J. Japan. Soc. Hort. Sci. 54 (3) : 379-387. 1985. New Means of Phalaenopsis Propagation with Internodal Sections of Flower Stalk' Yoshiyuki HOMMA' Faculty of Agriculture, and Kyoto Tadashi ASAHIRA University, Kyoto 606 Summary Internodal sections at upper parts of flower stalks were used for clonal propagation of Phalaenopsis tissue culture. Around 100 days after explanting, 50 to 80% of these explants produced PLBs (protocorm like bodies) at their basal end. During several times of subculturing, these PLBs proliferated and grew into plantlets. Supplement of 10% coconut milk, 5 mg/i a-naphtaleneacetic acid and 20 mg/l 6- benzylaminopurine to the basal medium consisting of macro elements of Thomale GD (1954), minor elements and organic addenda of Ringe and Nitsch (1968), increased formation rate of PLBs. The primordia of PLB seem to initiate in the inner part of cortex. In this method, at least 400 PLBs and shoots will be obtained from one stalk in the first year. Introduction Among many orchids, I'halaenopsis is one of the important genera from a horticultural viewpoint, because of its colorful big flowers attached to a long flower stalk. Although efficient systems of vegetative propagation have been investigated on this genus (1, 5, 19, 34), they are not well established as yet, unlike other ochid genera, Cyinbidium, Cattleya, Dendrobiunt etc. So, commercial growers have to use seedlings instead of clonal plant. Thus, commercially used hybrids of Phalaenopsis usually have very complicated names as listed in our study (Table 1), and flowers on the plants of the same name often show great variation. This is partly due to the negligence of breeders on the system of registration, and also to the difficulty of vegetative propagation. Many studies on in vitro propagation have been conducted using shoot tip (8), flower stalk nodes (10, 12, 20, 23, 32), excised axillary buds of flower stalk (37), leaves of plantlets obtained through flower stalk node culture 1 Received for publication January 14, 1985. 2 Present address : Shizuoka Prefectural Agriculture and Forestry Junior College, Toyodacho, Iwatagun, Shizuoka 438 (16, 31), and tip of flower stalk (6). But none of these methods are so effective for obtaining lots of plants in a short period, because of high frequency of contamination (2), low rate of PLB formation, long route for obtaining PLB (protocorm like body), and differences in culturing responces among hybrids or clonal plants. In this study, we tried to induce PLBs directly from internodal sections of a flower stalk. Propagation efficiency of this method is also discussed. Meterials and Methods Flower stalks (20-50 cm long, 2-3 months after emergence) with a few flower buds of I'halaenopsis hybrids (Table 1) were collected at an orchid nursery, and cut into 15 cm long from their tops. The cut surfaces were sealed with paraffin. Then excised flower stalks were soaked in 0. 1% chlorobenzalconium solution for 30 minutes, 70% ethanol for a few seconds, and sodium hypochlorite solution containing 1% active chlorine with 0.05% Tween 80 for 10 minutes. After removal of the basal end of the flower stalks, they were again soaked in sodium hypochlorite solution containing 0. 5% active chlorine with 0. 05% Tween 80, and sectioned into 379
380 HOMMA, Y. AND T. ASAIIIRA Table 1. Names of Phalaenop sis hybrids used for the experiments. Table 2a. Components of the basal medium. Fig. 1. Explants used in this study. A, B, --and 10 indicate the position in a flower stalk where explants were taken. Numerals are distances in cm from the top of the flower stalk to the basal end of each explant. Most of explants were taken from II, but few of them were taken from I in Experiment 1 and 2. 5 mm long explants (Fig. 1). Top section of flower stalk with terminal bud was named Explant A, and internodal sections without any axillary buds were named Explants B, C, D, -..and 10 from upper to lower, as shown in Fig. 1. In Experiment 4, modified Explants B, C, and D were used as follows. Bu : Explant B planted upside-down. Bh, Ch and Dh : Explants B, C and D cut in half longitudinally and planted with their quadrangle surfaces down on the medium. Bg, Cg and Dg : Explants 13, C and D of which epidermis were removed and planted with the side down on the medium. Media used in this study are shown in Table 2. The basal medium consisted of Thomale GD's major elements (33), minor elements and organic addenda of Ringe and Nitsch (21), and 8g/1 agar. All media were adjusted to ph 5. 8 prior to autoclaving (1. 3 kg/cm, 115 C, 13 minutes). Each explant was planted onto a 20 nil medium contained in a test tube (25 mm x 150 mm) without rinsing by sterilized water. The culture was maintained at 23±2 C under 1500 lux of 12-hour fluorescent light illumination. In Experiment 1 and 2, explants were subcultured a few times during 6 months onto fresh media of the same composition as the primary cultures. In Experiment 3 and 4, explants were subcultured biweekly The culture calendar is shown in Fig.2. Results In Experiment 1, six different types of me-
NEW MEANS OF PHALAENVOPSIS PROPAGATION WITH INTERNODAL SECTIONS 381 Table 2b. Supplements to basal medium in the experiment. o Explanting. X Transplanting 0 Transplanting + Investigating to the to the growth medium with the different med ium. of explants. same composition. Fig. 2. Culturing calendars. Fig. 3. Explant B, 31 days after explanting. Lower end of explant became larger than the top. Phal. ((Clyde x Malibu) x Joseph Hampton) x Anne Cavaco. Fig. 4. Explant Explant B, 120 days after explanting. produced PLBs at the bottom of it. dium plots (each comprises five test tubes) were used for culturing both Explants A and B. Explants, which were planted individually into each test tube, were transplanted to fresh media of the same composition as their primary cultures 27, 56 and 103 days afte explanting. Both Explants A and 13 began t( enlarge shortly after explanting. Tw( months later, the basal end of Explant I became larger than the upper end (Fig.3),
382 IIOMMA, Y. AND T. ASAFIIRA Fig. 5. Explant A, which produced PLB and differentiated shoots at the top 120 days after explanting. and a similar phenomenon was observed in Explant A. During 100-day culture, 42% of these explants produced PLBs at the bottom (Fig. 4), and 27% of terminal buds of Explant A developed to shoots (Fig. 5). The percentage of explants which produced PLBs was higher in Medium 6 (the basal mediurn supplemented with 20 mg/1 BA (6-benzylaminopurine), than in other media with lower BA concentrations (Table 3). at of the bottom, it. In Experiment 2, four different types of medium plots were used. Most of survived Explant As produced shoots at the top in 200- day culturing (Table 4). Through Experiment 1 and 2, Medium 6 seemed to be the best for inducing PLB. In Experiment 3 and 4, Medium 8, which was different from Medium 6 in sucrose content, was used as the control medium. In Experiment 3, 50 explants (25 from A and 25 from B) were cultured to determine the proliferation rate, and another 50 (25 from A and 25 from B) cultured explants were used for anatomical study to observe the localization of PLB primordia, respectively. Around 100 days after explanting, 80% of explants produced PLBs, which proliferated into 505 PLBs-r432 shoots in total during 227-day culturing. The epidermal layer of explants did not show any sign of meristematic activities. Meristematic activities were observed at the inner part of the cortex tissue (Fig.6), and cells in the outer part remained vacuolated. In Experiment 4, five explants each of several different kinds other than Explants A and B were cultured. A rather high percent- Table 3. Growth of explants in Experiment 1. 100 days after explatiti ng. Table 4. Growth of explants in Experiment 2. 206 days after explanting.
NEW MEANS OF PHALAENOPSIS PROPAGATION WITH INTERNODAL SECTIONS 383 Fig. 6. Vertical clays after section explanting. of Explant B at the lower end. 29 age of PLB production was observed on Explants C and D. And even in Explant 7, which were internodal sections taken from 7 cm below the top, two out of 5 explants produced PLBs in 130-day culturing (Fig. 7). Explants Bu, namely Explant B planted upside-down, did neither produce PLB nor grow in size 150 days after explanting. Explants Bh, Ch and Dh, which were cut into half longitudinally, grew faster and produced PLBs more rapidly (20-40 days earlier) than the controls without cutting. The percentage of explants having produced PLBs was also higher in those explants (Fig.8, Table 5). Explants whose epidermis had been removed (Bg, Cg and Dg) also produced PLBs. Fix1G hilt,-,f th PCa 7S, PVillAn+l tx1pre dead, but nine out of 10 survived explants produced PLBs. These explants also produced PLBs more rapidly than the controls whose epidermis were not removed. The media containing 50 or 100 mg/1 BA were not so good for PLB induction as the media containing 20 mg/1 BA (data not shown). Dead explants on Media 9-12 which have high BA concentrations, turned trans- T able 5. Growth of explants on Medium 8 in Experiment 4. 136 days after explanting. Fig. 7. Explant 7, which the stalk of Phal. produced PLB, taken from Mount Kaala x Hamaoka. Fig. 8. Explant Bh, taken from Mount Kaala x Hamaoka. planting. the stalk 115 days of Phal. after ex-
384 IIOMMA, Y. AND T. ASAIIIRA lucent white, unlike those on Medium 8 which turned black. On those high BA concentration media, however, some of explants grew normally as those on Medium 8 did. Discussion PLBs were successfully obtained adventitiously from the internodal section of the flower stalk. PLBs were always produced at the lower (basal) end of the explants, which faced media, but rarely at the top of explants taken from the uppermost internode (Explant B, data not shown). None of the PLBs were produced from axillary buds nor a terminal bud. In the liquid medium, PLBs were produced only at one side of Explant B (data not shown), and on the solid medium Explant B planted upside-down did not produce PLBs. Therefore, there seems to be a certain polarity in the flower stalk on adventitious PLB formation. Supplement of CM to the medium seemed to increase rate of PLB formation. The effect of CM has been considered to be mainly due to its internal cytokinin activities (4, 13, 26, 27, 28, 29). As shown in Table 3, the percentage of explants producing PLBs was elevated in both Explants A and B by supplementing BA. However, other plant growth substances (3, 17, 18, 25) and some nutritive values contained in CM might have some synergistic or additional effects on PLB formation. Macro elements of Thomale GD formula were used exclusively in the present experiment, because Phalaenopsis seedlings grew faster on the medium containing its macro elements than on that containing macro elements of Vacin and went (35) or Knudson C (11) in our preliminary experiments. Thomale GD formula is quite unique because it lacks calcium. In this tissue culture experiment, however, the influence of excluding calcium from the medium was unclear, because the final concentration of calcium in the medium was around 15 mg/1 due to supplement of CM. Explants of the internodal sections other than Explants A and B (including Explant 7 which was taken from internode in 7 cm be- Table 6. PLB producing rate of various position of flower are reffered to Fig.l. explants taken from stalk. The positions low the top) produced PI_Bs. According to the result shown in Table 6, however, flower stalk sections of the top 2 cm seemed to he available for practical propagation. Leaf culture of Phalaenopsis (31) is similar to our method on the point of obtaining adventitious PLB. Adventitious organogenesis often happens in this genus like Phalaenopsis stuartiana, which is popular with the ability of plantlet formation from its aerial roots (24, 30). We have, in some cases, observed PLB formation from the cultured bract of a cultivar in this genus (data not shown). Thus leaf, root, flower stalk and bract of this genus have abilities to form PLB adventitiously. The initiation of meristematic cells was observed at the inner part of the cortex in our experiment. This is quite different from the reports on inflorescence culture of sarcantine orchids (7), leaf culture of Phalaenopsis (31), and meristem culture of Cymbidium (14, 15), which showed that epidermal cells or subepidermal cells have the meristematic activity. In fact, that the explants from which epidermis were removed produced PLBs. Moreover, it is probable that the epidermal layer has an inhibitory effect on the development of PLBs with its tightness. The facts that both Explants Bh and Bg produced PLBs much earlier than normal B, and PLBs from Explant B usually grew downwards support this idea. A hypothetical proliferation rate was calculated in Experiment 3. Fifty explants (25 from A and 25 from 13) were cultured for
NEW MEANS OF PHALAENOPSIS PROPAGATION WITH INTERNODAL SECTIONS 385 Fig. 9. Diagram of flower of practical procedure for clorial stalk internodal sections. propagation of Phalaenopsis through the culture 227 days (subcultured 9 times), and 505 PLBs and 432 shoots were obtained in total. Around 100 days after explanting, 40 explants out of 50 produced initial PLBs. So, one PLB proliferated to 23. 4 PLBs and shoots in 177 days (I). 504+432 50-10 =23. 132...(I ) If one PLB will divide into two PLBs, and n is its dividing frequency during 117-day culture, then, 2't=23.4 71 =1092 23.4=4.55 277-100 177-38.9... (11 loge 23.4 1.55 It takes 38. 9 days for one PLB to divide into two PLBs. 365-1" 2 38.9 =112.4...(Ii) 365 112.4x233 =75, 048... (IV) 112.4x7x0.6=472.08...(V) In the first year, approximately 100 PLBs and shoots will be obtained (III), and in the next year 75,000 (IV) from one explant. Fig. 10. Plantlets obtained through the culture of flower stalk internodal section of Phal. ((Clvdex Malibu) x Joshph Hampton) x Anne Cavaco. 380 (lays after explanting. We can use seven explants (1 A, 2 13h, 2 Ch and 2 Dh) from one stalk in this method. If 60% of them produced PLBs after 100 days, we could obtain more than 400 PLBs or shoots in the first year from one stalk (V). This proliferation rate was obtained without subculturing the explants since the first 5 months, and without dividing aggregate of PLBs. So, higher rate than we obtained
386 IIOMMA, Y. AND T ASAIIIRA here would be expected, if proper operation was performed. The rare of contamination in this method was much lower than that of flower stalk node culture. Contamination was never observed in 400 of primary cultures. Only one or two out of 100 cultures were contaminated during 6-month subculture. Practical diagram of this method was shown in Fig. 9. PLBs begin to develop into plantlets spontaneously when they were cultured for a long time or transferred onto a hormone free medium. Plantlets 380 days after explanting were shown in Fig. 10. Necessity of subculturing seems to be a demerit of this method, for we subcultured biweekly in the Experiment 3 and 4. But in practice, subculturing once a month will be. enough. The merits of this method comparing to the culture of flower stalk node and leaves are as follows : 1) PLB will be obtained within a short period. 2) Rate of contamination is nearly 0%. 3) Special techniques are not necessary. 4) There is no injury to, or no sacrifice of the mother plant ; flowers at the lower position are able to be used for hybridizing. Acknowledgement Authors wish to thank Messrs. K. Hiramatsu for providing us with flower stalks and G. Peterson for his informative suggestion on writing of the manuscript. Literature Cited 1. ABDULLAH, M. and J. ARDITTI. 1983. Preparation of hormone pastes for plantlet induction on Phalaenopsis flower stalks. Orchid Rev. 91 : 291-292. 2. ARDITTI, J., J. A. JOHNSON and R. G. PERERA. 1981. Culture media which do not require sterilization : Phalaenopsis flower stalk nodes. Orchid Rev. 89 : 49-52. 3. DIX, L. and J. VAN STADEN. 1982. Auxin and gibberellin-like substances in coconut milk and malt extract. Plant Cell Tissue Organ Culture. 1 : 239-245. 4. ERNST, R. 1967. Effect of select organic nutrient additives on growth in vitro of phalaenopsis seedlings. Am. Orchid Soc. Bull. 36 : 694-704. 5. GREISBACH, R. J. 1983. The use of indolacetylamino acids on the in vitro propagation of Phalaenopsis orchids. Scientia Hortic. 19: 363-366. 6. INTUWONG, 0., T. KUNISAKI and Y. SAGAWA. 1972. Vegetative propagation of Phalaenopsis by flower stalk cuttings. Na Okika 0 Hawaii-Hawaii Orchid J. 1 : 13-18. 7. INTUWONG, O. and Y. SAGAWA. 1973. Clonal propagation of sarcantine orchids by aseptic culture of inflorescences. Am. Orchid Soc. Bull. 42 : 209-215. 8. INTUWONG, O. and Y. SAGAWA. 1974. Clonal propagation of Phalaenopsis by shoot tip culture. Am. Orchid Soc. Bull. 43 : 893-895. 9. INTUWONG, O. and Y. SAGAWA. 1974. Plantlet formation in Phalaenopsis. Na Okika 0 Hawaii-Hawaii Orchid J. 3 : 17-19. 10. KOCH, L. 1974. Ergleiche Vermehrung von Phalaenopsis in vitro. Gartenwelt. 22 : 482-484. 11. KNUDSON, L. 1946. A new nutrient solution for the germination of orchid seeds. Am. Orchid Soc. Bull. 15 : 214-217. 12. LAY, F. F. M. 1978. Studies on the tissue culture of orchids. 1 : Clonal propagation of Phalaenopsis by lateral buds from flower stems. Am. Orchid Soc. Bul. 86 : 308-310. 13. LETHAM, D. S. 1974. Regulators of cell division in plant tissues. XX. The cytokinins of coconut milk. Physiol. Plant. 32 : 66-70. 14. MOREL, G. M. 1971. The principles of clonal propagation of orchids. Proceedings of the sixth world orchid conference. p. 101-106. 15. MOREL, G. M. 1974. Clonal multiplication of orchids. In : The Orchids scientific studies. p. 169-222. 16. PIEPER, W. and K. ZIMMER. 1976. Clonal propagation of Phalaenopsis in vitro. Acta Horticulturae. 64 : 21-23. 17. POLLAND, J. K., E. M. SHANTZ and F. C. STE- WARD. 1961. Hexitols in coconut milk : Their role in nuture of dividing cells. Plant Physiol. 36 : 492-501. 18. RADLEY, M. and E. DEAR. 1958. Occurence of gibberellin -like substances in the coconut. Nature 182 : 1098. 19. RAO, A. N. 1977. Tissue culture in the orchid industry. In : Plant Cell, Tissue and Organ Culture. p. 44-69. Springer-Verlag. Berlin Heidelberg New York. 20. REISINGER, D. M., E. A. BALL and J. ARDITTI. 1976. Clonal propagation of Phalaenopsis by
NEW MEANS OF PHALAENOPSIS PROP GATION WITH INTERNODAL SECTIONS 387 21. 22. 23. 24. 25. 26. 27. 28. 29. means of flower-stalk node cultures. Orchid Rev. 84 : 45-52. RINGE, F. and J. P. NITSCH. 1968. Conditions leading to flower formation on excised Begonia fragments cultured in vitro. Plant Cell Physiol. 9 : 639-652. SAGAWA, Y. and J. T. KUNISAKI. 1982. Clonal propagation of orchids by tissue culture. Plant Tissue Culture 1982. (Proceedings of the 5 th International Congress of Plant Tissue and Cell Culture) 683-684. SCULLY JR., R. M. 1966. Stem propagation of phalaenopsis. Am. Orchid Soc. Bull. 35: 40-42. SCULLY JR., R. M. 1971. Febuary is phalaenopsis time in Florida. Am. Orchid Soc. Bull. 40 : 103-108. SHANTZ, E. M. and F. C. STEWARD. 1952. Coconut milk factor : The growth promoting substances in coconut milk. J. Am. Chem. Soc. 74 : 6133-6135. SHANTZ, E. M. and F. C. STEWARD. 1955. The identification of compound A from coconut milk as 1, 3-diphenylurea. J. Am. Chem. Soc. 77 : 6351-6353. VAN STADEN, J. and S. E. DREWES. 1974. Identification of cell division inducing compounds from coconut milk. Physiol. Plant. 32 : 347-352. VAN STADEN, J. and S. E. DREWES. 1975. Identification of zeatin and zeatinriboside in coconut milk. Physiol. Plant. 34 : 106-109. VAN STADEN, J. 1976. The identification of 30. 31. 32. 33. 34. 35. 36. 37. zeatin glucoside from coconut milk. Physiol. Plant. 36 : 123-126. STEWART, J. and J. BUTTON. 1978. Development of callus and plantlets from eqidendrum root tips cultured in vitro Am. Orchid Soc. Bull. 47 : 607-612. TANAKA, M., A. HASEGAWA and M. GUI. 1975. Studies on the clonal propagation of monopodial orchids by tissue culture. I. Formation of protocorm-like bodies from leaf tissue in Phalaenopsis and Vanda. J. Japan. Soc. Hort. Sci. 44 : 47-58. (in Japanese) TANAKA, M. and Y. SAKANISHI. 1978. Factors affecting the growth of in vitro cultured lateral buds from Phalaenopsis flower stalks. Scientia Hortic. 8 : 169-178. THOMALE, H. 1954. Vermehrung. In : Die Orchideen. p. 62-85. Euger Ulmer Verlag. Stuttgart. TSE, A. T., A. T. SMITH and W. P. HACKETT. 1971. Adventitious shoot formation on phalaenopsis nodes. Am Orchid Soc. Bull. 40: 807-810. VACIN, E. F. and F. W. WENT. 1949. Some ph changes in nutrient solutions. Bot. Gaz. 110: 605-613. WITFINER, C. L., P. K. NELSON and P. J. WEJKS- NORA. 1974. The anatomy of orchids. In : The Orchids scientific studies. p. 267-347. A Wiley-Interscience publication. ZIMMER, K. and W. PIEPER. 1978. Clonal propagation of Phalaenopsis by excised buds. Orchid Rev. 86 : 223-227.