Single-Leaf Plantlet Bioassays for the Study of Root Morphogenesis and Rhizobium-Legume Nodulation

Abstract

New plant bioassays have been developed to (a) study root morphogenesis in small seeded legumes, and (b) enable the routine examination of large numbers of independently transformed plants. In addition, these bioassays enable analysis of specific plant gene expression. The single leaf plantlet assay uses simplified plants that are without shoot meristems, have a photosynthetic organ, grow roots which can be nodulated by Rhizobium bacteria, and can form lateral roots. The rooted cotyledon assay (RCA) was developed to investigate root morphogenesis of clover plants. Studies showed that early in the development of the root there was a cotyledon-derived inhibitor that could inhibit the continued growth of Rhizobium-induced nodule primordia. However, the activity of this inhibitor diminished with continued growth and maturation of the root. Rhizobia that can synthesise wild-type Nod factors can partially overcome this inhibition of nodule formation, the Nod signals acting as 'anti-cotyledon inhibitors'. Comparison of the RCA plantlets with normal seedlings suggests that the rooted cotyledons have a slightly slower root maturation, which enables the experimentalist time to probe mechanisms underlying root development. The rooted leaf assay was developed so that the symbiotic and physiological properties of large numbers of transgenic plants could be studied. This is a rapid technique that can use either untransformed or recently transformed plant material, and avoids the potential problem of a disturbed hormone balance that is inherent in the use of 'hairy roots' generated by the use of the Agrobacterium rhizogenes-mediated transformation of small-seeded legumes.