El cultivo de protoplastos en cítricos y su potencial para el mejoramiento genético. Article (PDF Available) · July with 1, Reads. Export this citation. Results 80 – 90 Download Citation on ResearchGate | On Jan 1, , R.R. Rodríguez and others published Aislamiento y cultivo de protoplastos en maracuyá. Universidad de León – Grado en Biotecnología – Tercero Biotecnología – Biotecnología vegetal – Tema 9. Cultivo de protoplastos e hibridación somática.
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Somatic hybrids obtained by protoplast fusion between Solanum tuberosum L. New York, NY Genetic Resourses Conservation and Characterization Division. Interspecific somatic hybrids were obtained by polyethylene glycol fusion of protoplasts from tetraploid Solanum tuberosum L.
Tema 9. Cultivo de protoplastos e hibridación somática
Fusion-treated protoplasts were cultured in V-KM medium supplemented with bovine serum albumin. A rapid calli proliferation was observed after colonies developed. Nineteen somatic hybrid plants were obtained and confirmed by RAPD analysis. Chromosome observations indicated that cyltivo hybrids were aneuploids. The morphology of fusion-derived regenerants was intermediate between the donor parents.
This study shows that somatic hybrid potato plants can be obtained by the fusion method presented.
Solanum, protoplast fusion, polyethylene glycol, somatic hybrid, RAPD analysis. Potato, Solanum tuberosum L. The wild species S. Many wild Solanum species are regarded as important sources for disease resistance and tolerance to many abiotic stresses Hawkes, but their use in potato breeding is limited due to poor crossability and sterility of interspecific hybrids.
Isolamento e cultivo de protoplastos de porta-enxertos de citros
These barriers in classical breeding can be overcome using biotechnological methods such as somatic hybridization by protoplast fusion Carputo et al. Using chemical or electrical procedures Jones, protoplasts from different donor plants can be fused together and somatic hybrids regenerated from the fusion products. This technique has created novel cellular genome configurations by combining sexually incongruent species. Somatic potato prohoplastos have been produced by fusion of diploid wild species with tetraploid S.
Successful application of the fusion technique demands a protocol for plant regeneration from protoplasts. An analysis of putative fusion products is essential to confirm hybrid status Masuelli et al. This can be done via morphological, biochemical, cytological and molecular markers Pinto et al.
Cultivo y fusion de protoplastos 
In this paper we describe the fusion of leaf protoplasts of S. RAPD and chromosomal analysis of the hybrids are also presented. Plant material and protoplast isolation. The latter has a natural resistance to late blight, Phytophthora infestans Mont. The enzyme treatment was carried out overnight with gentle agitation. Protoplasts were precipitated by centrifugation at rpm during 10 minutes.
Final concentration was protoplasts per millilitre. Protoplast fusion, culture and regeneration. Prior to fusion, protoplasts from S. Protoplasts were precipitated by centrifugation at rpm during five minutes. After that, protoplasts settled down. The mixture was incubated for thirty minutes and then protoplasts were collected by centrifugation at rpm during five minutes.
The osmolality of this medium was fixed at mOsm.
After seven days, the culture medium was diluted 1: Then, at fifty days, cultivk culture media were replaced by a mOsm V-KM medium. When colonies had developed, alginate beads were despolimeryzed using 20 mM sodium citrate solution in sorbitol 0,3 M, pH 7,4 and gently shaking.
Then the suspension was centrifugated at rpm during ten minutes. After that, colonies were dispersed into prottoplastos with solid MS13K regeneration medium Benke, Regenerants from the fusion experiments were propagated in vitro using the Murashige and Skoog medium. The hybridity of the fusion regenerants was confirmed by RAPD markers. For this analysis, DNA extraction was performed according to Doyle and Doyle with some modifications.
Gels were analyzed and photographed under UV light. These samples were obtained from rooted plants maintained in a greenhouse. Root tips were stained by lacto-propionic orcein. Protoplaztos fusion and plant regeneration. Protoplasts fusion from S. Aggregations of more than two protoplasts were also observed.
Heterokaryons can not be identified because protoplasts of the parents were of the same type and shape. Due to that, fused and unfused protoplasts were cultured together. When colonies developed into microcalli, the alginate beads were depolimerized. Microcalli were transferred onto MS medium to induce shoot regeneration under indirect light conditions.
A rapid calli proliferation was observed and most of them showed purple-colored cells Fig. Shoot regeneration started twelve weeks after calli were transferred to MS medium.
Shoots also showed purple-colored meristematic zones. Nineteen plants were regenerated from calli Fig. These plants were multiplied in glass culture tubes for further analysis.
This value is different from that obtained by Mattheij et al. The other parent, the wild species S. Under these conditions, calli hybrids follow the developmental pathway of the morphogenic parents. To select hybrids, regeneration of in vitro plants was subjected to RAPD analysis.
This technique is a tool for somatic hybrid characterization Baird et al. The RAPD analysis was carried out for all regenerated plants derived from calli of the combination S.
The same primer amplified four specific bands of, and bp in S. These bands also appeared in the fusion regenerants Figs. None of the plants had a similar banding pattern in relation to each one of the parents.
Identification of somatic hybrids is a requisite for the effective exploitation of the protoplasts fusion in potato improvement. In this study, confirmation of fusion-derived regenerants by the use of RAPD markers represented an effective system for the detection of somatic hybrids from S. The numbers of chromosome of S. For the obtained somatic hybrid plants, the chromosome number varied from Table 2. These results showed that all hybrids were aneuploids Fig.
None of the regenerants had 72 chromosomes, which results from the protoplasts fusion of a tetraploid with a diploid genome. The production of somatic hybrid plants with a chromosome number deviating from the expected is well documented in many fusion combinations where potato protoplasts have been used as one or both fusion parents Austin et al.
After protoplasts fusion and during plant regeneration it frequently happens that genetic changes such as variations in chromosome number occur. The origin of this chromosome instability has been studied elsewhere in potato Kumar, It has been demonstrated that protoplast-derived plants show a higher degree of chromosomal number variation than those derived from tissue or organ culture.
It has been suggested that protoplasts are more prone to chromosome instability because they undergo a long period of greater stress during initial stages of cell division and dedifferentiation Pijnaker and Sree Ramulu ; Carrasco et al.
The ploidy of the genotype used in plant regeneration has been shown to profoundly affect the type of variation observed in morphological characters and chromosome numbers among regenerant plants.
Plant regeneration from mono- and dihaploid potato genotypes often results in ploidy changes, but little aneuploidy; on the other hand, regenerated plants from tetraploid genotypes produce a wide range of aneuploidy Kumar, For these reasons and considering the obtained results, it can be suggested that there is a high probability of fusion-derived regenerants which are aneuploids with an increase or loss of some chromosomes when one or both fusion partners have a high ploidy level.
Somatic hybrids were confirmed morphologically under greenhouse conditions, when intermediate characteristics between parent lines, such as shoot coloration, leaf shape and pubescence were observed. Tissue culture procedures to produce somatic hybrids and the application of molecular biological tools for their analysis are well established. In conclusion, somatic fusions between the cultivated potato Solanum tuberosum and the wild species S.
Nineteen somatic hybrid plants were obtained from fusion experiments using PEG in an effort to combine elite traits from both parents. Cytological and RAPD marker analysis confirm their hybrid nature.
Further work is needed to evaluate their resistance level to Phytophthora infestans Mont. The authors would like to thank the International Potato Center for financial support and to Martha Williams de Castro who revised the English version of this article. Transfer of resistance to potato leaf roll virus from Solanum brevidens into Solanum tuberosum by somatic fusion.
Interspecific somatic hybridization between Solanum tuberosum L. Molecular characterization of inter- and intra-specific somatic hybrids of potato using randomly amplified polymorphic DNA RAPD markers. Somatic hybridization in the genus Solanum: Plant Cell Rep 3: Regeneration in Gewebekulturen einiger Dihaploider Solanum tuberosum Z. Regeneration of isolated protoplasts to plants in Solanum dulcamara. Production of somatic hybrids between frost – tolerant Solanum commersonii and Solanum tuberosum: Tissue culture response in various wild and cultivated Solanum germplasm accessions for exploitation in potato breeding.
Plant Cell Tissue Organ Cult Resistance to Phytophthora infestans in Solanum tuberosum and wild Solanum species. Ruiz de Galarreta y E.