INSTYTUT DENDROLOGII

Biochemical and biophysical conditions of cryopreservation of embryonic axes of Acer tree seeds

Cryopreservation in liquid nitrogen (LN, -196°C) is method of long-term storage of plant material. This study was aimed to determine the influence of cryopreservation on viability, membranes, and oxidative stress in embryonic axes (EAs) excised from orthodox seeds of Norway maple (Acer platanoides L.) and recalcitrant seeds of sycamore (A. pseudoplatanus L.). Orthodox seeds tolerate desiccation and low temperature, while recalcitrant seeds are sensitive to both factors. The EAs were cryopreserved in three steps: (i) dehydration at room temperature; (ii) slow freezing to -40°C, at 0.25°C min^-1; ( iii) rapid freezing in LN at 600°C min^-1 for 24 h. After cryostorage EAs were rewarmed at 370°C min^-1 and their regrowth was assessed during in vitro culture.

In this study, differential calorimetry (DTA) showed that the freezing transition of water in EAs decreased with reduction of water content. Water crystallization, causing membrane degradation, was not observed in A. platanoides when moisture content was below 26.8%, while in A. pseudoplatanus, below 35.8%.

Embryonic axes and zygotic embryos of A. platanoides show similar tolerance to dehydration, as they can be dried to moisture content of 5% without viability loss. In A. pseudoplatanus, EAs tolerate water loss to 10% and are more tolerant to dehydration than zygotic embryos. Cryopreservation of EAs resulted in the highest viability and the highest number of well-developed plantlets on the medium for both species: A. platanoides (after dehydration to 15-10%) and A. pseudoplatanus (15-20%). Several months of cryopreservation ofEAs did not affect their viability in vitro culture, as compared to 24 h storage. EAs, which were still highly viable after cryopreservation, did not show any lethal damage to membranes. In embryo axes of A. platanoides, membranes maintained stability during cryopreservation after dehydration to less than 20%, because of stable content of two major phospholipids: phosphatidylcholine (PC) and phosphatidylethanolamine (PE). By contrast, in A. pseudoplatanus the loss of permeability of membranes to electrolytes during cryopreservation was accompanied by an increased de-esterification of membrane phospholipids and peroxidation of fatty acids, as compared to A. platanoides.

Dehydration followed by storage in LN of EA of both Acer species causes oxidative stress, reflected in accompanied by the formation of reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂) and superoxide anion radical (O₂^•-). ROS scavenging involves non-enzymatic antioxidants, e.g. ascorbic acid (AsA), glutathione and ?-tocopherol, as well as antioxidant enzymes: superoxide dismutase (SOD), guaiacol peroxidase (POX) and catalase (CAT). Glutathione was the antioxidant most strongly involved in cell protection in both studied species, as its concentration rapidly decreased in EAs after freezing to -40°C and in cryopreserved EAs, while their viability declined. Concentration of ?-tocopherol and activity of antioxidant enzymes (POX and SOD) were about 36% lower after the stress of dehydration to 10% and cryopreservation in both studied species, as compared to the control samples. AsA level in both studied species, as well as CAT activity in A. platanoides, did not change during cryopreservation.

The most dangerous step of cryopreservation for Acer embryonic axes is dehydration, which caused the increase of ROS production and membrane damage. EAs of A. platanoides are more tolerant than tissues of A. pseudoplatanusare able to protect membranes against damage caused by dehydration and low temperatures stress (-40°C and -196°C).