Hamptonforrest9313

Chloroplast transformation has been extremely valuable for the study of plastid biology and gene expression, but the tissue culture methodology involved can be laborious and it can take several months to obtain homoplasmic regenerated plants useful for molecular or physiological studies. In contrast, transformation of tobacco suspension cell plastids provides an easy and efficient system to rapidly evaluate the efficacy of multiple constructs prior to plant regeneration. Suspension cell cultures can be initiated from many cell types, and once established, can be maintained by subculture for more than a year with no loss of transformation efficiency. Using antibiotic selection, homoplasmy is readily achieved in uniform cell colonies useful for comparative gene expression analyses, with the added flexibility to subsequently regenerate plants for in planta studies. Plastids from suspension cells grown in the dark are similar in size and cellular morphology to those in embryogenic culture systems of monocot species, thus providing a useful model for understanding the steps leading to plastid transformation in those recalcitrant species.Stable plastid transformation in Nicotiana tabacum has been achieved by using two different methods, the biolistic method, using a particle gun, and the polyethylene glycol (PEG)-mediated transformation. PEG-mediated plastid transformation involves the treatment of isolated protoplasts (plant cells without cell wall) with PEG in the presence of DNA. We have previously shown that in Nicotiana tabacum both methods are equally efficient. The PEG-mediated transformation efficiencies range between 20 and 50 plastid transformants per experiment (106 viable treated protoplasts). One advantage of the PEG method is that no expensive equipment such as a particle gun is required. The only crucial points are the handling and the cultivation of protoplasts. Furthermore, markers for the selection of transformed plastids are required. One of the most often used selection markers is the aadA gene which encodes for spectinomycin and streptomycin resistance. Here we describe a simplified and inexpensive protocol for the transformation of plastids in Nicotiana tabacum using an optimized protoplast culture protocol. PEG-mediated plastid transformation has the potential to be developed into a high-throughput, automated pipeline.The protocol we report here is based on biolistic delivery of transforming DNA to tobacco leaves, selection of transplastomic clones by spectinomycin or kanamycin resistance and regeneration of plants with uniformly transformed plastid genomes. Because the plastid genome of Nicotiana tabacum derives from Nicotiana sylvestris, and the two genomes are highly conserved, vectors developed for N. tabacum can be used in N. sylvestris. The tissue culture responses of N. tabacum cv. Petit Havana and N. sylvestris accession TW137 are similar. Plastid transformation in a subset of N. tabacum cultivars and in Nicotiana benthamiana requires adjustment of the tissue culture protocol. We describe updated vectors targeting insertions in the unique and repeated regions of the plastid genome, vectors suitable for regulated gene expression by the engineered PPR10 RNA binding protein as well as systems for marker gene excision.While chlorophyll has served as an excellent label for plastids in green tissue, the development of fluorescent proteins has allowed their ready visualization in all tissues of the plants, revealing new features of their morphology and motility, including the presence of plastid extensions known as stromules. Gene regulatory sequences in nuclear transgenes that target proteins to plastids, as well as in transgenes introduced into plastid genomes, can be assessed or optimized through the use of fluorescent protein reporters. Fluorescent labeling of plastids simultaneously with other subcellular locations reveals dynamic interactions and mutant phenotypes. Transient expression of fluorescent protein fusions is particularly valuable to determine whether or not a protein of unknown function is targeted to the plastid. Fluorescent biosensors can assay molecules such as ATP, calcium, or reactive oxygen species. Particle bombardment and agroinfiltration methods described here are convenient for imaging fluorescent proteins in plant organelles. With proper selection of fluorophores for labeling the components of the plant cell, confocal microscopy and multiphoton microscopy can produce extremely informative images at high resolution at depths not feasible by standard epifluorescence microscopy.Excision of marker genes using DNA direct repeats makes use of the efficient native homologous recombination pathway present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and green algae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes (plastomes). Release of selection allows the accumulation of marker-free plastomes generated by marker excision, which is a spontaneous and unidirectional process. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Orantinib molecular weight Alternatively, accumulation of marker-free plastomes during growth, development and flowering of T0 plants allows for the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The procedure enables precise plastome engineering involving insertion of transgenes, point mutations and deletion of genes without the inclusion of any extraneous DNA. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.