Genetic transformation method taking tender stem segment of verbena salicifolia as explant

Abstract

The invention discloses a genetic transformation method taking a tender stem segment of verbena salicifolia as an explant, and relates to the field of plant genetic transformation, and the genetic transformation method specifically comprises the following steps: S1, preparing a verbena salicifolia aseptic seedling; s2, pre-culturing the explant; s3, preparing an agrobacterium infection solution; s4, performing explant infection and co-culture; s5, screening and culturing; s6, strong seedling culture; and S7, rooting culture. According to the method disclosed by the invention, the tender stem segment of the verbena salicifolia is taken as the explant, the agrobacterium tumefaciens-mediated verbena salicifolia genetic transformation system is established for the first time, and technical reference is provided for the research of molecular breeding of the verbena salicifolia.

Description

Technical Field

[0001] The invention relates to the field of plant genetic transformation, and in particular to a genetic transformation method using young and tender stem segments of Verbena officinalis as explants. Background Art

[0002] Verbena Verbena bonariensis ) is Verbenaceae ( Verbenaceae ), Verbena spp. Verbena ) is a perennial herbaceous plant, native to Brazil and Argentina in South America. It is a perennial herbaceous flower plant, also known as dragon bud grass, iron whip, wild thorn grass, wind neck grass, blood-removing grass and swallowtail grass. The whole plant is covered with white cilia, the stem is square, the leaf sequence is cross-opposite, and the shape is willow-leaf-shaped. The initial leaves are oval in shape with slightly notched edges. After the flower stems grow taller, the shape of the leaves becomes slender willow leaves, and the edges are still notched. The shape of the flowers is like a small tube, attached to the top of the stem, gathering and dispersing catkins, and the color is blue-purple or lavender. The flowering period is from May to September. The plant grows luxuriantly and can be as high as 1.5 meters after flowering. Although the plant has a high stem, its stalk is strong and not easy to fall over. Willow-leaf verbena is suitable for growing in sunny and warm areas. It is also drought-resistant and has strong soil adaptability. Willow-leaf verbena is favored by gardeners for its graceful posture, soft color and long flowering period. It is an ideal plant for group planting landscape construction. At present, a large number of verbena have been planted in many scenic spots such as the Yejiping Starry Sky Flower Sea in the Yeyuhai Scenic Area in Shuicheng District, Liupanshui City, Guizhou Province, the Gaopo Yunding Flower Sea in Huaxi District, Guiyang City, and the Yongle Flower Sea in Shuitang Village, Yongle Township, Nanming District, which have extremely high ornamental value.

[0003] In recent years, research on verbena has mainly focused on seed germination, seedling growth, and cultivation management techniques, while research on genetic transformation and variety improvement is very rare. For example, low temperatures in winter will accelerate the degradation of verbena and affect the seedling effect, leading to an increase in cultivation and seedling costs. To address the above problems, the most effective means is to use biotechnology to genetically improve verbena. However, since there is still a lack of a genetic transformation system for verbena, in-depth research on verbena is severely restricted. Therefore, the establishment of a genetic transformation system for verbena is of great significance to its variety improvement, promotion, and application. Summary of the invention

[0004] In view of the above problems, the present invention provides a genetic transformation method using young stem segments of Verbena officinalis as explants, filling the gap in the genetic transformation system of Verbena officinalis. Using young stem segments of sterile seedlings of Verbena officinalis as materials, a plant expression vector is constructed, and transgenic plants of Verbena officinalis are obtained by tissue culture technology, providing a technical reference for subsequent research on genetic transformation and molecular breeding of Verbena officinalis.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a genetic transformation method using young stem segments of Verbena officinalis as explants, which specifically comprises the following steps: S1: Preparation of sterile seedlings of Verbena officinalis: Select plump and uncontaminated Verbena officinalis seeds and sterilize them; inoculate the sterilized Verbena officinalis seeds into a germination induction medium for germination induction culture to obtain sterile seedlings of Verbena officinalis; S2: Explant pre-culture: 1 cm young stem segments were cut from sterile Verbena seedlings as explants, and the explants were inoculated into pre-culture medium for pre-culture; S3: Preparation of Agrobacterium infection solution: Pick a single Agrobacterium colony containing the target gene from the YEP solid medium, add it to 5 mL YEP liquid medium containing Kan and Rif, and shake and culture overnight; take 3 mL of overnight culture solution and add it to 50 mL of the above liquid medium and continue shaking and culturing until the OD value of the solution reaches 0. 600 When the concentration is 0.4-0.6, pour the bacterial solution into a 50mL centrifuge tube, centrifuge, and discard the supernatant; resuspend the bacteria with an equal volume of MS solution, which is the Agrobacterium infection solution; S4: Explant infection and co-cultivation: The explants pre-cultured for 2 days were immersed in the prepared Agrobacterium infection solution, infected for 8 minutes, taken out and placed on sterile filter paper to dry, and inoculated into the co-culture medium and cultured in the dark for 2 days; S5: screening culture: the explants after co-cultivation are transferred to screening medium and cultured until resistant callus grows and resistant buds differentiate; S6: Seedling cultivation: When the resistant buds extend to 2-3 cm, cut them from the base and inoculate them into seedling cultivation medium for seedling cultivation; S7: Rooting culture: The Verbena seedlings that have undergone seedling strengthening culture are transferred into rooting medium for rooting culture.

[0006] Furthermore, in step S1, the method for seed disinfection is: soak the seeds in pure water for 2 to 3 days in advance, put them in a clean bench, soak them in 10% H2O2 for 6 minutes, rinse them with sterile water, then disinfect them with 75% alcohol for 30 seconds, rinse the alcohol with sterile water, disinfect them with 20% sodium hypochlorite for 40 minutes, and rinse the sodium hypochlorite with sterile water; spread the disinfected seeds on sterilized absorbent paper, absorb the moisture on the surface of the seeds, and then inoculate them into germination induction medium to obtain sterile verbena seedlings, the composition of the germination induction medium is: 4.74g / LMS+30g / L sucrose+7g / L agar+1.5mg / LGA3, pH=6.0, and the culture time is 20 days.

[0007] Furthermore, in step S2, the preculture medium composition is: 4.74 g / L MS+30 g / L sucrose+7 g / L agar+1.5 mg / L TDZ+0.02 mg / L NAA, pH=6.0, and cultured in the dark in a 28° C. incubator for 2 days.

[0008] Furthermore, in step S3, the components of YEP solid culture medium are: 10g / L peptone + 10g / L yeast extract + 5g / LNaCl + 15g / L agar + 100mg / LKan + 10mg / LRif, pH=7.2; the components of YEP liquid culture medium are: 10g / L peptone + 10g / L yeast extract + 5g / LNaCl + 100mg / LKan + 10mg / LRif, PH=7.2; the components of MS solution are: 4.74g / LMS + 30g / L sucrose + 20mg / LAS, pH=6.0.

[0009] Furthermore, in step S4, the specific method of explant infection and co-cultivation is: in a clean bench, place the explant pre-cultured for 2 days in a sterilized culture dish, pour the Agrobacterium infection solution, soak for 8 minutes, use sterilized tweezers to take out the explant and place it on sterilized absorbent paper to absorb water; then place the explant neatly on the co-culture medium, the co-culture medium composition is: 4.74g / LMS+30g / L sucrose+7g / L agar+1.5mg / LTDZ+0.02mg / LNAA+20mg / LAS, pH=6.0, and place it in a 28°C incubator for dark incubation for 2 days.

[0010] Furthermore, in step S5, the screening culture medium composition is: 4.74g / L MS+30g / L sucrose+7g / L agar+1.5mg / L TDZ+0.02mg / L NAA+100mg / L Tim+2mg / L Hyg, pH=6.0, the culture environment is 28°C, and the light intensity is 12h / d.

[0011] Furthermore, in step S6, the composition of the seedling culture medium is: 4.74g / LMS+30g / L sucrose+7g / L agar+0.1mg / L NAA+100mg / L Tim, pH=6.0, the culture environment is 28°C, and the light intensity is 12h / d.

[0012] Furthermore, in step S7, the rooting medium composition is: 4.74 g / LMS+30 g / L sucrose+7 g / L agar+0.02 mg / L NAA+100 mg / L Tim, pH=6.0, the culture environment is 28° C., and the light intensity is 12 h / d.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The invention adopts young stem segments of sterile seedlings of Verbena officinalis with good growth as explants, infects with Agrobacterium liquid, and then uses tissue culture technology to induce callus differentiation to form regenerated buds, thereby establishing a stable Agrobacterium-mediated genetic transformation system of Verbena officinalis. The invention establishes a genetic transformation system method using young stem segments of Verbena officinalis as explants, fills the gap in genetic transformation of Verbena officinalis, has high transformation efficiency and short cycle, and lays a foundation for variety improvement and application of Verbena officinalis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The diagram shows the growth of callus induced and bud differentiation cultured at different times of young and tender stem segments of Verbena officinalis according to the present invention; in the diagram: A. co-culture of young and tender stem segments; B. screening and culture of young and tender stem segments on the 0th day; C. growth of bud differentiation on the 20th day of screening and culture of young and tender stem segments; D. growth of bud differentiation on the 35th day of screening and culture of young and tender stem segments; E. the state of adventitious bud differentiation of explant callus; F. growth of adventitious buds on the 40th day; the scale bars in the diagrams are all 1 cm.

[0015] Figure 2 The figures show the growth of resistant bud seedlings, rooting and transplanting of resistant plants of the present invention; in the figures: A. resistant bud seedlings culture; B. resistant bud rooting culture; C. 30d growth of resistant bud rooting culture; D. resistant plant transplanting culture; the scale bars in the figures are all 1 cm.

[0016] Figure 3 The results of GUS staining of the target gene of Verbena regenerated resistant plants by the method of the present invention; in the figure: A. GUS staining results of wild-type Verbena leaves (left) and transgenic Verbena leaves (right); B. GUS staining results of wild-type Verbena roots (left) and transgenic Verbena roots (right); the scale bars in the figures are all 1 cm. DETAILED DESCRIPTION

[0017] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0018] The instruments, reagents, materials, etc. involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0019] Example 1 1. Materials The Verbena seeds used in this example were purchased from Benary Seed Company in Germany, and were germinated under sterile conditions to obtain sterile seedlings. Young stem segments were then taken from the sterile seedlings as explants to establish a genetic transformation system.

[0020] 2. Methods 2.1 Genetic transformation vectors of Verbena officinalis The plasmid was extracted from the pCAMBIA1301-35S-GUS recombinant vector (provided by Wuhan Transduction Biological Laboratory Co., Ltd.) containing the GUS marker gene and then introduced into Agrobacterium GV3101 competent cells. After colony PCR detection and sequencing, the positive strains were saved for subsequent genetic transformation of Verbena willowii.

[0021] 2.2 Genetic transformation of Verbena officinalis by Agrobacterium tumefaciens (1) Pretreatment and disinfection methods of Verbena seeds Soak the seeds in pure water for 2 to 3 days in advance, put them in a clean bench, soak them in 10% H2O2 for 6 minutes, rinse them with sterile water several times, then disinfect them with 75% alcohol for 30 seconds, rinse the alcohol with sterile water, disinfect them with 20% sodium hypochlorite for 40 minutes, and rinse the sodium hypochlorite with sterile water; spread the disinfected seeds on sterilized absorbent paper, dry the moisture on the surface of the seeds, and inoculate them in a germination induction medium containing 1.5 mg / L GA3 for 20 days to obtain sterile seedlings of Verbena willowii. The leaves of the Verbena willowii seedlings can be used in the next experiment. The composition of the germination induction medium is: 4.74g / LMS+30g / L sucrose+7g / L agar+1.5mg / LGA3, pH=6.0.

[0022] (2) Explant pre-culture method In the clean bench, use sterilized tweezers and scissors to cut 1 cm tender stem segments from sterile seedlings of Verbena as explants, and inoculate the explants into the pre-culture medium at 28°C and culture in the dark for 2 days. The pre-culture medium composition is: 4.74 g / LMS + 30 g / L sucrose + 7 g / L agar + 1.5 mg / L TDZ + 0.02 mg / L NAA, pH = 6.0.

[0023] (3) Preparation of Agrobacterium infection solution First, a single colony of Agrobacterium containing the GUS gene was picked from the YEP solid medium containing Rif and Kan antibiotics, and added to 5 mL of YEP liquid medium containing Kan and Rif for overnight culture with shaking; 3 mL of overnight cultured bacterial solution was added to 50 mL of the above liquid medium and continued to be cultured with shaking until the bacterial solution OD reached 0. 600The pH value is 0.4-0.6, the bacterial solution is poured into a 50mL centrifuge tube, centrifuged, and the supernatant is discarded; finally, the bacteria are resuspended with an equal volume of MS solution, which is the Agrobacterium infection solution. The components of YEP solid culture medium are: 10g / L peptone + 10g / L yeast extract + 5g / L NaCl + 15g / L agar + 100mg / L Kan + 10mg / LRif, pH = 7.2; the components of YEP liquid culture medium are: 10g / L peptone + 10g / L yeast extract + 5g / L NaCl + 100mg / L Kan + 10mg / LRif, PH = 7.2; the components of MS solution are: 4.74g / L MS + 30g / L sucrose + 20mg / LAS, pH = 6.0.

[0024] (4) Explant infection with Agrobacterium and co-cultivation Under a sterile environment, the well-grown explant stem segments after pre-culture were added to the Agrobacterium infection solution of the plant expression vector pCAMBIA1301-35S-GUS for infection (OD 600 = about 0.5). The sterile stem segments were co-cultured with the Agrobacterium infection solution for 8 minutes. After the infection was completed, the explants were taken out by tweezers sterilized by burning with an alcohol lamp and placed on sterilized absorbent paper to dry the infection solution on the surface. The explants were then neatly placed and inoculated on the co-culture medium and cultured in the dark at 28°C for 2 days. The growth of the young stem segments in the co-culture was observed. Figure 1 A, the composition of co-culture medium is: 4.74g / LMS+30g / L sucrose+7g / L agar+1.5mg / LTDZ+0.02mg / LNAA+20mg / LAS, pH=6.0.

[0025] (5) Screening and cultivation After 2 days of co-cultivation, the explants were transferred to the screening medium in the clean bench for culture. The culture was carried out at 28°C with a light intensity of 12 h / d. During the culture process, the screening medium was replaced once a week. The growth of the screening culture on day 0 was observed. Figure 1 B, after about 8 days of culture, resistant callus tissue began to form, and after about 20 days of culture, the resistant callus gradually differentiated into adventitious buds (resistant buds). Figure 1 C, the growth trend of bud differentiation on the 35th day of screening culture Figure 1 D, the explant callus begins to differentiate into adventitious buds. Figure 1 E, the growth of adventitious buds on the 40th day Figure 1 F. The composition of the screening medium is: 4.74g / L MS + 30g / L sucrose + 7g / L agar + 1.5mg / L TDZ + 0.02mg / L NAA + 100mg / L Tim + 2mg / L Hyg, pH = 6.0.

[0026] (6) Cultivation of strong seedlings When the adventitious buds (resistant buds) differentiated from the resistant callus in the screening medium grow to 2-3 cm, cut the resistant buds from the base with a sterilized blade under a sterile environment, transfer and inoculate them into the seedling medium, and culture them for 7 days at 28℃ with 12h / d of light. Figure 2 A. The composition of the seedling medium is: 4.74g / L MS + 30g / L sucrose + 7g / L agar + 0.1mg / L NAA + 100 mg / L Tim, pH = 6.0.

[0027] (7) Rooting culture After the seedlings are strengthened, the resistant seedlings are transferred to the rooting medium and cultured at 28℃ with 12h / d light to about 8cm. Figure 2 B, 30 days of rooting culture of resistant buds Figure 2 C. After 2 days of hardening in the greenhouse, transplant the seedlings into nutrient soil for potted cultivation. Figure 2 D. The composition of the rooting medium is: 4.74g / L MS + 30g / L sucrose + 7g / L agar + 0.02mg / L NAA + 100mg / L Tim, pH = 6.0.

[0028] 3. Identification of positive transgenic plants Since the GUS gene of the plant expression vector is a marker gene, the resistant seedlings were stained using a GUS staining kit. Figure 3 A) and roots ( Figure 3 B) Obvious blue trait. A total of 60 resistant seedlings were tested, and the results showed that 9 resistant seedlings were stained blue, which was positive, and the transformation efficiency was 15%.

[0029] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A genetic transformation method using young stem segments of Verbena officinalis as explants, characterized in that: The specific steps include: S1: Preparation of sterile seedlings of Verbena officinalis: Select plump and uncontaminated Verbena officinalis seeds and sterilize them; inoculate the sterilized Verbena officinalis seeds into a germination induction medium for germination induction culture to obtain sterile seedlings of Verbena officinalis; S2: Explant pre-culture: 1 cm young stem segments were cut from sterile Verbena seedlings as explants, and the explants were inoculated into pre-culture medium for pre-culture; S3: Preparation of Agrobacterium infection solution: Pick a single Agrobacterium colony containing the target gene from the YEP solid medium, add it to 5 mL YEP liquid medium containing Kan and Rif, and shake and culture overnight; take 3 mL of overnight culture solution and add it to 50 mL of the above liquid medium and continue shaking and culturing until the OD value of the solution reaches 0. 600 When the concentration is 0.4-0.6, pour the bacterial solution into a 50mL centrifuge tube, centrifuge, and discard the supernatant; resuspend the bacteria with an equal volume of MS solution, which is the Agrobacterium infection solution; S4: Explant infection and co-cultivation: The explants pre-cultured for 2 days were immersed in the prepared Agrobacterium infection solution, infected for 8 minutes, taken out and placed on sterile filter paper to dry, and inoculated into the co-culture medium and cultured in the dark for 2 days; S5: Screening culture: the explants after co-cultivation are transferred to the screening medium and cultured until resistant callus grows and resistant buds differentiate; S6: Seedling cultivation: When the resistant buds extend to 2-3 cm, cut them from the base and inoculate them into seedling cultivation medium for seedling cultivation; S7: Rooting culture: The Verbena seedlings that have undergone seedling strengthening culture are transferred into rooting medium for rooting culture.

2. The genetic transformation method according to claim 1, wherein the young stem segments of Verbena officinalis are used as explants, is characterized in that: In the step S1, the method for seed disinfection is: soak the seeds in pure water for 2 to 3 days in advance, put them in a clean bench, soak them in 10% H2O2 for 6 minutes, rinse them with sterile water, then disinfect them with 75% alcohol for 30 seconds, rinse the alcohol with sterile water, disinfect them with 20% sodium hypochlorite for 40 minutes, and rinse the sodium hypochlorite with sterile water; spread the disinfected seeds on sterilized absorbent paper, absorb the moisture on the surface of the seeds, and then inoculate them into a germination induction medium to obtain sterile verbena seedlings, the composition of the germination induction medium is: 4.74g / LMS+30g / L sucrose+7g / L agar+1.5mg / LGA3, pH=6.0, and the culture time is 20 days.

3. The genetic transformation method using young stem segments of Verbena officinalis as explants according to claim 1, characterized in that: In step S2, the preculture medium composition is: 4.74 g / L MS+30 g / L sucrose+7 g / L agar+1.5 mg / L TDZ+0.02 mg / L NAA, pH=6.0, and is cultured in the dark in a 28° C. incubator for 2 days.

4. The genetic transformation method using young stem segments of Verbena officinalis as explants according to claim 1, characterized in that: In step S3, the components of YEP solid culture medium are: 10g / L peptone + 10g / L yeast extract + 5g / L NaCl + 15g / L agar + 100mg / L Kan + 10mg / LRif, pH=7.2; the components of YEP liquid culture medium are: 10g / L peptone + 10g / L yeast extract + 5g / L NaCl + 100mg / L Kan + 10mg / LRif, PH=7.2; the components of MS solution are: 4.74g / L MS + 30g / L sucrose + 20mg / LAS, pH=6.

0.

5. The genetic transformation method using young stem segments of Verbena officinalis as explants according to claim 1, characterized in that: In step S4, the specific method of explant infection and co-cultivation is: in a clean bench, the explant pre-cultured for 2 days is placed in a sterilized culture dish, the Agrobacterium infection solution is poured in, and the explant is soaked for 8 minutes, and the explant is taken out with sterilized tweezers and placed on sterilized absorbent paper to absorb the moisture; then the explant is neatly placed on the co-culture medium, the co-culture medium composition is: 4.74g / LMS+30g / L sucrose+7g / L agar+1.5mg / LTDZ+0.02mg / LNAA+20mg / LAS, pH=6.0, and placed in a 28°C incubator for dark incubation for 2 days.

6. The genetic transformation method using young stem segments of Verbena officinalis as explants according to claim 1, characterized in that: In step S5, the screening culture medium composition is: 4.74g / L MS+30g / L sucrose+7g / L agar+1.5mg / LTDZ+0.02mg / L NAA+100mg / L Tim+2mg / L Hyg, pH=6.0, the culture environment is 28°C, and the light intensity is 12h / d.

7. The genetic transformation method using young stem segments of Verbena officinalis as explants according to claim 1, characterized in that: In step S6, the composition of the seedling medium is: 4.74g / LMS+30g / L sucrose+7g / L agar+0.1mg / L NAA+100mg / L Tim, pH=6.0, the culture environment is 28°C, and the light intensity is 12h / d.

8. The genetic transformation method using young stem segments of Verbena officinalis as explants according to claim 1, characterized in that: In step S7, the rooting medium composition is: 4.74 g / LMS+30 g / L sucrose+7 g / L agar+0.02 mg / L NAA+100 mg / L Tim, pH=6.0, the culture environment is 28° C., and the light intensity is 12 h / d.

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