Indian Journal of Experimental Biology Vol. 37, November 1999, pp. 1109-1112 Regeneration of pea (Pisum sativum L.) from seedling explants via organogenic nodule-like structures G Franklin & S Ignacirnuthu Entomology Research Institute, Loyola College, Chennai 600 034, India Received 16 December 1998; revised 7 June 1999 Organogenic nodule-like structures were initiated from the shoots developing at the cotyledonary node and shoot tip of young seedling, when cultured on Murashige and Skoog medium containing B5 vitamins supplemented with benzylaminopurine and eventually formed many shoot buds. The best results were obtained at 13 .3111M benzylaminopurine when the 5 days old seedling was placed in an inverted position on the medium. Shoot buds elongated on the initial medium, then, rooted in the presence of 2.46 11M indole-3-butyric acid and formed viable plants. Hardened plants were successfully established in the soil. Pea with its rich seed protein globally recognised as one of the important grain legumes is cultivated in the temperate regions. Pea protein is preferred for human consumption and animal nutrition because of its low levels of sulphur-containing amino acids, higher levels of lysine and other essential amino acids I. Pea, reputed as a genetic tool, is being used as model plant for modern researches in plant scicnccs 2 • Although shoot regeneration from various explants via multiple shoot formation 3 , direct and indirect organogenesis 4 , androgenesis 5 , protoplast isolation and culture 6 and somatic embryogenesis7 have been reported already, recent transformation studies in Pisum sativum 8 . 12 and other grain legumes l3 have focussed on simple, reproducible shoot multiplication procedure from pre- existing meristem. The present paper is a report of regeneration of shoots from the cultured young seedling ex plants of Pisum sativum via organogenic nodule-like structures. Materials and Methods For in vitro germination of the variety PG5 (obtained from TNAU, Coimbatore, Tamil Nadu), the seeds were washed several times in running tap water and then treated with 70% ethyl alcohol for 30s. Following the alcohol treatment, the seeds were sterilised with a solution containing 0.1% of each mercuric chloride and sodium dodecyl sulphate for 3 min. and repeatedly washed with sterilised double distilled water. The seeds were allowed to imbibe in sterilised double distilled water overnight. Well imbibed and turgid seeds were selected and inoculated in each culture tube contaInIng 20 ml MS basal medium for germination. Five day old seedlings were placed on the medium either in an inverted position (shoot tip pointing downward and touching the medium). The nodules produced from the shoots were either excised and cultured on fresh medium or left in the culture as such. d · 14 . . 15 MS me lUm contaInIng B5 vltamms - supplemented with different concentrations of BAP (0.0, 1.33, 13.3, 22.5 and 44.3 11M) was used for induction of nodules and subsequent development of shoots. Half strength MS medium containing IBA was used for rooting. The pH was adjusted to 5.8 by using either O.IM NaOH or O.IM HC\. The medium was solidified with 0.7% Bacto agar (Hi-media, Mumbai) before autoclaving at 1.4 kg cm· 2 for 20 min . Each culture tube containing 20 ml of medium was inoculated with one explant and plugged with non- absorbent cotton. The cultures were incubated in 16 hr photoperiod of cool-fluorescent light providing a quantum flux density of 30 11 mol m· 2 s· 1 at 25° ± 2°e. For every concentration 20 replicates were maintained and each experiment was repeated twice. Rooted pi ant lets were transferred to plastic cups containing sterile vermiculite, watered with sterile half-strength Hoagland's solution l6 and covered with a plastic sheet. The plants were acclimatised by gradually exposing them to the external environment over a period of 10 days. Hardened plants were transferred to soil in the green house and then transplanted to the field.