Factors affecting the growth of in vitro cultured lateral buds from Phalaenopsis flower stalks

Scientia Horticulturae, 8 (1978) 169---178

169

Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

FACTORS AFFECTING THE GROWTH OF IN VITRO CULTURED LATERAL BUDS FROM PHALAENOPSIS FLOWER STALKS

MICHIO

TANAKA

and YOSHIHIRO

SAKANISHI

Laboratory of Floriculture, College of Agriculture, University of Osaka Prefecture, Mozu-umemachi, Sakai, Osaka 591 (Japan) (Received 19 July 1977)

ABSTRACT Tanaka, M. and Sakanishi, Y., 1978. Factors affecting the growth of in vitro cultured lateral buds from Phalaenopsis flower stalks. Scientia Hortic., 8: 169--178.

Phalaenopsis flower-stalk buds cultured in vitro show 3 modes of growth: dormant, vegetative and reproductive. The effects of bud position on the stalk, temperature, and benzyladenine (BA) on the mode of bud growth were examined. Flower stalks were cut into 1-node sections, each with 1 bud, and inserted into solid culture medium, top side up. Buds on the upper sections had a tendency to remain dormant regardless of temperature. Sprouting buds placed at 20°C or 25°C showed reproductive growth except for some on the basal sections which grew vegetatively. At 28°C, all buds developed vegetatively independent of their original position on the stalk. The buds on the sections taken from both stalks Which had elongated at rain 18°C and rain 28°C were subcultured; the mode of growth of the cultured buds was not affected by the temperature during flower-stalk elongation but was affected by the subsequent culture temperature. Cultured buds which had remained dormant were stimulated to sprout by addition of BA to the medium. At 5 p.p.m, and above, all buds began to develop, but malformations developed on the leaves of the shoots. All factors considered, BA at 2.5 p.p.m, was found to be the optimum level. The further development of the buds released from dormancy by BA was also affected by the position on the flower stalk and by cultural temperature.

INTRODUCTION T h e r e are several n o d e s o n t h e b a s a l p a r t o f t h e Phalaenopsis f l o w e r s t a l k w h i c h h a v e d o r m a n t l a t e r a l buds. I n d u c i n g p l a n t l e t s in v i t r o b y c u l t u r i n g t h e b u d , w i t h a p i e c e o f s t e m b e l o w a n d a b o v e it, w a s t r i e d f o r t h e f i r s t t i m e b y R o t o r (1949). This m e t h o d was m o d i f i e d b y m a n y later workers, and has b e e n u s e d as t h e m a j o r m e t h o d f o r t h e c l o n a l p r o p a g a t i o n of Phalaenopsis (Sagawa and Niimoto, 1960; Sagawa, 1961; K o t o m o r i and Murashige, 1965; U r a t a a n d I w a n a g a , 1 9 6 5 ; S c u l l y , 1 9 6 6 ; D a e r r , 1 9 6 7 ; T s e e t al., 1 9 7 1 ; I n t u w o n g e t al., 1 9 7 2 ; T e w s , 1 9 7 4 ) . H o w e v e r , in c u l t u r i n g t h e l a t e r a l b u d s e g m e n t s in v i t r o , a r a t h e r h i g h p e r centage of them remains dormant, while others may develop into flower stalks or vegetative shoots. These different growth responses of the cultured lateral

170

bud are shown in Fig. 1. Thus, this m e t h o d of propagation yields, in practice, a relatively small number of plantlets. In our studies on Phalaenopsis propagation by means of leaf tissue culture, we have attempted to obtain a high frequency of vegetative growth from nodal cuttings. The experiment included, first, p r o m o t i o n o f a flush of development in cultured lateral buds, and second, finding optimum cultural conditions to stimulate sprouting buds into vegetative growth. This paper reports our results, which were obtained from 1974 to 1976. GROWTH PATTERNS

wingvegetatively Flowerstalk ~

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Fig. 1. Diagrams showing different growth patterns in lateral buds of flower-stalk nodal cuttings cultured in vitro. MATERIAL AND METHODS

The stock plants used were 5--6 year old Phalaenopsis amabilis hybrids, grown in a greenhouse maintained at a m i n i m u m temperature of 18°C. Flower stalks were excised from stock plants during the period t h a t the first to fifth florets reached anthesis. T h e y were wiped 3 times with gauze previously dipped in 70% ethyl alcohol, and then sliced into nodal cuttings, each consisting of 1 lateral bud and 3 cm of flower stalk tissue above and below it. Each bud

171 section was surface~disinfected for 10 min in a filtered suspension of 7% calcium hypochlorite containing 0.1% of Tween-20. The materials were rinsed 3 times with sterile water and the bracts at the nodes were removed with forceps under sterile conditions. Subsequently, the bleached tissue, 0.5--1.0 cm long at both ends of the sections, was cut away with a razor blade. The nodal cuttings were inserted vertically with their morphological bases in the nutrient medium, about 1.5 cm of each section being immersed. The basal medium used was Vacin and Went's (1949) supplemented with 1.0% agar and 2.0% sucrose, and adjusted to pH 5.3 prior to autoclaving. When c o c o n u t water (CW) or benzyladenine (BA) was used, they were added to the medium prior to autoclaving (1.2 kg/cm 2 at 121°C for 15 min). C o c o n u t water was drained from locally purchased ripe nuts, filtered and stored at --18°C. BA was dissolved with 1 N NaOH. Test tubes (24 × 200 mm), each containing 16 ml of the medium, were used as culture vessels. After the bud sections were implanted, the tubes were closed with aluminium foil a t t a c h e d firmly with rubber bands. The cultures were maintained in a controlled-temperature r o o m u n d e r 16-hour light of approximately 500 lux provided by plant-growth fluorescent lamps (Plant-Lux, T o k y o Shibaura Electric Co., Ltd.). RESULTS E f f e c t o f cultural temperature. -- Lateral bud sections, obtained from different positions on the flower stalks, were cultured at 28, 25, or 20°C. The medium used was Vacin and Went's (VW) supplemented with CW 20% (v/v). At each temperature, some buds remained d o r m a n t (Fig. 2). A higher rate of d o r m a n c y occurred in those buds excised from the highest position on the flower stalk. In sprouting buds at 20°C, the buds showed reproductive growth except for a few taken from the lower position on the flower stalk. The results obtained at 25°C differed from those at 20°C in t h a t the proportion of buds growing vegetatively increased slightly. At 28°C all the developing buds were vegetative, irrespective of their original position on the flower stalk (Fig. 2). Phalaenopsis aphrodite Rchb. f. responded similarly to P. amabilis hybrid. Flower differentiation was observed in the shoots developing from nodal cuttings kept at 20°C. All shoots observed after 90 days of culture had florets at various stages of development. The node number of the first floret was lower, and the developmental stage of every floret differentiated was more advanced in shoots from upper nodal cuttings t h a n from lower ones. The average node number, from the base, of the first floret was 5.0, 6.0, 6.2 and 6.5 in the case of the uppermost, second, third and lowest nodal cutting, respectively. When reproductive shoots, obtained at 20°C or 25°C, were maintained at the same temperature for 1 year, branching occurred from the basal, second or third node of the shoot. At 20°C, 67% of the cultured reproductive shoots

172

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173

TABLE 1

Growth of cultured buds of nodal cuttings taken from flower stalks elongated at different temperatures. Results at end of 15 weeks culture. Minimum temperature

Temperature during bud culture (°C)

No. of buds cultured

18

28 20

24 24

28

28 20

20 20

during flower stalk elongation

No. of buds surviving

Percentage of buds Growing into

Remaining dormant

Vegetative shoot

Reproductive shoot

20 21

65 5

0 52

35 43

14 15

57 6

0 47

43 47

(°c)

the flower stalk. We have attempted to p r o m o t e sprouting of these buds by adding BA to the basic VW medium w i t h o u t CW. The cultures were maintained at 28°C. The results are shown in Fig. 3. The sprouting-rate of the buds with the addition of BA at 0.50 p.p.m, was similar to t h a t with the addition of CW. Furthermore, with increased concentrations of BA, the sprouting-rate of d o r m a n t buds increased progressively. High concentrations of BA were necessary for buds excised from a higher position on the flower stalk. When BA at 5.00 p.p.m, or 10.00 p.p.m, was added to the VW medium, almost all buds sprouted and grew vegetatively, except for a few taken from the upper position o f a flower stalk, which grew reproductively. However, m o s t of the vegetative shoots had either malformations on the leaves or in some cases unexpanded or twisted leaves. The development of more than 2 shoots from 1 node was occasionally observed in the medium containing 5.00 or 10.00 p.p.m. BA. Finally, buds were cultured at 20°C or 28°C on the medium which conrained BA at 2.5 p.p.m. At both temperatures, the percentage of buds which failed to sprout was very low. Buds sprouting at 20°C, which had been excised from the uppermost position on the flower stalk, were reproductive. Conversely, the growth was vegetative for buds excised from lower positions. On the other hand, at 28°C all the buds grew vegetatively (Fig. 4). These buds which grew vegetatively at 2.5 p.p.m. BA did n o t show any malformations as observed previously on the medium which contained BA at 5.0 p.p.m, or more. DISCUSSION

A d o r m a n t bud is present in each of 3--6 nodes of the flower stalk below the inflorescence of Phalaenopsis. In P. amabilis and P. aphrodite, most of the buds remain d o r m a n t unless the tip of the shoot is removed, although sometimes the upper buds will develop into secondary flowering shoots. When

174

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the tip is removed, the uppermost bud often grows to become a secondary flowering shoot. In P. schilleriana, however, some buds grow to be a secondary flowering shoot under appropriate conditions, w i t h o u t tip removal. Intuwong and Sagawa (1974) observed in P. cornu-cervi and P. lueddemanniana that a plantlet developed at the upper node of flowering shoots after anthesis. In past studies on flower-stalk node cultures of Phalaenopsis, it was shown that some o f the buds developed into vegetative or reproductive (flower stalks) shoots, while others remained dormant. However, the percentage of each m o d e of growth was different for different workers, and the effect of cultural conditions on the growth of the buds has n o t been previously studied in detail. Urata and Iwanaga (1965) and Koch (1974) showed that reproductive shoots usually developed from the upper nodes, while vegetative shoots developed from the lower nodes. They did n o t examine the effects of temperature. By culturing at 25°C or lower, we found the same tendency for the growth pattern to depend on the node position. However, all buds, including those of upper origin, tended to grow vegetatively when cultured at 28°C, and at all temperatures used, the increased rate of d o r m a n c y obtained was in proportion to the height of origin on the flower stalk. Although flower buds were n o t visible externally on the reproductive shoot

176 after 90 days in culture at 20°C, these shoots were defined as flower stalks, since flower development was observed after removing the secondary bracts. The lowest node of flower differentiation on the reproductive shoot which originated from an upper node was lower than t h a t from a node of lower origin. This suggests t h a t floret differentiation of the upper-origin shoot is more advanced than the lower-origin shoot. Tran Thanh Van (1974) reported t h a t the application of high temperature (27°C) to floral shoots ofP. amabilis and P. schilleriana, after the upper part had been excised, resulted in the development of an axillary bud, left intact at the base of the floral shoot, as a vegetative rosette. Previous studies on intact Phalaenopsis plants indicated t h a t the development of lateral buds into flower stalks was brought about by low temperature. For example, De Vries (1953) demonstrated t h a t a low night temperature (19°--21°C) was required for the flowering of P. schilleriana and t h a t high temperatures (29%-34°C) apparently inhibited the emergence of the flower stalk. Nishimura (1972) reported that flower stalk emergence of P. amabilis hybrid was dependent on low temperature (25°C during the light period and 15°C during the dark period), and Tran Thanh Van (1974) pointed o u t t h a t floral initiation t o o k place when the night temperature varied from 12 ° to 17°C and the day temperature did n o t exceed 27°C. According to Sakanishi and Imanishi (1977), a temperature of 25°C or lower was necessary for the emergence of a flower stalk from an intact plant of P. amabilis hybrid, and at 28°C it did riot occur. The temperature effect became more marked when the period of low temperature (25°C or lower) in a day exceeded 12 hours. These results indicate that the effect of temperature on the in vitro growth of lateral buds of flower stalks is similar to the effect on the growth of lateral buds of the main axis and of the flower stalk in intact Phalaenopsis. Based on our experiment with buds excised from flower stalks which had elongated at 18 ° C or 28 ° C, it is clear t h a t growth of excised buds was affected by culture temperature, irrespective of the temperature given to the stock plants. From these data, it seems reasonable to assume t h a t the buds, except for the uppermost one, are n o t predetermined to grow vegetatively or reproductively before culturing in vitro, and t h a t the same buds can produce a reproductive or a vegetative shoot, depending on the temperature after they are cultured in vitro. Many buds excised from lower positions on the flower stalk developed into vegetative shoots even at the temperature which induced formation of reproductive shoots from upper-position buds. Generally, a higher rate of reproductive growth was obtained in buds t h a t originated from a higher stalk position. These results suggest t h a t the upper buds m a y have had a greater floral induction than lower buds. The same phenomenon, in which the upper lateral buds are reproductive in t e n d e n c y and give rise to flowers earlier than the lower ones, can be observed on the flowering stems of carnations, roses, chrysanthemums and m a n y other plants. At 28°C, on VW medium with and w i t h o u t CW, the percentage of the cul-

177 tured buds which remained d o r m a n t was 35% and 76%, respectively. Similar results, obtained by K o t o m o r i and Murashige (1965), showed t h a t 53% of cultured buds remained d o r m a n t on VW medium containing only CW 15% (v/v) at an almost constant temperature of 29.4°C. It seems likely that the endogenous inhibition which was presented in an intact lateral bud remains to some degree even after it has been excised and placed in culture. There are some reports on the effect of BA in releasing the inhibition of lateral buds on intact plants. Basal branching was successfully obtained by using BA as a foliar spray on poinsettia (Carpenter et al., 1971), and by application o f BA and adenine in lanolin to buds o f roses (Parups, 1971). Similarly, Kender and Carpenter (1972) reported the positive effect of BA on the promotion of bud-sprouting in apple. Sawa (1972, 1977) f o u n d that plantlets could be obtained by application of BA in lanolin to lateral buds of intact flower stalks of Phalaenopsis, and that the lateral buds developed successfully into secondary flower stalks, which were later brought to flower by application of both BA and gibberellin A3 in lanolin in the summer season. The effect of BA on sprouting and development in cultured lateral buds has been studied in citrus (Altman and Goren, 1974; Navarro et al., 1975} and in apple (Dutcher and Powell, 1972). Reisinger et al. (1976) cultured the lateral buds of Phalaenopsis flower stalks on modified Knop's medium supplemented with trans-cinnamic acid, a known anti-auxin, in order to release t h e m from apical dominance, and reported 59% bud growth and plantlet formation. In our investigation, t h e percentage of the buds which sprouted was greater on VW medium with CW than w i t h o u t CW, which m a y have been caused by the presence of a cytokinin in CW. To further p r o m o t e sprouting of the buds, various concentrations of BA were included in the culture medium. High concentrations of BA were necessary to p r o m o t e sprouting in buds excised from an upper position on the flower stalk. An endogenous inhibition of buds excised from this upper position m a y be stronger than t h a t for the lower-position buds. Although the high concentrations of BA p r o m o t e d sprouting, malformation of the leaves was observed. Considering all factors, BA at 2.5 p.p.m, appeared to be the o p t i m u m concentration. Koch (1974) has examined the effect of BA on the growth and development of buds after sprouting, and reported that the optimal concentration was 1 p.p.m, of BA. We observed t h a t the plastochron of growing buds was reduced in proportion to increased concentrations of BA in the medium, b u t for the occurrence of leaf malformation at high levels of BA, it induced both d o r m a n t buds to grow and p r o m o t e d their further development. Buds released from d o r m a n c y by BA application were also influenced by the position on the flower stalk and by the in vitro temperature. Thus, BA was effective principally in promoting the growth of d o r m a n t buds, while further bud d e v e l o p m e n t was determined by cultural temperature. The young leaves o f the vegetative shoots, developed in vitro by BA-promotion of the lateral buds on the flower stalks, have been used as starting-material

178

for clonal propagation of Phalaenopsis. By appropriate tissue culture of these leaves we have obtained protocorm-like bodies, followed by plantlet formation. REFERENCES Altman, A. and Goren, P., 1974. Growth and dormancy cycles in Citrus bud cultures and their hormonal control. Physiol. Plant., 30: 240--245. Carpenter, W.J., Rodriguez, R.C. and Carlson, W.H., 1971. Growth regulator induced branching on non-pinched poinsettias. HortScience, 6: 457--458. Daerr, M., 1967. Vegetative Vermehrung bei Phalaenopsis. Die Orchidee, 18: 322--324. De Vries, J.T., 1953. On the flowering ofPhalaenopsts schillerfana Rchb.f. Ann. Bogor., 1 : 61--76. Dutcher, R.D. and Powell, L.E., 1972. Culture of apple shoots from buds in vitro. J. Am. Soc. Hortic. Sci., 97: 511--514. Intuwong, O. and Sagawa, Y., 1974. Plantlet (keiki) formation in Phalaenopsis. Na Okika O Hawaii -- Hawaii Orchid J., 3: 17--19. Intuwong, O., Kunisaki, J.T. and Sagawa, Y., 1972. Vegetative propagation of Phalaenopsis by flower stalk cuttings. Na Okika O Hawaii -- Hawaii Orchid J., 1: 13--18. Kender, W.J. and Carpenter, S., 1972. Stimulation of lateral bud growth of apple tree by 6-benzylamino purine. J. Am. Soc. Hortic. Sci., 97: 377--380. Koch, L., 1974. Erbgleiche Vermehrnng yon Phalaenopsis in vitro. Gartenwelt, 74: 482--484. Kotomori, S. and Murashige, T., 1965. Some aspects of aseptic propagation of orchids. Am. Orchid Soc. Bull., 34: 484--489. Navarro, L., Roistacher, C.N. and Murashige, T., 1975. Improvement of shoot-tip grafting in vitro for virus-free citrus. J. Am. Soc. Hortic. Sci., 100: 471--479. Nishimura, G., 1972. Researches on daylength and temperature affecting the flowering of Phalaenopsis. Jpn. Orchid Soc. Bull., 18 : 3--9 (in Japanese). Parups, E.V., 1971. Use of 6-benzylamino purine and adenine to induce bottom breaks in greenhouse roses. HortScience, 6: 456--457. Reisinger, D.M., Ball, E.A. and Arditti, J., 1976. Clonal propagation of Phalaenopsis by means of flower-stalk node cultures. Orchid Rev., 84: 45--52. Rotor, G., 1949. A method of vegetative propagation of Phalaenopsis species and hybrids. Am. Orchid Soc. Bull., 1 8 : 7 3 8 739. Sagawa, Y., 1961. Vegetative propagation of Phalaenopsis by stem cutting. Am. Orchid Soc. Bull., 30: 808--809. Sagawa, Y. and Niimoto, D., 1960. Vegetative propagation of Phalaenopsis. Fla. Orchidist, 3: 22. Sakanishi, Y. and Imanishi, H.~ 1977. Effects of temperature on growth and flowering of Phalaenopsis. II. Temperature in relation to reproductive development. Abstr. Spring Meeting Jpn. Soc. Hortic. Sci., 336--337, (in Japanese). Sawa, Y., 1972. Effect of benzyladenine on growth of axillary bud of ornamental plants. Abstr. A u t u m n Meeting Jpn. Soc. Hortic. Sci., 232--233, (in Japanese). Sawa, Y., 1977. Effect ~of benzyladenine and gibberel!in on flowering ofPhalaenopsts. Abstr, Spring Meeting Jpn. Soc. Hortic. Sci., 338--339, (in Japanese). Scully, R.M., 1966. Stem propagation of Pha~aenopsis. Am. Orchid Soc. Bull., 35: 40--42. Tews, G., 1974. Einfache vegetative Phalaenopsisvermehrung. Orchideen (Greifswald, DDR), 3: 14--15. Tran Thanh Van, M., 1974. Methods of acceleration of growth and flowering in a few species of orchids. Am. Orchid Soc. 'Bull., 43 : 699--707. Tse, A.T.-Y., Smith, R.J. and Hackett, W~P., 1971. Adventitious shoot formation on Phalaenopsis nodes. Am. Orchid Soc. Bull., 40: 807--810. Urata, U. and Iwanaga, E.T., 1965. The .use of Ito-type vials for vegetative propagation of Phalaenopsis. Am. Orchid Soc. Bull., 34: 410--413. Vacin, E.F. and Went, F.W., 1949. Some pH changes in nutrient solutions. Bot. Gaz., 110: 605--613.