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1 Journal of Coastal Research Fort Lauderdale, Florida Spring 1993 n Vitro Propagation of pomoea pes-caprae (Railroad-Vine)' ( Michael E. Kane[', Kimon T. Birdt and Tammera M. Leet tenvironmental Horticulture Department University of Florida Gainesville, FL 32611, U.S.A. *Center for Marine Science Research University of North Carolina at Wilmington 7205 Wrightsville Avenue Wilmington, NC 28403, U.S.A.!.tlllllll,:..,. '"d '*i ;,-- ABSTRACT. KANE, M.E.; BRD, K.T, and LEE, T.M., n vitro propagation of pomoea pes-caprae (Railroadvine). Journal of Coastal Research, 9(2), Fort Lauderdale (Florida), SSN A plant micropropagation protocol for in vitro establishment, plant multiplication, rooting, and ex vitro acclimatization of the dune species pomoea pes-caprae (Railroad-vine) was developed. Supplementation of agar-solidified Murashige and Skoog medium (MS) with either the cytokinin 2-isopentenyladenine (2iP) or zeatin significantly promoted shoot regeneration from nodal segments. Maximum shoot regeneration occurred from nodal segments cultured on agar-solidified MS supplemented with 5 mg/liter ZiP. Medium supplementation with either 2iP or zeatin did not promote axillary branching of the shoots produced. Microcuttings were effectively rooted (95 %) in vitro when cultured on agar-solidified fullstrength MS supplemented with 1.0 mg/liter indolebutyric acid (lba). Transplant survival of rooted. pes-caprae microcuttings was 100%. Approximately 446,000. pes-caprae plants can be produced per year from a single nodal segment using this procedure. ADDTONAL NDEX WORDS: Dune plants, micropropagation, cytokinins, coastal revegetation. t;l ;'1 NTRODUCTON Beach dune and spoil island stabilization by planting is an accepted practice to control erosion (DAVS, 1975). Stabilization usually involves planting bare areas with different species of dune plants. The root and rhizome systems hold sand particles together, while the vegetation above ground retards wind and water driven erosion (WOODHOUSE, 1982). n the Southeastern U.S., dunes are stabilized following planting with a variety of species such as sea-oats (Uniola paniculata L.), american beach grass (Ammophila breviligulata), and railroad-vine (pomoea pes-caprae (L.) R. Br.) (WOODHOUSE et al., 1968). The source of these species for use in dune stabilization and environmental restoration projects is of increasing concern (ROBERTSON, 1985; BROWN, 1988). Plant materials are generally obtained from either seed or vegetatively propagated nurserygrown materials or as transplants obtained from received 17 May 1992; accepted in revision 3 August Florida Agricultural Experiment Station Journal Series No. R This research was supported by the Florida Sea Grant College Program with support from the National Oceanic and Atmospheric Administration, Office of Sea Grant, U.S. Department of Commerce, Grant No. NA89AA D-SG053. Contribution No. 61 from the University of North Carolina at Wilmington Center for Marine Science Research. donor populations. There is increasing resistance to allowing damage to native plant donor populations for the benefit of these restoration activities. Consequently, more efficient and less environmentally damaging alternative methods are needed to provide plant materials for environmental restoration. Micropropagation (in vitro propagation) systems have been developed for the efficient commercial propagation of many horticultural crops (CHU and KURTZ, 1983; GEORGE and SHERRNGTON, 1984). Micropropagation of dune and other native species offers advantages of rapid year-round production of disease-eradicated plants, decreased production space requirements and selection of elite clonal lines exhibiting superior growth and enhanced disease resistance and stress tolerance (GARTON and MOSES, 1985; KANE et al., 1989). Little information is available on the feasibility of applying micropropagation techniques for production of dune and marsh species (STAUB et al., 1988; COOK et al., 1989). pomoea pes-caprae (Convolvulaceae), railroad-vine, is an important coastal species colonizing dune swales and high beaches (CRAG et al., 1978; WOODHOUSE, 1982; BARNETT and CREWZ, 1991). Plantings of this species form mats which serve to stabilize soil by j.f.,j &j'l : 't :1, j

2 357 Railroad-Vine n Vitro Propagation POMOEA PES-CAPAE 10 21P (mg/l) B WEEK 7 POMOEA PES-CAPRAE STAGE A 1x S POMOEA PES-CAPRAE 1/2MS mg/l BA 21P (mg/l) c DAY 18 D Figure 1. Micropropagation of pomoea pes-caprae. (A) Excised nodal explant with single axillary bud used to establish shoot cultures. Scale bar = 1.0 mm. (B) Stage production of shoots in polypropylene culture vessels. (C) Effect of the cytokinin 2-isopentenyladenine (2iP) on shoot regeneration from nodal segments after 7 weeks culture. Note presence of callus (arrow) at bases of microcuttings. (D) Rooting of 1.0 cm microcuttings in 1/2 strength and full-strength MS supplemented with the auxin indolebutyric acid (lba) after 18 days. Scale bars in Figure B and C = 1.0 em, reducing wind erosion. Although 1. pes-caprae produces seed, scarification is required to produce significant germination rates. However, survival of new seedlings on the dunes is low (DEVALL and THEN, 1989). n the present study, a micropropagation protocol for pomoea pes-caprae is described. MATERALS AND METHODS Plant Source Runners of pomoea pes-caprae were collected from flowering populations growing at Cedar Key, Florida, in June Approximately 20 em long runners were rooted in commercial potting soil and sand (1:1) and maintained in a greenhouse. These stock plants served as the tissue explant source for subsequent experiments. Stage. Culture Establishment Runners were defoliated and cut into singlenode segments. Each nodal segment consisted of a prominent axillary bud (Figure 1A). Nodal segments were rinsed for 25 minutes under flowing tap water. Segments were surface sterilized by successive immersion in 50 % (vv) ethanol for 1 min followed by a 12-min agitated soak in 1.05% (w/v) aqueous sodium hypochlorite. Segments were then rinsed three times in sterile deionized

3 358 Kane, Bird and Lee CYTOKNf\1 10 D2iP Zeatin... 8 ::... 0 :::... CJ Z Ẉ.oJ 6-0 :: en 4 2 D- O CONCENTRATON (MG/LTER) Figure 2. Effect of the cytokinins 2-isopentenyladenine (2iP) and zeatin on shoot growth of pomoea pes-caprae nodal segments after 7 weeks culture. Values represent the mean response of 16 replicate nodal segments. water. Prior to inoculation, the bleached cut ends of each nodal segment were removed with a sterile scalpel. Surface sterilized nodal explants were transferred individually into 150 x 25 mm culture tubes containing 12 ml sterile establishment (Stage ) medium consisting of Murashige and Skoog [MS] inorganic salts (MURASHGE and SKOOG, 1962) and organics [MS] and 30 g/liter sucrose. The antioxidant ascorbic acid (200 mg/liter) was also added to retard phenolic browning. The medium was solidified with 8.0 g/liter TCS agar (JRH Biosciences, Lenexa, Kansas). All media were adjusted to ph 5.7 with 0.1 N KOH before autoclaving at 1.6 Kg cm? for 20 min at 121 "C. Unless stated otherwise, all cultures were maintained at 25 ± 2 C under a 16-hr photoperiod provided by cool-white fluorescent lights at 90 J.lmol m 2 sec-oj as measured at culture level. Cultures were examined weekly for the presence of visible microbial contamination. State. Shoot Multiplication Medium Optimization Nodal explants (each 10 mm long) were excised from established Stage shoot cultures, defoliated and then transferred into 473 ml clear polypropylene culture vessels (Better Plastics, Kissimmee, Florida) containing 100 ml sterile MS supplemented with 30 g/liter sucrose and solidified with 7.5 g/liter TC agar (see Figure B). Consequently, medium optimization studies were supplemented with either of the naturally-occurring cytokinin 2-isopentenyladenine (2iP) or zeatin at five concentrations (0,0.1, 1, 5 and 10 mg/ liter). Nodal explants were oriented vertically with the basal cut ends embedded 3.0 mm into the medium. A culture vessel inoculated with four nodal segments served as the experimental unit. Each treatment was replicated 4 times using a completely randomized design. Treatment effects on shoot multiplication and length were evaluated after 7 weeks. Treatment effects were analyzed statistically using the General Linear Models Procedure (SAS nstitute, 1986). Stage Rooting Shoot microcuttings, 2.0 em long, were excised from Stage cultures and transferred into individual 150 x 25 mm culture tubes containing 10 ml either full-strength or half-strength MS in-

4 Railroad-Vine n Vitro Propagation CYTOKNN D 2iP Zeatin 6 a:5 w m :E 4 -' Z w 3-' c o 22 o-"---_ _------''---_--,----_ l --,-_---L. -,---_-----L.....,----_-----' o CONCENTRATON (MG/LTER) Figure 3. Effect of the cytokinins 2-isopentenyJadenine (2iP) and zeatin on node production from pomoea pes-caprae nodal segments after 7 weeks culture. Values represent the mean response of 16 replicate nodal segments. organic salts, 10 mg/liter sucrose, 100 mg/liter myoinositol and 0.4 mg/liter thiamine-hcl supplemented with 0, 0.5, or 1.0 mg/liter indolebutyric acid (BA) and solidified with 8.0 g/liter TC@> agar. Each treatment was replicated 20 times with a single microcutting serving as the experimental unit. Treatment effects on percent rooting, root number and length were recorded after 18 days culture. Treatment effects were analyzed statistically using the General Linear Models Procedure (SAS NSTTUTE, 1985). Stage V Establishment in Soil Rooted microcuttings were transferred into plastic Pro-Trayfs 38 cell trays (TLC Polyforrn, nc., Morrow, Georgia) containing Vergro Klay Mix A soilless growing medium (Verlite Company, Tampa, Florida). Growing medium was saturated weekly with 20N-13.9P-3.7K liquid fertilizer containing 150 mg N/liter and each tray was covered with a clear vinyl propagation dome to maintain high humidity. Microcuttings were maintained at 25 C under a 16-hr photoperiod provided by cool-white fluorescent lamps at 70 mol S--1 m", After two weeks, the propagation domes were removed during a 5 day period to gradually decrease humidity and acclimatize the plantlets. Trays were transferred to a lightly shaded greenhouse (950 to 1,150,umol sec-- 1 m 2) with temperature set points for heating and cooling at 18/29 DC, respectively. Plantlet survival and establishment was evaluated 4 weeks post-transplant. RESULTS AND DSCUSSON Stage Culture Establishment Early trials with field collected material resulted in almost 100C;o contamination. When nodal segments from greenhouse cultures were used to start Stage cultures, the contamination rate was reduced and ranged between 20 to 60%. nitial Stage medium screening trials were established using several different inorganic salt based media including MS, Gamborg B5 (GAMBORG, 1970) and Woody Plant Medium (LLOYD and MCCOWN, 1980). Optimal in vitro establishment of excised nodal segments occurred on MS with production of single unbranched shoots by the fourth week of culture. Preliminary plant growth regulator screening experiments indicated that MS supple-

5 360 Kane, Bird and Lee MEDUM 100-i D 1/2 MS ?ft 60 o z o 40 0 a: FULL MS BA (MG/LTER) Figure 4. Effects of Murashige and Skoog [MS] salt dilution and indolebutyric acid (lba) on percent rooting of pomoea pescaprae microcuttings after 18 days culture. Each value represents the mean response of 20 microcuttings. mentation with either the synthetic cytokinins benzyladenine or kinetin did not significantly promote shoot growth and branching. Concomitant addition of BA and a-naphthaleneacetic acid (NAA) resulted in extensive callus formation which inhibited subsequent shoot outgrowth from lateral buds. Stage Shoot Multiplication Medium Optimization The lack of unbranched shoots on Stage media indicated that pomoea pes-caprae exhibits strong apical dominance in vitro. n Stage media experiments, shoot regrowth from subcultured nodal segments required the presence of a cytokinin in the medium (Figure 2). Nodal segments subcultured onto MS without cytokinin supplementation were unresponsive. However, MS supplementation with either the naturally occurring cytokinins 2iP or zeatin significantly promoted growth [df = 8; MSE = ; F = 12.80; Pr > F = ] (Figure 2) and node number [df = 8; MSE = ; F = 15.58; Pr > F = ] (Figure 3) from cultured nodal segments. Cytokinin supplementation also promoted callus formation at the base of each nodal segment (Figure C). Other species of pomoea exhibit similar capacities for callus formation when cultured in vitro (LU et al., 1990). n comparison to zeatin, 2iP was significantly more effective in promoting growth in vitro. Generally, cytokinins promote axillary shoot proliferation in vitro by decreasing apical control (PERK, 1987). However, in. pescaprae, regardless of cytokinin type and level, axillary branching of the shoots produced from nodal segments in vitro was not observed (Figure lc). Propagators should use 2iP for shoot multiplication since it is significantly less expensive ($0.091/mg) than zeatin ($6.75/mg). Stage Rooting and Stage V Establishment Preliminary experiments indicated that unrooted Stage microcutting exhibited very low survival when transferred directly to greenhouse conditions. Thirty-two percent of the microcuttings rooted when cultured on either 1/2 MS or fullstrength MS for 18 days. Medium supplementation with BA increased percent rooting (Figure 4) and root number [df = 5; SS = ; Pr > F = ] (Figure 5). Maximum percent

6 Railroad-Vine n Vitro Propagation 361 MEDUM 4 D 1/2 MS FULL MS 3 a: w n 2 t O o a: O--" L L ' o BA (MG/LTER) Figure 5. Effects of Murashige and Skoog [MS] salt dilution and indolebutyric acid (BA) on number of roots produced on pomoea pes-caprae microcuttings after 18 days culture. Each value represents the mean response of 20 microcuttings. c Ị. MEDUM 35 D 1/2 MS "-'" ::: t- 20 C) z w...j t- O 10 0 a: 5 FULL MS 0-' BA (MG/LTER) Figure 6. Effects of Murashige and Skoog [MS] salt dilution and indolebutyric acid (lba) on length of roots produced on pomoea pes-caprae microcuttings after 18 days culture. Each value represents the mean response of 20 microcuttings.

7 362 Kane, Bird and Lee (95%) rooting was observed on microcuttings cultured on full-strength MS supplemented with 1.0 mg/liter BA (Figure 4). Average root length significantly (df = 5; SS = ; F = 2.26; Pr > F = 0.05) decreased on both 1/2 MS and fu11 strength MS supplemented with greater than 0.5 mg/liter BA (Figures 6 and D). Rooted microcuttings were readily established (100l} survival) when transferred to greenhouse conditions. Given a maximum node regeneration rate of 5.8 nodes/nodal segment/7 week culture cycle and 95 Cf microcutting rooting, approximately 446,000 rooted plants of 1. pes-caprae can be clonally propagated per year from a single nodal segment using this micropropagation procedure. This procedure could provide for rapid production of multiple elite genotypes selected for branched growth habit and increased resistance to environmental stress and disease. LTERATURE CTED BARNET', M.R. and CREWZ, D.W., An ntroduction to Planting and Maintaining Selected Common Coastal Plants in Florida. Florida Sea Grant Salt Tolerant Vegetation Advisory Panel. Florida Sea Grant Report No. 97, 108p. BROWN, K., By any other name. Aquaphyte, 8, 4-5. CHU, LY.E. and KURTZ, S.L., Commercialization of plant micropropagation. n: AMMRATO, P. V.; EVANS, D.A.; SHARP, W.R., and BAJAl, Y.P.S. (eds.), Handbook of Plant Cell Culture, Vol. 5, Ornamental Species. New York: McGraw-Hill Publishing Company, pp COOK, D.A.; DECKER, D.M., and GALLAGHER, J.L., Regeneration of Kosteletzkya oirginica (L.) Pres!' (Seashore Mallow) from callus cultures. Plant Cell, Tissue and Organ Culture, 17, CRAG, R.M.; SMTH, D.C., and OHLSEN, A.C., Changes occurring in coastal dune formation and plant succession along the Martin County coastline. Florida Soil and Crop Science Society Proceedings, 37, DAVS, J.H., JR., Stabilization of Beaches and Dunes by Vegetation in Florida. Florida Sea Grant Report No.7, 52p. DEVALL, M.S. and THEN, L.B., Factors influencing the reproductive success of pomoea pes-caprae (Convolvulaceae) around the Gulf of Mexico. American Journal of Botany, 76, l. GAMBORG, D.L., The effects of amino acid and ammonium on growth of plant cells in suspension culture. Plant Physiology, 45, GARTON, S. and MOSES,M., Production of native plants in tissue culture. Combined Proceedings nternational Plant Propagators' Society, 35, GEORGE, E.F. and SHERRNGTON, P.D., Plant Propagation by Tissue Culture: Handbook and Directory ofcommercial Laboratories. Reading, United Kingdom: Eastern Press, 709p. KANE, M.E.; SHEEHAN, T.J.; DEHGAN, B., and PHLMAN, N.L., n vitro propagation of Florida native plants: Styrax americana and Persea palustris. Proceedings Florida State Horticultural Society, 102, LU, Q.; KOKuBu, T., and SATO, M., Plant regeneration in stem, petiole and leaf explant cultures of pomoea triloba L. Japanese Journal of Breeding, 40, LLOYD, G. and MCCOWN, B., Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia by use of shoot-tip culture. Combined Proceedings nternational Plant Propagators' Society, 30, MUHASHGE, T. and SKOOG, F., A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15, PERK, R.L.M., n Vitro Culture of Higher Plants. Boston: Martinus Nijhoff, 344p. ROBERTSON, D.J., Freshwater Wetland Reclamation in Florida: An Overview. Bartow, Florida: Florida nstitute of Phosphate Research, 27p. SAS NSTTUTE, NC SAS Users Guide: Statistics. Cary, North Carolina, 956p. STRAUB, P.F.; DECKER, D.M., and GALLAGHER, J.L., Tissue culture and long-term regeneration of Phragmites austalis (Cav.) Trin ex Steud. Plant Cell Tissue and Organ Culture, 15, WOODHOUSE, W.W., JR., Coastal dunes of the U.S. n: LEWS, R.R. (ed.), Creation and Restoration of Coastal Plant Communities. Boca Raton, Florida: CRC Press, pp WOODHOUSE, W.W., JR.; SENECA, E.D., and COOPER, A.W., Use of sea oats for dune stabilization in the southeast. Shore and Beach, 35, 1521.