International Journal of Horticulture, 2017, Vol.7, No. 6, 40-46
44
efficiency of leaf explant of
N. plumbaginifolia
(Figure 1). Analysis of variance revealed significant difference
among regeneration efficiency on different regeneration media. LSD demonstrated the relationship between
number of shoots produced from each explant and the regeneration media used with highest mean value for RM
2
(Table 1; Table 2). (Rahman et al., 2010) showed similar results for different Bangladeshi tobacco cultivars with
maximum efficiency at IAA and Kinetin @ 2 mg/L each. However, (Li et al., 2003) evaluated shoot organogenic
potential of 115 Nicotiana accessions bu using Indole-3-acetic acid (IAA) 5.1 mM in combination with
6-Benzylaminopurine (BA) 11.1 mM. Our results showed a change in behavior of leaf explant on different media,
as RM
2
indicated higher regeneration as compared to other media as depicted in Table 2. RM
2
medium exhibited
regeneration on minimum concentration of IAA (0.25 mg/L), indicated that IAA in the culture medium greatly
influence organogenic response and development (Pierik, 1988).
3.2 Plant Transformation
Expression of β-glucuronidase (GUS) reporter gene is an efficient and reliable techniques studying gene
regulation (Fior and Gerola, 2009). For genetic transformation of
N. plumbaginifolia
, transformation vector
(Figure 2) coated gold particles (microcarriers) was bombardment under vacuum with a BioRad PDS 1000 (He)
gene gun, using 1100-psi rupture discs, stopping screens and macrocarriers having microcarriers. Fully expanded
lush green leaves were placed in the centre of a petri plate containing RM
2
medium with the help of sterile
foreceps in laminar air flow cabinet. Plates with bombarded leaf sections were wrapped and placed under dark
condition in growth room for 48 hrs, which were then transferred to normal light on RM
2
. The
gus
assay was
performed after 8 days of bombardment. All the leaves sections were dipped in 20 ml
gus
buffer (1 mM X-Gluc,
1M Na2HPO4, 0.1% Triton X-100, 0.5M EDTA) in a small beaker and leave the plants in assay solution at 37˚C
for 48 hrs. After 48 hrs, gus expressing cells showed blue colour as a result of chromogenic cleavage by X-Gluc,
representing successful integration of gus gene and its expression in leaf sections as depicted in Figure 3.
Figure 2 Plant transformation vector
p7i-UG
vector having
ubi
1 promotor,
uidA
gene and
nos
terminator
Figure 3 Histochemical assay of GUS gene
Note: A, B, C, D representing blue colour in leaf sections of
N.
plumbaginifolia
after biolistic transformation, E. Control
showing no blue colour
The
gus
assay was performed 8 days after the bombardment, this was done to minimize the transient expression
and increase the chances to see the stable transformation, as transient expression is maximum till 96 hrs of
bombardment and after that the chances to appear for the stable expression are more (Janssen and Gardner, 1990).
Gus gene showed sufficient expression 8 days after bombardment which means that there are enough chances for
the stable transformation. While our non-bombarded control did not show any colour. Similar study was
