Marie-Christine Chupeau

Characterization of the Early Events Leading to Totipotency in an Arabidopsis Protoplast Liquid Culture by Temporal Transcript Profiling

An efficient liquid medium gave high plating efficiencies of protoplasts. Transcription profiles of plantlets and protoplast-derived cells during the first week of culture were used to track major molecular processes of the reentry in the cell cycle as the plant cells transitioned toward a totipotent state. Candidate genes for plant cell reprogramming are highlighted. The molecular mechanisms underlying plant cell totipotency are largely unknown. Here, we present a protocol for the efficient regeneration of plants from Arabidopsis thaliana protoplasts. The specific liquid medium used in our study leads to a high rate of reentry into the cell cycle of most cell types, providing a powerful system to study dedifferentiation/regeneration processes in independent somatic cells. To identify the early events in the establishment of totipotency, we monitored the genome-wide transcript profiles of plantlets and protoplast-derived cells (PdCs) during the first week of culture. Plant cells rapidly dedifferentiated. Then, we observed the reinitiation and reorientation of protein synthesis, accompanied by the reinitiation of cell division and de novo cell wall synthesis. Marked changes in the expression of chromatin-associated genes, especially of those in the histone variant family, were observed during protoplast culture. Surprisingly, the epigenetic status of PdCs and well-established cell cultures differed, with PdCs exhibiting rare reactivated transposons and epigenetic changes. The differentially expressed genes identified in this study are interesting candidates for investigating the molecular mechanisms underlying plant cell plasticity and totipotency. One of these genes, the plant-specific transcription factor ABERRANT LATERAL ROOT FORMATION4, is required for the initiation of protoplast division.

Successful Gene Tagging in Lettuce Using the Tnt1 Retrotransposon from Tobacco

The tobacco (Nicotiana tabacum) element Tnt1 is one of the few identified active retrotransposons in plants. These elements possess unique properties that make them ideal genetic tools for gene tagging. Here, we demonstrate the feasibility of gene tagging using the retrotransposon Tnt1 in lettuce (Lactuca sativa), which is the largest genome tested for retrotransposon mutagenesis so far. Of 10 different transgenic bushes carrying a complete Tnt1 containing T-DNA, eight contained multiple transposed copies of Tnt1. The number of transposed copies of the element per plant was particularly high, the smallest number being 28. Tnt1 transposition in lettuce can be induced by a very simple in vitro culture protocol. Tnt1 insertions were stable in the progeny of the primary transformants and could be segregated genetically. Characterization of the sequences flanking some insertion sites revealed that Tnt1 often inserted into genes. The progeny of some primary transformants showed phenotypic alterations due to recessive mutations. One of these mutations was due to Tnt1 insertion in the gibberellin 3β-hydroxylase gene. Taken together, these results indicate that Tnt1 is a powerful tool for insertion mutagenesis especially in plants with a large genome.

Molecular and functional characterization of Slide, an Ac-like autonomous transposable element from tobacco.

A new transposable element of tobacco, Slide, was isolated from thetl mutant line, which shows somatic instability, after its transposition into a locus encoding nitrate reductase (NR). The Slide-124 element is 3733 bp long and its coding sequences show similarities with conserved domains of the transposases ofAc, Tam3 andhobo. Excision from the NR locus is detectable in somatic leaf tissues and Slide mobility is triggered by in vitro tissue culture. Slide excision events create footprints similar to those left byAc and Tam3. Tobacco lines derived from thetl mutant line seem characterized by unmethylated copies of a few members of the highly repetitive Slide family. Slide mobility was monitored in transient expression assays. In wild-type tobacco protoplasts, the complete Slide element, as well as a defective copy, is able to excise. The complete Slide element, but not the defective version, is able to excise in protoplasts of the heterologous species lettuce (Lactuca sativa). These results show that Slide carries the functions required for its own mobility, and represents the first autonomousAc-like element characterized inSolanaceae species.

The Isolation and Culture of Leaf Protoplasts of Cichorium intybus and Their Regeneration into Plants

Summary Enzymatically prepared protoplasts from leaves of various greenhouse grown Cichorium intybus cultivars did not show sustained divisions in a Murashige and Skoog salts-based medium. Replacement of ammonium and nitrate by glutamine as the sole nitrogen source improved plating efficiency and cell colony survival. Early transfer, before the second week of culture, to a low auxin medium was essential for development of microcalli. Plant regeneration from protoplast-derived calli was achieved through shoot formation.

Improved flax regeneration from hypocotyls using thidiazuron as a cytokinin source

SummaryThe effects of thidiazuron, benzyladenine and zeatin were tested with respect to bud regeneration of different flax explants from hypocotyls, cotyledons and apices of two fibre varieties (Ariane, Viking) and one linseed variety (Antarès). These three cytokinins were tested either alone or in combination with naphthalene acetic acid, indole acetic acid or 2,4-dichlorophenoxyacetic acid.Hypocotyls were the most responsive explants. Thidiazuron was significantly the most effective followed by benzyladenine, and then zeatin, in inducing organogenesis from hypocotyl segments. The optimal thidiazuron concentration for bud regeneration from hypocotyls was 0.1–0.3 μM in combination with 0.01 μM of naphthalene acetic acid. Six days after plating, shoot initials began to appear on hypocotyl sections compared with ten to fifteen days when using benzyladenine or zeatin.

Coat protein gene-mediated protection in Lactuca sativa against lettuce mosaic potyvirus strains.

Lettuce mosaic potyvirus (LMV) can be very destructive on lettuce crops worldwide. The LMV strain 0 (LMV-0) coat protein (CP) gene was engineered for expression in plants. It was introduced into three susceptible cultivars of Lactuca sativa using an improved procedure for transformation and regeneration of lettuce, by co-cultivation of leaf explants with Agrobacterium tumefaciens. Several transformants accumulated detectable levels of LMV CP. The R1 progeny of twelve R0 transformants (four plants per cultivar) with T-DNA integration at one single locus, was studied for protection against LMV. The progeny from five R0 transformants showed resistance to LMV-0, with the effectiveness of resistance depending on the development stage of the plants at the time of inoculation. The R1 and R2 progeny from one of these R0 transformants, Cocarde-9a, were more extensively analysed. The homozygous but not the hemizygous R1 plants displayed protection to LMV-0. The R2 progeny from one homozygous R1 plant were shown to be resistant to infection by LMV-0 and other LMV strains. As previously observed in other cases of potyvirus sequence-mediated protection, a phenomenon of recovery was observed in some plants, as well as complete resistance. However, this recovery phenotype was not always maintained, as opposed to the previous described cases, leading to a late progression of viral infection.

Requirement of thidiazuron for healthy protoplast development to efficient tree regeneration of a hybrid poplar (Populus tremula ß p. alba)

Summary Methods were developed for obtaining high yields of leaf protoplasts of a hybrid clone of white poplar, Populus tremula × P. alba (717-1-B4), as well as rapid and efficient plant regeneration. Protoplasts were isolated from leaves of shoot culture. A precise adaptation of liquid media for preparation and culture of protoplasts led to high rates of cell colony formation, on the average for 30% of initially plated protoplasts. The procedures employed avoided the formation of any exudate. Glucose (9%) as an osmoticum and carbon source was the only sugar leading to high viability. A precise balance of low concentrations of nitrate and ammonium in the culture medium was beneficial for development of healthy cell colonies. A combination of thidiazuron (0.05 µM) as the only cytokinin and 2,4-D (14 µM) as auxin was essential for sustained development of protoplast derived cell colonies. After dilution in liquid medium, about 10% of the colonies formed calli. Plants regenerated through bud formation on practically all protoplast-derived calli. Plants regenerated from 3% of the initially plated protoplasts. In vitro rooting and transfer to soil were also achieved with good efficiency.

Recovery of transgenic trees after electroporation of poplar protoplasts

Protoplasts from leaflets ofin vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying thenptII gene for resistance to neomycin; pGH1, carrying a mutant acetolactate synthase gene,als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 105 to 104 protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 μM paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS ofArabidopsis and 25 μM phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated.Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.

Importance of myo-inositol, calcium, and ammonium for the viability and division of tomato (Lycopersicon esculentum) protoplasts

Plants from four cultivars of Lycopersicon esculentum were grown under different conditions, in controlled environment chambers. Low light intensity, long photoperiod (16 h), 25° C/17°C temperature alternance (day/night) were found to be the most convenient conditions for obtaining viable protoplasts. The use of myo-inositol as an osmoticum in the digestion medium and the adjustment of the pH to 6.5, instead of the usual 5.8, for this medium increased the yield of viable protoplasts and enhanced their stability. Under these conditions neither pretreatment (dark and cold treatments), nor preplasmolysis of leaf tissues, were required before protoplast isolation. The concentrations of ammonium nitrate, calcium chloride, myo-inositol, and sucrose were found to be critical for the success of protoplast culture. A medium containing 5 mM ammonium nitrate, 40 mM calcium chloride, 10 mg l-1 adenine sulfate, 0.5% myo-inositol and 6% sucrose gave sustained protoplast divisions. Under these conditions, plating efficiency ranged from 5% for the cultivar Lukulus to 15% for the cultivar Golden Sunrise.

Transformation of Lycopersicon peruvianum and Lycopersicon esculentum mesophyll protoplasts by electroporation

Transformation of Lycopersicon peruvianum and Lycopersicon esculentum mesophyll protoplasts by electroporation