HORTSCIENCE, VOL. 30(5), AUGUST 1995 1074 PROPAGATION & TISSUE CULTURE HORTSCIENCE 30(5):1074–1077. 1995. opment of transgenic plants or the production of useful somaclonal variants, it is desirable to develop a tissue culture system that results in several shoots per explant and protocol appli- cable to various genotypes. Agrobacterium tumefaciens has been used successfully as a vector for the genetic trans- formation of dicotyledonous plants. Explants are usually wounded before Agrobacterium cocultivation, as the release of phenolic com- pounds from injured plant cells that activate vir genes are critical to T-DNA transfer (Horsch et al., 1986). To enhance transformation, the explant area exposed to wounding often needs to be increased. However, it is critical to en- sure that such explant wounding does not adversely affect regeneration potential. Ex- plant orientation during incubation also af- fects shoot induction and proliferation in other crops, such as soybean [Glycine max (L.) Merr.] (Kim et al., 1990) and apple (Malus domestica Borkh.) (Zimmerman and Fordham, 1989). The objective of this research was to de- velop an improved plant regeneration system for sweetpotato, specifically to 1) extend the regeneration protocol to many genotypes; 2) produce multiple shoots per explant; and 3) investigate the effect of surface-injury, com- bined with the orientation of explants, on shoot regeneration. Materials and Methods Plant material. The sweetpotato genotypes used in this study were obtained as in vitro shoot tips from the U.S. Dept. of Agriculture/ Agricultural Research Service Regional Plant Introduction Station, Griffin, Ga. (Jarret, 1989). As sweetpotato is vegetatively propagated, each genotype thus represents a clone. Plants were maintained as shoot cultures in vitro using a multiplication medium consisting of Murashige and Skoog (MS) (1962) salts with (in mg•liter –1 ) 100 myoinositol, 0.4 thiamine hydrochloric acid, 100 L-arginine, 200 ascor- bic acid, 2 pantothenic acid, 20 putrescine, and 20 gibberellic acid (Dodds et al., 1991) with 3.5 g Phytagel/liter (Sigma Chemical Co., St. Louis) used as a gelling agent. The pH of the media throughout this study was adjusted to 5.8 (before Phytagel addition). The medium was poured (40 ml) into GA 7 vessels (Magenta Co., Chicago) and autoclaved at 121C at 1.1 kg•cm –2 for 20 min. Stock plants were subcul- tured every 4 to 6 weeks using nodal sections (at least three nodes per explant) that were placed horizontally on the medium. The ves- sels were wrapped with parafilm. All plants were incubated in growth chambers at 25 ± 3C with a 16-h light/8-h dark photoperiod. Light (50 μmol•m –2 •s –1 ) was produced by cool-white fluorescent tubes combined with Grolux tubes (2:1). Explants and culture. Petiole pieces (5 to 10 mm) isolated from apical leaves were used as explants. The basal end of the petiole was removed (1 to 2 mm) to eliminate any potential axillary meristem. A two-stage protocol was followed throughout: the stage I culture con- sisted of basal medium supplemented with 2,4-D at 0.2 mg•liter –1 . The basal medium was composed of inorganic MS salts, myoinositol (100 mg•liter –1 ), thiamine-HCl (0.4 mg•liter –1 ), sucrose (3% w/v), and Phytagel (0.35% w/v). In stage II, the basal medium was supple- mented with 2iP, TDZ, or both, depending on the study. Control treatments included TDZ at 0.2 mg• liter –1 , as this treatment was found to result in the highest shoot regeneration re- sponse in genotype PI 318846-3 in our previ- ous study (Gosukonda et al., 1995). However, further preliminary studies showed that when 2iP was added to the stage II medium, it was necessary to reduce the concentration of TDZ from 0.2 to 0.05 mg•liter –1 . When 2iP was used without TDZ, shoot regeneration failed. Four 2iP levels (0.02, 0.05, 0.08 and 0.2 mg• liter –1 ) were tested in the stage II medium containing TDZ at 0.05 mg•liter –1 . All growth regulators were filter-sterilized and added to the auto- claved media before dispensing (25 ml) into 100 × 15-mm petri dishes. Most chemicals used in this study were obtained from Sigma Chemical Co., while TDZ was purchased from Crescent Chemical (Hauppauge, N.Y.). Petiole explants were placed horizontally on the nutrient medium in stage I, and the orientation of the explant on the stage II media varied with the type of experiment (Fig. 1). In the study on testing various levels of 2iP, explants of various genotypes were placed vertically with their bases embedded inside the nutrient medium. To assess the effect of surface injury of explants on shoot regenera- tion, petiole explants of PI 318846-3 were scraped using a fine scalpel to remove the epidermal (and possibly subepidermal) tis- sues across the petiole (Fig. 2A). The effect of There are several reports of in vitro regen- eration of sweetpotato plants either adventi- tiously or via somatic embryogenesis (Kuo, 1991). However, most of these approaches result in a low frequency of shoot regeneration and have been of limited use in the develop- ment of transgenic sweetpotato plants with normal phenotype (Al-Juboory and Skirvin, 1991; Otani et al., 1993; Prakash and Varadarajan, 1992). When a two-stage system of culture was employed with petiole explants pulsed with 2,4-D (0.2 mg• liter –1 ) for 2 to 3 days and then cultured on a medium contain- ing thidiazuron (TDZ; 0.2 mg•liter –1 ), most petiole explants of sweetpotato developed adventitious shoots (>80%) (Gosukonda et al., 1995). However, regeneration was limited to the production of one shoot per explant and only in a few sweetpotato genotypes. For the regeneration protocol to be useful in the devel- Received for publication 9 Dec. 1994. Accepted for publication 26 May 1995. Contribution no. 246 of the George Washington Carver Agricultural Ex- periment Station. We thank Korsi Dumenyo and Matand Kanyand for their technical assistance. Re- search supported by grants from U.S. Dept. of Ag- riculture (ALX-9201871), U.S. Agency for Interna- tional Development (DAN 5053G0000580 C), and National Aeronautics and Space Administration (NAGW-2940). The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. 1 Graduate Student. 2 Associate Professor; to whom reprint requests should be addressed. 3 Postdoctoral Research Associate. Shoot Regeneration in Vitro from Diverse Genotypes of Sweetpotato and Multiple Shoot Production per Explant Ramana M. Gosukonda 1 , C.S. Prakash 2 , and Ananta Porobo Dessai 3 Plant Molecular and Cellular Genetics Laboratory, School of Agriculture and Home Economics, Tuskegee University, Tuskegee, AL 36088-1641 Additional index words. Ipomoea batatas, biotechnology, organogenesis, tissue culture, thidiazuron, N 6 -(2-isopentenyl) adenine, explant variables Abstract. Studies were conducted to improve adventitious shoot regeneration in sweetpotato [Ipomoea batatas (L.) Lam.], specifically to extend the protocol to many genotypes and to elicit production of multiple shoots per explant. The use of a two-stage procedure where excised petioles were incubated on Murashige and Skoog (MS) (1962) medium with 2,4-D (0.2 mg•liter –1 ) for 3 days and transferred to a second medium containing MS salts with thidiazuron and 2iP (0.05 mg•liter –1 each) resulted in shoot regeneration from eight of 13 genotypes tested, including elite sweetpotato cultivars such as ‘Jewel’ and ‘Rojoblanco’. PI 318846-3 was the most regenerable genotype, with up to 77% of explants producing one to three shoots per explant. The orientation of the petiole on the nutrient medium was critical; those placed vertically inverted developed multiple shoots. Wounding explants through epidermal peeling with normal horizontal orientation of the explants during incubation also resulted in multiple shoot production (about three shoots per explant). Interference with auxin transport due to explant inversion or wounding may have stimulated increased shoot induction. Chemical names used: 2,4 dichlorophenoxyacetic acid (2,4-D); N-phenyl-N´-1,2,3-thiadiazol-5-ylurea (thidiazuron); N 6 -(2-isopentenyl) ad- enine (2iP).