CN112586357B - Method for establishing gleditsia sinensis tissue culture regeneration system - Google Patents
Method for establishing gleditsia sinensis tissue culture regeneration system Download PDFInfo
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Abstract
The invention belongs to the technical field of plant asexual propagation, and particularly relates to a method for establishing a gleditsia sinensis tissue culture regeneration system; the soap pod seeds are bred by utilizing a tissue culture technology, and the seeds are pretreated before tissue culture, so that the seed coats are thinned, sterile seedlings can grow out rapidly, the culture time is shortened, and the emergence rate is increased. A large number of in-vitro regenerated plants can be obtained in a short period by a tissue culture technology, the excellent characteristics of the female parent are maintained, and a scientific support is provided for the protection of soap pod resources, fine variety breeding and industrial development.
Description
Technical Field
The invention belongs to the technical field of plant asexual propagation, and particularly relates to a method for establishing a gleditsia sinensis tissue culture regeneration system.
Background
The gleditsia sinensis (Gymnocladus chinensis bail.) is also called as meat gleditsia sinensis, meat gleditsia sinensis and pig (quote (gleditsia sinensis and gleditsia sinensis)), and is a leguminous gleditsia tree, and is widely distributed in southern Shaanxi, sichuan, guizhou, hubei, hunan, northern Guangdong, jiangxi, anhui, southern Jiangsu, zhejiang, fujian and other places. The tree grows on hillsides, hillsides and miscellaneous woods with the altitude of 150-1500 meters, has larger crown shape, is particularly light-loving, is loving warm climate and fertile soil, has quicker growth when the tree is young, is not afraid of strong sunlight, and has slow growth in barren soil. The soap pods are rich in saponin and saponin, the seed oil can be used as industrial oil such as paint, the kernels are edible, the medical value is high, the soap pods are natural raw materials of foods, health products, cosmetics and washing products, are important tree species for landscaping and ecological engineering forestation, have extremely high economic and ecological values, are one of the sun-facing industries of helping hand, removing lean and hard mass and achieving the effect of developing villages, and are also important tree species for low-efficiency forest improvement. The soap pod is oblong, flat or thick, the fruiting amount is small, only 2-4 seeds are contained in one pod, the seeds are difficult to germinate into seedlings under natural conditions due to the special physiological structures of the pods and the seed coats, the seed amount is small, and natural updating is very slow. The resource amount is small, the population is endangered due to incomplete, and the collection, protection, development and utilization are urgently needed. At present, the traditional modes of sowing, cutting, grafting and the like are mainly adopted for seedling raising, and the defects of poor growth vigor, uneven spread, insufficient expression of improved variety potential and the like exist, so that the quantity of high-quality seedling resources is small at present. The tissue culture technology is used for breeding rare endangered species and excellent tree species, and the method has the characteristics of rapid breeding, short breeding period, annual production and the like. At present, no related report on the technical aspect of the tissue culture of the gleditsia sinensis exists, the invention takes seeds as explants, the influence of the combination of hormones with different concentrations on the induction, differentiation, proliferation and rooting of the callus of the gleditsia sinensis is explored through experiments, a tissue culture technical system of the gleditsia sinensis is established, the method has important significance on protecting the germplasm resources of the gleditsia sinensis, and a foundation is laid for the fine variety breeding, the mass production of excellent nursery stocks and the industrial development of the gleditsia sinensis.
At present, a few researches on tissue culture propagation of gleditsia sinensis exist, for example, patent document with application number of CN201610271956.4 discloses a method for establishing a tissue culture regeneration system of gleditsia sinensis, which adopts gleditsia sinensis seeds as explants to culture through disinfection, aseptic seedling culture, callus induction, adventitious bud proliferation, rooting culture and domestication transplantation. However, the research is carried out on the fructus gleditsiae, the fructus gleditsiae is different from the fructus gleditsiae, and the fruit of the fructus gleditsiae is one of Chinese medicinal materials and is different from the plant classification.
The patent document with the application number of CN201811429114.2 discloses a method for tissue culture and rapid propagation of gleditsia sinensis, which takes the tender branch of the base part of a gleditsia sinensis stump as an explant and comprises the steps of disinfection, induction culture, proliferation culture, rooting culture, seedling hardening and transplanting and the like. However, it adopts branches as explants and is induced to culture cluster buds, and gleditsia sinensis are two different plants.
Disclosure of Invention
The invention provides a method for establishing a soap pod tissue culture regeneration system for solving the problems.
The method is realized by the following technical scheme:
a method for establishing a gleditsia sinensis tissue culture regeneration system comprises the following steps:
1. explant collection: collecting mature pod of fructus Gleditsiae Abnormalis, and collecting seed.
2. Pretreatment of explants:
a. preparing a treatment solution by adopting 3-7% of potassium permanganate, 10-15% of dilute sulfuric acid solution with the concentration of 7% and 78-87% of water in percentage by mass;
b. soaking soap pod seeds in the treating liquid for 2-5min, and simultaneously treating with microwave radiation with frequency of 30-40W.
3. Explant sterilization: the collected soap pod seeds are placed in a beaker, washed clean with clear water for standby, soaked in 75% alcohol for 50-70s in an ultra-clean workbench, and soaked in 0.1% mercuric chloride for 17-22min.
4. Culturing aseptic seedlings: washing the sterilized explant with sterile water for 3-5 times, then sucking the surface water with sterile filter paper, inoculating on MS culture medium, performing germination test at 25+ -1deg.C, and culturing until the sterile seedling grows to 3-5 cm.
5. Induction culture: taking aseptic seedling cotyledon, cutting the cotyledon into 1cm pieces 2 The size is inoculated on a callus induction medium to induce callus and adventitious buds simultaneously.
Further, the composition of the callus induction medium is as follows: MS+6-BA 1.0-3.0 mg/L+TDZ 0.01-0.05 mg/L+NAA 0.1-0.5 mg/L+0.6-1.2% agar+2-5% sucrose.
6. Proliferation culture: inoculating the seedlings to a proliferation culture medium for culturing when the seedlings of the adventitious buds grow to 1-2 cm, and obtaining proliferation seedlings.
Further, the proliferation medium comprises the following components: MS+6-BA 0.1-1.0 mg/L+NAA 0.01-0.1 mg/L+0.6-1.2% agar+2-5% sucrose.
7. Rooting culture: when the proliferation-carrying seedling grows to 3-5cm, inoculating the proliferation-carrying seedling on a rooting induction culture medium for culture.
Further, the rooting induction culture medium comprises the following components: 1/2MS+IBA 0.1-0.5 mg/L+NAA 0.1-0.5 mg/L+0.6-1.2% agar+2-5% sucrose.
Further, the pH value of the culture medium is in the range of 5.5-6.0; the temperature is 25+/-1 ℃, the humidity is 70-80%, the illumination intensity is 2000-2200lx, and the illumination time is 12h each day in the culture process.
In summary, the beneficial effects of the invention are as follows: the invention breeds with soap pod seeds by utilizing a tissue culture technology, and pretreats the seeds before tissue culture to thin the seed coats, so that sterile seedlings can grow out rapidly, the cultivation time is shortened, and the emergence rate is increased. A large number of in-vitro regenerated plants can be obtained in a short period by a tissue culture technology, the excellent characteristics of the female parent are maintained, and a scientific support is provided for the protection of soap pod resources, fine variety breeding and industrial development.
Wherein, because of the special structure of the gleditsia sinensis seed coat, the explant is soaked by the treatment liquid, and is simultaneously assisted by microwave radiation treatment, plant cells can be effectively stimulated, the totipotency of the cells can be improved, the microbial cell membrane on the surface of the explant can be destroyed, the sterilization effect can be effectively improved, and the use of a sterilizing reagent can be reduced. And as the soaping pod is small in fruiting quantity, seeds are difficult to germinate into seedlings under natural conditions, compared with traditional seedling raising modes such as sowing, cutting, grafting and the like, the tissue culture technology can quickly obtain in-vitro regenerated plants, carry out large-scale germplasm, and simultaneously maintain the excellent properties of clone female parent.
Drawings
FIG. 1 is a graph showing the effect of culturing aseptic seedlings on a medium after seed sterilization treatment in example 1.
FIG. 2 is a graph showing the effect of callus induction culture on adventitious buds in example 2.
FIG. 3 is a graph showing the effect of the adventitious bud culture in example 2.
FIG. 4 is a graph showing rooting effects of the propagated seedlings in example 3.
Detailed Description
The following detailed description of the invention is provided in further detail, but the invention is not limited to these embodiments, any modifications or substitutions in the basic spirit of the present examples, which still fall within the scope of the invention as claimed.
Example 1
A method for establishing a gleditsia sinensis tissue culture regeneration system comprises the following steps:
1. explant collection: collecting mature pod of fructus Gleditsiae Abnormalis, and collecting seed.
2. Pretreatment of explants:
a. preparing a treatment solution by adopting 7%, 13% and 80% of potassium permanganate, a dilute sulfuric acid solution with the concentration of 7% and water in percentage by mass;
b. soaking soap pod seeds in the treatment solution for 4min, and simultaneously carrying out microwave radiation treatment with the frequency of 35W.
3. Explant sterilization: the collected soap pod seeds are placed in a beaker, washed clean with clear water for standby, soaked in 75% alcohol for 60s in an ultra-clean workbench, and soaked in 0.1% mercuric chloride for 20min.
4. Culturing aseptic seedlings: washing the sterilized explant with sterile water for 4 times, then absorbing the water on the surface with sterile filter paper, inoculating on MS culture medium, performing germination test at 25+ -1deg.C, and culturing until the sterile seedling grows to 4 cm.
5. Induction culture: taking aseptic seedling cotyledon, cutting the cotyledon into 1cm pieces 2 The size is inoculated on a callus induction medium to induce callus and adventitious buds simultaneously.
Further, the composition of the callus induction medium is as follows: MS+6-BA 2.0mg/L+TDZ0.03mg/L+NAA 0.3mg/L+0.8% agar+3% sucrose.
6. Proliferation culture: inoculating the seedlings to a proliferation culture medium for culturing when the seedlings of the adventitious buds grow to 2cm, and obtaining proliferation seedlings.
Further, the proliferation medium comprises the following components: MS+6-BA 0.6mg/L+NAA 0.05mg/L+0.8% agar+3% sucrose.
7. Rooting culture: when the proliferation-carrying seedlings grow to 4cm, inoculating the proliferation-carrying seedlings on a rooting induction culture medium for culturing.
Further, the rooting induction culture medium comprises the following components: 1/2MS+IBA 0.3mg/L+NAA0.3mg/L+0.8% agar+3% sucrose.
Further, due to the influence of error factors in the experimental process, the pH values of the three culture mediums cannot be precisely consistent, so that the pH values are all within the range of 5.5-6.0; the temperature in the culture process is 25+/-1 ℃, the humidity is 75%, the illumination intensity is 2100lx, and the illumination time is 12 hours per day.
Example 2
A method for establishing a gleditsia sinensis tissue culture regeneration system comprises the following steps:
1. explant collection: collecting mature pod of fructus Gleditsiae Abnormalis, and collecting seed.
2. Pretreatment of explants:
a. preparing a treatment solution by adopting potassium permanganate, a dilute sulfuric acid solution with the concentration of 7% and water according to the mass percentages of 3%, 10% and 87%;
b. soaking soap pod seeds in the treatment solution for 2min, and simultaneously carrying out microwave radiation treatment with the frequency of 30W.
3. Explant sterilization: the collected soap pod seeds are placed in a beaker, washed clean with clear water for standby, soaked in 75% alcohol for 50s in an ultra-clean workbench and soaked in 0.1% mercuric chloride for 17min.
4. Culturing aseptic seedlings: washing the sterilized explant with sterile water for 3 times, then absorbing the water on the surface with sterile filter paper, inoculating on MS culture medium, performing germination test at 25+ -1deg.C, and culturing until the sterile seedling grows to 3 cm.
5. Induction culture: taking aseptic seedling cotyledon, cutting the cotyledon into 1cm pieces 2 The size is inoculated on a callus induction medium to induce callus and adventitious buds simultaneously.
Further, the composition of the callus induction medium is as follows: MS+6-BA1.0 mg/L+TDZ0.01mg/L+NAA 0.1mg/L+0.6% agar+2% sucrose.
6. Proliferation culture: inoculating the seedlings to a proliferation culture medium for culturing when the seedlings of the adventitious buds grow to 1cm, and obtaining proliferation seedlings.
Further, the proliferation medium comprises the following components: MS+6-BA 0.1mg/L+NAA 0.01mg/L+0.6% agar+2% sucrose.
7. Rooting culture: when the proliferation-carrying seedlings grow to 3cm, inoculating the proliferation-carrying seedlings on a rooting induction culture medium for culturing.
Further, the rooting induction culture medium comprises the following components: 1/2MS+IBA 0.1mg/L+NAA0.1mg/L+0.6% agar+2% sucrose.
Further, due to the influence of error factors in the experimental process, the pH values of the three culture mediums cannot be precisely consistent, so that the pH values are all within the range of 5.5-6.0; the temperature in the culture process is 25+/-1 ℃, the humidity is 70%, the illumination intensity is 2000lx, and the illumination time is 12 hours per day.
Example 3
A method for establishing a gleditsia sinensis tissue culture regeneration system comprises the following steps:
1. explant collection: collecting mature pod of fructus Gleditsiae Abnormalis, and collecting seed.
2. Pretreatment of explants:
a. preparing a treatment solution by adopting potassium permanganate, a dilute sulfuric acid solution with the concentration of 7% and water according to the mass percentages of 5%, 15% and 80%;
b. soaking soap pod seeds in the treatment solution for 5min, and simultaneously carrying out microwave radiation treatment with the frequency of 40W.
3. Explant sterilization: the collected soap pod seeds are placed in a beaker, washed clean with clear water for standby, soaked in 75% alcohol for 70s in an ultra-clean workbench and soaked in 0.1% mercuric chloride for 22min.
4. Culturing aseptic seedlings: washing the sterilized explant with sterile water for 5 times, then absorbing the water on the surface with sterile filter paper, inoculating on MS culture medium, performing germination test at 25+ -1deg.C, and culturing until the sterile seedling grows to 5 cm.
5. Induction culture: taking aseptic seedling cotyledon, cutting the cotyledon into 1cm pieces 2 The size is inoculated on a callus induction medium to induce callus and adventitious buds simultaneously.
Further, the composition of the callus induction medium is as follows: MS+6-BA3.0mg/L+TDZ0.05mg/L+NAA0.5mg/L+1.2% agar+5% sucrose.
6. Proliferation culture: inoculating the seedlings to a proliferation culture medium for culturing when the seedlings of the adventitious buds grow to 2cm, and obtaining proliferation seedlings.
Further, the proliferation medium comprises the following components: MS+6-BA1.0mg/L+NAA 0.1mg/L+1.2% agar+5% sucrose.
7. Rooting culture: when the proliferation-carrying seedlings grow to 5cm, inoculating the proliferation-carrying seedlings on a rooting induction culture medium for culturing.
Further, the rooting induction culture medium comprises the following components: 1/2MS+IBA0.5mg/L+NAA0.5mg/L+1.2% agar+5% sucrose.
Further, due to the influence of error factors in the experimental process, the pH values of the three culture mediums cannot be precisely consistent, so that the pH values are all within the range of 5.5-6.0; the temperature in the culture process is 25+/-1 ℃, the humidity is 80%, the illumination intensity is 2200lx, and the illumination time is 12 hours per day.
The MS medium used in the present application has the following composition:
MS basic ingredient list
| Name of the name | mg/L | Name of the name | mg/L | Name of the name | mg/L | Name of the name | mg/L |
| NH 4 NO 3 | 1650 | FeSO 4 ·7H 2 O | 27.8 | ZnSO4·7H 2 O | 8.6 | VB6 | 0.5 |
| KNO 3 | 1900 | Na2-EDTA | 37.3 | Na 2 MoO 2 ·2H 2 O | 0.25 | Nicotinic acid | 0.5 |
| KH 2 PO 4 | 170 | KI | 1.03 | CuSO4·5H 2 O | 0.025 | Glycine (Gly) | 2 |
| MgSO 4 ·7H 2 O | 370 | H 3 BO 3 | 6.2 | CoCl 2 ·6H 2 O | 0.025 | Inositol (inositol) | 100 |
| CaCl 2 | 332.16 | MnSO 4 ·H 2 O | 16.9 | VB1 | 0.1 |
1. Screening comparative experiment
1.1 Experimental materials
Experiment 1: the experiment did not pretreat the explants under the same conditions as in example 1;
experiment 2: under the same conditions as those of the example 1, the experiment only adopts the treatment liquid to soak the explant during the pretreatment, and does not carry out microwave radiation;
experiment 3: under the same conditions as those of the embodiment 1, the experiment is carried out by only adopting 7% potassium permanganate solution for soaking and simultaneously assisting microwave radiation;
1.2 Experimental methods
The germination rate and the time required from seed to germination of the aseptic seedlings of experiments 1-3 were recorded and compared with examples 1-3, and the results are shown in Table 1.
Wherein, the seeds used in experiments 1-3 and examples 1-3 were 20 grains.
1.3 experimental results
TABLE 1
| Project | Example 1 | Example 2 | Example 3 | Experiment 1 | Experiment 2 | Experiment 3 |
| Bud ratio | 85％ | 80％ | 85％ | 55％ | 70％ | 60％ |
| Time/day | 12 | 11 | 13 | 17 | 14 | 16 |
From the experimental results, it is known that the experiment 1 did not pretreat the seeds, and the seed husks of the gleditsia sinensis seeds were thicker, the germination rate of the seeds which were not pretreated was lower, and the time required for culturing was longer. Experiment 2 was not irradiated with microwaves, but the germination rate and time were still inferior to examples 1-3, although they were comparable. Three groups of experiments show that the pretreatment of the soap pod seeds has the effects of accelerating germination rate and improving germination rate.
Claims (4)
1. The method for establishing the regeneration system of the gleditsia sinensis tissue culture comprises the steps of explant sterilization, aseptic seedling culture, induction culture and rooting culture; the method is characterized by comprising the steps of preprocessing the explant before disinfecting; the induction culture is to adopt a callus induction culture medium to induce callus and adventitious buds simultaneously, and then inoculate the adventitious buds on an adventitious bud proliferation culture medium for proliferation culture;
the pretreatment is to soak the soap pod seeds for 2-5min with the treatment liquid, and simultaneously, the soap pod seeds are treated by the microwave radiation with the frequency of 30-40W;
the treatment fluid comprises the following components in percentage by mass: 3-7% of potassium permanganate, 10-15% of dilute sulfuric acid solution with the concentration of 7% and 78-87% of water;
the callus induction culture medium comprises the following components: MS+6-BA 1.0-3.0 mg/L+TDZ 0.01-0.05 mg/L+NAA 0.1-0.5 mg/L+0.6-1.2% agar+2-5% sucrose;
the adventitious bud proliferation culture medium comprises the following components: MS+6-BA 0.1-1.0 mg/L+NAA 0.01-0.1 mg/L+0.6-1.2% agar+2-5% sucrose;
the rooting induction culture medium comprises the following components: 1/2MS+IBA 0.1-0.5 mg/L+NAA 0.1-0.5 mg/L+0.6-1.2% agar+2-5% sucrose.
2. The method for establishing a soap pod tissue culture regeneration system according to claim 1, comprising the steps of:
a. washing the collected soap pod seeds, sterilizing in an ultra-clean workbench, inoculating on an MS culture medium, and culturing aseptic seedlings to obtain aseptic seedling cotyledons;
b. c, synchronously inducing the callus and adventitious buds of the sterile seedling She Jiechong obtained in the step a on a callus induction medium;
c. inoculating adventitious buds on an adventitious bud proliferation culture medium for proliferation culture to obtain proliferation seedlings;
d. and inoculating the proliferation seedling on a rooting induction culture medium for rooting culture.
3. The method for establishing a regeneration system of gleditsia sinensis tissue culture according to claim 2, wherein the disinfection treatment is to soak the gleditsia sinensis tissue culture with 75% alcohol for 50-70s, soak the gleditsia sinensis tissue culture with 0.1% mercuric chloride for 17-22min, wash the gleditsia sinensis tissue culture with sterile water for 3-5 times, and suck the water.
4. The method for establishing a regeneration system for tissue culture of gleditsia sinensis as claimed in claim 1, wherein the pH value of the culture medium is in the range of 5.5-6.0; the temperature is 25+/-1 ℃, the humidity is 70-80%, the illumination intensity is 2000-2200lx, and the illumination time is 12 hours per day in the culture process.
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| US6360479B1 (en) * | 1999-07-15 | 2002-03-26 | Roger S. Webb | Method for deflowering sweet gum trees to eliminate seed and fruit debris |
| CN105766655B (en) * | 2016-04-28 | 2018-07-10 | 湖南师范大学 | The method for building up of villus Chinese honey locust Tissue Culture Regeneration System |
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