阅读说明：本技术 一种樟叶越桔悬浮细胞培养体系的建立方法 (Method for establishing suspension cell culture system of vaccinium camphorata ) 是由 赵平 唐军荣 刘云 阚欢 付羚 罗旭璐 张訸 于 2019-10-16 设计创作，主要内容包括：本发明公开一种樟叶越桔悬浮细胞培养体系的建立方法,属于植物细胞工程领域。本发明所述方法,本发明所述方法以樟叶越桔组培苗的幼嫩叶为外植体,将处理好的幼嫩叶放入诱导培养基中诱导出愈伤组织,将愈伤组织转移到增殖培养基中,生长成适合悬浮培养的状态,将适宜的愈伤组织转移到液体培养基中,成为稳定的悬浮培养体系。本发明所述方法可以根据实际生产选择相应的培养组合进行生产,从而降低生产成本,提高效率,为工厂化生产樟叶越桔悬浮培养体系提供指导依据。(The invention discloses a method for establishing a suspension cell culture system of vaccinium dunalianum, belonging to the field of plant cell engineering. According to the method, young leaves of the vaccinium camphorata tissue culture seedling are taken as explants, the processed young leaves are placed into an induction culture medium to induce callus, the callus is transferred into a multiplication culture medium to grow into a state suitable for suspension culture, and the suitable callus is transferred into a liquid culture medium to form a stable suspension culture system. The method can select corresponding culture combination for production according to actual production, thereby reducing production cost, improving efficiency and providing guidance for industrialized production of the vaccinium camphorate suspension culture system.)

1. A method for establishing a suspension cell culture system of vaccinium dunalianum, which is characterized by comprising the following steps: young leaves of the vaccinium camphorate tissue culture seedlings are used for inducing callus and establishing a cell suspension culture system.

2. The method for establishing the suspension cell culture system of vaccinium dunalianum according to claim 1, which comprises the following steps:

(1) induction of callus: cutting young leaves of the tissue culture seedling of the vaccinium dunalianum, marking strip-shaped wounds on the front side or the back side of the leaves, and enabling the back side of the leaves to be tightly attached to an induction culture medium for growing so as to induce callus;

the induction culture medium is a combination of a WPM minimal medium and a cell auxin: WPM + 1.0-2.0 mg/L6-BA + 0.05-1.0 mg/LNAA + 1.0-2.0 mg/L2,4-D + 28-42 g/L sucrose + 4.5-5.3 g/L agar;

(2) proliferation of callus: selecting non-browned and vigorously growing callus, transferring to WPM proliferation culture medium for culture, and growing to a state suitable for suspension culture;

the WPM proliferation culture medium is a combination of a WPM minimal medium and a cell auxin:

WPM + 1.0-2.0 mg/LZT + 0.2-0.6 mg/LNAA + 28-42 g/L sucrose + 4.5-5.3 g/L agar;

(3) establishing a suspension system: selecting the semitransparent callus with vigorous growth, transferring the selected semitransparent callus into a liquid culture medium, and removing large-particle callus after culturing for 7-10 days; after being sufficiently cleaned by sterile water, the screened small-particle callus is transferred to a fresh liquid culture medium for culture to form a stable suspension culture system, and suspension culture cells I are obtained by separation;

the culture medium is a combination of WPM culture medium and auxin: WPM culture medium + 1.5-2.5 mg/LZT + 0.3-0.6 mg/LNAA + 28-42 g/L sucrose.

3. The method for establishing the suspension cell culture system of vaccinium dunalianum according to claim 2, which is characterized in that: the culture conditions in the step (1) are as follows: 25 +/-2 ℃ and the illumination intensity of 30-50 mu mol/m²S, the illumination period is 10-12 h/d, and the culture time is 50-60 d.

4. The method for establishing the suspension cell culture system of vaccinium dunalianum according to claim 2, which is characterized in that: the conditions for the proliferation of the callus in the step (2) are as follows: and (4) dark culture at 25 +/-2 ℃ for 70-90 days.

5. The method for establishing the suspension cell culture system of vaccinium dunalianum according to claim 2, which is characterized in that: the culture conditions in the step (3) are as follows: dark culture is carried out at the temperature of 25 +/-2 ℃, the pH value of a culture medium is 4.8-5.2, the rotating speed of a shaking table is 120-160 rpm/min, and the culture time of the small-particle callus is 7-10 days.

6. The method for establishing the suspension cell culture system of vaccinium dunalianum according to claim 2, which is characterized in that: the screening process of the large and small particles in the step (3) is as follows: filtering with a 40-60 mesh stainless steel sieve, and obtaining the undersize tissue as the small-particle callus.

7. The method for establishing the suspension cell culture system of vaccinium dunalianum according to claim 2, which is characterized in that: the WPM culture medium in the step (3) is replaced by 3/5 organic modified WPM culture medium.

8. The method for establishing the suspension cell culture system of the vaccinium dunalianum according to any one of the claims 2 to 7, which is characterized in that: after the callus which normally grows in the step (3) is cultured, filtering out an original liquid culture medium by using a 40-60-mesh stainless steel sieve, absorbing water by using sterile paper, transferring the sterile paper to a fresh 3/5 organic improved WPM culture medium for culture to form a stable suspension culture system, and separating to obtain a suspension culture cell II;

the culture medium is 3/5 organic combination of improved WPM culture medium and auxin: 3/5 organic modified WPM medium + 1.0-2.0 mg/L ZT + 0.1-0.2 mg/LNAA + 28-42 g/L sucrose;

the culture conditions were: dark culture is carried out at 25 +/-2 ℃, the culture time is 7-10 days, the pH value of a culture medium is 4.8-5.2, the rotation speed of a shaking table is 120-160 rpm/min, the volume of the culture medium is 40-50 mL, and the inoculation amount is 20-40 g/L.

9. The method for establishing the suspension cell culture system of vaccinium dunalianum according to claim 2, which is characterized in that: the method is characterized in that: sieving the suspension culture cell I in the step (3) through a 180-200-mesh sieve, reserving tissues on the upper part of the sieve, sucking dry water by using sterile filter paper, inoculating the suspension culture cell I into 3/5 organic improved WPM culture medium, continuously culturing to form a stable suspension culture system, and separating to obtain a suspension culture cell II;

the culture medium is a combination of a WPM solid minimal medium and a cell auxin: 3/5 organic modified WPM medium + 1.0-2.0 mg/L ZT + 0.1-0.2 mg/LNAA + 28-42 g/L sucrose;

the culture conditions were: dark culture is carried out at 25 +/-2 ℃, the culture time is 10-20 days, the pH value of a culture medium is 4.8-5.2, the rotation speed of a shaking table is 120-160 rpm/min, the volume of the culture medium is 40-50 mL, and the inoculation amount is 20-40 g/L.

10. The method for establishing the suspension cell culture system of vaccinium dunalianum according to claim 8, which is characterized in that: the method is characterized in that: sieving the suspension culture cell II in the step (4) through a 180-200-mesh sieve, reserving tissues on the upper part of the sieve, sucking water by using sterile filter paper, inoculating the suspension culture cell II into 3/5 organic improved WPM culture medium, and continuously culturing;

the culture medium is 3/5 organic combination of improved WPM culture medium and auxin: WPM (3/5 organic) + 1.0-2.0 mg/L ZT + 0.1-0.2 mg/LNAA + 28-42 g/L sucrose;

the culture conditions were: dark culture is carried out at 25 +/-2 ℃, the culture time is 10-20 days, the pH value of a culture medium is 4.8-5.2, the rotation speed of a shaking table is 120-160 rpm/min, the volume of the culture medium is 40-50 mL, and the inoculation amount is 20-40 g/L.

Technical Field

The invention relates to a method for establishing a suspension cell culture system of vaccinium dunalianum, belonging to the field of plant cell engineering.

Background

Vaccinium dunalianum (Vaccinium dunalianum) is evergreen shrub of Vaccinium of Ericaceae (Ericaceae), and is mainly distributed in the northwest of Yunnan from the plateau of Yunnan to Yunnan and Yunnan east. The fruit is rich in nutrition, and the whole plant has the effects of relaxing tendons, activating collaterals, dispelling wind, removing dampness and the like, and has high medicinal and health-care values. In addition, the vaccinium dunalianum is also a special resource plant rich in caffeoyl arbutin substances, the melanin generation inhibiting activity of the main component of the vaccinium dunalianum is superior to that of arbutin, the toxicity is only 1/2 of arbutin, and the vaccinium dunalianum can be developed and utilized as a natural arbutin substitute resource. The vaccinium dunalianum is still in a wild state at present, and wild resources are reduced rapidly due to destructive picking in successive years, so that the establishment of a culture system by using a biotechnology means has important significance.

Plant cell culture includes the cultivation of young plant embryo, protoplast, cell, tissue, organ, production of important secondary metabolites, new plant individuals (rapid propagation of seedling), and a interdiscipline of engineering and biology for the physiological, biochemical and genetic studies of plant cells. In recent years, plant cell culture is favored by scientists due to its unique advantages, firstly, the cell culture process can be continuously and uniformly carried out in a culture dish and is not influenced by external factors such as climate, plant diseases and insect pests; secondly, the culture process can be carried out in a bioreactor in a large scale, the reaction condition can be artificially controlled, and the yield of the target product is improved. In recent years, domestic scholars have conducted many studies on production regulation of secondary metabolites of medicinal plants such as ginseng (Panax ginseng), American ginseng (p.quinquefolius), pseudo-ginseng (p.notogiseng), Lithospermum erythrorhizon (Lithospermum erythrorhizon), Taxus chinensis (Taxus chinensis), saussurea involucrata (saussurea involucrata), Ginkgo biloba (Ginkgo biloba) and the like by using a cell suspension culture technology, but the application of the cell suspension culture technology to vaccinium camphorate plants has not been reported yet. According to the invention, the induction and proliferation conditions of the callus are researched by taking the young leaves of the bilberry tissue culture seedlings of the camphor leaves as materials, a cell suspension culture system of the bilberry of the camphor leaves is established, and a foundation is further laid for the production regulation and control of the caffeoyl arbutin substances in the bilberry of the camphor leaves.

Disclosure of Invention

The invention aims to provide a method for establishing a suspension cell culture system of vaccinium camphorata, which uses young leaves of vaccinium camphorata tissue culture seedlings to induce callus and establish the cell suspension culture system.

Further, the method for establishing the suspension cell culture system of the vaccinium dunalianum disclosed by the invention specifically comprises the following steps of:

(1) induction of callus: cutting young leaves of the tissue culture seedling of the vaccinium dunalianum, marking strip-shaped wounds on the front side or the back side of the leaves, and enabling the back side of the leaves to be tightly attached to an induction culture medium for growing so as to induce callus;

the induction culture medium is a combination of a WPM minimal medium and a cell auxin: WPM + 1.0-2.0 mg/L6-BA + 0.05-1.0 mg/L NAA + 1.0-2.0 mg/L2,4-D + 28-42 g/L sucrose + 4.5-5.3 g/L agar.

The 1.0-2.0 mg/L6-BA in the culture medium refers to that 1.0-2.0 mg of 6-BA is added into 1L of WPM basic culture medium, the 28-42 g/L sucrose refers to that 30g of sucrose is added into 1L of WPM basic culture medium, and the meanings of other additives in the invention are the same as the above.

(2) Proliferation of callus: selecting and selecting calli which are not browned and grow vigorously, transferring the calli to a WPM enrichment medium for culture, and growing to a state suitable for suspension culture;

the WPM proliferation culture medium is a combination of a WPM minimal medium and a cell auxin: WPM + 1.0-2.0 mg/L ZT + 0.2-0.6 mg/L NAA + 28-42 g/L sucrose + 4.5-5.3 g/L agar.

(3) Establishing a suspension system: selecting and transferring the vigorously-growing semitransparent callus into a liquid culture medium (the callus generally presents dark yellow and is semitransparent and can be automatically dispersed into nearly powdery callus particles suitable for growing in the liquid culture medium, transferring the callus in the state to the liquid culture medium, slightly rolling to disperse the callus as much as possible), removing the large-particle callus after culturing for 7-10 days, fully cleaning the screened small-particle callus with sterile water, transferring the small-particle callus into a fresh liquid culture medium for culturing to form a stable suspension culture system, and separating to obtain suspension culture cells I;

the culture medium is a combination of WPM culture medium and auxin: WPM culture medium + 1.5-2.5 mg/L ZT + 0.3-0.6 mg/L NAA + 28-42 g/L sucrose.

Preferably, the culture conditions in step (1) of the present invention are: 25 +/-2 ℃ and the illumination intensity of 30-50 mu mol/m²S, the illumination period is 10-12 h/d, the culture time is 50-60 d, and the grown callus presents a good state of translucency and strong activity.

Preferably, the conditions for the proliferation of callus in step (2) of the present invention are: dark culture is carried out at the temperature of 25 +/-2 ℃, and the culture time is 70-90 days; the grown callus is in a semitransparent fluffy state, and can be automatically dispersed into small-particle callus at the edge, and the callus in the state meets the requirement of a suspension culture system.

Preferably, the culture conditions in step (3) of the present invention are: dark culture is carried out at the temperature of 25 +/-2 ℃, the pH value of a culture medium is 4.8-5.2, the rotating speed of a shaking table is 120-160 rpm/min, and the culture time is 7-10 days.

Preferably, the screening process of the large and small particles in step (3) of the present invention is: filtering with a 40-60 mesh stainless steel sieve, and obtaining the undersize tissue as the small-particle callus.

Preferably, the WPM medium in step (3) of the present invention is replaced with 3/5 organic modified WPM medium.

The WPM is called as a WPM woody plant basic culture medium completely, and comprises macroelements (400 mg/L of ammonium nitrate, 990mg/L of potassium sulfate, 370mg/L of magnesium sulfate and 170mg/L of monopotassium phosphate); calcium salt (calcium nitrate 684 mg/L); trace elements (manganese sulfate 22.5mg/L, zinc sulfate 8.6mg/L, boric acid 6.2mg/L, copper sulfate 0.25mg/L, sodium molybdate 0.25 mg/L); iron salt (ferrous sulfate 27.8mg/L, EDTA 37.3 mg/L); 3/5 organic four samples (thiamine hydrochloride 1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, glycine 2 mg/L); inositol (100 mg/L); the improved culture medium refers to that compared with a normal WPM culture medium, the content of the organic four substances is only common 3/5.

According to actual needs, the suspension system can be subjected to subculture: after the callus which normally grows in the step (3) is cultured, filtering out an original liquid culture medium by using a 40-60-mesh stainless steel sieve, absorbing water by using sterile paper, transferring the sterile paper to a fresh 3/5 organic improved WPM culture medium for culture to form a stable suspension culture system, and separating to obtain suspension culture cells II (filtering by using a 180-200-mesh stainless steel sieve, wherein the tissue above 180-200 meshes is the small-particle callus which is suitable for the suspension culture system and is marked as the suspension culture cells II);

the culture medium is 3/5 organic combination of improved WPM culture medium and auxin: 3/5 organic modified WPM medium + 1.0-2.0 mg/L ZT + 0.1-0.2 mg/L NAA + 28-42 g/L sucrose;

the culture conditions were: dark culture is carried out at 25 +/-2 ℃, the culture time is 7-10 days, the pH value of a culture medium is 4.8-5.2, the rotation speed of a shaking table is 120-160 rpm/min, the volume of the culture medium is 40-50 mL, and the inoculation amount is 20-40 g/L.

Furthermore, according to actual needs, the culture suspension culture system can be improved and then continuously cultured, and the method comprises two methods:

the first method comprises the following steps:

sieving the suspension culture cell I with a 180-200 mesh sieve, reserving tissues on the upper part of the sieve, sucking water by using sterile filter paper, inoculating the suspension culture cell I into 3/5 organic improved WPM culture medium, and continuously culturing;

the culture medium is a combination of a WPM solid minimal medium and a cell auxin: 3/5 organic modified WPM medium + 1.0-2.0 mg/L ZT + 0.1-0.2 mg/L NAA + 28-42 g/L sucrose;

the culture conditions were: dark culture is carried out at 25 +/-2 ℃, the culture time is 10-20 days, the pH value of a culture medium is 4.8-5.2, the rotation speed of a shaking table is 120-160 rpm/min, the volume of the culture medium is 40-50 mL, and the inoculation amount is 20-40 g/L.

The second method comprises the following steps:

sieving the suspension culture cell II with a 180-200-mesh sieve, reserving tissues on the upper part of the sieve, sucking water by using sterile filter paper, inoculating the suspension culture cell II into 3/5 organic improved WPM culture medium, and continuously culturing;

the culture medium is 3/5 organic combination of improved WPM culture medium and auxin: WPM (3/5 organic) + 1.0-2.0 mg/L ZT + 0.1-0.2 mg/L NAA + 28-42 g/L sucrose;

the culture conditions were: dark culture is carried out at 25 +/-2 ℃, the culture time is 10-20 days, the pH value of a culture medium is 4.8-5.2, the rotation speed of a shaking table is 120-160 rpm/min, the volume of the culture medium is 40-50 mL, and the inoculation amount is 20-40 g/L.

The WPM is a WPM solid culture medium, 6-BA is 6-benzylamino adenine, 2,4-D is 2, 4-dichlorophenoxyacetic acid, ZT is zeatin, and NAA is alpha-naphthylacetic acid.

The index for evaluating the suspension culture condition of the vaccinium dunalianum is biomass; the biomass refers to the net weight of the callus cells in suspension culture after the culture medium is filtered out and dried to remove water, and can reflect the influence of different hormone proportioning conditions and culture medium conditions on the growth condition of a suspension culture system.

The invention has the beneficial effects that:

(1) the method selects proper hormone proportion, and the callus cultured by induction presents semitransparent yellow, has no browning phenomenon, good quality, fluffy texture and strong embryogenic property, and is suitable for suspension culture.

(2) The proliferation culture time of the callus is limited by the method, so that the callus which is dark yellow and semitransparent and is self-dispersed into nearly powdery callus particles can be obtained, the callus particles can successfully survive in liquid and can be self-dispersed after being transferred to a liquid culture medium, the culture medium is light yellow and turbid, and the callus cells can be attached to the inner wall of a culture bottle but cannot agglomerate; the cells maintain a suspension state after passage without obvious browning; the cells cultured in the liquid are transferred in other states, and the culture medium is clear after passage and has no suspension cells.

(3) The invention selects auxin ZT to add into culture medium, not only is favorable to the multiplication of callus, but also can make the callus cell normally grow in liquid culture medium.

Drawings

FIG. 1 shows the callus induced under the condition A in example 1.

FIG. 2 shows the proliferation of calli in example 1.

FIG. 3 shows the growth of the suspension culture system established in example 3.

FIG. 4 is a graph showing the growth of the cells in suspension culture in example 4.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited to the examples.