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to the development of homoplasmic plants. Similarly,
chloroplast transformation using dual selection system
is achieved in sugarcane where heteroplasmic shoots
are recovered on streptomycin and kanamycin
(Mustafa, 2011). Nonetheless, these studies warrant
the development of a reproducible regeneration
system that allows recovery and purification of
homoplasmic shoots, without compromising the
regeneration with successive rounds. Further,
transplastomic sugarcane plants are recovered by
selecting calli only on streptomycin-containing
regeneration medium, expressing gfp in leaves (G.
Mustafa and Khan M.S., unpublished).
With regard to plastid transformation in wheat, stable
transformation of chloroplasts from immature scutella
and inflorescences is achieved by introducing
nptII
and
gfp
genes in the intergenic region between
atpB
and
rbcL
(Cui et al., 2011) and selection of
transformants on kanamycin. Stable inheritance of
transgenes,
nptII
and
gfp
in the progeny was
confirmed by PCR and by confocal microscopy,
respectively. Transformation efficiency reported (two
regenerants per 42 bombardments) was extra
ordinarily low, however was comparable with that
of sugar beet in which one transgenic per 36
bombardments and potato in which three transgenics
from 46 bombardments were recovered on selection
medium. Where this report encourages the use of
lethal selection genes for chloroplast transformation in
cereals, the article was retracted by the corresponding
author, stating that “After receiving the comments
from the readers, we checked the results carefully and
decided to repeat all the experiments. As the first
author has already left the lab, other members are still
trying to repeat these experiments. Because it takes a
long time to have these experiments redone, we
decide to retract our paper”. Hence, to date, only
report on chloroplast transformation in cereals is the
chloroplast transformation in rice, carried out by Khan
and Maliga (1999) and reproduced by Lee et al (2006).
6 Perspectives of engineering plastids
As explained above, chloroplast transformation after
the first report in tobacco using dominant selection
gene,
aadA
(Svab and Maliga, 1993) has been
extended to other dicotyledonous plants of agriculture
and horticulture importance. Contrary to dicotyledonous
plants, plastid transformation in monocotyledonous
cereal and sugar plants is at its early development,
conceivably because of above mentioned limitations
and due to the developmental differences. Hence,
developing routine plastid transformation protocols
for these crops remains a challenge. Efficient and
dominant selection is only one aspect that can be
developed, yet it warrants to be combined with
developing an
in vitro
cell culture protocol that allows
efficient recovery and purification of homoplasmic
shoots without compromising the regeneration with
successive subcultures.
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