Showing papers on "Protoplast published in 2000"

A mannose selection system for production of fertile transgenic maize plants from protoplasts.

Abstract: Maize (Zea mays L.) callus cultures cannot use mannose as a sole carbohydrate source, but can utilize fructose for that purpose. Phosphomannose isomerase (PMI) can convert mannose to fructose. Transgenic maize plants were obtained by selecting polyethylene glycol (PEG)-mediated transformed protoplasts on mannose (20 g/l) containing medium. Transgenic calluses and plants carrying the PMI structural gene, manA, were able to convert mannose to fructose. The PEG-mediated protoplast transformation frequency was 0.06%. Stable transformation was confirmed by PCR, PMI activity, germination tests, and by histochemical staining with 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc). Stable integration of the transgenes into the maize genome was demonstrated in T1 and T2 plants. Results indicate that the mannose selection system can be used for maize PEG-mediated protoplast transformation.

An alternative method for the genetic transformation of sweet orange.

Abstract: An alternative method for transforming sweet organe [Citrus sinensis (L.) Osbeck] has been developed. Plasmid DNA encoding the non-destructive selectable marker enhanced green fluorescent protein gene was introduced using polyethylene glycol into protoplasts of ‘Itaborai’ sweet organe isolated from an embryogenic nucellar-derived suspension culture. Following protoplast culture in liquid medium and transfer to solid medium, transformed calluses were identified via expression of the green fluorescent protein, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. Transgenic plantlets were recovered from germinating somatic embryos and by in vitro rooting of shoots. To expedite transgenic plant recovery, regenerated shoots were also micrografted onto sour orange seedling rootstocks. Presence of the transgene in calluses and regenerated sweet organe plants was verified by gene amplification and Southern analyses. Potential advantages of this transformation system over the commonly used Agrobacterium methods for citrus are discussed.

Iron Deficiency Decreases the Fe(III)-Chelate Reducing Activity of Leaf Protoplasts

Abstract: The ferric-chelate reductase (FC-R) activity of mesophyll protoplasts isolated from Fe-sufficient (control) and Fe-deficient sugar beet (Beta vulgaris L.) leaves has been characterized. Measurements were made in an ionic environment similar to that in the apoplastic space of the sugar beet mesophyll cells. The FC-R activity of Fe-sufficient and Fe-deficient protoplasts was dependent on light. Fe deficiency decreased markedly the FC-R activity per protoplast surface unit. The optimal pH for the activity of the FC-R in mesophyll protoplasts was in the range 5.5 to 6.0, typical of the apoplastic space. Beyond pH 6.0, the activity of the FC-R in mesophyll protoplasts decreased markedly in both Fe-sufficient and Fe-deficient protoplasts. These data suggest that both the intrinsic decrease in FC-R activity per protoplast surface and a possible shift in the pH of the apoplastic space could lead to the accumulation of physiologically inactive Fe pools in chlorotic leaves.

Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm.

Abstract: The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (Cm) to monitor membrane changes in protoplasts from Vicia faba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 microM) at constant ambient osmotic pressure (pi omicron) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (fFM,i) increased with reference to the total fluorescence (fFM,t) by 7.4 x 10(-4) min(-1). This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of pi omicron by 200 mosmol kg(-1) caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time fFM,i/fFM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of Cm and total fluorescence of a protoplast (fFM,p) showed that an increase in pi omicron evoked a decrease in Cm but no change in fFM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 microm) with FM 1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area.

Efficient intergeneric fusion of pea (Pisum sativum L.) and grass pea (Lathyrus sativus L.) protoplasts

Abstract: Large numbers of viable protoplasts of pea (Pisum sativum) and grass pea (Lathyrus sativus) were efficiently and reproducibly obtained and, for the first time, fused Different procedures for fusion were compared, based either on electrofusion (750, 1000, 1250 or 1500 V cm(-1)), or on the use of macro or micromethods with a polyethylene glycol (PEG 6000 or PEG 1540), or a glycine/high pH solution Over 10% of viable heterokaryons were obtained, with PEG as the most efficient and reproducible agent for protoplast fusion (>20% of viable heterokaryons) Both the division of heterokaryons and the formation of small calluses were observed

Improved resistance to bacterial soft rot by protoplast fusion between Brassica rapa and B. oleracea

Abstract: Erwinia soft rot is a destructive disease of Brassica rapa vegetables. Reliable sources of resistance and control methods are limited, so development of highly resistant breeding lines is desirable. Protoplasts from B. rapa and B. oleracea genotypes selected for resistance to soft rot were fused in order to combine different sources of resistance. Twelve somatic hybrids (synthetic B. napus) were obtained and confirmed by morphology, nuclear DNA content, and RAPD analysis. They were normal looking plants that easily set seeds following self-pollination and backcrossing to B. rapa. Assays of detached leaves or seedlings inoculated in a mist-chamber showed that most somatic hybrids had lower disease severity ratings than the B. rapa fusion partner and a commercial variety of B. napus. Some progeny from selfing or backcrossing of somatic hybrids to B. rapa showed much more resistance than either fusion partner. The offspring populations of the somatic hybrids (F1–S1 and F1–BC1) clearly moved to the resistant direction compared to the parents; the percentage of resistant plants increased from 21% (average of parents) to 36% (F1–S1) and 48% (F1–BC1). These results suggest that it may be possible to obtain highly resistant B. rapa lines by further backcrossing and selection.

Embryogenic Cell Culture, Protoplast Regeneration, Cryopreservation, Biolistic Gene Transfer and Plant Regeneration in Japanese Cedar (Cryptomeria japonica D. Don)

Abstract: Somatic embryogenesis in Cryptomeria japonica was initiated at a relatively high frequency from immature seeds collected from the end of June to mid-July. Induction of embryogenic cultures was possible on media with or without plant growth regulators, and the initiation frequency varied from 5 to 16%. Embryogenic cell lines have been maintained and proliferated for more than 2 years in solid and liquid media. For long-term storage embryogenic cells were cryopreserved using a simple freezing method. Cotyledonary embryos were obtained mostly on maturation media containing abscisic acid (ABA) and polyethylene glycol (PEG) as osmotic agent, however, the plant conversion rate was still low. Plants regenerated from somatic embryos continued growing in a greenhouse. Furthermore, a procedure for the individual culture of protoplasts isolates from embryonal masses, and an approach for microprojectile bombardment-mediated transformation using pIPT and pMAT vectors was also described.

A plasmolytic cycle: the fate of cytoskeletal elements

Abstract: In most plant cells, transfer to hypertonic solutions causes osmotic loss of water from the vacuole and detachment of the living protoplast from the cell wall (plasmolysis). This process is reversible and after removal of the plasmolytic solution, protoplasts can re-expand to their original size (deplasmolysis). We have investigated this phenomenon with special reference to cytoskeletal elements in onion inner epidermal cells. The main processes of plasmolysis seem to be membrane dependent because destabilization of cytoskeletal elements had only minor effects on plasmolysis speed and form. In most cells, the array of cortical microtubules is similar to that found in nonplasmolyzed states except that longitudinal patterns seen in some control cells were never observed in plasmolyzed protoplasts of onion inner epidermis. As soon as deplasmolysis starts, cortical microtubules become disrupted and only slowly regenerate to form an oblique array, similar to most nontreated cells. Actin microfilaments responded rapidly to the plasmolysis-induced deformation of the protoplast and adapted to its new form without marked changes in organization and structure. Both actin microfilaments and microtubules can be present in Hechtian strands, which, in plasmolyzed cells, connect the cell wall to the protoplast. Anticytoskeletal drugs did not affect the formation of Hechtian strands.

Somatic embryogenesis in the endemic black iris

Abstract: Somatic embryogenesis was achieved from callus, cell suspension and protoplast culture systems in the endemic black iris (Iris nigricans). Subculture of friable callus fragments on embryogenesis induction medium (EIM) containing 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 μM kinetin, 4.5 μM 1-naphthaleneacetic acid (NAA) and 300 mg l-1 proline in the dark was necessary before transfer to regeneration medium (RM). Regeneration was studied by transferring friable callus fragments from EIM to RM containing (0.0, 4.5, 9.0, 13.5 μM) of either 6-benzyladenine (BA), 2-isopentenyladenine (2iP), zeatin or thidiazuron (TDZ) in combination with 0.49 μM indole-3-butyric acid (IBA), 0.45 μM 2,4-D. Maximum embryogenesis was obtained at 4.5 μM BA while zeatin and TDZ were not effective and embryogenesis did not occur with these treatments. Sucrose at 0.2 M was more effective for embryogenesis when compared to glucose or fructose. Growing cells in suspension culture on EIM containing 4.5 μM 2,4-D in combination with 0.2 M sucrose for four weeks and transferring cells to RM (containing 4.5 μM BA) gave significant embryogenesis with maximum number of embryos (3568 embryos/g cells). Using 4.5 μM 2,4-D in protoplast culture was necessary for the best protoplast division and colony formation. In all experiments, embryos developed on RM were transferred to hormone-free medium (HFM) and 90% converted to rooted plantlets. Produced plantlets gave 95% survival ex vitro. Plantlets developed to whole plants in the greenhouse and flowered.

Protoplast preparation and regeneration from spores of the biocontrol fungus Pseudozyma flocculosa

Abstract: This paper describes a specific protocol for yielding and regenerating protoplasts from spores of the recently described powdery mildew biocontrol agent Pseudozyma flocculosa. With this protocol, a large quantity of protoplasts was obtained from β-mercaptoethanol-pretreated spores in 3-day-old cultures of P. flocculosa grown in YMPD. Enzymatic digestion was optimal with 0.5% Novozym 234 and 5% Glucanex prepared in 0.6 M KCl in 0.1 M citrate buffer. All liberated protoplasts fluoresced in the presence of fluorescein diacetate indicating that viability was nearly 100%. The regeneration rate was equally outstanding reaching 75% when 0.8 M sucrose was used as osmotic stabilizer in the regeneration medium. This protocol will find useful applications in genetic studies of this poorly characterized and understood biocontrol agent.

Fungal protoplast fusion – a revisit

Abstract: Protoplast fusion is a non-specific recombination technique used for transfer of cytosolic organelles including genetic material. The process involves cell wall breakdown, regeneration of protoplasts, chemofusion and electrofusion. This review article discusses all the stages involved in fusion of protoplasts and some of the applications of protoplast fusion technique in fungal systems.

Plant regeneration from protoplasts of mango (Mangifera indica L.) through somatic embryogenesis.

Abstract: This report describes a protocol for the regeneration of plants from protoplasts isolated from proembryogenic masses (PEMs) in a suspension culture derived from the nucellar callus of mango (Mangifera indica L. cv 'Amrapali'). The maximum yield (24.6±1.1×106), with 81.04±4.1% viable protoplasts per gram PEMs, was obtained with an enzyme mixture containing 1.2% cellulase, 1.0% hemicellulase and 0.6% pectinase. An optimum density of 5×104 cultured protoplasts per milliliter culture medium was required for the highest frequency (88.89±5.40%) of division. Dividing protoplasts developed into microcalli that proliferated on medium supplemented with growth regulators (auxins or kinetin alone, or auxins with kinetin) and produced somatic embryos after transfer to a growth regulator-free medium. The protocallus on 2,4-D-containing medium produced the maximum number (102.50±6.93) of somatic embryos. Maturation of somatic embryos depended upon the presence, and the nature and combination of growth regulators in the medium during proliferation of the callus. The mature somatic embryos germinated and developed into plants that were transferred to soil.

Characterization and molecular analysis of transgenic plants obtained by microprotoplast fusion in sunflower

Abstract: Asymmetric somatic hybrid (ASH) plants were obtained by PEG-mediated mass fusion of microprotoplasts from perennial Helianthus species and hypocotyl protoplasts of Helianthus annuus. The formation of micronuclei in perennial sunflower cell cultures was induced, at early log phase, by addition of the herbicides amiprophos-methyl or oryzalin. Sub-diploid microprotoplasts were isolated by high-speed centrifugation and the smallest enriched by sequential filtration through nylon sieves of decreasing pore size. Fusion products were cultured and the regenerated plants phenotypically, genetically and cytologically characterized. DNA analysis using RAPD markers revealed that 28 out of 53 regenerated plants were asymmetric hybrids. Subsequent nuclear-DNA flow cytometric analysis showed that these plants had a higher DNA content than the receptor H. annuus, suggesting that they represented addition lines. Cytological investigation of the metaphase cells of 16 hybrids revealed an addition of 2–8 extra chromosomes in these plants. The phenotype of most ASH plants resembled H. annuus. These results indicate that micronuclear induction and asymmetric somatic hybridization represent a potent tool for partial genome transfer aimed at the specific transfer of economically important traits in breeding programs.

Amylase hyper-producing haploid recombinant strains of Thermomyces lanuginosus obtained by intraspecific protoplast fusion.

Abstract: Amylase hyper-producing, catabolite-repression-resistant, recombinant strains were produced by intraspecific protoplast fusion of thermophilic fungus Thermomyces lanuginosus strains, using well-characterized, morphological, and 2-deoxy-D-glucose resistant markers. The fusant heterokaryons exhibited enhanced amylase activities as compared to the amylase hyper-producing parental strain (T2). Diploids derived from heterokaryons segregated to stable haploid recombinant strains. In the haploid strain (Tlh 4q), approximately 5-fold higher specific activities of alpha-amylase and glucoamylase in the culture filtrate were observed as compared to the wild-type strain (W0).

Somaclonal variation in transgenic plants.

Abstract: This paper summarises our experience of the molecular and field analysis of somaclonal variation within transgenic plant populations. Transgenic rice, poplar and sugarcane plants were produced with different transformation protocols. These were based on protoplast treatment, Agrobacterium tumefaciens infection, particle acceleration or intact cell electroporation, all having in common dedifferentiated cell culture, followed by plant regeneration and selection. Transgenic Arabidopsis thaliana plants have also been produced by an approach that apparently does not require cell dedifferentiation. This is based on in planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. The presence and activity of the foreign gene in the transgenic genonie has been verified by conventional methods (PCR, Southern. Northern and Western blotting). Genomic changes have been evaluated with PCR-based tools (RFLP. RAPD, AFLP, RAMP and AFRP). The statistical analysis of the polymorphic DNA bands produced by these tools provided the basis for answering some basic questions related to the conditions that favour induction, frequency, inheritance and commercial exploitation of transgenic somaclonal variants.

Genetic control of early events in protoplast division and regeneration pathways in sunflower

Abstract: Experiments were conducted to identify the genetic factors controlling protoplast division and to determine eventual relations between genetic factors involving organogenesis, somatic embryogenesis and protoplast division in sunflower. The present study involved protoplast culture and two traits: total division per 100 protoplasts (TOTD) and asymmetric division per 100 protoplasts (ASYD) were scored in 52 recombinant inbred lines (RILs) from a cross between PAC-2 and RHA-266. Asymmetric division is an early event in the formation of embryoids from protoplasts. Analysis of variance indicated the existence of highly significant differences among parental genotypes and their RILs. Heritability for the two protoplast division parameters (TOTD and ASYD) was high (0.87 and 0.89, respectively) and genetic gain expressed as percentage of the best parent for 10% of the selected RILs was significant. Twelve putative loci associated with total division per 100 protoplasts were identified. Eleven QTLs were also detected for asymmetric division per 100 protoplasts. The QTLs present high significant LOD scores and sum to a high percentage of phenotypic variance. The percentage of phenotypic variation explained by each QTL ranged from 2% to 24%. Some segments of the linkage groups I, XV and XVII are likely to contain genes important for organogenesis, somatic embryogenesis and protoplast division, as clustering of QTLs for these characters were described. The QTLs identified in these three linkage groups should be involved in cell division and in early events associated with cell differenciation.

Analysis of chromatin, nuclear DNA and organelle composition in somatic hybrids between Solanum tuberosum and Solanum sanctae-rosae

Abstract: A protoplast fusion strategy has been applied to advance aspects of a potato breeding programme. A sub-population of somatic hybrids, selected for agronomic potential, between tetraploid Solanum tuberosum cv. Brodick and a diploid EBN2 accession, S. sanctae-rosae was subjected to detailed molecular analysis. This study reports the use of simple sequence repeats (SSRs) to identify nuclear hybrid genomes and PCR and DNA-DNA analysis to determine organelle composition in somatic hybrids derived from these parents. SSR analysis revealed somatic hybrids containing the genetic background of S. tuberosum cv. Brodick with some specific markers from S. sanctae-rosae. One somatic hybrid contained the chloroplasts derived from S. sanctae-rosae, and several hybrids had detectable RFLP mitochrondrial DNA profiles, indicating genetic re-arrangements. We also examined the use of DNase I sensitivity to the genomic and ribosomal RNA sequences in these somatic hybrids as an indicator of changes in chromatin structure. Chromatin and DNAse I analysis showed differential sensitivity to increasing levels of nuclease; DNA from several somatic hybrids was found to be resistant to DNase I compared to the parental plants. The significance of the findings to somatic cell genetics and plant breeding studies is discussed.

Transformation of barley scutellum protoplasts: regeneration of fertile transgenic plants

Abstract: A system for barley transformation via polyethyleneglycol-mediated DNA uptake into protoplasts isolated directly from scutella and the regeneration of transgenic plants is reported. Scutellum protoplasts (cv. Clipper, an Australian malting cultivar) were co-transformed with plasmids Act 1-DGUS, containing the marker uidA gene, and pCaIneo, which contains the selectable marker neomycin phosphotransferase gene. Protoplast-derived calluses were selected on medium containing the antibiotic G418 (25 and 15 mg.l–1) and macroscopic antibiotic resistant colonies were recovered. Fertile plants were regenerated from a callus line and molecular analysis confirmed transgene integration.

Turning point article plant protoplasts

Abstract: What led me in 1960 to begin work to develop an enzymatic procedure for the isolation of plant protoplasts? The truth is that I initially had no idea that I wanted to isolate plant protoplasts! I arrived in 1959 at the University of Nottingham as a lecturer in plant physiology. I had already completed 3 yr as a Civil Service Commission postdoctoral fellow in bacterial chemistry, after receiving my Ph.D. in plant biochemistry from the University of Bristol in 1956 for studies on the synthesis of amino acids and proteins in barley. Two viewpoints had impacted on me from these 6 yr of research. The first was that the whole plant was too complex for detailed biochemical analysis and that experimentally it would be better to reduce it to the cell level and then reassemble it to relate biochemical studies to the whole plant. The second was an appreciation from studies with bacteria that single plant cells and small groups of cells would be much more amenable to biochemical analysis and cell biological studies. During this time, I had also been interested in the development of new analytical procedures and had experienced the way in which their introduction had opened up new research areas in the biological sciences. From the results of the first year of my Ph.D. I had published with my supervisor an improved procedure for the determination of amino acids with ninhydrin (Yemm and Cocking, 1955), which was to play a key role in the quantitative analysis of the amino acid composition of proteins. Also, by researching with bacteria, which produce results more quickly than plants, I had already published two significant papers and was keen to produce isolated cells from plants with the aspiration of developing a plant cell ‘bacterial’ type of culture system in which plant cells would divide, separate and produce a culture of single cells. The approach I adopted was to investigate the use of chelating agents to isolate cells from the rapidly elongating region of the roots of tomato seedlings. This established that cell separation was largely dependent on Ca in the middle lamella. However, although extensive cell separation was achieved, no division of the isolated tomato root cells was observed and it became evident that the chelating agents were negatively impacting on cell physiology and biochemistry. Chelating agents were therefore of little use; my thoughts turned to the possibility of breaking down not just the middle lamella but the cell wall itself in order to release the protoplast from within the cell wall. I had read about the isolation of bacterial and fungal protoplasts by the use of enzymes degrading their cell walls and, probably more importantly, I had had discussions with workers on bacterial protoplasts at the Microbiological Research Establishment, Porton when I had been a postdoctoral fellow. My training in plant biochemistry told me that I would have to use cellulases degrading cellulose rather than lysozyme. Also, I knew that it was possible to isolate plant protoplasts by physically breaking the cell wall, provided the protoplast had been plasmolyzed away from the cell wall. In my undergraduate practicals at the University of Bristol I had cut through pieces of plasmolized beetroot and observed the release of protoplasts from the ends of cut-through cells. As I have recounted, and illustrated with a picture of the first protoplasts isolated in May 1960 from tomato seedling root tips using Myrothecium verrucaria cellulase (Cocking, 1983), this led me to survey a wide range of commercially available cellulase preparations for their ability to isolate plant protoplasts. Seedling roots were chosen because material could thereby be readily obtained with cells at different known stages of differentiation, with minimal problems of penetration of the enzyme. Many commercially available enzyme preparations were tested, all without success. While this work was in progress, I read of the studies of D. R. Whitaker (National Research Laboratories, Ottawa) on the purification of cellulase from M. verrucaria. He generously provided me with a few grams of his enzyme preparation and, in his covering letter, dated 25 November 1959, said ‘In as much as the cellulose in cell walls tends to be highly crystalline, I should think its degradation would be a slow process—quite apart from accessibility factors due to other components of the wall acting as a physical barrier.’ Thinking that my idea of using cellulase might therefore be ruled out by such physiochemical factors, I put his sample at the bottom of the deep freeze. Only when everything else had failed did I test his preparation; it released protoplasts! (Cocking, 1960). Microscopically observing protoplasts being released from the rapidly elongating region of the roots and from the highly meristematic region was exciting, and it was possible to examine the inside of the protoplast with extra clarity because there was no cell wall. The more I looked at protoplasts the more I appreciated the indispensability and fundamental importance of the plasma membrane of the protoplast. In this respect it is interesting to recall that whether cells were inevitably bounded by a membrane was a question that arose because the solid walls that characterized plant cells could not with certainty be identified in animal cells. Also, as recalled by Henry Harris in his recent perceptive analysis and meticulous historiography of the cell doctrine (Harris, 1999), the distinction between the plant cell wall and the cytoplasmic membrane was only finally established by the classical plasmolysis

Fungal Protoplast: A Biotechnological Tool

Abstract: Isolation of Fungal Protoplasts of Filamentous Fungi Regeneration and Reversion of Protoplasts Protoplast Fusion Applications of Protoplast Fusion in Filamentous Fungi

Transfer of PLRV resistance from Solanum verrucosum Schlechdt to potato (S. tuberosum L.) by protoplast electrofusion.

Abstract: Somatic fusions between an accession of the diploid wild speciesSolanum verrucosum and a dihaploid S.tuberosum genotype were produced in order to incorporate resistance to potato leafroll virus (PLRV). In total 15 somatic hybrids out of 16 regenerants were obtained. Identification of hybrids was based on additive RAPD patterns, general morphological characteristics, chromosome numbers and chloroplast counts in stomata guard cells. A field trial was performed with the hybrids, their two parents and the control cultivar Kennebec to assess field performance and phenotypic variability. Yield parameters varied considerably among somatic hybrids. Some of the hybrids gave significantly higher yields, tuber numbers and tuber weights than both parents. Pollen fertility of hybrids ranged from 19 to 77%. Twelve hybrids were found to be resistant to PLRV.

Cryopreserved callus, a source of protoplasts for citrus improvement.

Abstract: SummaryThe availability and maintenance of embryogenic callus is a major limitation for large-scale application of somatic hybridization for citrus breeding. The suitability of cryopreserved callus as source of protoplasts was evaluated. Sweet orange callus were frozen by slow cooling and stored for two years in liquid nitrogen. Cryopreserved callus were fast thawed and used as source of material for protoplast isolation, protoplast fusion and plant regeneration, in comparison with control non-cryopreserved callus. No differences were found in protoplast yield, quality, growth and regeneration capacity between both callus types. Protoplasts isolated from cryopreserved callus were also successfully used in somatic fusion assays. Plants regenerated from protoplasts of the two sources had the same phenotypic characters and no differences were detected by microsatellite analysis. Availability of cryopreserved callus facilitates the development of breeding programmes based on somatic hybridization, avoiding th...

Protoplast isolation in lupin (Lupinus mutabilis Sweet): determination of optimum explant sources and isolation conditions.

Abstract: Effects of cultural factors on the yield, viability and division of protoplasts were investigated in Lupinus mutabilis Sweet containing a high protein content as well as a reasonable oil content which may make this species an alternative crop to soybean in Turkey. Explants from different in vitro seedling parts were evaluated on the suitability of protoplast isolation and viability. Leaf mesophyll was the most suitable tissue as a protoplast source. Pectinases as well as cellulases were essential for the isolation of protoplasts. Nine percent (w/v) mannitol was suitable to stabilise osmotic pressure together with low salt concentrations in washing and isolation solutions while 9% (w/v) glucose gave better results in culture medium. Shoot tip protoplasts exhibited a higher viability than other protoplast sources. Prolonged viability was observed when protoplasts were cultured in low density media. Techniques such as nurse cultures and electro-stimulation were ineffective. Sustained division of protoplasts in lupin (all sources) was not possible. However, techniques applied in this study may help other researchers, especially those studying protoplast culture of recalcitrant plant species, as well as further studies on this species.

Isolation of protoplasts from cardamom (Elettaria cardamomum Maton.) and ginger (Zingiber officinale Rose.)

Abstract: Protoplasts were isolated from leaf mesophyll tissue, collected from in vitro grown plantlets and cell suspension cultures of cardamom (Elettaria cardamomum) and ginger (Zingiber oJficinale). In cardamom, a protoplast yield of 3.5 x 105/ g of leaf tissue was obtained when incubated in an enzyme solution containing 0.5% macerozyme RIO, 2% cellulase Onozuka RIO and 9% mannitol for 18-20 h at 25°C in dark. The yield of protoplasts from cell suspension culture was 1.5 x 105 / g tissue, when incubated in I % macerozyme RIO and 2% cellulase Onozuka RIO for 24 h at 25°C with gentle shaking at 53 rpm in dark. The viability of leaf mesophyll protoplast was 75% and that of cell suspension was 40% on Evan's blue staining. In ginger, a protoplast yield of 2.5 x lOS / g of leaf tissue was obtained on digestion in an enzyme solution containing 0.5% macerozyme RIO, 3% hemicellulase and 5% cellulase Onozuka RIO, when incubated for 10 h at 15°C followed by 6 h at 30°C. The protoplast viability was 55%. Protoplast yield from cell suspension culture was 1 x lOS /g of callus when digested with an enzyme solution of I % macerozyme RIO, 3% hemicellulase and 6% cellulase Onozuka RIO and incubated for 10h at 15°C and later at 30°C for 8 h. Seventy two per cent of the protoplasts were viable. The protoplasts from both the species could be cultured and made to develop up to microcalli stage.

In vitro double fertilization in Nicotiana tabacum (L.): polygamy compared with selected single pair somatic protoplast and chloroplast fusions

Abstract: In vitro polygamy was studied mainly by using isolated sperm and central cells of tobacco in order to elucidate the mechanism that might be involved in preventing in vivo polygamy. In 17.5% 4000 M.W. polyethylene glycol, only when two sperm cells were made close enough to each other and adhered to a female cell simultaneously was polygamy possible. If one sperm cell fused with the egg or central cell, within 30 min another sperm cell could not fuse with the same egg or central cell. Similar phenomena were found in selected single somatic cell fusion. When more than two protoplasts adhered to each other simultaneously, fusion was always successful; after two protoplasts fused, within 30 min the fusion products could not fuse with another protoplast under the same conditions. This comparative study revealed this characteristic to be shared by both sexual and somatic cell fusion. However, after cytoplasm reorganization was complete in the fusion product, it was possible for the fusion product to fuse with the third protoplast. This indicates that the obstruction to additional fusion was present only during a certain period after the preceding fusion under certain condition. The possible reason for the effect is discussed.

Expression of green-fluorescent protein gene in sweet potato tissues

Abstract: Green-fluorescent protein (GFP) gene expression, transient and stable after electroporation and particle bombardment, was analyzed in tissues of sweet potato cv.Beauregard. Leaf and petiole tissues were used for protoplast isolation and electroporation. After 48 h, approximately 25–30% of electroporated mesophyll cell protoplasts regenerated cell walls, and of these, 3% expressed GFP. Stable expression of GFP after four weeks of culture was observed in 1.0% of the initial GFP positive cells. In a separate experiment, we observed 600–700 loci expressing GFP 48 h after bombarding leaf tissue or embryogenic calli, and stable GFP-expressing sectors were seen in leaf-derived embryogenic calli after four weeks of protoplast culture without selection. These results demonstrate GFP gene expression in sweet potato tissues. Screening for GFP gene expression may prove useful to improve transformation efficiency and to facilitate detection of transformed sweet potato plants.

Process of rapid variety-independent plant transformation

Abstract: Disclosed is a method of making transgenic plants. Heterologous DNA is first introduced into a donor plant, plant cell or protoplast to a plant cell or protoplast, and then moved from the donor to a recipient plant, plant cell or protoplast unaccompanied by any native genomic DNA of the donor. The donor and recipient are chosen that produce unstable progeny or demonstrate preferential segregation or sorting out. The DNA may be inserted randomly or at specific locations in the genome of the recipient plant. Also disclosed are transgenic plants produced by the methods, and plant progeny, plant parts and seeds and seed parts from the plants.