Micropropagation of Phalaenopsis and Doritaenopsis

Plant Cell Reports (1993) 13:7
11
Plant Cell Reports
9 Springer-Verlag 1993
Micropropagation of Phalaenopsis and Doritaenopsis by culturing shoot tips of flower stalk buds
Doritaenopsis.
Abbreviations:
PLB: protocorm-like body, PGR: plant growth
axillary buds (lntuwong et al. 1972, Reisinger et al. 1976), meristems (Intuwong and Sagawa 1974), shoot tips of flower stalk buds (Zimmer and Pieper 1978, Ichihashi 1992), internodal segments of flower stalks (Homma and Asahira 1985, Lin 1986), leaf segments (Tanmka and Sakanishi 1977, Hass-Von Schmude 1984) and root tips (Tanaka et al. 1976). However, not all of these methods can be used for commercial micropropagation because of differences in survival rate, PLB formation, and plantlet regeneration. Moreover, PLB obtained through some of these methods did not proliferate readily and their viability was low. In the present study, we examined the effects of several concentrations and combinations of PGR with a view toward developing a practical micropropagation method for these orchids.
A b s t r a c t . Green Protocorm-like Bodies (PLB) with high
multiplication capacity were induced from shoot tips of flower stalk buds having 1 or 2 leaf primordia using New Dogashima Medium (NDM) containing 0.1 mg 1~t o~naphthaleneacetic acid (NAA) and 1 mg 1-1 6 benzylaminopurine (BAP). These PLB were subcultured on the same medium. More than 10,000 PLBs were obtained from a few buds on a single flower stalk within one year. After transfer onto NDM containing no plant growth regulator (PGR), the PLB developed into plantlets. The micropropagation method formulated in this study was applicable to 12 different genotypes. These results suggest that the methodology could be used on a commercial scale for vegetative propagation of Phalaenopsis and
Ken Tokuhara 1 and Masahiro Mii 2
1 R e s e a r c h a n d D e v e l o p m e n t Center, Orchid S a n c t u a r y D o g a s h i m a , Nishina, Nishiizu-cho, K a m o - g u n , Shizuoka 410-35, J a p a n
Introduction
Pot plant and cut flower production of Phalaenopsis-orchids, including cultivars of Phalaenopsis and its intergeneric hybrid with Doritis, Doritaenopsis, have increased greatly in recent years. Unlike other orchids such as Cymbidium, Dendrobium and Cattleya, most of the commercially cultivated Phalaenopsis plants are produced from seeds and are heterozygous; micropropagation has not been feasible due to technical difficulties. Consequently, there is a great deal of variability in plant growth, flowering time and flower characteristics. This variability leads to increased production costs which reduce profits. Several micropropagation methods have been developed for Phalaenopsis, including the culture of flower stalks with
2 Faculty of Horticulture, Chiba University, Matsudo, Chiba 271, Japan
Received 1 F e b r u a r y 1993/Revised version received 13 A u g u s t ] 993 - C o m m u n i c a t e d by G. C. Phillips
regulator, NAA: ct-naphthateneacetic acid, BAP: 6-benzylaminopurine, NDM: New Dogashima Medium
Materials and m e t h o d s
Greep2~ouse-grown plants of 12 cultivars listed in Table 1 were used as materials. The scheme for isolating explants is shown in Fig. 1. Xdgetative buds bearing flower stalks were removed from the plants (Fig. 1A). And nodes were excised leaving 1.5 cm above and 2.5 cm below the bud (Fig. 1B). The bracts which covered the buds were removed (Fig. IC). And the segments were surface disinfected with the supernatant of 5% Ca(CIO)z solution (ca. 1% available chlorine) containing a few drops of Tween-20 and slirred for 15 rain. Following surface sterilization, the segments were washed once with sterile distilled water. After removing 2 or 3 scaly leaves from the buds (Fig. ID), shoot tip explants 1 mm wide and 0.5 mm high with 1 or 2 leaf primordia were excised from the flower slalk segments with a surgical knife (Fig. 1E). Explants were then placed with the cut side down on 15 ml of solid medium in 24 mm x 100 mm test tubes which were capped with aluminum foil. Two basal media were used; (1) NDM (Table 2), which was developed by us for the culture of several orchids, and (2) modified MS medium (Murashige and Skoog 1962) containing half-strength major elements (1/2 MS). These media were supplemented with 0.1 mg 1-1 NAA, 1 mg 1-1 BAP and 10 g 14 sucrose and solidified with 2 g 1-1 Gelrite. The pH of the media was adjusted to 5.4 prior lo sterilization in an autoclave for