In this chapter, we describe a new protocol for obtaining transgenic Arabidopsis thaliana plants by a so-called in planta transformation method. Such methods, with no in vitro culture or regeneration step, have already been described. Briefly, germinating seeds (Feldmann and Marks 1987) or wounded plants (Chang et al. 1994) are inoculated with an appropriate Agrobacterium strain; plants are then grown to maturity and their seeds collected. Transformants are selected at low frequency among the progeny of inoculated plants. The main advantages of these methods are the simplicity of the procedure and the low frequency of somaclonal variants in the transgenic lines, which is particularly useful forT -DNA mutagenesis strategies (Feldmann 1991).The mechanism(s) involved in these transformation procedures are largely unknown. However, it appears, based on the fact that all the transgenic individuals obtained are independent and hemizygous (Feldmann 1991;Bechtold et al. 1993;Bouchez et al. 1993), that the target cells for Agrobacterium transformation are likely to be the zygote itself or the gametes. Therefore, one can expect higher transformation efficiency if plants are inoculated as late as possible. A method largely used by plant pathologists to inoculate adult plants is vacuum infiltration with a bacterial suspension. Considering this, we set up a protocol to inoculate adult plants with an Agrobacterium suspension using vacuum infiltration (Bechtold et al. 1993). The plants are then transplanted to soil, and seeds are collected from these inoculated plants. This method has been used successfully in several laboratories to obtain transgenic Arabidopsis plants with various constructs. In our laboratory, we can routinely achieve a transformation frequency of up to ten transformants from one inoculated plant. Materials and ChemicalsGreenhouse materials: Aluminum alimentary trays 22 X 16 em (Bourgeat, 38490 Les Abrets, France), net pots diameter = 5.5 em (Teku, D2842 Lohne/Oldb, FRG), incubator for seed trays 45 X 33 X 3.5 em (BHR, 71370 St Germain du Plain, France), carrying tray 28 X 38 em (KIB NL5140 AD W aalwijk, Netherlands), plastic alimentary trays 13 X 10cm (Alphaform, 92100 Boulogne Equipment and instruments I. Potrykus et al. (eds.), Gene Transfer to Plants

Root formation in plants involves the continuous interpretation of positional cues. Physiological studies have linked root formation to auxins. An auxin response element displays a maximum in the Arabidopsis root and we investigate its developmental significance. Auxin response mutants reduce the maximum or its perception, and interfere with distal root patterning. Polar auxin transport mutants affect its localization and distal pattern. Polar auxin transport inhibitors cause dramatic relocalization of the maximum, and associated changes in pattern and polarity. Auxin application and laser ablations correlate root pattern with a maximum adjacent to the vascular bundle. Our data indicate that an auxin maximum at a vascular boundary establishes a distal organizer in the root.

The conversion of light to chemical energy by the process of photosynthesis is localized to the thylakoid membrane network in plant chloroplasts. Although several pathways have been described that target proteins into and across the thylakoids, little is known about the origin of this membrane system or how the lipid backbone of the thylakoids is transported and fused with the target membrane. Thylakoid biogenesis and maintenance seem to involve the flow of membrane elements via vesicular transport. Here we show by mutational analysis that deletion of a single gene called VIPP1 (vesicle-inducing protein in plastids 1) is deleterious to thylakoid membrane formation. Although VIPP1 is a hydrophilic protein it is found in both the inner envelope and the thylakoid membranes. In VIPP1 deletion mutants vesicle formation is abolished. We propose that VIPP1 is essential for the maintenance of thylakoids by a transport pathway not previously recognized.

Summary 3-ketoacyl-CoA thiolase (KAT) (EC: 2.3.1.16) catalyses a key step in fatty acid b-oxidation. Expression of the Arabidopsis thaliana KAT gene on chromosome 2 (KAT2), which encodes a peroxisomal thiolase, is activated in early seedling growth. We identi®ed a T-DNA insertion in this gene which abolishes its expression and eliminates most of the thiolase activity in seedlings. In the homozygous kat2 mutant, seedling growth is dependent upon exogenous sugar, and storage triacylglycerol (TAG) and lipid bodies persist in green cotyledons. The peroxisomes in cotyledons of kat2 seedlings are very large, the total peroxisomal compartment is dramatically increased, and some peroxisomes contain unusual membrane inclusions. The size and number of plastids and mitochondria are also modi®ed. Long-chain (C16 to C20) fatty acyl-CoAs accumulate in kat2 seedlings, indicating that the mutant lacks long-chain thiolase activity. In addition, extracts from kat2 seedlings have signi®cantly decreased activity with aceto-acetyl CoA, and KAT2 appears to be the only thiolase gene expressed at signi®cant levels during germination and seedling growth, indicating that KAT2 has broad substrate speci®city. The kat2 phenotype can be complemented by KAT2 or KAT5 cDNAs driven by the CaMV 35S promoter, showing that these enzymes are functionally equivalent, but that expression of the KAT5 gene in seedlings is too low for effective catabolism of TAG. By comparison with glyoxylate cycle mutants, it is concluded that while gluconeogenesis from fatty acids is not absolutely required to support Arabidopsis seedling growth, peroxisomal b-oxidation is essential, which is in turn required for breakdown of TAG in lipid bodies.