Chapter 21. Micropropagation of Cordyline terminalis. Tui Ray, Prasenjit Saha, and Satyesh C. Roy. Abstract. 1. Introduction

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1 Chapter 21 Micropropagation of Cordyline terminalis Tui Ray, Prasenjit Saha, and Satyesh C. Roy Abstract This protocol describes an ef fi cient and rapid method for large-scale multiplication of Cordyline terminalis in a cost-effective manner. Actively growing shoot tips were selected as explants. Murashige and Skoog (MS) basal medium was supplemented with different plant growth regulators at various developmental stages of C. terminalis. The highest percentage of regeneration (95 ± 2.8) and average number of shoot buds (60.2 ± 4.4) per explant were obtained in medium containing 80 mg /L adenine sulfate (AdSO 4 ), 2 mg/l 6-benzyladenine (BA), and 0.1 mg/l indole-3-acetic acid (IAA). Thousands of micropropagated plants were produced within 4 5 months using this protocol. Key words: Adenine sulfate, Cordyline terminalis, Micropropagation, Ornamental plants, Shoot apex explants 1. Introduction Cordyline terminalis, a native of Southeast Asia belonging to the family Agavaceae, is considered to be one of the most economically important ornamental indoor plants for its beautiful multicolored foliage. The economic value of ornamental plants has raised perceptibly worldwide and is still increasing to meet the steady demand of fl oriculture industry. To maintain the genetic stability of the variegated leaves, conventionally this cultivar is propagated by rhizomes or by terminal stem (tips) cuttings and is planted directly in pots. This method of vegetative propagation has reduced the interest due to very slow growth of the plant ( 1 ) and eventually long delay in commercial distribution. Moreover, there are newly introduced cultivars which do not develop rhizome ( 2 ). Micropropagation is the most extensively used and a routinely applied technique to mass propagate plants where the rate of conventional multiplication is otherwise very slow ( 3 6 ). Additionally, Maurizio Lambardi et al. (eds.), Protocols for Micropropagation of Selected Economically-Important Horticultural Plants, Methods in Molecular Biology, vol. 994, DOI / _21, Springer Science+Business Media New York

2 270 T. Ray et al. micropropagation gives the opportunity to produce a large number of clean and healthy uniform plants in small space in a timely manner and also reduces the need of mother plant ( 7 ). Here we describe a standardized, rapid, and ef fi cient method of micropropagation for large-scale production of C. terminalis plants. 2. Materials 2.1. Surface Sterilization of Source Material 2.2. Culture Media 2.3. Acclimatization of Regenerated Plants to Ex Vitro Conditions 1. Tap water. 2. Absolute ethanol and 70% (v:v) ethanol % (v:v) Tween 20 (Sigma, St. Louis, USA) % (w:v) Mercuric chloride (HgCl 2 ) (Qualigen, India) (s ee Note 1). 5. Autoclaved double distilled water in 500 ml screw-capped Duran glass bottles, (Schott AG, Mainz, Germany). 6. Measuring cylinders, 100 ml capacity (Borosil, India). 7. Beakers, 250 ml capacity (Borosil). 8. Autoclaved jam bottles (Sadana Brothers, India). 9. Autoclaved Whatman No.1 fi lter paper in Petri dishes (50 mm diameter; Borosil). 10. Tissue culture facilities Instruments (scalpel, forceps, spirit lamp to fl ame sterilize instruments), laminar air fl ow, culture room. 11. Two to three year-old ex vitro grown C. terminalis plants as source of explants. 1. Murashige and Skoog (MS) ( 8 ) medium containing different concentrations of various plant growth regulators is used for this protocol. Formulation of MS medium stock solutions (see Note 2) and plant growth regulators are given in Tables 1 and 2, respectively. 2. Measuring cylinders, 500 ml and 1 L capacity (Borosil). 3. Beakers, 500 ml and 1 L capacity (Borosil). 4. Erlenmeyer fl asks, 250 ml capacity (Borosil). 5. Culture tubes, mm (Borosil). 6. General tissue culture laboratory facilities Equipments (analytical scale, autoclave, distilling unit, magnetic stirrer with hot plate, microwave oven, ph-meter). 1. Plastic net pots (5 cm) and plastic pots (15 cm). 2. Soilrite (Keltech Energies Ltd. India).

3 21 Cordyline Micropropagation 271 Table 1 Stock solution compositions of Murashige and Skoog (MS) medium (8 ) Stock solution 1 (major salts) Concentration (20 ) (g/l) NH 4 NO 3 33 KNO 3 38 CaCl 2 2H 2 O 8.8 MgSO 4 7H 2 O 7.4 KH 2 PO Stock solution 2 (minor salts) Concentration (200 ) (g/l) KI H 3 BO MnSO 4 4H 2 O 4.46 ZnSO 4 7H 2 O 1.72 Na 2 MoO 4 2H 2 O 0.05 CuSO 4 5H 2 O CoCl 2 6H 2 O Stock solution 3 (Fe-EDTA) Concentration (200 ) ( see Note 2 ) (g/l) FeSO 4 7H 2 O 5.57 Na 2 EDTA 2H 2 O 7.45 Stock solution 4 (myo-inositol) Concentration (1,000 ) (mg/ml) Myo-inositol 50 Stock solution 5 (vitamins) Concentration (1,000 ) (mg/ml) Thiamine-HCl 0.1 Nicotinic acid 0.5 Pyridoxine-HCl 0.5 Table 2 Stock solutions of plant growth regulators Plant growth regulators Concentration (mg/ml) Adenine sulfate (AdSO 4 ) 2 6-benzyladenine (BA) 0.5 Indole-3-acetic acid (IAA) 0.5 Indole-3-butyric acid (IBA) 0.5 See Note 3 for plant growth regulators stock solutions preparation

4 272 T. Ray et al. 3. Methods 3.1. Preparation and Sterilization of Culture Media 1. Prepare full strength MS basal medium according to Table 3 by adding stock solutions of Table Make up the fi nal volume of media with double distilled water after dissolving the sucrose (Hi-media, India). Add stock solutions and sucrose according to the strength of media. 3. Supplement media with different concentrations of various plant growth regulators such as adenine sulfate (AdSO 4 ), 6- benzyladenine (BA), indole-3-acetic acid (IAA), and indole- 3-butyric acid (IBA) (Sigma) (Table 4 ) by using the stock solutions (Table 2 ) according to the developmental stages (s ee Note 3). 4. Adjust the ph of all the media to 5.8 using 0.1 N NaOH or 0.1 N HCl (see Note 4 ). 5. Add required amount of agar (Hi-media, India) to make the solid media (Table 3 ) after adjusting the ph. 6. Melt the agar by heating media. Dispense approximately 20 ml shoot initiation and multiplication (SIM) medium in culture tubes and 100 ml media in 250 ml Erlenmeyer fl asks or jam bottles for shoot elongation and root initiation, respectively. 7. Provide a support of fi lter paper bridge in the culture tubes for liquid root elongation (RE) medium (Table 4 ). 8. Autoclave media and all other required glass apparatus and instruments at 121 C and 15 lbs/in 2 for 15 min after proper plugging and wrapping. 9. Incline the SIM medium immediately after autoclaving to prepare slants. Table 3 Preparation of MS basal medium Components Stock solution 1 (major salts) Stock solution 2 (minor salts) Stock solution 3 (Fe-EDTA) Stock solution 4 (myo-inositol) Stock solution 5 (Vitamins) Sucrose Agar Amount required for 1 L of medium 50 ml 5 ml 5 ml 2 ml 1 ml 30 g 6 g

5 21 Cordyline Micropropagation 273 Table 4 Observation for optimum production of plants on media forti fi ed with different growth regulators Observation Resultsa Duration of culture (days) Name of media Type of media Concentration of growth regulator (mg/l) AdSO 4 BA IAA IBA Percentage of explants regeneration Average number of shoot buds 95 ± SIM MS (solid) ± SIM MS (solid) Average length of elongated shoots (cm) Average number of roots per plantlet 3.8 ± SE ½ MS (solid) 4.8 ± RI ½ MS (solid) Average length of root (cm) 4.5 ± RE ½ MS (liquid) SE shoot elongation medium; SIM shoot initiation and multiplication medium; RI root initiation medium; RE root elongation medium a Data presented as the mean value ± standard error 3.2. Surface Sterilization of Explants 1. Cut about 1 cm long apical part of shoot apex of C. terminalis plants. Remove sheathing leaves around the apical meristem, and wash shoot apices thoroughly under tap water (see Note 5). 2. For surface sterilization, fi rst rinse the shoot apices, with 0.01% (v:v) tween-20 solution for 10 min. Then wash 2 3 times with sterile distilled water to remove soapy traces of tween Under laminar air fl ow, treat explants with 0.1% (w:v) HgCl 2 for 10 min in an autoclaved jam bottle, followed by 3 4 washes in sterile distilled water to remove traces of HgCl Remove excessive water from the explants with sterile fi lter paper by placing in Petri dishes. By using a sterile scalpel, remove the exposed tissue of surface sterilized explants leaving the central cylinder of apical meristem. Before culture on the medium, cut explants into 2 3 mm small pieces Culture and Maintenance of Explants 1. During every culture and subculture, follow the routine aseptic steps of a tissue culture laboratory like swabbing hands and wiping the inside of laminar air fl ow with 70% (v:v) ethanol; switch on the UV lamp 30 min before starting culture.

6 274 T. Ray et al. 2. Place a piece of explants with a sterile forceps on the surface of the slant of SIM medium (Table 4 ) in each culture tube (see Note 6). 3. Maintain all the cultures under 39 m mol/m 2 /s white fl uorescent light (Philips, India) for 16 h photoperiod at 25 ± 2 C with 55 60% relative humidity until explants gradually swell, become enlarged in size, and turn light greenish. 4. Make shoot elongation (SE) medium (Table 4 ) after 20 days of initial culture when primary microshoots begin to appear (Fig. 1a ). Fig. 1. Different stages of micropropagation of C. terminalis from shoot apex meristem. ( a ) Multiple shoot bud formation from shoot apex explant after 20 days in culture. ( b ) Numerous shoots with profuse leaves and roots on root initiation (RI) medium. ( c ) Regenerated plants in half strength MS basal liquid (RE) medium. ( d ) Micropropagated plants, 20 days after transplanting in soil.

7 21 Cordyline Micropropagation Excise and subculture primary microshoots ( cm long) on SE medium containing half strength MS basal medium supplemented with 1 mg/l BA in 250 ml Erlenmeyer fl asks or in jam bottles. Eliminate callus tissues, if any, carefully from the base of newly formed shoots before subculture and place shoot clusters well separated on SE medium. Subculture the swelled explants back in a fresh slant of SIM medium for subsequent regeneration (see Note 7). 6. Separate the elongated shoots from the clusters after days and subculture individually on fresh SE medium with same composition in 250 ml Erlenmeyer fl asks or in jam bottles (see Note 8) Root Development on Regenerated Shoots In Vitro 3.5. Acclimatization of In Vitro Plants to Ex Vitro Conditions 1. Transfer shoots (3 5 cm long) with two or more leaves to root initiation (RI) medium amended with half strength MS basal medium forti fi ed with 0.5 mg/l IBA (Table 4 ) in 250 ml Erlenmeyer fl asks or sometimes in jam bottles for root induction (Fig. 1b ) (see Note 9). 2. Transfer plantlets with well-developed roots from solid RI medium to half strength MS basal liquid RE medium with a support of fi lter paper bridge for 14 days (Fig. 1c ) (see Note 10). 3. Transfer plantlets with profuse roots successively to one-fourth and one-eighth strength MS basal liquid RE media for 14 days in each, respectively (see Note 11). 4. Finally transfer the rooted plants in one-eighth strength of MS major and minor salt solutions (Table 1 ) for 7 days. 1. Transfer all the rooted plants to net pots containing autoclaved soilrite. At this stage, water plants with one-eighth strength of MS major and minor salt solutions. 2. Maintain the regenerated plants in growth room for 7 10 days inside jam bottles containing water so that the lower 1 cm of the net pots remain immerged (see Note 12 ). Leave the lid of jam bottles open for 1 h to whole day gradually increasing the time from the third day of transfer to net pots. 3. Finally transplant plants in 15 cm diameter pots in soil and maintain in the glasshouse for 14 days and then to the fi eld (Fig. 1d ). 4. Notes 1. Dissolve 100 mg HgCl 2 in 1 ml absolute ethanol and make up the volume with water to prepare 100 ml 0.1% HgCl 2 solution. 2. Dissolve 745 mg Na 2 EDTA 2H 2 O in 75 ml boiling double distilled water and add 557 mg FeSO 4 7H 2 O. Keep stirring on

8 276 T. Ray et al. a hot plate magnetic stirrer for at least 1 h at 100 C until the color of the solution turns to golden yellow. Finally make up the volume to 100 ml and store in amber-colored bottle at 4 C in the refrigerator. Except plant growth regulators, all other stock solutions are prepared by dissolving required amount of reagents in double distilled water and fi nally stored at 4 C. 3. Stock solutions of plant growth regulators should be prepared in small amount and stored at 0 C. Growth regulators should be stored maximum for 7 days. Add few drops of concentrated HCl to dissolve 20 mg AdSO 4 in 10 ml double distilled water before making up the fi nal volume. Always prepare fresh AdSO 4 solution just before media preparation. Similarly, dissolve 5 mg BA in 1 ml 1 N HCl and make up volume to 10 ml with double distilled water. Dissolve 5 mg IAA or IBA in 1 ml absolute ethanol and make up volume to 10 ml. 4. Adjust the ph with 0.1 N NaOH or 0.1 N HCl with continuous stirring. Addition of AdSO 4 increases the ph too high. Considering that keep the volume of media enough low, at least ml so that adequate HCl can be added to adjust the ph at We selected shoot apex meristem as an explant, since the organized meristems do not undergo much genetic change during cell division ( 9 ). 6. We tested various concentration of AdSO 4 ( mg/l) and BA (0.5 5 mg/l) in combination with auxin IAA ( mg/l) for shoot bud induction ( 6 ). Application of AdSO 4 (80 mg/l), BA (2 mg/l) with low concentration of IAA (0.1 mg/l) showed highest regeneration rate (95 ± 2.8%) and the highest number of shoot buds (60.2 ± 4.4) per explant (Table 4 ). Approximately after 7 days of culture, explants became enlarged in size, swelled, turned light greenish, and gradually within 20 days shoot buds began to appear. The high concentrations of BA and IAA were quite inhibitory on shoot bud formation. With the higher concentration of (0.5 mg/l) IAA, shoot apex explants swelled and turned into a yellowish hyaline mass of calli within days, which later turned brown ( 6 ). 7. Repeated subculture of shoot clusters in the same medium accelerated the formation of shoots in large numbers. The shoot multiplication could be repeated up to multiplication cycles with normal morphogenesis without returning to the original explant source. 8. Sometimes microshoots started rooting in the SE medium after being separated from clusters, but those roots were not well developed, so we cut those roots before transferring the individual microshoot in RI medium.

9 21 Cordyline Micropropagation We tried different concentrations (0.5 2 mg/l) of IAA or IBA or both in RI media. Of these, IBA (0.5 mg/l) was the most effective in inducing roots and showed normal rooting, whereas IAA produced deformed roots ( 6 ). 10. All rooted microshoots showed profuse growth and elongation in liquid half strength of MS basal medium. 11. It is very important to gradually eliminate sucrose from the liquid medium so that plants photosynthesize independently. 12. Immersing the lower part of the pots in water helped roots to come out of the net pot quickly and grow well into soil. References 1. DeMason DA, Wilson MA (1985) The continuity of primary and secondary growth in Cordyline terminalis (Agavaceae). Can J Bot 63: Khan S, Saeed B (2004) Establishment of in vitro technology for the large scale propagation of Cordyline (cv. Cordylin terminalis ). Pak J Bot 36: Murashige T (1974) Plant propagation through tissue cultures. Annu Rev Plant Physiol 25: Murashige T (1978) The importance of plant tissue culture on agriculture. In: Thorpe TA (ed) Frontiers of plant tissue culture, International Association for Plant Tissue Culture, University of Calgary 5. Miller LR, Murashige T (1976) Tissue culture propagation of tropical foliage plants. In Vitro 12: Ray T, Saha P, Roy SC (2006) Commercial production of Cordyline terminalis (L) Kunth. from shoot apex meristem and assessment for genetic stability of somaclones by isozyme markers. Sci Hortic 108: Ray T, Dutta I, Saha P, Das S, Roy SC (2006) Genetic stability of three economically important micropropagated banana ( Musa spp.) cultivars of lower Indo-Gangetic plains, as assessed by RAPD and ISSR markers. Plant Cell Tissue Organ Cult 85: Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: Larkin PJ, Scowcroft WR (1981) Somaclonal variation-a noval source of variability from cell culture for plant improvement. Theor Appl Genet 60: