CN112586357A

CN112586357A - Method for establishing gleditsia sinensis tissue culture regeneration system

Info

Publication number: CN112586357A
Application number: CN202011628134.XA
Authority: CN
Inventors: 李斌; 侯俊; 陈杰; 周应书; 毕宁; 王彩云; 肖艳; 周聿
Original assignee: Bijie Forestry Research Institute
Current assignee: Bijie Forestry Research Institute
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-04-02
Anticipated expiration: 2040-12-30
Also published as: CN112586357B

Abstract

The invention belongs to the technical field of plant asexual propagation, and particularly relates to a method for establishing a gleditsia sinensis lam tissue culture regeneration system; the soapberry seeds are bred by using a tissue culture technology, and the seeds are pretreated before tissue culture to thin seed coats, so that aseptic seedlings can grow rapidly, the culture time is shortened, and the emergence rate is increased. By the tissue culture technology, a large number of in-vitro regeneration plants can be obtained in a short time, the excellent characteristics of the female parent are maintained, and scientific support is provided for soap pod resource protection, fine breed breeding and industrial development.

Description

Method for establishing gleditsia sinensis tissue culture regeneration system

Technical Field

The invention belongs to the technical field of plant asexual propagation, and particularly relates to a method for establishing a gleditsia sinensis lam tissue culture regeneration system.

Background

The soap pod (Gymnocladus chinensis Baill), also known as Gleditsia sinensis Lam, and Ferris Sucus (Gleditsia sinensis Lam and Gleditsia sinensis Lam.), is a deciduous tree of the genus Gleditsia of the family Leguminosae, and is widely distributed in southern Shaanxi, Sichuan, Guizhou, Hubei, Hunan, northern Guangdong, Jiangxi, Anhui, southern Jiangsu, Zhejiang, Fujian, etc. The trees grow in hillside, waist and miscellaneous trees with elevation of 150-. The soap pod is rich in saponin and saponin, the seed oil can be used as industrial oil such as paint, the seed kernel is edible, the medicinal value is high, the soap pod is a natural raw material of food, health care products, cosmetics and washing products, is also an important tree species for landscaping and ecological engineering afforestation, has extremely high economic and ecological values, is one of the sunrise industry for assisting poverty and defficiency, attacking hardness and reviving countryside, and is also an important tree species for low-efficiency forest reconstruction. The soapberry pods are oblong, flat or thick, the fruiting amount is small, only 2-4 seeds exist in one pod, due to the special physiological structures of the pods and seed coats, the seeds are difficult to germinate and grow seedlings under natural conditions, the seed amount is small, and the natural renewal is very slow. The resource amount is small, the group is in imminent danger, and collection, protection, development and utilization are urgently needed. At present, the traditional modes of seeding, cuttage, grafting and the like are mainly adopted for seedling culture, and the defects of poor growth vigor, uneven growth, insufficient expression of good seed potential and the like exist, so that the quantity of high-quality seedling resources is small at present. The tissue culture technology is utilized to breed rare endangered species and excellent strains of tree species, and the method has the characteristics of quick breeding, short breeding period, annual production and the like. At present, no relevant report on the aspect of the soap pod tissue culture technology exists, the invention takes seeds as explants, researches the influence of hormone combinations with different concentrations on the induction, differentiation, proliferation and rooting of soap pod callus through experiments, establishes a soap pod tissue culture technical system, has important significance for protecting the germplasm resources of the soap pod, and lays a foundation for the fine variety breeding of the soap pod, the large-scale production of excellent seedlings and the industrial development.

At present, some researches on tissue culture propagation of gleditschia horrida are carried out, for example, patent document with application number of CN201610271956.4 discloses a method for establishing a tissue culture regeneration system of gleditschia horrida, which adopts gleditschia horrida seeds as explants to carry out cultivation through disinfection, aseptic seedling culture, callus induction, adventitious bud proliferation, rooting culture and domestication and transplantation. However, the study is carried out on the soapberry, the soapberry is different from the soapberry, and the soapberry fruit is one of the traditional Chinese medicinal materials and is different from the botanical classification.

Patent document CN201811429114.2 discloses a method for tissue culture and rapid propagation of Gleditsia sinensis, which takes tender branches at the base of Gleditsia sinensis stump as explants, and comprises the steps of disinfection, induction culture, proliferation culture, rooting culture, hardening seedling and transplanting and the like. But the branch is used as an explant and is induced and cultured aiming at multiple buds, and the gleditsia sinensis and the soap pod are also two different plants.

Disclosure of Invention

The invention provides a method for establishing a gleditsia sinensis pod tissue culture regeneration system to solve 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 ripe pods of fructus Gleditsiae Abnormalis, and collecting seeds.

2. Pretreatment of explants:

a. preparing a treatment solution from 3-7% by mass, 10-15% by mass and 78-87% by mass of potassium permanganate, a 7% dilute sulfuric acid solution and water;

b. soaking the seed of fructus Gleditsiae Abnormalis in the treating solution for 2-5min, and treating with microwave radiation at frequency of 30-40W.

3. Explant disinfection: cleaning collected soap pod seeds in a beaker with clear water for later use, soaking in 75% alcohol for 50-70s in a super clean bench, and soaking in 0.1% mercuric chloride for 17-22 min.

4. And (3) sterile seedling culture: and (3) cleaning the sterilized explant by using sterile water for 3-5 times, then, adsorbing water on the surface by using sterile filter paper, inoculating the water on an MS culture medium, carrying out a germination test at 25 +/-1 ℃, and carrying out the next step when the sterilized seedling grows to 3-5 cm.

5. And (3) induction culture: taking sterile seedling cotyledon, cutting cotyledon into 1cm²The size of the culture medium is inoculated on a callus induction culture medium to simultaneously induce callus and adventitious buds.

Further, the callus induction culture medium comprises the following components: 1.0-3.0 mg/L of MS +6-BA, 0.01-0.05 mg/L of TDZ, 0.1-0.5 mg/L of NAA, 0.6-1.2% of agar and 2-5% of cane sugar.

6. And (3) proliferation culture: and inoculating the seedlings of the adventitious buds to a proliferation culture medium for culturing when the seedlings of the adventitious buds grow to 1-2 cm, so as to obtain the proliferated 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% cane sugar.

7. Rooting culture: when the tape multiplication seedlings grow to 3-5cm, the tape multiplication seedlings are inoculated 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 values of the culture media are all within the range of 5.5-6.0; in the culture process, the temperature is 25 +/-1 ℃, the humidity is 70-80%, the illumination intensity is 2000-2200lx, and the illumination time is 12h every day.

In conclusion, the beneficial effects of the invention are as follows: the invention utilizes the tissue culture technology to breed the soap pod seeds, pretreats the seeds before tissue culture to thin seed coats, so that aseptic seedlings can grow out quickly, the culture time is shortened, and the emergence rate is increased. By the tissue culture technology, a large number of in-vitro regeneration plants can be obtained in a short time, the excellent characteristics of the female parent are maintained, and scientific support is provided for soap pod resource protection, fine breed breeding and industrial development.

Due to the special structure of the soap pod seed coat, the explant is soaked by the treatment fluid, and is assisted with microwave radiation treatment, so that plant cells can be effectively stimulated, the totipotency of the cells is improved, microbial cell membranes on the surface of the explant can be damaged, the sterilization and disinfection effects are effectively improved, and the use of disinfection reagents is reduced. And because the fertile soap pod bearing amount is few, the seed is difficult to germinate into seedlings under the natural condition, compared with the traditional seedling raising modes such as seeding, cuttage and grafting, the tissue culture technology can quickly obtain the in vitro regeneration plant, promote the large-scale germplasm, and simultaneously keep the excellent characters of the clone female parent.

Drawings

FIG. 1 is a graph showing the effect of the sterilization treatment of the seeds in example 1 on the culture of the sterilized seedlings in the medium.

FIG. 2 is a graph showing the effect of callus induction culture on adventitious bud formation in example 2.

FIG. 3 is a graph showing the effect of the adventitious bud culture and multiplication culture in example 2.

FIG. 4 is a graph showing the effect of rooting the proliferated shoots in example 3.

Detailed Description

The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.

Example 1

2. Pretreatment of explants:

a. preparing a treatment solution by using potassium permanganate, a dilute sulfuric acid solution with the concentration of 7% and water according to the mass percentage of 7%, 13% and 80%;

b. soaking the soap pod seeds in the treatment solution for 4min while treating with microwave radiation at 35W.

3. Explant disinfection: cleaning collected soap pod seeds in a beaker with clear water for later use, soaking in 75% alcohol for 60s in a super clean bench, and soaking in 0.1% mercuric chloride for 20 min.

4. And (3) sterile seedling culture: and (3) cleaning the sterilized explant by using sterile water for 4 times, then sucking water on the surface by using sterile filter paper, inoculating the explant on an MS culture medium, carrying out a germination test at 25 +/-1 ℃, and carrying out the next step when the sterilized seedling grows to 4 cm.

Further, the callus induction culture medium comprises the following components: MS +6-BA 2.0mg/L + TDZ0.03mg/L + NAA0.3mg/L + 0.8% agar + 3% cane sugar.

6. And (3) proliferation culture: inoculating the adventitious bud seedling to a proliferation culture medium when the adventitious bud seedling grows to 2cm, and culturing to obtain a proliferation seedling.

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 tape proliferated seedling grows to 4cm, inoculating the tape proliferated seedling on a rooting induction culture medium for culture.

Further, the rooting induction culture medium comprises the following components: 1/2MS + IBA 0.3mg/L + NAA0.3mg/L + 0.8% agar + 3% sucrose.

Furthermore, due to the influence of error factors in the experimental process, the pH values of the three culture media cannot be accurately consistent, so that the pH values are all within the range of 5.5-6.0; the temperature is 25 +/-1 ℃, the humidity is 75%, the illumination intensity is 2100lx, and the illumination time is 12h every day in the culture process.

Example 2

2. Pretreatment of explants:

a. preparing a treatment solution by using potassium permanganate, a dilute sulfuric acid solution with the concentration of 7% and water according to the mass percentage of 3%, 10% and 87%;

b. soaking the soap pod seeds in the treatment solution for 2min while treating with microwave radiation at a frequency of 30W.

3. Explant disinfection: cleaning collected soap pod seeds in a beaker with clear water for later use, soaking in 75% alcohol for 50s in a super clean bench, and soaking in 0.1% mercuric chloride for 17 min.

4. And (3) sterile seedling culture: and (3) cleaning the sterilized explant by using sterile water for 3 times, then, absorbing water on the surface by using sterile filter paper, inoculating the explant on an MS culture medium, carrying out a germination test at 25 +/-1 ℃, and carrying out the next step when the sterilized seedling grows to 3 cm.

Further, the callus induction culture medium comprises the following components: MS +6-BA1.0mg/L + TDZ0.01mg/L + NAA0.1mg/L + 0.6% agar + 2% cane sugar.

6. And (3) proliferation culture: inoculating the seedlings of the adventitious buds to a proliferation culture medium when the seedlings of the adventitious buds grow to 1cm, and culturing to obtain the proliferation seedlings.

Further, the proliferation medium comprises the following components: MS +6-BA 0.1mg/L + NAA0.01 mg/L + 0.6% agar + 2% sucrose.

7. Rooting culture: when the tape proliferated seedling grows to 3cm, inoculating the tape proliferated seedling on a rooting induction culture medium for culture.

Further, the rooting induction culture medium comprises the following components: 1/2MS + IBA 0.1mg/L + NAA0.1mg/L + 0.6% agar + 2% sucrose.

Furthermore, due to the influence of error factors in the experimental process, the pH values of the three culture media cannot be accurately 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 12h every day.

Example 3

2. Pretreatment of explants:

a. preparing a treatment solution by using potassium permanganate, a dilute sulfuric acid solution with the concentration of 7% and water according to the mass percentage of 5%, 15% and 80%;

b. soaking the seeds of fructus Gleditsiae Abnormalis in the treating solution for 5min while treating with microwave radiation at 40W.

3. Explant disinfection: cleaning collected soap pod seeds in a beaker with clear water for later use, soaking in 75% alcohol for 70s in a super clean bench, and soaking in 0.1% mercuric chloride for 22 min.

4. And (3) sterile seedling culture: and (3) cleaning the sterilized explant by using sterile water for 5 times, then, adsorbing water on the surface by using sterile filter paper, inoculating the water on an MS culture medium, carrying out a germination test at 25 +/-1 ℃, and carrying out the next step when the sterilized seedling grows to 5 cm.

Further, the callus induction culture medium comprises the following components: MS +6-BA3.0mg/L + TDZ0.05mg/L + NAA0.5mg/L + 1.2% agar + 5% cane sugar.

Further, the proliferation medium comprises the following components: MS +6-BA1.0mg/L + NAA0.1mg/L + 1.2% of agar + 5% of cane sugar.

7. Rooting culture: when the tape proliferated seedling grows to 5cm, inoculating the tape proliferated seedling on a rooting induction culture medium for culture.

Further, the rooting induction culture medium comprises the following components: 1/2MS + IBA0.5mg/L + NAA0.5mg/L + 1.2% agar + 5% sucrose.

Furthermore, due to the influence of error factors in the experimental process, the pH values of the three culture media cannot be accurately consistent, so that the pH values are all within the range of 5.5-6.0; the temperature is 25 + -1 deg.C, humidity is 80%, illumination intensity is 2200lx, and the illumination time is 12h per day.

The MS medium used in the present application has the following composition as shown in the following table:

MS basic ingredient list

Name (R)	mg/L	Name (R)	mg/L	Name (R)	mg/L	Name (R)	mg/L
								NH₄NO₃	1650	FeSO₄·7H₂O	27.8	ZnSO4·7H₂O	8.6	VB6	0.5
KNO₃	1900	Na2-EDTA	37.3	Na₂MoO₂·2H₂O	0.25	Nicotinic acid	0.5
								KH₂PO₄	170	KI	1.03	CuSO4·5H₂O	0.025	Glycine	2
MgSO₄·7H₂O	370	H₃BO₃	6.2	CoCl₂·6H₂O	0.025	Inositol	100
								CaCl₂	332.16	MnSO₄·H₂O	16.9	VB1	0.1

First, screening comparative experiment

1.1 Experimental materials

Experiment 1: in this experiment, explants were not pretreated under the same conditions as in example 1;

experiment 2: in the experiment, under the same conditions as those in example 1, only the treatment solution is adopted for soaking when the explant is pretreated, and microwave radiation is not carried out;

experiment 3: in the experiment, under the same conditions as those in example 1, only a potassium permanganate solution with the concentration of 7% is adopted for soaking, and microwave radiation is assisted;

1.2 Experimental methods

The germination rates and the time required from seed to germination of the tissue-cultured aseptic seedlings of experiments 1 to 3 were recorded and compared with those of examples 1 to 3, and the results are shown in Table 1.

In each of experiments 1 to 3 and examples 1 to 3, 20 seeds were used.

1.3 results of the experiment

TABLE 1

Item

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

According to the experimental results, the seeds are not pretreated in the experiment 1, and because the seed coats of the soap pod seeds are thick, the germination rate of the seeds which are not pretreated is low, and the time required for culturing is long. Experiment 2 did not use microwave for irradiation, and the germination rate and time were still not as good as those of examples 1-3, although they were relatively appreciable. Three groups of experiments are combined to see that the pretreatment of the soap pod seeds has the effects of accelerating the germination rate and improving the germination rate.

Claims

1. A method for establishing a gleditsia sinensis tissue culture regeneration system comprises the steps of explant disinfection, aseptic seedling culture, induction culture and rooting culture; it is characterized in that the pretreatment is carried out before the explant is disinfected; the induction culture adopts a callus induction culture medium to simultaneously induce the callus and the adventitious bud.

2. The method for establishing the tissue culture regeneration system of the soap pod as claimed in claim 1, wherein the pretreatment comprises soaking the soap pod seeds in the treatment solution for 2-5min while performing microwave radiation treatment at a frequency of 30-40W.

3. The method for establishing a gleditsia sinensis tissue culture regeneration system according to claim 2, wherein the treatment fluid comprises, by mass: 3-7% of potassium permanganate, 10-15% of dilute sulfuric acid solution with the concentration of 7% and 78-87% of water.

4. The method for establishing a gleditsia sinensis tissue culture regeneration system according to claim 1, wherein said callus induction medium comprises: 1.0-3.0 mg/L of MS +6-BA, 0.01-0.05 mg/L of TDZ and 0.1-0.5 mg/L of NAA.

5. The method for establishing a gleditsia sinensis tissue culture regeneration system according to claim 1, comprising the steps of:

a. washing collected soap pod seeds, then performing disinfection treatment in an ultra-clean workbench, and inoculating the seeds on an MS culture medium to culture aseptic seedlings to obtain aseptic seedling cotyledons;

b. inoculating the sterile seedling cotyledons obtained in the step a on a culture medium for inducing tissue, and synchronously inducing callus and adventitious buds;

c. inoculating the adventitious bud on an adventitious bud multiplication culture medium for multiplication culture to obtain a multiplication seedling;

d. and inoculating the proliferated seedling on a rooting induction culture medium for rooting culture.

6. The method for establishing a tissue culture regeneration system of Gleditsia sinensis as claimed in claim 5, wherein said sterilizing treatment comprises soaking in 75% ethanol for 50-70s, soaking in 0.1% mercuric chloride for 17-22min, washing with sterile water for 3-5 times, and removing water by suction.

7. The method for establishing a gleditsia sinensis tissue culture regeneration system according to claim 5, wherein said adventitious bud propagation medium is composed of: MS +6-BA 0.1-1.0 mg/L + NAA 0.01-0.1 mg/L.

8. The method for establishing a gleditsia sinensis tissue culture regeneration system of claim 5, wherein said rooting induction medium comprises: 1/2MS + IBA 0.1-0.5 mg/L + NAA 0.1-0.5 mg/L.

9. The method for establishing a tissue culture regeneration system for gleditschia horrida according to claims 4-8, wherein said culture medium further comprises agar 0.6-1.2% and sucrose 2-5%, and 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-.

10. A method for establishing a tissue culture regeneration system is characterized in that the method is used for tissue culture of soap pods.