Use of cytokinin pulse treatments and micrografting to improve sunflower (Helianthus annuus L.) plant recovery from cotyledonary tissues of mature seeds

Regeneration of sunflower plants from tissue culture can be problematic due to inefficiencies in shoot elongation and subsequent rooting. To minimize exposure to cytokinins, which can negatively impact shoot elongation and rooting, a cytokinin pulse treatment was evaluated by placing cotyledonary tissues from sunflower seeds onto a shoot induction medium containing 1.5 mg L-1 6-benzylaminopurine and 0.2 mg L-1 1-naphthaleneacetic acid for 0, 2, 4, 8, 12, and 16 d, prior to transfer to an elongation medium containing 0.1 mg L-1 gibberellic acid. The 16-d pulse treatment gave the highest numbers of shoot primordia, but shoot development was minimal and only 0.4 well-developed shoots could be obtained per explant. The 4-d pulse treatment followed by 14 d of culture on elongation medium led to the production of 5.5 well-developed shoots per explant. For plant recovery, well-developed shoots obtained with the 4-d pulse treatment followed by a 14-d elongation treatment were micrografted onto in vitro seedlings, with graft survival of over 60%. Short pulse treatments (4- and 8-d), followed by culture on elongation medium, generated well-developed shoots that seemed to be more responsive to micrografting than shoots obtained with the 16-d pulse treatment. The use of the 4-d pulse treatment with micrografting of well-developed shoots significantly improved the efficiency of whole-plant recovery of sunflower.