Sunflower (Helianthus annuus L.) organogenesis from primary leaves of young seedlings preconditioned by cytokinin

Abstract

Leaf tissue is widely used for regeneration of many different plants, but an efficient regeneration method using leaf tissue of sunflower is not yet available. In this report, adventitious shoots were obtained from primary leaves of 7-day-old sunflower seedlings. If seeds were germinated on a medium without cytokinin, only 25 % of the leaf explants produced shoots after 3 weeks of culture on a shoot induction medium (SIM) containing 1.5 mg L⁻¹ benzylaminopurine (BA), giving an average of three shoot primordia per leaf. Shoot induction was enhanced up to sixfold when seeds were germinated on media containing BA or 6-(γ, γ-dimethylallylamino)purine at 5 mg L⁻¹ and higher, or kinetin at 15 mg L⁻¹ and above, with an average of 12–19 shoot primordia per leaf. Shoots further developed and elongated following transfer to a medium containing 0.1 mg L⁻¹ gibberellic acid. The highest number of developed shoots was obtained from leaves preconditioned with 5 mg L⁻¹ BA, with 1.9 developed shoots per explant. Histological analysis of leaves from seedlings germinated on a medium containing 5 mg L⁻¹ BA revealed increased cell division primarily in the leaf surface tissue on the adaxial side of the mid vein. After the leaves were excised and plated on SIM, these cells continued to divide and differentiate to form adventitious shoots, suggesting that the cytokinin-containing germination medium preconditioned leaves for shoot organogenesis. An in vitro micrografting approach was developed using the elongated shoots as scions, and 57 % of grafted shoots survived to form plants.