CN114292870B - Agrobacterium-mediated kiwi fruit transformation method - Google Patents
Agrobacterium-mediated kiwi fruit transformation method Download PDFInfo
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Abstract
The invention discloses a method for transforming kiwi fruits mediated by agrobacterium, which belongs to the technical field of kiwi fruit genetic engineering, and has the technical scheme that: the kiwi fruit transformation method comprises stable transformation of hypocotyl and instantaneous transformation of fruits; the steps of stable transformation of hypocotyl are as follows: s1, sowing kiwi seeds; s2, removing the top of the hypocotyl and moisturizing; s3, constructing a genetic transformation vector; s4, agrobacterium transformation and infection; s5, identifying transgenic plants; s6, establishing a stable transformation hypocotyl system; the steps of the instant transformation of fruits are as follows: A. constructing a genetic transformation vector; B. agrobacterium transformation; C. transferring the agrobacterium liquid into kiwi fruits; D. transient transformed fruit system establishment. The method is mainly used for establishing a stable transformation hypocotyl system and a transient transformation fruit system, and the method is used for successfully realizing stable transformation and transient transformation of fruits, so that the current situation that the genetic transformation regeneration rate of kiwi fruits is generally low is effectively changed.
Description
Technical Field
The invention relates to the technical field of kiwi fruit genetic engineering, in particular to a method for agrobacterium-mediated kiwi fruit transformation.
Background
With the continuous development of modern biotechnology, genetic improvement of horticultural crops by using transgenic means will become an important development trend. The genotypes of the fruit tree crops are highly heterozygous, the childhood is long, and the defects of large population, large workload, long time consumption and the like of the traditional breeding mode are caused.
In the breeding practice process, how to maintain the excellent characters of the parents and modify individual inferior characters by adopting genetic engineering means, so that the rapid breeding of the excellent varieties meeting the breeding standards becomes a focus of attention of breeders. The targeted editing of genes by gene overexpression and silencing expression techniques is one of the important means for achieving rapid breeding. How to increase the genetic transformation efficiency becomes a key factor for success or failure of gene editing. However, studies have shown that perennial woody plants generally suffer from the troublesome problem of low genetic transformation efficiency.
With the development of research technology, the transformation efficiency of fruit crops such as grapes, oranges, apples and the like which are difficult to transform has been remarkably improved. However, the genetic transformation efficiency of the kiwi fruits is still generally low (generally below 1%), mainly because the in vitro regeneration capacity of the kiwi fruits is poor, the transformation system is not sound, and the application of the transgenic technology on the kiwi fruits is greatly hindered.
The genetic transformation system comprises stable transformation and transient transformation, the stable transformation is mostly seen in tissue culture seedling plants, the common method is a leaf disk transformation method, but the transformation efficiency of the method applied to kiwi fruits is still low, and the main reason is that the regeneration system and the genetic transformation system of kiwi fruits are not perfect, so that the traditional system needs to be optimized and a new genetic transformation system needs to be developed.
Hypocotyls are vegetative organs with certain embryology, are relatively vigorous in division and differentiation, are relatively good genetic transmission acceptor materials, and have been widely used in horticultural crops such as apples, peaches and tomatoes, but have not been reported in kiwi fruits. In addition, transient transformation is one of the common means for verifying the gene function, and the obtained transgenic plant needs to be flowering and fruiting after the childhood period, so that the function of the verification gene in fruit development is longer, and therefore, an effective kiwi fruit transient transformation system is very necessary to be established.
In order to solve the problems, a method for agrobacterium-mediated kiwi fruit transformation is provided on the basis of the prior art.
Disclosure of Invention
The invention aims to provide a method for agrobacterium-mediated kiwi fruit transformation, which establishes a stable transformation hypocotyl system and a transient transformation fruit system, and successfully realizes the stable transformation and the transient transformation of fruits by using the method; the method has the advantages of simple operation, short period, high conversion rate and the like, can establish a high-efficiency kiwi fruit genetic transformation system, and effectively changes the current situation that the kiwi fruit genetic transformation regeneration rate is generally low.
The technical aim of the invention is realized by the following technical scheme:
a method for agrobacterium-mediated kiwi fruit transformation, which comprises the following steps:
the kiwi fruit transformation method comprises stable transformation of hypocotyl and instantaneous transformation of fruits; the steps of the stable transformation of the hypocotyl are as follows:
s1, sowing kiwi seeds: sterilizing the laminated kiwi seeds with 75% alcohol for 1min, and washing with sterile water for 3 times; then sterilizing for 5min by using 2% sodium hypochlorite, and washing for 3-5 times by using sterile water; sowing the sterilized kiwi seeds in an MS culture medium, and placing the kiwi seeds in a tissue culture room until the kiwi seeds germinate and grow; the temperature in the tissue culture chamber is 22-25 ℃, the humidity is 70%, and the illumination period is 16 hours of illumination and 8 hours of darkness;
s2, removing the top of the hypocotyl and moisturizing: after the aseptic seedlings of the kiwi fruits with two cotyledons grow in the MS culture medium, cutting off the aseptic seedlings below the growing points of the two cotyledons by using an aseptic scalpel to obtain isolated hypocotyls; pre-culturing the isolated hypocotyl in an incubator for 1-2 d;
s3, genetic transformation vector construction: construction 35S: GUS (pRI 101-GUS) over-expression vector I, constructing a GUS gene sequence on a plant expression vector pRI101 by a seamless cloning technology, and driving the expression of the GUS gene sequence by a 35S promoter;
s4, agrobacterium transformation and infection: through the 35S: agrobacterium competent cells I were transformed with GUS (pRI 101-GUS) over-expression vector I to obtain 35S with successful transformation: GUS agrobacterium I; using GUS gene as an indication mark to verify a hypocotyl stable transformation system;
the 35S: centrifuging GUS agrobacterium tumefaciens I bacterial liquid, separating out supernatant liquid part, and then re-suspending the rest part to obtain the agrobacterium tumefaciens bacterial liquid I with the bacterial liquid concentration OD600 value of 0.6-1.0 for infection; 1-2 mu L of the agrobacterium tumefaciens liquid I, 6-BA and IBA are added at the section of the isolated hypocotyl, the concentration of the 6-BA is 0.2-1.0 mg/L, and the concentration of the IBA is 0.2-1.0 mg/L; and continuing to perform the moisture-preserving culture until leaves grow out, wherein the time of the moisture-preserving culture is 1 month, and the infection is repeated for 2-3 times during the time;
s5, identifying transgenic plants: extracting DNA and RNA of the leaf in the step S4, and identifying at genome and transcription level respectively; and GUS staining the leaves, and identifying at the protein level;
s6, establishing a stable transformation hypocotyl system: obtaining the kiwi fruits with stable transformation and transformation efficiency reaching more than 80 percent.
Further: in the step S2, the in-vitro hypocotyl is preserved and pre-cultured in an incubator for 1d; the concentration of 6-BA in the step S4 is 0.8mg/L, the concentration of IBA is 0.2mg/L, the concentration OD600 value of agrobacterium tumefaciens liquid is 0.8, the agrobacterium tumefaciens competent cells adopt GV3101, and the number of repeated infection during the moisturizing culture period is 2.
Further: the agrobacteria competence described in step S4 is EHA105 or GV3101.
The invention also provides a method for the agrobacterium-mediated instantaneous transformation of kiwi fruits, which comprises the following steps:
A. construction of genetic transformation vectors: construction 35S: GUS (pRI 101-GUS) over-expression vector II;
B. agrobacterium transformation: through the 35S: GUS (pRI 101-GUS) over-expression vector II is used for transforming agrobacterium competent cells II to obtain 35S with successful transformation: GUS agrobacterium II; using GUS gene as an indication mark to verify a fruit transient transformation system; preparing an agrobacterium tumefaciens bacteria solution II with the bacteria solution concentration OD600 value of 0.4-1.0;
C. transferring the agrobacterium liquid into kiwi fruits: b, transferring the agrobacterium tumefaciens liquid II in the step B into kiwi fruits, wherein the transferring mode is injection transferring or vacuumizing transferring, and the liquid amount of the fruit bacteria injected in the injection transferring is 20 mu L, 40 mu L or 60 mu L; the vacuum pressure in the vacuum transfer is 0.06Mpa, 0.08Mpa or 0.1Mpa, and the vacuum time is 2-5 min;
D. establishment of a transient transformation fruit system: and (3) standing for 3-5 d at 28 ℃ to obtain instant transformed fruits, and determining an instant transformed fruit system through GUS dyeing and enzyme activity.
Through the technical scheme, the agrobacterium-mediated transformation method has the advantages of simple operation, short period, high transformation rate and the like, and is an effective transgenic method; after agrobacterium infects a wound of a plant into cells, T-DNA can be inserted into the plant genome and can be stably inherited to offspring by meiosis; the establishment of a kiwi genetic transformation system has important significance for kiwi transgenesis and breeding practice; the obtained transgenic plant can bloom and fruit only after the childhood, and the function of the gene in the fruit development is verified to be longer, so that the development of the kiwi instant transformation system has important practical significance for researching the gene function in the fruit development process.
In summary, the invention has the following beneficial effects:
1. the method establishes a stable transformation hypocotyl system and a transient transformation fruit system, and the stable transformation and the transient transformation of the fruits are successfully realized by using the method.
2. The method has the advantages of simple operation, short period, high conversion rate and the like, can establish a high-efficiency kiwi fruit genetic transformation system, and effectively changes the current situation that the kiwi fruit genetic transformation regeneration rate is generally low.
Drawings
FIG. 1 is a flow chart of the transformation test of the hypocotyl of kiwi fruit in example 1 of the present invention;
FIG. 2 is a diagram showing the verification of the genetic transformation transgene of the hypocotyl of kiwi fruit in example 1 of the present invention;
FIG. 3 is a graph showing the experimental results of the effect of the Agrobacterium solution and 6-BA, IBA hormone concentration on the genetic transformation efficiency of hypocotyls in examples 1 to 13 of the present invention;
FIG. 4 is a graph showing transient transformation GUS staining of kiwi fruits of example 14 and comparative example 2 of the present invention;
FIG. 5 is a comparative graph showing the genetic transformation differences of the golden fruit and Xu Xiang kiwi fruits of example 14 and comparative example 2 of the present invention;
FIG. 6 shows the effect of the bacterial liquids and the vacuuming conditions of examples 14 to 21 and comparative example 1 on the instant transformation efficiency of kiwi fruits;
FIG. 7 shows the effect of the bacterial liquids and injection conditions of examples 22 to 28 on the instant transformation efficiency of kiwi fruits.
Detailed Description
The invention is described in further detail below with reference to the attached drawings and embodiments:
example 1: a method for agrobacterium-mediated kiwi fruit transformation comprises the following steps of:
s1, sowing kiwi seeds: sterilizing the laminated red-yang kiwi seeds with 75% alcohol for 1min, and washing with sterile water for 3 times; then sterilizing for 5min by using 2% sodium hypochlorite, and washing for 3-5 times by using sterile water; sowing the sterilized kiwi seeds in an MS culture medium, and placing the kiwi seeds in a tissue culture room until the kiwi seeds germinate and grow; the temperature in the tissue culture chamber is 22-25 ℃, the humidity is 70%, and the illumination period is 16 hours of illumination and 8 hours of darkness;
s2, removing the top of the hypocotyl and moisturizing: after the aseptic seedlings of the kiwi fruits with two cotyledons grow in the MS culture medium, cutting off the aseptic seedlings by a surgical knife below the growing points of the two cotyledons to obtain in-vitro hypocotyls; preserving the isolated hypocotyl in an incubator for pre-culturing for 1d;
s3, genetic transformation vector construction: construction 35S: GUS (pRI 101-GUS) over-expression vector I, constructing a GUS gene sequence on a plant expression vector pRI101 by a seamless cloning technology, and driving the expression of the GUS gene sequence by a 35S promoter;
s4, agrobacterium transformation and infection: by 35S: GUS (pRI 101-GUS) over-expression vector I was used to transform Agrobacterium competent cells IGV 3101 to obtain 35S with successful transformation: GUS agrobacterium I; using GUS gene as an indication mark to verify a hypocotyl stable transformation system;
35S: centrifuging GUS agrobacterium tumefaciens I bacterial liquid, separating out supernatant liquid part, and then re-suspending the rest part to obtain agrobacterium tumefaciens bacterial liquid I with the bacterial liquid concentration OD600 value of 0.8 for infection; 2 mu L of agrobacterium tumefaciens bacteria solution I, 6-BA and IBA are added at the section of the hypocotyl in vitro, the concentration of 6-BA is 0.8mg/L, and the concentration of IBA is 0.2mg/L; and continuing to perform the moisturizing culture until leaves grow out, wherein the time of the moisturizing culture is 1 month, and the period is repeated for 2 times of infection;
s5, identifying transgenic plants: extracting DNA and RNA of the leaf in the step S4, and identifying at genome and transcription level respectively; GUS staining the leaves, and identifying at the protein level;
s6, establishing a stable transformation hypocotyl system: obtaining the kiwi fruits with stable transformation and transformation efficiency reaching more than 80 percent.
Example 2: the difference from example 1 is that: in step S4, agrobacterium competent cell I was EHA105.
Example 3: the difference from example 1 is that: in the step S4, the OD600 value of the bacterial solution concentration of the infection agrobacteria solution I is 0.6.
Example 4: the difference from example 1 is that: in the step S4, the OD600 value of the bacterial solution concentration of the infection agrobacteria solution I is 1.0.
Example 5: the difference from example 1 is that: in step S4, the concentration of 6-BA was 0.2mg/L.
Example 7: the difference from example 1 is that: in step S4, the concentration of 6-BA was 0.4mg/L.
Example 8: the difference from example 1 is that: in step S4, the concentration of 6-BA was 0.6mg/L.
Example 9: the difference from example 1 is that: in step S4, the concentration of 6-BA was 1.0mg/L.
Example 10: the difference from example 1 is that: in step S4, the concentration of IBA is 0.4mg/L.
Example 11: the difference from example 1 is that: in step S4, the concentration of IBA is 0.6mg/L.
Example 12: the difference from example 1 is that: in step S4, the concentration of IBA is 0.8mg/L.
Example 13: the difference from example 1 is that: in step S4, the concentration of IBA is 1.0mg/L.
Example 14: the agrobacterium mediated kiwi fruit converting process includes the following steps:
A. construction of genetic transformation vectors: construction 35S: GUS (pRI 101-GUS) over-expression vector II;
B. agrobacterium transformation: by 35S: GUS (pRI 101-GUS) over-expression vector II is used for transforming agrobacterium competent cells II GV3101 to obtain 35S of which the transformation is successful: GUS agrobacterium II; using GUS gene as an indication mark to verify a fruit transient transformation system; preparing an agrobacterium tumefaciens bacteria solution II with the bacteria solution concentration OD600 value of 0.8;
C. transferring the agrobacterium liquid into kiwi fruits: transferring the agrobacterium tumefaciens liquid II in the step B into the fruit of the Chinese goosebeery, wherein the transferring mode is vacuum-pumping transferring, immersing the Chinese goosebeery fruit into the agrobacterium tumefaciens liquid II for vacuum pumping, and the vacuum-pumping pressure in the vacuum-pumping transferring is 0.08Mpa and the vacuum-pumping time is 2min;
D. establishment of a transient transformation fruit system: and standing at 28deg.C for 3d to obtain instant transformed fruit, determining instant transformed fruit system by GUS staining and enzyme activity, and instant transforming GUS staining results of fructus Actinidiae chinensis fruit are shown in figure 1.
Example 15: the difference from example 14 is that: in step B, agrobacterium competent cell II was EHA105.
Example 16: the difference from example 14 is that: in the step B, the bacterial liquid concentration OD600 value of the agrobacterium bacterial liquid II is 0.4.
Example 17: the difference from example 14 is that: in the step B, the bacterial liquid concentration OD600 value of the agrobacterium bacterial liquid II is 0.6.
Example 18: the difference from example 14 is that: in the step B, the bacterial liquid concentration OD600 value of the agrobacterium bacterial liquid II is 1.0.
Example 19: the difference from example 14 is that: in the step C, the vacuumizing pressure is 0.06Mpa.
Example 20: the difference from example 14 is that: in the step C, the vacuumizing pressure is 0.1Mpa.
Example 21: the difference from example 14 is that: in the step C, the vacuumizing time is 3min.
Comparative example 1: the difference from example 14 is that: in the step C, the vacuumizing time is 1min.
Comparative example 2: the difference from example 14 is that: the kiwi fruit variety used in the step C is delicious kiwi fruit Xu Xiang.
Example 22: the agrobacterium mediated kiwi fruit converting process includes the following steps:
A. construction of genetic transformation vectors: construction 35S: GUS (pRI 101-GUS) over-expression vector II;
B. agrobacterium transformation: by 35S: GUS (pRI 101-GUS) over-expression vector II is used for transforming agrobacterium competent cells II GV3101 to obtain 35S of which the transformation is successful: GUS agrobacterium II; using GUS gene as an indication mark to verify a fruit transient transformation system; preparing an agrobacterium tumefaciens bacteria solution II with the bacteria solution concentration OD600 value of 0.6;
C. transferring the agrobacterium liquid into kiwi fruits: transferring the agrobacterium tumefaciens liquid II in the step B into the fruit of the Chinese goosebeery, wherein the transferring mode is injection transferring, and the liquid amount of the fruit bacteria injected in the injection transferring is 40 mu L;
D. establishment of a transient transformation fruit system: the transiently transformed fruits were obtained by standing at 28℃for 5d, and the transiently transformed fruit system was determined by GUS staining and enzyme activity.
Example 23: the difference from example 15 is that: in step B, agrobacterium competent cell II was EHA105.
Example 24: the difference from example 15 is that: in the step B, the bacterial liquid concentration OD600 value of the agrobacterium bacterial liquid II is 0.8.
Example 25: the difference from example 15 is that: in the step B, the bacterial liquid concentration OD600 value of the agrobacterium bacterial liquid II is 1.0.
Example 26: the difference from example 15 is that: in the step B, the bacterial liquid concentration OD600 value of the agrobacterium bacterial liquid II is 0.4.
Example 27: the difference from example 15 is that: in step C, the amount of the fruit bacteria to be injected was 20. Mu.L.
Example 28: the difference from example 15 is that: in step C, the amount of the fruit bacteria to be injected was 60. Mu.L.
As shown in FIG. 1, the chart is a flow chart of a kiwi fruit hypocotyl transformation test.
As shown in FIG. 2, FIG. (a) shows the identification of hypocotyl transforming DNA; (b) identifying transcript levels following hypocotyl transformation.
As shown in fig. 3, panel (a) is the effect of GV3101 and EHA105 on hypocotyl genetic transformation; panel (b) shows the effect of GV3101 concentration on hypocotyl genetic transformation; panel (c) shows the effect of 6-BA concentration on hypocotyl genetic transformation; panel (d) shows the effect of IBA concentration on the genetic transformation of the hypocotyl. The figures are mean + standard deviation (n=3), the significance analysis is based on the danken new complex polar difference test, the same lowercase letters indicating no difference at p <0.05 level. Statistically significant differences were calculated from Student's t tests (< P <0.01, < P < 0.05). Fig. 7 shows: the agrobacteria competence adopts GV3101, the bacterial liquid concentration OD600 is 0.8, 1-2 mu L of bacterial liquid is added at the section of hypocotyl, 0.8mg/L6-BA and 0.2mg/L IBA are added on the section, the infection is repeated for 2 times, and the conversion efficiency reaches more than 80%.
Fig. 4 and 5 show that: the GUS dyeing condition of the Chinese goosebeery is better than that of the delicious Chinese goosebeery Xu Xiang, the GUS enzyme activity of the Chinese goosebeery is remarkable in Gao Yuxu aroma fruits, the Chinese goosebeery is easy to instantaneously transform, and the Chinese goosebeery is suitable to be used as an instantaneous transformation material.
As shown in fig. 6, wherein graph (a) is the effect of GV3101 and EHA105 on the transient transformation of kiwi fruits (vacuuming); panel (b) is the effect of GV3101 concentration on the transient inheritance of kiwi fruits (vacuuming); FIG. (c) is the effect of vacuum pressure on the transient inheritance of kiwi fruits; graph (d) is the effect of vacuuming time on the transient inheritance of kiwi fruits. The figures are mean + standard deviation (n=3), the significance analysis is based on the danken new complex polar difference test, the same lowercase letters indicating no difference at p <0.05 level. Statistically significant differences were calculated from Student's t tests (< P <0.01, < P < 0.05).
As shown in fig. 7, wherein panel (a) is the effect of GV3101 and EHA105 on the transient transformation of kiwi fruits (injection); panel (b) is the effect of GV3101 concentration on the transient inheritance of kiwi fruits (injection); FIG. (c) shows the effect of the amount of the injected bacterial strain on the transient inheritance of kiwi fruits. The figures are mean + standard deviation (n=3), the significance analysis is based on the danken new complex polar difference test, the same lowercase letters indicating no difference at p <0.05 level. Statistically significant differences were calculated from Student's t tests (< P <0.01, < P < 0.05).
In the above examples of the present invention, the method established a stable transformed hypocotyl system and a transient transformed fruit system, with which stable transformation and transient transformation of fruits were successfully achieved; the method has the advantages of simple operation, short period, high conversion rate and the like, can establish a high-efficiency kiwi fruit genetic transformation system, and effectively changes the current situation that the kiwi fruit genetic transformation regeneration rate is generally low.
The present embodiment is only for explanation of the present invention and is not to be construed as limiting the present invention, and modifications to the present embodiment, which may not creatively contribute to the present invention as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present invention.
Claims (3)
1. A method for agrobacterium-mediated kiwi fruit transformation, which is characterized by comprising the following steps: the kiwi fruit transformation method comprises stable transformation of hypocotyl and instantaneous transformation of fruits; the steps of the stable transformation of the hypocotyl are as follows:
s1, sowing kiwi seeds: sterilizing the laminated kiwi seeds with 75% alcohol for 1min, and washing with sterile water for 3 times; then sterilizing for 5min by using 2% sodium hypochlorite, and washing for 3-5 times by using sterile water; sowing the sterilized kiwi seeds in an MS culture medium, and placing the kiwi seeds in a tissue culture room until the kiwi seeds germinate and grow; the temperature in the tissue culture chamber is 22-25 ℃, the humidity is 70%, and the illumination period is 16 hours of illumination and 8 hours of darkness;
s2, removing the top of the hypocotyl and moisturizing: after the aseptic seedlings of the kiwi fruits with two cotyledons grow in the MS culture medium, cutting off the aseptic seedlings below the growing points of the two cotyledons by using an aseptic scalpel to obtain isolated hypocotyls; pre-culturing the isolated hypocotyl in an incubator for 1-2 d;
s3, genetic transformation vector construction: construction 35S: GUS over-expression vector pRI101-GUS, constructing a GUS gene sequence on a plant expression vector pRI101 by a seamless cloning technology, and driving the expression of the GUS gene sequence by a 35S promoter;
s4, agrobacterium transformation and infection: through the 35S: GUS over-expression vector pRI101-GUS was transformed into GV3101 to obtain 35S with successful transformation: GUS agrobacterium I; using GUS gene as an indication mark to verify a hypocotyl stable transformation system;
35S: centrifuging GUS agrobacterium tumefaciens I bacterial liquid, separating out supernatant liquid part, and then re-suspending the rest part to obtain agrobacterium tumefaciens bacterial liquid I with the bacterial liquid concentration OD600 value of 0.8 for infection; 2 mu L of agrobacterium tumefaciens bacteria solution I, 6-BA and IBA are added at the section of the hypocotyl in vitro, the concentration of 6-BA is 0.8mg/L, and the concentration of IBA is 0.2mg/L; and continuing to perform the moisturizing culture until leaves grow out, wherein the time of the moisturizing culture is 1 month, and the period is repeated for 2 times of infection;
s5, identifying transgenic plants: extracting DNA and RNA of the leaf in the step S4, and identifying at genome and transcription level respectively; and GUS staining the leaves, and identifying at the protein level;
s6, establishing a stable transformation hypocotyl system: obtaining the kiwi fruits with stable transformation and transformation efficiency reaching more than 80 percent.
2. The method for agrobacterium-mediated kiwi fruit transformation of claim 1, wherein the method comprises the following steps: in step S2, the isolated hypocotyl is maintained in an incubator for a pre-culture period of 1d.
3. The method for agrobacterium-mediated kiwi fruit transformation of claim 1, wherein the method comprises the following steps: the steps of the instant transformation of the fruits are as follows:
A. construction of genetic transformation vectors: construction 35S: GUS over-expression vector pRI101-GUS;
B. agrobacterium transformation: through the 35S: GUS over-expression vector pRI101-GUS was transformed into GV3101 to obtain 35S with successful transformation: GUS agrobacterium II; using GUS gene as an indication mark to verify a fruit transient transformation system; preparing an agrobacterium tumefaciens bacteria solution II with the bacteria solution concentration OD600 value of 0.4-1.0;
C. transferring the agrobacterium liquid into kiwi fruits: b, transferring the agrobacterium tumefaciens liquid II in the step B into kiwi fruits, wherein the transferring mode is injection transferring or vacuumizing transferring, and the liquid amount of the fruit bacteria injected in the injection transferring is 20 mu L, 40 mu L or 60 mu L; the vacuum pressure in the vacuum transfer is 0.06Mpa, 0.08Mpa or 0.1Mpa, and the vacuum time is 2-5 min;
D. establishment of a transient transformation fruit system: and (3) standing for 3-5 d at 28 ℃ to obtain instant transformed fruits, and determining an instant transformed fruit system through GUS dyeing and enzyme activity.
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| CN116103334A (en) * | 2022-11-16 | 2023-05-12 | 西北农林科技大学 | Instant transformation system special for screening anti-ulcer disease gene functions of kiwi fruits |
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