PRODUCTION OF ADVENTITIOUS SHOOTS AND PLANTLETS FROM THE HYPOCOTYL EXPLANTS OF SESBANIA ROSTRATA (BREMEK & OBREM.) AJAY KUMAR JHA 1 , SURYA PRAKASH 1 *, NEERU JAIN 1 , KANAN NANDA 2 , AND S. C. GUPTA 1 1 Department of Botany, University of Delhi, Delhi 110007, India 2 Department of Botany, Daulat Ram College, University of Delhi, Delhi 110007, India (Received 18 September 2001; accepted 12 March 2002; editor M. Madkour) Summary Adventitious shoots were induced from the hypocotyl explants derived from 12 – 15-d-old seedlings of Sesbania rostrata on Nitsch’s medium (Nitsch, 1969) supplemented with 1 mg l 21 (4.4 mM ) of N 6 -benzyladenine (BA). A maximum of 5:9 ^ 3:4 shoots per explant in 100% of cultures were obtained. The BA treatment for different time durations (1, 3, 5, 7, 10, 17, 21, or 30 d) exhibited significant variation in the caulogenic potential of the explants. BA treatment for 10 – 17 d proved optimum for the response. Although at all concentrations of kinetin the explants developed multiple shoots, they were malformed. Sucrose at 3% exhibited the development of the maximum of 3:5 ^ 0:9 shoots per explant with an average shoot length of 4:7 ^ 3:9 cm: Among the different carbon sources, i.e., fructose, galactose, maltose, mannose, and sucrose at 3% (w/v), sucrose supported the best caulogenic response. The role of various other factors (viz. size, orientation of explant, and seedling age) on the caulogenic response of the hypocotyl explants of S. rostrata were also studied. The shoot development in all cases was accompanied by the development of moderate to profuse callus at the basal cut end of the explant. The in vitro-regenerated shoots produced roots when transferred to half-strength MS medium (Murashige and Skoog, 1962) supplemented with 3% sucrose and 1 mg l 21 (4.9 mM ) indole-3-butyric acid (IBA). The developed plantlets were transferred to the field after an initial acclimatization period of 3 – 4 mo. Such transferred plants produced flowers and fruits in the field and exhibited the development of prominent and organized stem nodules. Key words: adventitious shoot development; forage crop; tissue culture; factors for caulogenesis; in vitro regeneration. Introduction Leguminous plants are next to members of Poaceae (Gramineae) in importance for the masses due to high seed protein content and wide eco-geographical distribution. Besides this, they are good sources of fuelwood, fodder and several industrial products. In addition, they are widely used in land reclamation, social forestry, and agroforestry programs (Isley, 1982). The genus Sesbania,a native of tropical regions of Africa, China, and South-East Asia, comprises over 50 species. Sesbania rostrata (Bremek & Obrem.) is mainly used as green manure. It also binds soil and is resistant to waterlogging. Besides this, it has a high fodder value (Kwon and Beevers, 1992). It exhibits a symbiotic association with rhizobial strain ORS 571, which is found in the stem nodules. It was introduced into India in the 1980s. Since then, it has gained immense importance due to its high capacity to assimilate both the soil and atmospheric nitrogen at a significantly high level. Vlachova et al. (1987) estimated that S. rostrata can assimilate nitrogen at 200 kg N 2 ha 21 in 50 d. The technique of micropropagation has proved very handy in mass propagation of plants in limited time and space with a cost-effective approach. Legumes are known for their recalcitrant nature, but still several studies have been carried out during the past decade to exploit the morphogenic potential for mass propagation of these plants (Babbar and Jain, 2000). The present study was undertaken to standardize the regeneration protocol for S. rostrata employing hypocotyl explants, as well as to assess the effect of various factors on the caulogenic potential of the hypocotyl explants. Materials and Methods The seeds of S. rostrata were procured from the Indian Agricultural Research Institute (IARI), New Delhi and Satellite Center of National Bureau of Plant Genetic Resources (NBPGR) at Amrawati, India. Seeds were treated with 0.1% (v/v) polysan (Polypharm Ltd., Mumbai, India) for 5 min followed by thorough washing in running tap water for half an hour. The seeds were pretreated with 80% sulfuric acid for 30 min, before surface-sterilization with freshly prepared chlorine water for 30 min. Subsequently, the seeds were rinsed four or five times with sterile distilled water and inoculated on Knop’s medium (Knop, 1865) containing 0.8% (w/v) bacteriological grade agar (Qualigens, Galxo Fine Chemical, India) and 1% (w/v) sucrose (Daurala, India), under aseptic conditions. Twelve to 15-d-old seedlings were used as the source of hypocotyl explants. Under aseptic conditions, the explants were placed on B5 (Gamborg et al., 1968), MS medium, N medium, and SH medium (Schenk and Hildebrandt, 1972) supplemented with 0.8% agar and 3% sucrose. The medium was supplemented with different concentrations (0, 0.1, 0.5, 1.0, 1.5, 2.0, 3.0, or 4.0 mg l 21 ) of N 6 -benzyladenine (BA), kinetin, 6(g,g-dimethylallylamino) purine (2iP), or zeatin (Sigma Chemical Co., St. Louis, MO). Different carbon *Author to whom correspondence should be addressed c/o Dr. David Mills, Institutes for Applied Research, Ben-Gurion University, Post Box 653, Beer- Sheva, Israel; Email psurya@lycos.com In Vitro Cell. Dev. Biol.—Plant 38:430–434, September–October 2002 DOI: 10.1079/IVP2002313 q 2002 Society for In Vitro Biology 1054-5476/02 $10.00+0.00 430