Method for improving cutting survival rate of transgenic alfalfa based on earth culture

Abstract

The invention discloses a method for improving the cutting survival rate of transgenic alfalfa based on earth culture. The method comprises the following five steps: cutting twigs of the transgenic alfalfa as cutting slips; cleaning the cutting slips and soaking the cutting slips in a rooting agent; the cutting slips are inserted into a cutting medium to be cultured; after the cuttings root, a nutrient solution is supplemented; and transplanting after the root system grows to a certain length. By improving the cutting material, the matrix formula, the environmental condition, the management measure and other links in the transgenic alfalfa soil culture cutting technology, the rooting rate and the survival rate of transgenic alfalfa cutting are remarkably increased, and the transplanting survival rate reaches 95% or above. The method is easy to operate, low in cost and suitable for large-scale production, and a feasible technical support is provided for rapid propagation and popularization of transgenic alfalfa.

Description

Technical Field

[0001] The present invention relates to a method for improving the cutting survival rate of alfalfa based on soil culture, and particularly to a method for improving the cutting survival rate of transgenic alfalfa based on soil culture, belonging to the technical field of alfalfa cutting. Background Art

[0002] Alfalfa has various values such as for food, medicine, ornament, soil and water conservation, and improving soil nutrients, and is widely planted around the world. By introducing foreign genes into alfalfa cells through genetic transformation methods, transgenic alfalfa can be obtained, which can change some agronomic traits of alfalfa, such as improving stress tolerance or nutritional value, etc. However, because the genetic transformation operation process is complex, the number of finally obtained alfalfa plants is small, and although many obtained alfalfa plants have excellent traits, they cannot be well preserved due to the inability to propagate.

[0003] Conventional seed propagation methods have limitations such as high production costs and long propagation cycles. Cutting is a simple, efficient, and easy-to-operate asexual propagation method, which can quickly propagate a small number of transgenic plants obtained through genetic transformation. Using cutting technology can propagate a large number of transgenic alfalfa with exactly the same genetic traits as the transformed parent in a short time, shorten the verification cycle of genetic transformation results, and facilitate the promotion and application of transgenic alfalfa.

[0004] There are two ways of alfalfa cutting: hydroponics and soil culture. The hydroponics method has strict requirements for nutrient solution, and this environment is prone to breeding bacteria. Once the alfalfa roots are infected, it will quickly spread throughout the hydroponics system. Compared with hydroponics, soil culture can play a role in blocking bacteria to a certain extent and reduce the harm of pathogenic bacteria. In addition, the advantages of using soil culture for alfalfa cutting also include: the soil contains abundant macronutrients such as nitrogen, phosphorus, and potassium, as well as micronutrients such as iron, manganese, and zinc; the slow release of nutrients in the soil can continuously meet the needs of alfalfa at different growth stages; the soil has the function of retaining and discharging water, which can provide a certain amount of water for alfalfa and will not cause the roots to rot due to lack of oxygen after being soaked in water for a long time.

[0005] However, for transgenic alfalfa, the existing cutting methods have problems such as low propagation rate, underdeveloped roots, and low transplanting survival rate, which limit the rapid propagation and large-scale promotion and application of high-quality transgenic alfalfa. Summary of the Invention

[0006] Object of the Invention: The object of the present invention is to provide a soil culture cutting method that can significantly improve the rooting rate and survival rate of transgenic alfalfa.

[0007] Technical Solution: A method for improving the cutting survival rate of transgenic alfalfa based on soil culture according to the present invention includes the following steps:

[0008] Step 1: Cut the tender branches of transgenic alfalfa with a length of 5 - 7 cm as cuttings.

[0009] Step 2: Wash the base of the cuttings and soak the base in a rooting agent.

[0010] Step 3: After washing off the rooting agent on the surface of the cuttings, insert the cuttings into the cutting substrate. The cutting depth is 1 / 3 - 1 / 2 of the cutting length. Water thoroughly and then cultivate. The cultivation conditions are 22 - 25 °C, air humidity of 70% - 80%, and light intensity of 1000 - 5000 lux.

[0011] Step 4: After the cuttings take root, supplement nutrient solution.

[0012] Step 5: Transplant after 45 - 50 days of cutting.

[0013] The present invention improves the cutting materials, substrate formula, environmental conditions, management measures and other aspects in the soil culture cutting technology of transgenic alfalfa, and obtains a method for improving the cutting survival rate of transgenic alfalfa.

[0014] Step 1 is the selection of cutting mother plants and the collection of cuttings. The growth state of the mother plant directly affects the quality and survival rate of the cuttings. Therefore, transgenic alfalfa in the vigorous growth period and free of diseases and pests is preferably selected as the mother plant. Cut the tender branches from the upper part of the mother plant as cuttings. Leave 1 - 2 leaves at the top of the cuttings to reduce water loss and ensure the photosynthetic area at the same time. Cut the bottom of the cuttings into a 45° inclined plane to increase the water absorption and rooting area.

[0015] Step 2 is the treatment of cuttings. After washing the cuttings, soak them in the rooting agent solution for about 30 minutes to facilitate the rooting of the cuttings.

[0016] Step 3 is the cutting operation and post - cutting management. First, wash the rooting agent on the cuttings to avoid inhibiting the normal growth of the regenerated seedlings. The cutting substrate is pre - watered sufficiently and holes are pressed out to facilitate the cutting of the cuttings. The cutting depth is preferably 1 / 2 of the cutting length (about 2 - 5 cm). Pay attention to keeping the cuttings upright during insertion to avoid damaging the epidermis of the cuttings. Immediately water thoroughly after cutting to keep the substrate moist but not waterlogged. To avoid water loss of the cuttings due to too strong light in the initial stage of cutting, the light intensity can be appropriately reduced. After one week, it can be adjusted to the normal light intensity. In subsequent management, spray water in a timely manner according to the substrate condition to keep the air humidity between 70% - 80% to promote the rooting of the cuttings. In the first two weeks after cutting, use a sprayer to spray water regularly to keep the leaves of the cuttings moist, thereby reducing water evaporation. In addition, a layer of transparent plastic film can be covered on the plug tray to maintain a higher air humidity. Regularly check the growth of the cuttings and deal with diseases and pests in time. Low - toxicity and high - efficiency biological pesticides can be used for prevention and control, such as Bacillus thuringiensis for pest control and carbendazim for disease control.

[0017] Step 4 is the nutrient supplement after the cuttings take root. After the cuttings take root (usually 2 - 3 weeks), nutrient solution can be appropriately supplemented. The nutrient solution is preferably 1 / 2 strength Hoagland nutrient solution, supplemented once a week to promote root growth and above-ground growth.

[0018] Step 5 is the transplanting management. When the cuttings are about 45 days after cutting and the roots grow to 5 - 10 cm, transplanting can be carried out. Before transplanting, harden the seedlings appropriately, gradually reduce the air humidity, and enhance the adaptability of the cuttings to environmental changes. Pay attention to protecting the roots during transplanting to avoid damage.

[0019] Preferably, in Step 1, the growth stage of the transgenic alfalfa is the budding stage, early flowering stage or full flowering stage. Most preferably, it is the transgenic alfalfa at the budding stage.

[0020] Preferably, in Step 3, the light intensity is 2000 - 4000 lux. Most preferably, it is 2000 - 3000 lux.

[0021] Preferably, in Step 3, the cutting substrate is peat soil.

[0022] The substrate is selected from soil that is loose, breathable and has good drainage. Peat soil can effectively retain water and fertilizers. Disinfecting the substrate before use can avoid the growth of pathogens. There are two ways to disinfect the substrate: high-temperature steam disinfection method can be used, such as using 121 °C for 20 min; or chemical disinfection method can be used, such as soaking in 0.1% potassium permanganate solution for 30 min for disinfection. After disinfection treatment, spread out the substrate and let it dry for later use.

[0023] Preferably, the cutting substrate also includes perlite or vermiculite.

[0024] Perlite and vermiculite can increase the air permeability and water permeability of the substrate. The mixture of peat soil, perlite and vermiculite provides a good rooting environment for the cuttings.

[0025] Preferably, the cutting substrate is peat soil:perlite:vermiculite = 1 - 2:1:1. Most preferably, it is peat soil:perlite:vermiculite = 2:1:1.

[0026] Preferably, in Step 2, the rooting agent is indolebutyric acid (IBA) or naphthaleneacetic acid (NAA).

[0027] Preferably, in Step 2, the concentration of the rooting agent is 50 - 300 mg / L. Most preferably, it is 100 - 200 mg / L.

[0028] Preferably, in Step 2, the cleaning is carried out successively with 75% alcohol, 10% sodium hypochlorite and sterile water.

[0029] Preferably, in Step 4, the nutrient solution is 1 / 2 strength Hoagland nutrient solution.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The present invention significantly improves the rooting rate and survival rate of transgenic alfalfa cuttings, and the transplanting survival rate reaches more than 95%. The present invention is simple to operate and has low cost, and is suitable for large-scale production, providing practical technical support for the rapid propagation and popularization of transgenic alfalfa. Description of the Drawings

[0031] Figure 1 It shows the growth status of the cuttings of transgenic alfalfa at the initial stage;

[0032] Figure 2 It shows the rooting situation of the cuttings of transgenic alfalfa two weeks after cutting;

[0033] Figure 3 It shows the growth status of the cuttings of transgenic alfalfa one month after cutting;

[0034] Figure 4 It shows the growth status of the cuttings of transgenic alfalfa during transplanting. Detailed Embodiments

[0035] The technical solution of the present invention will be further described below with reference to the drawings.

[0036] The embodiments involve a total of 5 kinds of female parent plants:

[0037] 1. Qingshui alfalfa: from Gansu Agricultural University.

[0038] 2. Suntory alfalfa: purchased from Beijing Zhengdao Seed Industry Co., Ltd.

[0039] 3. Overexpressing eGFP Qingshui alfalfa: obtained by genetic transformation in this laboratory.

[0040] 4. Overexpressing eGFP Suntory alfalfa: obtained by genetic transformation in this laboratory.

[0041] 5. Overexpressing WRKY71 Suntory alfalfa: obtained by genetic transformation in this laboratory.

[0042] Example 1: A method for improving the survival rate of transgenic alfalfa cuttings based on soil culture

[0043] In this example, the overexpressing eGFP Qingshui alfalfa is used as the cutting female parent plant, and the specific steps are as follows:

[0044] Step 1, cut 5-7 cm long tender branches of the overexpressing eGFP Qingshui alfalfa with strong growth, no diseases and pests, and at the budding stage as cuttings;

[0045] Step 2: Clean the base of the cuttings with 75% alcohol → 10% sodium hypochlorite → sterile water, and then soak the base of the cuttings in 150 mg / L IBA for 30 minutes;

[0046] Step 3: Rinse the base of the cuttings with sterile water;

[0047] The cutting substrate is mixed according to peat soil: perlite: vermiculite = 2:1:1; before use, the substrate is sterilized by high-temperature steam at 121 °C for 20 minutes; after the substrate is dried, it is filled into a 32-well plug tray with drainage holes at the bottom. After filling the substrate, gently tap it to ensure that the substrate can closely fit the base of the cuttings when inserting the cuttings; after watering the filled plug tray thoroughly, press out holes in each hole;

[0048] Insert the cuttings into the cutting substrate, and the cutting depth is about 1 / 2 of the length of the cuttings. Keep the cuttings upright when inserting;

[0049] Immediately water thoroughly after cutting, place the plug tray in a light incubator for cultivation, and the cultivation conditions are 22 - 25 °C, air humidity 70% - 80%, light intensity 2000 - 3000 lux; within the first two weeks after cutting, use a spray bottle to spray water regularly;

[0050] Step 4: After the cuttings take root, supplement 1 / 2 strength Hoagland nutrient solution once a week;

[0051] Step 5: When cutting for about 45 days and the roots grow to 5 - 10 cm, transplant them to a large flower pot.

[0052] In this example, the survival rate of transplanting overexpressing eGFP Medicago sativa is over 95%. The growth status of the cuttings of overexpressing eGFP Medicago sativa at the initial stage of cutting is as Figure 1 shown; the rooting situation of the cuttings two weeks after cutting is as Figure 2 shown; the growth status of the cuttings one month after cutting is as Figure 3 shown; the growth status of the cuttings at the time of transplanting is as Figure 4 shown.

[0053] Example 2: A method for improving the cutting survival rate of transgenic Medicago sativa based on soil cultivation

[0054] Based on the method of Example 1, in this example, the concentration of the rooting agent in Step 2 is changed to 200 mg / L, and the cutting substrate in Step 3 is changed to be mixed according to peat soil: vermiculite = 1:1.

[0055] The survival rate of transplanting overexpressing eGFP Medicago sativa in this example is 92%.

[0056] Example 3: Optimizing the light intensity in the cutting method of transgenic Medicago sativa based on soil cultivation

[0057] Based on the method of Example 2, in this example, the light intensity in Step 3 is changed to explore the effect of light intensity on the cutting survival rate.

[0058] The results are shown in Table 1. The light intensity is preferably 2000 - 4000 lux, and most preferably 2000 - 3000 lux.

[0059] Table 1: Effect of Light Intensity on Cutting Survival Rate

[0060] Light intensity (lux) Cutting medium Cutting shoot stage Rooting agent concentration (mg / L) Survival rate (%) 1000-2000 Peat soil + vermiculite (1:1) Bud emergence stage 200 77 2000-3000 Peat soil + vermiculite (1:1) Bud emergence stage 200 92 3000-4000 Peat soil + vermiculite (1:1) Bud emergence stage 200 86 4000-5000 Peat soil + vermiculite (1:1) Bud emergence stage 200 79

[0061] Example 4: Optimize the growth period of the mother plant in the cutting method of transgenic alfalfa based on soil cultivation

[0062] Based on the method of Example 2, in this example, the growth period of the mother plant in Step 1 is changed to explore its effect on the cutting survival rate.

[0063] The results are shown in Table 2. The growth period of the mother plant is preferably the budding stage.

[0064] Table 2: Effect of the Growth Period of the Mother Plant on Cutting Survival Rate

[0065] Growth stage Light intensity (lux) Cutting medium Rooting agent concentration (mg / L) Survival rate (%) Bud emergence stage 2000-3000 Peat soil + vermiculite (1:1) 200 92 Early flowering stage 2000-3000 Peat soil + vermiculite (1:1) 200 84 Full flowering stage 2000-3000 Peat soil + vermiculite (1:1) 200 72

[0066] Example 5: Optimize the cutting substrate in the cutting method of transgenic alfalfa based on soil cultivation

[0067] Based on the method of Example 2, in this example, the cutting substrate in Step 3 is changed to explore its effect on the cutting survival rate.

[0068] The results are shown in Table 3. The cutting substrate is preferably peat soil: perlite: vermiculite = 2:1:1.

[0069] Table 3: Effect of Cutting Substrate on Cutting Survival Rate

[0070] Cutting medium Light intensity (lux) Cutting shoot stage Rooting agent concentration (mg / L) Survival rate (%) Peat soil 2000-3000 Bud emergence stage 200 74 Peat soil + vermiculite (1:1) 2000-3000 Bud emergence stage 200 92 Peat soil:Perlite:Vermiculite (1:1:1) 2000-3000 Bud emergence stage 200 95 Peat soil:Perlite:Vermiculite (2:1:1) 2000-3000 Bud emergence stage 200 98

[0071] Example 6: Optimize the rooting agent concentration in the cutting method of transgenic alfalfa based on soil cultivation

[0072] Based on the method of Example 2, in this example, the rooting agent concentration in Step 2 is changed to explore its effect on the cutting survival rate.

[0073] The results are shown in Table 4. The concentration of the rooting agent (IBA) is preferably 100 - 300 mg / L, and most preferably 100 - 200 mg / L.

[0074] Table 4: Effect of Rooting Agent (IBA) Concentration on Cutting Survival Rate

[0075] Rooting agent concentration (mg / L) Light intensity (lux) Cutting medium Cutting shoot stage Survival rate (%) 0 2000-3000 Peat soil + vermiculite (1:1) Bud emergence stage 63 50 2000-3000 Peat soil + vermiculite (1:1) Bud emergence stage 74 100 2000-3000 Peat soil + vermiculite (1:1) Bud emergence stage 90 200 2000-3000 Peat soil + vermiculite (1:1) Bud emergence stage 92 300 2000-3000 Peat soil + vermiculite (1:1) Bud emergence stage 83

[0076] Example 7: Comparing the cutting survival rate of the present invention for different alfalfa plants

[0077] Based on the method of Example 1, in this example, the mother plant was changed to explore the cutting survival rate of the present invention for different alfalfa plants.

[0078] The results are shown in Table 5. The present invention can significantly improve the rooting rate and survival rate of transgenic alfalfa cuttings, and the transplanting survival rate can reach over 95%.

[0079] Table 5: Cutting survival rate of different alfalfa

[0080]

Claims

1. A method for improving the survival rate of transgenic alfalfa cuttings based on soil culture, characterized in that: The following steps are involved: Step 1: Cut 5-7 cm long shoots of transgenic alfalfa as cuttings; Step 2, cleaning the base of the cuttings and soaking the base in a rooting agent; Step 3, after washing off the rooting agent on the surface of the cuttings, insert the cuttings into the cutting medium, the cutting depth is 1 / 3-1 / 2 of the length of the cuttings, and culture after watering, the culture conditions are 22-25° C., air humidity 70%-80%, and light intensity 1000-5000 lux; Step 4: After the cuttings take root, add nutrient solution; Step 5: Transplant 45-50 days after cuttings.

2. The method according to claim 1, characterized in that In step 1, the growth period of the transgenic alfalfa is the budding period, the initial flowering period or the full flowering period.

3. The method according to claim 1, characterized in that In step three, the light intensity is 2000-4000 lux.

4. The method according to claim 1, characterized in that In step three, the cutting medium is peat soil.

5. The method according to claim 4, characterized in that The cutting matrix also includes perlite or vermiculite.

6. The method according to claim 5, characterized in that The cutting matrix is ​​peat soil: perlite: vermiculite = 1-2:1:

1.

7. The method according to claim 1, characterized in that In step 2, the rooting agent is indolebutyric acid or naphthylacetic acid.

8. The method according to claim 1, characterized in that In step 2, the concentration of the rooting agent is 50-300 mg / L.

9. The method according to claim 1, characterized in that: In step 2, the cleaning uses 75% alcohol, 10% sodium hypochlorite and sterile water in sequence.

10. The method according to claim 1, characterized in that In step 4, the nutrient solution is 1 / 2 concentration Hoagland nutrient solution.

Images