Plant Cell Reports (1993) 13:7 11
Plant Cell
Micropropagation of Phalaenopsis and Doritaenopsis
by culturing shoot tips of flower stalk buds
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
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
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
Doritaenopsis.
Abbreviations: PLB: protocorm-like body, PGR: plant growth
regulator, NAA: ct-naphthateneacetic acid, BAP: 6-benzylaminopurine,
NDM: New Dogashima Medium
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
Correspondence to: K. T o k u h a r a
Reports
9 Springer-Verlag 1993
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.
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
8

Table 1. List of culture media tested for the orchid cultivars used in the 'lhble 2. Components of NDM (mg 1-1) used for orchid tissue culture.
experiments.
Macro elements

Cultivarl) Major2~ PGR (mg 11) Sucrose G e l l i n g NFI4NO3 480

KNO3 200
elements NAA BA (g 1-t) agent
Ca(NO3)2.4H20 470
AD NDM, 1/2 MS 0.1 1- 5 10 Gelrite KCI 150
MA NDM, 1/2 MS 0.1 1 10 Gelrite MgSO4 9717120 250
PA NDM, 1/2 MS 0.1 1 10 Gelrite I ~ 21:~4 550
CS NDM, 1/2 MS 0.1 1 10 Gelrite Micro elements (modified Nitsch 1956)
OD NDM 0 - 20 0 - 20 20 Agar
MnSO4 94H20 3
CL NDM 0.1 1- 5 10 Gelrite ZnSO4,7H20 0.5
GP NDM 0.1 1- 5 10 Gelrite H3B04 0.5
GC NDM 0.1 1- 5 10 Gelrite ONO4 951-I20 0.025
C-O NDM 0.1 I - 5 10 Gelrite Na 2MOO4.2/-/20 0.025
CY NDM 0.1 1- 5 10 Gelrite COC12 96H20 0.025
CD NDM 0.1 1- 5 10 Gelrite conc, H2SO4 0.5 ml 1-I

GCi NDM 0.1 1- 5 10 Gelrite Organic compounds (modified Morel & Wetmore 1951)
1) AD: D. Amagi Dancer, MA: P. Mai, PA: P. Passion Art, CS: D. myo-Inositol 100
Chieko Serenity, OD: D. Odoriko cv. Nishiizu, CL: P. Crystal Lady, GP: Niacin 1.0
P. Grace Palm, GC: P. Grace Chinghua, GO: P. gigantea x D. Odoriko, Pyridoxine hydrochloride 1.0
CY: P. (Crescent x Yuuzuru), CD: P. Casablanca Dandy, GCi: P. Grand Thiamine hydrochloride 1.0
City. Calcium pantothenate 1.0
2) NDM = New Dogasfiima Medium (Table 2); 1/2 MS = half-strength Adenine 1.0
major elements of Murashige and Skoog (1962). 1-Cystein 1.0
d-Biotin, cryst. 0.1

Fe-EDTA 21

15 rain at 120 ~ To determine the optimum concentration and

combination of PGR, the NDM was supplemented with NAA (0 - 20 mg
14) and BAP (0 - 20 mg 1-t) singly or in combinations (Table 3). These
media were supplemented with 20 g 1-1 sucrose and solidified with 8 g 1 -t
agar. In another experiment designed to test genotypic difference, NDM
was supplemented with 10 g l -t sucrose, 2 g 1q Gelrite and 0.1 mg 1-1
/ NAA in combination with 1, 2 or 5 mg 1-1 BAR The cultures were
incubated at 23 + 1 ~ under 14 h photoperiods provided by fluorescent
lamps (Toshiba FLR40S W/M/36) at 33 Nnol m-Zs-1. After 3 and 5
months of culture, survival percentage and PLB formation of explants
were recorded. Throughout the experiments, 10 to 30 explants were
/1
cultured for each treatment. In the experiment to test genotypic
difference in PLB formation, each treatment was replicated 3 times.

Results and discussion

~ Effect o f basal medium

F o u r cultivars w e r e used to d e t e r m i n e the effects o f d i f f e r e n t

C D E e x p l a n t s ( T a b l e 4). M o s t e x p l a n t s o n 1/2 M S m e d i u m
Fig. 1. Procedures for removing explants from flower stalk buds of
Phalaenopsis. A: Plant excised with an elongating flower stalk, B:
Cutting of node with internodal segment 1,5 cm above and 2.5 cm
below bud, C: Removal of bract from flower stalk bud, D: Removing
scaly leaves from axillary bud of flower stalk, E: Shoot tip explant, 1
rrma wide and 0.5 mm high.
b a s a l m e d i a o n s u r v i v a l rate a n d P L B f o r m a t i o n o f s h o o t tip
showed necrosis within 1 month of the start of culture
e x c e p t for the c e n t r a l m e r i s t e m a t i c r e g i o n w h i c h r e m a i n e d
green. H o w e v e r , e x p l a n t s o n N D M r e m a i n e d m o s t l y g r e e n
without necrosis. After 3 months of culture, survival rates
w e r e m o r e t h a n 7 3 % o n N D M b u t 12 - 2 0 % o n 1/2 M S
(Table 4). P L B f o r m a t i o n o n N D M w a s also h i g h e r t h a n
t h a t o n 1/2 M S ; 7 - 4 0 % on N D M a n d less t h a n 1 0 % o n
 9

1/2 MS. Therefore, NDM was selected as a basal medium Table 3. Effects of NAA and BAP on survival and PLB formation of
orchid shoot tip explants after 5 months of culture.
for subsequent experiments.
In the previous study on Phalaenopsis, Zimmer and Pieper No. of Survival PLB Color 1) PLBs 2) Shoot
NAA BAP explants rate (%) formation of PLBs after formation
(1978) failed to induce PLB in cultures of axillary buds from (%) subculture (%)
flower stalks using Knudson C medium. In contrast, 0 8 100 38 P.G n 0
Ichihashi (1992) succeeded to induce PLB from the same 0.1 14 57 29 P.Y d 14
explants by systematic investigation of major ions, and 1 13 46 8 P.Y n 15
0
5 18 28 11 P.Y n 11
developed a medium with optimal mineral composition for
10 16 13 6 P.Y d 0
the culture. These results suggest that selection of 20 15 0 0 0
appropriate mineral composition of the media is essential ]'3- - - -38" - - ~ 1 - - - - ~.'~ - - "d . . . . O- - "
for the successful PLB formation in Phalaenopsis. 0.1 19 37 5 P.G m 0
1 16 100 33 G m 28
0.1
Effect of PGR 5 15 93 40 G m 27
10 20 20 15 G s 0
Table 3 shows the frequency of explant survival and PLB 2O 18 45 6 G s 6

induction in D. Odoriko cv. Nishiizu on media containing 0 i7- - - 47 - - -0- . . . . . . . . . 77- -"

different concentrations of NAA and BAR Survival rates 0.1 t0 100 0 40

1 8 1 O0 22 P.Y n 22
decreased with increasing BAP or NAA concentrations 1
5 19 42 26 G s 16
except for the combinations of 20 mg 1-t NAA and 0 - 0.1 10 15 40 20 P.Y d 20
mg 1 t BAR At high concentrations of both components 20 10 70 60 P.Y n 0
(combinatiots of 10 - 20 mg 1-1 NAA and 1 - 20 mg 1-1 0 17 18 18 P.Y n 0
BAP), most explants became necrotic and died. 0.1 13 46 15 P.Y d 15
The highest frequency of PLB formation was 60% on a 1 12 33 25 P.Y d 0
5
5 20 30 0 0
medium containing 0.1 mg 1-1 NAA and 20 mg 1-1 BAR
10 9 67 0 0
More than 30% of the explants produced PLB on media 2O 21 24 19 P.Y n 0
containing 0.1 mg 1-1 NAA with 0, 1 or 5 mg 1-1 BAR and 0 -9- -- -89-- - -0- . . . . . . . . . . 0- -"
containing 20 mg 1-1 NAA alone. PLB were induced from 0.1 7 86 0 0
cut surfaces on the basal part of explants and from inside of 10
1 8 63 25 P.Y n 0
the bases of leaf primordia. Greenish PLB were induced on 5 12 25 0 0
10 7 14 0 0
combinations of 0.1 mg 1-1 NAA with 1 - 20 mg 1-1 BAR
20 9 0 0 0
and 1 mg 1-1 NAA with 5 mg 11 BAR White and yellowish
7o- -- %& - - - - W- - . . . . o- -"
PLB were induced on other combinations. 0.1 10 60 30 W d 0
In the previous studies, natural organic compounds such as 1 10 0 0 0
20
coconut water or the bleeding sap of birch trees have been 5 10 0 0 0
10 10 0 0 0
used as important constituents of culture media for
2O 10 0 0 0
micropropagation of Phalaenopsis (Intuwong and Sagawa
1) RY: pale yellow, P.G: pale green, G: green, W: white.
1974, Zimmer and Pieper 1978, Homma and Asahira 1985,
2) n: no change, d: death, m: multiplication, s: direct shoot formation.
Tanaka and Sakanishi 1977, Hass-Von Schmude 1984). Very
recently, Ichihashi (1992) also reported the importance of
coconut water for inducing PLB formation from shoot tip
explants of Phalaenopsis without using PGR. However, the
rate of PLB formation he obtained was not as high as we Table 4. Influence of different basal media on survival rate and PLB
formation of orchid explants after 3 months of culture.
achieved in the present study. Moreover, he also observed
inhibitory effects of coconut water in.some cultivars. The Cultivar I) N) MA PA CS
use of natural organic compounds is also known to
Medium NDM 1/2MS NDM 1/2MS NDM 1/2MS NDM 1/2MS
influence reproducibility of the experimental results (George
and Sherrington 1984). Therefore, the use of PGR might be No. of
26 20 14 17 18 15 30 30
indispensable for the induction of PLB from shoot tip explants

explants in Phalaenopsis as shown in the present study. Survival

(%) 73.1 20 85.7 11.8 94.4 13.3 83.3 13.3

PLB proliferation PLB (%) 26.9 0 7.1 0 16.7 6.7 40 10

formation

Five months after initiation of shoot tip cultures, each PLB 1) See Table 1 for cultivar names.
10

Fig. 2. Micropropagation of Phalaenopsis from axillary buds of flower stalks. A: Explant with 1 - 2 leaf primordia, B" Induced PLB, C:
Proliferating PLB, D : Development of shoots from PLB, E: Plantlets before transplanting in pots, F: Flowering plants derived from a
flower stalk bud explant.
 11
'Ihble 5. Genotypic differences in survival rate and frequency of PLB formation in orchid tissue cultures.

BAP 1.0 BAP 2.0 BAP 5.0

Cultivar t)
Survival 2) PLB 3) Survival PLB Survival PLB

CL 83.3 +11.8 0 66.7 + 0 50.0+11.8 100 + 0 60.0+ 5.9*

GP 33.3 + 0 33.3+23.6 100 + 0 75.0+11.8 85.7 + 8.8 4 2 . 9 + 5.9
GC 75.0 + 17.7 50.0+ 0 75.0 + 17.7 50.0+ 0 50.0 + 0 50.0 + 0
(33 100 + 0 71.4+ 5.8 71.4 + 5.8 57.1 + 5.9 100 + 0 57.1 + 5.9
CY 75.0 + 5.9 58.3 + 5.9 76.9 + 4.2 46.2_.+ 2.5 64.3 + 5.1 21.4 + 5.1
133 85.0 + 3.5 3 5 . 0 + 3.5 100 + 0 60.0+ 7.1 t00 + 0 52.0+ 1.6"
GCi 77.4 + 2.0 51.6 + 4.8 29.4 + 1.6 5.9 + 2.4 32.4 + 2.2 11.8 + 2.6
AD 73.1 + 2.7 26.9 +_ 4.1 80.8 + 2.6 19.2 + 3.5* 80.8 + 8.0 3.8 + 3.4*
1) See Table 1 for cultivar names.
2) Frequency of surviving explants.
3) Percentage of explants producing PLBs with pale yellow color (*) and those with green (no *).
The values represent the mean (+ S.E) of 3 independent experiments. Ten explants were used for each experiment. NDM was used with 0.1
m g 1-1 N A A and with cytokinin as indicated.

mass (5 mm diameter) was transferred onto the same
medium as the one used for PLB induction and subcultured
monthly. After one month of subculture, white and yellow
PLB did not increase in fresh weight nor in number of
PLBs, and died following repeated subculture. In contrast,
green PLB survived after subculture and produced shoots
without PLB proliferation on most of the media. However,
when PLB induced on a combination of 0.1 mg 1-a NAA and
1 mg 1-1 BAP were cut into small pieces and subcultured
monthly on the same medium, more than 10,000 PLBs
were obtained from a few buds on a single flower stalk
within one year.
It is well known that micropropagation sometimes induces
somaclonal variations, which can cause severe problems in
commercial production systems. It has been considered that
the use of PGR m a y cause somaclonal variations in various
plant species (Vajrabhaya 1977, George and Sherrington
1984). However, we showed in a previous report that the
frequency of variations in micropropagated plants by
utilizing the method established in the present study was
extremely low (0 - 6%) (Tokuhara 1992). Therefore, the
rapid method of micropropagation with the use of PGR
established in the present study should be applicable to a
wide range of cultivars of Phalaenopsis.

Genotypic difference
Table 5 shows survival rates and frequency of PLB induction
of 8 cultivars on NDM supplemented with 0.1 mg 1-1 NAA
in combination with 1, 2 or 5 mg 1-t BAP. Optimum
concentration of BAP was similar for most cultivars at 1 - 2
mg 1-1. At 5 mg 1-I BAP, proliferation of pale yellow callus
occurred infrequently. All cultivars except AD showed more
than 50% PLB formation on the optimal BAP
concentration.
Plant regeneration and somaclonal variation
The PLB multiplied for more lhan one year on the optimum
culture medium showed differentiation of shoots and roots
following transfer onto a PGR-free medium. Thus, more
than 10,000 plantlets ready for transplanting to pots were
produced from a single flower stalk within 2 years after the
initiation of shoot tip cultures. The plantlets thus obtained
grew vigorously after transfer to sphagnum in pots, and
flowered one year after transfer to pots.
References
George EF, Sherrington PD (1984) Plant propagation by tissue culture.
Exegetics Ltd, Eversley, England
Hass-Von Schmude NF (1984) In: Tan KW (ed) Proc 11 th World Orchid
Conference, Miami, Florida, p 311
Homma Y, Asahira T (1985) J Japan Hort Sci 54:379-387
Ichihashi S (1992) Lindleyana 7:208-215
lntuwong O, Kunisaki JT, Sagawa Y (1972) Na Okika of Hawaii 1:13-18
lntuwong O, Sagawa Y (1974) Amer Orchid Soc Bull 43:893-895
Lin CC (1986) Lindteyana 1:158-163
Morel G, Wetmore RH (1951) Amer J Bot 38:141-143
MurashigeT, Skoog F (1962) Physiol Plant 15:473-497
Nitsch JP, Nitsch C (1956) Amer J Bot 43:839-851
Reisinger DM, Ball EA, Arditti J (1976) The Orchid Review 84:45-52
Tanaka M, Sakanishi Y (1977) Amer Orchid Soc Bull 46:733-737
Tanaka M, Senda Y, Hasegawa A (1976) Amer Orchid Soc Bull 45:1022-
1024
%kuhara K (1992) Ichihashi S, Nagata H (eds) Proc Nagoya Inlernat
Orchid Show '92, pp 317-319
Vajrabhaya T (1977) In: Orchid Biology 1. Arditti J (ed) Cornell Univ
Press, Ithaca, New York, pp 179-201
Zimmer K, Pieper W (1978) The Orchid Review 86:223-227