Jean-Pierre Bourgin

Valine-resistant plants from in vitro selected tobacco cells

SummaryGrowth inhibition of protoplasts and cells of tobacco (Nicotiana tabacum L.) occurs when 0.1 to 10 mM L-valine is present in the culture medium but not if L-isoleucine is simultaneously added. Mesophyll protoplasts from haploid tobacco were submitted to ultraviolet irradiation and cell colonies derived from these protoplasts were plated in a medium containing valine. Plants were regenerated from five presumptive resistant calli and retested for valine-resistance. Among the recovered plants two were definitely resistant to valine. This trait was transmitted in sexual crosses as a mendelian character.

Sexual and somatic hybridization in the genusLycopersicon

In recent years, a large number of reports have been published on the recovery of somatic hybrids in the genusLycopersicon and their potential use as a tool in plant breeding programs. Somatic hybridization as a way of enabling the incompatibility barriers which exist within the genusLycopersicon to be bypassed has attracted great interest. WildLycopersicon species harbor numerous interesting agronomic characteristics, which could be transferred to tomato by somatic hybridization. In particular, the production of asymmetric hybrids is explored as an approach to obtain the transfer of only a part of the nuclear genome of wildLycopersicon species. Considerable information is available on the fate of chloroplasts and mitochondria in fusion products inLycopersicon, and unfortunately, cybridization (transfer of chloroplasts and/or mitochondria) seems often difficult to achieve.

Isolation, culture and regeneration into plants of Ranunculus sceleratus L. leaf protoplasts

Abstract Viable protoplasts were isolated from leaves of Ranunculus sceleratus L. by a one-step mixed enzyme treatment. Cultured protoplasts regenerated cell walls and cell divisions were observed occasionally. Calli formed from the cell colonies differentiated into plantlets which were then grown to complete plants.

Transmission of paternal chloroplasts in tobacco (Nicotiana tabacum)

Medgyesy et al. (1986, Mol. Gen. Genet. 204, 195–198) have described in Nicotiana plumbaginifolia and in an interspecific cross involving N. plumbaginifolia and N. tabacum a procedure for selecting cell lines derived from seedlings carrying paternal chloroplasts by taking advantage of a plastid-encoded mutation which confers resistance to streptomycin. We have extended their demonstration of occasional transmission of chloroplasts through pollen to the case of an intraspecific cross in N. tabacum. The line used as maternal parent, ITB19(sua), displayed a cytoplasmic male sterility due to the presence of a cytoplasm originating from N. suaveolens. The line used as paternal parent, SR1, was fertile and possessed mutant chloroplasts conferring resistance to streptomycin. From cell lines derived from 204 seedlings, three were regenerated into streptomycin-resistant buds. The plants derived from these three clones were male-sterile. Their progeny, after crossing with a wild type tobacco line, XHFD8, was resistant to streptomycin. Tests of resistance of the seedlings to tentoxin and restriction analyses of the chloroplast DNA indicated that two clones still had the maternal chloroplasts and were thus probably new streptomycin-resistant mutants, whereas the third one had acquired the chloroplasts of the paternal parent, but had retained the mitochondria of the maternal parent.

Direct selection of cybrids by streptomycin and valine resistance in tobacco

SummaryDirect selection of cybrids by simultaneous selection for “donor” chloroplasts and for the “recipient” nuclei is described. Mesophyll protoplasts of two tobacco (Nicotiana tabacum) mutants, SR1 (streptomycin resistant) and Valr-2 (valine resistant), were fused by polyethylene glycol treatment. Streptomycin resistance in the SR1 mutant is a maternally inherited chloroplast trait while valine resistance is a Mendelian (nuclear) digenic recessive character. The fused protoplast population was cultured and colonies were selected for resistance to valine (1 mM) and streptomycin (343 μM). The efficiency of selection has been confirmed in three clones by demonstrating seed transmission of both streptomycin and valine resistances. In one subclone both streptomycin resistant and sensitive plants were obtained indicating that the streptomycin sensitive chloroplasts had not been totally eliminated by growth on the selective medium.

Regeneration and characterization of plants produced from mature tobacco pollen protoplasts via gametosomatic hybridization

SummaryMature pollen protoplasts (n) isolated from kanamycin resistant plants of Nicotiana tabacum (2n = 4x = 48) were fused with somatic mesophyll protoplasts (2n) of Nicotiana plumbaginifolia (2n = 20) to produce plants. A total of 3.6·106 mature pollen protoplasts were fused with 7·106 mesophyll protoplasts using a PEG/Ca2+ method. Mature pollen protoplasts did not divide in our culture conditions, and N. plumbaginifolia protoplasts stopped dividing when the protoplast-derived colonies were transferred to a selection medium containing paromomycine (20 mg·l-1). A total of 133 actively growing colonies were recovered on the selection medium containing kanamycin (100 mg·l-1). Plants from twenty resulting cell lines were confirmed as hybrids (17) or cybrids (3) based on leaf and floral morphology and fertility analysis. Isozyme pattern analysis confirmed the nuclear hybrid and cybrid nature, respectively, for 2 and 3 typical gametosomatic selected plants. Root tip squashes of 6 of the gametosomatic hybrid plants revealed chromosome numbers ranging from 44 to 68; the 3 selected cybrid plants had 48 chromosomes. Evidence for organelle transmission from the mesophyll partner in the gametosomatic plants is shown. From the analysis it can be concluded that the gametosomatic fusion involving mature pollen protoplasts (n) carrying a dominant selection marker can be convenient for synthesis of either hybrids or cybrids. Such gametosomatic fusion is therefore considered as a new approach towards the production of androgenetic plants with a choosen cytoplasm.

Preparation and fusion properties of protoplasts from mature pollen of Nicotiana tabacum.

SummaryA method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg-1 H2O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca2+ method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.

Cellular genetic study of a somatic instability in a tobacco mutant: in vitro isolation of valine-resistant spontaneous mutants.

SummaryA chlorophyll-deficient mutant line of tobacco (Nicotiana tabacum), named tl, displays spontaneously on leaves green, white, and twinned green/white somatic variations at high frequencies (10−3 to 10−2). The frequency of cell events leading to the somatic variations has been shown to be closely dependent on the stage of differentiation of cells during plant development. The activity of transposable elements is suspected in the tl genotype, and a study of its mutagenic ability was performed by selecting in vitro new mutant cellular types. The cellular marker chosen was the resistance to toxic doses of valine conferred by a permeation deficiency. A reproducible method allowing efficient selection of valine-resistant mutant clones from haploid tobacco mesophyll protoplast-derived cells was used. In 10 out of 12 experiments, the frequency of spontaneous valine-resistant clones obtained with the wild-type control was null for cell populations tested to the 106. On the other hand, spontaneous valine-resistant clones were repeatedly isolated at variable and sometimes high frequencies (greater than 10−3) from cell populations of the tl type. Valine resistance of plants regenerated from these clones was transmitted to the progeny as a single monogenic mutation. These results indicate an increased mutagenic ability of the tl genotype, as compared to the wild-type line.