Showing papers on "Protoplast published in 1989"

Metabolite levels in specific cells and subcellular compartments of plant leaves

Abstract: Publisher Summary This chapter focuses on the metabolite levels in specific cells and the subcellular compartments of plant leaves. Leaf material is difficult to fractionate because a typical plant cell contains several different, mechanically fragile subcellular organelles and is surrounded by a mechanically strong plant cell wall. Rather than attempting to isolate whole organelles or cells, leaves are frozen in liquid N 2 and then broken to small fragments that are enriched in material from a given compartment. These fragments are physically separated under conditions when the metabolic activity or redistribution of metabolites is prevented and subsequently their metabolism is quenched. The exclusion of water, or the use of extremely low temperatures, prevents metabolic activity during the fractionation procedures. The chapter describes the silicone oil centrifugation that allows chloroplasts to be separated from the remainder of the protoplast and to be quenched within 2–3 sec of disrupting the protoplast.

Analysis of Nuclear DNA content in plant cells by Flow cytometry

Abstract: Flow cytometry was used to analyse the DNA content of nuclei isolated from intact plant tissues and from callus and cell suspension cultures invitro. Cell nuclei were isolated either mechanically (chopping, syringing) or by a hypotonic lysis of isolated protoplasts. Although both methods gave similar results, a slight shift to lower ploidy levels was observed after protoplast isolation from intact tissues and calli. No differences were observed if the two methods were compared using cell suspension cultures. The results showed that flow cytometry is a rapid method of nuclear DNA content analysis in intact plant tissues and variousin vitro cultures.

Plant Protoplasts and Genetic Engineering V

Abstract: Section I Regeneration of Plants from Protoplasts.- I. 1 Regeneration of Plantlets from Protoplasts of Allium cepa (Onion).- I. 2 Regeneration of Plants from Protoplasts of Anthriscus sylvestris (L.) Hoffm. (Woodland Beakchervil).- I. 3 Regeneration of Plants from Protoplasts of Coffea spp. (Coffee).- I. 4 Direct Embryogenesis in Protoplasts of Ginkgo biloba (Maidenhair Tree).- I. 5 Regeneration of Plants from Protoplasts of Helianthus annuus L. (Sunflower).- I. 6 Regeneration of Plants from Protoplasts of Hordeum vulgare (Barley).- I. 7 Regeneration of Plants from Protoplasts of Lilium x formolongi.- I. 8 Regeneration of Plants from Protoplasts of Mentha piperita L. (Peppermint).- I. 9 Regeneration of Plants from Protoplasts of Mosses (Funaria hygrometrica and Physcomitrella patens).- I. 10 Regeneration of Plants from Protoplasts of Passiflora Species (Passion Fruit).- I. 11 Regeneration of Plants from Protoplasts of Poa pratensis L. (Kentucky Blue Grass).- I. 12 Regeneration of Plants from Protoplasts of Populus Species (Poplars).- I. 13 Regeneration of Plants from Protoplasts of Saintpaulia ionantha H. Wendl. (African Violet).- Section II Genetic Transformation and Transgenic Plants.- II. 1 Genetic Transformation in Agrostis palustris Huds. (Creeping Bentgrass).- II. 2 Genetic Transformation in Asparagus officinalis L..- II. 3 Genetic Transformation in Avena sativa L. (Oat).- II. 4 Genetic Transformation in Boehmeria nivea Gaud. (Ramie Fiber).- II. 5 Genetic Transformation of Carthamus tinctorius L. (Safflower).- II. 6 Genetic Transformation of Geraniums.- II. 7 Genetic Transformation of Gladiolus.- II. 8 Genetic Transformation of Lithospermum erythrorhizon for Increased Production of Shikonin.- II. 9 Genetic Transformation in Papaver somniferum L. (Opium Poppy) for Enhanced Production of Morphinan.- II. 10 Genetic Transformation of Panax ginseng (C. A. MEYER) for Increased Production of Ginsenosides.- II. 11 Genetic Transformation in Petunia.- II. 12 Genetic Transformation in Pinus elliottii Engelm. (Slash Pine).- II. 13 Genetic Transformation in Raspberries and Blackberries (Rubus Species).- II. 14 Genetic Transformation in Swertia japonica.

Isolation of an auxin-regulated gene cDNA expressed during the transition from G0 to S phase in tobacco mesophyll protoplasts

Abstract: A cDNA clone for an auxin-regulated gene was isolated from a tobacco mesophyll protoplast cDNA library by differential screening Nucleotide, sequence analysis showed that the deduced product of the gene, which we have designated par, is hydrophilic and is composed of 220 amino acids No significant homology to other known proteins was detected The mRNA of the par gene is approximately 900 bases long and its accumulation was detected in cultured mesophyll protoplasts as early as 30 min after the addition of 2,4-dichlorophenoxyacetic acid to the culture medium The par mRNA was not detected in leaves or freshly prepared protoplasts or in protoplasts in the absence of 2,4-dichlorophenoxyacetic acid Expression of the par gene was detected at a low level in actively dividing BY-2 tobacco suspension culture cells The conspicuous accumulation of par mRNA before the initiation of DNA synthesis in tobacco mesophyll protoplasts suggests that the par gene product could play a role in the initiation of meristematic activity in differentiated mesophyll cells

Plant regeneration from indica rice (Oryza sativa L.) protoplasts.

Abstract: Rice (Oryza sativa L.) plants of the indica cultivar IR54 were regenerated from protoplasts. Conditions were developed for isolating and purifying protoplasts from suspension cultures with protoplast yields ranging from 1·106 to 15·106 viable protoplasts/1 g fresh weight. Protoplast viability after purification was generally over 90%. Protoplasts were cultured in a slightly modified Kao medium in a Petri plate by placing them onto a Millipore filter positioned on top of a feeder (nurse) culture containing cells from a suspension culture of the japonica rice, Calrose 76. Plating efficiencies of protoplasts ranged from 0.5 to 3.0%; it was zero in the absence of the nurse culture. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the protoplasts. After three weeks the Millipore filter with callus colonies were transferred off feeder cells and onto a Linsmaier and Skoog-type medium for an additional three weeks. Selected callus colonies that had embryo-like structures were then transferred to regeneration medium containing cytokinins, and regeneration frequencies up to 80% were obtained. Small shoots emerged and were transferred to jars for root development prior to transferring to pots of soil and growing the plants to maturity in growth chambers. Of the cytokinins evaluated, N6-benzylaminopurine was the most effective in promoting shoot formation; however, kinetin was also somewhat effective. Regeneration medium could be either an N6 or Murashige and Skoog basal medium. Of 76 plants grown to maturity, 62 were fertile, and the plant heights averaged about three-fourths the height of seed-grown plants. Two other suspension cultures of IR54, one developed from the protoplast callus of the initial IR54 line, and the other developed from callus produced by mature seeds, have yielded protoplasts capable of regenerating plants when using cells of the Calrose 76 suspension as a nurse culture. In addition, protoplasts obtained from three-week-old primary callus of immature embryos of IR54 were capable of regenerating plants when using the same culture conditions.

Structural analysis of the cell walls regenerated by carrot protoplasts.

Abstract: A procedure was developed to isolate protoplasts rapidly from carrot (Daucus carota L. cv. Danvers) cells in liquid culture. High purity of cell-wall-degrading enzymes and ease of isolation each contributed to maintenance of viability and initiation of regeneration of the cell wall by a great majority of the protoplasts. We used this system to re-evaluate the chemical structure and physical properties of the incipient cell wall. Contrary to other reports, callose, a (1 → 3)β-d-glucan whose synthesis is associated with wounding, was not a component of the incipient wall of carrot protoplasts. Intentional wounding by rapid shaking or treatment with dimethyl sulfoxide initiated synthesis of callose, detected both by Aniline blue and Cellufluor fluorescence of dying cells and by an increase in (1 → 3)-linked glucan quantified in methylation analyses. Linkage analyses by gas-liquid chromatography of partially methylated alditol-acetate derivatives of polysaccharides of the incipient wall of protoplasts and various fractions of the cell walls of parent cells showed that protoplasts quickly initiated synthesis of the same pectic and hemicellulosic polymers as normal cells, but acid-resistant cellulose was formed slowly. Complete formation of the wall required 3 d in culture, and at least 5 d were required before the wall could withstand turgor. Pectic substances synthesized by protoplasts were less anionic than those of parent cells, and became more highly charged during wall regeneration. We propose that de-esterification of the carboxyl groups of pectin uronic-acid units permits formation of a gel that envelops the protoplast, and the rigid cellulose-hemicellulose frame-work forms along with this gel matrix.

Synthesis of male sterile, triazine-resistant Brassica napus by somatic hybridization between cytoplasmic male sterile B. oleracea and atrazine-resistant B. campestris

Abstract: Fusion of leaf protoplasts from an inbred line of Brassica oleracea ssp. botrytis (cauliflower, n=9) carrying the Ogura (R1) male sterile cytoplasm with hypocotyl protoplasts of B. campestris ssp. oleifera (cv “Candle”, n=10) carrying an atrazine-resistant (ATR) cytoplasm resulted in the production of synthetic B. napus (n=19). Thirty-four somatic hybrids were produced; they were characterized for morphology, phosphoglucose isomerase isoenzymes, ribosomal DNA hybridization patterns, chromosome numbers, and organelle composition. All somatic hybrids carried atrazine-resistant chloroplasts derived from B. campestris. The mitochondrial genomes in 19 hybrids were examined by restriction endonuclease and Southern blot analyses. Twelve of the 19 hybrids contained mitochondria showing novel DNA restriction patterns; of these 12 hybrids, 5 were male sterile and 7 were male fertile. The remaining hybrids contained mitochondrial DNA that was identical to that of the ATR parent and all were male fertile.

Stable co-transformation of maize protoplasts with gusA and neo genes

Abstract: An efficient co-transformation protocol using polyethylene glycol was developed for Zea mays L. (cv. A188 x BMS) protoplasts isolated from suspension culture cells. Co-transformation was accomplished by using plasmid constructions containing beta-glucuronidase (gusA) or neomycin phosphotransferase (neo) gene coding sequences; both were under control of the CaMV 35S promoter. Protoplast culture and transformation conditions were optimized to assure efficient recovery of transformed cells. The overall efficiency of transformation was 1 x 10(-4) (calculated per viable protoplast plated). Among kanamycin-resistant lines, 50% showed a high level of GUS activity (above one unit). Southern blot hybridization confirmed the presence of numerous gusA and neo coding sequences in the maize genome. In two analyzed lines, integrated sequences appeared to be organized in tandem head-to-tail repeats. Results also indicated that the integrated sequences were partially methylated.

Asymmetric somatic hybrids of Brassica: partial transfer of B. campestris genome into B. oleracea by cell fusion.

Abstract: To examine the possibility of producing asymmetric somatic hybrids of Brassica having a complete genome of one species and a part of the other, we fused inactivated B. oleracea protoplasts with X-irradiated B. campestris protoplasts. The plants obtained were studied with regard to their morphology, isozymes and chromosomes. The morphology of the hybrids was similar to B. oleracea in 9 out of 22 hybrids studied and the rest showed the intermediate phenotype of the parents. Analysis of three isozymes, leucine aminopeptidase, acid phosphatase and esterase indicated that ten hybrids lost B. campestris-specific bands in one or more of the three isozymes examined. The chromosome analysis showed that 90% of the hybrids were aneuploids. In addition, abnormal chromosomes were often found in root tip cells. These results suggested that the hybrids obtained were asymmetric in nature and resulted from elimination of B. campestris chromosomes by X-ray irradiation.

Plant regeneration from pea protoplasts via somatic embyogenesis.

Abstract: Plant regeneration via somatic embryogenesis was obtained from pea protoplasts. Strong auxins (picloram or 2.4-D) and increased osmolarity of the medium were necessary for embryo induction. Relatively high amounts of embryogenic calli could be obtained in 2 genotypes. After a period on hormone-free medium, a second induction of somatic embryos was possible. Further development of somatic embryos was accomplished on GA3 — containing medium.

A Comparison between Quin-2 and Aequorin as Indicators of Cytoplasmic Calcium Levels in Higher Plant Cell Protoplasts

Abstract: Assessment of the regulation of plant metabolism by the calcium ion requires a knowledge of its intracellular levels and dynamics. Technical problems have prevented direct measurement of the concentration of intracellular Ca2+ in plant cells in all but a few cases. In this study we show that electropermeabilized protoplasts of Daucus carota and Hordeum vulgare took up the Ca2+ indicating fluorescent dye methoxyquinoline(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (Quin-2) and the Ca2+ indicating photoprotein, aequorin. These protoplasts subsequently recovered their plasma membrane integrity. However, up to 10% of intracellularly trapped Quin-2 was associated with a protoplast vacuolar fraction. Also, Quin-2 loading reduced total ATP levels by approximately 60% and inhibited subsequent protoplast division whereas aequorin loading reduced ATP content by only 20% and did not prevent division. Therefore, the basal cytoplasmic Ca2+ level measured with aequorin (less than 200 nanomolar) may more reliably reflect that found in vivo in the unperturbed protoplast than that measured with Quin-2 (120-360 nanomolar). However, measurements made with aequorin were found to be inaccurate at Ca2+ levels below 200 nanomolar, Quin-2 proving complementary in indicating these low Ca2+ concentrations. Cytosolic Ca2+ was observed to increase on treatment with azide and silver ions.

Construction of rice cybrid plants

Abstract: The mitochondrial genomes of rice cells were transferred to a fertile rice variety (N8) from a cytoplasmic male sterile variety (CMS) by asymmetric protoplast fusion based on metabolic complementation. Protoplasts derived from CMS were X-irradiated (125 krad) and electrofused with protoplasts which had been treated with iodoacetamide. Metabolic complementation, presumably between nuclear and cytoplasmic compartments, enabled fused protoplasts to form colonies at high efficiency. Restriction digest analysis of mitochondrial DNA (mtDNA) indicated that hybrid cells carried mtDNA derived from both parents. Of the plants regenerated from hybrid calli, 68% carried a diploid chromosome set (2n=24) and the rest of them carried 48 chromosomes. All of them expressed the aryl acylamidase I deficient phenotype encoded by the recessive allele of the fertile N8 parent. These results indicate that the novel somatic hybrid plants regenerated were cybrids, deriving their nucleus from the iodoacetamide treated parent and their mitochondria from both parents.

Initiation of embryogenic callus and suspension cultures, and improved embryo regeneration from protoplasts, of white spruce (Picea glauca)

Abstract: Rapid and high frequency somatic embryo regeneration from protoplasts isolated from 10 embryogenic cell lines of white spruce (Picea glauca) is reported. Embryogenic callus was initiated from immature zygotic embryos as source material for protoplast isolation. Individual cell lines exhibited different capabilities for sustained growth. Protoplast plating efficiencies depended on the concentrations of macroelements included in the medium. Using a medium with reduced salts, individual protoplasts developed directly into embryos with no disorganized growth period. Protoplasts from newly established suspension cultures regenerated to recognizable somatic embryos within 8 days of culture. This embryo development was faster than that from protoplasts isolated from longer term suspension cultures. However, the latter suspensions yielded more protoplasts, displayed higher plating efficiencies, and differed in their response to media.

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Abstract: With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.

Plantlet regeneration from embryogenic protoplasts of white spruce (Picea glauca)

Abstract: We report plantlet regeneration from conifer protoplasts. White spruce protoplasts were isolated from previously cryopreserved embryogenic suspension cultures and cultured in PCM with an osmoticum containing myo-inositol. Protoplast derived embryos established in suspension culture, were used as source material for plantlet regeneration. Embryo maturation was induced on 1/2 strength LP medium containing 90 mM sucrose and 12 M abscisic acid. After 4 weeks, cultures were transferred to 1/2 strength LP medium, 60 mM sucrose and no growth regulators. Approximately 7–14 days after transfer to this medium, embryos bearing prominent cotyledons were separated from the main callus and cultured independently. These embryos underwent cotyledon and hypo-cotyl elongation and root development. The total time to plantlet recovery, following protoplast isolation was 15 weeks.

Recovery of somatic embryos and plantlets from protoplast cultures of Larix × eurolepis

Abstract: Somatic embryos and plantlets were regenerated from protoplasts of hybrid larch (Larix × eurolepis) isolated from two embryogenic callus and cell suspension culture lines (L1 and L2). L2, which was highly embryogenic, consistently yielded protoplasts that gave rise to somatic embryos. Centrifugation on a discontinuous medium/Percoll density gradient resulted in accumulation of embryogenic protoplasts in one of the Percoll interfaces. First division frequencies were in the range of 28–39% in line 1 and 18–20% in line 2 in both liquid and agarose-solidified culture media. The critical factor in maintaining high viability of cultures was lowering of osmotic pressure by dilution of the initial medium. The first somatic embryos were detected in 23- to 28-day-old cultures. Some of these developed into plants that were transferred to soil.

Plant tissue and protoplast culture: applications to stress physiology and biochemistry.

Abstract: Introduction Salinity and drought, two environmental stresses frequently found together, are major barriers to productivity of agricultural crops throughout the world. Crops exposed to these stressful environments are observed initially to have reduced growth rates. If the stress is more severe the response is manifested visually in a number of specific and recognisable symptoms, many of which are common to both salinity and drought. However, specific ion toxicity responses (e.g. marginal leaf burn) are observed in plants exposed to excess salinity. Since salinity and drought have many common responses some of the information presented in this review will be combined under the general umbrella of environmental stress. This chapter discusses the use of tissue and protoplast culture as a means to understand better the cellular processes related to stress tolerance with the expectation that these techniques will provide alternative methods for screening germplasm and assist in identifying useful material for incorporation into crop improvement programmes. Of equal importance is the ability to compare physiological and biochemical processes of cells and protoplasts selected for stress tolerance against unselected (wild-type) lines and to relate this to genetic regulation at the molecular level. The techniques of tissue and protoplast culture are important for application of molecular biology to genetic manipulation of plants. The field of plant pest-host interactions has been significantly advanced by the use of protoplasts and subsequent tissue culture and regeneration of pest-resistant plants (Harrison & Mayo, 1983). Somaclonal variation has provided important genetic material both for genetic studies and for selection of desired traits in plants (Scowcroft, Larkin & Brettell, 1983; Maddock & Semple, 1986).

Protoplast Isolation and Regeneration of Three Species of Ulva in Axenic Culture

Abstract: Several commercial and crude enzymes have been tested in various combinations to obtain isolated protoplasts from Ulvapertusa (mutant and wild type), U.fasciata and U. conglobata. Commercial enzymes together with abalone crude enzyme preparation, generally produced a high yield of protoplasts in all the species studied. Protoplasts which regenerated under the same culture conditions exhibited two developmental patterns. In one type protoplasts underwent repeated cell divisions and formed a thallus consisting of a single layer of cells. In the other type protoplasts, after cell wall regeneration, converted into reproductive cells and discharged many motile spores which on germination grew into normal plants. Protoplasts cultured in medium supplemented with mannitol showed abnormal cell division after regenerating cell walls.

Changes in DNA content and chromosomal size during cell culture and plant regeneration of Scilla siberica: selective chromatin diminution in response to environmental conditions

Abstract: The chromosomal complement and DNA content of cells of the monocotyledonous plant Scilla siberica were studied at various stages of growth, such as callus outgrowths from bulb tissue (“bulb callus”), callus grown from single protoplasts prepared from bulb callus (“protoplast callus”), regenerated plants kept for 2–4 years on agar medium and under constant climatic conditions, and regenerated plants grown first for 2 years on agar and then for 1 year in the garden. During callus culture, several different forms of chromatin loss were observed: (1) chromosome elimination early during cell culture, resulting in cells which were mostly diploid but still had large chromosomes similar to those of the original triploid plants (“type 1 cells”); from these cells no plants could be regenerated. (2) Dramatic reduction in heterochromatin containing the satellite DNA and, apparently subsequently, also of many other chromatin moieties, resulting in the formation of small chromosomes; frequent polyploidization in these cells, resulting in a variable number of chromosomes per cell (preferentially 30–40, in ca. 70% of the cells; “type 2 cells”). (3) Appearance of a large number of very small ( 95%) of the satellite sequences. Southern DNA blot analysis revealed that sequences hybridizing with certain protein-coding genes such as that for chalcone synthase were also drastically reduced in copy number whereas the proportion of rDNA was even somewhat increased. In plants regenerated from type 2 cells of protoplast calli, which were aneuploid at near-pentaploidy to hexaploidy, further conspicuous changes in chromosomal and DNA content were not observed, as long as they were kept on agar medium and under constant climatic conditions. However, when such plants were grown in the garden for at least 1 year, the satellite DNA as well as the sequences hybridizing with the chalcone synthase gene were disproportionately increased to 30%–40% of their normal proportion, whereas the total DNA had increased by only approximately 15%. The chromosome numbers remained near-pentaploid to hexaploid as in the cell cultures from which these plants had been regenerated. These phenomena of selective loss and regain of chromatin in response to environmental conditions (cell culture, regenerated plants on agar, regenerated plants grown outdoors) are discussed in relation to other forms of chromatin loss, including developmentally controlled chromatin diminution in certain animals.

Protoplast transformation of Bacillus stearothermophilus NUB36 by plasmid DNA.

Abstract: An efficient protoplast transformation system was established for Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tcr (4·5 kb) and mesophilic plasmid pLW05 Cmr (3 kb), a spontaneous deletion derivative of pPL401 Cmr Kmr. The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 107 to 4 x 108 transformants per µg DNA. The transformation frequency (transformants per regenerant) was 0·5 to 1·0. Chloramphenicol-resistant and tetracycline-resistant transformants were obtained when competent cells of Bacillus subtilis were transformed with pLW05 [2·5 x 105 transformants (µg DNA)-1] and pTHT15 [1·8 x 105 transformants (µg DNA)-1], respectively. Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cmr was not stably maintained in cultures growing at temperatures between 50 and 65 °C but the thermostable chloramphenicol acetyltransferase was active in vivo at temperatures up to 70 °C. In contrast, thermophilic plasmid pTHT15 Tcr was stable in cultures growing at temperatures up to 60 °C but the tetracycline resistance protein was relatively thermolabile at higher temperatures. The estimated copy number of pLW05 in cells of NUB3621 growing at 50,60, and 65 °C was 69,18, and 1 per chromosome equivalent, respectively. The estimated copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 °C was about 41 to 45 per chromosome equivalent and 12 in cells growing at 65 °C.

Tomato: a crop species amenable to improvement by cellular and molecular methods

Abstract: Tomato is a crop plant with a relatively small DNA content per haploid genome and a well developed genetics. Plant regeneration from explants and protoplasts is feasable which led to the development of efficient transformation procedures. In view of the current data, the isolation of useful mutants at the cellular level probably will be of limited value in the genetic improvement of tomato. Protoplast fusion may lead to novel combinations of organelle and nuclear DNA (cybrids), whereas this technique also provides a means of introducing genetic information from alien species into tomato. Important developments have come from molecular approaches. Following the construction of an RFLP map, these RFLP markers can be used in tomato to tag quantitative traits bred in from related species. Both RFLP's and transposons are in the process of being used to clone desired genes for which no gene products are known. Cloned genes can be introduced and potentially improve specific properties of tomato especially those controlled by single genes. Recent results suggest that, in principle, phenotypic mutants can be created for cloned and characterized genes and will prove their value in further improving the cultivated tomato.

Preferential digestion of chloroplast nuclei (nucleoids) during senescence of the coleoptile of Oryza sativa

Abstract: The coleoptile ofOryza sativa develops, grows and ages within 4 days that follow imbibition. It is, thus, a very useful system for experimental analysis of the life cycle of organelles, for example, the development, growth and aging of plastids in higher plants. We examined the behavior and levels of DNA and chlorophyll in the plastid by epifluorescence microscopy after staining with 4′-6-diamidino-2-phenylindole (DAPI), and by fluorimetry with a video-intensified-photon counting system (VIMPCS). The whitish yellow coleoptile appeared soon after imbibition and, between the first 24 and 60 h that followed imbibition, it grew markedly in a longitudinal direction, with concomitant elongation of the cells, and an increase in the volume of plastids and in the amount of DNA in the plastids. The chlorophyll content per plastid began to increase when the coleoptile turned green, 48 h after imbibition, and reached a plateau value when the coleoptile was 3.5 mm in length, 72 h after imbibition. More than 12 h later, the chlorophyll disappeared just before the breakdown of chloroplasts was initiated. Proplastids in young coleoptiles, contained a plastid nucleus which was located in the central area of the plastids and each nucleus consisted of approximately 6 copies of plastid DNA (ptDNA). The number of copies of ptDNA per plastid increased gradually, with a concomitant increase in the volume of the plastids after imbibition, and reached approximately 130 times the value in the young proplastids, 60 h after imbibition, when the plastid developed into a chloroplast. However, each plastid nucleus did not scatter throughout the entire interior region of each chloroplast. The disappearance of each plastid nucleus occurred more than 12 h before the degeneration of the chloroplasts. The number of plastids per cell increased from 10 to 15 in young coleoptiles within 12 h after imbibition. Yet the number remained constant throughout subsequent growth and aging of the coleoptile. Thus the preferential reduction in the amount of chloroplast DNA was not due to the division of the plastid but could, perhaps, be associated directly with the aging of the cells of the coleoptile which precedes senescence of the coleoptiles.

Chromosomal instability in cell- and tissue cultures of tomato haploids and diploids.

Abstract: The effect of the tissue culture system, the genotype and the ploidy level of the plant material used as explant source on the stability of the ploidy level of plants regenerated fromcell and tissue cultures of tomato was investigated. In addition the use of the chloroplast number in guard cells as a measure for ploidy level was evaluated. Haploids of tomato were very instable, which instability was observed already in somatic root tip and leaf cells. The number of regenerated plants that retained the original ploidy level differed significantly between the tested haploids. The plants that were regenerated from leaf explants of diploids were predominantly diploid in contrast to the plants regenerated from established callus cultures and protoplast where the majority was tetraploid.

The Role of Surface Components of the Entomopathogenic Fungus Entomophaga aulicae in the Cellular Immune Response of Galleria mellonella (Lepidoptera)

Abstract: SUMMARY: Larvae of Galleria mellonella (Lepidoptera) displayed a strong cellular reaction to the walled-stage hyphal bodies of the entomopathogenic fungus Entomophaga aulicae (Entomophthorales). In contrast, no reaction was observed against the wall-less protoplasts. This phenomenon was not due to an inhibition of the cellular response by secretion of a toxin by protoplasts. Furthermore, no molecular mimicry was observed between host and protoplast antigens. Host macromolecules which could protect them from being recognized as foreign did not attach to the protoplasts. Ultrastructural and chemical studies of hyphal body and protoplast surfaces of E. aulicae demonstrated the presence of 1,3-β-glucan and chitin on the hyphal wall, whereas these components were absent on the protoplast surface. Since purified 1,3-β-glucan and chitin isolated from E. aulicae and from another entomophthoralean species, Conidiobolus obscurus, induced their haemocytic encapsulation in vitro, it is suggested that these wall components are responsible for the recognition of hyphal body elements, and that their absence accounts for the ability of protoplasts to escape encapsulation.

Release of phytotoxic factors from plant cell walls during protoplast isolation.

Abstract: Isolation of protoplasts from plant cells involves a number of steps, the impact of which on the physiological response of the resulting protoplasts is not well understood. In particular, the usual treatment of cells with hydrolytic enzymes of fungal origin has received little attention, except for empirical selection of appropriate commercial preparations and, in some cases, their purification via gel filtration.

Transfer of radish (Raphanus sativus L.) chloroplasts into cabbage (Brassica oleracea L.) by protoplast fusion.

Abstract: Intergeneric hybrid plants were obtained through protoplast fusion between red cabbage (Brassica oleracea L.) and radish (Raphanus sativus L.). Protoplast fusion was carried out by the dextran method using protoplasts from hypocotyls and cotyledons of both cabbage and radish. The selection of somatic hybrids utilized the inactivation of cabbage protoplasts with iodoacetamide and the low cell-division ability of radish protoplasts. Ten plants were regenerated from selected colonies. Two of them developed to the flowering stage and formed male sterile flowers, which showed cytoplasmic inheritance. The plant and leaf morphology, chromosome number, acid phosphatase and peroxidase isozyme patterns, sexual compatibility, and SmaI cleavage patterns of chloroplast DNA indicated that these plants were intergeneric hybrids having the nucleus of cabbage and chloroplasts of radish.