Induction of in vitro flowering in Dendrobium Madame Thong-In (Orchidaceae) seedlings is associated with increase in endogenous N(6)-(Delta (2)-isopentenyl)-adenine (iP) and N (6)-(Delta (2)-isopentenyl)-adenosine (iPA) levels.

We analysed the endogenous cytokinin levels of Dendrobium Madame Thong-In seedlings grown in vitro during vegetative and flowering-inductive periods. HPLC was used to fractionate the extracts and radioimmunoassay (RIA) was used for assay of zeatin (Z), dihydrozeatin (DZ), N(6)-(Delta(2)-isopentenyl)-adenine (iP) and their derivatives. Coconut water used in experiments was found to contain high level (>136 pmol ml(-1)) of zeatin riboside (ZR). Protocorms and seedlings cultured in medium with coconut water were found to contain 0.5-3.9 pmol g(-1) FW of the cytokinins analysed. Seedlings (1.0-1.5 cm) cultured in flowering-inductive liquid medium containing 6-benzyladenine (BA, 4.4 muM) and coconut water (CW, 15%) contained up to 200 and 133 pmol g(-1) FW of iP and iPA, respectively. These levels were significantly higher than all other cytokinins analysed in seedlings of the same stage and were about 80- to 150-folds higher than seedlings cultured in non-inductive medium. During the transitional (vegetative to reproductive) stage, the endogenous levels of iP (178 pmol g(-1) FW) and iPA (63 pmol g(-1) FW) were also significantly higher than cytokinins in the zeatine (Z) and dihydrozeatin (DZ) families in the same seedlings. Seedlings that grew on inductive medium but remained vegetative contained lower levels of iPA. The importance of the profiles of iP and its derivatives in induction of in vitro flowering of D. Madame Thong-In is discussed.

High frequency early in vitro flowering of Dendrobium Madame Thong-In (Orchidaceae).

We have successfully developed a method to induce early in vitro flowering of the self-pollinated seedlings of a tropical orchid hybrid, Dendrobium Madame Thong-In. Transition of vegetative shoot apical meristem to inflorescence meristem was observed when young protocorms were cultured in modified KC liquid medium. In contrast, protocorms cultured on Gelrite-solidified medium only produced axillary shoots and roots. CW was required to trigger the transitional shoot apical meristem and BA enhanced inflorescence stalk initiation and flower bud formation. However, normal flower development was deformed in liquid medium but developed fully upon transferring to two-layered (liquid over Gelrite-solidified) medium. Under optimal condition, in vitro flowering was observed about 5 months after seed sowing. Segregation of flower colours was observed in these seedlings and seedpods formed upon artificial pollination of the in vitro flowers.