International Journal of Horticulture, 2017, Vol.7, No. 6, 40-46
42
2.2 Direct
in vitro
regeneration
In order to attain direct
in vitro
regeneration, six regeneration media containing 30 g/L sucrose, 4.33 g/L MS basal
medium supplemented with following six different combinations of IAA and kinetin were used. RM1: 0.125 mg/L
IAA and 1 mg/L Kinetin, RM2: 0.25 mg/L IAA and 1 mg/L kinetin, RM3: 0.75 mg/L IAA and 1 mg/L Kinetin,
RM4: 0.125 mg/L IAA and 2 mg/L Kinetin, RM5: 0.25 mg/L IAA and 2 mg/L Kinetin, RM6: 0.75 mg/L IAA and
2 mg/L Kinetin. All media were solidified with 2.6 g/L phytagel while pH was maintined at 5.8 before
autoclaving as described earlier. Regeneration efficiency was calculated as under.
2.3 Root induction
After 5 weeks of culturing the regenerated plantlets were transferred into jars having root induction media
medium {MS medium supplemented with different doses of IBA (0.00, 0.25 mg/L, 0.5 mg/L, 1 mg/L)}. Plantlets
with developed roots were successfully acclimatized to peat moss mixture in pots covered with polythene bags for
maintaining proper humidity and later placed in greenhouse.
2.4 Biolistic transformation of
Nicotiana plumbaginifolia
Explants cultured on regeneration media in the center of petri plates under aseptic conditions were used for
bombardment. The gene gun (Bio-Rad PDS-1000/He) was properly sterilized with ethanol to avoid contamination.
Bombardment was carried out under a partial vacuum (28 mm of Hg) using 1100-psi rupture disc. Gold particles
(0.4-0.8 µm) were coated with 5 µg of the plasmid DNA of the
p7i-UG
vector, following protocol described by
(Becker et al., 1994). The stopping screen and macro-carriers were placed into the vacuum chamber at 1
st
level
and the petri dish containing the target leaf sections were placed at 3
rd
level from the top. After bombardment,
plates containing bombarded leaves were sealed using cling film and placed at 25°C under dark condition in
growth room for 48 hrs.
2.5 Histochemical Gus assay
In order to check the efficiency of biolistic transformation histochemical assay was performed. These explant
were analyzed for Glucuronidase (Gus) activity (Jefferson et al., 1987) 8 days after bombardment. Explants were
incubated for 48 hrs at 37°C in buffer containing X-Gluc (5-Bromo-4-chloro-3-indolyl-D-glucuronic acid),
Potassium buffer (pH 7.0), 0.5% Triton X-100 as a substrate. Gus signals were visually observed and
photographed.
2.6 Statistical Analysis
A completely randomized design (CRD) with three replications per treatment was used in this study. Each
replication contained three plates of respective media. Mean value of three plates was considered as one
replication. Means and standard errors of means (SEM) were calculated and level of significance of the mean
values was checked by using LSD (Latin Square Design) test at 5% level of significance (Steel
et al., 1997).
3 Results and Discussion
3.1 Optimization of regeneration protocol
Efficient
in vitro
regeneration methodologies authorizing the development of complete plant from a single cell are
of vital significance to both clonal propagation and successful genetic engineering. Therefore, first and the
foremost endeavor should be the production of an immense number of regenerable cells as easily and quickly as
possible. Present research was designed to establish an efficient
in vitro
regeneration system which will help in
genetic transformation of
N. plumbaginifolia
L. The
in vitro
culture system was optimized exploiting leaf explant.
This study was focused on the development of direct regeneration for
N. plumbaginifolia
L.
on MS basal culture
media using different combinations of kinetin and IAA. Success in direct
in vitro
regeneration circumvented the
callogenesis step, accelerating the process of
in vitro
regeneration and increasing the chances of obtaining true to
type plants.
Fully expanded healthy 4 week old leaves from
in vitro
grown plants of
N. plumbaginifolia
were excised under
sterilized conditions and cultured on different regeneration media. For enhancing regeneration response different
combination of plant growth regulators (PGRs) were used and leaves from
in vitro
grown plants were cultured on
