Interspecific somatic hybrids between Solanum bulbocastanum and S. tuberosum and their haploidization for potato breeding.

Protoplast fusion between incongruent Solanum bulbocastanum and S. tuberosum haploids was accomplished to produce hybrids combining elite traits from both parents. We identified 11 somatic hybrids out of 42 regenerants analyzed through ISSR markers. Some hybrids had loss or gain of fragments compared to the parents, likely due to rearrangements and deletions of chromosome segments after fusion, and/or to somaclonal variation during hybrid regeneration. Increased heterotic vigor for some traits as well as high diversity was observed as the effect of both ploidy and fusion combination. Microsporogenesis analysis indicated the occurrence of multivalent configurations and several meiotic abnormalities, such as chromosomes bridges and various spindle orientations. Since all hybrids were sterile, in vitro anther culture was employed for haploidization as a possible strategy to overcome barriers to hybridizations. Haploids were obtained from all the tetraploid S. bulbocastanum (+) S. tuberosum somatic hybrids tested, although with differences in both the number of embryos per 100 anthers cultured and the number of differentiated green plantlets. This is the first report on the successful production of haploid plants from S. bulbocastanum (+) S. tuberosum hybrids

KARAKTERISTIK MINYAK NILAM HASIL FUSI PROTOPLAS ANTARA NILAM ACEH DENGAN NILAM JAWA

Oil characterization of somatic hybrids resulted from protoplast fusion between Aceh patchouli (Pogostemon cablin Benth.) Sidikalang and TT 75 and Java patchouli(Pogostemon heyneanus Benth.) Girilaya, was conducted at the laboratory of Indonesian Spices and Medicinal Crops Research Institute (ISMECRI) in March, 2003. The results showed that out of 13 somatic hybrids, two hybrids namely 9 II 8 and 9 II 21 (somatic hybrids between Girilaya and TT 75) have oil content (2,91% and 2,74% respectively), higher than that of parent TT 75 (2,09%). All somatic hybrids have higher than 30% patchouli alcohol content (minimum level of export quality). The number 9 II 21 showed the highest patchouli content (39,11%).

Extinction of gene expression in somatic cell hybrids. a reflection of important regulatory mechanisms?

Extinction in somatic cell hybrids is a multifactorial process that leads to loss of cell-type-specific gene expression. The underlying mechanisms are thought to mirror, at least in part, the repertoire of regulatory mechanisms controlling mammalian cell differentiation

Analysis of cytosine methylation in genomic dna of solanum × michoacanum (+) s. tuberosum somatic hybrids

Interspecific somatic hybridization is a noteworthy breeding strategy that allows the production of novel genetic variability when crossing barriers exist between two parental species. Although the genetic consequences of somatic hybridization have been well documented, little is known on its impact at the epigenetic level. The objective of our research was to investigate the epigenetic changes, in particular DNA methylation, occurring in a population of potato somatic hybrids. The analysis of 96 Solanum × michoacanum (+) S. tuberosum somatic hybrids from five fusion combinations and their parents was carried out by methylation-sensitive amplified polymorphism (MSAP) and high-performance liquid chromatography (HPLC) methods. Six MSAP primer combinations generated 622 unique bands, of which 295 were fully methylated. HPLC analysis showed from 15.5% to 16.9% total cytosine methylation within the parental forms. Overall, the MSAP and HPLC methods indicated an increase in DNA methylation in the somatic hybrids in comparison to their parents. Among the latter, a lower degree of DNA methylation in the wild S. × michoacanum species than S. tuberosum was found. Our findings indicated that somatic hybridization changed the level of cytosine methylation in the studied potato somatic hybrids

Impact of Transcription Factors on Fibroblast Specific Gene Silencing in Somatic Cell Hybrids

As totipotent cells differentiate to specific cell types in multicellular organisms, certain sets of genes are turned off (gene silencing/gene extinction) while others are turned on (gene activation). Cell type specific gene silencing and gene activation is the basis of cell differentiation. Somatic cell hybrids which are produced by fusing different types of somatic cells from two different tissues have been used as a valuable resource to discern the phenomenon of lineage- specific gene extinction. Identification of regulatory factors that mediate gene extinction in hybrids are key to understanding regulatory mechanisms that govern cell differentiation in a multicellular organism. Transcription factors (TFs) have been implicated as powerful activators and/or repressors of gene expression in mammalian cells. In our prior work on hepatoma-specific gene extinction we used a hepatoma X fibroblast somatic cell hybrid model to explore liver specific gene silencing and confirmed that loss of hepatoma phenotype was in large part a consequence of loss of hepatoma-specific TFs. Based on these prior findings we hypothesized that loss of fibroblast phenotype in somatic hybrids may be in part due to loss of fibroblast-specific TFs. Using a hepatoma X fibroblast somatic cell model, we subjected the parental hepatoma cells (FT02B), fibroblast cells (RA T-2) and somatic cell hybrids FR(2) to whole genome transcriptional microarray profiling and assigned a fivefold or more increase in expression of a gene as a selection criterion to identify fibroblast-enriched genes. We identified candidate fibroblast-specific genes from among the fibroblast-enriched genes by their decrease in fold expression in hybrids and validated the repression of the putative gene candidates (fibroblast-specific TFs like Prrx1, Snai2 and Shox2, signaling proteins like Bmp3, Opn, Co11a1 and Sema3a etc.) in somatic cell hybrids using q-RT-PCR. Our findings confirm that loss of these fibroblast-lineage specific TFs in hybrids likely affects fibroblast transcriptional regulatory networks (TRN\u27s) and contributes towards the loss of fibroblast phenotype in somatic cell hybrids. We also report that ectopic overexpression of our candidate TFs Prxx1 and Snai2 in somatic cell hybrid leads to induction of fibroblast-specific cell reprograming in the somatic hybrids and significant restoration of fibroblast-specific traits. Forced expression of Prrx1 in FR(2) hybrid cells and selection of clones that had at least five fold or more expression of Prrx1 than the parental hybrid demonstrated a significant change in expression of other key fibroblast associated TFs such as Shox2, c-Fos and Twist1. Individual overexpression of Snai2 in FR(2) hybrid cells led to a large fold increase of TFs c-Fos, Prrx1, Twist1 and Shox2. As a result of forced overexpression of Prrx1 and Snai2 individually in somatic cell hybrids FR(2) the cells re-acquired a spindle-shaped morphology and more significantly an enhanced migration capability, which is reminiscent of parental fibroblast cells. Thus it appears that ectopic expression of candidate fibroblast lineage specific transcription factors in somatic cell hybrids contributes to significant restoration of the fibroblast phenotype. In conclusion, in this study, we identify fibroblast-specific target genes as well as putative fibroblast lineage-specific TF regulatory genes. Our findings suggest that observed gene extinction in somatic cell hybrids may be attributed, in large measure, to the shutdown of fibroblast regulatory networks governed by master lineage-specific transcriptional regulators of the fibroblast phenotype