Structural and ultrastructural differences between field, micropropagated and acclimated leaves and stems of two Leucospermum cultivars (Proteaceae)

Abstract

The anatomy of field, in vitro and acclimatized shoots (leaves and stems) of two cultivars of Leucospermum (L. cordifolium ‘Flame Spike’ and L. ‘Tango’) was compared using light, scanning and transmission electron microscopy. Field plants showed several scleromorphic anatomical structures related to excess solar radiation such as: cuticle thickness, subepidermal collenchyma and sclerenchyma. Furthermore, a large quantity of phenolic deposits present in the cell lumen of various tissues is also a scleromorphic feature. The special conditions during in vitro culture result in plantlets with abnormal morphology and anatomy. These disorders are associated with the gaseous environment in the culture vessels, low irradiance in the incubation chamber and the addition of sucrose, nutrients and growth regulators to the culture medium. After transfer from in vitro to ex vitro conditions, substantial changes in leaf and stem anatomy were observed, above all in cuticle thickness, epidermal characteristics (stomatal and trichome index, and stomatal and pore size), differentiation of leaf mesophyll, chloroplast structure, and amount and localization of phenolic deposits. These changes allowed the plants to adapt to the new environmental conditions. The study of anatomical features of in vitro shoots facilitated adapting the acclimation protocol to predict which plantlet would survive the critical acclimation stage.