Tomato seedling raising method capable of resisting root diseases

Abstract

The invention discloses a tomato seedling raising method capable of resisting root diseases. The tomato seedling raising method capable of resisting the root diseases comprises the steps of pretreatment, pretreatment, seedling raising and transplanting. The tomato seedling cultivated by the method is a transgenic chimeric plant, the root system of the tomato seedling is a transgenic root tissue with a root disease resisting effect, and the overground stem, leaf, flower and fruit part is a native tissue which keeps the character characteristics of the original tomato variety, so that the method integrates the advantages of grafting and genetic engineering breeding, and has the advantages of simple and convenient operation of grafting, high yield and the like. The method also has the advantage of wide sources of disease-resistant DNA fragments of genetic engineering breeding, and also overcomes the limitation of grafting adaptability between scions and stocks in a grafting technology and the problem of food safety in genetic engineering breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of biology, and in particular relates to a tomato seedling raising method resistant to root diseases. Background Art

[0002] There are mainly two methods for crops to acquire resistance to root diseases: 1. Conventional breeding by hybridizing with disease-resistant parents and selecting disease-resistant plants from the offspring. The disadvantage of this method is that the cycle is extremely long and it takes many generations of screening to obtain stable inherited disease-resistant crop varieties. At the same time, the source of resistance is limited, and only natural disease-resistant genes can be used. Moreover, due to the limitation of hybrid fertility, it can only be used for the same species or closely related species; 2. Grafting by grafting with disease-resistant rootstocks to obtain plants resistant to root diseases has been successfully applied in tomato production. This method is simple and easy to operate, but it also has the disadvantages of limited sources of disease-resistant rootstocks and poor adaptability of some tomato varieties for grafting; 3. Transgenic breeding uses Agrobacterium tumefaciens to obtain plants with disease resistance. DNA fragments are transferred into plant callus tissue, and regenerated transgenic plants are obtained through tissue culture screening, induction of differentiation and rooting under sterile conditions. The advantage of this method is that the disease-resistant DNA fragments are widely sourced, which can be natural disease-resistant genes, toxic protein genes for pathogens, pathogen effect factors or gene interference fragments of plant disease-susceptible genes, etc. In theory, plants can obtain disease resistance to all diseases, but this method is difficult to operate, needs to be carried out under sterile conditions, and takes a long time. In addition, the introduction of exogenous DNA fragments may destroy the original functional genes of the plant and cause abnormal growth of the transgenic plant. More importantly, the transgenic plant obtained contains transgenic components throughout the plant, which poses a food safety hazard. Therefore, it is very necessary to develop a method that can solve the above technical problems. Summary of the invention

[0003] The invention aims to provide a tomato seedling raising method resistant to root diseases.

[0004] The object of the present invention is achieved in that the method for raising tomato seedlings resistant to root diseases comprises the steps of pretreatment, pretreatment, raising seedlings and transplanting, specifically comprising: A. Pretreatment: After the surface of tomato seeds is sterilized, they are sown in the seedling medium to germinate and grow seedlings; B. Pretreatment: When the cotyledons of the tomato seedlings are unfolded, the seedlings are cut 1-2 cm below the cotyledons, and the seedling cuts are immersed in the bacterial solution infected with Agrobacterium rhizogenes to obtain immersed tomato seedlings; C. Seedling cultivation: 1) Plant the soaked tomato seedlings in a seedling tray and keep them sealed and moist for 2 to 4 days to induce adventitious root germination; 2) After 2-4 days of cultivation, water with nutrient solution and move the seedling tray to the seedling greenhouse for seedling cultivation. During the seedling cultivation period, replenish nutrient solution every 7-10 days; D. Transplanting: After 20 to 25 days, when the tomato seedlings have 5 to 6 true leaves and are growing vigorously, they can be transplanted into the field.

[0005] Similar to Agrobacterium tumefaciens, Agrobacterium rhizogenes is also a type of engineered strain used for plant transgenics. The difference is that the bacteria can infect and induce the damaged parts of plants to form transgenic adventitious roots. Using this characteristic, the present invention designs a method for cultivating tomato seedlings resistant to root diseases. The specific steps are as follows: 1. After surface disinfection, sow the tomato seeds in the sterilized seedling medium to germinate and grow seedlings; 2. When the cotyledons of the seedlings unfold, cut the seedlings 1-2 cm below the cotyledons with a blade, and soak the seedling cut in a solution of Agrobacterium rhizogenes carrying disease-resistant DNA fragments for 8-10 minutes. During this period, use a sterile blade to gently scratch the stems of the seedlings; 3. After the soaked seedlings are placed on clean absorbent paper to dry the surface moisture, the seedlings are planted in a seedling tray with fully moistened sterilized vermiculite as the cultivation medium, and cultured in a sealed and moisturizing environment at 20-28℃ for 3 days to induce adventitious root germination; 4. After 3 days, water the seedlings with nutrient solution (such as Hoagland nutrient solution) and move the seedling tray to the seedling greenhouse. Replenish the nutrient solution every 7-10 days. 5. After 20-25 days, when the tomato seedlings have 5-6 true leaves and are growing vigorously, they can be transplanted into the field.

[0006] The tomato seedlings cultivated by the present invention are transgenic chimeric plants, whose root system is a transgenic root tissue with resistance to root diseases, while the above-ground stems, leaves, flowers and fruits are native tissues that retain the characteristics of the original tomato varieties. Therefore, the present invention combines the advantages of grafting and genetic engineering breeding, and has both the simple and fast operation of grafting and the advantage of a wide source of disease-resistant DNA fragments in genetic engineering breeding. At the same time, it also overcomes the grafting adaptability limitations between scion and rootstock in grafting technology and the food safety issues in genetic engineering breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 This is a schematic diagram of a tomato root transformed with the 35S:RUBY plasmid; Figure 2 Electrophoresis of DNA from different organs of tomato plants transformed with 35S:RUBY plasmid after amplification using 35S:RUBY sequence-specific primers. DETAILED DESCRIPTION

[0008] The present invention is further described below in conjunction with the embodiments, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention belong to the protection scope of the present invention.

[0009] The method for raising tomato seedlings resistant to root diseases of the present invention comprises the steps of pretreatment, pre-treatment, raising seedlings and transplanting, and specifically comprises: A. Pretreatment: After the surface of tomato seeds is sterilized, they are sown in the seedling medium to germinate and grow seedlings; B. Pretreatment: When the cotyledons of the tomato seedlings are unfolded, the seedlings are cut 1-2 cm below the cotyledons, and the seedling cuts are immersed in the bacterial solution infected with Agrobacterium rhizogenes to obtain immersed tomato seedlings; C. Seedling cultivation: 1) Plant the soaked tomato seedlings in a seedling tray and keep them sealed and moist for 2 to 4 days to induce adventitious root germination; 2) After 2-4 days of cultivation, water with nutrient solution and move the seedling tray to the seedling greenhouse for seedling cultivation. During the seedling cultivation period, replenish nutrient solution every 7-10 days; D. Transplanting: After 20 to 25 days, when the tomato seedlings have 5 to 6 true leaves and are growing vigorously, they can be transplanted into the field.

[0010] The surface disinfection described in step A is to soak the tomato seeds in a sodium hypochlorite solution for 3 to 5 minutes, discard the sodium hypochlorite solution and rinse the seeds with sterile water.

[0011] The mass percentage concentration of the sodium hypochlorite solution is 0.5-0.6%.

[0012] The seedling culture medium described in step A is composed of sterilized and decomposed plant straw, straw carbon and perlite.

[0013] The mass ratio of the decomposed plant straw, grass carbon and perlite is (3-5): (3-5): 1.

[0014] The Agrobacterium rhizogenes infection solution described in step B is obtained by activating the Agrobacterium rhizogenes strain (Ar.1193) carrying a functional structure connected to the 35S promoter, picking a single colony and placing it in 40-60 ml of YEB liquid culture medium containing spectinomycin and 90-110 mg / L streptomycin at a temperature of 25-30°C, and shaking and culturing at a speed of 200-250 r / min for 16-18 hours until the culture solution concentration reaches OD600=0.6-0.8, then transferring it to a sterile centrifuge tube and centrifuging it at 5000-7000 rpm for 2-4 minutes, discarding the supernatant, and adding 40-60 ml of MS liquid culture medium containing 18-20 g / L acetosyringone (AS) to suspend the precipitate. The suspension is the Agrobacterium rhizogenes infection solution.

[0015] The soaking time described in step B is 8 to 10 minutes.

[0016] The cultivation substrate in the seedling tray described in step C is fully moistened sterilized vermiculite.

[0017] C. The temperature of the sealed moisturizing culture in step 1) is 20-28°C.

[0018] C. The nutrient solution described in step 2) is Hoagland nutrient solution.

[0019] In step D, before transplanting to the field, the plant is first transplanted to a pre-installed sterilized cultivation matrix and cultured until it grows vigorously. The sterilized cultivation matrix is ​​composed of loam, straw carbon and sand.

[0020] The mass ratio of the loam, straw carbon and sand is 1:1:1.

[0021] The present invention is further described below with specific implementation cases: Example 1 Cultivation of transgenic chimeric tomatoes expressing betalain in roots 1. Wash the seeds of cherry tomato variety VFNT in 0.52% sodium hypochlorite solution for 4 minutes, then rinse with sterile water for no less than 4-5 times. Place them in a culture dish covered with moist filter paper at room temperature for germination, then transfer them to a seedling tray filled with moist seedling medium (rotted plant straw: grass carbon: perlite = 4:4:1) for cultivation until their cotyledons are fully expanded.

[0022] 2. After the activation of the Agrobacterium rhizogenes strain (Ar.1193) carrying the 35S promoter connected to the betalain synthesis expression cassette (RUBY) structure, pick a single colony and put it into 50mL YEB liquid culture medium containing 50mg / L spectinomycin and 100mg / L streptomycin at 28℃ and shake at 230r / min for about 16-18h. When the culture solution concentration reaches OD 600 =0.6-0.8, transfer to a sterile centrifuge tube, centrifuge at 6000rpm for 3min, discard the supernatant, add 50mL of MS liquid culture medium containing 19.26g / L acetosyringone (AS) to suspend the precipitate. The suspension is the infection liquid of Agrobacterium rhizogenes.

[0023] 3. When the cotyledons of tomato seedlings are fully expanded, remove the primary roots obliquely at the lower rachis axis and soak the incision in the infection solution of Agrobacterium rhizogenes for 8-10 minutes. During this period, use a sterile blade to lightly scratch the stem of the seedling in the infection solution, being careful not to cut it off.

[0024] 4. After soaking, place the seedlings on clean absorbent paper to dry the surface moisture, then plant the seedlings in a seedling tray with fully moistened sterilized vermiculite as the cultivation medium, and culture them in a sealed and moisturizing environment at 20-28℃ for 3 days to induce adventitious root germination.

[0025] 5. After 3 days, move the seedling tray out of the sealed moisturizing environment and water it with Hoagland nutrient solution every 7-10 days.

[0026] 6.21 days later, transplant the tomato seedlings that have grown 5-6 true leaves and are growing vigorously into pots pre-filled with sterilized cultivation medium (soil: straw carbon: sand = 1:1:1). At the same time, take some tomato seedlings, wash the roots, and check the expression of betalain. It can be seen that the tomato roots are red due to the synthesis of betalain (such as Figure 1 as shown).

[0027] 7. 60 days after transplanting, DNA was extracted from the roots, stems, leaves, flowers and fruits of the plants, and PCR amplification was performed using the specific sequence of the RUBY synthetic expression frame as primers to detect transgenic components. The results showed that the tomato plants only contained transgenic components in the roots, while the rest of the plants did not contain transgenic components (such as Figure 2 as shown).

[0028] Example 2 Breeding transgenic chimera tomatoes resistant to root-knot nematodes 1. Wash the seeds of the common tomato variety Zhongshu No. 4 in 0.52% sodium hypochlorite solution for 4 minutes, then rinse with sterile water for no less than 4-5 times. Place them in a culture dish covered with moist filter paper at room temperature for germination, then transfer them to a seedling tray filled with moist seedling medium for cultivation until their cotyledons are fully expanded.

[0029] 2. After the activation of the Agrobacterium rhizogenes strain (Ar.1193) carrying the 35S promoter connected to the silencing structure of the root-knot nematode effector protein gene 16D10, a single colony was picked and placed in 50 mL of YEB liquid culture medium containing 50 mg / L spectinomycin and 100 mg / L streptomycin at 28°C and cultured at a speed of 230 r / min for about 16-18 hours. When the culture solution concentration reached OD 600 =0.6-0.8, transfer to a sterile centrifuge tube, centrifuge at 6000rpm for 3min, discard the supernatant, add 50mL of MS liquid culture medium containing 19.26g / L acetosyringone (AS) to suspend the precipitate. The suspension is the infection liquid of Agrobacterium rhizogenes.

[0030] 3. When the cotyledons of tomato seedlings are fully expanded, remove the primary roots obliquely at the lower rachis axis and soak the incision in the infection solution of Agrobacterium rhizogenes for 8-10 minutes. During this period, use a sterile blade to lightly scratch the stem of the seedling in the infection solution, being careful not to cut it off.

[0031] 4. After soaking, place the seedlings on clean absorbent paper to dry the surface moisture, then plant the seedlings in a seedling tray with fully moistened sterilized vermiculite as the cultivation medium, and culture them in a sealed and moisturizing environment at 20-28℃ for 3 days to induce adventitious root germination.

[0032] 5. After 3 days, move the seedling tray out of the sealed moisturizing environment and water it with Hoagland nutrient solution every 7-10 days.

[0033] 6. After 21 days, transplant the tomato seedlings that have grown 5-6 true leaves and are growing vigorously into pots pre-filled with sterilized cultivation medium (loam: straw carbon: sand = 1:1:1).

[0034] 7. Seven days after transplanting, inoculate with 500 second-instar larvae of the root-knot nematode of the elephant ear bean ( M. enterolobii ) at the roots of tomato seedlings, and seedlings of the same variety of tomato seeds with the same inoculation intensity were set as a control.

[0035] 8. 60 days after inoculation, the aboveground growth indexes and underground pathological indexes of the experimental and control groups were investigated. The results showed that the growth indexes of the experimental group were better than those of the control group except for the plant height (Table 1), and the underground experimental group showed significant resistance to root-knot nematode disease (Table 2). .

Claims

1. A method for raising tomato seedlings resistant to root diseases, characterized in that: The root disease-resistant tomato seedling raising method comprises the steps of pretreatment, pre-treatment, seedling raising and transplanting, specifically comprising: A. Pretreatment: After the surface of tomato seeds is sterilized, they are sown in the seedling medium to germinate and grow seedlings; B. Pretreatment: When the cotyledons of the tomato seedlings are unfolded, the seedlings are cut 1-2 cm below the cotyledons, and the seedling cuts are immersed in the bacterial solution infected with Agrobacterium rhizogenes to obtain immersed tomato seedlings; C. Seedling cultivation: 1) Plant the soaked tomato seedlings in a seedling tray and keep them sealed and moist for 2 to 4 days to induce adventitious root germination; 2) After 2-4 days of cultivation, water with nutrient solution and move the seedling tray to the seedling greenhouse for seedling cultivation. During the seedling cultivation period, replenish nutrient solution every 7-10 days; D. Transplanting: After 20 to 25 days, when the tomato seedlings have 5 to 6 true leaves and are growing vigorously, they can be transplanted into the field.

2. The method for raising tomato seedlings resistant to root diseases according to claim 1, characterized in that: The surface disinfection described in step A is to soak the tomato seeds in a sodium hypochlorite solution for 3 to 5 minutes, discard the sodium hypochlorite solution and rinse the seeds with sterile water.

3. The method for raising tomato seedlings resistant to root diseases according to claim 2, characterized in that: The mass percentage concentration of the sodium hypochlorite solution is 0.5-0.6%.

4. The method for raising tomato seedlings resistant to root diseases according to claim 1, characterized in that: The seedling culture medium described in step A is composed of sterilized and decomposed plant straw, straw carbon and perlite.

5. The method for raising tomato seedlings resistant to root diseases according to claim 1, characterized in that: The Agrobacterium rhizogenes infection liquid described in step B is obtained by activating the Agrobacterium rhizogenes strain (Ar.1193) carrying the functional structure connected to the 35S promoter, picking a single colony and culturing it in 40-60 ml YEB liquid containing spectinomycin and 90-110 mg / L streptomycin. The liquid is shaken and cultured at a speed of 200-250 r / min for 16-18 hours at a temperature of 25-30 ° C until the concentration of the culture liquid reaches OD 600 =0.6~0.8, transfer to a sterile centrifuge tube and centrifuge at 5000~7000rpm for 2~4min, discard the supernatant, add 40~60ml of MS liquid culture medium containing 18~20g / L acetosyringone (AS) to suspend the precipitate. The suspension is the infection liquid of Agrobacterium rhizogenes.

6. The method for raising tomato seedlings resistant to root diseases according to claim 1, characterized in that: The soaking time described in step B is 8 to 10 minutes.

7. The method for raising tomato seedlings resistant to root diseases according to claim 1, characterized in that: The cultivation substrate in the seedling tray described in step C is fully moistened sterilized vermiculite.

8. The method for raising tomato seedlings resistant to root diseases according to claim 1, characterized in that: C. The temperature of the sealed moisturizing culture in step 1) is 20-28°C.

9. The method for raising tomato seedlings resistant to root diseases according to claim 1, characterized in that: C. The nutrient solution described in step 2) is Hoagland nutrient solution.

10. The method for raising tomato seedlings resistant to root diseases according to claim 1, characterized in that: In step D, before transplanting to the field, the plant is first transplanted to a pre-installed sterilized cultivation matrix and cultured until it grows vigorously. The sterilized cultivation matrix is ​​composed of loam, straw carbon and sand.

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