Plant Cell Reports (1992) 11:44 47 PlantCell Reports 9 Springer-Verlag 1992 High frequency adventitious shoot regeneration from immature cotyledons of pea (Pisum sativum L.) Sebahattin Ozcan, Mehdi Barghchi, Simon Firek, and John Draper Department of Botany, University of Leicester, Leicester LEI 7RH, UK Received September 26, 1991/Revised version received December 2, 1991 - Communicated by I. Potrykus ABSTRACT A procedure has been developed which allows high frequency adventitious shoot regeneration from immature cotyledons of pea. Prolific shoot regeneration occurred following an initial callus growth on a Murashige and Skoog (MS) medium containing 0.5 mg/l 6-benzyleminopurine (BAP) and 4 mg/l ~-naphthaleneacetic acid (NAA). Cotyledon explants proximal to the embryonic axis had the highest regeneration potential,however, the presence of an embryonic axis inhibited adventitious shoot regeneration. Addition of silver nitrate (AgNOs) to the medium did not promote the number of regenerated shoots but resulted in shoots with well developed tendrils and large stipules which had a reduced rooting capacity. Regenerated shoots rooted readily (80-90~) in half strength MS medium containing 1 mg/l indole-butyric acid (IBA) and further established well in compost. INTRODUCTION Conventional seed legume breeding programmes can be improved and complemented with in vitro genetic manipulation methods if an efficient plant regeneration is available. Adventitious shoot regeneration and somatic embryogenesis has been reported for pea in recent years (Gemborg et al. 1974, Mroginski and Kartha 1981, Hussey and Gunn 1984, Rubluo et al. 1984, Kysely et al. 1987, Natali and, Cavallani 1987, Puonti-Kaerlas and Erikson 1988, Lehminger-Martens and Jacobsen 1989, Kysely and Jacobsen 1990, De Kathen and Jacobsen 1990, Puonti-Kaerlas et al. 1990, Nauerby et al. 1991) from a range of explants (immature leaflets, shoot apices, epicotyls, protoplasts, nodal thin cell layer segments, immature embryos and cotyledons) cultured in vitro. However, in these reports regeneration was rather slow and had a low frequency. Although transgenic fertile pea plants have been reported previously (Puonti-Kaerlas et a2. 1990), the regeneration system they describe required many steps on media with different hormone combinations over several months. Therefore, inefficient regeneration systems seem to be the greatest barrier to the efficient production of transgenic pea plants. The present research aims are to provide tissue culture techniques for the potential genetic manipulation of pea through i~robacteriuu~mediated transformation or direct gene transfer techniques and for somaclonal variation studies in the future. This paper reports the morphogenic potential of cotyledon explants at various developmental stages. Offprint requests to: J. Draper MATERIALS AND METHODS Plant material: Pea cultivars "Orb" and "Consort", supplied by Sharpes International Limited, UK were raised in a greenhouse under a 18/6 hour light/dark (Osram high pressure sodium lamps, 400W) and a 22/16 "C day/night temperature regime. Seed pods were harvested at various stages of development and sterilized in 20~ commercial bleach ("Domestos") for 20 min in 500 ml glass jars and then washed 3 times in sterilized tap water. Seeds were removed from the sterile pods in a laminar air flow hood. After removal of the seed coat, the embryonic axis was gently prised off the split seed and axillary meristems at the area of attachment of the cotyledons to the embryo axis were removed under a dissecting microscope. The whole, or parts of the cotyledons, ranging in size from 1 to 9 mm and also the embryonic axes (Fig. i) were cultured on an appropriate medium for adventitious shoot regeneration. The cultures were grown at 24 "C under cool white fluorescent light (4000 lux) with a 16 h photoperiod. Shoots were excised and rooted in agar-solidified medium (xl/2 MS) supplemented with indole-butyric acid (IBA). For regeneration experiments each treatment had three replicates consisting of 100XI0 mm Petri dishes each containing 5-10 cotyledon explants. For rooting experiments each treatment had three replicates consisting of 125 ml glass jars containing 3-5 explants. All experiments were repeated at least once and the results were pooled. ~dia: MS mineral salts and vitamins (Murashige and Skoog 1962) containing 0.7% agar and 3~ sucrose were used as a basal medium. This basal medium was supplemented with 0-4 mg/1 6-benzylaminopurine (BAP), and 0-8 mg/l ~-naphthaleneacetic acid or 0-8 mg/l IBA. Silver nitrate (AgNO3) (5 and i0 mg/l) was also added to some media to study its effect on morphogenesis in pea cotyledons. The medium was adjusted to pH 5.6 with IN NaOH or IN HCI before autoclaving at 120 "C, 1.4 kg/cm z for 20 min. RESULTS Shoot regeneration potential of different pea organ ewplauts. Various organ explants (at different developmental stages) were investigated in order to establish efficient adventitious shoot regeneration. Hoot, epicotyl segments, immature leaflets and shoot tips from in vitro grown seedlings, and mature and immature embryos and cotyledons from green. house-grown plants were examined. The effect of varying the concentration of cytokinins and auxins in the growth media was also investigated.