Mechanisms of primordium formation during adventitious root development from walnut cotyledon explants.

In walnut (Juglans regia L.), an otherwise difficult-to-root species, explants of cotyledons have been shown to generate complete roots in the absence of exogenous growth regulators. In the present study, this process of root formation was shown to follow a pattern of adventitious, rather than primary or lateral, ontogeny: (i) the arrangement of vascular bundles in the region of root formation was of the petiole type; (ii) a typical root primordium was formed at the side of the procambium within a meristematic ring of actively dividing cells located around each vascular bundle; (iii) the developing root apical meristem was connected in a lateral way with the vascular bundle of the petiole. This adventitious root formation occurred in three main stages of cell division, primordium formation and organization of apical meristem. These stages were characterized by expression of LATERAL ROOT PRIMORDIUM-1 and CHALCONE SYNTHASE genes, which were found to be sequentially expressed during the formation of the primordium. Activation of genes related to root cell differentiation started at the early stage of primordium formation prior to organization of the root apical meristem. The systematic development of adventitious root primordia at a precise site gave indications on the positional and biochemical cues that are necessary for adventitious root formation.

Expression of antisense chalcone synthase RNA in transgenic hybrid walnut microcuttings. Effect on flavonoid content and rooting ability.

Walnut somatic embryos (Juglans nigra x Juglans regia) were transformed with a vector containing a neomycin phosphotransferase II, a beta-glucuronidase and an antisense chalcone synthase (chs) gene. This antisense construct included a 400 bp cDNA fragment of a walnut chs gene under the control of the duplicated CaMV-35S promoter. Molecular, biochemical and biological characterizations were performed both on transformed embryos propagated by secondary somatic embryogenesis and on microshoots developed by in vitro culture of embryonic epicotyls from somatic embryos. Thirteen transformed lines with the vector containing the antisense chs gene, one line with only the gus and nptII genes and one untransformed line were maintained in tissue culture. Six of the antisense lines were shown to be flavonoid-deficient. They exhibited a strongly reduced expression of chs genes, very low chalcone synthase activity and no detectable amounts of quercitrin, myricitrin, flavane-3-ols and proanthocyanidins in stems. Rooting tests showed that decreased flavonoid content in stems of antisense chs transformed lines was associated with enhanced adventitious root formation. Free auxin and conjugated auxin contents were determined during the latter phase of the micropropagation, and no variations were detected between control and antisense chs transformed lines. The in vitro plants developed a large basal callus and apical necrosis upon auxinic induction and the transformed lines highly deficient in flavonoids were more sensitive to exogenous application of indolebutyric acid (IBA).

Histological investigation of walnut cotyledon fragments for a better understanding of in vitro adventitious root initiation.

Rapid formation of adventitious roots by walnut cotyledon fragments in vitro was traced by light microscopy. It was shown that this plant model is characterized by two major developmental processes: a) confined elongation of the cotyledon petiole caused by a limited number of cell divisions and b) formation of a morphogenetic zone around each initially wounded vascular bundle within 36 h after detachment of the embryonic axis. During the first phase of development, granular storage protein bodies dissolved, and starch grains were deposited mainly in the distal portion of the cotyledon fragments. Rapidly, new globular protein bodies were formed, and phenolic inclusions accumulated in the vacuoles of epidermal and subepidermal cells and of individual cells close to the vascular bundles. Each adventitious root was found to be in continuity with a single vascular bundle of the cotyledon petiole. A short auxin treatment suppressed the formation of large roots and induced numerous tiny rootlets dispersed all over the surface of the cotyledons.