CN108085335B - Method for cultivating transgenic cotton with fruit branch included angle changed - Google Patents
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Abstract
The invention discloses a method for cultivating transgenic cotton with a changed fruit branch included angle, which belongs to the technical field of cotton breeding and comprises the steps of preparing embryonic callus, preparing recombinant agrobacterium containing target genes, preparing agrobacterium suspension, co-culturing and transforming, selecting and culturing, differentiating and culturing and the like.
Description
Technical Field
The invention belongs to the technical field of cotton breeding, and particularly relates to a method for cultivating transgenic cotton with a changed fruit branch included angle.
Background
The China has three cotton areas which are respectively (1) cotton areas in the yellow river basin, wherein the cotton areas comprise Shandong, Hebei, Henan and the like; (2) the cotton area of Yangtze river basin, this area includes areas such as Hunan, Hubei, Jiangsu, Anhui; (3) xinjiang cotton area. As an important economic crop and strategic resource, the cotton industry plays an important role in national economy, and cotton breeding achievements with various excellent properties are aggregated, so that the cotton has a vital scientific and technological contribution to yield increase and industrial prosperity. The yield, disease resistance and fiber quality are always the first choice targets for cotton breeding. As an economic agricultural and sideline product, the yield directly influences the income of the year, the disease resistance is a direct influence factor of the cotton yield, and the quality of the fiber is related to the quality and the commercial competitiveness of the cotton product.
The cotton has two types of long fruit branches and short fruit branches, and the cotton varieties used in production mainly comprise long fruit branch varieties and lack short fruit branch varieties. The short fruit branch variety not only can efficiently utilize light energy and soil fertility, but also can increase the number of plants in unit area, improve the yield of cotton, and in addition, the pruning process can be simplified by planting the short fruit branch variety. It has been reported that the tetraploid cotton has short fruit branch gene (cl) in both chromosomes 7 and 16 (Zhai flying, fine localization of cotton short fruit branch gene cl, university of southwest 2015). In addition, the zero fruit branch is also a mutation character of cotton fruit branches discovered in recent years, which is expressed in that cotton bolls directly grow on main stem leaves or only one fruit node, so that labor for pruning management of cotton can be reduced, and the zero fruit branch gene gb-nbl is important. The genes for changing the fruit branch types are transformed into other excellent cotton varieties by using a gene transformation method, which is beneficial to the development of new cotton varieties.
At present, the commonly used plant transgenic methods include vector-mediated methods and direct DNA introduction methods, wherein the vector-mediated methods further include Agrobacterium-mediated methods and virus-mediated methods. Agrobacterium mediated method is a commonly used gene transfer method for dicotyledonous plants at present. The target gene can be introduced into plant cells by utilizing agrobacterium carrying the target gene in the process of forming tumor by plants, and then a transgenic plant is constructed. Although the agrobacterium-mediated gene transformation method is widely applied to plants, the operation steps are complicated, and the transformation efficiency is low, so that the agrobacterium-mediated gene transformation method for cotton fruit branches is needed to be developed, and a foundation is laid for cultivating a new transgenic cotton variety with a changed fruit branch included angle.
Disclosure of Invention
In order to solve the problems, the method for cultivating the transgenic cotton with the changed fruit branch included angle provided by the invention utilizes agrobacterium to mediate gene transformation, improves the gene transformation efficiency and lays a foundation for cultivating a new transgenic cotton variety with the changed fruit branch included angle.
The invention aims to provide a method for cultivating transgenic cotton with a changed fruit branch included angle, which comprises the following steps:
s1, preparing embryogenic callus:
cleaning cotton seeds with sterile water, draining, soaking the cotton seeds in a seedling culture medium, performing dark culture at 20-28 ℃ for 3-4 days, and performing light culture at 20-28 ℃ for 2-3 days to obtain seedlings;
cutting the hypocotyl of the seedling into stem segments, spreading the stem segments into a callus culture medium for induction culture, subculturing for 3-4 weeks, selecting loose callus, inoculating the loose callus on the embryonic callus induction culture medium for culture, and inducing the generation of embryonic callus;
the preparation method of each liter of seedling culture medium comprises the following steps: adding 20-50mg of antibacterial agent into 1L of MS culture medium, and mixing;
the preparation method of each liter of callus culture medium is as follows: adding 20-50mg of vitamin B5 into 1L of MS culture medium, and mixing;
the method for preparing the culture medium for each liter of the embryogenic callus comprises the following steps: adding vitamin B5 20-50mg and antibacterial agent 10-20mg into YP culture medium 1L, and mixing;
s2, preparing the recombinant agrobacterium containing the target gene: transferring the recombinant plasmid containing the target gene into agrobacterium cells to obtain recombinant agrobacterium; wherein the recombinant agrobacterium contains a Kan gene;
s3, preparing agrobacterium tumefaciens suspension: the recombinant agrobacterium is propagated and cultured to the thallus concentration OD600Obtaining agrobacterium tumefaciens liquid at the culture temperature of 28 +/-1 ℃ and 0.4-0.6, centrifuging, and re-suspending the collected thallus with the culture liquid to obtain agrobacterium tumefaciens suspension for later use;
the preparation method of each liter of nutrient solution comprises the following steps: adding 10-20mg kanamycin into 1L MS culture medium, and mixing uniformly;
s4, co-culture transformation: soaking the embryonic callus in the agrobacterium tumefaciens bacterial suspension, dip-dyeing for 5-10min, taking out the embryonic callus, sucking out the residual bacterial suspension on the surface of the embryonic callus, then spreading the embryonic callus on a co-culture solution coated with a layer of filter paper, and culturing for 24-48h in a dark room at the temperature of 28 +/-1 ℃ to obtain the co-culture transformed embryonic callus;
the preparation method of the co-culture solution per liter is as follows: adding 1 mu L of 0.1mol/L calcium chloride solution and 100 mu L of 1mmol/L sodium chloride solution into 1L of MS culture medium, and uniformly mixing;
s5, selective culture: taking out the embryogenic callus transformed by co-culture, rinsing with sterile water, sucking water, transferring to a selective culture medium, culturing at 25 + -1 deg.C for 45-60d with photoperiod, wherein subculturing every 15-20d, and selecting new embryogenic callus;
the preparation method of each liter of selection medium is as follows: adding 30-50mg kanamycin into 1L MS culture medium, and mixing uniformly;
s6, differentiation culture: and (3) placing the embryonic callus selected for the last time in a differentiation culture medium for differentiation culture until differentiation seedling emergence to obtain a transgenic seedling, then carrying out gene identification on the transgenic seedling, screening out a positive seedling, and completing construction of a transgenic plant.
Preferably, in the method for cultivating transgenic cotton with a changed fruit branch angle, in S1, the length of the stem segment is 0.3-0.5 cm.
Preferably, in the method for cultivating transgenic cotton with a changed fruit branch angle, in S1, the antibacterial agent is streptomycin or cefamycin.
Preferably, in the method for cultivating transgenic cotton with a changed fruit branch angle, in S2, the vector selected by the recombinant plasmid construction is a pk7WG2D vector, and the agrobacterium is GV 3101.
Preferably, in the method for cultivating transgenic cotton with a changed fruit branch angle, in S5, the photoperiod cultivation conditions are as follows: illumination period 14h, intensity 2000 lux.
Preferably, in the method for cultivating transgenic cotton with a changed fruit branch angle, in S6, the differentiation medium is an MS medium.
Preferably, in the method for cultivating transgenic cotton with a changed fruit branch angle, in S2, the target gene is a cotton short fruit branch gene cl1, cl2, cl3 or a null fruit branch gene gb-nbl.
Compared with the prior art, the method for cultivating the transgenic cotton with the changed fruit branch included angle has the following beneficial effects:
1. in view of the fact that the agrobacterium-mediated transgenic method in the prior art has complicated steps and causes low transformation efficiency, the method takes the characteristics of cotton plants into consideration, adopts a special seedling culture medium formula, omits the step of soaking and sterilizing seeds, simplifies the seed treatment step, saves the trouble of preparing bactericides such as alcohol and the like, directly and fully cleans the seeds with sterile water, and prevents the seeds from being infected with bacteria and improves the seedling emergence rate of the seeds by relying on the action of an antibacterial agent in the process of culturing the seeds to seedlings.
2. The invention also adopts special callus culture medium, embryonic callus culture medium and co-culture solution; wherein, vitamin B5 is added into the callus culture medium to promote the generation of callus and shorten the time; the embryonic callus culture medium is also added with an antibacterial agent, so that the antibacterial effect is achieved in the process of generating the embryonic callus, and the bacterial contamination rate of the embryonic callus is reduced; the culture medium of the embryogenic callus is added with vitamin B5 to promote the generation of the embryogenic callus and shorten the time. The calcium chloride solution and the sodium chloride solution are added into the co-culture solution, so that the conversion rate of the target gene can be improved.
Detailed Description
The present invention is described in detail below with reference to specific examples, but the present invention should not be construed as being limited thereto. The test methods in the following examples, which are not specified in specific conditions, are generally conducted under conventional conditions, and the steps thereof will not be described in detail since they do not relate to the invention.
The invention provides a method for cultivating transgenic cotton with a changed fruit branch included angle, which specifically comprises the following steps:
s1, preparing embryogenic callus:
cleaning cotton seeds with sterile water, draining, soaking the cotton seeds in a seedling culture medium, performing dark culture at 20-28 ℃ for 3-4 days, and performing light culture at 20-28 ℃ for 2-3 days to obtain seedlings;
cutting the hypocotyl of the seedling into stem segments, spreading the stem segments into a callus culture medium for induction culture, subculturing for 3-4 weeks, selecting loose callus, inoculating the loose callus on the embryonic callus induction culture medium for culture, and inducing the generation of embryonic callus;
the preparation method of each liter of seedling culture medium comprises the following steps: adding 20-50mg of antibacterial agent into 1L of MS culture medium, and mixing;
the preparation method of each liter of callus culture medium is as follows: adding 20-50mg of vitamin B5 into 1L of MS culture medium, and mixing;
the method for preparing the culture medium for each liter of the embryogenic callus comprises the following steps: adding vitamin B5 20-50mg and antibacterial agent 10-20mg into YP culture medium 1L, and mixing;
the YP culture medium adopts the existing YP culture medium and has the formula as follows: 10g of yeast extract, 20g of peptone and distilled water are added until the volume is 1L;
s2, preparing the recombinant agrobacterium containing the target gene: transferring the recombinant plasmid containing the target gene into agrobacterium cells to obtain recombinant agrobacterium; wherein the recombinant agrobacterium contains a Kan gene;
s3, preparing agrobacterium tumefaciens suspension: the recombinant agrobacterium is propagated and cultured to the thallus concentration OD600Obtaining agrobacterium tumefaciens liquid at the culture temperature of 28 +/-1 ℃ and 0.4-0.8, centrifuging, and re-suspending the collected thallus with the culture liquid to obtain agrobacterium tumefaciens suspension for later use;
the preparation method of each liter of nutrient solution comprises the following steps: adding 10-20mg kanamycin into 1L MS culture medium, and mixing uniformly;
s4, co-culture transformation: soaking the embryogenic callus of S1 in Agrobacterium tumefaciens suspension of S3, dip-dyeing for 5-10min, taking out the embryogenic callus, sucking out the residual bacterial suspension on the surface of the embryogenic callus, then spreading the embryogenic callus on a co-culture solution coated with a layer of filter paper, and culturing for 24-48h in a dark room at 28 +/-1 ℃ to obtain the co-culture transformed embryogenic callus;
the preparation method of the co-culture solution per liter is as follows: adding 1 mu L of 0.1mol/L calcium chloride solution and 100 mu L of 1mmol/L sodium chloride solution into 1L of MS culture medium, and uniformly mixing;
s5, selective culture: taking out the embryogenic callus transformed by co-culture, rinsing with sterile water, sucking water, transferring to a selective culture medium, culturing at 25 + -1 deg.C for 45-60d with photoperiod, wherein subculturing every 15-20d, and selecting new embryogenic callus;
the preparation method of each liter of selection medium is as follows: adding 30-50mg kanamycin into 1L MS culture medium, and mixing uniformly;
s6, differentiation culture: and (3) placing the embryonic callus selected for the last time in a differentiation culture medium for differentiation culture until differentiation seedling emergence to obtain a transgenic seedling, then carrying out gene identification on the transgenic seedling, screening out a positive seedling, and completing construction of a transgenic plant.
The following examples are specifically included.
Example 1
The invention provides a method for cultivating transgenic cotton with a changed fruit branch included angle, which specifically comprises the following steps:
s1, preparing embryogenic callus:
cleaning cotton seeds with sterile water, draining, soaking the cotton seeds in a seedling culture medium, performing dark culture at 28 ℃ for 3 days, and performing light culture at 28 ℃ for 2 days to obtain seedlings;
cutting the hypocotyl of the seedling into 0.3-0.4cm stem segments, spreading the stem segments in a callus culture medium for induction culture, subculturing for 3 weeks, selecting loose callus, inoculating the loose callus on the embryonic callus induction culture medium for culture, and inducing the generation of embryonic callus;
the preparation method of each liter of seedling culture medium comprises the following steps: adding 20mg of antibacterial agent into 1L of MS culture medium, and mixing; the antibacterial agent is streptomycin;
the preparation method of each liter of callus culture medium is as follows: adding 20mg of vitamin B5 into 1L of MS culture medium, and mixing;
the method for preparing the culture medium for each liter of the embryogenic callus comprises the following steps: adding 20mg of vitamin B5 and 10mg of antibacterial agent into 1L of YP culture medium, and mixing; the antibacterial agent is streptomycin;
the YP culture medium adopts the existing YP culture medium and has the formula as follows: 10g of yeast extract, 20g of peptone and distilled water are added until the volume is 1L;
s2, preparing the recombinant agrobacterium containing the target gene: transferring the recombinant plasmid containing the target gene into agrobacterium cells to obtain recombinant agrobacterium; wherein the recombinant agrobacterium contains a Kan gene; the vector selected for the construction of the recombinant plasmid is a pk7WG2D vector, the agrobacterium is GV3101, and the target gene is a zero-type fruit branch gene gb-nbl (the information of the gene is described in Chinese patent CN 104561284B);
s3, preparing agrobacterium tumefaciens suspension: recombinant agrobacterium tumefaciensPerforming propagation culture to thallus concentration OD600When the culture temperature is 0.6, the culture temperature is 28 +/-1 ℃, agrobacterium liquid is obtained, the liquid is centrifuged for 10min at 1000r/min, and the collected thallus is subjected to nutrient solution resuspension to obtain agrobacterium suspension for later use;
the preparation method of each liter of nutrient solution comprises the following steps: adding 10mg kanamycin into 1L MS culture medium, and uniformly mixing;
s4, co-culture transformation: soaking the embryogenic callus of S1 in Agrobacterium tumefaciens suspension of S3, dip-dyeing for 5min, taking out the embryogenic callus, sucking out the residual bacterial suspension on the surface of the embryogenic callus, then spreading the embryogenic callus on a co-culture solution coated with a layer of filter paper (a layer of filter paper is spread in a culture dish, then the co-culture solution is contained, and finally a layer of embryogenic callus is spread), and culturing for 24h in a dark room at 28 +/-1 ℃ to obtain the co-culture transformed embryogenic callus;
the preparation method of the co-culture solution per liter is as follows: adding 1 mu L of 0.1mol/L calcium chloride solution and 100 mu L of 1mmol/L sodium chloride solution into 1L of MS culture medium, and uniformly mixing;
s4, selective culture: taking out the embryogenic callus transformed by co-culture, rinsing with sterile water, sucking water, transferring to a selective culture medium, culturing at 25 +/-1 ℃ for 45 days in a photoperiod manner, wherein each 15 days of subculture is carried out, and a new embryogenic callus is selected during each subculture; the photoperiod culture conditions were: the illumination period is 14h, and the intensity is 2000 lux;
the preparation method of each liter of selection medium is as follows: adding 30mg kanamycin into 1L MS culture medium, and mixing uniformly;
s5, differentiation culture: and (3) placing the embryonic callus selected for the last time in a differentiation culture medium for differentiation culture until differentiation seedling emergence to obtain a transgenic seedling, then carrying out gene identification on the transgenic seedling by using a molecular identification method, amplifying a target gene, screening out a positive seedling, and completing construction of a transgenic plant. Wherein the differentiation medium is MS medium.
The molecular identification method is the same as the identification method in the prior art, and the steps are complicated and are not the invention point of the invention, so the details are not repeated here.
Example 2
The invention provides a method for cultivating transgenic cotton with a changed fruit branch included angle, which specifically comprises the following steps:
s1, preparing embryogenic callus:
cleaning cotton seeds with sterile water, draining, soaking the cotton seeds in a seedling culture medium, performing dark culture at 20 ℃ for 4 days, and performing light culture at 20 ℃ for 3 days to obtain seedlings;
cutting the hypocotyl of the seedling into 0.4-0.5cm stem segments, spreading the stem segments in a callus culture medium for induction culture, subculturing for 4 weeks once, selecting loose callus, inoculating the loose callus on the embryonic callus induction culture medium for culture, and inducing the generation of embryonic callus;
the preparation method of each liter of seedling culture medium comprises the following steps: adding 50mg of antibacterial agent into 1L of MS culture medium, and mixing uniformly; the antibacterial agent is cefamycin;
the preparation method of each liter of callus culture medium is as follows: adding 50mg vitamin B5 into 1L MS culture medium, and mixing;
the method for preparing the culture medium for each liter of the embryogenic callus comprises the following steps: adding 50mg of vitamin B5 and 20mg of antibacterial agent into 1L of YP culture medium, and mixing; the antibacterial agent is cefamycin;
the YP culture medium adopts the existing YP culture medium and has the formula as follows: 10g of yeast extract, 20g of peptone and distilled water are added until the volume is 1L;
s2, preparing the recombinant agrobacterium containing the target gene: transferring the recombinant plasmid containing the target gene into agrobacterium cells to obtain recombinant agrobacterium; wherein the recombinant agrobacterium contains a Kan gene; the vector selected for the construction of the recombinant plasmid is a pk7WG2D vector, the agrobacterium is GV3101, and the target gene is cotton brachionia hainanensis gene cl1 (the information of the gene is described in a reference which indicates the fine positioning of the gene cl of the cotton brachionia hainanensis, university of southwestern 2015);
s3, preparing agrobacterium tumefaciens suspension: the recombinant agrobacterium is propagated and cultured to the thallus concentration OD600Culturing at 28 + -1 deg.C (0.4) to obtain Agrobacterium liquid, centrifuging at 1000r/min for 10min, resuspending the collected thallus with nutrient solution to obtain Agrobacterium suspension,standby;
the preparation method of each liter of nutrient solution comprises the following steps: adding 20mg kanamycin into 1L MS culture medium, and mixing uniformly;
s4, co-culture transformation: soaking the embryogenic callus of S1 in Agrobacterium tumefaciens suspension of S3, dip-dyeing for 10min, taking out the embryogenic callus, blotting the residual bacterial suspension on the surface of the embryogenic callus, then spreading the embryogenic callus on a co-culture solution coated with a layer of filter paper, and culturing for 24h in a dark room at 28 +/-1 ℃ to obtain the co-culture transformed embryogenic callus;
the preparation method of the co-culture solution per liter is as follows: adding 1 mu L of 0.1mol/L calcium chloride solution and 100 mu L of 1mmol/L sodium chloride solution into 1L of MS culture medium, and uniformly mixing;
s5, selective culture: taking out the embryogenic callus transformed by co-culture, rinsing with sterile water, sucking water, transferring to a selective culture medium, culturing at 25 + -1 deg.C for 60d with photoperiod, wherein each 20d is subcultured once, and selecting new embryogenic callus; the photoperiod culture conditions were: the illumination period is 14h, and the intensity is 2000 lux;
the preparation method of each liter of selection medium is as follows: adding 50mg kanamycin into 1L MS culture medium, and mixing uniformly;
s6, differentiation culture: and (3) placing the embryonic callus selected for the last time in a differentiation culture medium for differentiation culture until differentiation seedling emergence to obtain a transgenic seedling, then carrying out gene identification on the transgenic seedling by using a molecular identification method, amplifying a target gene, screening out a positive seedling, and completing construction of a transgenic plant. The differentiation culture medium is an MS culture medium; the molecular identification method is the same as the identification method in the prior art, and the steps are complicated and are not the invention point of the invention, so the details are not repeated here.
Example 3
The method for cultivating the transgenic cotton with the fruit branch included angle changed provided by the invention has the same operation steps as the example 2, and is different from the method in that the target gene is a cotton short fruit branch gene cl2 (the information of the gene is described in a reference document, namely Zhai flying, fine positioning of the cotton short fruit branch gene cl, university in southwest, 2015).
Example 4
The method for cultivating the transgenic cotton with the fruit branch included angle changed provided by the invention has the same operation steps as the example 2, and is different from the method in that the target gene is a cotton short fruit branch gene cl3 (the information of the gene is described in a reference document, namely Zhai flying, fine positioning of the cotton short fruit branch gene cl, university in southwest, 2015).
In view of the fact that the agrobacterium-mediated transgenic method in the prior art is complicated in steps and causes low transformation efficiency, the method takes the characteristics of cotton plants into consideration, adopts a special seedling culture medium formula, omits the step of soaking and sterilizing seeds, simplifies the seed treatment step, saves the trouble of preparing bactericides such as alcohol and the like, directly and fully cleans the seeds with sterile water, prevents the seeds from being contaminated by bacteria and improves the seedling emergence rate of the seeds by the action of an antibacterial agent in the process from culturing the seeds to the seedlings, and experiments prove that in examples 1-4, the bacterial contamination rate of the seeds is less than or equal to 1 percent, the seedling emergence rate of the seeds is more than 98 percent, and the effect is good.
The invention also adopts special callus culture medium, embryonic callus culture medium and co-culture solution; wherein, vitamin B5 is added into the callus culture medium to promote the generation of callus, shorten the time, and generate loose callus after 3-4 weeks of subculture, and the loose callus can be used for inducing the generation of embryogenic callus.
The embryonic callus culture medium is also added with an antibacterial agent, and plays a role in inhibiting bacteria in the process of generating the embryonic callus, so that the bacterial contamination rate of the embryonic callus is reduced, and experiments prove that in the examples 1-4, the bacterial contamination rate of the embryonic callus is less than or equal to 1 percent, and the calculation formula of the bacterial contamination rate of the embryonic callus is (the bacterial contamination number/the total number of the callus) multiplied by 100 percent; the culture medium of the embryogenic callus is added with vitamin B5 to promote the generation of the embryogenic callus and shorten the time.
The co-culture solution added with calcium chloride solution and sodium chloride solution can improve the transformation rate of target genes, in examples 1-4, 100 embryogenic calli are taken and used for agrobacterium-mediated transformation, and the transformation power is found to be more than or equal to 29.8% through molecular identification.
To verify the effect of sodium chloride and calcium chloride, we performed transformation experiments at different concentrations, and control experiments. 100 embryogenic calli of substantially the same size were prepared in four groups of 25 per group according to the method of example 1 and transformation experiments were performed using different methods. The specific method comprises the following steps:
experiment group
The preparation method of the co-culture solution per liter is as follows: 1 mu L of 0.1mol/L calcium chloride solution and 100 mu L of 1mmol/L sodium chloride solution are added into 1L of MS culture medium; co-culture transformation, selection culture and differentiation culture were carried out under the conditions of S3-S5 in example 1, and the success rate of transformation of the target gene was determined.
Two groups of experiments
The preparation method of the co-culture solution per liter is as follows: adding 20 mu L of 0.1mol/L calcium chloride solution and 100 mu L of 1mmol/L sodium chloride solution into 1L of MS culture medium; co-culture transformation, selection culture and differentiation culture were carried out under the conditions of S3-S5 in example 1, and the success rate of transformation of the target gene was determined.
Three groups of experiments
The preparation method of the co-culture solution per liter is as follows: 1 mu L of 0.1mol/L calcium chloride solution and 300 mu L of 1mmol/L sodium chloride solution are added into 1L of MS culture medium; co-culture transformation, selection culture and differentiation culture were carried out under the conditions of S3-S5 in example 1, and the success rate of transformation of the target gene was determined.
Control group
The co-culture solution is MS culture medium; co-culture transformation, selection culture and differentiation culture were carried out under the conditions of S3-S5 in example 1, and the success rate of transformation of the target gene was determined.
The results showed that the conversion success rate for the experimental group was 30.5%, the conversion success rate for the experimental group was 8.6%, the conversion success rate for the experimental group was 7.1%, and the conversion success rate for the control was 5.2%. The results show that the addition of sodium chloride and the addition of calcium chloride solution can improve the gene conversion rate, but the dosage is limited, the more the sodium chloride and the calcium chloride are added, the better the sodium chloride and the calcium chloride are, the dosages of the sodium chloride and the calcium chloride are respectively increased in the two experimental groups and the three experimental groups, but the conversion rate is not very high. The control group did not use sodium chloride solution and calcium chloride solution, and the conversion power was not very high.
In the above embodiment, the formula of the MS medium is shown in table 1, and first, mother liquor i, mother liquor ii, mother liquor iii, and mother liquor iv are prepared according to the formula shown in table 1, where mother liquor i is a 10-fold concentrated solution, and mother liquor ii, mother liquor iii, and mother liquor iv are 100-fold concentrated solutions, and when preparing MS medium per liter, 100mL of mother liquor i is taken, 10mL of each of mother liquor ii, mother liquor iii, and mother liquor iv is taken, and then water is added to 1L.
TABLE 1 formulation of MS culture Medium
It should be noted that the preferred embodiments and effects of the present invention have been described for the purpose of preventing redundancy, and although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (6)
1. The application of the combination of vitamin B5, calcium chloride and sodium chloride in cultivating transgenic cotton plants with changed fruit branch included angles is characterized in that the method for cultivating the transgenic cotton plants with changed fruit branch included angles comprises the following steps:
s1, preparing embryogenic callus:
cleaning cotton seeds with sterile water, draining, soaking the cotton seeds in a seedling culture medium, performing dark culture at 20-28 ℃ for 3-4 days, and performing light culture at 20-28 ℃ for 2-3 days to obtain seedlings;
cutting the hypocotyl of the seedling into stem segments, spreading the stem segments into a callus culture medium for induction culture, subculturing for 3-4 weeks, selecting loose callus, inoculating the loose callus on the embryonic callus induction culture medium for culture, and inducing the generation of embryonic callus;
the preparation method of each liter of seedling culture medium comprises the following steps: adding 20-50mg of antibacterial agent into 1L of MS culture medium, and mixing;
the preparation method of each liter of callus culture medium is as follows: adding 20-50mg of vitamin B5 into 1L of MS culture medium, and mixing;
the method for preparing the culture medium for each liter of the embryogenic callus comprises the following steps: adding vitamin B5 20-50mg and antibacterial agent 10-20mg into YP culture medium 1L, and mixing;
s2, preparing the recombinant agrobacterium containing the target gene: transferring the recombinant plasmid containing the target gene into agrobacterium cells to obtain recombinant agrobacterium; wherein the recombinant agrobacterium contains a Kan gene;
s3, preparing agrobacterium tumefaciens suspension: the recombinant agrobacterium is propagated and cultured to the thallus concentration OD600Obtaining agrobacterium tumefaciens liquid at the culture temperature of 28 +/-1 ℃ and 0.4-0.6, centrifuging, and re-suspending the collected thallus with the culture liquid to obtain agrobacterium tumefaciens suspension for later use;
the preparation method of each liter of nutrient solution comprises the following steps: adding 10-20mg kanamycin into 1L MS culture medium, and mixing uniformly;
s4, co-culture transformation: soaking the embryonic callus in the agrobacterium tumefaciens bacterial suspension, dip-dyeing for 5-10min, taking out the embryonic callus, sucking out the residual bacterial suspension on the surface of the embryonic callus, then spreading the embryonic callus on a co-culture solution coated with a layer of filter paper, and culturing for 24-48h in a dark room at the temperature of 28 +/-1 ℃ to obtain the co-culture transformed embryonic callus;
the preparation method of the co-culture solution per liter is as follows: adding 1 mu L of 0.1mol/L calcium chloride solution and 100 mu L of 1mmol/L sodium chloride solution into 1L of MS culture medium, and uniformly mixing;
s5, selective culture: taking out the embryogenic callus transformed by co-culture, rinsing with sterile water, sucking water, transferring to a selective culture medium, culturing at 25 + -1 deg.C for 45-60d with photoperiod, wherein subculturing every 15-20d, and selecting new embryogenic callus;
the preparation method of each liter of selection medium is as follows: adding 30-50mg kanamycin into 1L MS culture medium, and mixing uniformly;
s6, differentiation culture: placing the embryonic callus selected for the last time in a differentiation culture medium for differentiation culture until differentiation seedling emergence is realized to obtain transgenic seedlings, then carrying out gene identification on the transgenic seedlings, screening out positive seedlings, and completing construction of transgenic plants;
the target gene is a cotton short fruit branch gene cl1, cl2, cl3 or a zero fruit branch gene gb-nbl.
2. Use of a combination of vitamin B5, calcium chloride and sodium chloride in cultivating a transgenic cotton plant with altered fruit shoot angle as claimed in claim 1, wherein in S1 the stem section is 0.3-0.5cm in length.
3. The use of a combination of vitamin B5, calcium chloride, and sodium chloride in cultivating a transgenic cotton plant with altered fruit shoot angle as in claim 1, wherein the antibacterial agent is streptomycin or cephamycin in S1.
4. The use of the combination of vitamin B5, calcium chloride and sodium chloride in cultivating a transgenic cotton plant with a modified fruit branch angle as claimed in claim 3, wherein in S2, the vector selected for the construction of the recombinant plasmid is pk7WG2D vector, and the Agrobacterium is GV 3101.
5. The use of a combination of vitamin B5, calcium chloride, and sodium chloride in cultivating a transgenic cotton plant with altered fruit shoot angle as claimed in claim 2, wherein the photoperiod culture conditions in S5 are: illumination period 14h, intensity 2000 lux.
6. The use of a combination of vitamin B5, calcium chloride, and sodium chloride in cultivating a transgenic cotton plant with altered fruit shoot angle as in claim 2, wherein the differentiation medium is MS medium in S6.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101421404A (en) * | 2006-02-10 | 2009-04-29 | 马哈拉施特拉杂交种子有限公司 | Express the transgenosis eggplant (SOLANUM MELONGENA) of CRY1AC gene |
| CN103667336A (en) * | 2012-08-31 | 2014-03-26 | 新疆农业大学 | Cultivation method of transgenic sea island cotton |
| CN104293824A (en) * | 2013-07-15 | 2015-01-21 | 丰益(上海)生物技术研发中心有限公司 | Transformation method of Crypthecodinium cohnii |
| CN104745523A (en) * | 2015-04-03 | 2015-07-01 | 天津吉诺沃生物科技有限公司 | Separation, transformation and regeneration system for oilseed rape protoplast |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120084884A1 (en) * | 2010-10-05 | 2012-04-05 | University Of Tennessee Research Foundation | Stably transformed ferns and related methods |
-
2017
- 2017-12-29 CN CN201711486088.2A patent/CN108085335B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101421404A (en) * | 2006-02-10 | 2009-04-29 | 马哈拉施特拉杂交种子有限公司 | Express the transgenosis eggplant (SOLANUM MELONGENA) of CRY1AC gene |
| CN103667336A (en) * | 2012-08-31 | 2014-03-26 | 新疆农业大学 | Cultivation method of transgenic sea island cotton |
| CN104293824A (en) * | 2013-07-15 | 2015-01-21 | 丰益(上海)生物技术研发中心有限公司 | Transformation method of Crypthecodinium cohnii |
| CN104745523A (en) * | 2015-04-03 | 2015-07-01 | 天津吉诺沃生物科技有限公司 | Separation, transformation and regeneration system for oilseed rape protoplast |
Non-Patent Citations (3)
| Title |
|---|
| Inducing osmotic stress leads to better genetic transformation efciency in cotton (Gossypium hirsutum L.);Surendra BARPETE et al.;《Turkish Journal of Biology》;20151124(第40期);摘要，第831页第3.4节，表4 * |
| 农杆菌介导外源基因在棉花中的表达;赵俊侠等;《棉花学报》;20011231;第13卷(第3期);第146-148页 * |
| 提高植物农杆菌转化效率辅助策略研究进展;叶兴国等;《中国农业科学》;20121231;第45卷(第15期);第3007-3019页 * |
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