Abstract
BackgroundGarlic has lost its ability to form seeds in the course of its domestication. Therefore, the germplasm storage via cryopreservation is increasingly applied. The progression of the various steps within the cryopreservation procedure is accompanied by declining survival rates of the explants. Much of the recent work on cryo-stress has been focussed on osmotic and cold stress components. However, two decades after invention of garlic cryopreservation, the function of metabolites and oxygen in and around the cryopreserved tissues is still largely obscure.MethodsIn this study, hypoxia was characterized in cryopreservation of garlic with oxygen sensors and amino acid metabolism. Furthermore, malondialdehyde, soluble sugars and ammonium were quantified to demonstrate the influence of cryo-stress in declining survival rates.ResultsTo better understand the possible reasons for a reduction in the survival rate at the subsequent steps of cryopreservation, the concentration of amino acids, ammonium, γ-aminobutyric acid (GABA), soluble sugars, malondialdehyde (MDA), and oxygen were measured in garlic shoot tips undergoing cryopreservation. Using microsensors, a very low oxygen concentration (<0.1 μM) was detected within the central meristem region of the shoot apex. When apices were immersed in cryoprotectant solution, the well-oxygenated peripheral regions (foliage leaf bases) became likewise hypoxic within a few minutes, probably resulting from strongly restricted gaseous diffusion.ConclusionsTissue level oxygen measurements supported the occurrence of hypoxia while biochemical analysis indicated adaptive responses, in particular the modulation in alanine and glutamate metabolism. The possible role of serine and glycine metabolism during cryopreservation is also discussed.
Highlights
Garlic has lost its ability to form seeds in the course of its domestication
We demonstrated that some of the metabolites are regulated during dehydration and after storage in liquid nitrogen (LN)
In vitro cultures were maintained via cyclic micropropagation using standard medium consisting of MS [13] with 0.1 mg/L naphthalene acetic acid (NAA), 0.5 mg/L N6-(2-isopentenyl) adenine (2iP), 30 g/L sucrose, and 10 g/L agar, pH 5.8 [14, 3] and maintained at 25 °C and 16 h illumination of 60 μmol m−2 s−1
Summary
The germplasm storage via cryopreservation is increasingly applied. Two decades after invention of garlic cryopreservation, the function of metabolites and oxygen in and around the cryopreserved tissues is still largely obscure. Cryopreservation, the storage of germplasm at ultra-low temperature in liquid nitrogen, was successfully applied to many plant species [1, 2]. Shoot apices are the preferred tissue source for garlic cryopreservation [3]. Meristem cells located in the apical region of shoot tips have higher tolerance towards cryo-storage protocols [4]. Vitrification is characterized by initiating glass transition of the cellular water content.
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