Potato (Solanum tuberosum) is an important crop in the world. It is an important dual-purpose crop for food and vegetable because of its short growth period, high yield, wide adaptability, rich nutrition, and storage and transportation resistance. Potatoes are very susceptible to viruses during the planting process, and there are as many as 17 viruses that harm potatoes. Potatoes are asexually propagated crops. Viruses proliferate and transport in the mother body, accumulate in the tubers, and are passed down from generation to generation, increasing year by year, reducing its yield and quality. The use of stem apical meristem culture technology to detoxify infected varieties and obtain virus-free potato plants has a very significant effect on increasing potato yield.
Viruses parasitic in potato tubers also replicate and reproduce virus particles in potato plants as the tuber buds germinate and grow into plants, but the distribution of viruses in potato plants is uneven. According to research, there are no viruses in the metabolically active stem apical meristem. It may be because the cell division rate in the stem apical meristem is very fast, exceeding the replication rate of virus particles, so that the virus particles cannot get nutrition during the replication process and are inhibited. It may also be due to the high concentration of certain hormones in the meristem that inhibit the virus. The mechanism of the above reasons has not yet been clarified, but through virus detection of stem apex (with 1-2 leaf primordia, less than 0.2 mm) tissue culture seedlings, no virus was found, while stem apex greater than 0.2 mm can often detect viruses. This has become an important basis for the propagation of virus-free seedlings in stem apex detoxification tissue culture. In order to improve the detoxification efficiency of potatoes, heat treatment of explant materials can be used.
During the potato growing season, select plants with vigorous growth and no obvious diseases, pests and insects, and take their axillary buds and terminal buds. The stem tips of the terminal buds grow faster than those taken from the axillary buds, and the survival rate is also high. In order to easily obtain sterile stem tips, the test plants should be planted in sterile potting soil and cultivated in a greenhouse. For materials grown in the field, cuttings can also be grown in the nutrient solution in the laboratory. The branches grown from the axillary buds of these cuttings are much less contaminated than those taken directly from the field.
Cut off 2-3 cm strong buds, remove all leaves, wash them thoroughly with water, and rinse continuously for 0.5-1 h. Then rinse with 75% alcohol for 1 min, rinse with sterile water 3 times, and 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 tool dish.
Under a 10x dissecting microscope, cut off 1-1.5 mm stem tips with a scalpel, inoculate the removed stem tips on MS culture medium, and culture them in a culture room at 25°C with 16 h of light per day. When the stem tips grow to 1 cm, transfer them to a light incubator for culture, and treat them at 36°C with 16 h of light per day for 6-8 weeks.
On the clean bench, use a dissecting needle under a 40x dissecting microscope to carefully remove the small leaves and leaf primordia around the stem tip, exposing the smooth growth point at the top, and carefully cut the required stem tip meristem with a scalpel, leaving only the growth point with a leaf primordium, which is 0.1-0.2 mm in size. The cut stem tip meristem is then inoculated into potato stem tip culture medium (MS + 0.5 mg/L NAA + 3% sucrose + 0.7% agar, pH 5.8), with the cut surface in contact with agar, and the bottle is sealed and placed in the culture room for culture.
Shoot tip culture conditions: temperature 23-25°C, light intensity 25-50 µmol/(m2·s), light duration of about 16h per day. Under normal conditions, after 30-40 d of culture, the shoot tip can be seen to have obvious growth. Subculture 2-3 times, and then move it into rooting medium (1/2 MS + 1.5% sucrose + 0.7% agar, pH 5.8) for rooting.
When the roots of the sterile seedlings grow to 1-2 cm long, remove the cover on the culture 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 on the roots and transplant them into a culture medium containing 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 potato 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.
C. Serological detection.
ELISA virus detection technology is used. This method is fast, simple and highly sensitive.
