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Molecular Plant Breeding 2012, Vol.3, No.9, 91
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102
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1 Plastid genome engineering: state of the art
Plastid genome encodes approximately 120 protein
and RNA genes in various plant species (Sugiura,
1992; Yang et al., 2010), suggesting that most of their
ancestral genes have been either lost or transferred to
the nuclear genome during evolution. When comes to
plastid genome copy number, it reaches to 10,000
copies per cell because a single leaf mesophyll cell of
flowering plants carries 100 chloroplasts, and each
chloroplast contains 100 plastome copies. Further, the
number of genes, located on the plastome in the
inverted repeat region is doubled because of
duplication of a large region of plastome in an
inverted orientation.
Engineering genome of plastids involves a series of
steps to develop homoplasmic transplastomic clones.
During the process, transformations vectors that
harbor a selectable marker gene, a reporter gene
and the passenger gene (s) flanked by homologous
targeting sequences, are used. Successful transformation
requires; a totipotent explant, an efficient DNA
delivery method, selection agents (antibiotics) and a
reproducible regeneration protocol to efficiently
recover transgenic plants. Transgenes are incorporated
into the plastome through homologous recombination
events. In practice, the inserted transgene has short
DNA sequence tails added at each end, the tails are
homologous to sequences on the chloroplast target
gene, which thus initiate homologous recombination.
Antibiotics used in selecting transgenic plants inhibit
chlorophyll accumulation and shoot formation on
plant regeneration media, however, cells resistant to
these antibiotics regenerate into green shoots, and
resistance to these antibiotics is conferred by the
expression of genes in plastids. Lethal (kanamycin)
and non-lethal antibiotics (streptomycin, spectinomycin)
are used to select transformants. As different species
have differing sensitivities to selective agents, hence,
the successful recovery of the transplastomic lines
is dependent on selection concentration that how
carefully the concentration of the selection agent was
titrated that permitted growth and development of the
transformed cells while limiting the growth of the
non-transformed cells, and the regeneration strategy
adopted. Further, same selective agent cannot be
universally used in selecting the transformation events.
For example, spectinomycin is routinely used in
plastid transformation experiments to recover green
shoots on regeneration media from tobacco, and has
also been used for tomato, potato, cabbage, oil rape
seed and carrot. But, rice, sugarcane and few other
monocots are naturally resistant to spectinomycin;
therefore selection for transplastomic lines was
carried out on medium containing streptomycin,
which inhibits growth of embryogenic cells on
regeneration media, because
aadA
gene confers
resistance to streptomycin as well.
Initially, only few plastome copies are transformed,
and stable homoplasmic lines are produced with
continued selection pressure in which each and every
plastome contains the transgene. The process involves
cultivation of the cells on a selective medium, during
which the cells divide at least 20 times, transformed
and non-transformed plastids and plastome copies
gradually sort out, yielding chimeric shoots consisting
of sectors of transgenic and non-transgenic cells. Both
transgenic and non-transgenic cells in a chimeric
shoot are green in color because of phenotypic
masking by the transgenic cells (Khan and Maliga,
1999), referring that antibiotic resistance is not cell
autonomous. Nevertheless, transgenic and non-transgenic
sectors can be readily identified in knockout
transgenic plants lacking a photosynthesis gene
(Figure 1A~D; Khan et al., 2007) or by green
fluorescent protein (GFP) accumulation (Khan and
Maliga, 1999). The GFP allows direct imaging of the
fluorescent gene product in living cells without the
need for prolonged and lethal histochemical staining
procedures. Its chromophore forms autocatalytically
in the presence of oxygen and fluoresces green when
absorbing blue or ultraviolet (UV) light (Khan, 1997).
The preferred strategy to develop homoplasmic shoots
is to regenerate subsequent shoots from the
homoplasmic sector, which are rooted, shifted to soil
and hardened for growth and seed setting. Homoplasmic
transgenic plants can be recovered from embryogenic
cells from suspensions or calli but plant regeneration
is delayed until plastome and plastid segregation is
complete. However, extended propagation of cells on
regeneration media is undesirable because it results in
either scanty or no regeneration of embryogenic cells
into shoots.
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