Plastid Transformation in Potato: Solanum tuberosum
Although plastid transformation has attractive advantages and potential applications in plant biotechnology, for long time it has been highly efficient only in tobacco. The lack of efficient selection and regeneration protocols and, for some species, the inefficient recombination using heterologous flanking regions in transformation vectors prevented the extension of the technology to major crops. However, the availability of this technology for species other than tobacco could offer new possibilities in plant breeding, such as resistance management or improvement of nutritional value, with no or limited environmental conc...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Stable Plastid Transformation of Petunia
Petunia hybrida is a commercial ornamental plant and is also an important model species for genetic analysis and transgenic research. Here we describe the steps required to isolate stable plastid transformants in P. hybrida using the commercial Pink Wave cultivar. Wave cultivars are popular spreading Petunias sold as ground cover and potted plants. Transgenes introduced into P. hybrida plastids exhibit stable expression over many generations. The development of plastid transformation in P. hybrida provides an enabling technology to bring the benefits of plastid engineering, including maternal inheritance and stable express...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Plastid Transformation in Tomato
This article describes the basic methods involved in the generation and analysis of tomato plants with transgenic chloroplast genomes and summarizes current applications of tomato plastid transformation. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Nicotiana tabacum : PEG-Mediated Plastid Transformation
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 expensiv...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Plastid Transformation in Nicotiana tabacum and Nicotiana sylvestris by Biolistic DNA Delivery to Leaves
We describe updated vectors targeting insertions in the unique and repeated regions of the plastid genome as well as systems for marker excision. The simpler genetics of the diploid N. sylvestris, as opposed to the allotetraploid N. tabacum, make it an attractive model for plastid transformation. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Plastid Transformation for Rubisco Engineering and Protocols for Assessing Expression
The assimilation of CO2 within chloroplasts is catalyzed by the bi-functional enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco. Within higher plants the Rubisco large subunit gene, rbcL, is encoded in the plastid genome, while the Rubisco small subunit gene, RbcS is coded in the nucleus by a multi-gene family. Rubisco is considered a poor catalyst due to its slow turnover rate and its additional fixation of O2 that can result in wasteful loss of carbon through the energy requiring photorespiratory cycle. Improving the carboxylation efficiency and CO2/O2 selectivity of Rubisco within higher plants has been a ...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Quantification of Organellar DNA and RNA Using Real-Time PCR
Quantitative (real-time) polymerase chain reaction (PCR) allows the measurement of relative organellar gene copy numbers as well as transcript abundance of individual mitochondrial or plastidial genes. Requiring only minute amounts of total DNA or RNA, the described method can replace traditional analyses like Southern or Northern hybridization which require large amounts of organellar nucleic acids and usually provide only semiquantitative data. Here we describe prerequisites, reaction conditions, and data analysis principles, which should be applicable for a wide range of plant species and experimental situations where c...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Determination of the Half-Life of Chloroplast Transcripts in Tobacco Leaves
The amounts of specific transcripts that accumulate in chloroplasts are determined by the rates of synthesis and degradation of the transcripts. The 3′ untranslated region of transcripts is a major determinant of the stability of transcripts in chloroplasts. The half-lives of specific transcripts can be determined by northern blot analysis of a time course of transcripts in detached tobacco leaves incubated with actinomycin D, a potent transcription inhibitor. This analysis may be applied to transcripts of endogenous genes or of transgenes introduced into the chloroplast genome in transplastomic plants. Sequence dete...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Plastid Marker Gene Excision in Greenhouse-Grown Tobacco by Agrobacterium-Delivered Cre Recombinase
Uniform transformation of the thousands of plastid genome (ptDNA) copies in a cell is driven by selection for plastid markers. When each of the plastid genome copies is uniformly altered, the marker gene is no longer needed. Plastid markers have been efficiently excised by site-specific recombinases expressed from nuclear genes either by transforming tissue culture cells or introducing the genes by pollination. Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. Agrobacterium encoding the recombinase on its T-DNA is injected at an axill...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Tryptophan and Indole Analog Mediated Plastid Transformation
A nonantibiotic/herbicide-resistance selection system for plastid transformation is described here in technical detail. This system is based on the feedback-insensitive anthranilate synthase (AS) α-subunit gene of tobacco (ASA2) as a selective marker and tryptophan (Trp) or indole analogs as selection agents. AS catalyzes the first reaction in the Trp biosynthetic pathway, naturally compartmentalized in the plastids, by converting chorismate to anthranilate and is subjected to feedback inhibition by Trp. In addition to Trp, various Trp analogs and indole compounds that can be converted to Trp analogs can also inhibit...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Plastid Transformation of Tobacco Suspension Cell Cultures
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 o...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Fluorescent Labeling and Confocal Microscopic Imaging of Chloroplasts and Non-green Plastids
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. Gene regulatory sequences in plastid transgenes can be 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 i...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Excision of Plastid Marker Genes Using Directly Repeated DNA Sequences
Excision of marker genes using DNA direct repeats makes use of the predominant homologous recombination pathways present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and microalgae. 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 recombinas...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Engineering Chloroplasts for High-Level Foreign Protein Expression
Expression of transgenes from the plastid genome offers a number of attractions to biotechnologists, with the potential to attain very high protein accumulation levels arguably being the most attractive one. High-level transgene expression is of particular importance in resistance engineering (e.g., via expression of insecticidal proteins) and molecular farming. Over the past years, the production of many commercially valuable proteins in chloroplast-transgenic (transplastomic) plants has been attempted, including pharmaceutical proteins (such as subunit vaccines and protein antibiotics) and industrial enzymes. Although, i...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news

Plastid mRNA Translation
Overall translational machinery in plastids is similar to that of E. coli. Initiation is the crucial step for translation and this step in plastids is somewhat different from that of E. coli. Unlike the Shine-Dalgarno sequence in E. coli, cis-elements for translation initiation are not well conserved in plastid mRNAs. Specific trans-acting factors are generally required for translation initiation and its regulation in plastids. During translation elongation, ribosomes pause sometimes on photosynthesis-related mRNAs due probably to proper insertion of nascent polypeptides into membrane complexes. Codon usage of plastid mRNA...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news