New Plantlet Proliferation and Bulbing Promotion in In Vitro Culture of Ornithogalum Hybrid

New Plantlet Proliferation and Bulbing Promotion in In Vitro Culture of Ornithogalum Hybrid Jeung-Keun Suh, Wan-Hee Lee and Ae-Kyung Lee Lab. of Floriculture, College of Bio-Resources Science, Dankook University San 29 Anseo Dong, Cheonan, Choongnam, 330-714 South Korea Keywords: tissue culture, bulblet formation Abstract The in vitro culture on Ornithogalum hybrid cultivar was investigated for mass propagation. The highest development of shoot number was observed when bulb scale explants cultured on MS medium under continuous fluorescent light. In general, culturing under light condition promoted shoot and root development more than darkness. The number of shoot was significantly increased when perianth as a source of explant was cultured at 0.5 mg L -1 NAA and 1.5 mg L -1 BA combination. The proximal position of leaf explants showed superior responses, increasing of shoot number to 11.2. A temperature of 19 C was found to be most effective in the increasing of shoot number at 5.4. The development of shoot and root was inhibited at lower culture temperature. The number of shoot and root were promoted on the medium of mixed with 60g L -1 sucrose and 0.8 mg L -1 ancymidol. However, development of plantlet was not affected by high concentration of 60 g L -1 sucrose. The diameter, fresh and dry weight of bulb to tend promote at 35/15 (D/N). In conclusion, when proximal position of leaf explants were cultured on MS basic medium, supplemented with 30 g L -1 sucrose, 0.8 mg L -1 ancymidol, and 0.5 mg L -1 NAA and 1.5 mg L -1 BA, and incubate at 19 C under continuous white light condition, the highest mean number of shoots was showed. And those results with 35/15 C treatment at the stage of bulbing in vitro culture were considered that rapid bulb production is possible. INTRODUCTION Ornithogalum spp. belong to the subclass Monocotyledonae and the family Liliaceae (Van Scheepen, 1991). There are over 150 species that are indigenous to Europe, Western Asia, and South Africa (Brayan, 1989; Du Plessis and Duncan, 1989; Liberty Hyde Baily Horturium, 1976). Ornithogalum species, including O. arabicum L. and O. thyrsoides Jacq, are mainly produced as cut flowers in greenhouse. However, O. dubium Houtt. was cultured as potted plant (De Hertogh, 1997). The bulbs consist of scales, leaf bases and submerged leaves of three seasons (Rees, 1985). Some species in South Africa is evergreen, but most are deciduous. Scape lengths range to over 1 meter in some species. The inflorescence is a simple raceme consisting of many 6-petaled florets. Flower colors are white, white with green stripes on the outside, yellow, orange or orange-red (De Hertogh and Le Nard, 1993). Dorothea (1981) reported that propagation by in vitro culture with various plant parts, such as bulb scales, scapes, florets, and leaf segments. From the results, lots of contaminations were occurred in bulb scales and also scape and floret segments showed poor development of bulblets. But leaf segments induced lots of bulblets comparatively. Numerous adventitious shoot formation from O. maculatum leaf explants was occurred on media containing al combination of BA at 0.5~2.0 mg L -1 and NAA at 0.01~0.2 mg L -1. Shoots growing on media with low concentrations of the growth regulators (BA at 0.5 mg L -1 or NAA at 0.01 mg L -1 ) tended to form bulblets (Van Rensbur et al., 1988). Some researches were mainly on cultural techniques or physiology were conducted in Korea. Most of bulbs are imported and some growers enlarge bulblets for 1 year and then force them for cut flowers, but those bulbs, enlarged in Korea, were revealed some problem, such as infected virus and poor growth and development in field. Proc. V th IS on New Flor. Crops Eds.: A.F.C. Tombolato and G.M. Dias-Tagliacozzo Acta Hort. 683, ISHS 2005 155

Although several studies have reported concerning on vegetative propagation, method of rapid mass proliferation with in vitro culture and bulb enlargement for rapid bulb production were not established. Therefore, this research was conducted to investigate the rapid mass propagation of Ornithogalum hybrid. With in vitro culture techniques, we intend to examine optimum light condition, plant part as explants, concentrations of plant growth regulators, culture temperatures and efficient medium for rapid bulbing. MATERIALS AND METHODS The various plant parts of unknown Ornithogalum thyrosides hybrid were used in the experiments. Explants were cultured on MS (Murashige and Skoog 1962) basic medium, supplemented with 0.4 mg L -1 thiamin-hcl, 0.5 mg L -1 nicotinic acid, 0.5 mg L -1 pyridoxin-hcl, 2 mg L -1 glycine, 100 mg L -1 myo-inositol, 30g L -1 sucrose, adjusted to ph5.5, and solidified with 8% agar. All cultures were incubated at 24±1 C in continuous white light provided from fluorescent tube at light irradiation level of 40 μmol m -2 sec -1 for 8 weeks. Effect of Media, Plant Parts, and Light/Dark Condition To investigate the influence of media on development of shoot, leaves and bulb scales were cultured on MS, Knudson, and 1/2 MS media (50% macro elements of MS basic medium). All media were contented 0.1 mg L -1 NAA and 2.0 mg L -1 kinetin. And also the darkness and continuous white light were treated in order to investigate the influence on shoot growth. Effect of Plant Growth Regulator, Plant Part as Explants Stalk(middle part with 2mm), leaf (15cm) and immature perianth segments were considered experimental factors, to detect the most useful explants for in vitro culture. In this experiment, the combinations of NAA (0.25, 0.5, 0.75 mg L -1 ) and BA (1.5, 3.0, 4.5 mg L -1 ) were supplemented in MS basic media. Effect of Leaf Position as Explants The leaves were divided into 3 positions as distal, middle, and proximal position. It was conducted to investigate the ability of shoot inducing according to leaf position. All explants were placed on MS basic medium with 0.1 mg L -1 NAA and 2.0 mg L -1 kinetin. Effect of Culture Temperature The influences of different temperatures was investigated with incubating at 16, 19, and 23 C. The middle positions of leaves, as explants, were cultured on MS medium with 0.1 mg L -1 NAA and 2.0 mg L -1 kinetin. Effect of Sucrose Concentration and Plant Growth Retardant To investigate the effect of the plant growth retardant and sucrose concentration on shoot development, Ancymidol (0, 0.8, 1.6, 2.4 mg L -1 ) and sucrose (30g L -1 and 60g L -1 ) were supplemented into MS basic medium. Simultaneously, leaf segments was used as explants, to observe the influence of plant parts. And also all media were contented 0.1 mg L -1 NAA and 2.0 mg L -1 kinetin. Effect of Support Material, Explant Type, and Temperature on Bulb Development In Vitro Materials (agar or vermiculite) was supplemented in MS basic medium with 0.1 mg L -1 NAA and 2.0 mg L -1 kinetin. And two types of explant (single or multiple shoot) were cultured on them. In addition, to observe the influence of temperature on shoot development, temperature conditions were controlled with ambient temperature, 35/15 C, 15/35 C, and 25/15 C(day/night). 156

RESULTS AND DISCUSSION Effect of Media, Plant Parts, and Light/Dark Condition The highest development of shoot number was observed when bulb scales explants cultured on MS medium under continuous fluorescent light (Table 1). When leaves explants were cultured on MS medium in darkness, showed longest shoot with 4.2cm. In general, culturing under light condition promoted shoot and root development more than darkness (Fig. 1). Concerning the plant parts as explants, although bulb scales induced the most shoot, lots of contamination were revealed. Therefore, leaf segment is may be more useful as explants. These results are in agreement with those of Dorothea (1981). Effect of Plant Growth Regulator, Plant Part as Explants The number of shoot was significantly increased when perianth as a source of explant was cultured at 0.5 mg L -1 NAA and 1.5 mg L -1 BA combination with 15.0 (Table 2). Explants that did not regenerate (dead) and callus without organogenesis was observed at higher concentration of NAA and BA. Also, shoot regeneration was induced effectively by lower concentrated hormonal combination of NAA and BA, and the mortality was increased by higher concentration (Fig. 2). These results are in agreement with those of Van Rensbur et al., (1988) who reported that adventitious shoot formation on O. maculatum leaf explants occurred on media containing lower concentrated hormonal combination of BA at 0.5~2.0 mg L -1 and NAA at 0.01~0.2 mg L -1. Concerning the plant part as explants, leaf segment was the best explants for length and developments of shoot in vitro culture (Fig. 3). The number of root was increased when leaf as explant was cultured at 0.5 mg L -1 NAA and 3.0 mg L -1 BA combination with 13.2 ea (Table 3). Effect of Leaf Position as Explants The proximal position of leaf explants showed superior responses, increasing of shoot number to 11.2. But the distal position of leaf explants shortening the number of days to initiate shoot formation to 14.0 days (Table 4). This result was similar to that explants close to proximal of leaf was the best explant on development of shoot of Lachenalila (Suh et al., 1996). Effect of Culture Temperature A temperature of 19 C was found to be most effective in the increasing of shoot number at 5.4 (Table 5). And other temperature conditions, 16 and 23 C, showed lower regeneration producing 3.8 and 4.0, respectively, shoots. However, concerning the days to initiate shoot formation, 23 C condition required the least with 37 days. Number of root was also significantly decreased at 19 C. Therefore, development of shoot and root was inhibited at lower culture temperature. Effect of Sucrose Concentration, Plant Growth Retardant, and Plant Part The number of shoot (3.0) was significantly increased on the medium, supplenmented with 60 g L -1 g/l sucrose and ancymidol 0.8 g L -1 (Table 6). And the number of shoot and root were promoted on the medium of mixed with 60g L -1 sucrose and 0.8 mg L -1 ancymidol treatment. However, development of plantlet was not affected by high concentration of 60 g L -1 sucrose. These results are in agreement with those of Van Rensbur et al., (1988). Coolbaugh and Harmhon (1976) reported that ancymidol is an inhibitor of gibberllin (GA) synthesis and has been shown to block the oxidation of entkaurenol, and ent-kaurenal. Incorporation of ancymidol in culture medium accelerated the production of plantlets, promoted development of stronger roots and shoots, and suppressed undesirable proliferation of callus of Asparagus officinalis L. (Chin, 1982). 157

Effect of Support Material, Explant Type, and Temperature on Bulb Development In Vitro The diameter of bulb was significantly increased at 35/15 C(D/N) on agar and vermiculite medium as single explant with 6.26 and 6.34mm, respectively (Table 7). Also, the diameter of bulb was remarkably enlarged when at 35/15 C(D/N) treatment than 20/15 C(D/N) (Fig. 4). The fresh and dry weight to tend promote at 35/15 C (D/N). The result, no significant differences were observed among support materials or explant types treatments. Accordingly, bulbing, temperature differential is may be more affective factor than support materials or explant types. In conclusion, when proximal position of leaf explants were cultured on MS basic medium, supplemented with 30 g L -1 sucrose, 0.8 mg L -1 ancymidol, and 0.5 mg L -1 NAA and 1.5 mg L -1, and incubate at 19 C under continuous white light condition, the highest mean number of shoots was showed. Those results with 35/15 C treatment at the stage of bulb development were considered that rapid bulb production. Literature Cited Bryan, J.E. 1989. Bulbs. Timber Press, Portland, Oregon. 2 : 451. Chin Chee-kok. 1982. Promotion of Shoot and Root Formation in Asparagus in vitro by Ancymidol. Hortscience 17(4): 590-591. Coolbaugh, R.C. and Harmhon, R. 1976. Inhibition of ent-kaurene oxidation and growth by α-(p-methoxyphenyl)-5-α-eyelopropyl-α(p-methoxyphenyl)-5-pyrimidin methyl alcohol. Plant Physiol. 57: 245-248. De Hertogh, A.A. and Gallitano, L. 1997. Basic forcing requirements for Israeli-grown Ornithogalum. Acta Hort. 430:227-232. De Hertogh, A.A. and Le Nard, M. 1993. The Physiology of Flower Bulbs. Elsevier. pp. 761-764. Dorothea, D. NEL. 1981. Rapid Propagation of Ornithogalum Hybrid in vitro. Agronlantae 13: 83-84. Du Plessis, N. and Duncan, G. 1989. Bulbous Plants of Southern Africa-A Guide to their Cultivation and Propagation. Tafelberg Publishers Limited, Cape Town. p. 1952. Liberty Hyde Bailey Hortorium. 1976. Hortus Third: A Concise Dictionary of Plants Cultivated in the United States and Canada. 3rd Edition. Macmillian Publishing Company, New York. p. 1290. Rees, A.R. 1985. Miscellaneous bulbs. Handbook of Flowering, CRC Press, Boca Raton, Florida, 1: 306-308. Suh, J.K., Lee, J.S. and Jeung, S.K. 1996. Studies on Growth, Development, Propagation, and Cultural Techniques of Lachenalia. Rural Development Administration. Van Rensburg, J.G.J., Vcelar, B.M. and Landby, P.A. 1988. Micropropagation of Ornithogalum maculatum. South African Jour. of Botany. 55: 137-139. Van Scheepen, J. 1991. International checklist for Hyacinths and Miscellaneous Bulbs. Royal General Bulb Growers Association(KAVB), Hillegom, The Netherlands. p. 409. 158

Tables Table 1. Effect of media and plant parts on shoot regeneration of Ornithogalum hybrid in vitro culture. Light Dark Treatment Shoot Root Shoot Root Number Length Number Length Number Length Number Length (ea) (cm) (ea) (cm) (ea) (cm) (ea) (cm) MS Leaf 3.8 bc z 0.7 d 4.0 c 0.8 d 3.1 a 4.2 a 2.9 c 1.8 bc Scale 6.1 a 2.4 a 5.3 b 5.4 bc 3.0 ab 3.9 b 3.0 b 3.4 a 1/2MS Leaf 4.5 b 1.7 b 8.6 a 4.0 c 2.0 b 0.8 d 4.2 a 2.4 b Scale 2.9 cd 1.8 ab 8.0 a 2.2 d 1.9 c 1.2 cd 2.0 d 1.3 d Knudson Leaf 2.1 d 1.1 c 4.5 bc 5.9 b 3.0 ab 1.9 c 3.5 ab 1.5 c Scale 3.0 c 1.1 c 1.5 d 7.6 a 2.0 c 0.5 d 2.0 d 1.3 d z Means separation within columns by Duncan's multiple range test, 5% level. Means with same letter are not significantly different from each other. Table 2. Effect of NAA/BA concentration, plant parts and light condition on shoot number and length of Ornithogalum hybrid in vitro culture. NAA (mg L -1 ) BA (mg L -1 ) 1.5 3.0 4.5 ST z LE PE ST LE PE ST LE PE ------------- Shoot number(ea) -------------- 0.25 8.2 *y 2.9 9.3 * 8.0 * 1.8 10.0 * 2.0 1.5 1.7 0.50 2.5 2.1 15.0 * - 1.8 - - C - 0.75 - C x 12.0 * - W W - W W -------------- Shoot length(cm) -------------- 0.25 1.1 3.4 * 2.1 1.6 2.6 * 1.5 0.3 0.9 0.4 0.50 1.3 1.7 1.2-1.8 - - C - 0.75 - C 1.3 - W W - W W z ST : stalk, LE : leaf, PE : perianth. y Significant at 5% level by LSD as compared to the respective treatment. Means with same letter are not significantly different from each other. x C : only callus, W : wilting 159

Table 3. Effect of NAA/BA concentration, plant parts and light condition on root number and length of Ornithogalum hybrid in vitro culture. BA (mg L -1 ) NAA (mg L -1 ) ST z 1.5 LE PE ST 3.0 LE PE ST 4.5 LE PE --------------- Root number(ea) -------------- 0.25-11.4 *y - 8.0 * 7.8 - - 5.0-0.50-11.3 * - - 13.2 * - - C - 0.75 - C x - - W - - W - --------------- Root length(cm) --------------- 0.25-0.9 * - 1.2 * 0.7 - - 0.5-0.50-0.6 - - 0.6 - - C - 0.75 - C - - W - - W - z ST : stalk, LE : leaf, PE : perianth. y Significant at 5% level by LSD as compared to the respective treatment. Means with same letter are not significantly different from each other. x C : only callus, W : wilting Table 4. Effect of leaf position on shoot regeneration of Ornithogalum hybrid in vitro culture. Shoot Root Position of leaf Number Days z Number (ea) (ea) Days Distal 7.0 c y 14.0 c 4.8 a 14.0 b Middle 9.2 b 14.6 b 5.0 a 14.2 b Proximal 11.2 a 15.2 a 5.2 a 15.6 a z Days from inoculation to initial plantlet formation. y Means separation within columns by Duncan's multiple range test, 5% level.. Means with same letter are not significantly different from each other. Table 5. Effect of temperature on shoot regeneration of Ornithogalum hybrid in vitro culture. Shoot Root Temperature ( C) Number Days z Number Days (ea) (ea) 16 3.8 c y 54.7 a 4.4 c 68.7 a 19 5.4 a 46.8 b 8.8 a 50.0 b 23 4.0 b 37.0 c 5.4 b 45.8 c z Days from inoculation to initial plantlet formation. y Means separation within columns by Duncan's multiple range test, 5% level.. Means with same letter are not significantly different from each other. 160

Table 6. Effect of sucrose/ancymidol concentration on shoot regeneration of Ornithogalum hybrid in vitro culture. 30 g L -1 60 g L -1 Sucrose Shoot Root Shoot Root Treatment Number Length Number Length Number Length Number Length (ea) (cm) (ea) (cm) (ea) (cm) (ea) (cm) 0 - - 11.0 *z 0.2 1.5 0.4 6.1 0.8 0.8 2.8 * 0.3 5.3 1.1 3.0 * 0.4 9.5 * 2.3 * 1.6 1.7 0.6 12.7 * 1.7 2.5 0.4 9.5 * 1.5 2.4 1.5 0.5 1.7 1.9 2.0 1.1 14.0 * 0.9 z Significant at 5% level by LSD as compared to the respective treatment.. Means with same letter are not significantly different from each other. Table 7. Effect of support materials, explant type and temperature treatment on bulb development of Ornithogalum hybrid in vitro culture. Treatment Medium Agar medium Vermiculite medium Explant type single explant multiple explant Single explant multiple explant Bulblet diameter (mm) Fresh weight (g/10ea) Dry weight (g/10ea) Temperature (day/night, C) Ambient z 3.85 bc y 0.62 b 0.061 ab 35/15 6.26 a 1.58 a 0.148 a 25/15 4.67 b 0.84 ab 0.075 ab 15/35 5.43 a 0.93 ab 0.076 ab Ambient 3.76 bc 0.47 b 0.061 ab 35/15 5.79 a 1.16 a 0.163 a 25/15 4.71 b 0.90 ab 0.165 a 15/35 5.14 ab 0.92 ab 0.158 a Ambient 3.55 bc 0.43 b 0.027 b 35/15 6.34 a 1.56 a 0.118 a 25/15 4.12 bc 0.81 ab 0.046 b 15/35 4.72 b 1.01 a 0.055 ab Ambient 3.71 bc 0.66 b 0.036 b 35/15 5.21 ab 1.22 a 0.070 ab 25/15 4.38 b 1.07 ab 0.048 ab 15/35 5.59 a 1.27 a 0.061 ab z Ambient : 2025 C. y Means separation within columns by Duncan's multiple range test, 5% level.. Means with same letter are not significantly different from each other. 161

Figures Fig. 1. Effect of light condition on shoot regeneration of Ornithogalum hybrid in vitro culture(left : light, right : dark). Fig. 2. Effect of NAA/BA concentration on shoot regeneration of Ornithogalum hybrid in vitro culture(from left to right : 1.5, 3.0, 4.5mg L-1, from top to bottom : 0.25, 0.5, 0.75mg L-1). 162

Fig. 3. Effect of plant parts on shoot regeneration of Ornithogalum hybrid in vitro culture(left : perianth, middle : leaf, right : stalk). Fig. 4. Effect of temperature on bulb development of Ornithogalum hybrid (left : ambient(20/25 C), right : 35/15 C(day/night)). 163