We have developed an efficient rooting protocol using half-strength Lepoivre medium for studying AR formation in stems, traced back the cellular AR origins in stems, and correlated expression of rooting-related genes with root initiation sites.

Regeneration from Anthurium scherzerianum leaf segments was found to be highly dependent on genotype and leaf age. Of the various medium factors tested, the NH4NO ~ level had the most significant effect on callus and shoot formation from leaf tissue. A low level of NH4NO 3 (200 mg/1) proved beneficial to the induction of regeneratio n in all genotypes investigated. As compared to induction, multiplication of callus and shoots in subcultures was less susceptible to the action of various medium factors. After isolation from callus, shoots readily formed roots in the absence of growth regulators. High NH4NO3 level (720 rag/l) strongly accelerated root formation. Rooting ability progressively decreased as a consequence of repeated shoot multiplication in the presence of benzyladenine (BA) as sole growth regulator. The advantages and limitations of different mieropropagation schemes are discussed.

This chapter describes the origin, structure and properties of chimaeras in plants, and presents the methods of chimaera dissociation using plant regeneration via adventitious buds or somatic embryos.

KNOX (knotted1-like homeobox) genes have a widely conserved role in the generation of dissected leaves. Ectopic KNOX activity in leaves in various angiosperm lineages causes leaf form changes that can elucidate how the configuration of leaf development evolved. We present an analysis of leaf morphology and morphogenesis in transgenic Kohleria lines overexpressing a heterologous KNOX gene. Kohleria, like many members of Gesneriaceae, has simple-serrated leaves with pinnate venation. KNOX overexpression causes prolonged segment proliferation in proximal, but not distal, parts of leaf blades. Elaborate dissected segments reiterate the zonation of the whole leaf, with organogenic activity persisting between a distal maturation zone and a proximal intercalary elongation zone. The architecture of vascular bundles is severely altered, with a reduced midvein and a more palmate venation. The initial establishment of organogenically competent primordial margins (marginal blastozones) and the onset of tissue differentiation in early stages of leaf development were similar in wild-type and KNOX overexpressing lines. However, leaves overexpressing KNOX often failed to fully mature, and persistent marginal blastozones were found at the base of blades in mature portions of the shoot. We conclude that KNOX-mediated perpetuation of marginal blastozones in Kohleria is sufficient to induce a set of processes that result in highly dissected leaflets, which are unusual in this plant family. Spatial confinement of blastozones between an early maturing tip and a late elongating petiole zone reflects the presence of distinct maturation processes that limit the ability of the leaf margins to respond to ectopic KNOX gene expression.

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Internode explants of Kohleria sp. (Gesneriaceae) are capable of regenerating large numbers of adventitious shoots. Regeneration of green shoots from explants of an albino periclinal chimera with genetically green L1, as well as microsurgical removal of the epidermis revealed that shoots originate only from the epidermis. Histological studies further showed that shoots arise from a particular epidermal cell type, viz the basal cell of young glandular trichomes. On the other hand, cells competent for Agrobacterium-mediated transformation are mainly located in vascular tissues, as could be shown by histochemical localization of ß-glucuronidase (GUS) expression in explants that had been inoculated with A. tumefaciens strains carrying binary plasmids with GUS and kanamycin resistance (NPTII) genes. Only 3% of GUS expression events took place in the epidermis. Consequently, shoot regeneration in the presence of kanamycin was very poor. Moreover, most of those shoots proved GUS-negative and did not survive subcultivation on kanamycin-containing medium. Six regenerants, however, were most probably transgenic, as suggested by the ability to produce adventitious shoots in the presence of kanamycin and by polymerase chain reaction (PCR) analysis. To our knowledge, this is the first positive result towards genetic transformation in a taxon of the Gesneriaceae.

Production and rooting behaviour of rolB-transgenic plants of grape rootstock 'Richter 110' (Vitis berlandieri 3 V. rupestris) Abstract Genetic improvement of grape rootstocks is aimed at protection against grape phylloxera and other soil-borne pests and diseases, good rooting and graft compatibility as well as adaptability to a wide range of soil and climatic conditions. Apart from the long evaluation period required, breeding is complicated by the high heterozygosity in grapes. As an alternative to traditional crossing, gene transfer permits addition of single traits, largely without affecting the genetic background of existing valuable cultivars. Here we report on the production and rooting behaviour of transgenic grape rootstock 'Richter 110' carrying the Agrobacterium rhizogenes rolB gene, which is known to promote rooting. Transformation was achieved by co-cultivation of somatic embryogenic callus with Agrobacterium tumefaciens LBA4404 harbouring plasmid pHrB. The T-DNA of pHrB contains the hpt gene, conferring hygromycin resistance, and the rolB gene under control of its own promoter. PCR using transgene-specific primers verified the presence of hpt in all 36 hygromycin resistant clones selected, while only 24 clones also possessed the rolB gene. Rooting behaviour was examined in vitro, using tip, node and internode explants, and in aeroponic culture in the greenhouse, using single-node cuttings. Compared to internodes of non-transgenic 'Richter 110', those of rolB-transgenic clones in general showed significantly higher rooting ability and, in contrast to the former, were able to root profusely even in the absence of auxin. Cuttings of three rolB-transgenic clones in aeroponic culture produced almost twice as many primary roots as those of the non-transgenic control.