International Journal of Horticulture, 2017, Vol.7, No. 6, 40-46
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A proficient and consistent plant regeneration protocol is essential for the application of routine plant
transformation techniques. Exploitation of totipotent genotypes, suitable explants, appropriate media contents and
culture conditions are of core importance in optimizing plant tissue culture procedures (Joyia and Khan, 2013).
Additionally, the process of dedifferentiation and regeneration under
in vitro
culture conditions has long been
recognized to prompt somaclonal variations (Filipecki and Malepszy, 2006; Sun et al., 2013) which may also be
an efficient source of crop improvement. Development of whole plants from single cells under
in vitro
conditions
leads to various perspectives in order to manipulate somatic cells (Conner and Meredith, 1989). Consequently, it
provides a powerful tool for molecular, biochemical and physiological studies of different plant characters.
Nicotiana plumbaginifolia
is the most fascinating model systems which allowed a variety of genetic
manipulations at the somatic cell level (Maliga, 1982; Nikova et al., 1991). The establishment of protoplast and
cell cultures of this specie is simple and can promptly regenerate into whole plant. Monoploid (n =10) cells can be
obtained and easily diploidized to restore fertility. The large progenies of this small plant can be grown in a
limited space and can allow rapid genetic analysis.
Genetic transformation is the insertion of foreign gene in genome of an organism. It is the genetic alteration of
plant which provides a powerful way to manipulate different characteristic and conservation of desired traits for
crop improvement (Paszkowski et al., 1984). A varied range of techniques has been established for DNA transfer
into plant cells. Genetic transformation can be accomplished with Agrobacterium transformation method or by
electroporation in protoplast or direct DNA uptake using gene gun. Agrobacterium has limited host range and
regeneration from protoplast is difficult in some species. Direct DNA uptake through gene gun is valuable for
both transient as well as stable gene expression without any limitation of host specificity. Biolistic method is the
most powerful method of plant transformation. In spite of different variety of specialized techniques for plant
genetic transformation, particle bombardment is considered to be the most rapid, simple and versatile. It can
deliver DNA directly to intact cells and tissues, therefore avoids the limitation of host range and sometimes tissue
culture (Sanford et al., 1987; Klein et al., 1988). Adequate number of reports has been published on
in vitro
propagation of
N. plumbaginifolia
using leaf explant; however there is no report on utilization of these explants
for biolistic genetic transformation studies of
N. plumbaginifolia
. (Li et al., 2003) evaluated potential of shoot
organogenesis in 53
Nicotiana
species including
Nicotiana plumbaginifolia
. Similarly, leaf explant-based direct
organogenesis in five wild
Nicotiana
species has been reported by (Sarala
et al., 2008).
To the best of our
knowledge, this is the first report of biolistic transformation of
N. plumbaginifolia
using gene gun. Hence, we
report here, the expression of the
Gus
reporter gene in
N. plumbaginifolia
using
in vitro
grown mature leaves as
explant.
2 Materials and Methods
2.1 Plant material
Stem cuttings were obtained from
Nicotiana plumbaginifolia
plants grown as a weed in the fields of University of
Agriculture Faisalabad - Pakistan. Stem cuttings were surface sterilized by successive immersions in tap water,
70% (v/v) ethanol for few seconds followed by washing with 5% commercial bleach with few drops of tween 20
for 15 minutes. Finally, the explant was rinsed 3-4 times with sterile distilled water. The cuttings were cultured in
glass jars having solid MS medium without plant growth regulators (PGRs) and incubated at 25˚C ±2˚C under
16/8 hrs light and dark condition.
The basal medium used for
in vitro
regeneration, root formation and further
in vitro
multiplication was MS
medium (Murashige and Skoog, 1962). All the stock solutions of vitamins and plant growth regulators (PGRs)
were prepared in double distilled water. Sucrose was added as a carbon source at the rate of 30 g/L. pH of culture
medium was adjusted to 5.8 and solidified with 2.6 g/L gellan gum powder. The screw capped glass jars of 175 ml
capacity were used accommodating 50 ml of culture medium in each jars. The culture medium for all
investigations was sterilized by autoclaving at 121˚C at 15psi for 20 minutes. Leaves of the
in vitro
grown plants
were employed as explant in further experimemtation. Explants were cut into small pieces and placed onto
regeneration media containing various plant hormones.
