Viability loss of neem (Azadirachta indica) seeds associated with membranephase behaviour

Storage of neem (Azadirachta indica) seeds is difficult because of their se nsitivity to chilling stress at moisture contents (MC) greater than or equa l to 10% or imbibitional stress below 10% MC. The hypothesis was tested tha t an elevated gel-to-liquid crystalline phase transition temperature (T-m) of membranes is responsible for this storage behaviour. To this end a spin probe technique, Fourier transform infrared microspectroscopy, and electron microscopy were used. The in situ T, of hydrated membranes was between 10 degreesC and 15 degreesC, coinciding with the critical minimum temperature for germination. During storage, viability of fresh embryos was lost within two weeks at 5 degreesC, but remained high at 25 degreesC. The loss of via bility coincided with an increased leakage of K+ from the embryos upon imbi bition and with an increased proportion of cells with injured plasma membra nes. Freeze-fracture replicas of plasma membranes from chilled, hydrated ax es showed lateral phase separation and signs of the inverted hexagonal phas e. Dehydrated embryos were sensitive to soaking in water, particularly at l ow temperatures, but fresh embryos were not. After soaking dry embryos at 5 degreesC (4 h) plus 1 d of further incubation at 25 degreesC, the axis cel ls were structurally disorganized and did not become turgid. In contrast, c ells had a healthy appearance and were turgid after soaking at 35 degreesC. Imbibitional stress was associated with the loss of plasma membrane integr ity in a limited number of cells, which expanded during further incubation of the embryos at 25 degreesC. It is suggested that the injuries brought ab out by storage or imbibition at sub-optimal temperatures in tropical seeds whose membranes have a high intrinsic T-m (10-15 degreesC), are caused by g el phase formation.