Initiation medium supplemented with 4 to 6 mg/l

Initiation of organogenesis

After surface sterilization, the explants were aseptically
inoculated on MS (Murashige & Skoog, 1962) basal medium   augmented  
with   160mg/l   Adenine  
sulfate (ADS), 100mg/l Tyrosine, 0.8 % Agar agar and 3 % (W/V)   sucrose.  
The   growth regulators consisted
of different concentrations and combinations of BAP (6 –Benzyl amino purine) at
the range of 1.0 – 10.0 mg/l. The pH of the media was adjusted 5.8 and thirty
replicates were used for each of the concentrations.

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Growth condition

A clean culture incubation room with provision for
temperature, light and humidity control is preferred. The cultures should be incubated
in the basal MS media supplemented with plant growth regulators. These cultures
were  induced  for 
four  weeks  at 
25±2ºC,  with  16  h
photoperiod and 40% relative humidity in growth chamber, illuminated  by 
cool,  white  fluorescent 
lamps  (40  Watts). The light intensity thus obtained is
in the order of 30 to 50µ E.m2/sec. 
There upon the healthy, contamination free explants should be taken for
next multiplication stage.

Mass multiplication of propagules

The regenerated shoots obtained from initiation cultures were
vertically marked into smaller pieces (usually 2 to 4) and transferred to MS
medium supplemented with 4 to 6 mg/l BAP, 0.1 to 1.2 mg/l Thidiazuron (TDZ) and
0.1 to 0.9 ml/l coconut water for the induction of multiple shoot buds. The
cultures were incubated for 4 to 5 weeks. The shoot buds were organized from
the transversely   cross initiated shoot
buds. At each cycle, organized multiple propagules were transformed to new
organogenic media supplemented with the same concentration of growth
regulators.  To  minimize 
further  blackening,  the meristematic  tissues 
were  transferred  to  the  similar 
fresh medium frequently every 10 to 12 days for about one month by  removing 
the  blackish  tissues 
using  sterilized  scalpel during  the 
initial  phase. 

During subculture, the cultures were checked for bacterial
and fungal contamination which may appear within 15 days of incubation. At each
cycle, organized multiple propagules were transformed to new organogenic media
supplemented with   the same
concentration of growth regulators. 
The  clumps  of 
shoot propagules  developed  on 
multiplication  media  were transformed  to 
elongation  media.  The shoot elongation media   composed of 5 mg/l BAP.  At 
this  stage  the regenerants  were 
maintained  for  2 
-3  weeks  and transformed to rooting media.

Rhizogenesis

At the end of multiple shoot generation cycles, individual
shootlets  attained  the 
height  of  4-5 
cm  were  carefully isolated from the shoot clump and
aseptically transferred to rooting  
media  composed   of  
MS   basal   nutrients augmented with 0.5  –  1.5
mg/l IBA, 0.5  –  1.5 mg/l IAA, 0.5 – 1.5 mg/l NAA and 0.2%
activated charcoal to induce root formation. The observations were made on the
development pattern of plantlets after four weeks of incubation and the data
were recorded.

2.1.2.
Indirect organogenesis:

Plant material and disinfection:

The
leaves of Musa paradisiaca Monthan
cv. Karibale were obtained from plants growing at farmyards of Shivamogga
District, Karnataka, India. These were
treated with 5% teepol (w/v) for 5 min, and rinsed three times with distilled
water. This was followed by surface sterilization with 0.1% (w/v) Mercuric Chloride
(HgCl2) for 5 min followed by washing with sterile double distilled water
inside the laminar airflow chamber to remove traces of HgCl2.

 

Organogenic
callus induction:

The leaf segments (1.0–1.5 cm) were excised
aseptically and transferred to sterile agar-solidified medium. The basal medium
containing MS (Murashige and Skoog, 1962) nutrients agumented with 3% (w/v)
sucrose, 0.8% (w/v) agar and the plant growth regulators (PGRs), pH 5.8
adjusted with 1 N NaOH before autoclaving at 121°C for 15 min. The leaf
segments were cultured on callus induction MS media containing 2, 4-dichlorophenoxyacetic
acid (2, 4-D)              (2 – 4mg/l) and Benzyl amino purine (BAP) (0.0
– 0.9 mg/l). For shoot organogenesis four weeks old leaf derived calli were
transferred onto the regeneration media amended with different concentrations
BAP (2 – 4 mg/l) and Thiadiazuran (TDZ) (0.4 – 0.6 mg/l).

All the cultures were maintained in a growth
room with a 16 h photoperiod (cool, white fluorescent light – 3000 lux light
intensity) and the temperature was maintained at 25 ± 2ºC, with 50 – 80% relative
humidity. The percent explants forming callus, the number of regenerated shoots
per unit callus (callus piece 10 × 10 mm) and shoot lengths were recorded after
8 weeks. The percent induction was calculated using the following equation

Frequency = No. of explants showing response   X100

                                                 Total no. of explants

In
vitro differentiated shoots
measuring 3.0 – 4.0 cm in length were excised and cultured on rooting medium
containing IBA (0.25-1.0 mg/l). The data collected in this study were
subjected to the statistical analysis by using
ezANOVA tool (0.98 versions).

Hardening

Once the
plantlets are ready for shifting outside the laboratory, they are carefully acclimatized  to 
adapt  to  the 
green  house  and 
later  to  least  protected 
field  conditions. During  hardening, 
the  plantlets  undergo 
physiological  adaptation  to 
changing  external factors  like 
water,  temperature,  relative 
humidity  and  nutrient 
supply.   Hardening of plantlets
were carried   out in two steps

i) 
Primary Hardening

            The  plantlets 
from  culture  bottles 
are  shifted  from 
the  laboratory  to 
a  room  at ambient 
temperature  and  kept 
aside  for  four 
days  so that  relative 
humidity  was  less promoting the formation of better
cuticular wax. The plantlet having roots were isolated from the bottle and
washed thoroughly with water to remove the media contents. The roots  were 
trimmed  to  required 
length  that  is, 
to  about  6 
to  7  cm.  
The  plantlets  were dipped 
in  0.1%  Bavistin 
and  placed  in 
portrays  containing  peat 
mixture  as  a 
growth matrix. The trays were covered with polythene and  allowed to harden in green house, optimized  at  27°C,  70% 
RH  and  15,000 
lux2 Periodic  water  spraying 
was  done  on leaves. After ten days plants were ready
to shift to secondary hardening.