Tobacco Micropropagation Technology

Tobacco (Nicotiana tabacum) belongs to the Solanaceae plant, is an important industrial raw material, and is also an important one of the four typical experimental plants (tobacco, petunia, carrot, and turmeric) for plant tissue culture. It is the most widely studied and always ahead of other plant materials.

Principle

Two methods can be used for micropropagation of plants: the transfer propagation of clustered buds and the propagation of lateral buds and terminal buds. The inoculum material for the transfer propagation of cluster buds should be cluster buds grown aseptically. When propagation, each bud is separated and inoculated into the medium separately, and after a period of cultivation, it is transferred to the rooting medium to take root, or it can be directly inoculated into the medium that can grow both buds and root. The inoculation material for the propagation of lateral buds and terminal buds is sterile plants. When propagation, cut the plant into several sections, keep a side bud for each section, then inoculate the stem section and terminal bud into the culture medium, after a period of cultivation, the side buds will grow from the axillary buds, and the other steps are the same as the first method.

Figure 1. Tissue culture of Nicotiana tabacum. (Hussain, A.; et al. 2012)

Procedures

1. Preparation and Distribution of Medium
   Prepare MS-2 medium (MS basic medium + 0.2 mg/L NAA + 1 mg/L KT + 8 g/L agar, pH 5.8) and sterilize. Melt the sterilized culture medium (MS-2) for the micropropagation of tobacco seedlings, cool it down to 60°C, and dispense it into sterilized Erlenmeyer flasks under aseptic conditions. It is also possible to directly dispense the sterilized culture medium that has not been cooled into Erlenmeyer flasks.
2. Preparation of Aseptic Tobacco Seedlings
   Sterile tobacco seedlings can come from seedlings germinated from aseptic seeds, and can also be propagated from terminal buds and lateral buds of aseptic seedlings. A medium that favors bud proliferation should be used. The recommended medium formulations for tobacco micropropagation are: MS basic medium + 0.5 mg/L IAA + 0.5 mg/L BA + 8 g/L agar, pH 5.8, this medium is conducive to the growth of tobacco seedlings and roots, but less bud proliferation; MS basal medium + 0.2 mg/L NAA + 1 mg/L KT + 8 g/L agar, pH 5.8, this medium is conducive to the proliferation of tobacco buds, but less root growth; MS basic medium + 8 g/L agar, pH 5.8, this medium is conducive to the growth of buds and roots, but not the proliferation of buds.
3. Inoculation and Cultivation of Plant Material
   Take sterile tobacco plants, cut off the roots in a sterile Petri dish (or on sterile filter paper), and cut off large leaves at the same time. Cut the tobacco stalks into sections, leaving one node per section. When cutting, it is required to leave less of the upper part of the stem node and more of the lower part of the stem node, and the length of the stem segment is 5-15 mm. The lower part of the stem segment was inserted into the culture medium, and one stem segment was connected to each bottle. The plant materials were cultivated at 25°C, with a light intensity of 30 µmol/(m²·s), and a light time of 12 hours per day. After 1 week, lateral buds could be observed growing from the axillary buds.
4. Follow-up Work
   The newly grown seedlings can be continued to proliferate and subcultured, and the seedlings can also be transferred to the rooting medium to induce rooting.