RESUMEN
The rapid development of tissue culture and recombinant-DNA technology in recent years has enabled plants of many species to be regenerated from cultured cells and their genetic information to be manipulated in various ways. Gene transfer has already resulted in the production of a wide range of transgenic dicotyledons, particularly in the Solanaceue. Although success with monocotyledons is more limited, transgenic rye (1), maize (2), and rice (3) plants have been reported.
RESUMEN
Using a modification of the alginate film culture technique we show that it is possible to prepare and culture tobacco mesophyll and barley cell suspension protoplasts without centrifugation. Comparable division frequencies and colony development were observed from protoplasts embedded with enzyme and protoplasts purified by centrifugation. A 3 × 30 min washing regime was found to be the minimum time necessary to remove the enzyme from the gelled alginate matrix. The procedure provides a more gentle method for isolating protoplasts. It has the additional benefit of recovering all of the cells released from the starting tissue. In particular, the smaller protoplasts that are frequently lost during conventional isolation, are maintained. In barley, we illustrate the use of the system for recovering plants from embryogenic protoplast-derived calli from the cultivars Dissa and Igri. Finally, using small volumes of enzyme (50 µl) single cell aggregates were used to isolate and culture protoplasts.
RESUMEN
Freshly isolated cell suspension protoplasts ofSolanum dulcamara were mixed withAgrobacterium rhizogenes, allowed to settle for 2 h, exposed to electrical pulses and further incubated for 2h. Two pulses of 600 V cm(-1) for 2 msec separated by 15 sec produced transformed colonies at relative and absolute transformation frequencies which were 3-4 and 10 fold greater than those obtained by co-cultivation of 3 days old protoplast-derived cells with bacteria. Transformed colonies were not produced when freshly isolated protoplasts were mixed withAgrobacterium but not electroporated. Biochemical analysis confirmed the transgenic nature of plants regenerated from protoplast-derived tissues.
RESUMEN
Here we report on the development of a new dominant selection marker for plastid transformation in higher plants using the aminoglycoside phosphotransferase gene aphA-6 from Acinetobacter baumannii. Vectors containing chimeric aphA-6 gene constructs were introduced into the tobacco chloroplast using particle bombardment of alginate-embedded protoplast-derived micro colonies or polyethylene glycol (PEG)-mediated DNA uptake. Targeted insertion into the plastome was achieved via homologous recombination, and plastid transformants were recovered on the basis of their resistance to kanamycin. Variations in kanamycin resistance in transplastomic lines were observed depending on the 5' and 3' regulatory elements associated with the aphA-6 coding region. Transplastomic plants were fertile and showed maternal inheritance of the transplastome in the progeny.
Asunto(s)
Genes Bacterianos/genética , Kanamicina Quinasa/genética , Kanamicina/farmacología , Nicotiana/genética , Plastidios/genética , Selección Genética , Transformación Genética , Acinetobacter baumannii/enzimología , Acinetobacter baumannii/genética , Biolística , Resistencia a Medicamentos/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Marcadores Genéticos/genética , Kanamicina Quinasa/metabolismo , Hojas de la Planta/genéticaRESUMEN
Seedling hypocotyl explants ofGlycine canescens were inoculated withAgrobacterium rhizogenes carrying a chimaeric NPTII gene cointegrated into the TL-DNA of pRiA4. Transformed roots produced shoots on B5 based medium with 10.0 mgl(-1) BAP, 0.05 mgl(-1) IBA and 50 µgml(-1) kanamycin. Cultured roots and regenerated plants expressed NPTII enzyme activity which was correlated with the presence of Ri TL-DNA and the structural sequence of the NPTII gene.
RESUMEN
Transgenic rice plants have been regenerated by somatic embryogenesis from cell suspension derived protoplasts electroporated with plasmid carrying the NPTII gene under the control of the 35S promoter from cauliflower mosaic virus. Heat shock of protoplasts prior to electroporation maximised the throughput of kanamycin resistant colonies. Omission of kanamycin from the medium for plant regeneration was essential for the recovery of transgenic rice plants carrying the NPTII gene. This report of the production of kanamycin resistant transgenic rice plants establishes the use of protoplasts for rice genetic engineering.