Showing papers on "Protoplast published in 2007"

Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis.

Abstract: The transient gene expression system using Arabidopsis mesophyll protoplasts has proven an important and versatile tool for conducting cell-based experiments using molecular, cellular, biochemical, genetic, genomic and proteomic approaches to analyze the functions of diverse signaling pathways and cellular machineries. A well-established protocol that has been extensively tested and applied in numerous experiments is presented here. The method includes protoplast isolation, PEG-calcium transfection of plasmid DNA and protoplast culture. Physiological responses and high-throughput capability enable facile and cost-effective explorations as well as hypothesis-driven tests. The protoplast isolation and DNA transfection procedures take 6-8 h, and the results can be obtained in 2-24 h. The cell system offers reliable guidelines for further comprehensive analysis of complex regulatory mechanisms in whole-plant physiology, immunity, growth and development.

Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells

Abstract: Transient expression of fluorescent fusion proteins in plant cells has dramatically facilitated our study of newly identified genes and proteins. This protocol details an in vivo transient expression system to study the subcellular localization and dynamic associations of plant proteins using protoplasts freshly prepared from Arabidopsis or tobacco BY-2 suspension cultured cells. The method relies on the transformation of DNA constructs into protoplasts via electroporation. The whole protocol is comprised of three major stages: protoplast generation and purification, transformation of DNA into protoplasts via electroporation and incubation of protoplasts for protein analysis. Similar to stably transformed cell lines, transformed protoplasts are compatible with protein localization studies, pharmaceutical drug treatment and western blot analysis. This protocol can be completed within 11-24 h from protoplast production to protein detection.

Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts.

Abstract: Vacuoles are very prominent compartments within plant cells, and understanding of their function relies on knowledge of their content. Here, we present a simple vacuole purification protocol that was successfully used for large-scale isolation of vacuoles, free of significant contamination from other endomembrane compartments. This method is based on osmotic and thermal disruption of mesophyl-derived Arabidopsis protoplasts, followed by a density gradient fractionation of the cellular content. The whole procedure, including protoplast isolation, takes approximately 6 h.

Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato.

Abstract: A tomato (Lycopersicon esculentum Mill.) glycerol-3-phosphate acyltransferase gene (LeGPAT) was isolated. The deduced amino acid sequence revealed that LeGPAT contained four acyltransferase domains, showing high identities with GPAT in other plant species. A GFP fusion protein of LeGPAT was targeted to chloroplast in cowpea mesophyll protoplast. RNA gel blot showed that the mRNA accumulation of LeGPAT in the wild type (WT) was induced by chilling temperature. Higher expression levels were observed when tomato leaves were exposed to 4°C for 4 h. RNA gel and western blot analysis confirmed that the sense gene LeGPAT was transferred into the tomato genome and overexpressed under the control of 35S-CaMV. Although tomato is classified as a chilling-sensitive plant, LeGPAT exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT increased total activity of LeGPAT and cis-unsaturated fatty acids in PG in thylakoid membrane. Chilling treatment induced less ion leakage from the transgenic plants than from the WT. The photosynthetic rate and the maximal photochemical efficiency of PS II (Fv/Fm) in transgenic plants decreased more slowly during chilling stress and recovered faster than in WT under optimal conditions. The oxidizable P700 in both WT and transgenic plants decreased obviously at chilling temperature under low irradiance, but the oxidizable P700 recovered faster in transgenic plants than in the WT. These results indicate that overexpression of LeGPAT increased the levels of PG cis-unsaturated fatty acids in thylakoid membrane, which was beneficial for the recovery of chilling-induced PS I photoinhibition in tomato.

Effect of enzyme concentrations on protoplast isolation and protoplast culture of Spathiphyllum and Anthurium

Abstract: Vital protoplasts from Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were isolated from both somatic embryos and leaves. The highest yields were obtained when 1.5% cellulase, 0.5% macerase and 0.5% driselase were used for Spathiphyllum wallisii leaves and 0.5% cellulase, 0.3% macerase and 0.5% driselase for Anthurium scherzerianum embryos. About 1 × 106 protoplasts g−1 and 1 × 105 protoplasts g−1 could be isolated from leaves and embryos, respectively. For protoplast fusion Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were mixed in a 1:1 ratio in a fusion solution containing 1 mM CaCl2·2H2O, 1 mM MES and 0.5 M mannitol. Fusion was performed by protoplast alignment under 500 V cm−1 alternating current for 60 s and subsequent generation of two pulses of 4500 V cm−1 direct current during 50 μs. Development until colony stage was achieved using agarose beads for protoplast culture.

Interspecific potato somatic hybrids between Solanum berthaultii and Solanum tuberosum L. showed recombinant plastome and improved tolerance to salinity

Abstract: In this study three somatic hybrid lines originating from protoplast fusion between Solanum tuberosum cv. BF15 and Solanum berthaultii were subjected to a detailed molecular analysis using the I-SSR-PCR technique based on 5′-anchored microsatellite primers. The data obtained revealed a polymorphism between the different lines, suggesting that they correspond to symmetric hybrids. The analysis of chloroplast genome of these hybrids showed that they are resulting from a recombination between parental plastomes. When transferred to a greenhouse, these hybrid lines displayed an improved vigour compared to the cultivated potato BF15 parent. Indeed, an important growth rate and high tuber yield and weight were obtained for these hybrids compared to the parent. Some of these hybrids showed also an improved ion homeostasis control and they seem to display a better tolerance to salt stress compared to the potato BF15 parent.

Introduction of Xa21 ,a Xanthomonas-resistance gene from rice, into 'Hamlin' sweet orange (Citrus sinensis (L.) Osbeck) using protoplast- GFP co-transformation or single plasmid transformation

Abstract: SummaryPlasmid DNA (pARS108) containing the non-destructive selectable marker Green Fluorescent Protein (GFP) gene, and a plasmid containing a cDNA of the Xa21 gene from rice (pXa21-mtaq) were co-transformed into ‘Hamlin’ orange protoplasts using polyethylene glycol (PEG). Alternatively, plasmid DNA (pAO3), containing both genes (GFP and Xa21) was directly transformed into ‘Hamlin’ orange protoplasts. Over 1,000 transgenic plantlets were regenerated from approx. 80 independent transformation events. The transgenic plants showed normal growth and stable GFP expression over more than 2 years in the greenhouse. This is the first report of a large population of transgenic ‘Hamlin’ sweet orange plants containing one or more target gene(s), using a protoplast-GFP transformation system. Polymerase chain reaction (PCR) revealed the presence of the Xa21 cDNA and the GFP genes in all single plasmid transformed plants, and in 35% of the co-transformed plants. Southern blot analysis showed the integration of the cDNA...

The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of Gentiana kurroo (Royle)

Abstract: Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH4NO3 was replaced with 3.0 g l−1 glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l−1 2,4-D + 1.0 mg l−1 kinetin or 2.0 mg l−1 BAP + 1.0 mg l−1 dicamba + 0.1 mg l−1 NAA + 80 mg l−1 adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l−1 2,4-D + 1.0 mg l−1 kinetin or 1.0 mg l−1 kinetin + 0.5 mg l−1 GA3 + 80.0 mg l−1 adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to half-strength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.

Production and Evaluation of Somatic Hybrids Derived from Monoploid Potato

Abstract: Biotechnological approaches to hasten hybrid plant breeding are available, including anther culture to halve the genome and protoplast fusion to create hybrids between incompatible partners. We applied these techniques to potato to evaluate their potential for breeding a highly heterozygous, cross-pollinating species. Four families of monoploids (2n=1x=12), developed from diploid hybrids with various genomic constitutions favoringSolarium phureja, an ancestral cultivated potato subsequently selected for tuberization under long photoperiod, were used in electrofusion experiments to create intermonoploid somatic hybrids. The “monoploid sieve” results in the survival of only those gametes free of lethal and deleterious genes, but it generates sterile sporophytes, necessitating protoplast fusion for developing hybrids from differentially derived monoploids. From six intermonoploid electrofusion combinations, 178 plants, including both somaclones and somatic hybrids confirmed by microsatellite analysis, were regenerated over six to nine months. Fusion conditions of 8 V for 45 sec with two pulses and 12 V for 30 sec and one pulse were optimum. Ploidy of the fusion products as determined by flow cytometry ranged from 2x to 8x with only somaclones at the 2x level and both 4x and 6x somatic hybrids. Field evaluations over three years revealed that intermonoploid somatic hybrids were inferior toSolarium tuberosum cultivars. Dihaploids derived by anther culture of a tetraploid intermonoploid somatic hybrid were reduced in vigor with the increase in homozygosity, while 2x X 2x sexually derived populations were similar in yield to the somatic hybrids. The biotechnological breeding scheme to reduce generation time will require selection for horticultural traits during inbreeding in order to produce potatoes more similar to cultivars. The elimination of epistatic and/or intra-allelic interactions through reduc-tion of ploidy may underlie the poor performance of monoploids, doubled monoploids and dihaploids as well as most somatic hybrids compared to conventionally bred potato germplasm.

Arabinogalactan-proteins stimulate the organogenesis of guard cell protoplasts-derived callus in sugar beet

Abstract: Arabinogalactan proteins (AGPs) represent a class of proteoglycans implicated in the development and differentiation of cells and tissues both in planta and in vitro. Here we report that AGP-rich extracts isolated from media of embryogenic and non-embryogenic suspension cultures of sugar beet (Beta vulgaris L.) are able to enhance the organogenesis of guard protoplast-derived callus and to increase the number of shoots formed, in comparison to control cultures. Immunocytochemical detection of carbohydrate antigens in the extracts revealed the presence of epitopes that typify both AGP and pectin, the latter being frequently bound to AGPs or, in some cases, even contributing to the polysaccharide structure of proteoglycan molecules. The most abundant epitopes proved to be those recognized by the JIM13, LM2, and MAC207 antibodies, whereas some others could be found only in relatively small or trace amounts—these included epitopes recognized by JIM16, JIM5, and LM6. Surprisingly, the JIM4- and JIM8-binding epitopes that are expressed in the course of in vitro morphogenetic processes of many species could not be detected at all in sugar beet AGPs. This is the first report of the improvement of sugar beet protoplast-derived callus organogenesis by exogenous AGP-rich extracts, an achievement that will have great impact on the biotechnological applications of protoplast technology in this species.

Plant regeneration from leaf protoplasts of Solanum virginianum L. (Solanaceae)

Abstract: Leaves of Solanum virginianum plants were used for protoplast isolation. To support cell wall formation and cell division, protoplasts were cultured in thin alginate layers floated in liquid medium. When protoplasts were plated at a density of 1.0 × 106/ml in Kao and Michyaluk (KMp8) medium supplemented with 0.5 mg/l zeatin, 1.0 mg/l 2,4-dichlorophenoxyacetic acid, and 1.0 mg/l α-naphthaleneacetic acid, 42.3% of the dividing cells developed microcalli in 3–4 weeks. Shoot formation via organogenesis of protoplast-derived calli was achieved for 28% of calli transferred to solidified KMp8 medium supplemented with 2.0 g/l zeatin and 0.1 mg/l 3-indol acetic acid in about 2 weeks. Further shoot development was observed in Murashige and Skoog (MS) medium without growth regulators and roots were induced after transfer to MS medium containing 1.0 mg/l 3-indol butyric acid. Regenerated plants have normal morphology.

A proteome study of the proliferation of cultured Medicago truncatula protoplasts.

Abstract: A proteome study of the first five days of Medicago truncatula protoplast cultures was done to investigate molecular changes taking place during protoplast proliferation. A total of 1556 protein spots were analysed, of which 886 protein spots showed significant (p<0.005) changes in abundance at some time during the first five days of protoplast culture. Of the 886 significantly changing protein spots, 89 proteins were identified by MALDI-TOF MS. The majority of the identified proteins were part of four main cellular processes that may be involved in protoplast proliferation: energy metabolism, defence or stress response, secondary metabolism and protein synthesis and folding. The accumulation pattern of these proteins indicates extensive changes in the energy metabolism of the cells, accompanied by the activation of stress response pathways and modifications of the cell wall. In addition, seven PR10-like (pathogenesis related) proteins were identified. The accumulation pattern of these seven PR10-like proteins suggests that they could have a developmental role during protoplast proliferation.

Genotyping of somatic hybrids between Festuca arundinacea Schreb. and Triticum aestivum L.

Abstract: In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.

Involvement of arabinogalactan proteins in the regeneration process of cultured protoplasts of Marchantia polymorpha

Abstract: Protoplasts of Marchantia polymorpha L. (liverwort) regenerated new cell walls in initial culture. However, the survival rate of regenerated cells decreased rapidly after this stage. The decrease in survival rate was suppressed by the β-glucosyl Yariv reagent (pglcY), which binds to arabinogalactan proteins (AGPs), only when it was added to culture medium during the period of incipient cell wall regeneration. The addition of βglcY after the period of incipient cell wall regeneration had no effect on the survival rate. These results suggested the involvement of AGPs in the cell wall regeneration process. After cell wall regeneration, the regenerated cells started to divide actively after being transferred to a medium with 1% activated charcoal (AC). Protoplasts that had been cultured with βglcY during the period of incipient cell wall regeneration and then transferred to the AC medium divided vigorously, and the cell division rate was remarkably increased (>80%). However, without transfer to the AC medium, βglcY at concentrations higher than 20 μg ml -1 inhibited cell division. No effect on cell survival nor cell division was observed with the α-galactosyl Yariv reagent. Staining of p-1,3-glucan (callose) with aniline blue (AB) showed that a large amount of β-1,3-glucan was deposited in the regenerated cell walls of the protoplasts cultured without βglcY, while little or no β-1,3-glucan was stained by AB in protoplasts cultured with βglcY. These results suggest that AGPs and β-1,3-glucan play important roles in the survival and subsequent cell division of regenerated cells of M. polymorpha protoplast cultures.

Tetraploid somatic hybrids of potato ( Solanum tuberosum L.) obtained from diploid breeding lines

Abstract: Intraspecific somatic hybrids between 16 different diploid breeding lines of Solanum tuberosum L. were produced by PEG-induced fusion. Manually selected heterokaryons were cultured in a Millicells-CM using a post-fusion protoplast mixture. Plants were regenerated from calli derived from heterokaryons obtained from 10 out of 38 combinations of diploid lines. Of the tested putative somatic hybrids, 14.2% were diploid, 72.8% were tetraploid and 13% pentaploid. The DNA amplification pattern obtained with RAPD or semi-random primers confirmed that 6 fusion combinations were hybrids. In most cases, the morphological traits were intermediate to those of the diploid fusion partners. About 23.0% of the tested somatic hybrids showed variation in their morphology. Of the tested somatic hybrids, 78.0% flowered and 86.0% tuberized. The cytoplasm of 9 diploid lines and 6 somatic hybrid combinations was analysed. Two of the diploid lines had W/S chloroplasts and α or e mitochondria; the remainder contained T chloroplasts and β mitochondria. All the analysed somatic hybrids carried T chloroplasts and β mitochondria.

Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis.

Abstract: Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0–7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.

Production and characterization of asymmetric hybrids between upland cotton Coker 201 (Gossypium hirsutum) and wild cotton (G. klozschianum Anderss)

Abstract: Asymmetric somatic hybrids were obtained between Gossypium hirsutum Coker 201 and wild cotton G. klozschianum Anderss. An investigation on the effect of ultraviolet (UV) irradiation on donor protoplasts was carried out, and the lethal dose was determined to be 38.7 J cm−2. We firstly screened the putative hybrids by the color of the calli produced, followed by morphological, cytological, and molecular analysis of putative hybrid plants. Most regenerated plants derived from fused protoplasts displayed a recipient-like morphology, while some showed an intermediate phenotype between Coker 201 and G. klozschianum. Chromosome numbers in these somatic hybrids ranged from 54 to 74. The hybrids were verified by random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR). Absence or co-existence of parents’ genome DNA fragments was identified through molecular analysis. The heredity of cytoplasm was investigated by cleaved amplified polymorphic sequence (CAPS) analysis using mitochondrial and chloroplast universal primer pairs. The results indicated that recombination and rearrangements might have occurred in some regions of mitochondria (mt) and chloroplast (cp) DNA. To our knowledge, this is the first report about asymmetric protoplast fusion in cotton, and the hybrids obtained would be useful for breeding programs.

Measuring the osmotic water permeability of the plant protoplast plasma membrane: implication of the nonosmotic volume.

Abstract: Starting from the original theoretical descriptions of osmotically induced water volume flow in membrane systems, a convenient procedure to determine the osmotic water permeability coefficient (Pos) and the relative nonosmotic volume (β) of individual protoplasts is presented. Measurements performed on protoplasts prepared from pollen grains and pollen tubes of Lilium longiflorum cv. Thunb. and from mesophyll cells of Nicotiana tabacum L. and Arabidopsis thaliana revealed low values for the osmotic water permeability coefficient in the range 5–20 μm · s−1 with significant differences in Pos, depending on whether β is considered or not. The value of β was determined using two different methods: by interpolation from Boyle-van’t Hoff plots or by fitting a solution of the theoretical equation for water volume flow to the whole volume transients measured during osmotic swelling. The values determined with the second method were less affected by the heterogeneity of the protoplast samples and were around 30% of the respective isoosmotic protoplast volume. It is therefore important to consider nonosmotic volume in the calculation of Pos as plant protoplasts behave as nonideal osmometers.

Protoplast Isolation, Salt Stress and Callus Formation of Two Date Palm Genotypes

Abstract: This study reports for the first time protoplast isolation, salt stress and c allus formation from protoplasts in date palm (Phoenix dactylifera L.). Protoplasts were isolated from young leaves of offshoots in B arhee and Zaghloul genotypes. The p rotoplast yield depended on genotype, several combinations and concentrations of hydrolytic enzymes, incubation t ime, different stainless steel s ieves, salt stress and different agarose concentrations were used. The optimum condition for protoplast isolation was established by using 4% cellulase, 1% hemicellulase and 2% pectinase, incubate f or t wo days in t he d ark. Maximum protoplast yields we re 6 .40 x10 per g ram in B arhee. The best yield 5.80 x10 and viability of protoplast 5 5

Generation of intracellular reactive oxygen species during the isolation of Brassica napus leaf protoplasts

Abstract: The isolation of leaf protoplasts is a stress-inducing procedure that generates reactive oxygen species (ROS). We found that a cellulase-pectinase enzyme solution used for isolating protoplasts contained peroxidase (POX), catalase (CAT), and superoxide dismutase (SOD) activity, and effectively scavenged externally added hydrogen peroxides. During the isolation of Brassica napus leaf protoplasts, the protoplasts accumulated 1.5 times more intracellular H2O2 than normal leaf cells, but the H2O2 released into the medium increased only negligibly during culture. H2DCF-DA and DAF2-DA were used as probes for the intracellular localization of H2O2 and NO with a confocal laser scanning system. H2O2 and NO had different intracellular localizations in freshly prepared B. napus leaf protoplasts. These results indicate that intracellular ROS are generated by the stresses of protoplast isolation and will induce the apoptosis-like cell death of the cultured protoplasts.

Biological Control Agent for Rice Weeds from Protoplast Fusion between Curvularia lunata and Helminthosporium gramineum

Abstract: The advancement of biological weed control is limited by the slow development of effective, broader-spectrum biological control agents. Protoplast fusion was carried out between the Helminthosporium gramineum subsp. echinochloae (HGE) strain, HM1, and Curvularia lunata (CL) to breed new strains with improved biocontrol efficiency. The HM1 strain was derived from HGE by ultraviolet (UV) treatment. Conditions for protoplast fusion were optimized, including lytic enzyme mixtures and incubation time. The efficacy of lytic enzyme mixtures on cell wall digestion was also compared. The most effective lytic enzyme mixture for CL was 2% lywallzyme plus 2% snailase and for HM1, 2% cellulase plus 2% snailase. The optimum incubation time was 16 h for CL and 24 h for HM1. All fusant strains exhibited similar morphological and conidial properties to the HM1 parent. A total of 1,360 fusant strains were produced, 136 of which were randomly selected for characterization. Seven fusant strains showed improved spore...

Novel Method for Preparing Spheroplasts from Cells with an Internal Cellulosic Cell Wall

Abstract: Protoplast and spheroplast preparations allow the transfer of macromolecules into cells and provide the basis for the generation of engineered organisms. Crypthecodinium cohnii cells harvested from polyethylene glycol-containing agar plates possessed significantly lower levels of cellulose in their cortical layers, which facilitated the delivery of fluorescence-labeled oligonucleotides into these cells.

Plant regeneration from Gossypium davidsonii protoplasts via somatic embryogenesis

Abstract: Protoplasts isolated from wild cotton Gossypium davidsonii were cultured in KM8P medium supplemented with different phytohormones. The most effective combination was 0.45 µM 2,4-dichlorophenoxyacetic acid, 2.68 µM α-naphthaleneacetic acid and 0.93 µM kinetin and the division percentage at the 8th day was 30.78 ± 3.04 %. The density of protoplasts at 2–10 × 105 cm−3 was suitable for protoplast division and calli formation, with a division percentage of 32.21 ± 3.64 % and a plating efficiency of 9.12 ± 2.61 % at the 40th day. The optimal osmotic potential was achieved using 0.5 M glucose or 0.1 M glucose plus 0.5 M mannitol. Protoplasts were cultured in three ways, a double-layer culture system, with liquid over solid medium was proved to be the best way. Embryo induction was further increased by addition of 0.14 µM gibberellic acid.

Wild relatives and biotechnological approaches

Abstract: Wild species of the genus Lens are an important source of genetic variation for breeding lentil varieties adaptable to new environments and tolerant of biotic and abiotic stresses The wild species are endemic to a wide range of environments and possess many diverse characteristics Lens species can be divided into three groups, a primary, secondary and tertiary gene pool, according to their inter-crossability Crosses between members of the different genepools generally fail because the hybrid embryos abort However, embryo rescue has been used successfully to obtain viable hybrids between groups It is possible to intercross most of the wild Lens species with cultivated lentils using plant growth regulators and/or embryo rescue to allow the growth of hybrid plants Other biotechnology techniques which may impact on lentil breeding include, micropropagation using meristamatic explants, callus culture and regeneration, protoplast culture and doubled haploid production Micropropagation and regeneration from callus culture are relatively well established techniques with further research required for the development of reliable protoplast regeneration and doubled haploid protocols

Protoplast Technology and Citrus Improvement

Abstract: Citrus is one most important fruit crop in China and worldwide. Protoplast fusion has been an effective and successful technique for citrus improvement by circumventing reproductive barriers such as nucellar polyembryony, pollen/ovule sterility, sexual incompatibility and long juvenility encountered in conventional breeding. Protoplasts isolated from embryogenic callus line, as source material are convenient and available at any time for genetic transformation of seedless citrus cultivars since most commercial cultivars are seedless, and routinely used epicotyl seedling segments are only available for seedy cultivars. Strategies and recent progress by protoplast fusion and protoplast transformation in our citrus improvement program will be provided.