Georges Ducreux

Influence of donor material and genotype on protoplast regeneration in banana and plantain cultivars (#Musa# spp.)

Among the various strategies for genetic improvement in banana, somatic hybridisation appears to be a promising complement to classical breeding since protoplasts are amenable to plant regeneration. Therefore, the present investigation was undertaken to improve protoplast regeneration in banana. Protoplasts were isolated from young leaves, calli and embryogenic cell suspensions. The highest protoplast yield was obtained with cell suspensions. Mesophyll protoplasts and callus-derived protoplasts did not divide. However, protoplasts isolated from cell suspension developed into plants. Feeder cells and protoplast culture at high density were required for plant regeneration from protoplasts. Plant regeneration through somatic embryogenesis was achieved in Grande Naine and Gros Michel (Musa spp. Sub-Group cavendish AAA), Currare Enano and Dominico (Sub-Group plantain AAB), SF265, IRFA903 and Col49 (Sub-Group AA). The frequency of embryo formation and plant regeneration was genotype-dependent. Large number of somatic embryos and regenerated plants were produced in SF265 (AA) followed by Dominico (AAB) and Grande Naine (AAA).

Interspecific somatic hybridization in potato by protoplast electrofusion

Abstract Somatic hybrid plants between a dihaploid potato, BF15, and one of its wild tuber-bearing relatives, Solanum berthaultii, were produced after protoplast electrofusion with the objective of transferring insect resistance, which is associated with the presence of glandular hairs, into potato lines. Early selection of the putative hybrids was based on differences in the cultural behaviour of the parental and hybrid calli, and in morphological markers. A total of 25 somatic hybrid plants was regenerated from 70 selected calli. The regenerants exhibited strong hybrid vigour and had several morphological traits intermediate to both parents including plant habit, leaf form, and particularly short four-lobed glandular trichomes inherited from the wild parent. DNA analysis of the hybrids using flow cytometry, associated with cytological analysis revealed that 14 were tetraploids, 1 aneuploid, 8 hexaploids and 2 mixoploids. The hybrid nature of the 25 selected plants was confirmed by examining isoenzyme patterns for esterase and peroxidase. Fifteen somatic hybrids were transplanted in soil under a plastic tunnel for a preliminary evaluation of agronomic traits, particularly insect resistance through analysis of browning enzyme activity from four-lobed glandular hairs, and tuber characteristics. The selected somatic hybrids were classified into four groups according to increasing browning enzyme activity. Most somatic hybrids from group 1 were hexaploids, and exhibited a comparable level of browning enzyme activity to that of potato BF15. Their phenotype and tuber characteristics were very close to potato BF15. In groups 3 and 4 all somatic hybrids but one were tetraploids. They showed levels of browning enzyme activity as high as those from the wild species. Some of these hybrids had very high yields, but with tuber characteristics close to those of S. berthaultii. Interestingly, two hexaploid somatic hybrids from group 2 exhibited high activity of browning enzymes and high yields. These traits were associated with the expression of the cultivated phenotype including plant morphology and tuber characteristics. Most somatic hybrid plants tested set fruit with viable seeds.

Plant regeneration from protoplast culture of sweet potato (Ipomoea batatas Lam.).

This is the first report on successful plant regeneration from protoplasts of sweet potato. Two cultivars (Guyana and Duclos XI) of sweet potato plants propagated under in vitro conditions were used as the source of protoplasts. Green compact calli with meristematic areas were induced in the medium supplemented with 2mg1−1 zeatin, and plant regeneration occurred when these calli were transferred onto the medium with zeatin level reduced to 0.25mg1−1. Plant regeneration was found to be genotype-dependent, since it was only obtained for cultivar Duclos XI.

Immunological Detection of Potential Signal-Transduction Proteins Expressed during Wheat Somatic Tissue Culture

An immunochemical approach was used to detect the expression of putative guanine nucleotide-binding proteins (G-proteins), arrestin, and nucleoside diphosphate kinases during wheat (Triticum aestivum) tissue culture initiated from immature embryos. Both the soluble and membrane extracts from the immature embryos revealed bands of 58, 40, and 16 kD with antibodies to G-protein ([alpha] subunit), arrestin, and nucleoside diphosphate kinase, respectively. These proteins were overexpressed in vitro in both nonembryogenic callus and embryogenic cultures. An additional soluble protein (32 kD) was detected by anti-G[alpha] antibodies in cultured tissues but not in immature embryos, suggesting a possible function in cell multiplication. Moreover, somatic embryogenesis was associated with the appearance of a 29-kD protein reactive with anti-arrestin antibodies, both in soluble and membrane fractions. Tissue-cultured genetic stocks of Chinese Spring wheat, including the disomic, 36 ditelosomic, and 6 nullisomic-tetrasomic wheat lines, were used to ascertain the chromosomal location of the genes encoding the 29-kD arrestin-like protein. The lack of a signal with the nonembryogenic ditelosomic 3 D short chromosome arm line suggests that the 3 D long chromosome arm possesses at least one gene involved in the expression of the 29-kD protein. The putative role of the 29-kD protein in signal-transduction regulating embryogenesis is discussed.

Total protein pattern expression during induction and development of carrot somatic embryos

Abstract Differential gene expression revealed by the analysis of total protein patterns was investigated in somatic embryogenesis of Daucus carota L. The aim of this work was to identify markers, and to assess the relationships between induction and different developmental stages of somatic embryogenesis. Proteins extracted from an embryogenic cell suspension and five developmental stages of somatic embryos were separated by two-dimensional gel electrophoresis. Both quantitative and qualitative changes were detected, and the relationships between the protein patterns were visualized using an unrooted tree. The different organs of somatic plantlets, namely root, hypocotyl and cotyledon, were analyzed separately for spatial differences in protein distribution. A comparison of six protein patterns revealed that the variable proteins were modulated according to five profiles, three of which are most relevant. Among the variable proteins, ten were specific to the cell suspension stage and might be assigned either to callogenesis or to embryogenesis induction. Six proteins found only in somatic embryos were shown to fluctuate at different developmental stages and represent morphogenesis markers. Proteins absent during the early stages of embryo development but expressed in the cell suspension stage as well as in the later stages, might be correlated with the presence of 2,4-D. In fact, the unrooted tree revealed that the cell suspension protein pattern was closer to that of somatic plantlets than to earlier stages of embryogenesis. Furthermore, the CS pattern was closer to that of roots, which are auxin inducible, than to the other organ of the plantlet. Therefore, possible effects of exogenous and endogenous auxins on protein synthesis are discussed.

Cultural behavior of protoplasts from different organs of eggplant (Solanum melongena L.), and plant regeneration

Plants of Solanum melongena were propagated under in vitro conditions (27°C, 12h/day illumination at 62 μEm-2s-1, 60% humidity) by subculture of terminal and lateral cuttings on MS medium +20 gl-1 sucrose + Morel and Wetmore vitamins at 1/8 strength and 7 gl-1 agar. Lamina, petioles and stems of 3-week-old cuttings were used as sources of protoplasts. The best mean yield of protoplasts was obtained from the lamina with 9,030×103 protoplasts per gram of tissue. Petioles and stems yielded respectively 3,144×103 and 1,220.4×103 protoplasts per gram of tissue. first division of petiole and stem protoplasts occurred within 48 h, while lamina protoplasts underwent division after 3–4 days of culture in KM8p medium +2,4-D(0.2 gl-1) + zeatin (0.5 mgl-1) + NAA (1 mgl-1) and 0.35M glucose as osmoticum. The highest percentage of dividing cells was obtained from petiole material, estimated at 33.4% after 7 days, compared to 23.8% and 19.4% respectively for stem and lamina protoplasts. When BAP replaced zeatin in KM8p, the division percentage of lamina protoplasts was reduced to 10–15%. When transferred to regeneration medium, all calli derived from KM8p + zeatin formed deep-green spots identified as embryo-like structures, while only few calli from KM8p + BAP underwent shoot organogenesis without formation of green spots. Some of embryo-like structure developed into plantlets with a frequency of 1–2 plantlets per callus especially on MS medium + zeatin (4 mgl-1) + IAA (0.2 mgl-1). Maintaining protoplast-derived calli on MS + BAP (0.5 mgl-1) + NAA (0.5 mgl-1) for more than 3 weeks resulted in a decrease and loss of cell totipotency.

Variations of morphogenetic behavior and plant regeneration in cultured protoplasts of Solanum nigrum

Abstract This paper reports variations of morphogenetic behavior induced by zeatin, and their consequence in the improvement of plant regeneration in cultured protoplasts of Solanum nigrum . Protoplasts were first induced to divide in KM8p medium supplemented with 0.2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg/l zeatin and 1 mg/l naphthaleneacetic acid (NAA), then they underwent various treatments with zeatin, either singly used or combined with auxins, to induce morphogenetic changes. A lower dilution of the culture with a new medium containing zeatin either single or combined with NAA resulted in development of organized structures in the form of elongated bodies witha great number of cells. A higher dilution to about 10 times of the volume of culture with a new medium supplemented with 0.25–4 mg/l zeatin alone promoted the formation of meristematic nodules which exhibited a high regenerative capacity. In the latter, 605 shoots could be potentially recovered from 1000 cultured protoplasts within 42 days, meanwhile the original calli, continuously maintained in the initial medium, gave a potential recovery of 29 shoots from 1000 cultured protoplasts after at least a culture period of 100 days. The results were statistically analyzed.

Isozyme patterns in zygotic and somatic embryogenesis of carrot

Isozyme patterns of carrot (Daucus carota L.) zygotic embryos between the torpedo stage up to 5-day-old seedlings have been compared with those of the similar stages from the embryogenic cell suspension culture to the late somatic plantlet. Somatic embryos blocked at the torpedo stage by β-cyclodextrine have also been analyzed. All these stages have been analyzed with respect to seven different enzyme systems: arylesterase, glucosephosphate isomerase, phosphogluconate dehydrogenase, alcohol dehydrogenase, isocitrate dehydrogenase, aspartate aminotransferase and phosphoglucomutase (EC 2.7.5.1, PGM). The relationships between the different stages of both types of embryogenesis have been visualized using an unrooted tree. Generally, profiles of somatic embryos were different from those of zygotic embryos. Interestingly however, a typical zygotic embryo pattern was found in the cyclodextrine-blocked somatic embryos. Only aspartate aminotransferase patterns revealed a similarity between zygotic and somatic torpedo embryos. Both plantlet types showed close patterns with common isozymes. Moreover, similarities were evident between somatic plantlets and cell suspensions. A few isozymes appeared to be stage specific markers: esterase 10–11 were specific to achenes and early germination, phosphogluconate dehydrogenase 8 was specific to 4–5 day-old seedlings and phosphoglucomutase 1 and 7 and alcohol dehydrogenase 4 were markers for zygotic embryos. No somatic embryogenesis specific isozyme could be found. We show that patterns can be associated with particular tissue formation: mainly, aspartate aminotransferase 2 and 1, phosphoglucomutase 8 and 9 and phosphogluconate dehydrogenase 7 coincided with apical meristem initiation and phosphoglucomutase 4 and 5, zones “b” and “d” of esterase and zone “b” of phosphogluconate dehydrogenase coincided with vascular bundle formation.

EXPERIMENTAL MODIFICATION OF THE MORPHOGENETIC BEHAVIOR OF THE ISOLATED SUB-APICAL CELL OF THE APEX OF SPHACELARIA CIRROSA (PHAEOPHYCEAE)1

Experimental isolation of the terminal cells of the apex of Sphacelaria cirrosa (Roth) C. Agardh shows that the isolated apical cell retains a self‐maintained mode of functioning. The isolated sub‐apical cell, however, gives rise to a newly‐formed axis after undergoing a different type of developmental sequence. An experimental scheme allowing one to defer isolation of the sub‐apical cell after killing the apical cell shows that the maintenance, for a few hours only, of connections with subjacent cells is sufficient to induce an apical type mode of functioning in the sub‐apical cell. The sequential analysis of the first cytological events in this phenomenon allows one to relate this modification of the morphogenetic program to a functional and polarized arrangement of organelles within the cell reconstituting the apical. The morphogenetic program is characterized by a migration of the nucleus to a distal position prior to an asymmetrical mitosis characteristic of the apical mode of functioning.

Connexions cortex–paroi chez la cellule apicale de Sphacelaria

Abstract The apical cell of Sphacelaria (Fucophyceae) exhibits a permanent polarized organization throughout asymmetric divisions. The apex organization was studied by immunolocalization of tubulin, vitronectin, α-actinin and β1 integrin. Microfilaments were stained directly by fluorescein phalloidin. The apex was highly organized around a patch of microfilaments densely packed at the tip, where vitronectin-like and α-actinin-like proteins colocalized. In the same area, an actin-dependent targeted secretion of sulfated polysaccharides was shown. The permanent localization of these components throughout cell elongation suggests that a cortical site involving transmembrane connections between the cytoskeleton and the extracellular matrix is required for cell polarity. A model of the organization of the tip is proposed.