Plant Cell Repor ts (1993) 13:7 11 Plant Cell Reports �9 Springer-Verlag 1993

Micropropagation of Phalaenopsis and Doritaenopsis by culturing shoot tips of flower stalk buds

Ken Tokuhara 1 and Masahiro Mii 2

1 Research and Deve lopment Center , Orchid Sanctuary Dogash ima , Nishina , Nishi izu-cho, K a m o - g u n , Shizuoka 410-35, Japan 2 Faculty of Horticulture, Chiba University, Matsudo, Chiba 271, Japan

Received 1 Februa ry 1993/Revised vers ion 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.

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 b r e v i a t i o n s : PLB: protocorm-like body, PGR: plant growth

regulator, NAA: ct-naphthateneacetic acid, BAP: 6-benzylaminopurine,

NDM: New Dogashima Medium

I n troduc t ion

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

Materials and methods

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 experiments.

Cultivarl) Major2~ PGR (mg 1 1) Sucrose Gel l ing

e lements NAA BA (g 1 -t) agent

AD NDM, 1/2 MS 0.1 1 - 5 10 Gelrite

MA NDM, 1/2 MS 0.1 1 10 Gelrite

PA NDM, 1/2 MS 0.1 1 10 Gelrite

CS NDM, 1/2 MS 0.1 1 10 Gelri te

OD NDM 0 - 20 0 - 20 20 Agar

CL NDM 0.1 1 - 5 10 Gelrite

GP NDM 0.1 1 - 5 10 Gelri te

GC NDM 0.1 1 - 5 10 Gelrite

C-O NDM 0.1 I - 5 1 0 Gelrite

CY NDM 0.1 1- 5 10 Gelrite

CD NDM 0.1 1 - 5 10 Gelrite

GCi NDM 0.1 1 - 5 1 0 Gelri te

1) AD: D. Amagi Dancer, MA: P. Mai, PA: P. Passion Art, CS: D. Chieko Serenity, OD: D. Odoriko cv. Nishiizu, CL: P. Crystal Lady, GP: P. Grace Palm, GC: P. Grace Chinghua, GO: P. gigantea x D. Odoriko, CY: P. (Crescent x Yuuzuru), CD: P. Casablanca Dandy, GCi: P. Grand City.

2) NDM = New Dogasfiima Medium (Table 2); 1/2 MS = half-strength major elements of Murashige and Skoog (1962).

'lhble 2. Components of NDM (mg 1-1) used for orchid tissue culture.

Macro elements

NFI 4NO3 480

KNO3 200

Ca(NO3)2.4H20 470

KCI 150 MgSO4 �9 717120 250 I ~ 21:~4 550

Micro elements (modified Nitsch 1956)

MnSO4 �9 4H20 3 ZnSO4,7H20 0.5

H3B04 0.5

ONO4 �9 51-I20 0.025 Na 2MOO4.2/-/20 0.025 COC12 �9 6H20 0.025

conc, H2SO4 0.5 ml 1 -I

Organic compounds (modified Morel & Wetmore 1951)

myo-Inositol 100 Niacin 1.0 Pyridoxine hydrochloride 1.0 Thiamine hydrochloride 1.0 Calcium pantothenate 1.0 Adenine 1.0 1-Cystein 1.0 d-Biotin, cryst. 0.1

Fe-EDTA 21

/

/1

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 1 q 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 cultured for each treatment. In the experiment to test genotypic difference in PLB formation, each treatment was replicated 3 times.

~

C D E

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.

Results and discussion

Effect o f basal medium

F o u r cul t ivars were used to d e t e r m i n e the effects o f d i f fe ren t

basa l m e d i a o n surv iva l ra te and P L B f o r m a t i o n of shoo t t ip

e x p l a n t s (Tab le 4). M o s t e x p l a n t s on 1/2 M S m e d i u m

s h o w e d n e c r o s i s w i t h i n 1 m o n t h o f the s ta r t o f cu l t u r e

e x c e p t for the cen t ra l m e r i s t e m a t i c r eg ion w h i c h r e m a i n e d

green. H o w e v e r , exp lan t s on N D M r e m a i n e d m o s t l y g r e e n

w i t h o u t nec ros i s . Af t e r 3 m o n t h s o f cu l ture , su rv iva l ra tes

we re m o r e t han 7 3 % on N D M bu t 12 - 2 0 % on 1/2 M S

(Table 4). P L B f o r m a t i o n on N D M was a lso h i g h e r t han

tha t on 1/2 M S ; 7 - 4 0 % on N D M a n d less than 10% on

1/2 MS. Therefore, NDM was selected as a basal medium

for subsequent experiments. In the previous study on Phalaenopsis, Zimmer and Pieper

(1978) failed to induce PLB in cultures of axillary buds from flower stalks using Knudson C medium. In contrast, Ichihashi (1992) succeeded to induce PLB from the same explants by systematic investigation of major ions, and developed a medium with optimal mineral composition for the culture. These results suggest that selection of appropriate mineral composition of the media is essential for the successful PLB formation in Phalaenopsis.

Effect of PGR

Table 3 shows the frequency of explant survival and PLB

induction in D. Odoriko cv. Nishiizu on media containing

different concentrations of NAA and BAR Survival rates decreased with increasing BAP or NAA concentrations except for the combinations of 20 mg 1 -t NAA and 0 - 0.1 mg 1 t BAR At high concentrations of both components (combinatiots of 10 - 20 mg 1-1 NAA and 1 - 20 mg 1-1 BAP), most explants became necrotic and died.

The highest frequency of PLB formation was 60% on a medium containing 0.1 mg 1-1 NAA and 20 mg 1-1 BAR More than 30% of the explants produced PLB on media containing 0.1 mg 1-1 NAA with 0, 1 or 5 mg 1-1 BAR and

containing 20 mg 1-1 NAA alone. PLB were induced from cut surfaces on the basal part of explants and from inside of the bases of leaf primordia. Greenish PLB were induced on combinations of 0.1 mg 1-1 NAA with 1 - 20 mg 1-1 BAR

and 1 mg 1-1 NAA with 5 mg 1 1 BAR White and yellowish

PLB were induced on other combinations.

In the previous studies, natural organic compounds such as

coconut water or the bleeding sap of birch trees have been used as important constituents of culture media for

micropropagation of Phalaenopsis (Intuwong and Sagawa 1974, Zimmer and Pieper 1978, Homma and Asahira 1985,

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 achieved in the present study. Moreover, he also observed inhibitory effects of coconut water in.some cultivars. The

use of natural organic compounds is also known to influence reproducibility of the experimental results (George and Sherrington 1984). Therefore, the use of PGR might be indispensable for the induction of PLB from shoot tip

explants in Phalaenopsis as shown in the present study.

PLB proliferation

9

Tab le 3. Effects of NAA and BAP on survival and PLB formation of

orchid shoot tip explants after 5 months of culture.

No. of Survival PLB Color 1) PLBs 2) Shoot NAA BAP explants rate (%) formation of PLBs after formation

(%) subculture (%)

0

0.1

1 0

5 10

20

0.1

1 0.1

5

10

2O

0

0.1 1

1 5

10

20

0

0.1

1 5

5

10 2O

0

0.1

1 10

5

10

20

0.1

1 20

5

10

2O

8 100 38 P.G n 0

14 57 29 P.Y d 14

13 46 8 P.Y n 15

18 28 11 P.Y n 11

16 13 6 P.Y d 0

15 0 0 0

] '3 - - - -38" - - ~1 - - - - ~. '~ - - "d . . . . O- - "

19 37 5 P.G m 0

16 100 33 G m 28

15 93 40 G m 27

20 20 15 G s 0

18 45 6 G s 6

i7- - - 4 7 - - - 0 - . . . . . . . . . 7 7 - - "

t 0 100 0 40 8 1 O0 22 P.Y n 22

19 42 26 G s 16

15 40 20 P.Y d 20

10 70 60 P.Y n 0

17 18 18 P.Y n 0

13 46 15 P.Y d 15

12 33 25 P.Y d 0

20 30 0 0

9 67 0 0 21 24 19 P.Y n 0

- 9 - - - -89- - - - 0 - . . . . . . . . . . 0 - - "

7 86 0 0

8 63 25 P.Y n 0

12 25 0 0

7 14 0 0

9 0 0 0

7o- -- %& - - - - W- - . . . . o- -"

10 60 30 W d 0

10 0 0 0

10 0 0 0

10 0 0 0

10 0 0 0

1) RY: pale yel low, P.G: pale green, G: green, W: white.

2) n: no change, d: death, m: mult ipl icat ion, s: direct shoot formation.

Tab le 4. Influence of different basal media on survival rate and PLB

formation of orchid explants after 3 months of culture.

Cult ivar I) N ) MA PA CS

Medium NDM 1/2MS NDM 1/2MS NDM 1/2MS NDM 1/2MS

No. of 26 20 14 17 18 15 30 30

exp lan t s

Survival (%) 73.1 20 85.7 11.8 94.4 13.3 83.3 13.3

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.

'Ihble 5. Genotypic differences in survival rate and frequency of PLB formation in orchid tissue cultures.

11

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 3 3 . 3 + 2 3 . 6 100 + 0 75 .0+11 .8 85.7 + 8.8 42 .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 35 .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 mg 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.

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 format ion 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.

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 may 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.

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

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%kuhara K (1992) Ichihashi S, Nagata H (eds) Proc Nagoya Inlernat

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