The was dispensed into culture bottles, these bottles

 

The
success of plant tissue culture largely depends on the type of nutrient medium,
including its chemical composition and physical form (Murashige, 1974). Various types
of nutrient media  have been reported for
banana shoot tip culture viz.,  B5 media (Gamborg et al., 1968), N6 media (Chu
et al., 1975), Linsmaier and Skoog (LS) (Linsmaier and Skoog, 1975) media etc.
but most of the investigators used modified Murashige and Skoog (MS) media
(Brown et al., 1995).  The microbial
contaminants are normally invade the medium right from the beginning and the
exposure time varies with the volume of the liquid to be sterilized.

In
the present study, the high grade chemicals were used (Hi-Media, India) for
stock solution preparation and the growth regulators were of Sigma made (Sigma
Research Laboratory, America) that are comparatively free from impurities. Stock
solutions of major and minor salts, vitamins and plant growth regulators were
prepared in double distilled water at required concentrations and kept in refrigerators
at 4oC until subsequent use. The stock solutions are
examined periodically for possible precipitation or contaminants. In the
present investigation MS (Murashige and Skoog, 1962) nutrient medium, was used to
standardize the banana micropropagation protocol.

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The
MS nutrient media was prepared by adding appropriate quantity of nutrients and
grow regulators into a one liter standard flask containing double distilled
water.  The carbon source, 3% sucrose was
added and dissolved in the media. The pH of the medium was adjusted between 5.6
– 5.8 by adding 0.1 N NaOH or 0.1 N HCl using electronic digital pH meter. The
media was polymerized with 0.8 % bacteriological grade Agar – Agar (Hi-Media,
Mumbai) on hot plate, well whirled for proper mixing and homogenized. After
homogenization about 40 ml of media was dispensed into culture bottles, these
bottles were closed with ebonite screw caps and autoclaved at 15 lb/in2 (1200C)
for 15 minutes, care must be taken not prolong the autoclaving beyond times as
it may bring about degradation of sugars, vitamins and growth regulators and
even of agar – agar. The media is stored for about six days after sterilization
to observe the presence of contamination by microorganisms.

3.2.
Direct
organogenesis: 3.2.1. Material collection and maintenance The healthy four week
old sword suckers from Musa paradisiaca Monthan cv. Karibale weighing about one
kilogram uniformly sized were identified from elite plants of superior
qualities such as bunch weight of above 40 kg’s, more than ten number of hands
in a bunch, more than eighteen number of fingers in second hand from top and
length of peduncle more than 20 to 24 inches was selected and cut from the
pseudostem 15 cm above the base level, weighing 500 to 1500 g were collected from
the banana fields of a progressive farmer and used as starting material for
micropropagation.

These
suckers were transported to the Department of Biotechnology and Bioinformatics,
Kuvempu University, Shankaraghatta and arranged on sand bed under shade and
watered regularly. Periodically fungicides/ bactericides are applied to these
suckers till they are taken for initiation process. The disinfection of suckers
was carried out thoroughly washed with tap water, soaked in a solution of 0.2%
bavistin and 0.1% streptocyclin for overnight. The corm portion was trimmed to
a width of 1 inch and height about 2 inches, and sterilized using 0.001 M
teepol solution for an hour and washed 3-4 times using sterile distilled water to
remove traces of teepol.

3.2.2.
Surface sterilization: The trimmed explants after removing a whorl of leaf were
surface sterilized in laminar air flow chamber with 70% ethanol for six min and
0.1% mercuric chloride solution (with few drops of tween 20) for ten minutes. Rinse
the explants 3-4 times repeatedly with sterile distilled water for 5 minutes to
remove the traces of sterilant. The process was repeated for once again and the
corm portion was sterilized with 1 % sodium hypochlorite (with few drops of tween
20) for 15 min. and rinsed 3-4times repeatedly with sterile distilled water for
5 minutes. Then the cut portion of the sucker was treated with antibiotic
solution containing Cifotaxime 0.1% and Gentamicin 0.05 % for 5 min. After
surface sterilization suckers were treated with ascorbic acid (100mg/l) for 10
min to avoid tissue blackening due to phenolic exudation.

3.2.3.
Initiation of organogenesis:

The
surface sterilized explants were aseptically inoculated on MS (Murashige &
Skoog, 1962) basal medium supplemented with 160mg/l Adenine sulfate (ADS), 0.8
% Agar agar, 3 % sucrose along with the growth regulator BAP (6 –Benzyl amino
purine) at the range of 1.0 – 10.0 mg/l. The pH of the media was adjusted to 5.8
and thirty replicates were used for each of the concentrations.

 

 

3.2.4.
Growth condition:

The
cultures were maintained in incubation room with controlled temperature, light
and humidity conditions. The cultures were incubated for four
weeks at 25±2ºC, with 16 h photoperiod and 40% relative humidity illuminated by
cool, white fluorescent lamps (40 Watts). After shoot elongation the cultures
were taken for multiplication stage.

3.2.5. Mass
multiplication of propagules

The regenerated shoots were vertically marked into
smaller pieces (usually 2 to 4) and transferred on to the 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 multiple shoots formed were transferred to
new organogenic media supplemented with the same concentration of growth
regulators. To minimize tissue blackening due to phenol exudation, 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 subculture, the multiple shoots were
transferred to the fresh media supplemented with the same concentration of
growth regulators. The clumps of shoot propagules developed on multiplication
media were transferred to shoot elongation media supplemented with 5 mg/l BAP. At
this stage the regenerants were maintained for 2 -3 weeks and transferred on to
the rooting media.

3.2.6. 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.

 3.3.
Indirect organogenesis:

3.3.1. 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 washed three times with sterile 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.

 3.3.2.
Organogenic callus induction:

The leaf segments (1.0–1.5 cm) were excised
aseptically and transferred onto the callus induction MS media augmented with
3% (w/v) sucrose, 0.8% (w/v) agar (PGRs), pH 5.8 and the plant growth
regulators 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 and shoot lengths were recorded after 8 weeks of incubation.

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 augmented with 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).

 3.3.3. Hardening

Once the plantlets are
fully developed they are carefully acclimatized 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 about 6 to 7 cm and the plantlets were dipped in 0.1% Bavistin to protect
from fungal infections. Then the plantlets were placed in portrays containing
cocopeat mixture along with growth nutrients. The trays were covered with
polythene and allowed to harden in green house, optimized at 27°C, 70% RH and
15,000 lux2. After ten days, plants were transferred to secondary hardening process.

ii)                 
Secondary
Hardening

After primary hardening, the plantlets are
transferred from micropots to 18 x 15 cm polybags. Base substrate is generally the
mixture of sand, red soil and cow dung in 1:2:1 ratio. Initially,         in vitro plantlets are maintained in low
light intensity under shade nets and 70% RH (Relative humidity). After 15days of
hardening the light intensity has been increased and 40% RH maintained. After 5
to 6 weeks, the plants become ready for field planting having 3 to 5 well
developed leaves and a good mass of fibrous roots. During the process of
hardening, the stocks were evaluated for phenotypic variations at weekly
intervals. These could include vegetative deformities like dwarfism, leaf
variegation, rosette foliage and leaf crinkiness.

3.3.4. Manuring and plant protection in
nursery

Plantlets
should be off two to three weeks old before any fertilizer is applied. In 1000
ml of water, 0.5 g of NPK (19:19:19) was applied twice a week till three to
four weeks. After 3-4 weeks, doubling the dosage of manure helps better growth
of the plantlets in nursery and filed condition. Regular applications of
fungicide or bactericide avoid the risk of damage by pest and diseases.

To protect the plant from the pathogens and
other disease, 25 g of copper oxy chloride, 1 g streptocycline and 10 g of
bavistin was mixed in ten litres of water and sprayed regularly.

3.3.5. Field preparation and planting of in
vitro raised banana plants

Well-developed plants were acclimatized in
green house for about two months and transferred to the field to evaluate the
agro-morphological variations along with vegetatively propagated Karibale
plants of the same age at the experimental farm.

Cultivation practices such as, fertilizer
application and irrigation followed conventional protocols (Chattopadhyay et
al., 2001). Planting of tissue culture banana can be done throughout the year
as per the market demand except in severe
winter and during heavy rains. The land was ploughed 2-4 times and levelled
using Ratovator to break the clump and bring the soil to a fine tilt. During
soil preparation basal dose of FYM is added and thoroughly mixed into the soil.
A pit size of 60cmx 60cm x 60cm was prepared and filled with 8kg of FYM, 200 gm
of Neem cake and 20 gm of carbofuran. When the plants are 30 cm, polybag was
separated from the plant without disturbing the root ball of the plant and then
plants are planted in the pits keeping the pseudo-stem 4cm below the ground level.

3.4. Evaluation of morphoagronomic characters
of the in vitro regenerants

To evaluate the phenotypic variations, the in vitro raised plants after planting in
farmyard were subjected to field trails at standing crop stage. For field
evaluation, following yield parameters were recorded after two months of
bunching such as height of the plants (in ft), number of leaves, length of the
leaves (in cm), width of the leaves (in cm), length of the bunch (in cm),
weight of the bunch (in kg), number of hands in a bunch, number of fingers in a
hand, length of the finger (in cm), girth of the finger (in cm) and girth of
the pseudo stem (in cm) were recorded.

All the data were analyzed by using ezANOVA
software (version – 0. 98).

 i)
Plant height: The height of the plants was measured from base of the plant to
the tip of main shoot from five plants and mean plant height was calculated and
expressed in feet.

ii) Girth of the pseudostem: The girth of the
pseudostem of the plants was measured at the base above two feet from the
ground level using the tape from five plants and expressed in cm.

iii) Number of leaves per plant: The number
of leaves emerging from the main pseudostem of plants from five plants were
counted and the mean value was worked out.

iv) Length of the leaves:  The length from base of the leaf to the tip
was measured and expressed as length of the leaves and mean length of the
leaves was expressed in cm.

v) Width of the leaves: The width of each old
leaves of the plants was recorded and mean value was worked out.

vi) Length of the bunch: The length from base
of the bunch to the point where inflorescence ends was measured from five
plants expressed as length of the bunch and mean value was expressed in cm.

 

vii) Weight of the bunch: The weight of the
bunch from five plants was recorded at the harvesting stage and the value was
expressed in Kilograms.

viii) Number of hands in a bunch: The mean
value of hands in a bunch from five plants were computed and recorded in this
parameter.

ix) Number of fingers in a hand: From each
hand of a bunch the fingers were calculated and the mean value of fingers was
computed.

x) Length of the finger: In this parameter
the length of the finger from each bunch of five plants was calculated and the
mean value was expressed in cm.

xi) Girth of the finger: The girth of the
finger was calculated by using Vernier caliper and the data is recorded and
expressed in cm.

 

x

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