Literatura científica selecionada sobre o tema "Putative hybrid colonies"

Background The coral genus Acropora contains more than 150 species with very high morphological diversity. This high diversity may have been caused by repeated hybridization via mass spawning. However, we have little information whether hybrids are formed in these corals. Identifying morphological differences between hybrids and their parental species would provide an opportunity to find wild hybrids in the field and to understand how colony shapes of Acropora have become highly diversified throughout evolutionary history. In the two morphologically distinctive coral species Acropora florida and A. intermedia in the Indo-Pacific, their gametes show high rates of bi-directional intercrossing in vitro, and thus these two species are ideal species to investigate the morphological traits of the hybrids. Methods We examined morphological characters of F1 hybrids from A. florida to A. intermedia, which were produced from in vitro crossing experiments. To compare morphological differences, we grew juveniles and mature colonies of reciprocal F1 hybrids (FLOint: A. florida eggs × A. intermedia sperm, and INTflo: A. intermedia eggs × A. florida sperm) and of the parental species (purebreds of A. intermedia and A. florida). We analyzed skeletal morphology such as colony size, branch length, and branching number, and compared them with those of a putative F1 hybrid between A. florida and A. intermedia found in the field. We also confirmed the molecular phylogenetic position of F1 hybrids, parental species, and a putative F1 hybrid using the mitochondrial non-coding region. Results Our morphological analysis revealed that branching number of the F1 hybrids was intermediate relative to the parental species. Moreover, the FLOint hybrids were morphologically more closely related to the maternal species A. florida, and the INTflo hybrids were to A. intermedia. Molecular data showed that A. florida and A. intermedia were clearly divided into two clades, and that F1 hybrids grouped in the clade based on their maternal parent. A very similar pattern to the INTflo hybrids was obtained for the putative F1 hybrid in nature. Discussion Our results revealed that F1 hybrids between two Indo-Pacific species A. florida and A. intermedia had intermediate morphology relative to their parent species but reflected the maternal parent more. Similarity to maternal species in hybrids is opposite to the Caribbean Acropora species that had more paternal morphological characters in hybrids. These results further suggest that some genetic factor in eggs is likely to affect determination of colony shape in the Indo-Pacific. At present, we have considered colonies with intermediate morphs between different species to be intra-specific morphological variation, but they may be real F1 hybrids. Indeed, a putative F1 hybrid represented similar morphological and molecular features to the F1 hybrids, and thus it is plausible to be attributed as a “real” F1 hybrid in nature.

ABSTRACT A 3.6-kb endogenous plasmid was isolated from aPropionibacterium freudenreichii strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of anEscherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli andP. freudenreichii. Whereas electroporation of P. freudenreichii with vector DNA isolated from an E. coli transformant yielded 10 to 30 colonies per μg of DNA, use of vector DNA reisolated from a Propionibacteriumtransformant dramatically increased the efficiency of transformation (≥108 colonies per μg of DNA). It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of Propionibacterium with DNA ligation mixtures.

The effects of altered leader sequences on the secretion and localization of restrictocin expression in Aspergillus nidulans and Aspergillus niger were investigated. The region encoding the leader sequence of the Aspergillus resirictus restrictocin (res) gene was altered and variants were expressed under the glucoamylase (glaA) promoter in A. nidulans and A. niger. The entire restrictocin leader sequence was replaced by the glaA leader sequence in one variant. In another, the signal sequence of restrictocin was replaced with the glaA signal, leaving a hybrid with the putative restrictocin pro region in place of the glaA pro region. The putative pro region was deleted from the restrictocin leader of a third variant. Toxic effects, such as reduced transcript levels and cellular lysis, were minimal when restrictocin was expressed with the native leader sequence, but became more pronounced as the leader sequence was varied. These toxic effects were inversely proportional to the level of restrictocin secreted. In all transformed strains, restrictocin secretion appeared at the periphery of colonies and was observed to occur at the tips of hyphae. Localization of restrictocin to differentiated structures (conidiophores), as occurs in A. resirictus, was observed only in transformed strains containing the complete restrictocin leader sequence.Key words: localization, secretion, targeting, translocation, development.

ABSTRACT The oral biofilm community consists of >800 microbial species, among which Streptococcus mutans is considered a primary pathogen for dental caries. The genomic island TnSmu2 of S. mutans comprises >2% of the genome. In this study, we demonstrate that TnSmu2 harbors a gene cluster encoding nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and accessory proteins and regulators involved in nonribosomal peptide (NRP) and polyketide (PK) biosynthesis. Interestingly, the sequences of these genes and their genomic organizations and locations are highly divergent among different S. mutans strains, yet each TnSmu2 region encodes NRPS/PKS and accessory proteins. Mutagenesis of the structural genes and putative regulatory genes in strains UA159, UA140, and MT4653 resulted in colonies that were devoid of their yellow pigmentation (for strains UA140 and MT4653). In addition, these mutant strains also displayed retarded growth under aerobic conditions and in the presence of H2O2. High-performance liquid chromatography profiling of cell surface extracts identified unique peaks that were missing in the mutant strains, and partial characterization of the purified product from UA159 demonstrated that it is indeed a hybrid NRP/PK, as predicted. A genomic survey of 94 clinical S. mutans isolates suggests that the TnSmu2 gene cluster may be more prevalent than previously recognized.

Abstract The metalloprotease ADAMTS13 regulates the size of released von Willebrand factor (VWF) multimers by cleaving a peptide bond in the VWF A2 domain. Unlike the other ADAMTS family members, ADAMTS13 contains two C-terminal CUB domains, and mutations in this region have been found in patients with congenital thrombotic thrombocytopenic purpura (TTP). In vitro studies indicate that CUB domains are dispensable for ADAMTS13 catalytic activity under static conditions with denatured VWF substrate, but they may be crucial for recognition of native VWF under flow. CUB domains in many proteins mediate protein-protein interactions, and to identify potential binding partners we conducted yeast two-hybrid screening using MATCHMAKER GAL4 two-hybrid system 3 (Clontech Laboratories Inc., CA, USA). A cDNA fragment encoding both CUB domains was inserted into the pGBKT7 vector. The resulting GAL4BD-CUB fusion protein was used as bait to screen a human liver cDNA library. Both bait and library vectors were simultaneously introduced into Saccharomyces cerevisiae AH109 and double transformants were selected on minimal synthetic dropout media lacking Trp, Leu and His. A total of approximately 3 × 106 clones were screened. Putative positive colonies were re-streaked and tested for β-galactosidase activity. DNA fragments from positive colonies were sequenced and 26 proteins were identified, 10 of which were plasma proteins including factor XII (FXII), β2-glycoprotein I, and fibrinogen β chain. Interactions were further characterized with a binding assay employing purified ADAMTS13 immobilized on microtiter plates. The FXII-ADAMTS13 interaction was saturable and reversible, with Kd = 17 nM, which is significantly lower than the plasma concentration of FXII (∼ 375 nM). Deletion of CUB domains in ADAMTS13(ΔCUB) reduces its affinity toward FXII by ∼10-fold (Kd = 164 nM). Activated FXII (FXIIa) bound saturably and reversibly to ADAMTS13 with Kd = 19 nM. Binding of FXIIa to ADAMTS13(ΔCUB) was undetectable, suggesting that the FXIIa-ADAMTS13 interaction is more specific than the FXII-ADAMTS13 interaction. This interaction of FXIIa and ADAMTS13 was totally blocked by a heat and acid stable inhibitor in plasma, whereas the interaction of FXII and ADAMTS13 was decreased only 50% by plasma. These data suggest that FXII can bind either to ADAMTS13 CUB domains or with lower affinity to a distinct site that does not involve the CUB domains; FXIIa, a two-chain enzyme formed by a single cleavage of FXII, only retains the structure involved in high affinity binding to ADAMTS13 CUB domains. Fibrinogen and β2-glycoprotein 1 also bound to ADAMTS13 in vitro, but with much lower affinity. However, the plasma concentrations of both proteins (about 9 μM and 4 μM, respectively) are at least 10 times higher than their apparent Kd values. Whether these CUB domain-interacting proteins are important for the regulation of ADAMTS13 activity in vivo remains to be established.

ABSTRACT Mycoplasma gallisepticum, the cause of chronic respiratory infections in the avian host, possesses a family of M9/pMGA genes encoding an adhesin(s) associated with hemagglutination. Nucleotide sequences of M9/pMGA gene family members indicate extensive sequence similarity in the promoter regions of both the transcribed and silent genes. The mechanism that regulates M9/pMGA gene expression is unknown, but studies have revealed an apparent correlation between gene expression and the number of tandem GAA repeat motifs located upstream of the putative promoter. In this study, transposon Tn4001was used as a vector with the Escherichia coli lacZ gene as the reporter system to examine the role of the GAA repeats in M9/pMGA gene expression in M. gallisepticum. A 336-bp M9 gene fragment (containing the GAA repeat region, the promoter, and the translation start codon) was amplified by PCR, ligated with alacZ gene from E. coli, and inserted into the Tn4001-containing plasmid pISM2062. This construct was transformed into M. gallisepticum PG31. Transformants were filter cloned on agar supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) to monitor lacZ gene expression on the basis of blue/white color selection. Several cycles of filter cloning resulted in cell lineages in which lacZ gene expression alternated between the On and Off states in successive generations of progeny clones. The promoter regions of the M9-lacZ hybrid genes of individual progeny clones were amplified by PCR and sequenced. The only differences between the promoter regions of the blue and white colonies were in the number of GAA repeats. Clones that expressedlacZ had exactly 12 tandem copies of the GAA repeat. Clones that did not express lacZ invariably had either more than 12 (14 to 16) or fewer than 12 (5 to 11) GAA repeats. Southern analysis of M. gallisepticum chromosomal DNA confirmed that the phase-variable expression of the lacZ reporter gene was not caused by Tn4001 transposition. These data strongly indicate that changes in the length of the GAA repeat region are responsible for regulating M9/pMGA gene expression.

In October 2010, a Colletotrichum species was isolated from white Phalaenopsis flowers growing in a greenhouse in San Francisco, CA. This Phalaenopsis is a common commercial orchid hybrid generated mostly likely from Phalaenopsis amabilis and P. aphrodite. The white petals showed anthracnose-like lesions where necrotic tissue is surrounded by a ring of green tissue. The green halo tissues around the necrotic tissue contain functional chloroplasts. One-centimeter disks were cut around the necrotic sites and surface-sterilized with 95% ethanol and 0.6% sodium hypochlorite. The disks were placed on potato dextrose agar (PDA) medium to establish cultures. Pure cultures were obtained by subculturing hyphal tips onto fresh PDA plates. The generated colonies had white aerial mycelia and orange conidial mass. The color of the reverse colony varies between colorless and pale orange. Microscopic observations identified the conidia as cylindrical, straight, and rounded at both ends. In addition, the conidia were approximately 15.0 to 18.0 μm long and 5.0 to 6.5 μm in diameter. These observed morphological features suggested that these isolates possessed the same characteristics as previously described for Colletotrichum karstii, a species considered as part of the C. boninense species complex (1). Four putative independent Colletotrichum isolates were recovered (DED9596, DED9597, DED9598, and DED9599). To confirm the Colletotrichum isolates as the causative pathogen, healthy white Phalaenopsis flowers (five total) in a whole plant were sprayed with a conidial suspension (approximately 1.2 × 106 conidia/ml) of the isolates and incubated at 20°C and 100% relative humidity with cycles of 16 h light and 8 h of darkness. Approximately 1 ml of conidial suspension solution was used for each flower. The plants were watered regularly and flowers were sprayed with sterile double-distilled water daily. As negative controls, five flowers in a whole plant were sprayed with water. Fifteen to twenty days after inoculation, lesions started to form on the petals sprayed with the putative Colletotrichum isolates. All controls remained healthy. The Colletotrichum-inoculated flowers remained alive and did not die as a result of the infection. This same experiment was repeated and the same results were obtained. DNA was extracted from the necrotic regions of the petals infected by the pure cultures of the four isolates and used to sequence the 18S rRNA ITS (internal transcribed spacer) region. All four isolates gave identical ITS sequences. Analysis of the obtained representative sequences (GenBank Accession No. JQ277352) suggested that the isolated pathogen as C. karstii. Using the published ITS data for the C. boninense species complex (1), a phylogenetic tree was generated via the maximum likelihood method. This created tree places the isolates in the same group as C. karstii. This type of C. karstii infection in Phalaenopsis orchid petals was not documented in the U.S. before, although it has been reported in China and Thailand (2). To our knowledge, this is the first report of infection and green island formation caused by C. karstii on orchid flower in the United States. References: (1) Damm et al. Studies in Mycology 73:1, 2012. (2) Yang et al. Cryptogamie Mycologie 32:229, 2011.

Zoantharians are sessile marine invertebrates and colonial organisms possessing sexual and asexual reproductive ability. The zooxanthellate zoantharian genus Palythoa is widely distributed in coral reef ecosystems. In the Ryukyu Archipelago, Japan, sympatric Palythoa tuberculosa and P. mutuki are the dominant species of this genus in the intertidal zone. Previous phylogenetic analyses have shown that these two species are closely related, and additionally revealed a putative sympatric hybrid species (designated as Palythoa sp. yoron). In this study, we attempted to delineate Palythoa species boundaries and to clarify the relationships among these three groups plus another additional putative sympatric species (P. aff. mutuki) by multiple independent criteria. The morphology of these four lineages was clearly different; for example the number of tentacles was significantly different for each species group in all pairwise comparisons. From observations of gonadal development conducted in 2010 and 2011, P. sp. yoron and P. aff. mutuki appear to be reproductively isolated from P. tuberculosa. In the phylogenetic tree resulting from maximum likelihood analyses of the ITS-rDNA sequence alignment, P. tuberculosa and P. sp. yoron formed a very well supported monophyletic clade (NJ = 100%, ML = 95%, Bayes = 0.99). This study demonstrates that despite clear morphological and/or reproductive differences, P. tuberculosa and P. sp. yoron are phylogenetically entangled and closely related to each other, as are P. mutuki and P. aff. mutuki. Additionally, no single molecular marker was able to divide these four lineages into monophyletic clades by themselves, and a marker that has enough resolution to solve this molecular phylogenetic species complex is required. In summary, the morphological and reproductive results suggest these lineages are four separate species, and that incomplete genetic lineage sorting may prevent the accurate phylogenetic detection of distinct species with the DNA markers utilized in this study, demonstrating the value of morphological and reproductive data when examining closely related lineages.

The origin of the words $lsquo;cacao$rsquo; and $lsquo;chocolate$rsquo; and their use in the reconstruction of the early history of Mesoamerica, remain very controversial issues. Cambell and Kaufman (1976, American Antiquity 41:80–89), for example, proposed that the word $lsquo;cacao$rsquo; originated from Mixe–Zoque languages, thus possibly representing Olmec traditions. According to this argument, other Mesoamerican languages, including Nahuatl, borrowed the word as a symbol of prestige and Olmec influence. Other researchers claim the word $lsquo;chocolate$rsquo; represents a more recent neologism, a possible Maya–Nahuatl hybrid, due to the late appearance of the word in central Mexico's Colonial sources. We refute the putative Mixe–Zoque origin of $lsquo;cacao$rsquo; and provide linguistic evidence to propose that $lsquo;cacao,$rsquo; like $lsquo;chocolate,$rsquo; is a Uto-Aztecan term. Analysis of these words highlights general and particular evolutionary trends that originate from the Uto-Aztecan language family. In addition, we show that these two words were initially used as descriptive terms to refer to the shape of the plant's bean and the techniques of drink preparation. Etymological evidence verifies the use of a Mayan term for cacao as early as the Classic period (fourth century a.d.). This early appearance of the term in Mayan and the later diffusion of the Nahua word throughout all of Mesoamerica correlate with additional data to support the conclusion that Teotihuacanos spoke Nahuatl.

Maize (Zea mays L.), an important food and feed crop worldwide, can be infected by Fusarium pathogens that can contaminate grain with mycotoxins. From August to October in 2018 and 2019, a field survey for maize ear rot was conducted in 76 counties of Guizhou province. The incidence ranged from 3% to 15% at individual fields in different areas. A total of 175 diseased maize ears with similar symptoms, including kernels covered with white, pink or salmon-colored mold or exhibiting a white streaking (“starburst”) symptom, were collected from fields. Symptomatic kernels were surface-sterilized by soaking for 30 s in 70% alcohol and for another 2 min in 2% sodium hypochlorite solution, followed by five rinses with sterile water. Each kernel was cut into half and placed on potato dextrose agar (PDA). After incubation at 28 °C in the dark for 5 days, colonies displaying morphological characteristics of Fusarium were transferred to fresh PDA (Leslie and Summerell 2006). Single-sporing was conducted to purify the putative Fusarium colonies. A total of 120 isolates belonged to 16 Fusarium species were determined and F. meridionale was the dominant species. Five isolates from Huaxi district of Guiyang City were identified as F. miscanthi (Gams et al. 1999). Colonies on PDA were white and floccose, and pigmentation as viewed from the underside of the Petri dish was violet. The average growth rate was 7.5-8.0 mm/day at 28 °C in the dark. In cultures grown on PDA, 0-1-septate microconidia were produced in slimy heads. Microconidia were clavate to fusiform with a truncate base and a broadly rounded tip, 4.8-13.3 μm × 1.8-3.3 μm (n=110). In cultures grown on half-strength CMC broth (Xu et al. 2010), macroconidia were mostly 3-septate, almost straight for most of the length, with a slightly foot-shaped basal cell and curved apical cell that gradually tapered, 17.8-71.3 μm × 2.0-4.3 μm (n=78). The identity of the fungus was confirmed by sequence comparison of the partial translation elongation factor-1α (TEF-1α), RNA polymerase II subunit (RPB2), mitochondrial small subunit rDNA (mtSSU) and β-tubulin genes (Mirete et al. 2004; O’Donnell et al. 2010; O’Donnell and Cigelnik 1997). BLASTn searches of GenBank, using the partial TEF-1α (MN750829), RPB2 (MN750834), mtSSU (MT594104) and β-tubulin (MT584781) sequences of representative isolate GYHXB03 as the queries, revealed 99.84%, 99%, 100% and 100% sequence identity, respectively, to F. miscanthi NRRL 26231 accessions AF324331, JX171634, AF060371 and AF060384. Inoculum of isolate GYHXB03 was prepared (Xu et al. 2010), and a pathogenicity test was conducted on maize hybrid “Shundan7” and repeated twice. A 106/mL spore suspension (2 mL) or sterile water was injected into each of 8 maize ears through the silk channel at the blister stage of reproductive development in field (Duan et al. 2019). After three weeks, typical Fusarium kernel rot symptoms the same as those previously shown in the field was observed on all pathogen-inoculated plants, while the controls were asymptomatic. The pathogens re-isolated from two diseased kernels were identified as F. miscanthi based on morphology and TEF-1α and mtSSU analyses. F. miscanthi was first isolated from Miscanthus sinensis in Denmark (Gams et al. 1999), and also identified from M. × giganteus rhizomes in Belgium (Scauflaire et al. 2013). To our knowledge, this is the first report of F. miscanthi causing maize ear rot in China. This disease should be monitored in Guizhou due to its threat to maize production.

Protoplast fusion of Lolium perenne and Lotus corniculatus for gene introgression by Sanjeev V. Raikar Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×106 g-1FW was obtained when cell suspensions were used as the tissue source, with enzyme combination ‘A’ (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×106 g-1FW) of L. corniculatus was achieved from cotyledons also with enzyme combination ‘A’ (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm2 for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×10⁶ g⁻¹FW was obtained when cell suspensions were used as the tissue source, with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×10⁶ g⁻¹FW) of L. corniculatus was achieved from cotyledons also with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm² for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.