenon, plant tissue culture technology is applied to the production of ornamental bamboos, the tissue culture research of the brocade bamboos has been reported at present (the research on the tissue culture technology of the brocade bamboos has been reported (the research on the technologies of remai, wangxahfeng, linxinchun, myrcia, Zhancui, Huanglichun, the research on the test tube rapid propagation technology of the phyllostachys albopictus, the academy of southwestern forest, 2010,30 (3): 38-41), but the cluster buds of the brocade bamboos are easy to brown in the rapid propagation process, explants are easy to brown and die, and the establishment of a rapid propagation system.
The browning of the explant is one of three problems in plant tissue culture and is an important factor influencing the success of tissue culture. It has been found that the main cause of browning is caused by the action of polyphenol oxidase on the natural substrate phenolics to form quinones, which is better controlled by the addition of antioxidants, adsorbents and by adjustment of the osmotic pressure.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a method for effectively inhibiting the browning of the cluster buds in the tissue culture process of the phyllostachys pubescens, wherein the browning resistant agent is added into the culture medium and the concentration of sucrose is adjusted, so that the browning phenomenon of the tissue culture seedlings of the phyllostachys pubescens is reduced, the browning resistant capability of the cluster buds of the phyllostachys pubescens is effectively improved, a large number of healthy cluster buds of the phyllostachys pubescens are obtained in a short time, and a foundation is laid for the establishment of a rapid propagation system of the.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
a method for effectively inhibiting cluster bud browning in a tissue culture process of Phyllostachys pubescens comprises the following steps:
(1) selecting explants: selecting healthy and disease-free semi-lignified buds of the Jinzhu in the current year in 6-8 months in summer, and pretreating for later use;
(2) and (3) disinfection and sterilization: sterilizing the pretreated explant by using a sodium hypochlorite solution and alcohol to obtain a sterile phyllostachys pubescens explant;
(3) primary induction culture: inoculating the aseptic brocade bamboo explant to an induction culture medium for cluster bud induction to obtain an aseptic bud; wherein the induction culture medium is MS +4 mg/L6-BA (6-benzylamino adenine) +0.5mg/L KT (kinetin) +0.5mg/L TDZ (thiadiazolyl phenylurea);
(4) subculture multiplication culture: after healthy sterile seedlings are obtained in primary culture, cutting the seedlings into 3-5 buds as a cluster, and transferring the cluster to an adventitious bud multiplication culture medium for subculture multiplication culture for 30 d; the proliferation culture medium is MS +5 mg/L6-BA +0.2mg/L KT, and an anti-browning agent or sucrose is added into the proliferation culture medium; the anti-browning agent is PVP (polyvinylpyrrolidone), L-GLu (L-glutamine) or activated carbon, and the concentration of sucrose is adjusted to change the osmotic pressure in the culture medium.
According to the method for effectively inhibiting cluster bud browning in the tissue culture process of the phyllostachys pubescens, the concentration of PVP is 0.2-2 g/L, the concentration of L-GLu is 0.05-1 g/L, and the concentration of activated carbon is 0.2-2 g/L; the concentration of the sucrose is 10-30 g/L;
the method for effectively inhibiting cluster bud browning in the tissue culture process of the phyllostachys pubescens comprises the following steps of 1) explant pretreatment: cutting the current-year-old branches collected outdoors into stem sections with buds with the length of 0.5-1.5 cm, cleaning for 1min by using a detergent, and showering for 3-4h by using tap water.
The method for effectively inhibiting cluster bud browning in the tissue culture process of the phyllostachys pubescens comprises the following steps of explant disinfection and sterilization treatment in step 2): soaking the workpiece on a superclean workbench for 45-60 s by using alcohol with the mass fraction of 70%, washing the workpiece for 3-5 times by using sterile water, disinfecting the workpiece for 12-17min by using sodium hypochlorite with the mass fraction of 0.5%, and washing the workpiece for 4-5 times by using the sterile water.
According to the method for effectively inhibiting cluster bud browning in the tissue culture process of the phyllostachys pubescens, the concentration of L-glutamine in the step 5) is 50 mg/L; the sucrose concentration was 10 g/L.
According to the method for effectively inhibiting cluster bud browning in the tissue culture process of the phyllostachys pubescens, the pH of the induction culture medium and the pH of the proliferation culture medium are both adjusted to 5.80 before sterilization, and the sterilization condition is 15min at 121 ℃.
According to the method for effectively inhibiting cluster bud browning in the tissue culture process of the phyllostachys pubescens, the temperature of a culture room is 25 +/-1 ℃ during primary induction culture and secondary multiplication culture, the photoperiod is 14 h/dark 10h, and the illumination intensity of the surface of a culture is about 3000 lx.
Has the advantages that: compared with the prior art, the invention has the advantages that:
(1) the method optimizes the operation method of the brocade bamboo tissue culture at each stage, effectively reduces the browning rate to be below 5 percent, and solves the problem of high tissue culture browning rate of the brocade bamboo.
(2) The invention adopts three anti-browning agents of PVP (polyvinylpyrrolidone), L-glutamine and active carbon, which are respectively added into a culture medium, and the effect of preventing browning is achieved by adsorbing phenolic substances released by explants and quinone substances formed after oxidation.
(3) The invention further optimizes the culture medium formula in the tissue culture and proliferation stage of the phyllostachys pubescens, adjusts the concentration of sucrose in the culture medium, and effectively reduces the browning problem in the tissue culture process of the phyllostachys pubescens.
(4) The phenolic substances accumulated on the wound of the brocade bamboo explant can cause browning after too long time, and the subculture time is shortened to be within 20 days, so that the cluster bud browning phenomenon in the tissue culture process of the brocade bamboo can be reduced.
Drawings
FIG. 1 is a diagram of a Phyllostachys bambusicola explant;
FIG. 2 is a diagram of induced clump buds;
FIG. 3 is a diagram of the proliferation of multiple shoots of Phyllostachys bambusoides;
FIG. 4 is a 30d cluster bud map of brocade bamboo proliferation;
FIG. 5 is a diagram of browned Phyllostachys bambusoides bud;
FIG. 6 is a comparison chart of the growth of the brocade bamboo bud with different concentrations of PVP;
FIG. 7 is a comparison chart of the growth of the Phyllostachys pubescens buds added with L-glutamine of different concentrations;
FIG. 8 is a comparison chart of the growth of the brocade bamboo bud with different concentrations of active C;
FIG. 9 is a comparison chart of the growth of the Phyllostachys pubescens buds with different sucrose contents.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
Example 1
A method for effectively inhibiting cluster bud browning in the tissue culture process of phyllostachys pubescens. The method comprises the following steps:
(1) selecting explants: selecting healthy and disease-free semi-lignified branch with buds of the current-year Jinzhu in 6-8 months in summer, shearing the current-year branch collected outdoors into stem sections with buds of about 1cm, cleaning with detergent for 1min, and showering with tap water for 3-4 h.
(2) And (3) disinfection and sterilization: soaking the pretreated explant in 70% alcohol for 1min on a clean bench, washing with sterile water for 3-5 times, disinfecting with 0.5% sodium hypochlorite for 15min, and washing with sterile water for 4-5 times; placing in a starting medium: MS +4 mg/L6-BA +0.5mg/L KT +0.5mg/L TDZ, and after the brocade bamboo rhizome buds treated by the disinfection method are inoculated, the aseptic rate is the highest, and the bud induction survival rate is high (Table 1).
TABLE 1 Effect of different disinfectant treatments on explant culture at different times
(3) Primary induction culture: inoculating the obtained aseptic chinlon bamboo explant to an induction culture medium for cluster bud induction, and forming cluster buds after one week; wherein the induction culture medium is MS +4 mg/L6-BA +0.5mg/L KT +0.5mg/L TDZ; MS in the present invention means MS medium components (Murashige & Skoog medium, 1962, Physiol. plant 15: 473-;
(4) subculture multiplication culture: after healthy sterile seedlings are obtained in primary culture, cutting the seedlings into 3-5 buds as a cluster, and transferring the cluster to an adventitious bud multiplication culture medium for subculture multiplication culture; in 9 formulas, the optimal proliferation medium is MS +5 mg/L6-BA +0.2mg/L KT from the aspects of proliferation coefficient and seedling growth height.
TABLE 2 Effect of different phytohormone concentrations on the proliferation of Phyllostachys pubescens shoots
Note: the different lower case letters represent that the same column differs significantly at the 0.05 level
(5) Anti-browning culture: adding PVP, L-glutamine or active carbon with different concentrations into a proliferation culture medium MS +5 mg/L6-BA +0.2mg/L KT (30 g/L of sucrose), wherein the concentration of the PVP is 0.2g/L, 1g/L or 2 g/L; the concentration of L-glutamine is 50mg/L, 0.2g/L and 1 g/L; the concentration of the active carbon is 0.2g/L, 1g/L and 2 g/L; the sucrose concentration was 10g/L, 20g/L, 30 g/L. Through research, the effect of adding PVP with different concentrations and active carbon on inhibiting the browning of the brocade bamboo tissue culture seedlings is found to be poor. The browning rate of the brocade bamboo shoot buds is different from that of the L-glutamine with different concentrations, and the browning rate is only 4.81% under the condition of adding 50mg/L L-glutamine, but the browning rate is increased along with the increase of the concentration. Meanwhile, the osmotic pressure of the culture medium is changed by adjusting the concentration of sucrose in the culture medium, the culture medium suitable for the multiplication growth of the Phyllostachys pubescens clumps is further screened, the concentration of the sucrose is 10g/L, 20g/L and 30g/L, and experiments show that when the concentration of the sucrose is 10g/L, the browning rate of the Phyllostachys pubescens clumps is only 3.05%, the clumps are good in growth vigor, multiple flowers and leaves are tender and green, and the leaves are large and unfolded.
TABLE 3 Effect of different anti-browning agents on the proliferation of Phyllostachys Pubescens clumps
Claims (7)
1. A method for effectively inhibiting cluster bud browning in a tissue culture process of Phyllostachys pubescens is characterized by comprising the following steps:
(1) selecting explants: selecting healthy and disease-free semi-lignified buds of the Jinzhu in the current year in 6-8 months in summer, and pretreating for later use;
(2) and (3) disinfection and sterilization: sterilizing the pretreated explant by using a sodium hypochlorite solution and alcohol to obtain a sterile phyllostachys pubescens explant;
(3) primary induction culture: inoculating the aseptic brocade bamboo explant to an induction culture medium for cluster bud induction to obtain an aseptic bud; wherein the induction culture medium is MS +4 mg/L6-BA +0.5mg/L KT +0.5mg/L TDZ;
(4) subculture multiplication culture: after healthy sterile seedlings are obtained in primary culture, cutting the seedlings into 3-5 buds as a cluster, and transferring the cluster to an adventitious bud multiplication culture medium for subculture multiplication culture for 30 d; the proliferation culture medium is MS +5 mg/L6-BA +0.2mg/L KT, and an anti-browning agent or sucrose is added into the proliferation culture medium; the anti-browning agent is PVP, L-GLu or activated carbon.
2. The method for effectively inhibiting cluster bud browning in the tissue culture process of the phyllostachys pubescens as claimed in claim 1, wherein the concentration of PVP is 0.2-2 g/L, the concentration of L-GLu is 0.05-1 g/L, and the concentration of activated carbon is 0.2-2 g/L; the concentration of the sucrose is 10-30 g/L.
3. The method for effectively inhibiting cluster bud browning in the tissue culture process of the phyllostachys pubescens as claimed in claim 1, wherein in the step 1), the pretreatment of the explant comprises the following steps: cutting the current-year-old branches collected outdoors into stem sections with buds with the length of 0.5-1.5 cm, cleaning for 1min by using a detergent, and showering for 3-4h by using tap water.
4. The method for effectively inhibiting the cluster bud browning in the tissue culture process of the phyllostachys pubescens as claimed in claim 1, wherein the explant sterilization treatment in the step 2): soaking the workpiece on a superclean workbench for 45-60 s by using alcohol with the mass fraction of 70%, washing the workpiece for 3-5 times by using sterile water, disinfecting the workpiece for 12-17min by using sodium hypochlorite with the mass fraction of 0.5%, and washing the workpiece for 4-5 times by using the sterile water.
5. The method for effectively inhibiting cluster bud browning in the tissue culture process of the phyllostachys pubescens as claimed in claim 1, wherein the concentration of L-glutamine in the step 5) is 50 mg/L; the sucrose concentration was 10 g/L.
6. The method for effectively inhibiting the cluster bud browning of the brocade bamboo during the tissue culture process as claimed in claim 1, wherein the pH of the induction medium and the proliferation medium are adjusted to 5.80 before sterilization, and the sterilization condition is 121 ℃ for 15 min.
7. The method for effectively inhibiting the cluster bud browning during the tissue culture of Phyllostachys pubescens according to claim 1, wherein the temperature of the culture chamber during the primary induction culture and the secondary proliferation culture is 25 ± 1 ℃, the photoperiod is 14 h/dark 10h, and the illumination intensity on the culture surface is about 3000 lx.
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Cited By (2)
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| CN114793892A (en) * | 2022-02-24 | 2022-07-29 | 南京林业大学 | Primary culture method for reducing yellow-heart nocturnal explant pollution and browning |
| CN114793892B (en) * | 2022-02-24 | 2023-08-01 | 南京林业大学 | Primary culture method for reducing pollution and browning of yellow heart night-integrated explants |
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