J. Japan. Soc. Hort. Sci. 52(1) : 45-50. 1983. In Vitro Formation of Gerbera (Gerbera hybrida Hort.) Plantlets through Excised Scape Cultures Chien-young CHU and Min-chang HUANG Department of Horticulture, National Chung Hsing University, Taiwan, Republic of China Summary Four cultivars (`Arendsoog', `Beatrix', `Continent' and `Super Giant Yellow') of gerbera were used in this experiment. The flower stem, also referred to as scape, was used in tissue culture. When the 2 nd or 3 rd whorl of the disc florets started to unfold, the upper 4 cm solid part of the scape just below the head was cut off and sterilized for 2 or 3 seconds in 70% ethanol, followed by soaking in 2% NaOC1 for 30 minutes, and then rinsed 3 times with sterilizedistilled water. After sterilization, both ends of the scape section were removed 0.5 cm in length and discarded. It was divided longitudinally into identical strips. Each strip was placed with the wounded side down on the culture medium composed of MS (Murashige and Skoog, 1962) macroelements at 1/2 strength, Heller's microelements, Na2FeEDTA 21.4 mg/1, MS organiconstituents, 1% sucrose, 6-benzyl amino purine. (BA) 10 mg/l and Bacto-agar 0.8%. The ph of all nutrient media was adjusted to 5.6 prior to autoclaving. The explants were first maintained complete darkness for 2 weeks, and then subjected to light at intensity of 800 lx for 16 hours daily. At the end of 16 weeks, 5 or more shoots were formed per scape. Introduction Clonal propagation has the benefit of producing plantlets with the same genetical characters. In gerbera division propagation is quite inefficient as only 5 plantlets on the average are produced per year from a single 1-year-old plant. Even with the improved rhizome cutting method (11, 14), only 30-50 plantlets are produced per year from a singl 1-year-old plant. In recent years, tissue culture as an alternative has been used in asexual propagation. Pierik et al. (8, 9, 10) showed that excised gerbera capitulum-explants could be forced to form shoots in vitro. Unfortunately, the excised capitulum-explants of yellow flowered cultivars were unable to form shoot.) Murashige et al. (4) showed that shoot tip culture could also propagate gerbera plantlets in vitro. In the shoot tip culture system, however, shoot tips were lost by microbial infection at the ' Received for publication February 1, 1983 rate as high as 80%. Roest and Bokelmann (12) succeeded in promoting in vitro shoot formation from excised pedicels of chrysanthemum. In this paper, the experimental result with excised scape-explants of gerbera used to form shoots for propagation is presented. Materials and Methods Three clonal cultivars, `Arendsoog', `Beatrix' and `Continent' from Holland and a seedling cultivar, `Super Giant Yellow', from Japan were used in this experiment. `Arendsoog' was used throughout if not otherwise mentioned. Plants were grown on raised bed under PE tent in the garden of Horticulture Department of National Chung Hsing University. The flower (inflorescence) stem of gerbera, also referred to as scape, was used in tissue culture. When the 2nd or 3rd whorl of the disc florets started to unfold (commercial cut flower stage), the upper 4 cm solid part of 45
46 CHIEN-YOUNG CHU AND MIN-CUANG HUANG the scape just below the head was removed from the plant and the flower head was cut off. The scape segments were sterilized for 2 or 3 seconds in 70% ethanol, followed by soaking in 2% NaOCI for 30 minutes, and then rinsed 3 times with sterilized distilled water. After all these procedures, both ends of the scape segment 0.5cm in length were cut off. The remaining 3 cm section was divided longitudinally into two equal strips. were subsequently placed with the wounded side down on the 10 ml solide medium in 50 ml Erlenmeyer flask. The flasks were plugged with two layers of aluminum foil. The basic medium was composed of MS (5) macroelements at 1/2 strength, Heller's Nat FeEDTA 21.4 mg/1, MS organic constituents, 1% sucrose, BA 10 mg/1 and Bacto-agar 0.8. The ph of all nutrient media was adjusted to 5.6 prior to autoclaving. Scape-explants were first placed in darkness in 27±3 C for 2 weeks and subsequently placed under 16-hour daily illumination at 8001x (Taiwan Toshiba FL 40 SBR) at 27±3 C. After 16 weeks, the number of shoots per scape-explant was counted. Results 1. General observation. Scape-explants were cultured on the basic medium. After 8 weeks, many shoots with leaves (Fig. l) and buds without leaf (Fig. 2) could be found at the edge of the unbrowning part (Fig.3). At the end of the 16th week, 5 or more shoots were found per scapeexplant (2 strips) (Fig. 4). Shoots could be subcultured on the medium for gerbera shoot tip culture(4) but buds could not be subcultured easily. 2. Factors influencing the regeneration of scape-explants Scape age The stapes of various ages of `Super Giant Yellow' were collected in January, 1979. In this test, scape-explants were placed in darkness for 4 weeks and subsequently placed under 16-hour illumination at 8001x. Shoot Fig. 1. Shoot developed on the scape-ex plant. Fig. 3. Development of scape-explant 8 weeks culturing. Arrow indicates shoots and formed on the scape-explant. after buds Fig. 2. Bud developed on the scape-explant. Fib. 4. Development of scape- xplant after culturing. Brown no part by arrow was the scape-explant. 16 weeks in dicated
IN VITRO FORMATION OF GERBERA (GERBERA HYBRIDA HORT.) PLANTLET 47 Table 1. Influence of dark period of scape-explantsz. on adventitious shoot formation Table 2. Influence of length of scape-explant on formation of scape-explantsz. adventitious shoot Table 3. Influence of sucrose of scape-explantz. concentration on adventitious shoot formation formation was found on the scape-explants of commercial cut flower stage only. The scape-explants from tight bud stage (scape about 5-10 cm in length) formed more callus. There was no survival in the scape-explants culture from full bloom stage (all disc florets unfolded). Darkness and illumination Two-week and 4-week dark periods could induce the scape-explants to form shoot. The former induced the explants to form shoots after 9-week incubation, and the latter after 8-week incubation. And at the end of 16- week incubation, the former induced 4.5 shoots per scape-explant and the latter induced 1 shoot per scape-explant (Table 1), The scape-explants could not f ormany shoot when they were placed under light at 3000 lx, instead of 800 lx, for 16 hours after various dark periods. Explant length The scapes of `Super Giant Yellow' were tested in September, 1980. The upper 4 cm segments of the scapes were sterilized. After both ends 0.5 cm in length were removed, they were further cut into segments 1 cm, 2 cm or 3 cm or 3 cm in length. Table 2 shows the number of shoots formed as influenced by the explant-length. The longer the explant, the greater the number of shoots formed. Sucrose The effect of sucrose concentration in the basic medium was examined. As shown evidently in Table 3, presence of 1/ sucrose in the culture medium was optimum for shoot formation. When sucrose concentration was increased in the medium, callus formation was
48 CHIEN-YOUNG CHU AND MIN-CHANG HUANG Table 4. Influence of IAA concentration of scape-explans2. on adventitious shoot and root formation Table 5. Influence of cytokinin on adventitious shoot formation of scape-explantsz Table 6. Adventitious shoot formation of scapes of different cultivars 3 months after explanting. induced instead of shoot formation. IAA The response of scape-explants to various concentrations of IAA was summarized in Table 4, representing the results obtained after 8 weeks. Shoot-formation was induced on the media containing IAA at 0.1-0.5 mg/l. Explants produced an increasing quantity of callus with increasing IAA concentration. Root formation was induced on the medium containing 5 mg/1 IAA. Cytokinins The effects of two cytokinins in different concentrations on the differentiation of scapeexplants are shown in Table 5. Shoot formation was found in explants cultured on the medium with 10mg/1 BA. Cultivars Scapes of 4 cultivars were cultured on the basic medium with IAA 0.1 mg/1 and BA 10 mg/l. After 12 week incubation, scapes of `Arendsoog' and `Super Giant Yellow' formed shoots. The ratio for `Super Giant Yellow' was slightly higher than the ratio for `Arendsoog'. The scape-explants of `Beatrix' formed more callus. There was no shoot and little callus-formation on the scape-explants of `Continent' (Table 6).
IN VITRO FORMATION OF GERBERA (GERBERA HYBRIDA HURT) PLANTLET 49 Discussion In recent years, many papers have shown that gerbera can be propagated vegetatively by in vitro culture of shoot tip (1, 4), capitulum (8, 9, 10), inflorescence (7), leaf (2) and pith of rhizome (13). Each method, however has its advantages and disadvantages, and not any of them is the ideal method for commercial production of gerbera plantlets. The present results showed that shoot formation could be induced in the upper 4 cm part of the scape at commercial cut flower stage. Plant organogenesis is affected by auxin/ cytokinin ratio ; high auxin level induces root formation, and high cytokinin level induces shoot formation. Novak and Makova (6) considered that the combination of auxin and cytokinin in some ranges could regulate the organogenesis in tomato shoot tip culture. Kamat and Rao(3) investigated the combined effects of auxin and cytokinin on shoot, bud and root formation. Shoot formation of scape-explants of gerbera was induced by a combination of a high cytokinin level (BA 10 mg/1) and a low auxin level (IAA 0.1 mg/1), which agrees with the results of capitulum (8, 9, 10) and shoot tip (4) cultures. On the other hand, increasing IAA level (5 mg/1) induced root formation of scape-explants (Table 4). This result proved that the organogenesis of gerbera in vitro was also affected by the ratio of auxin and cytokinin. Capitulm-explants of yellow flowered cultivars failed to produce shoots (8, 9). In the present experiment, scape-explants of yellow flowered gerbera produced shoots, but the other cultivars failed. As Roest and Bokelmann(12) showed that the conditions for shoot formation of various chrysanthemum cultivars differed markedly, further detailed investigations are necessary for gerbera to find favorable conditions for shoot formation in scapeexplants of the cultivars that failed. At present, scape culture system as well as capitulum culture system is important for the initial phase of commercial production of gerbera plantlets. Literature Cited 1. GREGORINI, G., R. LORENZI and G. LANCIONI. 1976. The propagation of gerbera by in vitro culture of vegetative apices. Rivista della Ortoflorofirutti Coltura Italiana 60: 282-288. (Abstr.). 2. HEDTRICH, C. M. 1979. Production of shoots from leaves and production of Gerbera jamesonii. Gartenbawwissenschaf t 44 : 1-3. (Abstr.). 3. KAMAT, M. G. and P. S. RAO. 1978. Vegetative multiplication of eggplants using tissue culture techniques. Plant Science Letters 13: 57-65. 4. MURASHIGE, T., M. SERPA and J. B. JONES. 1974. Clonal multiplication of gerbera through tissue culture. HortScience 9 : 175-180. 5. MURASHIGE, T. and G. SKOOG. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497. 6. NOVAK, F. J. and I. MAKOvA. 1979. Apical shoot tip culture of tomato. Scientia Hortic. 10 : 337-344. 7. PAWLOWSKA, H. 1977. Trials on gerbera propagation in vitro. Hod. Rosl. Aklimat. Nasienn. 21:177-181. (Abstr.) 8. PIERIK, R. L. M., J. L. M. JANSEN, A. MAASDAM and C. M. BINNENDIJK. 1975. Opitimalization of gerbera plantlet production from excised capitulum explants. Scientia Hortic. 3 : 351-357. 9. PIERIK, R. L. M., H. H. M. STEEGMANS and J. J. MARELIS. 1973. Gerbera plantlets from in vitro cultivated capitulum explants. Scientia Hortic. 1 : 117-119. 10. PIERIK, R. L. M., H. H. M. STEEGMANS, A. N. WOUTER and J. VEZHEAGH. 1979. New developments in the vegetative propagation of gerberas in testtubes. Vakbladvoor de Bloemisterij 34(25) : 36-37. (Abstr.) 11. RAALTE, D. VAN. 1978. Gerbera breeding and propagation. Deutscher Gartenbau 32(42) 1754-1755. (Abstr.) 12. ROEST, S.. and G. S. BOKELMANN. 1975. Vegetative propagation of Chrysanthemum morifolium Ram. in vitro. Scientia Hortic. 3 317-330. 13. SAWA, K. 1977. The culture of pith from rhizome of gerbera in vitro. Agriculture and Horticulture. Seibundo Sinkoshia. 32(3) 50-51. 14. SCHIVA, T. 1975. Vegetative propagation in gerbera improvement. Annali deli' Istituto Sperimentale per la Floricoltura 6(1) : 133-135. (Abstr.)