Somatic embryogenesis and cryopreservation of South African bread wheat (Triticum aestivum L.) genotypes

• Established in vitro regeneration protocols for Southern African wheat cultivars
• Immature embryos, isolated 12 days post anthesis, regenerated the best in vitro.
• Maltose, MS vitamins and silver nitrate increased explant in vitro regeneration.
• A novel cryopreservation protocol was developed for immature wheat seeds.
• Desiccation of tissue prior to cryopreservation ensured successful preservation.

The present study focused on the development of efficient in vitro regeneration protocols for six southern African bread wheat genotypes (Triticum aestivum L.). The tested wheat genotypes showed variation in their in vitro coefficiency abilities, with efficiencies ranging from 0 to 36.5%. Gamtoos and Tugela genotypes displayed the highest and lowest regeneration efficiencies, respectively on the tested growth media. The data indicated that immature embryos, isolated 12 days post-anthesis, resulted in embryogenic calli formation 4 to 6 days after initiation in the presence of picloram and/or 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious shoots emerged from 3 to 4 week old calli in the presence of 6-benzylaminopurine (BAP) or zeatin. Maltose as carbon source, MS vitamin mix and the addition of 50 μM silver nitrate also increased the in vitro regeneration abilities of the wheat explant material. Rooting of emerged in vitro plantlets was established on MS or half strength MS without the addition of any phytohormones. Furthermore, a novel cryopreservation protocol was successfully developed by encapsulation/vitrification/dehydration of immature wheat seeds. Immature seeds encapsulated in alginate beads, vitrified in 0.5 M sucrose, desiccated for 72 h, cryoprotected in 80% glycerol and flash frozen in liquid nitrogen resulted in the highest survival rate of immature embryos isolated after cryopreservation and regenerated in vitro. Desiccation of tissue prior to cryopreservation seems to be the singularly most important step to ensure successful preservation of wheat tissue.