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Molecular Plant Breeding 2012, Vol.3, No.9, 91
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102
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98
room, leaves of the transgenic shoots showed early
development of chlorophyll pigments compared to the
wild type shoots. When cuttings were grown in the
light, similar to dark, transgenic shoots also showed
significant difference in root development from
untransformed wild type shoots (S. Nazir and M. S.
Khan, unpublished). Two more set of chloroplast
transgenic plants were developed by engineering
plastome with expression cassettes; one carrying
chlL
and
chlN
genes and the other
chlLN
&
B
genes in an
operon to express under a constitutive promoter from
tobacco chloroplast ribosomal RNA operon. Plants
are screened and under investigations to generate
physiological and biochemical data, supporting the
early findings on gymnosperms that three subunit
enzyme is required for developing functional green
chloroplasts in the dark. Our attempts are first known
efforts to engineering the three subunit pathway in
plastid genome of tobacco, a representative model
plant from angiosperms. This will help understanding
the molecular biology of transgenic angiosperms.
5 Engineering plastids of monocotyledonous
plants
Chloroplast transformation is recently achieved in a
number of plant species (Table 1) in which transformed
cells has been regenerated from tissues containing
green plastids, the chloroplasts. Salient examples are:
tobacco, potato, cotton, tomato, carrot, oilseed rape,
petunia, sugar beet, lettuce, cabbage, egg plant and
soybean. However, extending plastid transformation
to monocotyledonous sugar and cereal crops including
rice, wheat and sugarcane is still at its early stage of
development. Amongst major hurdles, one barrier to
developing plastid transformation in cereals, has been
their regeneration from non-green embryonic cells,
containing undifferentiated plastids (the proplastids)
rather chloroplasts. Other impediment in developing
homoplasmic transgenic plants, particularly of rice,
might be the low level of marker gene expression in
non-green plastids in embryogenic cells because of
low genome copy number and low rates of protein
synthesis, this has been discussed at length elsewhere
(Daniell et al., 2002). Identification of promoters and
UTRs active in non-green tissues can help to
overcome this limitation.
The rRNA operon has two
promoters, one for the eubacterial-type plastid-encoded
plastid RNA-polymerase (PEP) and one for the
phage-type nuclear-encoded plastid RNA polymerase
(NEP). Expressing transgene under ribosomal
RNA promoter, recognized by both polymerases
may accumulate transprotein to high levels, leading to
development of stable homoplasmic lines.
Yet, another limitation is the availability of a
single dominant marker gene,
aadA
that encodes
aminoglycoside adenylyltransferase and confers
resistance to spectinomycin and streptomycin. The
aadA
has been used predominantly to transform
plastids and the selection was carried out on
spectinomycin in dicotyledonous plants. Cells from
monocotyledonous or cereal crops are naturally
resistant to spectinomycin but are sensitive to
streptomycin. Hence, streptomycin can be used as a
selection agent to recover transgenic clones on media,
as demonstrated in rice (Khan and Maliga, 1999). In
these studies, embryogenic suspensions were developed
from calli derived from scutellum of rice seeds.
Embryogenic cells were bombarded using chloroplast
transformation vector harboring
aadA
and
gfp
(encoding a modified green fluorescent protein) genes
which were translationally fused and expressed under
ribosomal RNA promoter recognized by plastid
encoded RNA polymerase (PEP) and heteroplasmic
shoots were recovered. Encouraged from these results;
Lee and his colleagues (2006) transformed rice
plastids using same selectable marker
aadA
gene and
a reporter gene,
gfp
. Both genes were expressed under
ribosomal RNA promoter in an operon. Again,
heteroplasmic shoots were recovered on streptomycin
containing regeneration medium. Whether heteroplasmic
progeny plants could further be purified to develop
homoplasmic plants was not reported. These results
have reconfirmed the findings of Khan and Maliga
(1999). It may be argued that streptomycin may delay
the isolation of resistant shoots, hence it may hinder in
the recovery and purification of homoplasmic plastid
transformants in cereals when used as a primary
selection means. Hence, adding a second marker for
dual or stepwise selection on streptomycin and on
a second antibiotic may facilitate selection and
purification of transplastomic cells/shoots of cereals.
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