WUSCHEL Overexpression Promotes Callogenesis and Somatic Embryogenesis in Medicago truncatula Gaertn.

The induction of plant somatic embryogenesis is often a limiting step for plant multiplication and genetic manipulation in numerous crops. It depends on multiple signaling developmental processes involving phytohormones and the induction of specific genes. The WUSCHEL gene (WUS) is required for the production of plant embryogenic stem cells. To explore a different approach to induce somatic embryogenesis, we have investigated the effect of the heterologous ArabidopsisWUS gene overexpression under the control of the jasmonate responsive vsp1 promoter on the morphogenic responses of Medicago truncatula explants. WUS expression in leaf explants increased callogenesis and embryogenesis in the absence of growth regulators. Similarly, WUS expression enhanced the embryogenic potential of hairy root fragments. The WUS gene represents thus a promising tool to develop plant growth regulator-free regeneration systems or to improve regeneration and transformation efficiency in recalcitrant crops.

Cryopreservation of Quercus robur L. embryogenic calli.

Embryogenic calli of pedunculate oak (Quercus robur L.) were cryopreserved using direct immersion in liquid nitrogen. The pretreatment consisted of culture on a solid medium with increasing sucrose concentrations (0.25 M for 1 day, 0.5 M for 1 day, 0.75 M for 2 days, and 1.0 M for 3 days), followed by air desiccation of embryogenic calli to 17.3 percent (fresh weight basis). This method of cryoprotection was compared to a liquid cryoprotection treatment using high concentrations of sucrose solutions, followed by glycerol solutions. Regrowth of frozen tissue pretreated on a solid medium was significantly higher than those pretreated in the liquid solutions.

Cryopreservation of Prunus avium L. embryogenic tissues.

Embryogenic tissues from wild cherry (Prunus avium L.) were successfully cryopreserved by using a one-step freezing procedure. Cryoprotection consisted of a pretreatment on solid medium with increasing sucrose concentrations (0.25 M for 1 day, 0.5 M for 1 day, 0.75 M for 2 days, and 1.0 M for 3 days), followed by air desiccation to about 20 percent moisture content (fresh weight basis). This method was compared with a pretreatment on solid medium containing 5 percent DMSO and 2 percent proline, followed by immersion in a modified PVS2 cryoprotective solution. Pretreatment on solid medium with increasing concentrations of sucrose led to regrowth of frozen embryogenic tissues, and after 6 weeks of culture, growth was comparable to that of non-dehydrated and non-frozen tissues. By contrast, no regrowth was observed when embryogenic tissues were submitted to the solid/liquid pretreatment with DMSO/proline and a modified PVS2 solution.

Transcriptional regulation of proline biosynthesis in Medicago truncatula reveals developmental and environmental specific features.

The model legume plant Medicago truncatula accumulates free proline in response to hyperosmotic stress as do many other organisms. In order to analyse the transcriptional regulation of proline biosynthesis in M. truncatula, three cDNAs encoding Delta(1)-pyrroline-5-carboxylate synthetase (P5CS1, P5CS2; EC not assigned) and ornithine delta-aminotransferase (OAT; EC 2.6.1.13) were isolated. The cDNAs shared high homologies with the other plant sequences and genomic organization analysis indicated the presence of two P5CS and two putative OAT genes. The two P5CS genes showed differing transcript level regulation according to organs and in response to osmotic stress. MtP5CS1 steady-state transcript levels in the different plant organs were correlated with proline levels but transcript abundance was unaffected by osmotic stresses. MtP5CS2 transcripts were poorly detected in all organs but were strongly accumulated in shoots of salt-stressed plants. We suggest a specific of MtP5CS1 and MtP5CS2 as a housekeeping product and as a stress specific isoform, respectively. MtOAT transcripts were predominantly detected in roots and shoots of unstressed plants. Salt-stress treatment induced the accumulation of MtOAT transcripts in the whole plant whatever the developmental stage. In salt-stressed roots, a positive correlation was found between proline and MtOAT transcript accumulation. These results suggest that both ornithine and glutamate biosynthesis pathways contribute to the osmotic stress-induced proline accumulation in M. truncatula.