Regeneration of fertile interspecific hybrids from protoplast fusions between Helianthus annuus L. and wild Helianthus species.

The use of interesting characteristics from wild Helianthus species in sunflower breeding is limited by poor crossability or sterility of interspecific hybrids. To overcome this barrier, mesophyll protoplasts of Sclerotinia sclerotiorum-resistant clones of Helianthus maximiliani, H. giganteus and H. nuttallii were fused with hypocotyl protoplasts of H. annuus in the presence of polyethyleneglycol and dimethylsulfoxide. Fusion products were embedded in agarose and subjected to a regeneration protocol developed for sunflower protoplasts. Organogenic calli were transferred onto solid medium and emerging shoots were elongated in the absence of plant growth regulators. Rooting of shoots was induced by a 1-naphthaleneacetic acid treatment and putative hybrid plants from fusions between H. annuus + H. maximiliani and H. annuus + H. giganteus were transferred into the greenhouse. All of them exhibited a hybrid phenotype with a high percentage of rhizome producing plants. Their hybrid origin was confirmed by random amplified polymorphic DNA analysis. Plants flowered after 3-4 months and set seeds, of which 70-80% germinated.

Regeneration of fertile plants from Helianthus nuttallii T&G and Helianthus giganteus L. mesophyll protoplasts.

Interspecific hybridisation in the genus Helianthus via somatic cell fusion is thought to play an important role in future sunflower breeding programs. The establishment of this technique requires, however, the development of single-cell-regeneration protocols. For this purpose, we applied a regeneration protocol recently developed for Helianthus annuus L. to mesophyll protoplasts of two wild sunflowers (H. nuttallii T&G, H. giganteus L). Protoplasts of both species were embedded in agarose droplets and covered by liquid mKM medium. After 4-5 weeks, callus was transferred onto solid differentiation medium yielding plating efficencies of 1.5% (H. nuttallii) and 2.5% (H. giganteus). Emerging shoots were elongated on hormone-free medium, and root formation was induced by an NAA treatment. Regenerated plants were transferred to the greenhouse where they grew up to a height of 2 m and flowered after 3 months. Seeds were harvested from regenerated plants of both species.

A regeneration protocol for sunflower (Helianthus annuus L.) protoplasts.

Hypocotyl protoplasts of four different Helianthus annuus genotypes were cultivated for 22-28 days in agarose droplets covered with liquid medium. In the first week, supplementation of the medium with plant growth regulators was at a 0.8/1 ratio of cytokinin and auxin followed by a high auxin concentration in the second week and a cytokinin to auxin ratio of 8/1 in the third and fourth week. Following transfer onto solid medium containing cytokinin and auxin in a proportion of 40/1 morphogenic callus started to form globular structures that developed into leaf primordia. Subsequent shoot elongation and rooting were obtained on hormone free medium after dipping the cut shoots into high auxin solution. Thirteen weeks after protoplast isolation, plantlets could be transferred to the greenhouse. Shoot regeneration was obtained for all four cultivars (Florom-328, Cerflor, Euroflor, Frankasol) at different rates reflecting their regenerative potential.