CN108085335A - A kind of method for cultivating the transgene cotton that fruit branch angle changes - Google Patents
A kind of method for cultivating the transgene cotton that fruit branch angle changes Download PDFInfo
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Abstract
Cotton breeding technical field of the present invention, a kind of method for cultivating the transgene cotton that fruit branch angle changes is disclosed, including preparing embryo callus, prepares the recombinational agrobacterium containing target gene, prepare Agrobacterium bacteria suspension, conversion is co-cultured, selection is cultivated, differentiation culture and etc., using special seedling culture medium prescription, callus tissue culture base, embryo callus culture medium, co-culture media formula, seed treatment step is simplified, callus contamination rate is reduced, improves genetic transformation efficiency.
Description
Technical field
The invention belongs to cotton breeding technical fields, and in particular to a kind of transgene cotton cultivated fruit branch angle and changed
Method.
Background technology
There are three cotton regions in China, is (1) Yellow River basin cotton region respectively, which includes the ground such as Shandong, Hebei, Henan；(2)
Yangtze river basin cotton region, the region include the ground such as Hunan, Hubei, Jiangsu, Anhui；(3) Xinjiang cotton.Cotton is as one important
Industrial crops and strategic resource, Cotton Industry occupy an important position in national economy, and the cotton for polymerizeing a variety of merits educates
Kind achievement, the prosperity of volume increase and industry for cotton have vital scientific and technological contribution portion.Yield, disease resistance, fiber
Quality is always the breeding preferred object of cotton.As an economical agricultural and sideline product, yield height directly affects warp then
Ji income, disease resistance is the direct acting factor of output of cotton, and the height of fiber quality is related to the quality and business of cotton product
Competitiveness.
Cotton has two kinds of long fruit branch and fruit spur, and the cotton variety used in production is mainly long fruit branch kind, lacks short
Fruit branch kind.Fruit spur kind can not only efficiently utilize luminous energy, soil fertility, and can increase grown per area strain number, improve
Output of cotton, training process can be simplified by addition planting fruit spur kind.Display is had been reported, tetraploid cotton the 7th, 16 dyes
There are fruit spur gene (cl) (Zhai Tengfei, the finely positioning of cotton fruit spur gene cl, Southwest University, 2015) for body.In addition,
Zero formula fruit branch is also a kind of mutant character of cotton fruit branch of discovered in recent years, show as cotton boll directly it is raw at stem leaf
Or only there are one fruit sections, it is possible to reduce the recruitment to cotton training management, zero formula fruit branch gene gb-nbl is one important.
Using gene transformation method by these change fruit branch types genetic transformation into some other elite cotton kind, contribute to cotton
The exploitation of flower new varieties.
At present, common plant transgenic method includes Direct gene transfer and DNA direct guiding methods, wherein Direct gene transfer
Include agrobacterium-mediated transformation, virus-mediated methods again.Agrobacterium-mediated transformation is the common gene transfer method of current dicotyledon.
It is to be formed in neoplastic process in plant using the Agrobacterium for carrying target gene and can target gene be imported plant cell
In, and then construct transfer-gen plant.Although agriculture bacillus mediated gene transformation method is had been widely used in plant,
Since its is complex for operation step, transformation efficiency is not high, and it is therefore necessary to develop a kind of agriculture bacillus mediated cotton fruit branch type
Gene transformation method, the transgene cotton new varieties to cultivate the change of fruit branch angle lay the foundation.
The content of the invention
To solve the above-mentioned problems, a kind of method for cultivating the transgene cotton that fruit branch angle changes provided by the invention,
It is converted using Agrobacterium-mediated gene, improves genetic transformation efficiency, it is new to cultivate the transgene cotton of fruit branch angle change
Kind lays the foundation.
The object of the present invention is to provide a kind of method for cultivating the transgene cotton that fruit branch angle changes, including following step
Suddenly：
S1 prepares embryo callus：
Cotton seeds are taken, sterile water wash drains away the water, and then cotton seeds is soaked in seedling culture medium, 20-28 DEG C
Light culture 3-4d, then 20-28 DEG C of optical culture 2-3d, obtains seedling；
The hypocotyl of seedling is cut into stem section, tiles and Fiber differentiation is carried out into callus tissue culture base, 3-4 weeks subculture one
It is secondary, it then chooses loose callus and is seeded on embryonic callus induction culture medium and cultivate, induced embryonic callus
Generation；
The preparation method of wherein every liter seedling culture medium is as follows：20-50mg antiseptics, mixing are added in 1L MS culture mediums；
The preparation method of every liter of callus tissue culture base is as follows：20-50mg vitamin B5s are added in 1L MS culture mediums,
Mixing；
The preparation method of every liter of embryo callus culture medium is as follows：20-50mg vitamins are added in 1L YP culture mediums
B5 and 10-20mg antiseptics, mixing；
S2 prepares the recombinational agrobacterium containing target gene：It is thin that recombinant plasmid containing target gene is transferred to Agrobacterium
In born of the same parents, recombinational agrobacterium is obtained；Wherein recombinational agrobacterium contains Kan genes；
S3 prepares Agrobacterium bacteria suspension：Recombinational agrobacterium is expanded into numerous culture to cell concentration OD600=0.4-0.6, culture
Temperature is 28 ± 1 DEG C, obtains Agrobacterium bacterium solution, is centrifuged, and the thalline of collection is resuspended through nutrient solution, obtains Agrobacterium bacteria suspension, standby
With；
The preparation method of every liter of nutrient solution is as follows：10-20mg kanamycins, mixing are added in 1L MS culture mediums；
S4 co-cultures conversion：Embryo callus is soaked in Agrobacterium bacteria suspension, disseminates 5-10min, takes out embryo
Callus blots the bacteria suspension of embryo callus remained on surface, and then embryo callus tiles to being covered with one layer of filter
On the co-culture media of paper, 24-48h is cultivated in 28 ± 1 DEG C of darkrooms, obtains co-culturing the embryo callus of conversion；
The preparation method of every liter of co-culture media is as follows：1 μ L 0.1mol/L calcium chloride solutions are added in 1L MS culture mediums
With 100 μ L 1mmol/L sodium chloride solutions, mixing；
S5, selection culture：The embryo callus that conversion will be co-cultured takes out, with rinsed with sterile water, suck dry moisture, then
It is transferred on Selective agar medium, 25 ± 1 DEG C, photoperiod culture 45-60d, wherein, when each subculture is selected per 15-20d subcultures once
Select the embryo callus newly grown；
The preparation method of every liter of Selective agar medium is as follows：30-50mg kanamycins, mixing are added in 1L MS culture mediums；
S6, differentiation culture：The embryo callus of last time selection, which is placed in differential medium, breaks up culture, until point
Seedling is dissolved, obtains transgenic seedling, identified for genes then is carried out to transgenic seedling, positive seedling is filtered out, completes transfer-gen plant
Structure.
Preferably, the method for the transgene cotton that above-mentioned cultivation fruit branch angle changes, in S1, the length of stem section is 0.3-
0.5cm。
Preferably, the method for the transgene cotton that above-mentioned cultivation fruit branch angle changes, in S1, the antiseptic is streptomysin
Or cephalosporin.
Preferably, the method for the transgene cotton that above-mentioned cultivation fruit branch angle changes, in S2, what construction of recombinant plasmid was selected
Carrier be pk7WG2D carriers, Agrobacterium GV3101.
Preferably, the method for the transgene cotton that above-mentioned cultivation fruit branch angle changes, in S5, the condition of photoperiod culture
For：Periodicity of illumination 14h, intensity 2000lux.
Preferably, the method for the transgene cotton that above-mentioned cultivation fruit branch angle changes, in S6, the differential medium is MS
Culture medium.
Preferably, the method for the transgene cotton that above-mentioned cultivation fruit branch angle changes, in S2, the target gene is cotton
Fruit spur gene cl1, cl2, cl3 or zero formula fruit branch gene gb-nbl.
Compared with prior art, the method provided by the invention for cultivating the transgene cotton that fruit branch angle changes has following
Advantageous effect：
1st, in view of agriculture bacillus mediated transgenic method complex steps in the prior art, cause transformation efficiency not high, this hair
The bright characteristic in view of cotton plants using special seedling culture medium prescription, the step of the seed soaking sterilization of omission, simplifies
Seed treatment step is saved the trouble for preparing the fungicide such as alcohol, is directly fully cleaned seed with sterile water, seed culture is extremely
The effect of antiseptic is relied on during seedling, prevents seed microbiological contamination, improves seed hair seedling rate.
2nd, the present invention additionally uses special callus tissue culture base, embryo callus culture medium, co-culture media；Its
In, vitamin B5 is with the addition of in callus tissue culture base, promotes callus generation, shortens the time；Embryo callus culture
Base is also with the addition of antiseptic, during embryo callus generates, plays bacteriostasis, reduces embryo callus microbiological contamination
Rate；Vitamin B5 is with the addition of in embryo callus culture medium, promotes embryo callus generation, shortens the time.Co-culture media
Middle addition calcium chloride solution and sodium chloride solution, can improve target gene conversion ratio.
Specific embodiment
With reference to specific embodiment, the present invention is described in detail, but should not be construed as the limitation of the present invention.It is following
The test method of actual conditions is not specified in embodiment, is usually operated according to normal condition, due to not being related to inventive point, thus it is not right
Its step is described in detail.
A kind of method for cultivating the transgene cotton that fruit branch angle changes provided by the invention, specifically includes following steps：
S1 prepares embryo callus：
Cotton seeds are taken, sterile water wash drains away the water, and then cotton seeds is soaked in seedling culture medium, 20-28 DEG C
Light culture 3-4d, then 20-28 DEG C of optical culture 2-3d, obtains seedling；
The hypocotyl of seedling is cut into stem section, tiles and Fiber differentiation is carried out into callus tissue culture base, 3-4 weeks subculture one
It is secondary, it then chooses loose callus and is seeded on embryonic callus induction culture medium and cultivate, induced embryonic callus
Generation；
The preparation method of wherein every liter seedling culture medium is as follows：20-50mg antiseptics, mixing are added in 1L MS culture mediums；
The preparation method of every liter of callus tissue culture base is as follows：20-50mg vitamin B5s are added in 1L MS culture mediums,
Mixing；
The preparation method of every liter of embryo callus culture medium is as follows：20-50mg vitamins are added in 1L YP culture mediums
B5 and 10-20mg antiseptics, mixing；
YP culture mediums use existing YP culture mediums, and formula is：10g yeast extracts, 20g peptones, distilled water are settled to
1L；
S2 prepares the recombinational agrobacterium containing target gene：It is thin that recombinant plasmid containing target gene is transferred to Agrobacterium
In born of the same parents, recombinational agrobacterium is obtained；Wherein recombinational agrobacterium contains Kan genes；
S3 prepares Agrobacterium bacteria suspension：Recombinational agrobacterium is expanded into numerous culture to cell concentration OD600=0.4-0.8, culture
Temperature is 28 ± 1 DEG C, obtains Agrobacterium bacterium solution, is centrifuged, and the thalline of collection is resuspended through nutrient solution, obtains Agrobacterium bacteria suspension, standby
With；
The preparation method of every liter of nutrient solution is as follows：10-20mg kanamycins, mixing are added in 1L MS culture mediums；
S4 co-cultures conversion：The embryo callus of S1 is soaked in the Agrobacterium bacteria suspension of S3, disseminates 5-10min,
Embryo callus is taken out, blots the bacteria suspension of embryo callus remained on surface, then embryo callus tiles to covering
On the co-culture media for having one layer of filter paper, 24-48h is cultivated in 28 ± 1 DEG C of darkrooms, obtains co-culturing the embryo callus of conversion；
The preparation method of every liter of co-culture media is as follows：1 μ L 0.1mol/L calcium chloride solutions are added in 1L MS culture mediums
With 100 μ L 1mmol/L sodium chloride solutions, mixing；
S5, selection culture：The embryo callus that conversion will be co-cultured takes out, with rinsed with sterile water, suck dry moisture, then
It is transferred on Selective agar medium, 25 ± 1 DEG C, photoperiod culture 45-60d, wherein, when each subculture is selected per 15-20d subcultures once
Select the embryo callus newly grown；
The preparation method of every liter of Selective agar medium is as follows：30-50mg kanamycins, mixing are added in 1L MS culture mediums；
S6, differentiation culture：The embryo callus of last time selection, which is placed in differential medium, breaks up culture, until point
Seedling is dissolved, obtains transgenic seedling, identified for genes then is carried out to transgenic seedling, positive seedling is filtered out, completes transfer-gen plant
Structure.
Specifically include following embodiment.
Embodiment 1
A kind of method for cultivating the transgene cotton that fruit branch angle changes provided by the invention, specifically includes following steps：
S1 prepares embryo callus：
Cotton seeds are taken, sterile water wash drains away the water, and then cotton seeds are soaked in seedling culture medium, and 28 DEG C dark
3d is cultivated, then 28 DEG C of optical culture 2d, obtain seedling；
The hypocotyl of seedling is cut into 0.3-0.4cm stem sections, tiles and Fiber differentiation is carried out into callus tissue culture base, 3
Zhou Jidai once, then chooses loose callus and is seeded on embryonic callus induction culture medium and cultivate, and induces embryo
The generation of callus；
The preparation method of wherein every liter seedling culture medium is as follows：20mg antiseptics, mixing are added in 1L MS culture mediums；Institute
Antiseptic is stated as streptomysin；
The preparation method of every liter of callus tissue culture base is as follows：20mg vitamin B5s are added in 1L MS culture mediums, are mixed
It is even；
The preparation method of every liter of embryo callus culture medium is as follows：20mg vitamin B5s are added in 1L YP culture mediums
With 10mg antiseptics, mixing；Antiseptic is streptomysin；
YP culture mediums use existing YP culture mediums, and formula is：10g yeast extracts, 20g peptones, distilled water are settled to
1L；
S2 prepares the recombinational agrobacterium containing target gene：It is thin that recombinant plasmid containing target gene is transferred to Agrobacterium
In born of the same parents, recombinational agrobacterium is obtained；Wherein recombinational agrobacterium contains Kan genes；The carrier that construction of recombinant plasmid is selected is pk7WG2D
Carrier, Agrobacterium GV3101, target gene are that (information of the gene has been documented in Chinese patent to zero formula fruit branch gene gb-nbl
In CN104561284B)；
S3 prepares Agrobacterium bacteria suspension：Recombinational agrobacterium is expanded into numerous culture to cell concentration OD600=0.6, cultivation temperature
For 28 ± 1 DEG C, Agrobacterium bacterium solution, 1000r/min centrifugation 10min are obtained, the thalline of collection is resuspended through nutrient solution, obtains Agrobacterium
Bacteria suspension, it is spare；
The preparation method of every liter of nutrient solution is as follows：10mg kanamycins, mixing are added in 1L MS culture mediums；
S4 co-cultures conversion：The embryo callus of S1 is soaked in the Agrobacterium bacteria suspension of S3,5min is disseminated, takes
Go out embryo callus, blot the bacteria suspension of embryo callus remained on surface, then embryo callus tiles to being covered with
(one layer of filter paper of paving, then contains co-culture media in culture dish, is finally cured in the one layer of embryo that tile on the co-culture media of one layer of filter paper
Injured tissue), 28 ± 1 DEG C of darkroom cultures for 24 hours, obtain co-culturing the embryo callus of conversion；
The preparation method of every liter of co-culture media is as follows：1 μ L 0.1mol/L calcium chloride solutions are added in 1L MS culture mediums
With 100 μ L 1mmol/L sodium chloride solutions, mixing；
S4, selection culture：The embryo callus that conversion will be co-cultured takes out, with rinsed with sterile water, suck dry moisture, then
It is transferred on Selective agar medium, 25 ± 1 DEG C, photoperiod culture 45d, wherein, selection is new long during each subculture per 15d subcultures once
The embryo callus gone out；Photoperiod culture condition be：Periodicity of illumination 14h, intensity 2000lux；
The preparation method of every liter of Selective agar medium is as follows：30mg kanamycins, mixing are added in 1L MS culture mediums；
S5, differentiation culture：The embryo callus of last time selection, which is placed in differential medium, breaks up culture, until point
Seedling is dissolved, obtains transgenic seedling, identified for genes then is carried out to transgenic seedling using method for identifying molecules, amplifies purpose base
Cause filters out positive seedling, completes the structure of transfer-gen plant.Wherein differential medium is MS culture mediums.
Wherein method for identifying molecules is identical with the identification method of the prior art, due to complex steps, and is not of the invention
Where inventive point, therefore it will not go into details herein.
Embodiment 2
A kind of method for cultivating the transgene cotton that fruit branch angle changes provided by the invention, specifically includes following steps：
S1 prepares embryo callus：
Cotton seeds are taken, sterile water wash drains away the water, and then cotton seeds are soaked in seedling culture medium, and 20 DEG C dark
4d is cultivated, then 20 DEG C of optical culture 3d, obtain seedling；
The hypocotyl of seedling is cut into 0.4-0.5cm stem sections, tiles and Fiber differentiation is carried out into callus tissue culture base, 4
Zhou Jidai once, then chooses loose callus and is seeded on embryonic callus induction culture medium and cultivate, and induces embryo
The generation of callus；
The preparation method of wherein every liter seedling culture medium is as follows：50mg antiseptics, mixing are added in 1L MS culture mediums；Institute
Antiseptic is stated as cephalosporin；
The preparation method of every liter of callus tissue culture base is as follows：50mg vitamin B5s are added in 1L MS culture mediums, are mixed
It is even；
The preparation method of every liter of embryo callus culture medium is as follows：50mg vitamin B5s are added in 1L YP culture mediums
With 20mg antiseptics, mixing；Antiseptic is cephalosporin；
YP culture mediums use existing YP culture mediums, and formula is：10g yeast extracts, 20g peptones, distilled water are settled to
1L；
S2 prepares the recombinational agrobacterium containing target gene：It is thin that recombinant plasmid containing target gene is transferred to Agrobacterium
In born of the same parents, recombinational agrobacterium is obtained；Wherein recombinational agrobacterium contains Kan genes；The carrier that construction of recombinant plasmid is selected is pk7WG2D
Carrier, Agrobacterium GV3101, for cotton fruit spur gene cl1, (information of the gene has been documented in bibliography to target gene
In, which is Zhai Tengfei, the finely positioning of cotton fruit spur gene cl, Southwest University, 2015)；
S3 prepares Agrobacterium bacteria suspension：Recombinational agrobacterium is expanded into numerous culture to cell concentration OD600=0.4, cultivation temperature
For 28 ± 1 DEG C, Agrobacterium bacterium solution, 1000r/min centrifugation 10min are obtained, the thalline of collection is resuspended through nutrient solution, obtains Agrobacterium
Bacteria suspension, it is spare；
The preparation method of every liter of nutrient solution is as follows：20mg kanamycins, mixing are added in 1L MS culture mediums；
S4 co-cultures conversion：The embryo callus of S1 is soaked in the Agrobacterium bacteria suspension of S3,10min is disseminated, takes
Go out embryo callus, blot the bacteria suspension of embryo callus remained on surface, embryo callus tiling is then covered with one
On the co-culture media of layer filter paper, 28 ± 1 DEG C of darkroom cultures for 24 hours, obtain co-culturing the embryo callus of conversion；
The preparation method of every liter of co-culture media is as follows：1 μ L 0.1mol/L calcium chloride solutions are added in 1L MS culture mediums
With 100 μ L 1mmol/L sodium chloride solutions, mixing；
S5, selection culture：The embryo callus that conversion will be co-cultured takes out, with rinsed with sterile water, suck dry moisture, then
It is transferred on Selective agar medium, 25 ± 1 DEG C, photoperiod culture 60d, wherein, selection is new long during each subculture per 20d subcultures once
The embryo callus gone out；Photoperiod culture condition be：Periodicity of illumination 14h, intensity 2000lux；
The preparation method of every liter of Selective agar medium is as follows：50mg kanamycins, mixing are added in 1L MS culture mediums；
S6, differentiation culture：The embryo callus of last time selection, which is placed in differential medium, breaks up culture, until point
Seedling is dissolved, obtains transgenic seedling, identified for genes then is carried out to transgenic seedling using method for identifying molecules, amplifies purpose base
Cause filters out positive seedling, completes the structure of transfer-gen plant.Differential medium is MS culture mediums；Wherein method for identifying molecules with
The identification method of the prior art is identical, due to complex steps, and is not the inventive point place of the present invention, therefore it will not go into details herein.
Embodiment 3
A kind of method for cultivating the transgene cotton that fruit branch angle changes provided by the invention, operating procedure and embodiment 2
Identical, difference lies in target gene, for cotton fruit spur gene cl2, (information of the gene has been documented in bibliography, the ginseng
Document is examined as Zhai Tengfei, the finely positioning of cotton fruit spur gene cl, Southwest University, 2015).
Embodiment 4
A kind of method for cultivating the transgene cotton that fruit branch angle changes provided by the invention, operating procedure and embodiment 2
Identical, difference lies in target gene, for cotton fruit spur gene cl3, (information of the gene has been documented in bibliography, the ginseng
Document is examined as Zhai Tengfei, the finely positioning of cotton fruit spur gene cl, Southwest University, 2015).
In view of agriculture bacillus mediated transgenic method complex steps in the prior art, cause transformation efficiency not high, the present invention
In view of the characteristic of cotton plants, using special seedling culture medium prescription, the step of the seed soaking sterilization of omission, kind is simplified
Subprocessing step is saved the trouble for preparing the fungicide such as alcohol, is directly fully cleaned seed with sterile water, seed culture to children
The effect of antiseptic is relied on during seedling, prevents seed microbiological contamination, seed hair seedling rate is improved, by experimental verification, embodiment 1-4
In, seed contamination rate≤1%, and seed sends out seedling rate more than 98%, works well.
The present invention additionally uses special callus tissue culture base, embryo callus culture medium, co-culture media；Wherein,
Vitamin B5 is with the addition of in callus tissue culture base, promotes callus generation, shortens the time, 3-4 weeks subculture is once, you can production
Raw loose callus, and for the generation of induced embryonic callus.
Embryo callus culture medium is also with the addition of antiseptic, during embryo callus generates, plays antibacterial
Effect reduces embryo callus contamination rate, by experimental verification, in embodiment 1-4, and embryo callus contamination rate≤1%,
The calculation formula of embryo callus contamination rate is (microbiological contamination number/callus sum) × 100%；Embryo callus culture medium
In be with the addition of vitamin B5, promote embryo callus generation, shorten the time.
Calcium chloride solution and sodium chloride solution are added in co-culture media, target gene conversion ratio, embodiment 1-4 can be improved
In, 100 embryo callus is taken to carry out Agrobacterium-medialed transformation using the co-culture media, find to convert by Molecular Identification
Success rate >=29.8%.
In order to verify the effect of sodium chloride and calcium chloride, transformation experiment and control experiment We conducted various concentration.
Method according to embodiment 1 prepares 100 essentially identical embryo callus of size, is divided into four groups, every group 25, uses
Distinct methods carry out transformation experiment.Specific method is as follows：
Test one group
The preparation method of every liter of co-culture media is as follows：1 μ L 0.1mol/L calcium chloride solutions are added in 1L MS culture mediums
With 100 μ L 1mmol/L sodium chloride solutions；Co-cultivation conversion, selection culture are carried out using the condition of S3-S5 in embodiment 1 and divided
Change culture, measure the conversion success rate of target gene.
Test two groups
The preparation method of every liter of co-culture media is as follows：20 μ L 0.1mol/L calcium chloride solutions are added in 1L MS culture mediums
With 100 μ L 1mmol/L sodium chloride solutions；Co-cultivation conversion, selection culture are carried out using the condition of S3-S5 in embodiment 1 and divided
Change culture, measure the conversion success rate of target gene.
Test three groups
The preparation method of every liter of co-culture media is as follows：1 μ L 0.1mol/L calcium chloride solutions are added in 1L MS culture mediums
With 300 μ L 1mmol/L sodium chloride solutions；Co-cultivation conversion, selection culture are carried out using the condition of S3-S5 in embodiment 1 and divided
Change culture, measure the conversion success rate of target gene.
Control group
Co-culture media is MS culture mediums；Using the condition of S3-S5 in embodiment 1 carry out co-cultivation conversion, selection culture and
Differentiation culture measures the conversion success rate of target gene.
The results show that the conversion success rate of one group of experiment is 30.5%, the conversion success rate of two groups of experiment is 8.6%, real
The conversion success rate for testing three groups is 7.1%, and the conversion success rate of control is 5.2%.The result shows that the addition and chlorination of sodium chloride
The addition of calcium solution can improve genetic transformation rate, and still, dosage is restricted, be not the The more the better of addition, test two groups
In three groups of experiment, the dosage of sodium chloride solution and calcium chloride solution is added respectively, but it is not very high to convert success rate.
Control group is not using sodium chloride solution and calcium chloride solution, and conversion success rate is nor very high.
It should be noted that in above-described embodiment, the formula of MS culture mediums is as shown in table 1, first respectively according to described in table 1
Formula prepare mother liquor I, mother liquor II, mother liquor III and mother liquor IV respectively, wherein mother liquor I is 10 times of concentrates, mother liquor II, mother liquor III
It is 100 times of concentrates with mother liquor IV, when every liter of MS culture medium is prepared, takes I 100mL of mother liquor, take mother liquor II, mother liquor III and mother liquor
IV each 10mL, then adds water to 1L.
The formula of table 1MS culture mediums
It should be noted that repeat in order to prevent, description of the invention preferred embodiment and effect, although having described
The preferred embodiment of the present invention, but those skilled in the art once know basic creative concept, then it can be to these
Embodiment makes other change and modification.So appended claims are intended to be construed to include preferred embodiment and fall into
All change and modification of the scope of the invention.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
God and scope.In this way, if these modifications and changes of the present invention belongs to the scope of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to comprising including these modification and variations.
Claims (7)
- A kind of 1. method for cultivating the transgene cotton that fruit branch angle changes, which is characterized in that comprise the following steps：S1 prepares embryo callus：Cotton seeds are taken, sterile water wash drains away the water, and then cotton seeds are soaked in seedling culture medium, 20-28 DEG C of dark training 3-4d is supported, then 20-28 DEG C of optical culture 2-3d, obtains seedling；The hypocotyl of seedling is cut into stem section, tiles and Fiber differentiation is carried out into callus tissue culture base, 3-4 weeks subculture once, Then choose loose callus and be seeded on embryonic callus induction culture medium and cultivate, the production of induced embryonic callus It is raw；The preparation method of wherein every liter seedling culture medium is as follows：20-50mg antiseptics, mixing are added in 1L MS culture mediums；The preparation method of every liter of callus tissue culture base is as follows：20-50mg vitamin B5s, mixing are added in 1L MS culture mediums；The preparation method of every liter of embryo callus culture medium is as follows：In 1L YP culture mediums add in 20-50mg vitamin B5s and 10-20mg antiseptics, mixing；S2 prepares the recombinational agrobacterium containing target gene：Recombinant plasmid containing target gene is transferred in agrobatcerium cell, Obtain recombinational agrobacterium；Wherein recombinational agrobacterium contains Kan genes；S3 prepares Agrobacterium bacteria suspension：Recombinational agrobacterium is expanded into numerous culture to cell concentration OD600=0.4-0.6, cultivation temperature For 28 ± 1 DEG C, Agrobacterium bacterium solution is obtained, is centrifuged, the thalline of collection is resuspended through nutrient solution, obtains Agrobacterium bacteria suspension, spare；The preparation method of every liter of nutrient solution is as follows：10-20mg kanamycins, mixing are added in 1L MS culture mediums；S4 co-cultures conversion：Embryo callus is soaked in Agrobacterium bacteria suspension, disseminates 5-10min, takes out embryo callus subculture Tissue blots the bacteria suspension of embryo callus remained on surface, and then embryo callus tiles to being covered with one layer of filter paper On co-culture media, 24-48h is cultivated in 28 ± 1 DEG C of darkrooms, obtains co-culturing the embryo callus of conversion；The preparation method of every liter of co-culture media is as follows：1 μ L 0.1mol/L calcium chloride solutions and 100 are added in 1L MS culture mediums μ L 1mmol/L sodium chloride solutions, mixing；S5, selection culture：The embryo callus for co-culturing conversion is taken out, with rinsed with sterile water, then suck dry moisture is transferred Onto Selective agar medium, 25 ± 1 DEG C, photoperiod culture 45-60d, wherein, selection is new during each subculture per 15-20d subcultures once The embryo callus grown；The preparation method of every liter of Selective agar medium is as follows：30-50mg kanamycins, mixing are added in 1L MS culture mediums；S6, differentiation culture：The embryo callus of last time selection, which is placed in differential medium, breaks up culture, until differentiating Seedling obtains transgenic seedling, then carries out identified for genes to transgenic seedling, filters out positive seedling, complete the structure of transfer-gen plant.
- 2. the method according to claim 1 for cultivating the transgene cotton that fruit branch angle changes, which is characterized in that in S1, The length of stem section is 0.3-0.5cm.
- 3. the method according to claim 1 for cultivating the transgene cotton that fruit branch angle changes, which is characterized in that in S1, The antiseptic is streptomysin or cephalosporin.
- 4. the method according to claim 3 for cultivating the transgene cotton that fruit branch angle changes, which is characterized in that in S2, The carrier that construction of recombinant plasmid is selected is pk7WG2D carriers, and the Agrobacterium is GV3101.
- 5. the method according to claim 2 for cultivating the transgene cotton that fruit branch angle changes, which is characterized in that in S5, Photoperiod culture condition be：Periodicity of illumination 14h, intensity 2000lux.
- 6. the method according to claim 2 for cultivating the transgene cotton that fruit branch angle changes, which is characterized in that in S6, The differential medium is MS culture mediums.
- 7. the method according to claim 1 for cultivating the transgene cotton that fruit branch angle changes, which is characterized in that in S2, The target gene is cotton fruit spur gene cl1, cl2, cl3 or zero formula fruit branch gene gb-nbl.
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