What are the precautions for walnut tissue culture

  (1) Contamination The tissue culture process requires a relatively highly sterile environment. During tissue culture, it is often infected by microorganisms such as fungi and bacteria, which multiply in the culture vessels and affect the normal growth and development of the culture material. At every step of culturing, attention must be paid to adequate disinfection and sterilization. Inoculation tools, inoculation materials, and operators must all be strictly sterilized as required before inoculation operations, including used inoculation bottles, bottle stoppers, and so on, which must be carefully cleaned and sterilized before they can be used again. The inoculation room, culture room, and ultra-clean workbench should be regularly fumigated with formalin and frequently sterilized with ultraviolet lamp irradiation.

Pictured: walnut

  (2) Vitrification Vitrification is a physiological disorder symptom of test-tube plantlets, manifested as leaves and young shoots of test-tube plantlets appearing water-soaked, transparent, or semi-transparent, with leaves becoming smaller and deformed. Vitrified plantlets root with difficulty and are not easy to survive after transplanting. Its occurrence causes test-tube plantlets to grow slowly and the propagation coefficient to decline. The main factors causing vitrification of test-tube plantlets include the agar and sucrose concentrations of the medium, culture temperature, growth regulator concentration, ethylene concentration in the culture bottle, light, and nitrogen content in the medium, among others, and attention should be paid to these during the culture process.

  (3) Browning The various tissues of walnut have a relatively high content of phenolic substances. During tissue culture, polyphenol oxidase in the tissues is activated, and phenolic compounds are oxidized to form brown quinone-like substances. Quinone substances polymerize with proteins, causing other enzyme systems to become inactivated, leading to metabolic disorder and hindered growth. The browning phenomenon is relatively severe and is a major bottleneck problem in walnut tissue culture propagation.

  The commonly used measures to prevent browning are: selecting young and tender explants, which have strong meristematic ability and low phenolic substance content; selecting a suitable medium; reducing temperature and light without affecting normal growth and differentiation; adding antioxidants or adsorbents to the medium, such as ascorbic acid, sodium thiosulfate, citric acid, polyvinylpyrrolidone, activated carbon, and so on.