Jean-Eric Chauvin

Chromosome doubling of 2x Solanum species by oryzalin: method development and comparison with spontaneous chromosome doubling in vitro

A potato breeding scheme implies the possibility of ploidy level manipulation either by reducing the chromosome number of cultivars from 48 to 24 to be able to cross them with diploid related species or by doubling diploid material to reach the generally optimal tetraploid level. In vitro spontaneous chromosome doubling is widely used but can lead to somaclonal variation. Since oryzalin has proven to be efficient as a chromosome doubling agent on potato cell suspension cultures, we tried this herbicide on various Solanum species and interspecific diploid hybrids. A 24 h dip in a 28.8 μM aqueous oryzalin solution applied on apical buds was the most efficient treatment in terms of tetraploid plant production (mean = 4.1 tetraploid plants for 10 treated buds over 4 genotypes). However 50–100% of the regenerated tetraploid plants acclimatized after in vitro treatment proved to be chimaeric. Consequently, a selection procedure in the progeny was necessary to obtain real and stable doubled clones and final yields were low. This technique is easy to apply and could be a good alternative to chromosome doubling by spontaneous in vitro regeneration in the case of refractory genotypes especially where somaclonal variation is problematic. Percentage of tetraploids among the regenerated plants varied from 6 to 29% with the oryzalin doubling technique while it varied from 20 to 78% by in vitro spontaneous doubling for five diploid genotypes. An observation of the progeny indicated that chimaeras were more frequent using oryzalin (50–100% of the initially supposed tetraploid plants) than when chromosomes doubled spontaneously (4–67% of the initially supposed tetraploid plants).

Introduction and expression of a deregulated tobacco nitrate reductase gene in potato lead to highly reduced nitrate levels in transgenic tubers.

Twenty transformed Solanum tuberosum plants issued from five different varieties and carrying a chimeric tobacco nitrate reductase gene (a truncated tobacco Nia2 coding sequence fused to the CaMV 35S promoter) were cultivated in field conditions at INRA Ploudaniel in 1999 and 2000. In 60% of the transgenic plants, the presence of the tobacco Nia2 transcript was detected by RT-PCR. These clones exhibited a drastic decrease in the nitrate content in tubers. Indeed the nitrate content decreased by about 95% in the tubers of transformed plants compared to non-transformed potato plants from the same variety. This decrease was correlated with a modified regulation of NR expression as revealed by a higher chlorate sensitivity of these transgenic lines. Two methods of nitrate content determination in tubers were also compared and were found to give similar results.

Expression of a deregulated tobacco nitrate reductase gene in potato increases biomass production and decreases nitrate concentration in all organs

We investigated the physiological consequences for nitrogen metabolism and growth of the deregulated expression of an N-terminal-deleted tobacco nitrate reductase in two lines of potato (Solanum tuberosum L. cv Safrane). The transgenic plants showed a higher biomass accumulation, especially in tubers, but a constant nitrogen content per plant. This implies that the transformed lines had a reduced nitrogen concentration per unit of dry weight. A severe reduction in nitrate concentrations was also observed in all organs, but was more apparent in tubers where nitrate was almost undetectable in the transgenic lines. In leaves and roots, but not tubers, this nitrate decrease was accompanied by a statistically significant increase in the level of malate, which acts as a counter-anion for nitrate reduction. Apart from glutamine in tubers, no major changes in amino acid concentration were seen in leaves, roots or tubers. We conclude that enhancement of nitrate reduction rate leads to higher biomass production, probably by allowing a better allocation of N-resources to photosynthesis and C-metabolism.

Effects of gelling agents on in vitro regeneration and kanamycin efficiency as a selective agent in plant transformation procedures

Successful plant transformation requires effective regeneration and selection systems. The regeneration of tulip scape segments, gladiolus cormel slices, and tobacco leaf pieces were compared on media solidified with different gelling agents and with kanamycin at various concentrations. Increasing concentration of kanamycin generally resulted in full or partial inhibition of regeneration. However, regeneration was observed with one of the gelling agents, a κ-carrageenan, and 200 mg l−1 kanamycin in the medium. With other gelling agents, 50% of this concentration was generally sufficient to totally inhibit regeneration. Therefore, the choice of the gelling agent is critical when establishing a plant transformation procedure.

Major-effect QTLs for stem and foliage resistance to late blight in the wild potato relatives Solanum sparsipilum and S. spegazzinii are mapped to chromosome X

To find out new resistance sources to late blight in the wild germplasm for potato breeding, we examined the polygenic resistance of Solanum sparsipilum and S. spegazzinii by a quantitative trait locus (QTL) analysis. We performed stem and foliage tests under controlled conditions in two diploid mapping progenies. Four traits were selected for QTL detection. A total of 30 QTLs were mapped, with a large-effect QTL region on chromosome X detected in both potato relatives. The mapping of literature-derived markers highlighted colinearities with published late blight QTLs or R-genes. Results showed (a) the resistance potential of S. sparsipilum and S. spegazzinii for late blight control, and (b) the efficacy of the stem test as a complement to the foliage test to break down the complex late blight resistance into elementary components. The relationships of late blight resistance QTLs with R-genes and maturity QTLs are discussed.

Modeling of yield losses caused by potato late blight on eight cultivars with different levels of resistance to phytophthora infestans

The Shtienberg model for predicting yield loss caused by Phytophthora infestans in potato was developed and parameterized in the 1990s in North America. The predictive quality of this model was evaluated in France for a wide range of epidemics under different soil and weather conditions and on cultivars different than those used to estimate its parameters. A field experiment was carried out in 2006, 2007, 2008, and 2009 in Brittany, western France to assess late blight severity and yield losses. The dynamics of late blight were monitored on eight cultivars with varying types and levels of resistance. The model correctly predicted relative yield losses (efficiency = 0.80, root mean square error of prediction = 13.25%, and bias = -0.36%) as a function of weather and the observed disease dynamics for a wide range of late blight epidemics. In addition to the evaluation of the predictive quality of the model, this article provides a dataset that describes the development of various late blight epidemics on potato as a function of weather conditions, fungicide regimes, and cultivar susceptibility. Following this evaluation, the Shtienberg model can be used with confidence in research and development programs to better manage potato late blight in France.

Marker-assisted breeding for disease resistance in potato.

Sub-project 5 of BIOEXPLOIT aims to design durable disease resistance through marker-assisted breeding by converting existing markers for high-throughput application, developing and validating high-throughput marker technologies and pyramiding major R genes and/or quantitative trait loci into elite material. Activities include (1) the fine mapping of the quantitative trait locus PiXspg which accounts for a large proportion of the variation in late blight resistance, (2) converting SNP-based markers and an AFLP marker to easy-to-use-markers, (3) testing of progenies with combined sources of late blight resistance for presence of R genes and agronomic features, (4) backcrossing new sources of resistance to S. tuberosum and molecular screening of breeding materials with marker GP94 linked with gene Rpi-phu1 conferring late blight resistance, (5) evaluating potato clones with enhanced resistance against Phytophthora infestans under field conditions of Toluca (México), and (6) developing populations and marker-assisted breeding for disease resistance.

Integration of cryopreservation in French plant genetic resource collections: the CRYOVEG project

This book represents contributions, oral as well as posters, of the final meeting of COST Action 871, CRYOPLANET (Cryopreservation of crop species in Europe) held in Angers. Local organizers of the meeting were Dr. Agnes Grapin (AGROCAMPUS OUEST – Angers) and Dr. Florent Engelmann (Institut de Recherche pour le Developpement). COST Action 871 started in December 2006 with a Kick-off meeting at the COST office in Brussels and officially ended in December 2010. Twenty-one COST Action Countries (see figure 1) and 3 non-COST institutes (New Zealand Institute for Crop & Food Research (New Zealand), Vavilov Institute of Plant Industry (Russian Federation), Faculte des Sciences de Sfax (Tunisia) participated actively in this initiative. The Action was created because plant cryobiologists realized that plant cryopreservation was hardly applied in Europe. This was mainly due to the fact that efficient and robust cryopreservation protocols applicable to many plant species and diverse germplasm types were not available, plant researchers were unacquainted to recent developments in cryogenic storage methods and there was a lack of coordinated research in Europe on plant cryopreservation. The main objective of this action was therefore “to improve and apply technologically advanced techniques for plant genetic resources conservation of crops that are grown/ and or conserved in Europe with main emphasis on long-term conservation through cryopreservation”. In order to achieve this, 2 working groups (WGs) were established