International Journal of Aquaculture, 2016, Vol.6, No.8, 1-10
2
aquarium plants namely
Aponogeton
sp. (Kukulczanka et al., 1980),
Cryptocoryne lucens
(Kane et al., 1990),
Anubias barteri
(Huang et al., 1994),
Cryptocorine wendtii
(Kane
et al., 1999; Mo and Jiang, 2003),
Ludwigia
repens
(Ozturk et al., 2004),
Cryptocorine becketti
,
Cryptocorin lutea and Rotala rotundifolia
(Micheli et al.,
2006). However, there has been no report on the effect of medium on the shoot proliferation of
Lindernea
sp. The
objective of the present study is to establish an efficient micropropagation protocol for the mass production of
L.
antipoda
(L.) plantlets.
2 Materials and Methods
2.1 Explants collection and preparation
Disease free, young and healthy nodal explants were collected from Malabar Botanical Garden, Olavanna,
Kozhikkode, Kerala state, India.
Terminal shoots bearing 4 to 5 nodes were cut off from plants growing in the
field. They were washed under running tap water for 10 minutes followed by washing with few drops of Cleansol
solution. Finally, thorough washing was done for several times using sterile distilled water.
2.2 Explants sterilization
The leaves were removed from the explants and washed under running tap water for 30 minutes in order to wash
off the external dust/contaminants. After this treatment
,
the explants were surface disinfected using a strong
surface sterilant, sodium hypochlorite (4% Chlorine) of different concentrations for various time intervals (Table
1)
.
Then the explants were removed from the sterilizing solution and rinsed thoroughly twice with sterile double
distilled water. In the next step, explants were soaked in an aqueous solution containing 150 mg/l bavistin (BASF,
India Limited) and 0.03 % Ampicillin for 10 minutes in a laminar flow hood. Then the explants were thoroughly
washed twice with sterile double distilled water for 5 minutes.
Table 1 Response of
L. antipoda
(L.) for surface sterilization with various concentration of NaOCl
Sl. No
Treatment
Time
Rate of Explants survival after 14 days (%) Rate of Contamination
1.
Control
-
0
100%
2.
T0.5% NaOCl
10 min
20%
80%
3.
T1.5% NaOCl
15 min
24.5%
75.5%
4.
T2. 5% NaOCl
20 min
39.4%
60.6%
5.
T3.10% NaOCl
5 min
45.6%
54.5%
6.
T4.10% NaOCl
10 min
60.2%
39.8%
7.
T5.10% NaOCl
20 min
10.4%
9.6%
2.3 Initiation of cultures
Sterilized explants were transferred aseptically to sterilized glass plate and the material was cut in to both basal as
well as the top portion to remove undesirable/dead portions after surface sterilization. For shoot initiation from
explants mainly two different strength of MS media were used, Full strength MS (Full MS) and half strength MS
(Half MS). Each nodal explant was aseptically placed in an erect position in the test tube containing medium.
Tubes were kept in the growth room with temperature conditions 25±2°C, with a photoperiod of 16 hours daylight
and 8 hrs night break under the cool white fluorescent light of average 2500 lux (cool white fluorescent tube light
40 W GE).
2.4 Establishment of cultures
After approximately 9-10 days of inoculation, the axillary bud break was seen in some explants. When the
explants attained bud proliferation, these cultures were transferred to jars containing fresh medium. After 21- 25
days of incubation, the initiated plants were taken out of the test tube using clean and sterilized forceps, medium
adhered to the plants was removed, undesirable/brownish leaves were removed from the plants and were put into
the culture bottles containing autoclaved semi-solid media having the same combinations as that for the culture
initiation. The bottles were then placed in culture room under the standard
conditions of temperature (25 ±2°C)
