chapter 3 materials and methods -...

Thesis by N C Vora Research Guide: Dr. Y T Jasrai

CHAPTER 3

MATERIALS AND

METHODS


III. Materials and Methods

The investigation was carried out at the Department of Botany,

University School of Sciences, Gujarat University, Ahmedabad (Gujarat),

during the year 2007-2010. The chapter contains the details regarding the

material and methodology for the experiments carried out to find cost-

reducing alternatives for the micropropagation of banana var Grand

naine.

3.1 Source of explants

One hundred suckers of banana var Grand naine were brought

from Gujarat Green Revolution Company, Umareth, Gujarat, in two

batches in 2007-08. The shoot tips as explants were isolated from these

suckers to follow the micropropagation protocol (Cronauer and Krikorian,

1984). During shoot-multiplication cycles, multiple-shoots containing

approx 5 shoots were utilized to conduct each experiment on the cost-

reducing alternatives. For the experiment on regeneration of banana

plantlets through somatic embryogenesis, male flowers were isolated as

the explants. For this, three inflorescence of banana (var Grand naine)

Materials and Methods


40

were brought from Bharuch, Gujarat, to follow the published protocol

(Strosse et al, 2003).

3.2 Culture media

The most widely accepted MS medium (Murashige and Skoog,

1962) was used at each stage including initiation, multiplication and

rooting for the shoot-tip culture. The composition of MS basal medium as

stock solutions is presented in Table-4. The requirement of PGR varies at

each stage, the details of which are given at the appropriate places.

3.2.1 Chemicals

Analytical grade chemicals from various companies including

Thomas and Baker, SD Fine Chemical Ltd, Astron, Merk and SRL were

used in the preparation of media.

3.2.2 Media preparation for in vitro multiplication of banana

The stock solutions were prepared for 10 liter media (Table-4), prior

to the media preparation by dissolving specified amount of each

constituent one by one in double DW. The final volume of major, minor

and iron stock solutions was made to 500 ml and that of vitamin stock was



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Table-4: Composition and components for MS basal medium (Murashige and Skoog, 1962)

Stock Constituents Quantity Stock/l

medium 1 liter 10 liter

A Major Stock gm gm 50 ml

- Ammonium nitrate (NH4NO3) 1.650 16.5

- Potassium nitrate (KNO3) 1.900 19.0

- Calcium chloride (CaCl2 ּ 2H2O) 0.440 4.4

- Magnecium sulphate (MgSO4ּ7H2O)

0.370 3.7

- Monobasic potassium phosphate (KH2PO4)

0.170 1.7

B Minor Stock mg mg 50 ml

- Potassium iodide (KI) 0.830 8.3

- Boric acid (H3BO3) 6.200 62.0

- Manganese sulphate (MnSO4 ּ 4H2O)

22.30 223.0

- Cobalt chloride (CoCl2 ּ 6H2O) 0.025 0.25

- Zinc sulphate (ZnSO4 ּ 7H2O) 8.600 86.0

- Sodium molybdate (Na2MoO4 ּ 2H2O)

0.250 2.5

- Copper sulphate (CuSO4 ּ 5H2O) 0.025 0.25

C Iron Stock mg mg 50 ml

- Sodium EDTA (Na2 EDTA ּ 2 H2O)

37.3 373

- Ferric sulphate (FeSO4 ּ 7H2O) 27.8 278

D Vitamin Stock mg mg 10 ml

- Nicotinic Acid 0.5 5

- Pyridoxine HCl 0.5 5

- Thymine HCl 0.1 1

E Myo Inositol 100 mg

F Glycine 2 mg

G Agar-agar 8 gm

H Sucrose 30 gm



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made up to 100 ml. These stock solutions were stored in the refrigerator in

the amber coloured glass bottles, each with 500 ml capacity with a label,

depicting the name of the stock solution and date of preparation. For

preparation of 1000 ml of MS medium, the major stock (50 ml), minor

stock (50 ml), iron stock (50 ml) and vitamin stock (10 ml) were taken in

series in a flask/beaker of 1000 ml capacity and diluted with double DW.

Cytokinin, known weight of BA (4-5 mg/ l), was dissolved in 2-3

drops of 0.1 N KOH and added in to the medium for multiple-shoot

formation. 2 mg of Glycine, 100 mg of Myo Inositol and 30 gm of Sucrose

TC was added. The medium was stirred in between to dissolve each of

these completely. The pH was adjusted to 5.8 + 0.1 with a pH meter

(Testronix, India) using either 0.1 N HCl or 0.1 N KOH. The medium was

slightly heated to dissolve Agar-agar (8 gm). Finally the volume was made

up to 1000 ml with double DW, stirred and dispensed into the culture

bottles (10X5 cm), which were cleaned and pre-heated in oven at 70 ºC for

approx 2 h. The media was sterilized in a vertical autoclave (Lab Line,

India) at 121 ºC and 15 psi pressure for 20 min. The media was then stored

in the culture room for 1-2 days prior to its use for inoculation.



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3.3 Aseptic measures (Razdan, 2006; Dodds and Roberts, 1985)

The entry of the microorganisms hindering the growth of the

explants was prevented during the manipulation work by employing

certain measures. The surfaces of the working area, treys, test-tube stands,

etc were swabbed with ethyl alcohol prior to its use every time. All the

glassware were washed with soap solution (5 %, Laboratory detergent,

Burgoyne Co), followed by a thorough wash with tap-water, rinsed with

distilled water and dried in hot-air oven at 70 ºC (2 h). The washed and

dried glassware were stored in dust-free cabinet till their use. Sterilization

in autoclave was carried out for the media dispended in glass culture

bottles (10X5 cm), paper-wrapped stainless steel plates (14 cm) or glass

petridishes (10 cm) for manipulation, Lab-coat (apron) etc prior to their

respective use.

Laminar Air-flow cabinet (Labline, India) was used for the

inoculation and related manipulation process to maintain the surrounding

air free from the microorganisms. The surface of the hood was swabbed

with 70 % ethanol. All the required articles during manipulation except

the explants, like media-vessels, petridishes, cotton, forceps, scalpels,

ethanol, glass-bead sterilizer (Yorco Co, India) or spirit lamp etc were



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placed in the laminar air-flow cabinet for UV sterilization (20 min) prior to

aseptic manipulations. Forceps and scalpels were first dipped in ethanol

and heated for a while in the glass-bead sterilizer before their use during

inoculation each time. Similarly, thoroughly washed hands were

frequently sterilized with ethanol during aseptic manipulation. Apron,

mask and cap as a precautionary measure were used during all the

transfers.

Fumigation (overnight) was carried out in the inoculation room on

regular intervals with 4 gm of Potassium Permanganate (KMnO4) mixed

with 8 ml of formaldehyde (Connolly and Fletcher, 2007). The floor of

inoculation room as well as culture room was cleaned regularly with

phenyl solution (Lysol, Nice Chemical Co) to keep the area dust and germ-

free.

3.4 Culture conditions

The cultures were maintained at 25 + 1 ºC temperature in the

culture room (air conditioner, Samsung Co). The photoperiod of 16 h light

and 8 h dark with 55 µmol m-2s-1 PPFD was regulated with the help of

fluorescent light (Phillips Co) for the cultures.



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3.5 In vitro propagation of banana var Grand naine

3.5.1 Initiation of cultures

Suckers were washed with tap water and their outer coverings and

undesired sucker portions were carefully removed with knife. These were

then kept submerged in clean water to prevent browning (oxidation) of

the cut tissues that occurs due to phenolic compounds. The suckers were

then trimmed till shoot-tip of approx 3-4 cm length was obtained, attached

with its basal sucker portion bearing active meristematic zone. They were

then washed with double DW and treated (20 min) with 0.05 % of

Carbendazim (Bavistin), a fungicide and 0.1 % of activated charcoal by

constant rotating the solution in a beaker (1000 ml) with 6 shoot tips at a

time. The solution was removed and DW wash was given (2-3 times). A

wash of soap solution (10 %, Teepol) was given (10 min) by frequent

shaking. All the traces of soap solution were removed by repeated rinsing

in double DW. Further sterilization procedure was carried out under

aseptic conditions in the laminar airflow hood. The leaf scales were

removed further using scalpel, forceps and pertridishes. The base was also

trimmed in accordance with the diameter of the attached shoot-tip,

maintaining the cubic shape of the base. The explants were then treated (8

min) with Mercuric chloride (HgCl2) solution (0.1 %) prepared in sterile



46

double DW by continuously shaking in a conical flask (500 ml). The

solution was then drained out in a beaker kept for discarding solutions

and wash of sterile double DW was given (3-4 times). The explants were

now again trimmed carefully with scalpel and forceps till the shoot-tip

appear to be as thin as a needle, without causing any injury, so that it

could remain intact with the cubic base. The explants were then quickly

inoculated in the test tubes (15.5X3 cm) with the help of forceps to initiate

the cultures on MS medium (Fig. 2 A) containing BA (3 mg/l).

3.5.2 Shoot proliferation

The explants started showing greening and enlargement within first

2 weeks of inoculation (Fig. 2 B). The shoot-tip continued to grow in a

single shoot at the end of the 4th week (Fig. 2 C, D). Incisions were made

on the shoot tip at the time of the first subculture to break the apical

dominance (Fig. 2 E). The explants were then transferred to the glass

culture bottles (10X5 cm) having wider mouth as compared to the test

tubes (Fig. 2 I). Single multiple-shoot explant was maintained per bottle on

MS medium with BA (4-5 mg/l) during the shoot-multiplication cycles.



47

Fig. 2: Establishment and growth of shoot-tip explant of banana (var Grand

naine) A. Shoot-tip explant showing greening after a week of inoculation; B. Enlargement of shoot-tip after 2 weeks of inoculation; C, D. Development of shoot after 4 weeks; E. Superficial incisions on the shoot after 1st subculture; F to H. Successive development of multiple-shoots after 1st subculture; I. Initial multiple-shoots during shoot-multiplication cycles; J. Growth and multiplication of multiple-shoots after 4 weeks; K. Elongation growth of shoots; L. Rooting of shoots; M. Acclimatization of banana plantlets showing successive growth after ex vitro transfer in thermocol glasses containing coco-peat (Horizontal bar = 1 cm)

The multiple-shoots were sub-cultured at every four week intervals.

During this, the clumps of multiple-shoots (Fig. 2 J) were separated into 2-

A B C D E

F G H

KJ L

I

M



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3 smaller bunches, each containing approx 5 shoots. These smaller

bunches were transferred to the fresh medium so that each culture bottle

contained one clump of multiple-shoots.

3.5.3 Rooting and acclimatization

Single shoots of 7-8 cm length were transferred to half-strength MS

media with IBA (3mg/l) for rooting (Fig. 2 K, L). The rooted shoots were

transferred for acclimatization in thermocol-glasses (8X5 cm) containing

coco-peat. The coco-peat was treated (overnight) with Bavistin solution

(0.05 %). During ex vitro transfer for hardening, the plantlets were first

dipped in 0.05 % of Bavistin solution (2 min) to remove the adhered media

with Agar-agar; planted in thermocol glasses (8X5 cm) containing coco-

peat and placed under the shade cover. Initially, Bavistin solution (0.01 %)

and ¼ strength of liquid MS medium were sprayed on these plantlets.

After 4-6 weeks of ex vitro transfer, plantlets attaining a height of around

20 cm with approx. 8 leaves (Fig. 2 M) were transferred to black perforated

polythene bags (20X8 cm) containing a mixture of soil: vermiculite: sand

(1: 2: 1, v/v/v; desirably yellow soil with pH value 7-8 and high P, K

content) without disturbing the roots. Plantlets were irrigated every 4th

day or as per the need. After 12-14 weeks of ex vitro transfer, gradually the



49

covering was taken off for short span of time and time for the exposure to

the outer environment was successively increased.

3.6 Experiments on cost-reducing alternatives for shoot

proliferation

Several alternatives were evaluated as cost-reducing component

during in vitro shoot-multiplication of banana. For each experiment, MS

medium (Table-4) with BA (4 mg/l) was used as the control. For each

experiment, 15 replicates were used per each alternative. The effect of

different alternatives on in vitro shoot-multiplication was recorded after

four weeks in terms of increase in number of shoots, length of the largest

shoot and number of leaves.

3.6.1 Alternatives for the gelling agent

Different alternatives were used to substitute Agar-agar in the

medium. These included different gums, starches, support matrices and

synthetic gelling agents. These substances included gums, such as Guar

gum (3 %), Tragacanth gum (3 %), Karaya gum (3 %), Xanthan gum (6 %),

Carraggenan gum (2 %); various starches like Corn starch (14 %), Tapioca

Starch (22 %), Potato Starch (7 %), Starch soluble (7 %), which were

obtained from HiMedia and Astron Co and various support matrices like



50

Cotton (2gm/40ml liquid medium), Polystyrene foam, Plastic scrubber,

Luffa sponge, Coir, Hemp, Glass beads, Plastic beads, Marbles, Stones

(differently coloured pebbles) and Blotting papers (in layer-form as well as

pulp-form). In addition to these, Isabgol (3 %), Wheat flour (8 %), Rice

flour (11 %), Acid-washed sand, Vermicompost; Synthetic gelling agents

such as beads, named as Crystal-mud (1 ml liquid Medium/ 2 mm sized

bead), ready-made Polymer gel (both of them swell by absorbing water,

which are utilized for the indoor plants) and Acrylic (20 %) were also

used. Moreover, Agarose (0.7 %) and Gelrite (0.2 %) were also utilized to

compare their effect on shoot-multiplication with Agar-agar. Certain

agents, in addition to their sole usage, were also tried in combination with

Agar-agar (0.4 %), which included Corn starch (8 %), Guar gum (1.6 %),

Karaya gum (5 %) and Tragacanth gum (4 %). The appropriate

concentrations of these agents at which the media could remain firm

enough to inoculate the explant within, even after their sterilization in

autoclave were first decided by trying their various amounts in DW.

3.6.1.1 Procedure for using different alternatives of gelling agent

The medium containing various gums, starches, rice flour, wheat

flour, Agarose, Gelrite etc were boiled for a while on the gas stove before



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their sterilization in autoclave, in a same manner as Agar-agar, to dissolve

them properly and to obtain their gelling ability. But this was not required

for Tragacanth gum and Karaya gum, where weighed samples of

coagulated gum particles were directly added to each culture bottle

separately containing 40 ml liquid MS medium for the shoot-

multiplication. Because powdered form of these two gums were found to

loose the capacity of gelling. All the gums were readily soluble in water

but Guar gum was first dissolved in 2-3 ml of Acetone (pure) to prevent

undesired coagulation. The pH values of the media containing Tragacanth

gum and Karaya gum were adjusted to 7.2 and 7.3 to prevent the fall in

post-autoclave pH.

Various support matrices like Cotton (absorbent), Polystyrene foam,

Plastic scrubber, Luffa sponge and Blotting paper layers were cut in a

round shape, keeping the diameter (5 cm) same as the culture bottle. The

pieces of the Polystyrene foam, scrubbers, beads and marbles were

washed in Teepol soap solution, followed by thorough wash of tap water

and DW and then dried in air. Coir and hemp were sterilized in DW first

for several times until it remain clear without turning brown or yellow in

colour. Sand was treated with 10 % of hydrochloric acid (overnight) and



52

washed thoroughly with water, until it showed neutral pH value (7) and

then dried in air prior to its use. All these support matrices, including

Vermicompost, Blotting paper and Sand (acid-washed) were first

sterilized in autoclave alone and then again with the liquid medium for

the shoot-multiplication. Liquid medium was poured in each culture

bottle containing various support matrices just to cover their upper surface

with medium. Explants were inoculated on these support matrices so that

their lower surface remained in contact with the liquid medium.

The Synthetic Crystal-mud beads at specified amount were added

separately in the culture bottles containing the liquid medium for shoot-

multiplication. These were allowed to swell till the medium was absorbed

by them completely (2 h) and then sterilized in the autoclave.

Liquid medium (10 ml/culture bottle) without any substratum was

utilized for static cultures in both culture bottles (10X5 cm) and polythene

bags (8X5 cm). Rotary shaker was used to agitate culture bottles with

liquid medium (85-90 rpm) for in vitro shoot-multiplication.



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3.6.2 Alternatives for the Carbon-source

3.6.2.1 Different sugar-forms

Media was prepared for shoot proliferation with 12 different types

of sugars, each at 3 %, namely Sucrose TC (as control), Sucrose AR,

Sucrose LR, Sucrose GR, Sucrose Extra-pure, Glucose (Dextrose), Fructose,

Table-sugar, Crystal Sugar, Refined Jaggery, Organic Jaggery and Honey.

For this, different grades of sucrose, dextrose and fructose were obtained

from HiMedia, Burgoyne and Thomas and Baker Co respectively. The

experiment was repeated thrice, each time with five replicates of each

sugar-form.

3.6.2.2 CO2-enriched air

Elevated CO2 (1%) was used as a supplementary carbon-source with

reduced level of Sucrose, viz 1 % and 2 % so as to make lesser

consumption of sucrose for the purpose of cost-reduction. For CO2-

enrichment, 100 ml of beaker (Borosil, India) containing 3 M solutions of

KHCO3 and K2CO3 mixed in the ratio of 62/38 (v/v) was placed in air-

tight chambers (Solarova et al, 1989) of 28X28X20 cm3 size. CO2-free

atmosphere was maintained in the chamber by placing a 100 ml beaker

containing 10 % of KOH solution. Each chamber contained 5 replicates of

the cultures. Another set was simultaneously maintained under ambient



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condition with 0.03 % of CO2 outside the chamber. The effects of

photomixotrophic conditions were studied after four weeks of incubation,

in terms of increase in the number of shoots, length of shoot, number of

leaves, chlorophyll content, leaf area of newly emerged leaf and total fresh

and dry weight. Chlorophyll content was determined from the leaf

samples (Hiscox and Isaraelstam, 1979). A portable leaf area meter

(Systronics, India) was used to measure the leaf area.

In another experiment, elevated CO2 (1 %) was provided to cultures

with 3 % fructose instead of sucrose in the medium to detect its advantage,

if any, as it was found to provide maximum rate of shoot-multiplication

when used as a sole carbon-source over sucrose. During this experiment,

elevated CO2 was provided to the culture in the air-tight chambers of

(25X25X18 cm3) in the same way as described earlier, each containing 5

replicates of the cultures.

3.6.3 Alternatives for culture vessels

Four different culture vessels were evaluated. The culture vessels

included vented-culture bottles (10X5 cm), non-vented culture bottles

(10X5 cm), conical flasks (500 ml) with cotton-plugs and Polythene bags



55

(8X5 cm). Both the polythene bags and media were sterilized separately in

autoclave and the sterilized media was poured in the bags under laminar

airflow hood. The opening of the polythene bag was sealed with the help

of a sealing machine (Vijay Heat India Co) and then kept in standing

position in the culture room for solidification of media. After inoculation,

again their opening was sealed with the help of a sealing machine.

3.6.4 Alternatives for sterilization of media

In place of using autoclave, different ways were tried to sterilize the

media effectively, which can reduce the consumption of electricity, energy

and time. These included the sterilization of media in a microwave oven

(Samsung Co), use of sterilizing agents- chlorine tablet (C3Cl2N3NaO3) and

Sodium hypochlorite (NaOCl). The use of chlorine (0.0003 %) and sodium

hypochlorite (0.0003%) for media sterilization was reported earlier

(Teixeira et al, 2006). The culture bottles containing media (40 ml/bottle)

were subjected to different wattages (180-900 W) in a microwave oven

(Samsung Co) for different periods of time (10 sec to 5 min) to standardize

the sterilization process. The sterilized media was used for shoot

proliferation.



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3.6.5 Alternatives for DW

Media was prepared with Rain water, RO water, Mineral water and

Tap water in place of double DW for in vitro shoot-multiplication. For each

treatment 15 replicates were used and inoculation was carried out in all of

them at a time.

3.6.6 Alternatives for Plant growth regulator

The extracts of fresh fruits were used at different concentrations (3

%, 5 % and 10 %) in place of BA. These included Sweet-lime juice, Tomato

extract and Sweet corn juice. The pH of the media containing these fruit

juices was adjusted to 5.8 prior to sterilization.

3.6.7 Alternatives for light

Monochromic lights of different wavelengths were provided to the

cultures during in vitro shoot-multiplication using cellophane papers of

different colours viz Red, Blue, Green and Yellow. Cultures provided with

normal cool white fluorescent light were used as the control,

simultaneously. The cultures were also maintained in complete dark and

its effect on shoot-multiplication was evaluated.



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3.6.8 Alternatives for culture room

The cultures were kept under a glass chamber in a green house

(without temperature control) to avail natural diffused Sun-light. Shoot-

multiplication of banana was evaluated twice in different seasons with 6

replicates, each time.

3.6.9 Alternatives for hardening area

In place of Greenhouse that consume electricity to maintain

humidity and temperature, besides exhibiting higher installation cost,

various low-cost options were evaluated for acclimatization of banana

plantlets.

a. Tunnels (92X64X52 cm3) of iron mesh covered with transparent

polythene sheet (4 mm) was used. The in vitro raised plantlets were kept

for hardening as per the protocol reported earlier (Jasrai et al, 1999). The

floor of the tunnel was prepared with the sand bed, which was kept wet

by pouring water. Moisture was maintained by sprinkling water with the

help of a sprayer towards the roof 3-4 times a day or when needed.

b. Similar tunnel covered with jute sheet was also used to maintain high

humidity during summer. The lower ends of the jute sheet were dipped in



58

water tanks from both the sides. Moreover, water was sprinkled on the

tunnel with sprinkler and also drip-wise from a tube. The relative

humidity of 85-90 % was maintained in the tunnel, which was regularly

checked using dry-wet thermometer.

c. The in vitro raised plantlets were kept in the air-tight boxes (43X34X30

cm3) made up of thermocol sheets with transparent (polythene sheet) lids

for acclimatization. The humidity was maintained inside by sprinkling

water with a sprayer at regular intervals.

The protocol for ex vitro transfer for acclimatization of banana

plantlets was repeated as described in 3.5.3 every time. Moreover, the

plantlets were also transferred to soil mixture directly in the thermocol

glasses (8X5 cm) with soil: vermicompost: sand (1: 1: 1, v/v/v) and their

survival rate was compared with those using coco-peat as the substratum

in the thermocol glasses.

3.6.10 Favourable alternatives in a combined manner

Among all the above mentioned alternatives, those which showed

the best result and found effective for the purpose of cost-reduction were



59

used altogether to verify their combined effect on in vitro shoot-

multiplication of banana.

3.7 RAPD Analysis

The in vitro raised banana plantlets were screened for clonal fidelity

using RAPD markers.

3.7.1 Plant Material

Young leaves from the mother plant and randomly selected in vitro raised

banana shoots were utilized for genomic DNA extraction following CTAB

method (Doyle and Doyle, 1990) with some modifications (Patel and

Jasrai, 2010).

3.7.2 DNA extraction protocol

Leaves (500 mg) were crushed in CTAB buffer (1 ml) using pre-

chilled mortar and pestle. These leaves were stored at -20° C in a deep

freezer (Blue star Co) prior to its use. The samples were transferred to

microcentrifuge tubes (1.5 ml size) and 2 µl each of Protease K, RNAase A

were added. The tubes were incubated (1h) in water-bath (65° C) with

frequent shaking at intervals (every 15 min). Equal volume of Phenol,

Chloroform and Isoamyl alcohol (24: 24: 1, v/v/v) was added and mixed



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by gentle inversion (2 min) and centrifuged at 1000 rpm (10 min). The

supernatant was transferred to fresh microcentrifuge tube and 350 µl of

IPA (pre-chilled) was added and again centrifuged (10,000 rpm) for 10 min

(4° C). The supernatant was discarded. The obtained pellet was air-dried

until the alcohol was removed and dissolved in 100 µl of TE buffer (10 mM

Tris HCl-1mM EDTA buffer, pH 8.0). The DNA pellet was then incubated

at room temperature for 30 min and stored at -20° C for further use.

3.7.3 RAPD analysis

The quantity of DNA was checked with the help of UV

Spectrophotometer (wavelengths 260 and 280 nm). The quality of

extracted DNA was ascertained by agarose gel electrophoresis (0.8 %) with

ethidium bromide. To check the suitability of extracted DNA for

downstream analysis, RAPD analysis was carried out with three different

primers (Operon Technologies Inc, USA), ie OPB 04 (5’-GGA CTG GAG T-

3’), GLF 03 (5’-CCT GAT CAC C-3’) and OPE 04 (5’-GTG ACA TCC C-3’).

Each PCR reaction mixture of 20 µl consisted of 1 µl of genomic DNA, 10

µl of 2X PCR reaction buffer, 1 µl of primer and 8 µl of nanopure water for

dilution. The reaction mixture was centrifuged at 6,000 rpm (1 min). DNA

amplification was carried out in a thermal cycler (Bio-Rad, USA). The



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modified PCR conditions were optimized. The first step consisted of

holding the samples at 94 °C (5 min) for complete denaturation of

template DNA, followed by 44 cycles, each involving: 94 °C for 1 min

(denaturation), 36 °C for 1 min (annealing) and 72 °C for 2 min (extension)

followed by a final extension temperature at 72 °C for 10 minutes and

being held at 4 °C until electrophoresis (El-Dougdoug, 2007; Akhare et al,

2008). The amplification products were revealed using the Agarose gel

electrophoresis system (Bio-Rad, USA), where PCR products

electrophoresed on 1.2 % (w/v) agarose gel containing 0.1 µg/ml

Ethidium bromide at 100 Volts in 1X TAE buffer for 1 h and visualized in a

UV trans-illuminator at 300 nm. Along with the samples, known

molecular weight supermix DNA ladder (Fermentas, USA) was also

loaded. The polymorphic bands were recorded for each locus for

comparisions.

3.8 Somatic Embryogenesis

The protocol for the technique of somatic embryogenesis was

followed according to Strosse et al (2003). This report depicted the

production of 35,000 plantlets per ml of plated cells for Grand naine

variety of banana, which is remarkably high regeneration capasity. This



62

technique was examined for its utility in cost-reduction of in vitro

regeneration of banana, using male flowers as the explants from the same

variety. Establishment of the explants was carried out on MA1 medium

(Strosse et al, 2003) in total 180 culture bottles, each with 5-7 male flowers

of 0.4-2 cm sizes, which were situated at 8 to 16 rows from the tip of the

inflorescence (Fig. 18 C). For the induction of callus, cultures were

maintained in dark condition in the culture room (Fig. 18 L). The ideal

callus (white coloured) was transferred in the fresh media after removing

yellowish-brown friable callus (19 M, N). The embryogenic calli were

transferred to MA2 medium (Strosse et al, 2003) in flasks (100 ml) and

agitated constantly on a rotary shaker (70-100 rpm) provided with 16 h

light in the culture room for the initiation of cell suspention (Fig. 20 A). A

part of medium was refreshed every 8-10 days, during total 8 month

period of mainainance of cell suspension culture. Regular observation of

cell suspension culture was carried out under the stereomicroscope until

the round somatic proembryos formed (Fig. 20 R-U). To test their viability,

samples of embryogenic cell suspension was taken from 3 flaks of total 11

and stained with fluorescein diacetate (FDA) stock to observe them under

the fluorescent microscope (Fig. 20 U, V). After confirming the viability,

the embryogenic cell suspension was taken on the MA3 medium (Strosse et



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al, 2003) for the induction of embryo. Each culture bottle contained 1 ml of

embryogenic cell suspension on to the MA3 medium with a Whatman

filter paper. The cultures were maintained in light condition in the culture

room (Fig. 20 X-Z).

3.9 Alternative for Room Sterilization

3.9.1 Requirements

Petridishes prepared with bacteriological media, beakers,

formaldehyde, KMnO4, Incense sticks and match-box

3.9.2 Procedure

Fumigation with KMnO4 (4 gm) and formaldehyde (8 ml) mixture

was carried out in a room (300X300X240 cm3) used for in vitro transfers.

Sterile petridishes (10 cm) with bacteriological media (Nutrient-Agar

media) were kept open (2 min) in the room after 18 h of fumigation.

Similarly, the room was fumigated with 8 Incense sticks (5.6 gm) of Sandal

wood (Cottage Industries, India). Again, the sterile petridishes (10 cm)

with bacteriological medium were exposed (2 min) in the same manner.

Observations were carried out to check the bacterial growth in form of

colonies after 24 h of incubation (37 °C) period. The experiment was

repeated thrice for both these ways of area sterilization.

chapter 3 materials and methods -...

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