Luisa Moysset

EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations

BackgroundTILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin.ResultsHere, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified.ConclusionsThis new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction.

Effects of Calmodulin Inhibitors and Blue Light on Rhythmic Movement of Robinia pseudoacacia Leaflets

Abstract— The effect of several calmodulin (CAM) antagonists, blue light and an intracellular calcium inhibitor, on the circadian rhythm of Robinia pseudoacacia leaflet movement has been studied. The CAM antagonists, chlorpromazine (CPZ), trifluoperazine (TFP), calmidazolium and N‐(6‐aminohexyI)‐5‐chloro‐1‐naphthalenesulfonamide (W7) shifted the phase of the circadian rhythmic movement while W5, an inactive analogue of W7, had no effect. Two hour pulses of calmidazolium (10–50 μM) gave rise to a phase‐response curve with maximum advances (up to 9 h) at circadian time (CT) 6 and maximum delays (up to 7 h) at CT 22. No effect was found on transition from subjective day to subjective night and vice versa. The TFP (10–50 μM), applied as 2 h pulses during the circadian cycle, shifted the phase of the circadian leaflet movement and also produced maximum advances in the middle of subjective day. Two hour blue light pulses shifted the phase of leaflet rhythmic movement. The phase‐response curve obtained showed maximum advances (up to 5 h) in the middle of subjective day and maximum delays on transition from subjective day to subjective night. Two hour pulses of 50 μM 8‐(diethylamino)octyl 3,4,5‐trimethoxybenzoate hypochloride (TMB‐8), an intracellular calcium inhibitor, caused the same type of phase‐response curve, with maximum advances and delays occurring at the same time as those produced by blue light. These results indicate that CAM might be involved in controlling the circadian oscillator that drives Robinia leaflet movement. The relationship between CAM and calcium with red and blue light is discussed.

Microautoradiographic localisation of [3H]sucrose and [3H]mannitol in Robinia pseudoacacia pulvinar tissues during phytochrome-mediated nyctinastic closure.

Summary.We have analysed the incorporation of [3H]sucrose and [3H]mannitol in pulvinar motor cells of Robinia pseudoacacia L. during phytochrome-mediated nyctinastic closure. Pairs of leaflets, excised 2 h after the beginning of the photoperiod, were fed with 50 mM [3H]sucrose or [3H]mannitol, irradiated with red (15 min) or far-red (5 min) light and placed in the dark for 2–3 h. Label uptake was measured in whole pulvini by liquid scintillation counting. The distribution of labelling in pulvinar sections was assessed by both light and electron microautoradiography. [3H]Sucrose uptake was twice that of [3H]mannitol incorporation in both red- and far-red-irradiated pulvini. In the autoradiographs, [3H]sucrose and [3H]mannitol labelling was localised in the area from the vascular bundle to the epidermis, mainly in vacuoles, cytoplasm, and cell walls. Extensor and flexor protoplasts displayed a different distribution of [3H]sucrose after red and far-red irradiation. Far-red light drastically reduced the [3H]sucrose incorporation in extensor protoplasts and caused a slight increase in internal flexor protoplasts. After red light treatment, no differences in [3H]sucrose labelling were found between extensor and flexor protoplasts. Our results indicate a phytochrome control of sucrose distribution in cortical motor cells and seem to rule out the possibility of sucrose acting as an osmoticum.

Fundamental and Applied Aspects of Somatic Embryogenesis in Araujia sericifera

Araujia sericifera belongs to the Asclepiadaceae family. It has twining stems up to 10 m high, and the leaves are ovate-oblong, acurate, truncate at the base and white-tomentose beneath. Cymes are few-flowered with long peduncles. The flowers which are in axillary cymes with single corola and five segments, are white and pink striped and give off a delicate fragance. Flowers are always insect pollinated. The fruit is oblong and pruinose, containing many seeds with terminal filaments. More botanical information can be attained from Cabrera and Zardini (1978), Fiori (1925) or Flora Europea, vol.III.

Calcium, Calmodulin and Phosphoinositides in Leaflet Movements Mediated by Phytochrome

Calcium transduction pathways are involved in plant signaling, including light and hormonal responses (Sanders et al 1999). Calmodulin and phosphoinositide metabolism may also be other components of the transduction pathway (Yang 1996). Cytoplasmic Ca2+ has been related with some phytochrome-mediated responses such as protoplast swelling, germination of fern spores and seed germination (Tretyn et al 1991). Microinjection experiments reported by Neuhaus et al (1993) strongly support this involvement. Thus, a pre-eminent role of Ca2+ in phytochrome transduction pathways has been proposed (Roux 1994). Protein phosphorylation and calcium-binding proteins such as calmodulin, could be downstream in the phytochrome transduction pathway (Schafer et al 1997).