阅读说明：本技术 棉花遗传转化中产生正常苗的培养基及方法 (The culture medium and method of field run plant are generated in Cotton Transformation ) 是由 罗晓丽 肖娟丽 张安红 王志安 刘传亮 马丹 孙瑞斌 刘志红 王少辉 于 2018-05-25 设计创作，主要内容包括：本发明公开了棉花遗传转化中产生正常苗的培养基及方法,通过使用无激素的增殖培养基对诱导筛选培养后的愈伤组织进行增殖培养,缩短胚性愈伤组织继代培养时间,增加胚性愈伤组织继代培养透气性,提高分化培养基固化剂含量,胚状体形成后降低培养基中糖含量,进而保证正常苗的产生率达90％以上。(The invention discloses culture mediums and method that field run plant is generated in Cotton Transformation, Multiplying culture is carried out to the callus after induction screening and culturing by using the proliferated culture medium of no hormone, shorten the embryo callus squamous subculture time, increase embryo callus squamous subculture gas permeability, improve differential medium curing agent content, embryoid reduces sugared content in culture medium after being formed, and then guarantees the generation rate of field run plant up to 90% or more.)

1. generating the culture medium of field run plant in Cotton Transformation, which is characterized in that the culture medium includes proliferated culture medium, institute Stating proliferated culture medium includes following component: vitamin B1 8-12mg/L, glucose 28-32g/L, Phytagel 2.8-3.0g/L And inorganic salts, pH 6.8；Wherein, the inorganic salts are the inorganic salts in MS culture medium, and the nitric acid potassium concn in inorganic salts It doubles.

2. generating the culture medium of field run plant in Cotton Transformation according to claim 1, which is characterized in that the culture Base further includes differential medium, and the differential medium removes the ammonium nitrate in inorganic salts based on the proliferated culture medium Ingredient adds asparagine 0.45-0.55g/L, glutamy ammonium 0.8-1.2g/L, and Phytagel concentration is 3.5-3.9g/L, Concentration of glucose is 20-25g/L.

3. generating the culture medium of field run plant in Cotton Transformation according to claim 2, which is characterized in that the culture Base further includes seedling culture medium, and the seedling culture medium is based on the differential medium, and Phytagel concentration is 3.2-3.3g/L, concentration of glucose 15-25g/L.

4. in Cotton Transformation generate field run plant cultural method, which is characterized in that including following methods any one or It is several:

(1) Multiplying culture is carried out to the callus after induction screening and culturing using the proliferated culture medium without hormone；

(2) shorten the embryo callus squamous subculture time；

(3) increase embryo callus squamous subculture gas permeability；

(4) differential medium curing agent content is improved；

(5) sugared content in culture medium is reduced after embryoid is formed.

5. generating the cultural method of field run plant in Cotton Transformation, which comprises the following steps:

S1. aseptic seedling is cultivated, aseptic seedling hypocotyl segment is taken；

S2. dip dyeing liquid for shell is prepared；

S3. hypocotyl segment is infected using dip dyeing liquid for shell, is co-cultured；

S4. induction screening and culturing is carried out to the hypocotyl segment after co-cultivation, obtains callus；

S5. the proliferated culture medium of no hormone is used to carry out Multiplying culture to the callus after induction screening and culturing, until callus group Differentiation is knitted, embryo callus is formed；

S6. embryo callus is transferred to differential medium and carries out ventilative culture, 10-15d subculture is primary, continuous subculture 3-5 times Until there is embryoid；

S7. sugared content in culture medium is reduced after embryoid is formed, and is continued squamous subculture, is obtained regeneration plant.

6. generating the cultural method of field run plant in Cotton Transformation according to claim 5, which is characterized in that the S3 Specific step is as follows:

Hypocotyl segment 5-10min is disseminated using dip dyeing liquid for shell, the hypocotyl segment after dip dyeing is put into co-culture medium, 22-24 DEG C of dark culture 48h；

The co-culture medium adds 2,4-D 0.08-0.12mg/L, KT 0.08- based on the proliferated culture medium 0.12mg/L。

7. generating the cultural method of field run plant in Cotton Transformation according to claim 6, which is characterized in that the S4 Specific step is as follows:

S41. the hypocotyl section after co-culturing is put into the first screening and culturing medium, is cultivated 2 months, is obtained callus；

S42. the callus of diameter 1-2cm is transferred to postsearch screening culture 1 month in the second screening and culturing medium；Less than being cured for 1cm Injured tissue is transferred to chain induction screening and culturing 1 month in the first screening and culturing medium again；

First screening and culturing medium adds 2,4-D 0.08-0.12mg/L, KT 0.08- based on the proliferated culture medium 0.12mg/L, kanamycins 90-110mg/L, cephalosporin 450-550mg/L；

Second screening and culturing medium adds kanamycins 90-110mg/L, cephalosporin based on the proliferated culture medium 450-550mg/L。

8. generating the cultural method of field run plant in Cotton Transformation according to claim 6, which is characterized in that the S5 Middle 25d subculture is primary, continuous subculture 2-3 times, until callus breaks up.

9. generating the cultural method of field run plant in Cotton Transformation according to claim 6, which is characterized in that the S7 Middle regeneration plant, which is grown to after 5-8cm, can be used as scion and is grafted on rootstock seedling.

Technical field

The present invention relates to technical fields, more particularly in Cotton Transformation generate field run plant culture medium and Method.

Background technique

Cotton is a kind of important industrial crops, however serious pest and disease damage is subject in its planting process, so that Annual lint yield loss reaches the 10%-20% of total yield.

With the development of biotechnology, researcher is by many beneficial economical characters, Resistant and the property such as degeneration-resistant Shape is transformed into cotton, to improve its yield and quality, increases insect resistace, disease resistance, antiweed and resistance, to improve The plantation efficiency of cotton reduces cost, and mitigates environmental pollution.Agrobacterium_mediated method can be efficiently whole by foreign gene It closes in cotton gene group, and the stable heredity of foreign gene and high efficient expression must be based on tissue cultures.Cotton body is thin Blastula occurs and, often along with a large amount of Embryos and lopsided seedling, Vitrification, half glass especially often occurs in regenerative process The phenomenon that glass, these problems have become Cotton Transformation, the verifying of cotton functional gene and cotton Cloning of Genes Related Etc. research fields obstacle and bottleneck.

Therefore, how to reduce the generation of lopsided seedling during Cotton Transformation is that those skilled in the art are urgently to be resolved Technical problem.

Summary of the invention

In view of this, the present invention provides the culture mediums and method that generate field run plant in Cotton Transformation, by this hair Bright culture medium and cultural method can make the incidence of field run plant up to 90% or more.

To achieve the goals above, the present invention adopts the following technical scheme:

The culture medium of field run plant is generated in Cotton Transformation, the culture medium includes proliferated culture medium, the proliferation training Feeding base includes following component: vitamin B1 8-12mg/L, glucose 28-32g/L, Phytagel2.8-3.0g/L and inorganic salts, pH 6.8；Wherein, the inorganic salts are the inorganic salts in MS culture medium, and the nitric acid potassium concn in inorganic salts doubles.

Preferably, the culture medium further includes differential medium, and the differential medium is using the proliferated culture medium as base Plinth removes the ammonium nitrate component in inorganic salts, adds asparagine 0.45-0.55g/L, glutamy ammonium 0.8-1.2g/L, and Phytagel concentration is 3.5-3.9g/L, concentration of glucose 20-25g/L.

Preferably, the culture medium further includes seedling culture medium, and the seedling culture medium is using the differential medium as base Plinth, and Phytagel concentration is 3.2-3.3g/L, concentration of glucose 15-25g/L.

The cultural method of field run plant is generated in Cotton Transformation, any one or a few including following methods:

(1) Multiplying culture is carried out to the callus after induction screening and culturing using the proliferated culture medium without hormone.

(2) shorten the embryo callus squamous subculture time.

(3) increase embryo callus squamous subculture gas permeability.

(4) differential medium curing agent content is improved.

(5) sugared content in culture medium is reduced after embryoid is formed.

Lopsided seedling can be reduced by taking out hormone immediately after chain induction screening and culturing, when callus is transferred to proliferated culture medium Quantity.Embryo callus squamous subculture overlong time on differential medium will appear callus browning death and later period The phenomenon that lopsided seedling increases, therefore, is adjusted the embryo callus squamous subculture time, shortens embryo callus from body Cell maturation can reduce the generation of browning death and lopsided seedling to the time of emergence.In addition, embryo callus squamous subculture mistake Increase gas permeability in journey, improve differential medium curing agent content, embryoid reduces sugared content in culture medium after being formed can subtract Few vitrifying, the generation of semivitreous deformity seedling.

The cultural method of field run plant is generated in Cotton Transformation, comprising the following steps:

S1. aseptic seedling is cultivated, aseptic seedling hypocotyl segment is taken；

S2. dip dyeing liquid for shell is prepared；

S3. hypocotyl segment is infected using dip dyeing liquid for shell, is co-cultured；

S4. induction screening and culturing is carried out to the hypocotyl segment after co-cultivation, obtains callus；

S5. the proliferated culture medium of no hormone is used to carry out Multiplying culture to the callus after induction screening and culturing, until more Injured tissue differentiation, forms embryo callus；

S6. embryo callus is transferred to differential medium and carries out ventilative culture, 10-15d subculture is primary, continuous subculture 3- 5 times until there is embryoid；

S7. sugared content in culture medium is reduced after embryoid is formed, and is continued squamous subculture, is obtained regeneration plant.

Preferably, specific step is as follows by the S3:

Hypocotyl segment 5-10min is disseminated using dip dyeing liquid for shell, the hypocotyl segment after dip dyeing is put into co-culture medium On, 22-24 DEG C of dark culture 48h；

The co-culture medium adds 2,4-D 0.08-0.12mg/L, KT based on the proliferated culture medium 0.08-0.12mg/L。

Preferably, specific step is as follows by the S4:

S41. the hypocotyl section after co-culturing is put into the first screening and culturing medium, is cultivated 2 months, is obtained callus；

S42. the callus of diameter 1-2cm is transferred to postsearch screening culture 1 month in the second screening and culturing medium；Less than 1cm Callus be transferred to chain induction screening and culturing 1 month in the first screening and culturing medium again；

First screening and culturing medium adds 2,4-D 0.08-0.12mg/L, KT based on the proliferated culture medium 0.08-0.12mg/L, kanamycins 90-110mg/L, cephalosporin 450-550mg/L；

Second screening and culturing medium adds kanamycins 90-110mg/L, cephalo based on the proliferated culture medium Mycin 450-550mg/L.

Preferably, 25d subculture is primary in the S5, continuous subculture 2-3 times, until callus breaks up.

Preferably, regeneration plant grows to after 5-8cm and can be used as scion and be grafted on rootstock seedling in the S7.

It can be seen via above technical scheme that compared with prior art, the invention discloses generate in Cotton Transformation The culture medium of field run plant reduces lopsided seedling by the adjustment to ingredients such as hormone, curing agent, sugars in different culture period culture mediums Generation rate.Further, disclosed herein as well is the methods that field run plant is generated in Cotton Transformation, by different cultures The adjustment of phase culture medium, gas permeability, incubation time makes the generation rate of field run plant up to 90% or more.

Detailed description of the invention

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis The attached drawing of offer obtains other attached drawings.

Fig. 1 attached drawing is to induce screening and culturing state diagram in 1 month for the first time in screening and culturing medium M3；

Fig. 2 attached drawing is to induce screening and culturing state diagram in 2 months for the first time in screening and culturing medium M3；

Fig. 3 attached drawing is second of induction screening and culturing state diagram in 1 month in screening and culturing medium M3；

Fig. 4 attached drawing is programmed screening culture state diagram in 1 month in screening and culturing medium M4；

Fig. 5 attached drawing is first time proliferation and subculture in proliferated culture medium M1；

Fig. 6 attached drawing is second of proliferation and subculture in proliferated culture medium M1；

Fig. 7 attached drawing is third time proliferation and subculture in proliferated culture medium M1；

Fig. 8 attached drawing is ventilative culture；

Fig. 9 attached drawing is to break up subculture for the first time in differential medium M5；

Figure 10 attached drawing is second of differentiation subculture in differential medium M5；

Figure 11 attached drawing is to break up subculture for the third time in differential medium M5；

Figure 12 attached drawing is the 4th differentiation subculture in differential medium M5；

Figure 13 attached drawing is the embryoid culture in differential medium M6；

Figure 14 attached drawing is the embryoid culture in differential medium M6；

Figure 15 attached drawing is the seedling culture in seedling culture medium M7；

Figure 16 attached drawing is the seedling culture in seedling culture medium M7；

Figure 17 attached drawing is the field run plant that present invention culture obtains；

Figure 18 attached drawing is the embryo callus improved after curing agent content；

Figure 19 attached drawing is the embryoid improved after curing agent content；

Figure 20 attached drawing is not improve curing agent content lopsided seedling obtained；

Figure 21 attached drawing is influence of the different Subculture Times to embryo callus plant regeneration.

Specific embodiment

Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other Embodiment shall fall within the protection scope of the present invention.