Showing papers on "Protoplast published in 1995"

A periplasm in Bacillus subtilis.

Abstract: The possibility of there being a periplasm in Bacillus subtilis, in the distinct cell compartment bounded by the cytoplasmic membrane and the thick cell wall, has been investigated quantitatively and qualitatively. Cytoplasmic, membrane, and protoplast supernatant fractions were obtained from protoplasts which were prepared isotonically from cells grown under phosphate limitation. The contents of the protoplast supernatant fraction represent an operational definition of the periplasm. In addition, this cell fraction includes cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from, or lysis of a fraction of, protoplasts. The latter, measured by assay of enzyme markers and by radiolabeled RNA and protein, was found to represent 7.6% of total cell protein, yielding a mean of 9.8% +/- 4.8% for B. subtilis 168 protein considered periplasmic. Qualitatively, after subjection of all cell fractions to polyacrylamide gel electrophoresis, RNase and DNase, zymographs revealed that (i) each cell fraction had a unique profile of nucleases and (ii) multiple species and a major fraction of both nucleases were concentrated in the periplasm. We conclude that the operationally defined periplasmic fraction corresponds closely, both quantitatively and qualitatively, to the contents of the periplasm of Escherichia coli. We discuss evidence that the maintenance of the components of this surface compartment in B. subtilis is compatible with the thick negatively charged cell wall acting as an external permeability barrier.

Auxins induce clustering of the auxin-binding protein at the surface of maize coleoptile protoplasts.

Abstract: The predominant localization of the major auxin-binding protein (ABP1) of maize is within the lumen of the endoplasmic reticulum. Nevertheless, all the electrophysiological evidence supporting a receptor role for ABP1 implies that a functionally important fraction of the protein must reside at the outer face of the plasma membrane. Using methods of protoplast preparation designed to minimize proteolysis, we report the detection of ABP at the surface of maize coleoptile protoplasts by the technique of silver-enhanced immunogold viewed by epipolarization microscopy. We also show that ABP clusters following auxin treatment and that this response is temperature-dependent and auxin-specific.

A Simple and Rapid Method of Transformation of Streptomyces rimosus R6 and Other Streptomycetes by Electroporation.

Abstract: Usually plasmid DNA is introduced into Streptomyces strains by polyethylene glycol-mediated transformation of protoplasts. However, many Streptomyces strains are only poorly or not at all transformable via protoplasts. Therefore, we have optimized the parameters critical for the application of electrotransformation of plasmid DNA into Streptomyces species. The most critical parameters evaluated for electrotransformation of the model strain Streptomyces rimosus R6 were the pretreatment of mycelia, buffer composition, and electric field strength. The electrocompetent mycelia were prepared from 24-h-old cultures, treated mildly with lysozyme, resuspended in sucrose-glycerol-polyethylene glycol buffer, and stored in aliquots at -70 deg C. The electric field strength of 10 kV/cm at 400 (Omega) and a capacitance of 25 (mu)F was applied. The method is simple and rapid, yielding transformant colonies in 48 to 72 h. Efficiencies of 10(sup5) to 10(sup6) transformants per (mu)g of plasmid DNA were reproducibly achieved for S. rimosus R6 and its mutants, and these numbers were 10(sup2) to 10(sup3) higher than those attained by polyethylene glycol-assisted transformation of protoplasts. In addition, we show that electroporation can be applied to other Streptomyces species, such as S. lividans 66, S. coelicolor A3(2), and an S. venezuelae strain. This last one could not be transformed by the standard protoplast procedure. Our data suggest that, because of the diversity of streptomycetes, the conditions have to be optimized for each strain.

Transfer of resistance to Xanthomonas campestris pv campestris into Brassica oleracea L. by protoplast fusion

Abstract: Black rot caused by the bacterium Xanthomonas campestris pv campestris is one of the most serious diseases of Brassica oleracea. Since sources of resistance to the disease within B. oleracea are insufficient and control means are limited, the development of resistant breeding lines is extremely desirable. Certain lines of B. napus contain very high resistance controlled by a dominant gene, but crossing the two species sexually is very difficult. Therefore, somatic hybrids were produced by protoplast fusion between rapid cycling B. oleracea and a B. napus line highly resistant to X. campestris pv campestris. Hybrid identity was confirmed by morphological studies, flow cytometric estimation of nuclear DNA content, and analysis of random amplified polymorphic DNA (RAPD). Inoculations with the pathogen identified four somatic hybrids with high resistance. The resistant hybrid plants were fertile and set seed when selfed or crossed reciprocally to the bridge line ‘15’ (Quazi 1988). Direct crosses to B. oleracea were unsuccessful, but embryo rescue facilitated the production of a first-backcross generation. The BC1 plants were resistant to the pathogen. Progeny from the crosses to ‘line 15’ were all susceptible. Embryo rescue techniques were not obligatory for the development of a second-backcross generation, and several resistant BC2 plants were obtained.

Sexual and somatic hybridization in the genus Lactuca

Abstract: Various genes for disease resistance identified in wild Lactuca are difficult, even impossible to exploit in lettuce breeding, due to sexual incompatibility between L. sativa and wild Lactuca sp. We adapted two cellular biology techniques to overcome these interspecific barriers: in vitro embryo rescue and protoplast fusion. In vitro rescue of immature embryos was used successfully for sexual hybridization between L. sativa and L. virosa. Vigorous hybrid plants were produced between L. sativa and seven accessions of L. virosa. Protoplast fusion permitted the regeneration of somatic hybrids between L. sativa and either L. tatarica or L. perennis. Hybrids between L. sativa and L. tatarica were backcrossed to L. sativa.

An improved procedure for plant regeneration from indica and japonica rice protoplasts

Abstract: Plant regeneration from protoplasts of two commercially cultivated Indian indica rice varieties, Pusa Basmati 1 and Java, has been accomplished by plating embryogenic cell suspension-derived protoplasts on the surface of filter membranes overlying agarose-embedded feeder cells of Lolium multltiflorum and Oryza ridleyi, combined with the use of a maltose-containing shoot regeneration medium. Embryogenic cell suspension cultures of Pusa Basmati 1 and Jaya were initiated from mature seed scutellum-derived calli in liquid R2 medium modified by the addition of 560 mg l−1 of proline and 1.0 % (w/v) maltose. In both varieties, protoplast plating efficiencies up to 0.4 % were obtained, depending on the nature of the feeder cells. L. multiflorum feeder cells induced a 6-fold higher plating efficiency than feeder cells of O. ridleyi. In combination, O. ridleyi and L. multiflorum feedercells further enhanced protoplast plating efficiency. Protoplast-derived cell colonies were not obtained from protoplasts of either indica varieties in the absence of feeder cells. MS-based medium containing kinetin (2.0 mg l−1) and α-naphthaleneacetic acid (0.5 mg 1−1), together with sucrose and maltose both at 1.5 % (w/v), induced green shoot regeneration in 44 % of protoplast-derived tissues, depending on the feeder cells used for protoplast culture. In both varieties, tissues obtained using O. ridleyi feeder cells were more morphogenic than tissues obtained using L. multiflorum feeder cells, either alone or in combination with cells of O. ridleyi. In the japonica rice variety Taipei 309, this new procedure resulted in a 30-fold increase in plant regeneration from protoplasts compared to previous published procedures.

A Tool for Monitoring Trichoderma harzianum: I. Transformation with the GUS Gene by Protoplast Technology

Abstract: To obtain a genetically marked strain of Trichoderma harzianum that can be used as a tool for studies of population dynamics, T. harzianum was cotransformed with the Escherichia coli uidA β-glucuronidase (GUS) gene and the hygromycin B (hygB) gene as the selective marker. To improve the efficiency of conditions used for the transformation, the isolation, reversion, and germination of mycelial protoplasts were studied by scanning electron microscopy (SEM). Hexamethyldisilzane was useful for preparing protoplasts from suspensions for SEM. It was essential to obtain a transformant that phenotypically resembled the wild-type. After mitotic stabilization of the transformants by single-spore isolations, three transformants were compared to the wild-type by measurement of spore germination and mycelial growth rates, identification of secondary metabolite profiles, and studies of extracellular proteins. One transformant, T3c, was dissimilar to the wild-type in most tests, whereas transformant T3a was physiologically very similar to the wild-type. Furthermore, transformant T3a remained genetically stable during nonselective cultivation on plates and in sterile peat-bran.

Thidiazuron-induced high frequency of shoot induction and plant regeneration in protoplast derived pea callus.

Abstract: Protoplasts isolated from lateral shoot buds of cotyledon-free pea embryo axes were regenerated to callus. Protoplast derived calluses with a diameter of about 1cm were transferred to shoot induction media, containing different concentrations (1–50µM) of thidiazuron. Shoot formation was observed after 16 weeks up to 12% efficiency. Thidiazuron (10µM) was the most effective concentration in all experiments. Shoot buds elongated in medium supplemented with N-isopentenyl adenine and indole-3-butyric acid. Since rooting was almost impossible in these thidiazuron-induced shoots, shoots were grafted onto young pea seedlings and regenerated to fertile plants.

A simple method for isolation, liquid culture, transformation and regeneration of Arabidopsis thaliana protoplasts

Abstract: An efficient technique was developed for the isolation, culture, transformation and regeneration of protoplasts derived from auxin conditioned Arabidopsis root cultures. On an average 30% of root protoplasts underwent cell division in liquid culture and formed somatic embryolike structures which regenerated to plants without embedding in Ca(2+)-alginate. The protoplast protocol was applicable to different landraces of Arabidopsis thaliana (L.) Heynh., such as RLD, Columbia or C24. PEG-mediated DNA uptake into protoplasts using different uidA reporter gene constructs yielded transient gene expression in over 25% of treated cells indicating that root-derived protoplasts are suitable recipients for transformation.

Plant regeneration from mesophyll protoplasts of lisianthus (Eustoma grandiflorum) by adding activated charcoal into protoplast culture medium

Abstract: Plant regeneration from isolated protoplasts of 8 cultivars of lisianthus, Eustoma grandiflorum (Griseb.) Schinners, has been established by using activated charcoal. Protoplasts were isolated from lisianthus leaves grown in vitro and started to divide within 3–4 days of culture, but successful colony formation was only achieved by adding gellan gum blocks containing 1% (w/v) activated charcoal immediately after culture. Colonies consisting of as many as 50–100 cells formed after 30 days of culture and were transferred to fresh medium for callus proliferation and shoot regeneration, respectively. These shoots rooted on MS medium containing 0.5 mg l−1 indolebutyric acid(IBA) and the plantlets were finally transplanted to pots. Morphological characteristics, growth habit and pollen fertility of protoplast-derived plants of one cultivar were not different from those of seed-grown plants as control.

Inheritance of mitochondrial DNA in sexual crosses and protoplast cell fusions in Lentinula edodes

Abstract: By using mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) as genetic markers, the modes of mitochondrial inheritance in sexual crosses and protoplast cell fusions of the higher basidiomycete Lentinula edodes were examined. All newly established dikaryons from reciprocal crosses between compatible monokaryons carrying different mtDNA RFLP phenotypes retained mtDNA genotypes from one of the monokaryons, suggesting that mitochondrial inheritance is principally uniparental. In contrast, it was shown that recombinant mtDNA genomes arose in some dikaryons obtained after protoplast cell fusion. Based on these results, a possible mechanism for mitochondrial inheritance in L. edodes is discussed.

Zea mays (L.) with capability of long term, highly efficient plant regeneration including fertile transgenic maize plants having a heterologous gene, and their preparation

Abstract: Protoplasts which regenerate reproducibly in a short time to normal, fertile plants can be regenerated from an auxin-autotrophic genotype of Zea mays (L.). Starting from immature embryos on hormone-free media, an auxin-autotrophic, embryogenic callus is formed on the shoot basis of the seedlings, which callus retains its embryogenic potential over a substantial period of time when subcultured on hormone-free medium. In addition to fully-developed embryos, adventitious embryos are also formed under suitable culture conditions (6-9% of sucrose in the medium). When the sucrose content is reduced to 2-3% and 2,4-dichlorophenoxyacetic acid is added, soft, granular calli are formed which consist of embryogenic cell aggregates (type II callus). After subculturing the type II callus in the form of a cell suspension culture, totipotent protoplasts can be isolated. From these protoplasts, the maize plants according to the invention are regenerated.

Protoplast Isolation and Culture

Abstract: Protoplasts can be isolated from plant tissues or cultured cells by enzymatic digestion to remove the cell walls. The enzymes for this purpose are preparations which are commercially available. The success of protoplast isolation depends especially on the condition of the tissue and the combination of enzymes being used. There is no standard method for the isolation and culture of protoplasts. However, the procedure follows a general pattern. The individual cell or tissue source may require special conditions for successful isolation or for culture. Protoplasts from cell suspension cultures generally are the most readily obtained and usually regenerate into dividing cells at a reasonable frequency. Moreover, if plant regeneration is possible from the cells of the suspension culture, the same is often true for the cells regenerated from the protoplasts derived from the culture. Leaf mesophyll cells from a wide range of plants also have been used as protoplast sources with success.

Production of fertile somatic hybrid plants of sunflower and Helianthus giganteus L. by protoplast fusion.

Abstract: Protoplasts of Helianthus giganteus and Helianthus annuus were fused using polyethylene glycol. Before fusion H.giganteus protoplasts were subjected to iodoacetic acid treatment to render them unable to divide. Fused protoplasts were cultured in V-KM medium containing benzylaminopurine and naphtaleneacetic acid. Hybrid calli were identified on the basis of their ability of embryogenic development contributed by the Helianthus giganteus parent. Fifty embryogenic calli were cultured on MS based medium without growth regulators to induce further development of somatic embryos. Elongated shoots were removed, rooted and transferred into growth chambers. Overall morphology of the plants was intermediate between the two parents. Their hybrid nature was confirmed by chromosome counting and by the analysis of esterase isozymes. The plants flowered within two to three months and later died. Thus the perennial nature of H.giganteus is a recessive trait in this interspecific hybrid. Seeds were obtained from two of the regenerated plants. From these seeds normal fertile F2 plants could be grown.

Plant regeneration from explant and protoplast derived calluses of Medicago littoralis

Abstract: Plant regeneration from explant and protoplast derived callus has been achieved in Medicago littoralis cv. Harbinger 1886, an annual legume resistant to the fungus Pseudopeziza medicaginis. Callus was induced from different tissue explants and the fastest growth rate was observed for hypocotyls in B5 medium with 2 mg l−1 2,4-dichlorophenoxyacetic acid and 0.5 mg l−1 N6-benzyladenine. Protoplasts were isolated from cotyledons and leaves of sterile plants and from callus; the first two kinds of protoplasts showed a plating efficiency of 5.6% and 5%, respectively, when embedded in agarose. Plant regeneration occurred on media containing % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9qq-f0-yqaqVeLsFr0-vr% 0-vr0db8meaabaqaciGacaGaaeqabaWaaeaaeaaakeaacaqGobWaaW% baaSqabeaacaqG2aaaaOGaaeOVfiaabs5adaahaaWcbeqaaiaaikda% aaGccaqG+waaaa!3F97!\[{\text{N}}^{\text{6}} {\text{\Delta }}^2 {\text{}}\]isopentenyl-adenine combined with indole-3-acetic acid or 1,2-benzisoxazole-3-acetic acid, and on media with N6-benzyladenine plus α-naphtaleneacetic acid; a cytokinin/auxin ratio higher than 1 induced embryos while a ratio around 1 stimulated shoot formation. Embryo development and rooting of shoots were performed in RL medium without growth regulators.

Industrial yeast strain improvement: construction of a highly flocculent yeast with a killer character by protoplast fusion

Abstract: Conditions were optimized for rapid release and improved regeneration of protoplasts of Saccharomyces cerevisiae NCIM 3458. Rapid protoplast release was also obtained with representatives of several other yeast genera under the modified conditions of treatment. The application of the procedure in construction of a highly flocculent Saccharomyces cerevisiae with a killer character is described. Fusion was effected between UV-killed protoplasts of S. cerevisiae NCIM 3578 with a killer character and live protoplasts of the highly flocculent S. cerevisiae NCIM 3528 in the presence of polyethylene glycol (PEG) 6000. Fusants were selected using benomyl resistance as marker, the killer toxin producer rather than the highly flocculent yeast being resistant to the fungicide at a concentration of 100 micrograms ml-1. Fusants were also characterized by their DNA contents, capacity for ethanolic fermentation of molasses sugar and levels of invertase, alcohol dehydrogenase and pyruvate decarboxylase activities.

Cryopreserved callus: a source of protoplasts for rice transformation

Abstract: We cryopreserved whole rice calli (Oryza sativa L cv Taipei 309) to investigate the ability of the surviving cells to regenerate plants and yield protoplasts competent for genetic transformation. Four out of six callus lines cryopreserved after four months in culture contained small sectors able to continue cell division and subsequently regenerate fertile plants. Both cryopreservation efficiency and regeneration ability decreased when using eight month old cultures. High yields of protoplasts were obtained from different cryopreserved callus lines. Protoplasts were transfected with chimeric genes consisting of the maize ubiquitin 1 promoter, first exon and first intron fused to the coding region of either the GUS or BAR marker genes. Levels of transient gene expression from both marker genes were similar to those previously obtained using protoplasts derived from callus that had not been frozen. Stable transformants were selected by their resistance to Bialaphos and could be identified with the pH indicator chlorophenol red. Southern blot analysis confirmed the integration of the BAR gene into the rice genome. Therefore, cryopreservation does not affect the ability of rice cells to integrate and express foreign genes.

Plant regeneration from protoplast fusion inPassiflora spp.

Abstract: Protoplasts were isolated from leaf explants ofPassiflora edulis var.flavicarpa (the yellow passion fruit) and from cell suspensions of fivePassiflora species. Chemical fusion was performed using polyethylene glycol and the microcolonies obtained were transferred to growth medium to produce calli. Electrophoresis of soluble proteins and analysis of isoenzymes from calli produced from the fusion experiments were performed to select somatic hybrids. Specific polypeptide bands allowed the identification of somatic hybrids betweenP. edulis var.flavicarpa (+)P. alata, P. edulis var.flavicarpa (+)P. amethystina, P. edulis var.flavicarpa (+)P. cincinnata, P. edulis var.flavicarpa (+)P. giberti andP. edulis var.flavicarpa (+)P. coccinea. An average of 3 to 5% hybrid calli were obtained. With the exception of theP. edulis var.flavicarpa (+)P. coccinea, whole plants were recovered from all hybrids. These somatic hybrids showed 4n=36 chromosomes, which represents a further evidence of their hybridity.

Efficient plant regeneration from Indica (group 1) rice protoplasts of one advanced breeding line and three varieties.

Abstract: Regenerable embryogenic suspensions were established from one Indica (group 1) rice advanced breeding line and 9 Indica (group 1) rice varieties in 6–8 weeks. Four were chosen for protoplast culture and plant regeneration. About 4–7×107 protoplasts were isolated from one gram of 8-week-old cell suspension. High plating efficiency (30.5%) and colony formation (13.7%) were obtained using nurse culture methods. A high plant regeneration frequency (67.5%) was observed for line IR57311-95-2-3. In total, 322 plants were regenerated. All the regenerated plants were fertile.

A simple method for the isolation of plant protoplasts

Abstract: A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO4), and a protectant (CaCl2 2H2O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.

Ultrahigh-resolution low-voltage SEM reveals ultrastructure of the glucan network formation from fission yeast protoplast.

Abstract: The refined field emission SEM, S-900 LV which gives better resolution especially at low voltages below 5 kV was developed for ultrahigh resolution scanning electron microscopy. A visualization test at x 300,000 was made using a gold-evaporated magnetic tape, and the resolution was found to be about 1 nm at 2.5 kV. The ultrastructure of the cell wall, especially the reverting glucan network, from the protoplast of Schizosaccharomyces pombe was studied using this improved ultrahigh-resolution low-voltage SEM (UHR-LVSEM). The results with uncoated reverting protoplasts observed with this microscope revealed that the network was originally formed as secreted particles scattered on the protoplast surface and these were subsequently stretched to microfibrils about 2 nm thick. The microfibrils were twisted around each other and joined together so that they developed into 8-nm-thick fibrils, forming a ribbon-shaped network of glucans about 16-nm-thick which covered the entire protoplast surface. The UHR-LVSEM images of reverting protoplasts treated with glucanase confirmed that the particles scattered on the protoplast surface in the initial stage of regeneration were glucan in nature.

Regeneration of Medicago truncatula from protoplasts isolated from kanamycin-sensitive and kanamycin-resistant plants.

Abstract: Medicago truncatula (barrel medic) is an annual legume of agricultural and biological interest. In this report regeneration from isolated mesophyll protoplasts is described. A specifically developed, highly regenerable seed line is essential for regeneration. Other critical requirements for regeneration are the starting plant material, the use of agarose droplets incubated in a shallow layer of liquid medium, and protoplast density. Plants are grown in controlled environment conditions. Protoplasts are purified using a Percoll-based flotation procedure, then embedded in 100 μl agarose droplets containing a basal medium plus 25 μM NAA and 4 μM BAP (the same medium as in the surrounding shallow liquid layer) to induce protoplast division. A protoplast density of 6-8×10(5) ml(-1) is required for maximum colony formation. M. truncatula plants previously transformed for kanamycin resistance yielded embryogenic callus and also regenerated plants. Protoplasts from other annual Medicago (M.intertexta and M.scutellata) species readily form calli by the procedure we have described.

Intraspecific protoplast fusion of Candida tropicalis for enhancing phenol degradation

Abstract: Auxotrophic mutants and phenol-degrading defective mutants were separately isolated in a phenol-utilizing strain of Candida tropicalis M4, and were hybridized through protoplast fusion. Some protoplast fusants with phenol-degrading ability were obtained and were very stable. Two of the fusants exhibited slightly higher rates of growth than did the wild strain when the cells were grown on phenol medium, and they possessed about 1.9 and 2.2 times respectively higher phenol hydroxylase activity than the wild strain.

In vitro culture of Cucumis sativus L. XVIII. Plants from protoplasts through direct somatic embryogenesis

Abstract: A procedure is described for the isolation and culture of protoplasts from embryogenic callus (gel-like callus — GLC) and embryogenic suspension cultures (ESC) of Cucumis sativus c.v. Borszczagowski. Maximal protoplast yields from GLC and ESC were 5×106 and 1×107 protoplasts/g tissue respectively. They were obtained following 14–16 h digestion with 1.2% Cellulase Onozuka R-10, 1.2% Macerozyme R-10 and 0.3% Driselase. At a plating density of 2×105 / ml, first divisions occurred in 4–5 days and 7–8 days in ESC-and GLC-derived protoplasts respectively. The highest percentage of direct embryogenesis (over 80%) was observed with ESC. It was possible to obtain approximately 5000 embryo structures / g tissue. Some embryos converted into plants after 6 weeks, but most of them after 2 months of culture. ESC-derived plants, when transferred into the glasshouse, bloomed normally, and set seeds.

Synthesis of high erucic acid rapeseed (Brassica napus L.) somatic hybrids with improved agronomic characters.

Abstract: Novel Brassica napus somatic hybrids have been created through protoplast fusion of B. oleracea var. botrytis and B. rapa var. oleifera genotypes selected for high erucic acid (22:1) content in the seed oil. Fifty amphidiploids (aacc) and one putative hexaploid (aacccc) hybrid were recovered in one fusion experiment. Conversely, only one amphidiploid and numerous regenerates with higher DNA contents were produced in a similar fusion using a different B. rapa partner. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Analysis of organellar DNA revealed a distinct bias toward the inheritance of chloroplasts from the B. rapa (aa) genome. All amphidiploids set self-pollinated seed. A erucic acid content as high as 57.4% was found in the seed oil of one regenerated plant. Fatty acid composition was stable in the R1 generation and was coupled with increased female fertility. Other novel agronomic characters in the hybrids recovered include large seed size, lodging resistance, and non-shattering seed pods.

Developmental patterns during direct somatic embryogenesis in protoplast cultures of european larch ( Larix decidua Mill.)

Abstract: Protoplasts isolated from embryogenic suspension cultures of European larch (Larix decidua Mill.) were cultured in thin alginate layers using a nylon mesh to enable a monitoring of the development of single cells. The patterns of cell division and differentiation are characterized and compared with zygotic embryogenesis to which homologies can only be drawn to some extent when the protoplasts grow in an auxinfree environment. Already at 2.5 μM both 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid cause vacuolation and elongation of individual cells, thus disturbing the process of somatic embryogenesis which generally lacks the precise quantitative patterns occurring in vivo. Prior to the formation of an embryo, a proembryonal mass develops. Oligonucleated products of spontaneous protoplast fusions are able to cellularize even without preceding karyokinesis and perform a normal embryogenic program.