Strawberry (Fragaria ananassa) is a perennial herb with high economic benefits. It mainly reproduces by stolons and is easily infected by one or more viruses during cultivation. At present, more than 20 viruses that can infect strawberries have been reported. After the virus infects strawberries, the main phenomena are leaf chlorosis, deformity, reduced growth, dwarfing, reduced yield, and deterioration of quality. In severe cases, it can cause devastating effects. Therefore, using stem apex meristem culture to obtain virus-free seedlings is of great significance to the yield and quality of strawberry production.
The distribution of viruses on infected plants is not consistent. The virus content is higher in old leaves and mature tissues and organs, while the virus content is lower in young and immature tissues and organs, and the growth point (0.1-1.0 mm area) contains almost no or very little virus. Because the proliferation and transportation speed of the virus is different from the division and growth speed of the stem tip cells, the virus is transported upward slowly, while the meristem cells reproduce quickly, so that the cells in the stem tip part are free of viruses. This is the principle of using the stem tip meristem to detoxify strawberries. Therefore, the effect of detoxification of stem tip culture is negatively correlated with the size of the stem tip. The smaller the stem tip, the better the detoxification effect. The survival rate of the cultured stem tip is positively correlated with the size of the stem tip. The smaller the stem tip, the lower the survival rate. In specific applications, both the detoxification effect and the survival rate should be considered. Therefore, it is generally better to cut a 0.2-0.3 mm stem tip with one or two leaf primordia as a culture material.
The material was taken during the peak growing season of strawberry stolons, i.e., in August each year. The mother plant needs to be sterilized and treated with 500 times thiophanate-methyl once a week. Take the 4-5 cm long buds at the top of the stolons growing healthily in the field, and pay attention to choosing disease-free and insect-free plants. If the amount of material is relatively large, the lower end of the stem can be immersed in clean water and placed in a 4°C refrigerator for storage. It can be stored for up to 1 week, and the water needs to be changed several times in the middle.
Peel off the outer large leaves of the top buds of the stolons by hand and rinse them underwater for 0.5-1 h. Then rinse in 75% alcohol for 1 min and rinse with sterile water 3 times. Then disinfect with 2% sodium hypochlorite for 10 min. During the disinfection process, it is necessary to shake continuously, and then rinse with sterile water 3-5 times. Place on a sterilized glass dish.
After the material is disinfected, place it under a binocular dissecting microscope on a clean bench and it can be seen at 30 times. Use a dissecting needle to peel off the young leaves and scales layer by layer to expose the growth point. Generally, one or two leaf primordia are retained, cut with a scalpel to 0.2-0.3 mm, and immediately put into the triangular flask culture medium.
The differentiation medium of strawberry stem apex is MS + 0.5 mg/L 6-BA + 3% sucrose + 0.7% agar. The light intensity is 40-55 µmol/(m2·s), and the light time is 14 h/d. The culture temperature is (23±2)°C. Culture for 26 d, and the reproduction coefficient is calculated. Subculture once every 26 d. Transfer to rooting medium (1/2MS + 0.2 mg/L IAA + 1.5% sucrose + 0.7% agar), pH 5.8, and root.
When the roots of sterile strawberry seedlings grow to 1-2 cm long, remove the cover on the triangular bottle and harden the seedlings in the light culture room for 1-2 d. During this process, pay attention to maintaining the humidity in the culture room. Then wash the culture medium of the roots and transplant them into a culture pot filled with vermiculite and peat (1:2). Pay attention to maintaining the air humidity, and they will survive in 1 week.
A. Use indicator plants to detect viruses
The method of small leaf grafting is generally used to detect viruses using indicator plants. 1-2 months before grafting, plant a single healthy indicator plant in a pot. After it survives, pay attention to the prevention and control of aphids. Take 1-3 g of young leaves from the identified plant, grind it into a homogenate in a phosphate buffer solution of pH.7.0, filter it with two layers of gauze, and remove the residue. Apply or spray the filtrate on the leaf surface of the indicator plant and keep it warm at 15-25°C. Observe whether there are any symptoms of virus disease 2-6 d after inoculation.
B. Molecular biology to detect viruses
That is, use RT-PCR to detect the presence of viruses. Use the isolated viruses and their gene sequences to synthesize primers for RT-PCR reaction, extract RNA from the virus-free strawberry plants to be tested, use reverse transcriptase to obtain its cDNA, and then perform PCR reaction, agarose gel electrophoresis, and observe whether there are target fragments. If there are target bands, the virus removal is unsuccessful.
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