The present invention relates to a secondary proliferation culture method of color osmanthusfragrans test-tube plantlets. The method comprises: adopting the newly-germinating shoots of the color osmanthusfragrans as the explants, carrying out disinfection sterilization on the explants,
cutting into the small segments with the length of 1-1.5 cm under a sterile condition, inoculating into an
axillary bud induction culture medium, culturing for 4-5 weeks to produce axillary buds,
cutting the axillary buds into the terminal
bud or stem segment with the length of 1-1.5 cm, and inoculating into the same culture medium to carry out secondary proliferation culture for 4-5 weeks to obtain the clustered shoots, wherein the culture process is performed under the conditions of the lighting time of 14 h each day, the photosynthetically active
photon flux density of 40-50 [mu]mol / m<2>.s and the temperature of 24-26 DEG C, the
axillary bud induction culture medium comprises a base culture medium, 0.1-0.5 mg / L of TDZ, 0.1-1.0 mg / L of KT, 5.5 g / L of
agar, and 30 g / L of white granulated
sugar, and the pH value is adjusted to 5.8. The secondary proliferation culture method of the present invention has characteristics of rapid breeding and no limitation of the season change on the production. In addition, the defects of the
cutting seedlings are effectively overcome.