Random mutagenesis was applied to produce a new wheat mutant (RYNO3926) with superior characteristics regarding tolerance to water deficit stress induced at late booting stage. The mutant also displays rapid recovery from water stress conditions. Under water stress conditions mutant plants reached maturity faster and produced more seeds than its wild type wheat progenitor. Wild-type Tugela DN plants died within 7 days after induction of water stress induced at late booting stage, while mutant plants survived by maintaining a higher relative moisture content (RMC), increased total chlorophyll, and a higher photosynthesis rate and stomatal conductance. Analysis of the proteome of mutant plants revealed that they better regulate post-translational modification (SUMOylation) and have increased expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) proteins. Mutant plants also expressed unique proteins associated with dehydration tolerance including abscisic stress-ripening protein, cold induced protein, cold-responsive protein, dehydrin, Group 3 late embryogenesis, and a lipoprotein (LAlv9) belonging to the family of lipocalins. Overall, our results suggest that our new mutant RYNO3936 has a potential for inclusion in future breeding programs to improve drought tolerance under dryland conditions.

Drought response in wheat is considered a highly complex process, since it is a multigenic trait; nevertheless, breeding programs are continuously searching for new wheat varieties with characteristics for drought tolerance. In a previous study, we demonstrated the effectiveness of a mutant known as RYNO3936 that could survive 14 days without water. In this study, we reveal another mutant known as BIG8-1 that can endure severe water deficit stress (21 days without water) with superior drought response characteristics. Phenotypically, the mutant plants had broader leaves, including a densely packed fibrous root architecture that was not visible in the WT parent plants. During mild (day 7) drought stress, the mutant could maintain its relative water content, chlorophyll content, maximum quantum yield of PSII (Fv/Fm) and stomatal conductance, with no phenotypic symptoms such as wilting or senescence despite a decrease in soil moisture content. It was only during moderate (day 14) and severe (day 21) water deficit stress that a decline in those variables was evident. Furthermore, the mutant plants also displayed a unique preservation of metabolic activity, which was confirmed by assessing the accumulation of free amino acids and increase of antioxidative enzymes (peroxidases and glutathione S-transferase). Proteome reshuffling was also observed, allowing slow degradation of essential proteins such as RuBisCO during water deficit stress. The LC-MS/MS data revealed a high abundance of proteins involved in energy and photosynthesis under well-watered conditions, particularly Serpin-Z2A and Z2B, SGT1 and Calnexin-like protein. However, after 21 days of water stress, the mutants expressed ABC transporter permeases and xylanase inhibitor protein, which are involved in the transport of amino acids and protecting cells, respectively. This study characterizes a new mutant BIG8-1 with drought-tolerant characteristics suited for breeding programs.

a b s t r a c t Edited by J Van StadenThe 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.