Protocols for Micropropagation of Woody Trees and Fruits

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C. LIU ET AL.
6. Excise the shoots developing from the petiole cultures with sterile scalpels
and place them in Magenta boxes each containing 50 ml CS culture medium.
The shoots should be pushed 0.5–1.0 cm deep into the medium with shoot
tip well above the surface of the medium.
7. Maintain stock cultures by subculturing shoots to fresh medium every 3
weeks. Up to four shoots can be cultured per Magenta box.
2.6. Large-scale Propagation
Mass production of rooted C. cujete plants can be achieved by placing differentiated
nodes from aseptic stock cultures on a solid medium, in liquid medium or in a
bioreactor. The solid, liquid, and bioreactor culture systems can also be utilized in
combination to maximize the plantlet proliferation and growth rates.
2.6.1. Solid Culture System for Plant Production
1. Excise 10 ± 2 mm long plant nodes from the aseptic stocks. Leave one
intact leaf at the top of the node and remove all other leaves of the node.
2. Place up to four plant nodes in Magenta boxes containing 50 ml of solid C.
cujete rooting (SCR) medium supplemented with 1.0 µM kinetin. Ensure
that the nodes are in contact with the culture medium by pushing the bottom
portions (0.5–1.0 cm) of the explants gently into the medium. Incubate the
cultures under the same growth conditions as the stock cultures.
3. Examine the cultures for rooting response after 2 weeks. Normally, the
roots differentiate from the nodal section in contact with the medium within
2 weeks of culture.
4. Whole plantlets with well established root systems develop after 5 weeks of
culture (Figure 2E). Representative samples of plantlets are rinsed, blotted
dry, and growth parameters including weight, shoot height, leaf number, and
rooting frequency are measured (Figure 4). Transfer single plantlets to Magenta
boxes each containing 50 ml SCR culture medium for future culture.
2.6.2. Flask-liquid Culture System for Plant Production
1. Prepare 10 ± 2 mm long plant nodes from the aseptic stocks. Leave one
intact leaf at the top of the node removing the other leaves of the node.
2. Place up to four nodes in flasks containing 50 ml of liquid C. cujete rooting
(LCR) medium with 1.0 µM kinetin. Ensure that the node explants are not
entirely submerged in the liquid culture medium.
3. Place the cultures on a shaker at a speed of 80 rpm and incubate under the
same conditions as the stock cultures.
4. Examine the cultures for rooting response after 2 weeks. Normally, the
roots differentiate from the nodal section in contact with the medium within
2 weeks of culture.