ISHS Acta Horticulturae 131: In Vitro Culture, XXI IHC
SURFACE-STERILIZATION FOR IN VITRO CULTURE OF PHALAENOPSIS FLOWER-STALK CUTTINGS USING ANTIMICROBIALS |
| Authors: | M. Tanaka, M. Kumura, M. Goi |
| Abstract:
The in vitro culture of leaf segments of young shoots derived from flower-stalk cuttings cultured in vitro is recommended for clonal propagation of Phalaenopsis. However, surface-sterilization of the cuttings is a serious problem because of microbial contamination of the cultures. To overcome this problem we have used several antimicrobials for the surface-sterilization. Excised flower-stalks were wiped 3 times with 70% (v/v) ethanol, and then cut into nodal cuttings. The cuttings were immersed in 70% (v/v) ethanol for 10 sec, then rinsed 3 times in sterile water. Thereafter, the cuttings were immersed in the solution which contained several antimicrobials for 30 min in the dark. After the cuttings were planted, 1 ml of the same prepared antimicrobials solution was poured on the agar media. The cultures were maintained in the dark for first 10 days and then placed under light at 25°C. Among several combinations of antimicrobials examined, the solution contained 10 ppm Benlate, 25 ppm PCNB, 100 ppm TBZ, 10 ppm rifampicin, 500 ppm ampicillin and 50 ppm vancomycin (solution C) brought about complete aseptic cultures. In this case, pretreatment with 70% (v/v) ethanol was essential to asepsis of the cultures. The cuttings from the flower-stalks whose florets had fallen showed high percentage of contamination. A higher percentage of aseptic cultures occured in those cuttings from higher positions on the flower-stalks. When the cuttings were cultured on the media in which antimicrobials incorporated previously, many of them died and the growth of the lateral buds on aseptic cuttings was inhibited. On the other hand, the survival rate was high in the standard procedure using solution C. | |
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