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Advantages of chloroplast as transformation system
1. plastid genes are maternally inherited
This makes plastid transformation a valuable tool for the creation and cultivation of genetically
modified plants that are biologically contained, thus posing lower environmental risks
2. plastids provide a viable alternative to conventional production systems such as microbial
fermentation or mammalian cell culture
3. ability to accumulate large amounts of foreign protein (up to 46% of total leaf protein)
4. Site specific integration into the chloroplast genome by homologous recombination of flanking
chloroplast DNA sequences present in the chloroplast vector eliminates the concerns of position
effect, frequently observed in nuclear transgenic lines
5. lack of transgene silencing despite the accumulation of transcripts at a level 169-fold higher
than in nuclear transgenic plants
6. Chloroplast genetic engineering also offers the unique advantage of transgene stacking, i.e.
simultaneous expression of multiple transgenes, creating an opportunity to produce multivalent
vaccines in a single transformation step.
7. Polycistrons are translated without processing into monocistrons
8. Foreign proteins synthesized in chloroplasts are properly folded with appropriate
posttranscriptional modifications, including disulfide bonds
Plastid transformation
DNA delivery by the biolistic process
polyethylene glycol (PEG) treatment of protoplasts
transgene integration into the chloroplast genome via homologous recombination
facilitated by a RecA-type system between the plastid-targeting sequences of the
transformation vector and the targeted region of the plastid genome.
Both flanking sequences are essential for homologous recombination
integration of the transgene into a few genome copies, followed by 25 to 30 cell divisions
under selection pressure to eliminate untransformed plastids, thereby achieving a
homogeneous population of plastid genomes.
Transgenes were first integrated into transcriptionally silent spacer regions.
transcriptionally
active spacer regions offer unique advantages, including insertion of transgenes
without 5 or 3 untranslated regions (UTRs) or promoters.
the most commonly used site of integration is the transcriptionally active intergenic
region between the trnI-trnA genes, within the rrn operon, located in the IR regions of the
chloroplast genome.
Rubisco engineering obtained by integrating the rbcS gene at this site
The aadA gene encodes the enzyme aminoglycoside 3# adenylyltransferase that
inactivates spectinomycin and streptomycin by adenylation and prevents binding to
chloroplast ribosomes
The neo gene is another alternative marker for plastid transformation that confers
kanamycin resistance. A different kanamycin resistance gene (aphA6) with relatively
high transformation efficiency was reported later
The cotton plastid transformation vector contained two different genes (aphA6 and
nptII) coding for two different enzymes. The aphA6 gene was regulated by the 16S rRNA
promoter and gene 10 UTR capable of expression in the dark and in nongreen tissues.
The nptII gene was regulated by the psbA promoter and UTR capable of expression in the
light. Both genes with different regulatory sequences facilitated detoxification
of the same selection agent (kanamycin) during day and night as well as in developing
plastids and mature chloroplasts. The double barrel transformation vector was reported
to be at least 8-fold more efficient than single gene (aphA6)-based chloroplast vectors.
The spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (badh) gene has been
developed as a plant-derived selectable marker gene to transform chloroplast genomes
(Daniell et al., 2001b). The selection process involved conversion of the toxic compound
betaine aldehyde to beneficial Gly betaine by the chloroplast-localized gene-encoding
enzyme BADH. Because the BADH enzyme is present only in chloroplasts of a few plant
species adapted to dry and saline environments
A negative selection scheme has also been employed for plastid transformation based on
expression of the bacterial gene codA (Serino and Maliga, 1997). Cytosine deaminase (codA)
catalyzes the deamination of cytosine to uracil. 5-Fluorocytosine is toxic to cells that express
cytosine deaminase because this enzyme converts 5-fluorocytosine to toxic 5-fluorouracil.
This negative selection scheme was utilized to identify seedlings on 5-fluorocytosine medium
from which codAwas removed by the P1 bacteriophage site-specific recombinase CRE-lox
(Corneille et al., 2001).
REPORTER GENES USED IN PLASTIDS
GUS, chloramphenicol acetyl transferase, and GFP have been used as plastid reporters
The GFP chromophore forms autocatalytically in the presence of oxygen and
fluoresces green when absorbing blue or UV light
GFP has also been fused with AadA and used as a bifunctional visual and selectable
marker
GFP has been used to test the concept of receptor-mediated oral delivery of foreign
proteins. Cholera toxin B-subunit (CTB)-GFP fusion protein with a furin cleavage site in
between CTB and GFP has been used to elucidate the path of CTB and GFP in the circulatory
system(Limaye et al., 2006). Mice were fed with CTB-GFP-expressing plant leaf material.
GFP was detected in the intestinal mucosa and submucosa, the hepatocytes of the liver, as
well as various cells of spleen utilizing fluorescence microscopy and anti-GFP antibodies. In
mice fed with untransformed leaf material or IFN-GFP fusion protein-expressing plant leaf
material, no GFP fluorescence was observed. This confirmed the receptor-mediated oral
delivery of a foreign protein (GFP) across the intestinal lumen into the systemic circulation.
TYPES OF CMS IN MAIZE
There are four major types of cytoplasm, namely N (normal), C (Charrua), T (Texas) and
S (USDA), which are classified according to differential sterility expression in response to
restorer (Rf) genes (Laughnan, 1983), mitochondrial DNA restriction digest patterns (Pring
and Levings, 1978), and compliments of low MW plasmids (Kemble and Bedrock, 1980).
Even though testcrossing is the most conclusive method of categorizing maize cytoplasms,
they are time-consuming and labor-intensive (Liu et al., 2002). In many plant species,
mutations responsible for CMS have been shown to reside in mitochondrial DNA (Schnable
and Wise, 1998). Therefore, various features of mitochondria, especially its genome structure
and gene expression, have been used to distinguish major types of maize cytoplasm (Levings
and Pring, 1977). Pring et al. (1977) were the first to show that mitochondrial preparations
(not digested by restriction enzymes) from lines carrying S cytoplasm possessed unique low
(molecular weight) MW plasmids not found in N, T or C cytoplasms. Later on Kemble et al.
(1980) studied low MW mitochondria DNA from 31 separately discovered sources of
cytoplasms. Unique banding patterns to each of four classes were identified which were in
conformity to the classification of these sources done by Beckett (1971). A major advantage of
this analysis was that no within-group heterogeneity was found as had been in earlier
studies (Levings and Pring, 1977). Similarly, Forde et al. (1978) used SDS-PAGE to analyze
mitochondrial translation products of CMS cytoplasm and detected additional or variant
polypeptides in T and C cytoplasm. More recently, Liu et al. (2002) developed a rapid PCR
assay of sterile cytoplasm in maize using three pairs of primers designed corresponding to
chimeric regions of mt-DNA sequences. The primer sets used amplified unique 440 bp
(CMS-T), 398 bp (CMS-C) and 799 bp (CMS-S) cytoplasm, whereas no unique fragment was
amplified in N cytoplasm, possibly because no such fragment existed in N cytoplasm.
CMS-T
Rogers and
Turf 13 is a chimeric region gene which is a
recombination product of 5 region of the atp 6 gene and 3 region of the
265 ribosomal gene
(rrn 26). Its transcription is presumably under the control of the atp 6
promoter (Stamper et
al., 1987). It is located in 3547-nucleotide mt DNA sequence that contains
two open reading
frames, one coding for urf 13 and the other for orf 221, which codes for a
25 kd polypeptide
consisting of 221 amino acids and is 77 nucleotides downstream of urf 13
(Levings, 1990). The
orf 221 encodes a membrane bound protein that has been identified as
ATP4 (Heazlewood et
al., 2003).