Plant virus diseases are one of the important factors limiting agricultural production. After most vegetatively propagated plants are infected with viruses, their vegetative propagation characteristics allow viruses to accumulate for a long time, resulting in low crop yield, poor quality and variety degradation. Effective detection and control of plant viruses, cultivating virus-free seedlings, and implementing virus-free cultivation of crops are the fundamental ways to prevent plant virus diseases. Plant shoot apical meristem culture is an important means for eliminating viruses in many plants, and it is also the main technology used to prevent and control plant viral diseases in production.
Viruses transfer over long distances in plants through vascular bundles and between cells through plasmodesmata. There are no vascular bundles in the plant shoot apical meristem region, and the virus can only be transmitted through plasmodesmata. The auxin concentration in this area is high, the metabolism is strong, and the virus multiplication and movement speed is not as fast as the cell division and growth of the shoot apical meristem tissue. The closer to the shoot tip area, the less virus infection occurs, and the shoot tip growth point (0.1~1 mm) area contains almost no or very little virus.
The essence of shoot apex meristem culture is to use 0.1 to 0.5 mm shoot apex meristems that do not contain virus particles or contain very few virus particles as explants for micropropagation to obtain virus-free plants. Some viruses can also infect the shoot apical meristem region of plants. By heat-treating the materials used for shoot apical meristem culture, that is, treating them for a period of time at a suitable constant high temperature or variable temperature and under certain light conditions, the virus can be inactivated. The combination of heat treatment and shoot tip meristem culture detoxification can improve the detoxification rate.
The detoxified plant materials were germinated indoors and cultured in pots at 36-38°C with 55 µmol/(m2·s) light for 2 weeks.
Select appropriate terminal buds and lateral buds, cut them with clean scissors and clean them. Sterilize with 75% alcohol for 30 s, and remove the outer bud sheaths with a sterilized scalpel. After peeling off several pieces of bud sheaths, cut off the upper part of the new shoots together, and then treat them with 2.5% calcium hypochlorite or sodium hypochlorite solution for 15 min. After taking it out, wash it with sterile distilled water 3-5 times, and use sterile filter paper to absorb excess water and set aside.
Under a binocular dissecting microscope, cut off the leaves to expose the buds, and peel off the young leaves with a scalpel. The top part is the meristem at the stem tip growth point. Use a scalpel to cut off the growth point and generally retain 1-2 leaf primordia.
Place the stripped stem tips immediately into liquid culture medium or inoculate them onto solid culture media for culture. The culture conditions are 25°C and 16 h of light. For liquid culture, the culture container needs to be placed on a shaker at 80-120 r/min.
On average, subculture is performed once every 2-3 weeks, and a single small plant can be obtained in 5-7 weeks. The shoot apex meristem culture varies according to the cultured gene detoxification target. MS medium is often used as the basic medium plus a suitable hormone combination.
Commonly used media for sweet potato shoot tip meristem culture: MS+0.2 mg/L IAA+0.5 mg/L6-BA; MS+0.5 mg/L KT+0.2 mg/L IAA; MS+1.0 mg/L 6-BA+0 .01 mg/L NAA + 1.0 mg/L GA.
Virus-free seedlings obtained through shoot apical meristem culture must be rigorously tested for specific viruses. Only after confirmation of detoxification can further rapid propagation be carried out for production practice.
Commonly used plant virus detection techniques include indicator plant methods and serological methods.
A. Indicator plant method
Use the virus characteristics that appear on other plants as the standard for identifying virus species. This host specifically used to produce virus symptom characteristics is an indicator plant, also known as an identifying host. There are two types of symptoms. One is that the plant produces systemic symptoms after inoculation and expands to non-inoculated parts. The other is to produce local lesions only at the inoculated site, with necrosis, chlorosis or annular lesions appearing depending on the type of virus. Indicator plants include Nepeta, Divan, Quinoa and various tobaccos. The indicator plant method is simple and easy to operate, has low cost, and the results are accurate and reliable. However, it takes a long time and is difficult to detect a large number of samples. For example, periwinkle and tangerine seedlings can be used as indicator plants for citrus greening disease. Brazilian morning glory, amaranth, etc. are used as indicator plants for sweet potato viruses.
Testing procedure: Take 1-3 g of young leaves from the identified plants, grind them in a pH 7.0 phosphate buffer until homogenized, filter them with two layers of gauze, and remove the residue. Apply or spray the filtrate on the foliage of indicator plants. Instruct plants to be cultured in an aphid-free environment and kept at 15-25°C. Observe whether symptoms of viral disease appear 2-6 days after plant inoculation.
B. Serological method
Plant viruses can be used as an antigen and can produce antibodies when injected into animals. Antibodies present in serum are called antisera. Antisera produced by different viruses have their own specificities. Use antisera of known viruses to identify unknown viruses. This antiserum becomes a highly specific reagent with high specificity. Commonly used serological detection methods include ELISA or Dot-ELISA detection methods. Serological detection is the most widely used and feasible method and can be used for the detection of large quantities of samples.
In addition, biological technologies such as electron microscopy, quantitative polymerase chain reaction (Q-PCR) and visual biochip technology can also be used for plant virus detection.
