Clonal Propagation In Vitro of Paphiopedilum Hybrids from Adult Plants

in HortScience

The aim of this study was to develop an efficient protocol for shoot tip culture from adult plants of Paphiopedilum Pfitzer. A considerable seasonal effect on explant collection was observed in the aseptic cultures established from adult plants, including the survival and microbial contamination of explants. The shoot tip explants excised from adult plants in February and May showed higher survival and had less contamination than those explants excised in August and November. Moreover, the season of explant collection also affected the subsequent shoot forming capacity and multiplication of axillary buds. In Paphiopedilum ‘In-Charm Silver Bell’, higher shoot forming capacity was observed in February and May, whereas higher shoot multiplication was observed only in February. In Paphiopedilum ‘Hsinying Maudiae Leopard’, both February and May were optimal timing for shoot forming capacity and multiplication. We also demonstrated the effectiveness of transcinnamic acid (tCA), an antiauxin chemical in diminishing the apical dominance of shoot tip explant and thus improving the axillary bud outgrowth. In P. ‘In-Charm Silver Bell’, the addition of 100 μM tCA plus 13.3 μM 6-benzylaminopurine (BA) for 1 month promoted axillary shoot bud formation from shoot tip explants as compared with the control.

Contributor Notes

This work was supported by grant 106AS-8.6.3-FD-Z1 from the Council of Agriculture, Executive Yuan, ROC, to Shan-Te Hsu.

Corresponding author. E-mail: leeyungi@hotmail.com.

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    Micropropagation of Paphiopedilum ‘In-Charm Silver Bell’. (A) After surface sterilization, the lateral bud with one leaf attached was used as the explant. Bar = 1 cm. (B) After 2 months of culture, the basal axillary buds (arrows) started to grow. Bar = 1 cm. (C) After 5 months of culture, two axillary buds had arisen from the basal part of explants. Bar = 1 cm. (D) The newly formed axillary shoots (as indicated in C) were excised for multiplication. After 3 months of subculture, three new shoots were visible. Bar = 1 cm. (E) The newly formed axillary shoots (as indicated in D) were excised again for multiplication. After 3 months of subculture, more shoot multiplication can be observed. Bar = 1 cm.

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    Micropropagation of Paphiopedilum ‘Hsinying Maudiae Leopard’. (A) After the surface sterilization, the lateral bud with one leaf attached was used as the explant. Bar = 1 cm. (B) After 2 months of culture, the basal axillary buds (arrow) started growing. Bar = 1 cm. (C) After 5 months of culture, a number of axillary buds had arisen from the basal part of explants. Bar = 1 cm. (D) The newly formed axillary shoots (as indicated in C) were excised for multiplication. After 3 months of subculture, several new shoots were visible. Bar = 1 cm. (E) The newly formed axillary shoots (as indicated in D) were excised again for multiplication. After 3 months of subculture, more shoot multiplication can be observed. Bar = 1 cm.

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    Rooting and acclimatization of plantlets. (A) Shoots were excised and cultured on the rooting medium in the Magenta GA-7 plant culture box. Bar = 1 cm. (B) After 4 months of culture, plantlets with well-developed roots were ready to take out of flasks. Bar = 2 cm. (C) After 3 months of culture in the greenhouse, the vigorous plantlets with newly emerged leaves could be observed. Bar = 2 cm.

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