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Adventitious Shoot Regeneration in Chrysanthemum

Adventitious Shoot Regeneration in Chrysanthemum - J Kor...

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Unformatted text preview: J. Kor. Soc. Hort. Sci. 46(5):335—340. 2005. Adventitious Shoot Regeneration in Chrysanthemum as Affected by Plant Growth Regulators, Sucrose, and Dark Period So Hyeon Parkl, Gyeong Hee Kim‘, and Byoung Ryong Jeongz’” [Agricultural Technology Center of Busan Metropolitan City, Busan 618440, Korea ZDepartmenl of Horticulture, Division of Applied Life Science, Graduate School, Gyeongsang National University, Jinju 660-701, Korea jlnstitute of Agriculture and Life Science, Gyeongsang National University, Jinju 660- 701, Korea (*Corresponding author) Abstract. Adventitious shoots were regenerated from three Chrysanthemum (Dendranthema grandiflora) cultivars ‘Orlando’, ‘Klondike’, and ‘Pink Pixie Time’. Explants were taken from petals, stems, and leaves. Adventitious shoot formation from petal explants was greatly influenced by plant growth regulators, sucrose, and dark period. Shoots were regenerated on modified MS medium (MS salts, B5 vitamins, and 0.8% agar) supplemented with various combinations of 6—benzylamino purine (BAP), kinetin, and indole-3—acetic acid (1AA). ‘Orlando’ gave the greatest number of shoots per explant on modified MS medium containing 57.1 uM 1AA, 44.2 uM BAP, and 0.4 uM kinetin (regeneration medium). Number of shoots per petal explant increased in the dark as compared to the light condition. The greatest number of shoots in ‘Orlando’ and ‘Klondike’ was observed on the regeneration medium containing 6% (w/v) sucrose. Regenerated shoots could be easily rooted and transferred to a glasshouse condition. All regenerated plants showed the same morphological characteristics of vegetative organs in comparison to those of the stock plant. Additional key words: Dendrantlzema grandiflorum, micropropagation, morphological characteristics, organogenesis Introduction Regeneration in Chrysanthemum was achieved mainly through organogenesis (Kaul et al., 1990). Hill (1968) reported the formation of shoots on callus cultures, derived from stem and leaf explants. Hattori (1992) used sections of the receptacle for the production of adventitious shoots and Ben-Jacov and Langhans (1970) and Earle and Langhans (1974a, b) obtained adventitious shoots via callus, derived from a stem tip. Since then, regeneration in Chrysanthemum has been successful by using various explants such as flower buds (Dabin et al., 1983), stem (Annadana et al., 2000; Lu et al., 1990), and leaf (Slusarkiewicz et al., 1981). Regeneration depends on concentration and type of plant growth regulators, especially auxins and cytokinins. The optimum ratio of auxin and cytokinin for shoot organogenesis in Chrysanthemum was contradictory among investigators. In spite of many researches on plant regeneration in Chrysanthemum, it still has many pro- blems. A drawback of the most protocols is cultivar specificity. Therefore, plant regeneration protocol of new cultivars has usually been found by trials and errors. Sucrose has a role to provide energy which is necessary for the organ initiation (Amirato and Steward, 1971). In addition, the beneficial effect of high sucrose concen- Received September 1, 2005; accepted October 25, 2005. 335 trations in the regeneration medium has been attributed to an osmotic effect (Kong and Yeung, 1992). Cultural environment was also important for successful plant regeneration. In callus formation from most explants and cell cultures, consecutive dark condition was more efficient. When the explant cultured in dark condition was moved to light condition, many shoots were supposed to be formed (Rugini, 1988). Plantlet production via the process of organogenesis is now firmly established as an important tool for micro- propagation and gene transfer techniques. However, it needs effective condition according to species and cultivar. Materials and Methods Preparation of three type explants for shoot regeneration Leaf disc explants excised from leaves on 6-week—old plant were grown on modified MS medium supple- mented with MS salts (Murashige and Skoog, 1962), B5 vitamins (Gamborg et al., 1968), 30 g-L’l sucrose, and 8 g-L'1 agar with pH 5.8 adjusted before autoclaving. The leaves were out about 5 mm away from the midrib. The slices were cut across along their length to produce explants of about 36 mm2 in size (Annadana et al., 2000). The leaf explants were then planted with the 336 So Hyeon Park, Gyeong Hee Kim, and Byoung Ryong Jeong adaxial side up on the regeneration medium. Modified MS medium with 3% (w/v) sucrose, 0.8% (w/v) agar, 57.1 uM indole-3-acetic acid (1AA), 44.2 uM 6-benzyl- amino purine (BAP), and 0.4 uM kinetin was used as the regeneration medium. Internodal stem segments were sliced into pieces of about 5 mm in length. The slices were placed horizontally on the regeneration medium. Flowering was induced by short day treatments in a greenhouse. After floral initiation, young flower petals were isolated and sterilized in 1.0% (w/v) NaOCl solution for 10 min, followed by rinsing three times in sterile distilled water. After sterilization, ovaries and the other terminal parts were excised from the petal using a surgical blade and the remaining petals were cut into 6 mm long segments. The petal explants were then planted on the regeneration medium with the abaxial sides of the petal to face down (Tanaka et al., 2000). One month after initiation of culture, adventitious shoots were elongated on modified MS medium containing 0.46 uM kinetin. Adventitious shoots were planted in Magenta boxes (GA7, Sigma Chemical Co., St. Louis, MO, USA) and incubated under 16 h photoperiod of 40 umol-m‘Z-s'l photosynthetic photon flux (PPF) provided by cool white fluorescent lamps, at 25 i 1°C and 70—80% RH. Number of adventitious shoots and roots were counted 1 month after the initiation of the shoot elongation culture. Effect of combination of plant growth regulators and of sucrose on shoot regeneration The percentage shoot regeneration from stem and leaf explants was low in experiment of explant type. Hence, petal segments were selected as experimental materials for determination of the influence of plant growth regulators. Nine treatment combinations were used in the experi- ment including combination of IAA, BAP, and kinetin (Table 1). Modified MS medium was used as the control. Four explants were planted in each petri dish (90 X 150 mm). Each treatment was repeated three times. Adventi- tious shoots and roots were counted after 1 month of shoot elongation. The effect of sucrose concentration was also examined. Petal explants were incubated on modified MS medium (0.8% agar and B5 vitamins) supplemented with 57.1 uM IAA, 44.2 “M BAP, 0.4 pM kinetin, and 3, 6, 9 or 12% (w/v) sucrose. Modified MS medium supplemented with 57.1 uM IAA, 44.2 uM BAP, and 0.4 uM kinetin without sucrose was used as the control. Five explants were planted in each petri dish (90 X 150 mm). Each treatment was repeated three times. After 1 month of shoot elongation, adventitious shoots and roots were counted. Effect of dark period on shoot regeneration To investigate the effect of dark incubation period on the induction of shoots, petal explants were cultured for the initial 6, 12, or 18 days in darkness and then were transferred to a standard illumination condition where they were kept for additional 3 weeks. The explants in the control were kept under 16 h photoperiod of 40 umol-m‘Z-s‘1 PPF provided by cool white fluorescent Table 1. Effect of plant growth regulators on the numbers of adventitious shoots and roots regenerated per petal explant of three Chrysanthemum cultivars. Plant growth regulator (uM) No. of shoots per explant No. of roots per explant 1AA BAP Kinetin ‘Orlando’ ‘Pink Pixie Time’ ‘Klondike’ ‘Orlando’ ‘Pink Pixie Time’ ‘Klondike’ 0 0 0 0.0 0.0 0.0 0.0 0.0 0.0 57.1 44.2 0.4 8.7 8.2 3.2 0.0 0.0 0.0 57.1 44.2 0 6.7 5.4 3.2 0.0 0.0 _ 0.0 57.1 0 0.4 0.0 0.3 0.0 1.5 0.6 10.9 57.1 0.4 0 3.1 2.0 0.0 3.8 2.7 8.1 5.7 44.2 0.4 0.6 0.9 0.5 0.0 0.0 0.0 5.7 44.2 0 0.4 0.5 0.0 0.0 1.3 0.0 5.7 0 0.4 0.1 0.3 0.0 1.4 1.6 2.8 5.7 0.4 0 0.1 0.3 0.0 0.3 2.0 0.0 Plant growth regulator *** Cultivar * * * plant growth regulator X cultivar *** **’***Significant at P: 0.01 or 0.001, respectively. *** ** *** J. Kor. Soc. Hort. Sci. 46(5):335~340. 2005. 337 lamps for 5 weeks. Modified MS medium with 3% (w/v) sucrose, 0.8% (w/v) agar, 57.1 uM 1AA, 44.2 uM BAP, and 0.4 pM kinetin was used as the culture medium. Subsequent establishment ex vitro After 2 months of culture, 3—4 cm long plantlets derived from petal explants were washed in tap water to remove agar and then were transferred to lOO-cell plug trays containing a commercial plug medium (Tosilee medium, Shinan Grow C0,, Jinju, Korea). The plants were kept on a greenhouse bed with a fogging system for ex vitro acclimatization for 2—3 weeks before they were transferred to a greenhouse bench. A complete nutrient solution was supplied daily through an overhead irrigation system. Results and Discussion Effect of explant type on shoot regeneration The first morphogenetic change, observed after 2 weeks in culture in all cultivars and explant types, was the development of callus at the cut surfaces (Fig. 1). In 3 weeks in culture the first adventitious shoot arising from explants was visible. Shoots elongated rapidly over the next 4 weeks following the initiation. When experiment was conducted to find out the most efficient explant type on modified MS medium sup- plemented with 57.1 pM 1AA, 44.2 pM BAP, and 0.4 pM kinetin, the greatest number of shoots per explant was formed from petal explant (Table 2). Petal explants of ‘Orando’ gave the greatest number, 9.30 per explant, of shoots. Stern and leaf explants showed poor regeneration percentage in all cultivars (Table 2). Effect of combination of lAA, BAP, and kinetin and of sucrose concentration Petal explants of three cultivars formed the most adventitious shoots in the combination containing 57.1 uM 1AA, 44.2 uM BAP, and 0.4 pM kinetin (Table 1). On the medium containing the same concentrations of IAA and BAP but without 0.4 uM kinetin, ‘Klondike’ showed 3.2 shoots per explant. But most shoots did not develop well. ‘Klondike’ showed the greatest, 10.9 roots per explant, in the medium containing 57.1 uM IAA and Fig. l. The established organogenetic procedure of plant regeneration from petal explants in Chrysanthemum cultivar ‘Orlando'. A, Petal explant removed of ovary and the other terminal parts; B, development of callus of a transparent color at the cut surfaces; C and D, primordia formation in 2—3 weeks in culture and the first adventitious shoot arising from explants, respectively; E, rapidly elongated shoots over the next 3—4 weeks; F, plantlets with entangled shoots and roots; G, plantlets having well~developed roots and shoots, and greater than 2 cm in size; and H, the regenerated plant with flowers bloomed in a greenhouse. 338 S0 Hyeon Park, Gyeong Hee Kim, and Byoung Ryong Jeong Table 2. Effect of explant type on shoot regeneration in three Chrysanthemum cultivars on modified MS medium sup- plemented with 57.1 uM IAA, 44.2 ”M BAP, and 0.4 11M kinetin. No. of shoots Cultlvar Explant formed per explan’t ‘Orlando’ Stem 1.1 Leaf 3.4 Petal 9.3 ‘Pink Pixie Time’ Stem 0.4 Leaf 0.2 Petal 6.1 ‘Klondike’ Stem 0.2 Leaf 0.2 Petal 1.3 t-test in each cultivar *** ***Significant at P = 0.001. 0.4 uM kinetin. In the medium containing 57.1 uM IAA and 0.4 uM BAP, ‘Orlando’ and ‘Klondike’ showed simultaneous rooting. The explant planted on the medium containing no plant growth regulators turned brown or died in 3—4 weeks. Lu et a1. (1990) reported 2.22 uM BAP and 5.37 uM NAA were most. effective for shoot regeneration in ‘Royal Purple’, while Khehra et a1. (1995) reported 4.44 “M BAP and 2.69 uM NAA were most effective in ‘Early Charm’. Kaul et a1. (1975) reported 5 uM NAA and 5 uM BAP were optimum in eleven Chrysanthemum cultivars. These results may reflect cultivar specificity. Based on the previous studies, Chrysanthemum cultivars may be classified into three groups depending on their regeneration responses to plant growth regulators. Some cultivars showed effective regeneration in higher concen- trations of auxin than cytokinin (Lu et al., 1990), some cultivars showed effective regeneration in higher concen- trations of cytokinin than auxin (Bush and Pueppke, 1991; Earle and Langhans, 1974a; Khehra et al., 1995; Ledger et al., 1993; Low et al., 1993; Slusarkiewicz et al., 1981), and the other cultivars showed effective regeneration in similar concentrations of auxin and cyto- kinin (Kaul et al., 1990). Also, leaf explants needed higher auxin concentrations as compared to stem ex- plants and that means different parts of the plant reacted differently to plant growth regulators added to the medium (Kaul et al., 1990). Our experiment showed petal explants needed similarly high concentrations of auxin and cytokinin and they were more responsive than stem or leaf explants. These differential responses among different types of explants to exogenously applied plant .- a + 'Orlando' —0* 'Pluk Pixie Time' + ‘Klondike' a N l _. o .7 No. of shoots formed per explant Sucrose concentration (%) Fig. 2. Effect of sucrose concentration on adventitious shoot regeneration from petal explant of three chrysanthemum cultivars. Vertical bar means SE of the mean. growth regulators may have been caused by different concentrations of endogenous plant growth regulators. Many shoots were regenerated from petal explants on the medium containing high concentrations of auxin and cytokinin as compared to the general tendency in which high auxin and low cytokinin are required for rooting and low auxin and high cytokinin are required for shooting. The effect of sucrose concentration of the medium containing 57.0 uM IAA, 44.0 uM BAP, and 0.4 uM kinetin was evaluated for ‘Orlando’, ‘Pink Pixie Time’, and ‘Klondike’ (Fig. 2). Two to three percent (w/v) sucrose has been commonly used for the optimum shoot development in Chrysanthemum (Earle and Langhans, 1974b; Hill, 1968; Khalid et al., 1989). Roest and Bokelmann (1975) reported that the presence of sucrose in the culture medium is required for the initiation and development of adventitious shoots. The greatest number of shoots in ‘Orlando’ and ‘Klondike’ was observed on the medium containing 6% (w/v) sucrose. The greatest number of adventitious shoots was produced in ‘Pink Pixie Time’ which, unlike the other two cultivars, showed more adventitious shoots in the medium containing up to 9% (w/v) sucrose. However, on the medium containing 9 or 12% (w/v) sucrose, the regenerated shoots of all three cultivars turned black. Hammersley-Straw and Thorpe (1988) reported that adventitious shoot formation from callus cultures of tobacco (Nicotiana tabacum) was completely suppressed in the presence of 12% (w/v) sucrose. In this experi- ment, expression of organogenesis from petal explants of Chrysanthemum on the medium containing high concen- trations of sucrose might have been due to osmotic effects rather than to the absolute nutritional requirement. Effect of dark period on shoot regeneration To find out what influence dark incubation period has J. Kor. Soc. Hort. Sci. — 'Orlando' :1?an Pixie Time' T _ ‘Klondlke' No. of shoots formed per explant 0 6 12 18 Dark period (Day) Fig. 3. Effect of dark period on adventitious shoot regeneration from petal explant of three Chrysanthemum cultivars. Vertical bar means SE of the mean. on shoot regeneration, dark incubation period of 0, 6, 12, or 18 days at the beginning of culture was experimented (Fig. 3). The number of adventitious shoots produced was significantly influenced by the initial dark incubation period. Cultures maintained under a continuous light condition from the beginning without any dark incubation showed no shoot differentiation. ‘Orlando’ and ‘Klondike’ showed greater number of shoots formed in 12 days as compared with that in 6 or 18 days of dark incubation treatment. However, ‘Pink Pixie Time’ showed much more shoot formation in 18 days of dark incubation treatment unlike the other two cultivars. Although there was a slight difference among cultivars. generally 12 to 18 days of dark incubation gave the greatest number of shoots regenerated on the medium with the same concentrations of plant growth regulators. Explants in no dark incubation treatment did not form any callus. From this result, it is considered that callus formation in organogenesis is accelerated by dark incubation at the initial culture period in the same way as embryogenic callus formation in embryogenesis is enhanced by the dark incubation (Tanaka et al., 2000). This research suggests an efficient plant regeneration method in Chrysanthemum, through the shoot organo- genesis from petal explants. The regeneration efficiency was improved by high concentrations of auxin and cytokinin, increasing the initial dark incubation period and sucrose concentration. Acknowledgment: This work was financially support— ed by the Brain Korea 21 project. Literature Cited Annadana, S., M. Ademaker, M. Ramanna, M. Udayakumar, and J. Jong. 2000. Response of stem explants to screening and explant source as a basis for methodical advancing of 46(5):335—340. 2005. 339 regeneration protocols for Chrysanthemum. Plant Cell Tiss. Organ Cult. 62:47—55. Ben-Jacov, J. and R.W. Langhans. 1970. A tissue culture tech- nique for rapid multiplication of Chrysanthemum morifolium. 19th Int. Hort. Congr., Tel Aviv. p. 211—225. (Abstr.) Bush, AL. and SO. Pueppke. 1991. Cultivar—strain specificity between Chrysanthemum morz'jblium and Agrobacterium tumefaciens. Physiol. Mol. Plant Pathol. 39:309—323. Dabin, P., D. Choisez-Givron, and A. Dekeyer. 1983. In vitro culture of buds applied to the vegetative propagation of chry- santhemum cv. White Spider. Bulletin du Societe Royale and de Botani Belgique 116:161—166. Earle, ED. and R.W. Langhans. 1974a. Propagation of Chrysan- themum in vitro. 1. Multiple plantlets from shoot tips and the establishment of tissue cultures. J. Amer. Soc. Hort. Sci. 99:128—132. Earle, ED. and R.W. Langhans. 1974b. Propagation of Chrysan— themum in vitro. 11. Production, growth, and flowering of plantlets from tissue cultures. J. Amer. Soc. Hon. Sci. 99:352—358. Gamborg, O.L., R.A. Miller, and K. Ojima. 1968. Nutrient requirements of suspension of soybean root cells. Exp. Cell Res. 50:151—158. Hammersley-Straw, D.R.H. and TA. Thorpe. 1988. Use of osmotic inhibition in studies of shoot formation in tobacco callus cultures. Bot. Gaz. l49:303~310. Hattori, K. 1992. The process during shoot regeneration in receptacle culture of Chrysanthemum, Chrysanthemum mori— folz‘um Ramat. Jpn. J. Breed. 42:227—234. Hill, GP. 1968. Shoot formation in tissue culture of Chrysanthe- mum ‘Bronze Pride’. Physiol. Plant. 21:386—389. Kaul, V., R.M. Miller, J.F. Hutchinson, and D. Richards. 1990. Shoot regeneration from stem and leaf explants of Dendran- thema grandiflora Tzvelev (syn. Chlysanthemum morifo/ium Ramat.). Plant Cell Tiss. Organ Cult. 21:21—30. Khalid, N., M.R. Davey, and J.B. Power. 1989. An assessment of somaclonal variation in Chrysanthemum morz'foh'um: The generation of plants of potential commercial value. Sci. Hort. 38:287—294. Khehra, M., K.C. Lowe, M.R. Davey, and J.B. Power. 1995. An improved micropropagation system for Chrysanthemum based on pluronic F-68-supplemented media. Plant Cell Tiss. Organ Cult. 41:87—90. Kong, L. and EC Yeung. 1992. Development of white spruce somatic embryos. II. Continual shoot meristem development during germination. In Vitro Cell. Dev. Biol. 28:125—131. Ledger, S.E., S.C. Deroles, and N.K. Given. 1993. Regeneration and Agrobacterium-mediated transformation of Chrysanthe— mum. Plant Cell Rpt. 10:195—199. Low, J.M., M.R. Davey, J.B. Power, and KS. Blundy. 1993. A study of some factors affecting transformation of Chrysanthe- mum and plant regeneration of Dendranthema grandz'flom Tzvelev. Plant Cell Tiss. Organ Cult. 33:171—180. Lu, C., G Nugent, and T. Wardley. 1990. Efficient, d...
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