CN115094083A - Construction method of agrobacterium-mediated genetic transformation system of Tamarix hispida - Google Patents
Construction method of agrobacterium-mediated genetic transformation system of Tamarix hispida Download PDFInfo
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Abstract
The invention relates to a construction method of an agrobacterium-mediated Tamarix hispida genetic transformation system, belonging to the technical field of plant genetic engineering. The invention provides a method for constructing an agrobacterium-mediated genetic transformation system of Tamarix hispida, aiming at solving the problem that the current transformation system of Tamarix hispida cannot obtain stable genetic characters. The invention optimizes the tissue propagation system of Tamarix hispida, improves the transformation efficiency, establishes a stable genetic transformation system of Tamarix hispida, lays a solid foundation for the stable transformation of Tamarix hispida by agrobacterium, and lays a solid foundation for the further research of gene functions and regulation mechanisms of Tamarix hispida and the acquisition of new transgenic lines.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a construction method of an agrobacterium-mediated genetic transformation system of Tamarix hispida.
Background
Tamarix hispida is a shrub or small tree of the genus Tamarix (Tamariacia) of the family Tamaricaceae (Tamaricaceae). The rice has the advantages of light pleasure, drought resistance, cold resistance, salt and alkali resistance, sand wasteland tolerance, developed root system, strong germination capacity and good pruning and cutting resistance. Widely distributed in gravel, gobi, clay, sandy soil, quicksand and various salinized soils with different degrees and various typical salinized soils. Has excellent stress resistance, and becomes an ideal material for researching plant stress resistance mechanism and stress resistance gene cloning.
In the research of the stress resistance mechanism and the stress resistance gene function, a high-efficiency genetic transformation system is indispensable, while in the existing research of the gene function of the tamarix setifera, only by means of a transient transformation technology, a series of physiological index analysis can be carried out after the gene is transiently overexpressed and inhibited in the tamarix setifera, but the transient transformation system cannot obtain stable genetic characters which can be subcultured. The asexual rapid propagation system based on callus differentiation solves the problem of difficult callus differentiation and regeneration in the tamarix Briggi tissue culture technology, but the system can not obtain stable genetic characters capable of subculture.
The existing setaria tamarix transformation systems can not obtain stable genetic characters and can not visually judge the growth and phenotype conditions of plants, so that a stable and efficient setaria tamarix genetic transformation method needs to be established.
Disclosure of Invention
The invention provides a construction method of an agrobacterium-mediated genetic transformation system of tamarix setifera, aiming at solving the problem that the current transformation system of tamarix setifera cannot obtain stable genetic characters.
The technical scheme of the invention is as follows:
a construction method of an agrobacterium-mediated Tamarix hispida genetic transformation system comprises the following steps:
and 3, infection: immersing the explant of the tamarix hispida pre-cultured in the step 1 in the infection solution containing the agrobacterium prepared in the step 2 under a dark condition, and completing infection under the dark condition;
and 5, screening resistant buds: and transferring the tamarix setifera explants subjected to co-culture to an adventitious bud induction differentiation medium, transferring the differentiated adventitious buds to an elongation medium for inducing the adventitious buds to elongate, transferring the adventitious buds to a rooting medium for inducing the adventitious buds to root after the adventitious buds grow to a certain length, and finally obtaining the tamarix setifera genetic transformation strain.
Further, the tamarix setifera explants in the step 1 are stem tissue of well-grown tamarix setifera tissue culture seedlings, the pre-culture is carried out for 1 day at 25 +/-2 ℃, and the pre-culture medium is 1/2MS +22.50g/L sucrose +6.50g/L agar and has the pH value of 5.8-6.0.
Further, in the step 2, the agrobacterium rhizogenes is 35S of agrobacterium rhizogenes, pBI21-GFP, and the culture method of the agrobacterium rhizogenes 35S of pBI21-GFP is as follows: selecting 35S Agrobacterium rhizogenes, namely pBI21-GFP, and inoculating the single clone in an LB liquid culture medium to culture to OD 600 2.0, then transferred to LB liquid medium for secondary culture to OD 600 0.8; the cells were collected by centrifugation at 3000g for 10 min.
Further, the staining solution in the step 2 consists of 1/2MS +22.50g/L sucrose +150 μ M acetosyringone + 0.04% (v/v) Tween 20; OD of Agrobacterium in the infection solution containing Agrobacterium 600 ＝0.8。
Further, the infection in the step 3 is infection at 90r/min for 15min at the temperature of 25 ℃.
Further, the co-culture medium in the step 4 is 1/2MS +22.50g/L sucrose +1.006-BA medium, and the co-culture is carried out for 2-3 d under the condition of 25 +/-3 ℃.
Further, the formula of the differentiation medium in step 5 is as follows: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder + 0.50-2.00 mg/L6-BA + 0.10-1.00 mg/LNAA +200mg/LTimentin +100mg/LCef +35 mg/LKan.
Further, the formula of the elongation medium in the step 5 is as follows: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder + 0.50-1.00 mg/L6-BA + 0.05-0.20 mg/LNAA +200mg/LTimentin +100mg/LCef +35 mg/LKan.
Further, the formula of the rooting medium in the step 5 is as follows: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder + 0.50-2.00 mg/LIBA +200mg/LTimentin +100mg/LCef +45 mg/LKan.
Further, the explant of tamarix hispida which is co-cultured in the step 5 is washed by a washing culture medium containing a bacteriostatic agent, and then is placed on an adventitious bud differentiation induction culture medium, wherein the washing culture medium is 1/2MS, 200mg/LTimentin and 100mg/LCef liquid culture medium, and the washing treatment time is 10-20 min.
The invention has the beneficial effects that:
the construction method of the agrobacterium-mediated Tamarix hispida genetic transformation system provided by the invention optimizes the Tamarix hispida tissue propagation system, improves the transformation efficiency, can obtain Tamarix hispida transgenic lines with stable inheritance, lays a solid foundation for stable transformation of Tamarix hispida by agrobacterium, and lays a solid foundation for further research on gene functions and regulation mechanisms of the Tamarix hispida and acquisition of new transgenic lines.
The method directly transforms the stem tissue of the Tamarix hispida without a callus stage, is simple and shortens the transformation period; the scheme of a culture medium for differentiation, extension and rooting of adventitious buds is optimized, the regeneration efficiency of the Tamarix hispida is improved, and the genetic transformation system of the Tamarix hispida is further perfected.
Drawings
FIG. 1 is a photograph of the Agrobacterium rhizogenes 35S of Tamarix hispida in example 1 showing the induction of adventitious bud formation, adventitious bud elongation and adventitious bud rooting after being infected with pBI121-GFP,
panel A is a photograph of Tamarix hispida stem segments cultured for 15d after infection with Agrobacterium rhizogenes 35S, pBI21-GFP,
panel B is a photograph of Tamarix hispida stem segments cultured for 30 days after being infected with Agrobacterium rhizogenes 35S, pBI21-GFP,
FIG. C is a photograph of Tamarix hispida stem segments cultured for 60 days after infection with Agrobacterium rhizogenes 35S, pBI21-GFP,
panel D is a photograph of stem segments of Tamarix hispida cultured for 75D after infection with Agrobacterium rhizogenes 35S, pBI21-GFP,
panel E is a photograph of Tamarix hispida induced to adventitious bud rooting culture for 7d,
FIG. F is a photograph of Tamarix hispida subjected to root-growth-inducing culture for 30 days for adventitious buds;
FIG. 2 is a photograph showing the results of gene detection electrophoresis of the genetic transformation lines of Tamarix hispida obtained in example 1;
FIG. 3 is a confocal laser micrograph of the genetic transformation line of Tamarix hispida obtained in example 1;
FIG. 4 is a diagram showing the structure of an expression unit of pBI21-GFP in a plant expression vector 35S;
FIG. 5 is a plasmid map of the plant expression vector 35S pBI 21-GFP.
Detailed Description
The technical solutions of the present invention are further described below with reference to the following examples, but the present invention is not limited thereto, and any modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention. The process equipment or apparatus not specifically mentioned in the following examples are conventional in the art, and if not specifically mentioned, the raw materials and the like used in the examples of the present invention are commercially available; unless otherwise specified, the technical means used in the examples of the present invention are conventional means well known to those skilled in the art.
Example 1
The embodiment provides a method for constructing an agrobacterium-mediated genetic transformation system of tamarix setifera, which comprises the following specific steps:
adopting sterile tissue culture seedlings of Tamarix hispida for subculture for 2 months, selecting healthy and strong stem tissue as an experimental material, cutting the stem tissue of Tamarix hispida into about 1cm stem segments by using a sterile scalpel after shearing along the stem base, paving the stem segments on a basic culture medium in a sterile operating platform, and pre-culturing for 1d under the dark condition of 25 +/-2 ℃.
The basic medium formulation for the preculture in this example was 1/2MS +22.50g/L sucrose +6.50g/L agar, pH 5.8-6.0.
the Agrobacterium tumefaciens strain used in this example was EHA105, containing the plant expression vector 35S shown in FIGS. 4 and 5, pBI21-GFP, carrying a reporter gene GFP under the control of the CaVM35S promoter, hereinafter collectively referred to as Agrobacterium rhizogenes 35S, pBI 21-GFP. Control WT was tamarix bristlegrass wild type.
The agrobacterium rhizogenes 35S comprises a pBI21-GFP bacterial liquid preparation method which comprises the following steps:
selecting 35S Agrobacterium rhizogenes, namely pBI21-GFP, and inoculating the single clone in an LB liquid culture medium to culture to OD 600 2.0, then transferred to LB liquid medium for secondary culture to OD 600 0.8; the cells were collected by centrifugation at 3000g for 10 min.
In the embodiment, the staining solution consists of 1/2MS +22.50g/L sucrose +150 mu M acetosyringone + 0.04% (v/v) Tween 20; the collected 35S of the agrobacterium rhizogenes is centrifuged and resuspended in infection liquid at 3000g for 10min by pBI21-GFP, and the OD of the agrobacterium in the infection liquid containing the agrobacterium is obtained 600 ＝0.8。
And 3, infection:
immersing the stem segments of the Tamarix hispida which is pre-cultured in the step 1 in the infection solution containing the agrobacterium prepared in the step 2 under the dark condition, and infecting at the temperature of 25 ℃ for 15min at 90r/min to finish infection;
and taking out the infected stem tissue, transferring the stem tissue to sterile filter paper, sucking excess bacterial liquid, then placing the stem tissue on a co-culture medium, co-culturing for 2d under the dark condition of 28 ℃, and taking the condition that the agrobacterium grows out of the culture medium visible to naked eyes as the best culture stopping time.
The formula of the co-culture medium in this example is: 1/2MS +22.50g/L sucrose +1.006-BA medium.
And 5, screening resistant buds:
and transferring the stem segments of the Tamarix hispida subjected to co-culture to a cleaning culture medium containing a bacteriostatic agent for cleaning for 10-20 min. The cleaning culture medium is 1/2MS +200mg/LTimentin +100mg/LCef liquid culture medium.
Transferring the cleaned stem segments of Tamarix hispida to an adventitious bud differentiation induction culture medium at a culture temperature of 25 + -2 deg.C and a light intensity of 250 μ Em -2 s -1 And the illumination time is 14 h. During which the medium was changed every 15 d.
The formulation of the differentiation medium in this example was: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder +2.00mg/L6-BA +0.10mg/LNAA +200mg/LTimentin +100mg/LCef +35 mg/LKan.
FIG. 1 is a photograph of Tamarix hispida stem segments after 15d of cultivation after infection with Agrobacterium rhizogenes 35S: (pBI 21-GFP), and a photograph of Tamarix hispida stem segments after 30d of cultivation after infection with Agrobacterium rhizogenes 35S: (pBI 21-GFP); it can be seen from the figure that significant enlargement of the wound of the stem section occurs.
Culturing for 75 days, transferring stem of Tamarix hispida to elongation culture medium for inducing adventitious bud elongation when the resistant adventitious bud grows to 0.5cm in height, culturing at 25 + -2 deg.C under illumination intensity of 250 μ Em -2 s -1 And the illumination time is 14 h. During which the medium was changed every 15 d.
The formulation of the elongation medium in this example was: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder +1.00mg/L6-BA +0.10mg/LNAA +200mg/LTimentin +100mg/LCef +35 mg/LKan.
FIG. 1, Panel C, is a photograph of a stem section of Tamarix hispida after culturing for 60 days after infecting Agrobacterium rhizogenes 35S with pBI21-GFP, and Panel D, is a photograph of a stem section of Tamarix hispida after culturing for 75 days after infecting Agrobacterium rhizogenes 35S with pBI 21-GFP; it can be seen from the figure that the resistant adventitious bud shoots, growing gradually to 1-2 cm.
Culturing for 90 days to obtain 2 cm-long resistant adventitious bud, transferring stem of Tamarix hispida to rooting medium for inducing adventitious bud to root, culturing at 25 + -2 deg.C under illumination intensity of 250 μ Em -2 s -1 And the illumination time is 14 h.
The formula of the rooting medium in the embodiment is as follows: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder +1.00mg/LIBA +200mg/LTimentin +100mg/LCef +45 mg/LKan.
FIG. 1, panel E is a photograph of Tamarix hispida subjected to adventitious bud rooting-inducing culture for 7 days, and panel F is a photograph of Tamarix hispida subjected to adventitious bud rooting-inducing culture for 30 days; it can be seen from the figure that Tamarix hispida adventitious buds gradually root until seedlings are formed.
And (4) inducing adventitious buds to grow roots and culturing for 45 days to obtain a Tamarix hispida genetic transformation strain.
Aiming at polysaccharide polyphenol plants of Tamarix Briggi, browning phenomenon is easy to occur. Therefore, in the present embodiment, carbon powder is added to the differentiation medium, the elongation culture medium and the rooting medium to prevent browning during the whole plant tissue culture process.
Example 2
The difference between this example and example 1 is that the formulation of the differentiation medium in this example is: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder +2.00mg/L6-BA +0.50mg/LNAA +200mg/LTimentin +100mg/LCef +35 mg/LKan.
Example 3
The present example differs from example 1 only in that the formula of the differentiation medium in the present example is: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder +1.00mg/L6-BA +0.10mg/LNAA +200mg/LTimentin +100mg/LCef +35 mg/LKan.
Example 4
The present example differs from example 1 only in that the formula of the differentiation medium in the present example is: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder +1.00mg/L6-BA +0.50mg/LNAA +200mg/LTimentin +100mg/LCef +35 mg/LKan.
Example 5
The present example differs from example 1 only in that the formula of the differentiation medium in the present example is: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder +0.50mg/L6-BA +0.10mg/LNAA +200mg/LTimentin +100mg/LCef +35 mg/LKan.
Example 6
The present example differs from example 1 only in that the formula of the differentiation medium in the present example is: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder +0.50mg/L6-BA +1.00mg/LNAA +200mg/LTimentin +100mg/LCef +35 mg/LKan.
Example 7
This example differs from example 1 only in that the formulation of the elongation medium in this example is: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder +0.50mg/L6-BA +0.20mg/LNAA +200mg/LTimentin +100mg/LCef +35 mg/LKan.
Example 8
This example differs from example 1 only in that the formulation of the elongation medium in this example is: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder +1.00mg/L6-BA +0.05mg/LNAA +200mg/LTimentin +100mg/LCef +35 mg/LKan.
Example 9
The present example is different from example 1 only in that the formulation of the rooting medium in the present example is: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder +0.50mg/LIBA +200mg/LTimentin +100mg/LCef +45 mg/LKan.
Example 10
The difference between this example and example 1 is only that the formulation of the rooting medium in this example is: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder +1.50mg/LIBA +200mg/LTimentin +100mg/LCef +45 mg/LKan.
Example 11
The difference between this example and example 1 is only that the formulation of the rooting medium in this example is: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder +2.00mg/LIBA +200mg/LTimentin +100mg/LCef +45 mg/LKan.
The genetic transformation lines of tamarix hispida obtained in this example 1 were subjected to genetic testing, plant DNA was extracted, and PCR detection was performed using the obtained DNA as a template. The PCR amplification system is shown in Table 1.
TABLE 1
35S, the PCR detection primer of the pBI121-GFP vector is as follows:
pBI121-F：GACCTAACAGAACTCGCCGT，
pBI121-R：GGTCTTGTAGTTGCCGTCGT。
the amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 59 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10min, wherein the amplified target band size is as follows: 870 bp.
Running the PCR product on agarose gel electrophoresis, and as can be seen from an electrophoresis gel chart shown in figure 2, the No. 1-6 are all transformation positive single plants, and the sizes of PCR gel electrophoresis strips are consistent with the size of a target strip, which indicates that an exogenous 35S gene pBI21-GFP gene is integrated into a Tamarix hispida line, and a transgenic plant is obtained by preliminary verification.
The young root tip tissue (non-root-crown part) of the genetic transformation line of Tamarix hispida obtained in example 1 is subcultured for 4 times, the young root tip tissue of the genetic transformation line of Tamarix hispida which still survives is selected and subcultured for 4 times on a selected medium, and the genetic transformation line 35S of Tamarix hispida is observed under LSM700 confocal laser microscope (Zeiss, Jena, Germany) at an excitation wavelength of 488nm and an emission wavelength of 525nm, pBI21-GFP and WT lines are photographed, fluorescence images under dark background, images in bright field and superimposed images of dark background and bright field are respectively photographed, and as shown in FIG. 3, the young root tip tissue of the clone line appears bright green as can be seen from FIG. 3, and the conclusion of PCR analysis is further confirmed, and the genetic transgenic line of Tamarix hispida is obtained.
The differentiation state and the induction rate of adventitious buds obtained by inducing adventitious buds in examples 1 to 6 were examined, and the results are shown in Table 2.
TABLE 2
| Processing number | Inductivity (%) | Adventitious bud differentiation status |
| Example 1 | 34.00±0.02 a | The cut is enlarged, the differentiation rate is high, the adventitious bud is many and is strong |
| Example 2 | 23.22±0.02 cd | The cut is enlarged, the differentiation rate is high, and the growth vigor of the adventitious bud is weak |
| Example 3 | 27.67±0.01 b | The incision is enlarged, the differentiation sign is obvious, and the adventitious bud differentiation rate is high |
| Example 4 | 25.00±0.03 bc | The cut is enlarged and the growth of the adventitious bud is weak |
| Example 5 | 27.00±0.04 b | The cut has swelling, obvious differentiation sign and high adventitious bud differentiation rate |
| Example 6 | 26.00±0.02 b | Adventitious buds are more differentiated but grow more slowly |
Note that the difference is significant in different lower case letters, P < 0.05
As can be seen from the data in Table 2, the construction method of the agrobacterium tumefaciens-mediated Tamarix hispida genetic transformation system does not involve a callus stage, is simple, can obtain a genetic transformation system rapidly, and shortens the transformation period, and the differentiation rate of the adventitious bud obtained by induced differentiation is high, and the adventitious bud is more and robust.
The elongation and survival rate of adventitious bud elongation induced by example 1 and examples 7 and 8 were examined, respectively, and the results are shown in Table 3.
TABLE 3
| Processing number | Elongation (%) | Survival rate (%) |
| Example 1 | 67.00％±0.03 a | 99.33％±0.01 a |
| Example 7 | 32.67％±0.01 c | 89.33％±0.04 ab |
| Example 8 | 51.00％±0.02 b | 98.67％±0.01 a |
Note that different little-case letters show significant difference, and P is less than 0.05
As can be seen from the data in Table 3, the method for constructing the agrobacterium-mediated genetic transformation system of Tamarix hispida has the advantages that the elongation and survival rate of adventitious buds of Tamarix hispida are improved, the transformation period is shortened, and the transformation efficiency is improved by optimizing the adventitious bud elongation culture medium scheme.
The rooting rate, the average number of roots and the average root length of the adventitious bud induced rooting in example 1 and examples 9, 10 and 11 were examined, respectively, and the results are shown in Table 4.
TABLE 4
| Processing number | Rooting percentage (%) | Average number of roots | Average root length (cm) |
| Example 1 | 98.15％±0.03 | 2.63±1.13 a | 0.36±0.10 a |
| Example 9 | 100.00％±0.00 | 2.33±1.46 ab | 0.27±0.08 b |
| Example 10 | 82.41％±0.06 | 2.42±1.17 ab | 0.28±0.11 b |
| Example 11 | 79.17％±0.00 | 2.00±0.67 b | 0.16±0.07 c |
Note that different little-case letters show significant difference, and P is less than 0.05
The data in table 4 show that the method for constructing the agrobacterium-mediated Tamarix hispida genetic transformation system improves the rooting rate and regeneration efficiency of the Tamarix hispida genetic transformation system by optimizing a culture medium scheme for adventitious bud differentiation, elongation and rooting, further improves the Tamarix hispida genetic transformation system, lays a solid foundation for stable transformation of Tamarix hispida by agrobacterium, and lays a solid foundation for further deep research on gene functions and regulation mechanisms of the Tamarix hispida and acquisition of new transgenic strains.
Claims (10)
1. A construction method of an agrobacterium-mediated Tamarix hispida genetic transformation system is characterized by comprising the following steps:
step 1, obtaining an explant of Tamarix hispida and pre-culturing the explant under dark conditions;
step 2, preparing an infection liquid containing agrobacterium tumefaciens;
and 3, infection: immersing the explant of the tamarix hispida pre-cultured in the step 1 in the infection solution containing the agrobacterium prepared in the step 2 under a dark condition, and completing infection under the dark condition;
step 4, co-culture: taking out the infected explants, placing the explants on a co-culture medium, and finishing co-culture under dark conditions;
and 5, screening resistant buds: and transferring the tamarix setifera explants subjected to co-culture to an adventitious bud induction differentiation medium, transferring the differentiated adventitious buds to an elongation medium for inducing the adventitious buds to elongate, transferring the adventitious buds to a rooting medium for inducing the adventitious buds to root after the adventitious buds grow to a certain length, and finally obtaining the tamarix setifera genetic transformation strain.
2. The method for constructing an agrobacterium-mediated genetic transformation system of Tamarix hispida according to claim 1, wherein the Tamarix hispida explant in step 1 is stem tissue of a well-grown tissue culture seedling of Tamarix hispida, the pre-culture is performed at 25 ± 2 ℃ for 1 day, and the pre-culture medium is 1/2MS +22.50g/L sucrose +6.50g/L agar and has a pH of 5.8-6.0.
3. The method for constructing the agrobacterium-mediated genetic transformation system for Tamarix hispida according to claim 1 or 2, wherein the agrobacterium is Agrobacterium rhizogenes 35S:: pBI21-GFP in step 2, and the method for culturing Agrobacterium rhizogenes 35S:: pBI21-GFP comprises the following steps: selecting 35S Agrobacterium rhizogenes, namely pBI21-GFP, and inoculating the single clone in an LB liquid culture medium to culture to OD 600 2.0, then transferred to LB liquid medium for secondary culture to OD 600 0.8; the cells were collected by centrifugation at 3000g for 10 min.
4. The method for constructing an agrobacterium-mediated genetic transformation system for Tamarix hispida according to claim 3, wherein the staining solution in the step 2 consists of 1/2MS +22.50g/L sucrose +150 μ M acetosyringone +0.04 v/v% Tween 20; OD of Agrobacterium in the infection solution containing Agrobacterium 600 ＝0.8。
5. The method for constructing an agrobacterium-mediated genetic transformation system for Tamarix hispida according to claim 4, wherein the infection in step 3 is at 25 ℃ for 15min at 90 r/min.
6. The method for constructing an agrobacterium-mediated genetic transformation system for Tamarix hispida according to claim 5, wherein the co-culture medium in step 4 is 1/2MS +22.50g/L sucrose +1.006-BA medium, and the co-culture is performed at 25 ± 3 ℃ for 2-3 days.
7. The method for constructing an agrobacterium-mediated genetic transformation system for Tamarix hispida according to claim 6, wherein the formula of the differentiation medium in step 5 is: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder + 0.50-2.00 mg/L6-BA + 0.10-1.00 mg/L NAA +200mg/L Timentin +100mg/L Cef +35 mg/Llan.
8. The method according to claim 7, wherein the elongation medium of step 5 is formulated as follows: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder + 0.50-1.00 mg/L6-BA + 0.05-0.20 mg/L NAA +200mg/L Timentin +100mg/L Cef +35 mg/Llan.
9. The method for constructing an agrobacterium-mediated genetic transformation system for Tamarix hispida according to claim 8, wherein the rooting medium in step 5 comprises the following formula: 1/2MS +22.50g/L sucrose +6.50g/L agar +1.50g/L carbon powder + 0.50-2.00 mg/L IBA +200mg/L Timentin +100mg/L Cef +45mg/L Kan.
10. The method for constructing an agrobacterium-mediated genetic transformation system of tamarix setifera according to claim 9, wherein the explants of tamarix setifera which are co-cultured in step 5 are washed with a washing medium containing bacteriostatic agents, and then placed on a culture medium for inducing adventitious bud differentiation, wherein the washing medium is 1/2MS +200mg/L Timentin +100mg/L Cef liquid medium, and the washing time is 10-20 min.
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