International Journal of Aquaculture, 2016, Vol.6, No.8, 1-10
7
After sufficient rooting, plantlets were transferred to the pots having Neopeat (Sterlings Farm Research Services
Pvt. Ltd.) for acclimatization. Agar was removed from the rooted plantlets and washed with tap water. The roots
of each plant were dipped in 0.1% Bavistin solution to avoid future fungal attack and then roots of explants were
inserted carefully into the Neopete. Then potted plants were transferred to the tanks with 2-3 inches water level.
The system is completely covered with polythene and humidity maintained is approx 80% and temperature was
28–30°C and kept there for 15 days. 90% plant survived in the polythene propagator system.
4 Discussion
In vitro
establishment of
L. antipoda
(L.), through vegetative explants presented complications due to excessive
contamination, as has been recorded for other aquatic plants (Balestri et al., 1998; Jenks et al., 2000). The
establishment of aseptic shoot explants of
L. antipoda
(L.) was proved difficult due to the bacterial contamination
which was found to retard the shoot growth and rooting and eventually led to the death of the young plantlets.
Difficulties in the establishment of axenic aquatic plant cultures have
been reported (Dore Swamy and Mohan
Ram, 1969; Madsen, 1985; Godmaire and Nalewajko, 1986). Difficulty in obtaining aseptic cultures has also been
reported for another aquatic plant
, Nympiodes indica
(Jenks et al., 2000).
For surface sterilization of aquatic plants sodium hypoclorite (NaOCl) was effectively used than HgCl
2
. NaOCl
was successfully used in the surface sterilization of
Bacopa monniera
(Tiwari et al., 1998). Our results indicate 10%
NaOCl for 10 min as the most effective method for surface sterilization of
Lindernia
sp. Fungal contamination
was a major problem in the study, so that a systemic fungicide, bavistin was used for inhibiting the growth of
fungus on explants. Pre-soaking the explants in fungicide solution for reducing fungal contamination has been
suggested by Broome and Zimmerman (1972) in Black berry, Sharma et al. (2010) in
Bacopa monneri
and
Thomas et al. (2006) in Papaya (
Carica papaya
). 0.2%
bavistin
is effectively used to kill endophytic fungus. The
results obtained with these studies agree with these reports in the case of bavistin concentration as a fungicide.
MS media with half ionic strength was the suitable media for culture initiation of
Lindernia
sp. Half strength
MS medium has been successfully reported to be the most suitable media for
Ipomea
(Priya et al., 2005),
Myriophyllum
(Sumitha et al., 2005) and
Gymnema
(Anu et al., 1994). The media tried to initiate the axillary bud
development including growth regulator free medium and medium supplemented with various concentrations of
BAP, Kinetin and IAA. Thus, BAP was found to be very effective medium for culture initiation and establishment
when compared to Kinetin and IAA. BAP was reported to be the effective medium in the culture establishment in
Myriophyllum
sp. (Kane and Gilman, 1991) and
Spilanthes acmella
(Haw and Keng, 2003). Shrivastva and Rajani
(1999) reported that out of two cytokinins, BAP was found to be more suitable than Kn. As BAP resulted in
quicker and better response then the latter while, addition of NAA (0.2 mg/l) proved synergistic (Srivastava et al.,
2002). Hence, different combination of BAP and NAA was selected for the multiplication of
L. antipoda
L
explants. Among the different combinations of BAP and NAA was tried, the maximum shoot proliferation and
shoot length was obtained on BM
3
Media (MS+1mg/l BAP+0.2 mg/l NAA).
The stimulatory effects of BAP on bud break multiple shoot formation reported earlier in
Ocimum sanctum
(Patnaik and Chand, 1996). However, further increase in concentration of BAP above the optimum level reduced
the rate of shoot multiplication. According to Hongrat et al.
(2005), maximum number of shoots of
Cryptocoryne
cordata
was regenerated in MS medium supplemented with BAP at 1 mg/l.
In BM
3
media 1mg/l BAP & 0.2 mg/l NAA stimulates high multiple shoot proliferation and shoot length. In
Paederia foetida
and
Centella asiatica
multiple shoots were obtained in MS medium supplemented with BAP 1.0
mg/l (Singh et al., 1999), and for
Rauwolfia serpentina
MS medium supplemented with BAP and NAA (Sehrawat
et al., 2001) was the best
.
In
Aponogeton madagascariensis
2 mg/l BAP and 0.5 mg/l NAA was the optimum
formula that could increase the number of plantlet (Van Bruggen, 1987).
