Plant Regeneration from Hypocotyl‐ and Anther‐Derived Callus of Berseem Clover1

Abstract

Plants were regenerated from hypocotyl and anther explains of berseem clover (Trifolium alexandrinum L.) on Murashige and Skoog (MS) medium containing various combinations of plant growth regulators. The most efficient production of plants from hypocotyl explants involved: callus induction on MS medium with 1.0 mg/liter of naphthaleneacetic acid (NAA) and 1.5 mg/liter 6‐furfurylaminopurine (KIN); callus increase on MS medium with 2.0 mg/liter of NAA and 0.1 mg/liter of N6‐(Δ2‐isopentenyl) adenine (2iP); induction of shoots on MS medium with 0.5 mg/liter each of NAA and KIN followed by induction of roots on MS medium with 1.0 mg/liter of indoleacetic acid (IAA) and 0.1 mg/liter of 6‐benzylaminopurine (BAP). Suspension cultures in liquid MS medium containing 2.0 mg/liter of NAA and 0.2 mg/liter of 2iP provided filterable cell preparations with 45% viable cells, 4% of which gave rise to colonies within 3 weeks after transfer to agar plates. Shoot development was observed when callus from the colonies was cultured on MS medium with 0.5 mg/liter of NAA and KIN. Twenty percent of uncontaminated anthers from a single plant cultured on MS medium containing 1.0, 0.1, and 0.01 mg/liter of NAA, 2,4‐D, and 2iP, respectively, produced callus which could be maintained and from which plants could be regenerated by culturing on MS medium with 0.5 mg/liter each of NAA and KIN. Preliminary results indicate that cells of root tips from hypocotyl‐ and antherderived callus have the expected diploid and haploid number of chromosomes (2n = 16 and n = 8, respectively).