An in vitro system for callus induction and plant regeneration from explants-bud, stem, leaf and cotyledon pieces as well as protoplasts isolation enzymatically from leaves and their culture in different media is described using six wild diploid species (2n:2x:34) of sunflower viz.,Helianthus nuttallii Tats., H.mollis Lam., H divaricatus L., H. debilis Nutt., H. maximilliani S., and H. praecox E & G. All explants formed calli on a modified MS medium (MSD4). Plant regeneration was obtained in all species from calli of the above explants except cotyledons on R mnedium. Maximum regeneration (81.25%) was obtained from stem-induced calli of H. nuttallii whereas it was only 4.80% from bud-induced calli of H. mollis. Protoplasts from all species were examined for division, colony and subsequent callus formation. Colonies were formed in all species, however, callus formation was obtained only in H. praecox. Freshly isolated protoplasts of these species have shown variability for yield, size and viability.