Module 1 Knowledge of Plant Tissue Culture
  Main Content
  Work Tasks
  Summary
  Review Test
Module 2 Plant Tissue Culture Laboratory Design and Equipment
  Work Tasks
  Summary
  Review Test
Module 3 Basic Operation of Plant Tissue Culture
  Main Content
  Work Tasks
  Summary
  Review Test
Module 4 Tissue Culture of Virus-free Plantlets
  Work Tasks
  Summary
  Review Test
Module 5 Tissue Culture of Ornamental Flowers
  Source
  Work Tasks
  Summary
  Review Test
Module 6 Tissue Culture of Fruit Trees
  Source
  Work Tasks
  Summary
  Review Test
Module 7 Tissue Culture of Valuable Medicinal Herbs
  Main Content
  Source
  Work Tasks
  Summary
  Review Test
Module 8 Management of Industrialized Production of Tissue Culture Plantlets
  Work Tasks
  Summary
  Review Test
Module 9 Application of Tissue Culture Technique in Transgenic Engineering
  Main Content
  Work Tasks
  Summary
  Review Test

    Task 4 Aseptic Operation Techniques and Post-inoculation Management
    Knowledge Points
    In the inoculation room for tissue culture, sterilization is conducted through fumigation. The strong oxidizing potassium permanganate is used to volatilize formaldehyde in the formalin solution. Since formaldehyde can denature and coagulate microbe protein and dissolve the microbe lipoid, it can kill the bacterial propagules, brood cell fungus, and viruses on the surface of an object and in the air, thus killing the pathogenic microorganisms after a certain period of time. During fumigation, first close the doors and windows of the inoculation room and then add formaldehyde and potassium permanganate in sequence in a beaker at a ratio of 10 ml/m3 to 5 g/m3 ; leave the room immediately with the windows closed for 20–30 min of fumigation. Then, open the windows; turn on the UV lamp and fan for 30 min before inoculation to ensure that the inoculation room is thoroughly sterilized.
    I. Preparations before Inoculation
    The following steps are generally followed before inoculation.
    (1) One day before inoculation, fumigate the inoculation room with formaldehyde and keep the ultraviolet lamp on for sterilization for 30 min.
    (2) Put the primary culture medium and inoculation instruments, sterile paper, sterile water, sterilants, and other inoculation supplies on the clean bench for later use. Turn on the fan of the clean bench as well as the UV lamp on the bench 30 min before inoculation.
    (3) Wash hands, put on a special lab coat and change to slippers in the buffer room.
    (4) Before operating on the bench, wipe hands, especially the nails, with absorbent cotton balls, and then wipe the bench surface.
    (5) Clean the inoculation tools with absorbent cotton balls, sterilize the tweezers and scissors thoroughly, and then repeatedly burn their tips for sterilization.
    (6) During inoculation, the inoculator must keep his or her hands on the bench and may not talk, walk around, or cough, as these actions can increase microbes while keeping quiet helps to reduce the microbes.
    II. Inoculation
    1. The aseptic operation of primary culture
    (1) Put the preliminarily washed and cut explants into a beaker. place the beaker on the clean bench, disinfect it and rinse it with sterile water several times. drain the water, take out the explants, and put them on sterilized filter paper.
    (2) After the material is dried, hold tweezers in one hand and scissors or scalpel in the other hand to cut the material properly. For example, cut the leaves into 0.5 cm tubes and the stems into segments with 1 node; strip the micro shoot tip so that it has only 1–2 young leaves. Burn inoculation instruments frequently during inoculation to prevent cross-infection.
    (3) Insert or place cut explants onto the medium using burned instruments.
    Specific operations are as follows.
    First, remove the sealing film, hold and tilt the culture flask or test tube to make its mouth close to the flame of the alcohol burner, and rotate and burn the mouth over the flame for several seconds. Second, gently insert a piece of cut explant into the medium with tweezers. If leaves are taken as explants, directly attach 1–3 pieces (with the leaf back upward) to the medium. There are no requirements as to how the materials should be inoculated, except that the shoot tips and stem segments should be inoculated upright (with the tip upward). It is advisable to inoculate only a small number of materials, i.e. one piece of material in one inoculation flask each time, which can save medium and manpower. Once the culture is contaminated, it can be discarded.
    (4) After inoculation, the mouth of the culture flask and the sealing film should be rotated and burned over the flame for several seconds, and then the flask should be sealed with the sealing film tied up. indicate the medium number, culture material, and inoculation date on the culture flasks and send the flasks to the culture room.
    (5) Clean the bench and sterilize it with a UV lamp for 30 min after inoculation work is completed. In the case of continuous inoculation, high-level disinfection is required every 5 days.
    2. The aseptic operation of subculture
    (1) Put primary culture medium, inoculation instruments, aseptic paper, sterile water, disinfectants, and other inoculation supplies onto the clean bench, and turn on the fan of the clean bench as well as the UV lamp on the bench 30 min before inoculation.
    (2) Wash hands, wear a lab uniform and then enter the inoculation room. wipe hands, the bench surface, and inoculation tools with absorbent cotton balls.
    (3) Place the alcohol burner about 30 cm from the edge of the clean bench directly in front of the chest, and note that all subsequent operations should be completed within a 10 cm radius around the alcohol burner. Ignite the alcohol burner, burn the scissors and tweezers from top to bottom, then burn their tips repeatedly. place the explant flasks to the left of the lamp and the blank medium flasks to the right; place the petri dish for inoculation about 15 cm from the edge of the clean bench directly in front of the chest to facilitate operation.
    (4) Open the explant flask and burn the inner and outer walls of its mouth. Plantlets are first transferred to Petri dishes with aseptic paper inside. Hold the culture flask horizontally with the left hand, pinch a piece of cut inoculum material with tweezers in the right hand, and put it into the flask. gently insert it into the medium. then burn the flask mouth over the flame of the alcohol burner for a few seconds, and quickly cover the flask with sealing film and tie the mouth up. Frequently wipe (or spray) both hands and the bench with 70% alcohol during operation; repeatedly soak inoculation instruments in 95% alcohol and sterilize them over the flame (or with a dry heat sterilizer) to prevent cross-contamination. After inoculation, burn both the mouth and sealing film of the explant flask when needed. For a skilled operator, suspension inoculation may be adopted, i.e. holding the tip of the test-tube plantlet with tweezers to pull it out of the flask, cutting off the tip, and inserting it into the blank medium.
    (5) After inoculation of a flask of stock plant material, wipe both hands with absorbent cotton balls, discard the used Petri dish, and replace it with a new sterile one to prevent cross-infection.
    (6) After inoculation of 10 flasks of stock plant material, label them, move them down the clean bench into a plastic basket, and inoculate the next batch.
    (7) When all the inoculation work is finished, put out the alcohol burner, clean up the clean bench, put plantlet flasks, and then use Petri dishes, empty explant flasks, inoculation tools, and other inoculation supplies into plastic baskets. take them out of the inoculation room and clean them.
    (8) Transfer them to the culture room for culture after inoculation. Check contamination after 3 days and remove any contaminated culture flasks in time.