RESUMO
Tomato plastid transformants were obtained using two vectors containing cloned plastid DNA of either Nicotiana tabacum or Solanum nigrum and including point mutations conferring resistance to spectinomycin and streptomycin. Transformants were recovered after PEG-mediated direct DNA uptake into protoplasts, followed by selection on spectinomycin-containing medium. Sixteen lines contained the point mutation, as confirmed by mapping restriction enzyme sites. One line obtained with each vector was analysed in more detail, in comparison with a spontaneous spectinomycin-resistant mutant. Integration of the cloned Solanum or Nicotiana plastid DNA, by multiple recombination events, into the tomato plastome was confirmed by sequence analysis of the targeted region of plastid DNA in the inverted repeat region. Maternal inheritance of spectinomycin and streptomycin resistances or sensitivity in seedlings also confirmed the transplastomic status of the two transformants. The results demonstrate the efficacy in tomato of a selection strategy which avoids the integration of a dominant bacterial antibiotic resistance gene.
Assuntos
Plantas Geneticamente Modificadas/genética , Plastídeos/genética , Mutação Puntual/genética , Ribossomos/genética , Solanum lycopersicum/genética , Sequência de Bases/genética , DNA de Plantas/genética , Resistência a Medicamentos/genética , Regulação da Expressão Gênica de Plantas/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Solanum lycopersicum/crescimento & desenvolvimento , Recombinação Genética/genética , Plântula/genética , Solanum nigrum/genética , Nicotiana/genética , Transformação Genética/genéticaRESUMO
Data are presented on the potential of gynogenesis for the production of monohaploids and on factors affecting their frequency and relative vigour. Diploid Solanum tuberosum L. and S. tuberosumxS. phureja Juz et Buk hybrids were used as maternal parents and selected S. phureja clones as prickle pollinators with embryo-spot and nodal band as dominant seed and plant marker. About 2 million seeds were screened for absence of embryo-spot. After raising plants from phenotypically spotless seeds, further screening for absence of nodal bands and for ploidy level was carried out. Finally more than 500 monohaploid plants from three genetically different groups of maternal parents were obtained. Frequency and vigour of the monohaploids were clearly dependent on their maternal genotypes. The data also indicated an effect of the pollinator genotype, the physiological stage of the maternal plant and the environment on monohaploid frequency. On the basis of these results the possibility of breeding for a higher monohaploid production rate and for more stable and vigorous monohaploids is discussed. Furthermore, gynogenesis and androgenesis are compared. It is suggested that both should be used in order to obtain monohaploids from sufficiently various diploid breeding material.
RESUMO
Plant growth performance was studied in 118 potato monohaploids and in their diploid parents. Of these monohaploids 76 were also investigated at the protoplast level and eight of these were used in protoplast fusion experiments as well. No correlation was found between relative performance of greenhouse grown and in vitro grown plants. No or only weak correlations were found between different in vitro characteristics such as plant growth, protoplast yield per gram plant material, plating efficiency and callus growth. This indicates the unpredictability of these characters.The protoplast fusion experiments indicated that only in some genotype combinations increased callus growth rates may be found. However, it is not clear whether such calli were hybrids or not. In protoplast monocultures only diploid and tetraploid regenerants were obtained. After fusion, tetraploids but also some triploids could be regenerated. The finding of triploids indicates that monoploid protoplasts were involved in fusion. Isozyme analysis and morphological assessment of the plants pointed out that the majority of the fusion regenerants were hybrids. The implications of these results are discussed.
RESUMO
The DNA distributions of 23 different monohaploid potato clones were investigated by flow-cytometric measurements. All monohaploid clones differed in DNA distribution but none of them contained only monoploid cells in the leaves. All were highly stable on the monohaploid level for 2-3 years. Investigation of the influence of different factors on the DNA distribution in leaf cells showed that the material derived from in vitro shoot tip propagation contained a lower proportion of polyploidized cells than greenhouse grown plants. With protoplast isolation the enzyme treatment of in vitro cultured plant material induced a striking shift of DNA distribution towards the lower C-value whereas the mechanical purification steps caused a selective loss of monoploid nuclei. Seasonal influence on the DNA patterns could be detected.
