J. De Greef

Construction of a heat-inducible chimaeric gene to increase the cytokinin content in transgenic plant tissue

The ipt gene of Agrobacterium tumefaciens T‐DNA encodes an isopentenyltransferase which causes cytokinin overproduction and developmental alterations in transformed plants. A chimaeric gene constructed by positioning the ipt coding region under the control of the hsp70 gene from Drosophila melanogaster allows heat‐regulated expression in transgenic plant tissue. Heat‐shock treatment of tobacco calli transgenic for the chimaeric hsipt gene increases the endogenous cytokinin concentration and enables these calli to grow on cytokinin‐free medium. Transgenic plants regenerated from calli transformed with the hsipt gene and grown at normal temperature are phenotypically normal.

Agrobacterium T-DNA gene 1 codes for tryptophan 2-monooxygenase activity in tobacco crown gall cells

Cloned tobacco crown gall tissue induced by the Agrobacterium tumefaciens C58 T‐DNA mutant pGV3132, defective for the T‐DNA‐encoded amihydrolase (iaaH), accumulates about 1000‐times more indole‐3‐acet‐amide (IAM) when compared to untransformed tobacco callus and crown gall tissue induced by a T‐DNA mutant defective for gene 1. In vitro experiments demonstrated that this IAM accumulation is correlated with the active conversion of Trp into IAM. The results presented in this report provide biochemical evidence that the T‐DNA gene 1 encodes a tryptophan 2‐monooxygenase (iaaM) activity in transformed plant cells. This gene cooperates with the gene 2‐encoded amidohydrolase (iaaH) in the T‐DNA‐controlled indole‐3‐acetic acid (IAA) biosynthesis pathway in crown gall cells.

Tobacco plants transformed with the Agrobacterium T-DNA gene 1 contain high amounts of indole-3-acetamide

The T‐DNA genes 1 and 2 of the Ti plasmid of Agrobacterium tumefaciens are involved in the biosynthesis of IAA in transformed plant cells. Previously, it has been shown that gene 2 codes for an amidohydrolase able to convert IAM into IAA. We have isolated Nicotiana tabacum regenerates transformed with either gene 1 or genes 6a and 6b of the T‐DNA. The tobacco plants transformed with gene 1 contain 500–1000‐times more IAM as compared to plants transformed with genes 6a and 6b, and as compared to untransformed control plants. No drastic differences in endogenous IAA concentrations were observed between the three plant types analyzed.

Phytohormone-receptors from tobacco crown gall tissues

Plant cell and tissue cultures are useful tools for the study of plant hormone receptors as it is clearly illustrated by the work of the ‘Leiden-group’ on different aspects of auxin binding in tobacco callus tissue. In this contribution we will review some physiological and genetical aspects of the tobacco crown gall system, illustrating not only the possibilities offered by this model to investigate the early mode of action of phytohormones, but also the necessity to integrate the study of plant hormone receptors as an essential complementation of data on the endogenous phytohormonal levels.