A method for obtaining transgenic plants of Myriophyllum spicatum

Abstract

The present invention discloses a method for obtaining transgenic Myriophyllum var. tumefaciens plants, comprising the steps of obtaining clustered buds from Myriophyllum var. tumefaciens, co-cultivating with Agrobacterium tumefaciens, screening for resistant buds, and rooting and culturing resistant seedlings. The method directly induces a large number of small bud clusters from stem segments, avoiding the process of callus induction and redifferentiation, significantly saving time and improving the efficiency of rapid propagation. Using a bacterial liquid co-cultivation instead of a solid co-cultivation ensures sufficient contact between the Agrobacterium and the explant, thereby improving transformation efficiency.

Description

Technical Field

[0001] This invention belongs to the technical field of transgenic Myriophyllum spicatum, specifically relating to a method for obtaining transgenic Myriophyllum spicatum plants. Background Technology

[0002] Myriohpyllum verticillatum, a plant belonging to the genus Myriohpyllum in the family Myriohpylaceae, is an aquatic herb widely distributed throughout the world. Myriohpyllum verticillatum not only serves as food, refuge, and spawning grounds in fish, shrimp, and crab farming, but it is also a water purification species and a pioneer species for vegetation restoration in current lake ecological restoration projects. Furthermore, due to its high crude protein content, Myriohpyllum verticillatum has the potential to be processed into silage for poultry and livestock feed.

[0003] Transgenic technology refers to the use of molecular biology methods to introduce beneficial genes from different or the same organisms into a target organism, thereby adding new functional characteristics to the target organism's original genetic traits, resulting in new varieties and the production of new products. The development of new transgenic crop varieties has shifted from first-generation products such as insect resistance and herbicide resistance to second-generation products that improve nutritional quality and increase yield. Currently, there are three common methods for plant transgenic transformation: Agrobacterium-mediated transformation, gene gun-mediated transformation, and pollen tube pathway transformation. Agrobacterium-mediated transformation is currently the most common method, but it has a limiting factor: the plants to be transformed must first undergo aseptic culture to induce callus tissue, followed by Agrobacterium infection and co-culture, then selection of resistant callus and induction of plant regeneration and rooting. This results in a low transformation rate and is not applicable to all plants. Gene gun-mediated transformation is also widely used in current transgenic research, but this method is costly, has a high chimerism rate, and poor genetic stability. The pollen tube pathway transformation has a wider range of applications than the above two methods, but its transformation technology is imperfect, lacks systematic research on the transformation mechanism, and is limited to the flowering period. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art and provide a method for obtaining transgenic plants of Myriophyllum spicatum that is simple and quick to operate, has a short operation cycle, high conversion efficiency, and low cost.

[0005] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0006] The method for obtaining transgenic plants of Myriophyllum spicatum includes the following steps:

[0007] (1) Obtaining clustered shoots of Myriophyllum spicatum: Select stem segments of superior Myriophyllum spicatum plants as explants. After disinfection, the explants are cultured on MS agar solid medium supplemented with 6-BA, KT, IAA, and NAA for 20-30 days to induce explants with clustered shoots. The concentrations of 6-BA, KT, IAA, and NAA in MS agar solid medium are 1-2 mg / L, 0.1-0.5 mg / L, 0.1-0.5 mg / L, and 0.1-1 mg / L, respectively, preferably 1 mg / L, 0.5 mg / L, 0.3 mg / L, and 0.5 mg / L.

[0008] (2) Co-culture of Agrobacterium tumefaciens: The stem segments of Myriophyllum sp. cut from explants with small bud clusters were inoculated with Agrobacterium tumefaciens bacterial solution and then co-cultured.

[0009] (3) Screening of resistant shoots: Stem segments of *Myriophyllum spicatum* after co-culture were inoculated into MS screening medium supplemented with 6-BA, KT, IAA, NAA, cephalosporin, carbenicillin, and hygromycin to screen and obtain resistant shoots. The positive shoot emergence rate reached 8%–15%. The concentrations of 6-BA, KT, IAA, NAA, cephalosporin, carbenicillin, and hygromycin in the MS screening medium were 1–2 mg / L, 0.1–0.5 mg / L, and 0.5 mg / L, respectively.

[0010] 0.1–0.5 mg / L, 0.1–1 mg / L, 500 mg / L, 400 mg / L, 50 mg / L; preferably 1 mg / L, 0.5 mg / L,

[0011] 0.3mg / L, 0.5mg / L, 500mg / L, 400mg / L, 50mg / L;

[0012] (4) Rooting culture of resistant seedlings: The resistant seedlings were inoculated into MS agar solid medium supplemented with NAA and IAA for rooting culture; the concentrations of NAA and IAA in MS agar solid medium were 0.1-1.5 mg / L and 0.1-1 mg / L, respectively, preferably 0.5 mg / L and 0.5 mg / L;

[0013] (5) Transplant the resistant rooted seedlings from step (4) to a greenhouse, then transplant them to the field and test them to screen out the successfully transgenic plants.

[0014] Preferably, the explant disinfection treatment in step (1) involves cutting the stem of the Myriophyllum spicatum plant into a length of 2-4 cm, cleaning it with laundry detergent, washing it 5-7 times with sterile water on a sterile operating table, soaking it in 70% alcohol for 30 seconds, sterilizing it in 0.1% mercuric chloride solution for 10-15 minutes, rinsing it with sterile water 5-6 times, and then setting it aside for use.

[0015] Preferably, step (1) further includes a subculture step of the induced explants with clustered buds: the old stems are cut into 2-4 cm pieces and the clustered buds are cut into 0.5 cm × 0.5 cm pieces and then transferred into the MS agar solid medium containing BA1, KT, IAA and NAA as described in step (1), and subcultured once every 28-30 days.

[0016] Preferably, the co-culture steps of Agrobacterium tumefaciens in step (2) are as follows:

[0017] ① Prepare Agrobacterium tumefaciens bacterial suspension three days in advance: After sterilizing LB solid medium, cool it to 60℃, then add Kan, Chl, and Rif, with concentrations of 100 mg / L, 34 mg / L, and 100 mg / L respectively in the LB solid medium; spread the Agrobacterium tumefaciens bacterial suspension on LB solid medium containing Kan, Chl, and Rif, and incubate at 28℃ upside down for 2-3 days. Wash the cultured LB plates into MS liquid co-medium containing As, and adjust OD600 to 0.5, with the As concentration in the MS liquid co-medium being 0.1 mM; thus obtaining the Agrobacterium tumefaciens bacterial suspension.

[0018] ② Agrobacterium-mediated transformation: The explants with clustered buds after subculture in step (3) were cut into 2-4 cm stem segments and transferred to Agrobacterium tumefaciens solution for co-culture for 3 days. The co-culture conditions were dark culture at 25℃-28℃.

[0019] Preferably, the screening and culturing time in step (3) is 10 to 15 days.

[0020] Preferably, the rooting culture time in step (4) is 15 to 20 days, and the plants are transplanted after rooting.

[0021] Preferably, the culture temperature in steps (1), (3) and (4) is 24±2℃, and the culture is carried out in a low light environment of 1000~2000lx for 10~12h of light per day.

[0022] Preferably, step (5) involves loosening the cap of the culture bottle containing the resistant seedlings from step (4) and opening it by 1 / 4, placing it at room temperature for 2 days, then opening the cap completely and placing it at room temperature (18-25℃) for another 2 days, then rinsing off the culture medium with running water, culturing it in tap water at room temperature for 5-7 days, and finally transplanting it into the paddy field. After 20-30 days, samples are taken for preliminary PCR testing, and the success rate of transgenic transplantation in positive seedlings is over 90%.

[0023] More preferably, the method further includes a step of performing real-time quantitative PCR detection on the successfully transgenic plants that have undergone preliminary PCR testing, and finally obtaining transgenic plants.

[0024] The present invention will be further described below:

[0025] The culture medium formula for inducing small bud clusters from stem segments in this invention was obtained through extensive exploration and experimentation. Unlike the existing technology where callus tissue is first induced from Myriophyllum sp. explants and then transferred to the culture medium for differentiation and proliferation, this invention directly induces a large number of small bud clusters from stem segments, which significantly saves time and improves the efficiency of rapid propagation.

[0026] Based on the characteristics of Myriophyllum spicatum as an aquatic plant, this invention utilizes the high water absorption capacity of its stem segments and boldly adopts a method of co-culturing the stem segments in liquid Agrobacterium tumefaciens solution. This allows Agrobacterium tumefaciens to come into close contact with the cut Myriophyllum spicatum stem segments, thereby achieving high-efficiency transformation. It was found that both the infection and transformation efficiencies were ideal.

[0027] The culture medium formula obtained by the inventors through extensive exploration and experimentation is suitable not only for inducing small shoot clusters from stem segments in step (1) of this invention, but also for stem segment culture after Agrobacterium transfection, because the physiological and biochemical characteristics of stem segments will be changed after Agrobacterium transformation. Step (3) of this invention directly induces small shoot clusters or seedlings from stem segments without first inducing callus tissue and then differentiating, which also saves time and improves the efficiency of rapid propagation.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. This invention eliminates the need for callus induction and differentiation, directly inducing small bud clusters from stem segments, significantly saving culture time and improving culture efficiency.

[0030] 2. Directly immersing the stem segments of *Myriophyllum spicatum* into Agrobacterium bacillus solution can induce new shoots to emerge from the wounds and utilize the high water absorption capacity of the *Myriophyllum spicatum* stem segments to allow Agrobacterium bacillus to come into close contact with the cut *Myriophyllum spicatum* stem segments, thereby achieving efficient transformation.

[0031] 3. This invention uses bacterial co-culture instead of solid co-culture, and after the bacterial co-culture is completed, there is no need to rinse with sterile water, but the culture medium can be directly added, which simplifies the operation steps.

[0032] 4. In addition, the present invention has low cost, low chimerism rate, high conversion rate, good genetic stability, and is not affected by the flowering period. Therefore, the present invention further expands the application scope of plant transgenic technology.

[0033] After screening with hygromycin, the positive seedling rate of this invention is as high as 8%-15%. Preliminary PCR detection shows that the success rate of transgenic transformation in positive seedlings is over 90%. Real-time quantitative PCR detection shows that the probability of plants expressing the target gene can reach over 75%.

[0034] In summary, the method described in this invention does not require callus induction. Instead, it involves directly immersing *Myriophyllum spicatum* stem segments in Agrobacterium-mediated transformation solution (*Myriophyllum spicatum* liquid differentiation medium eluted with Agrobacterium OD600 = 0.5). This rapidly induces new shoots from the wound and utilizes the efficient water absorption capacity of the *Myriophyllum spicatum* stem segments, allowing for close contact between the Agrobacterium and the cut stem segments, thus achieving efficient transformation. Furthermore, this invention is low-cost, has a low chimerism rate, good genetic stability, and is unaffected by the flowering period. Therefore, this invention further expands the application scope of plant transgenic technology. The rapid and efficient *Myriophyllum spicatum* transgenic method provided by this invention fills a gap in the history of *Myriophyllum spicatum* transgenic research and lays the foundation for future studies. This method can further improve the nutritional quality and yield of *Myriophyllum spicatum*, increasing its value as silage. Attached Figure Description

[0035] Figure 1 Growth of induced clustered buds at the wound site of sterile short branches of Myriophyllum spicatum;

[0036] Figure 2 Growth of seedlings that tested positive for hygromycin after co-culture was completed;

[0037] Figure 3 Rooting status of positive *Myriophyllum spicatum* seedlings;

[0038] Figure 4 : The indoor hardening-off status of positive Myriophyllum spicatum seedlings;

[0039] Figure 5 The growth of newly transplanted Myriophyllum spicatum test-tube seedlings;

[0040] Figure 6 The growth of *Myriophyllum spicatum* test-tube seedlings 30 days after transplanting;

[0041] Figure 7 : Detection of hygromycin resistance genes in positive seedlings using PCR technology;

[0042] Lane 1 serves as a positive control;

[0043] Lane 2 served as a negative control.

[0044] Lanes 3-10 were PCR-positive plants. Detailed Implementation

[0045] The method for obtaining transgenic plants of Myriophyllum spicatum includes the following steps:

[0046] (1) Obtaining shoot clusters of Myriophyllum spicatum: Cut the explant stem segments into lengths of 2-4 cm and then sterilize them using the following methods: First, clean them with laundry detergent, then wash them 5-7 times with sterile water on a sterile operating table, soak them in 70% alcohol for 30 seconds, then sterilize them in 0.1% mercuric chloride solution for 10-15 minutes, and rinse them 5-6 times with sterile water for later use; After sterilization, the explants are cultured on MS medium containing 1-2 mg / L 6-BA, 0.1-0.5 mg / L KT, 0.1-0.5 mg / L IAA, and 0.1-1 mg / L NAA to induce a large number of small shoot clusters.

[0047] The above culture process was compared in different culture media: Culture medium 1 (i.e., the culture medium formula of this invention): MS + 6-BA 1 mg / L + KT 0.5 mg / L + IAA 0.3 mg / L + NAA 0.5 mg / L; Culture medium 2: MS + 6-BA 1 mg / L + NAA 0.5 mg / L;

[0048] The proliferation coefficient of medium No. 1 is at least 1:40. Each induced lateral bud can be cut into 2-3 sections for subculture, and the base of the remaining bud bulb can also be cut into 0.5cm × 0.5cm sections for further subculture. A single sterile short branch can multiply into hundreds of millions of sterile seedlings in six months. The proliferation coefficient of medium No. 2 is about 1:10, significantly lower than that of medium No. 1. The above experimental comparison shows that the proliferation coefficient of the medium of this invention is significantly higher than that of medium No. 2, and also superior to the proliferation coefficients of other existing technology media.

[0049] (2) Transformation and co-culture preparation of Agrobacterium tumefaciens: LB plates (after sterilization of LB solid medium and cooling to 60°C, add Kan 100mg / L + Chl 34mg / L + Rif 100mg / L, pour plates), spread bacterial solution and invert for 2 days, wash Agrobacterium with liquid co-culture medium (MS + As 0.1mM) to obtain Agrobacterium bacterial solution, which is used for the inoculation step.

[0050] (3) Immerse the 2-4 cm long Myriophyllum spicatum stem segments obtained in the culture at 25-28℃ in the Agrobacterium tumefaciens solution in step (2) and culture in the dark for 3 days. After co-culture, the part of Agrobacterium tumefaciens immersed in the culture turns brown but does not die, and new shoots emerge from the cut. Without rinsing with sterile water, directly inoculate the Myriophyllum spicatum stem segments into the screening medium for hygromycin screening.

[0051] (4) Screening of resistant shoots: After co-culture, the stem segments of Myriophyllum spicatum were inoculated into...

[0052] Resistant seedlings were screened in a selection medium consisting of MS + 6-BA 1 mg / L + KT 0.5 mg / L + IAA 0.3 mg / L + NAA 0.5 mg / L + 500 mg / L cephalosporin + 400 mg / L carbenicillin + 50 mg / L hygromycin. Positive seedlings appeared in 10-15 days, with a emergence rate as high as 8%-15%.

[0053] (5) Rooting culture of resistant seedlings: The resistant seedlings in step (4) are inoculated into the following agar solid medium to root.

[0054] Culture medium No. 3:

[0055] MS + NAA 0.5 mg / L + IAA 0.5 mg / L;

[0056] Culture medium #4: (Reference)

[0057] MS + 6-BA 0.05 mg / L + NAA 0.1 mg / L;

[0058] In medium No. 3, each *Myriophyllum spicatum* stem segment rooted within 5 days, with a rooting rate of 100%. After 10 days, each segment produced 10-20 roots, demonstrating rapid and abundant rooting. After 7 days of hardening-off following 20 days, the seedlings could be transplanted into the field. No sterilization of the transplanting substrate was required, and the survival rate was 100%. In medium No. 4, rooting took 10 days, and after 20 days, each segment produced 5-15 roots. After sterilization of the substrate, the survival rate reached 96%. The above experimental comparisons show that the rooting speed and number of roots in the medium of this invention are higher than those in existing technologies such as medium No. 4.

[0059] (6) Transplant the resistant rooted seedlings from step (5) to a greenhouse or field, ensuring a 100% survival rate. Perform preliminary PCR testing (see [link to relevant documentation]). Figure 7 The successful transgenic plants were selected, and the success rate of transgenic transplantation in positive seedlings reached over 90%.

[0060] (7) Real-time quantitative PCR was performed on the transgenic plants obtained in step (6) to obtain transgenic plants in which the target gene was expressed. Among the successfully transgenic plants, the probability of the target gene being expressed was as high as 75% or more.

Claims

1. A method for obtaining transgenic plants of Myriophyllum spicatum, characterized in that, The method includes the following steps: (1) Obtaining clustered shoots of Myriophyllum spicatum: Select stem segments of superior Myriophyllum spicatum plants as explants. After disinfection, the explants are cultured on MS agar solid medium supplemented with 6-BA, KT, IAA and NAA for 20-30 days to induce explants with clustered shoots. The concentrations of 6-BA, KT, IAA and NAA in MS agar solid medium are 1-2 mg / L, 0.1-0.5 mg / L, 0.1-0.5 mg / L and 0.1-1 mg / L, respectively. (2) Co-culture of Agrobacterium tumefaciens: The stem segments of Myriophyllum sp. cut from explants with small bud clusters were inoculated with Agrobacterium tumefaciens solution and then co-cultured. (3) Screening of resistant shoots: The stem segments of Myriophyllum spicatum after co-culture were inoculated into MS screening medium supplemented with 6-BA, KT, IAA, NAA, cephalosporin, carbenicillin and hygromycin to screen and culture resistant shoots; the concentrations of 6-BA, KT, IAA, NAA, cephalosporin, carbenicillin and hygromycin in MS screening medium were 1~2 mg / L, 0.1~0.5 mg / L, 0.1~0.5 mg / L, 0.1~1 mg / L, 500 mg / L, 400 mg / L and 50 mg / L, respectively; (4) Rooting culture of resistant seedlings: The resistant seedlings were inoculated into MS agar solid medium supplemented with NAA and IAA for rooting culture; the concentrations of NAA and IAA in MS agar solid medium were 0.1~1.5 mg / L and 0.1~1 mg / L, respectively; (5) Transplant the resistant rooted seedlings from step (4) to a greenhouse, then transplant them to the field and test them to screen out the successfully transgenic plants.

2. The method for obtaining transgenic *Myriophyllum spicatum* plants according to claim 1, characterized in that, The explant disinfection process described in step (1) involves cutting the stem of the Myriophyllum spicatum plant into a length of 2-4 cm, cleaning it with laundry detergent, washing it 5-7 times with sterile water on a sterile operating table, soaking it in 70% alcohol for 30 seconds, sterilizing it in 0.1% mercuric chloride solution for 10-15 minutes, rinsing it with sterile water 5-6 times, and then setting it aside for use.

3. The method for obtaining transgenic *Myriophyllum spicatum* plants according to claim 1, characterized in that, Step (1) also includes the subculture of the induced explants with clustered buds: cut the old stems into 2-4cm pieces and the clustered buds into 0.5cm×0.5cm pieces, and then transfer them into the MS agar solid medium containing 6-BA, KT, IAA and NAA as described in step (1), and subculture once every 28-30 days.

4. The method for obtaining transgenic *Myriophyllum spicatum* plants according to claim 3, characterized in that, The specific steps for co-culturing Agrobacterium tumefaciens in step (2) are as follows: ① Prepare Agrobacterium tumefaciens bacterial solution three days in advance: After sterilizing LB solid medium, cool it to 60℃, and then add Kan, Chl and Rif. The concentrations of Kan, Chl and Rif in LB solid medium are 100mg / L, 34mg / L and 100mg / L, respectively. Spread Agrobacterium tumefaciens bacterial solution on LB solid medium with Kan, Chl and Rif added, and incubate upside down at 28℃ for 2-3 days. Wash the cultured LB plate bacteria into MS liquid co-culture medium with As added, and adjust OD600=0.

5. The concentration of As in MS liquid co-culture medium is 0.1mM. Obtain Agrobacterium tumefaciens bacterial solution; ② Agrobacterium-mediated transformation: Cut the explants with clustered buds after subculture in step (3) into 2-4cm stem segments, transfer them to Agrobacterium tumefaciens bacterial solution for soaking and co-culturing for 3 days. The co-culturing conditions are dark culture at 25℃-28℃.

5. The method for obtaining transgenic *Myriophyllum spicatum* plants according to claim 1, characterized in that, The screening and culture time in step (3) is 10 to 15 days.

6. The method for obtaining transgenic plants of Myriophyllum spicatum according to claim 1, characterized in that, The rooting culture time in step (4) is 15 to 20 days, and the plants are transplanted after rooting.

7. The method for obtaining transgenic plants of Myriophyllum spicatum according to any one of claims 1 to 6, characterized in that, The culture temperature for steps (1), (3) and (4) is 24±2℃, and the culture is carried out in a low light environment of 1000~2000lx for 10~12h of light per day.

8. The method for obtaining transgenic plants of Myriophyllum spicatum according to claim 1, characterized in that, Step (5) involves loosening the cap of the culture bottle containing the resistant seedlings from step (4) and opening it by 1 / 4. The bottle is placed at 18-25℃ for 2 days. Then, the cap is fully opened and the bottle is placed at room temperature for another 2 days. The culture medium is then rinsed off with running water and cultured in tap water at room temperature for 5-7 days. Finally, the seedlings are transplanted into paddy fields. Samples are taken for preliminary PCR testing 20-30 days later.

9. The method for obtaining transgenic plants of Myriophyllum spicatum according to claim 8, characterized in that, The method also includes a step of performing real-time quantitative PCR detection on the successfully transgenic plants that have undergone preliminary PCR testing, ultimately obtaining transgenic plants.

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