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3. MATERIALS AND METHODS

The field experiments to undertake the research endeavour on Imperata

cylindrica mediated haploid production in various cereals and efficiency

enhancement of the system in wheat by colchicine manipulation technique were

conducted at the Experimental Farm, Department of Crop Improvement,

Chaudhary Sarwan Kumar Himachal Pradesh Krishi Vishvavidyalaya (CSK

HPKV), Palampur during 2007-08 to 2009-10, while all the in vitro experiments

were conducted in the Molecular Cytogenetics & Tissue Culture Laboratory

under controlled environmental conditions. The experiments were undertaken so

as to see the possibilities of using Imperata cylindrica as pollen source for

production of doubled haploids (DHs) in various other cereals and handling the

hurdles encountered during colchicine treatment, thereby increasing the

efficiency of DH production in the existing system. The experimental materials

used and the methods employed are described as under:

3.1 Experimental materials

3.1.1 Induction of haploids through wide hybridization using Imperata cylindrica as pollen source

The materials involved in the induction of haploids through wide

hybridization with I. cylindrica consists five important cereal crops i.e. wheat, rice,

maize, barley and oat. Various elite and diverse genotypes/ lines of each crop

were used during the investigation and their sources are given in Table 3.1

3.1.1.1 Manipulation in the application of 2, 4-dichlorophenoxy acetic acid (2, 4-D)

2, 4-D (C6H6Cl2O3) having molecular weight 221.04 and grade ‘high’ was

used in the investigation. Different concentrations ranging from 30 ppm-250 ppm

were applied in vivo to the uppermost internodes in the form of injection in all the

different five cereal crops at 24, 48 and 72 hrs after pollination. In case of wheat,

only the standard dose of 100 ppm was used.

Table 3.1 Materials used in wide hybridization

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Sr. No.

Crops Lines/Genotypes Source

1. Wheat KWS-29 ,C-306, Durum wheat (HW-896)

CSKHPKV, Palampur

2. Rice Bhrigu dhan,Varun dhan,Kunjan, HPR-1068, HPR-2143, HPR-1156, Deku, Amdeng, Yasing Boling, Tatum ,Kabder

CSKHPKV, Palampur and Arunachal Pradesh

3 Maize Early Composite, Bajaura Makka CSKHPKV, Palampur

4 Barley Dolma, Karpat local ,Purthi barley, Barot barley, Tindi barley

CSKHPKV, Palampur

5 Oat Palampur-1, Naked oat CSKHPKV, Palampur

3.1.2 Enhancement of doubled haploid production efficiency

For the execution of doubled haploid production efficiency enhancement

experiments at both in vivo and in vitro levels, first of all F1 hybrids were

generated by crossing DH-100 and DH-40 at crossing block of the Department of

Crop Improvement, CSKHPKV, Palampur, during rabi 2007-08 and 2008-09. The

F1 hybrids were then utilized for the afore mentioned endeavour .

3.1.2.1 Colchicine application

Colchicine (C22 H25 NO6) having molecular weight 399.45 of ‘high’ grade

was utilized for both in vivo and in vitro colchicine dose manipulation experiments

for enhancing doubled haploid production efficiency in wheat. Various dose

ranging from 100 ppm to 10,000 ppm were administered in vivo to the uppermost

internodes of the wheat plants in the form of injection (1 to 3 injection of each

dose) with and without regular doses of 2, 4-D at various intervals just after 24

hrs of pollination up to 120 hrs. Different doses of colchicine ranging from 100-

300 ppm were applied along with culture media in the in vitro experiments.

3.1.3 Media used for the embryo rescue

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3.1.3.1 Medium for embryo regeneration

Murashige and Skoog (1962) medium supplemented with essential amino

acids (Table 3.2 and 3.3) was used for the rescue of haploid embryos and the

embryos developed after in vivo colchicine manipulation.

Table 3.2 Composition of MS medium stock solutions

S. No

Quantity Strength Salts Quantity Use ml/L

Amount in

culture (mg)

I (2 L) X 20 Ammonium Nitrate 66.0 g 50 1650

Potassium Nitrate 76.0 g 1900

Potassium Dihydrogen Phosphate

6.8 g 170

Boric acid 0.248 g 6.2

Manganese Sulphate 0.892 g 22.3

Zinc Sulphate 0.344 g 8.6

Potassium Iodide 0.033 g 0.825

Copper Sulphate * 1.0 ml 0.025

Cobalt chloride * 1.0 ml 0.250

Sodium Molybdate * 1.0 ml 0.025

II (1/2 L) X 50 Calcium Cholride 11 g 20 440

III (1/2 L) X 50 Magnesium Sulphate 9.25 g 20 370

IV (1 L) X 100 Ferrous Sulphate 2.78 g 10 27.8

Disodium EDTA 3.728 g 37.28

V (1 L) X 100 Thiamine HCl 0.010 g 10 0.1

Nicotinic acid 0.050 g 0.5

Pyridoxine HCl 0.050 g 0.5

Glycine 0.200 g 2

VI Myoinositol 100

VII Sucrose 30000

VIII Agar 8000

IX Glutamine 150

X Kinetin ** 0.5

* Dissolve 100 mg of copper sulpate and cobalt chloride and 1 g of sodium molybdate in 100 ml of water separately. Then take 1 ml each and add to stock solution I.

** Dissolve 0.1g in 1 N solution of NaOH (5 ml) and water 9.5 m

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Table 3.3 Composition of MS medium (1 litre)

S. no. Constituents Quantity

1 Sucrose 30 g

2 Agar- Agar 8 g

3 Myoinositol 0.10 g

4 Glutamine 0.15 g

5 Kinetin 0.50 ml

6 Stock solution I 50 ml

7 Stock solution II 20 ml

8 Stock solution III 20 ml

9 Stock solution IV 10 ml

10 Stock solution V 10 ml

3.1.3.2 Medium for rooting

Liquid rooting medium consisted of half strength MS salts, 1mg

naphthalene acetic acid per litre (NAA) and 1mg indole-3-butyric acid (IBA) per

litre was used for inducing profuse rooting in regenerated plants (Table 3.4).

Table 3.4 Composition of rooting medium (1 litre)

S. no. Constituents Quantity

1 Glutamine 0.15 g

2 Myoinositol 0.10 g

3 IBA * 1 ml

4 IAA * 1 ml

5 Stock solution I 50 ml

6 Stock solution II 20 ml

7 Stock solution III 20 ml

8 Stock solution IV 10 ml

9 Stock solution V 10 ml

* Dissolve 100 mg in 5 ml 0.5 N NaOH (Heat slightly in test tube till complete dissolution)

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3.1.4 Materials for cross- sectional studies

3.1.4.1 Dehydration of the tissue

i. Ethyl alcohol

ii. t-butyl alcohol

iii. Eosin

iv. Paraffin

v. Muslin cloth

vi. Thread, Scissor

vii. Plastic basket

3.1.4. 2. Embedding to paraffin

i. Plastic box /ring (1x1x0.5 cm)

ii. Paraffin block

3.1.4. 3 Attach paraffin to the wooden block

i. Wooden block

ii. Spirit lamp

iii. Spatula

3.1.4. 4 Trimming of the paraffin

i. Blade (Fine, high quality, surgical)

3.1.4. 5 Section cutting of the tissue

i. Slide

ii. Slide baskets

iii. Slide markers (Diamond glass cutter)

iv. Microtome

v. Hot plate

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3.1.4.6 Staining of the tissue

i. Slide baskets

ii. Xylene

iii. Ethyl alcohol

iv. Double distilled water

v. Safranine in 2-Ethoxy ethanol

vi. Fast green

vii. Clove oil

viii. Entellan (Microscopy)

ix. Cover glass (24x60 mm)

x. Hot air oven

xi. Magnetic stirrer

xii. 2- Ethoxyethanol

3.1.4. 7 For visualization/ Observation of slide

Phase contrast microscope

3.1.5 Materials for Cytological Investigation

Cytological investigation was carried out so as to check whether

chromosome doubling had occurred or not by various colchicine treatments. The

materials used in each step of cytology were as follows:

3.1.5.1 Cold treatment

i. Crushed ice

ii. Distilled water

iii. Eppendorf tubes (1ml)

iv. 4o C Refrigerator

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3.1.5.2 Fixation of roots

i. Glacial acetic acid

ii. Ethanol (99.9 %)

3.1.5.3 Slide preparation

i. Acetocarmine solution (0.2 %)

ii. Slides

iii. Coverslips

iv. Whatman filter paper

v. Forceps

vi. Surgical blade with holder

vii. 45 % Acetic acid

viii. Slide rack

ix. Spirit lamp

x. Tooth- pick sticks

3.1.5.4 Observation

i. Phase Contrast Microscope (Olympus make)

ii. TFT Screen

3.2 Methods

3.2.1 Induction of haploids through wide hybridization with Imperata cylindrica as pollen source

3.2.1.1 Wheat x Imperata cylindrica

Haploids in wheat were produced following the protocol standardized for

wheat x I. cylindrica (Chaudhary et al. 2002 and 2005). The staggered sowing of

wheat genotypes KWS-29, Durum wheat (HW-896) and C-306 was done in the

Experimental Farm of the Department during rabi 2008-09 and 2009-10 so as to

synchronize flowering time of Imperata cylindrica for successful hybridization

programme. Procedure involved the following steps (Plate 1):

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23

(i) Emasculation

At ear emergence, wheat spikes were emasculated by removing anthers

with the help of forceps without disturbing lemma and palea. The central florets,

apical and basal spikelets of each spike were removed. The emasculated spikes

were covered with butter paper bags for maintaining humidity and avoid stray

pollen and labeling of tags was done with the information viz., date of

emasculation and genotype name.

(ii) Pollination

Preferably next day, emasculated spikes were pollinated with the fresh

pollen of I. cylindrica using camel hair brush carefully. The pollen of I. cylindrica

was available in abundance during the morning hours from 7.30 to 9.30 am. The

pollinated spikes were covered immediately with butter paper bags and tagged

accordingly.

(iii) 2, 4-D application

The uppermost internodes of the wheat culms were injected with a

solution of 100 ppm 2, 4-D for three consecutive days at 24 hrs, 48 hrs and 72

hrs after pollination. The injection holes were sealed by using petroleum jelly.

(iv) Embryo rescue

After 18-20 days of pollination, the crossed spikes were harvested. The

embryo carrying pseudoseeds were identified under a source of light (Bains et al.

1998). The embryo was seen floating in the fluid (aqueous solution) instead of

solid endosperm as found in selfed seeds. The embryo carrying pseudo seeds

were then washed thoroughly using Tween- 20 under tap water to avoid any sort

of infection/contamination. At the time of culturing these embryos in Laminar Air

Flow Chamber, surface sterilization of seeds was done using 0.1% HgCl2 for 3-5

minutes followed by two washing with autoclaved distilled water. The embryos

were then excised from sterilized seeds. These excised embryos were

transferred to the test tubes containing MS medium supplemented with essential

amino acids.

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25

25

(v) Growth conditions

Embryos were excised out using two fine forceps under laminar air flow

chamber and placed on the culture medium in the test tubes. Cultured embryos

were given cold treatment at 4oC in the dark for 24 hours immediately after

embryo rescue followed by incubation in the dark at 20 ± 20C for regeneration for

about a week or till the roots and shoot initiation. The regenerated plantlets were

then shifted to Growth Chamber at 20 ± 20C with 10 hours day length regime

75% relative humidity (RH), until they developed properly into complete green

plantlets.

(vi) Transferring to liquid media

The green haploid plantlets developed through embryo culture were then

subjected to rooting medium (liquid) for profuse rooting. Rooting medium

comprising half strength of MS salts 1 mg/ L each of NAA and IBA (Indole 3

butyric acid) and devoid of sucrose and agar. M-shaped filter paper immersed in

test tube for support and continuous supply of nutrients through capillary action.

(vii) Transfer of haploid plantlets to soil

Potting mixture was prepared by mixing 2:1:1 proportion of soil, sand and

compost. The soil and sand mixture was sterilized by autoclaving at 15 pounds/

inch2 pressure for 15 minutes at 1210C temperature. The plantlets were then

transferred from liquid medium to the soil in small pots (10 cm diameter).

Conditions were maintained for proper growth until maturity.

(viii) Chromosome doubling

For colchicine treatment, the plants with 4-5 leaves were removed from

liquid media and the roots trimmed to about 2 cm length. The trimmed and

tagged plants were placed in a solution of 0.1% colchicine and 2% DMSO

sufficient to cover the crown portion of the plants in a container. After 4-5 hours

treatment at 200 C, the plants were removed from the colchicine solution, rinsed

for ½ to 1 hour in running tap water and planted into pots with potting mixture.

The plants were kept in proper growth region condition with 10 hour to 14 hour

light /dark photoperiod, 75% RH and 20± 20C temperature upto the period before

shifting to the cage house under natural conditions.

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3.2.1.2 Rice x Imperata cylindrica

Ten lines of rice were used for carrying out hybridization with Imperata

cylindrica viz., Bhrigu dhan, Varun dhan, Kunjan, HPR-1068, HPR-2143, HPR-

1156, Deku, Amdeng, Yasing Boling, Tatum and Kabder. The crossing

programme was executed during kharif 2008-09, rabi 2009-2010 as well as in

kharif 2009-10 at the Experimental Farm of the Department of Crop

Improvement, CSKHPKV, Palampur. As the flowering time of I. cylindrica and

rice falls on different seasons, the crossing was carried out in two different

seasons (rabi and kharif), with natural and preserved pollen of Imperata

cylindrica. For rabi season, the materials (rice germplasm) were raised in

polyhouse whereas during kharif season, the materials were raised normally in

the experimental field. But the preserved pollen (at -20oC) of I. cylindrica was

used due to non-synchronization of flowering time. The steps involved in the

crossing programme were as follows:

(i) Emasculation

Panicles on which slight blooming had occurred in the uppermost florets

were selected for emasculation. Self fertilized florets visible through the enclosing

lemma and palea were removed with scissors. Remaining each floret was

clipped across the lemma and palea through the six anthers slightly above the

attachments of the anthers to the filaments. Although the clipping effectively

emasculated florets yet the stamen remnants were removed with forceps

immediately following clipping. Removal of six stamens/ anthers was ensured

before covering the panicles with butter paper bag. Emasculated panicles were

then covered with butter paper bag fastened to the peduncles with paper clips

and labeled.

(ii) Pollination

Pollination of individual florets was practiced by gently applying the pollen

of I. cylindrica on feathery stigma with the help of fine camel brush, followed by

tagging and labeling. The pollination operation was done preferably between

9.30 to 10.30 am.

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27

(iii) 2, 4-D application

Various doses of 2,4-D viz., 30 ppm, 50 ppm, 70 ppm and 100 ppm were

applied in vivo to the uppermost internodes of the rice plants in the form of

injection consecutively for 3 days after pollination.

(iv) Embryo culture

After 8-10 days of pollination, the crossed panicles were harvested. The

embryo carrying seeds were separated from panicle and washed thoroughly

using Tween-20 under tap water. Culturing activities were carried out in Laminar

Air Flow Chamber. Surface sterilization of seeds was done using 0.1% HgCl2 for

3-5 minutes and was given subsequently two washings with autoclaved distilled

water. The embryos were then dissected out from sterilized seeds under

dissecting microscope. These excised embryos were transferred to the test tubes

containing MS medium supplemented with essential amino acids. These test-

tubes were then given cold treatment at 4oC for 24 hours followed shifting to dark

room for further regeneration/ germination.

3.2.1.3 Maize x Imperata cylindrica

The potential of doubled haploids (DHs) in maize breeding has been

recognized. By the use of such techniques various gene combinations can be

fixed in homozygous and homogeneous form in a short time and also the DH

lines possessing high fertility and favourable agronomic characteristics can be

utilized directly in heterosis breeding to produce new promising hybrids. The

variety, Early Composite and Bajauara Makka of maize were grown in polyhouse

during rabi 2009-10 for synchronization of flowering time with I. cylindrica. The

steps followed were as below:

(i) Emasculation

Detasseling was operated by removing the tassels prior to silk emergence

and pollen shed to prevent self pollination followed by bagging of ears (cob) with

brown paper bags which were just emerged from stalks.

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28

(ii) Pollination

When there was emergence of pale yellow silks from leaf whorl at the end

of the ear, it was cut opened with the help of surgical blade and fresh collected

pollen of I. cylindrica were dusted on it with the help of camel brush. The timing

for pollination of maize was 10.30 to 11.30 am.

(iii) 2, 4-D Application

Two doses of 100ppm and 150ppm were injected at the base of cob for

inducing development of haploid seed.

(iv) Embryo culture

The cobs were harvested after 10 to 15 days of pollination and swollen

seeds were excised.

3.2.1.4 Barley x Imperata cylindrica

Five lines of barley (Dolma, Karpat, Purthi, Barot and Tindi) were used for

attempting crossing with I. cylindrica. The crop was raised in the Experimental

Field of the Department of Crop Improvement during rabi 2008-09 and 2009-10.

The steps followed were as below:

(i) Emasculation

Young spike which was half emerged from flag leaf was selected for

emasculation. With the help of scissors and forceps, one fourth of the top of the

florets were cut and the three anthers were removed. Each emasculated spike

was covered with butter paper bag and tagged.

(ii) Pollination

I. cylindrica pollen was collected in the morning by gently tapping the

spikes over a glass petridish. Using camelbrush, the collected pollen was gently

applied over stigma of each floret. Pollinated spikes were covered with butter

paper bag followed by tagging & labeling.

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29

(iii) 2,4-D injection

Various dosages viz., 50ppm, 100ppm, 150ppm and 200ppm of 2, 4-D

were injected to the spike at uppermost internodes in the form of injection

consecutively for 3 days after pollination.

(iv) Embryo rescue

Embryos were rescued after 10-12 days after pollination. All the activities

were carried out in laminar air flow chamber under aseptic conditions. Fine

forceps were used to remove the seed from the tissue covering it by giving the

seed a sharp jerk. Seeds were placed in a sterile petridish and surface sterilized

in a solution of 0.1% mercuric chloride and rinsed three times with sterile distilled

water. Embryos were carefully dissected out under the dissecting microscope

stage and were placed in embryo culture medium in test tubes. It was then

shifted to 4o C for 24 hours and was further shifted to dark chamber for

regeneration.

(v) Haploid regeneration

Haploid plants could not be regenerated during the investigation of

hybridization between barley and I. cylindrica. Needs to work more and

standardized the technique. Further investigation in respect of embryo culture

media and growth hormone application is needed to be executed.

3.2.1.5 Oat x Imperata cylindrica

Two types of oat, that is, Palampur-1 and Naked Oat were used for

performing hybridization with I. cylindrica. The crop was raised in the

Experimental Field of the Department of Crop Improvement during rabi 2008-09

and 2009-10. The steps involved were:

(i) Emasculation

Emasculation of oat florets was done by clipping the florets with scissors,

followed by removal of anthers (three in each floret).

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30

Oat florets are produced in spikelets of a panicle with each spikelet

composed of an outer glume overlapping an inner glume, with surrounding, basal

floret, a small secondary floret and sometimes a tertiary floret. Because spikelet

in an oat panicle develops over several days in an apical to basal fashion,

emasculation is done when the primary floret in the apical (most mature) spikelet

is at anthesis. The uppermost & lowermost florets were removed. Care was

taken not to injure the developing stigma.

(ii) Pollination

Freshly collected I. cylindrica pollen is distributed lightly over the

emasculated florets with the aid of a camel brush while using forefinger and

thumb to gently position the individual floret upward.

(iii) 2, 4-D application

For promotion of caryopsis development 2, 4-D injection of 50 ppm,

100ppm and150 ppm was administered in the form of injection consecutively for

three days after pollination.

(iv) Embryo culture

The pollinated spikes were harvested at 10 to 12 days after pollination.

3.2.2 Protocols for cross sectional studies (Plate 2)

The section cutting protocol of plant tissue consisted of six steps:

3.2.2 .1 Dehydration

3.2.2 .2 Embedding to paraffin

3.2.2.3 Attaching paraffin to the wooden stock

3.2.2 .4 Trimming of the paraffin

3.2.2 .5 Section cutting and

3.2.2 .6 Staining

31

a

b c

g i

d

e f

h j

k l m

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32

3.2.2.1 Dehydration of tissue

The fertilized ovules of rice and barley were preserved in the 1:3 acetic

acid: ethanol solution and carried to the Lab of Plant Molecular Genetics, Osaka

Kyoiku University, Osaka, Japan for undertaking cross-sectional studies and

investigated the fertilization status and development of embryos.

The tissue was dehydrated in

(i) 85% ethyl alcohol (300ml) for 24 hours

(ii) 80% ethyl alcohol (195ml) + t-butyl alcohol (105ml) =300ml for 4 hours

(iii) 90% ethyl alcohol (135 ml) +t- butyl alcohol (165ml) =300ml for 4 hours

(iv) 100% ethyl alcohol (75ml) + t butyl alcohol (225ml) =300 ml for 4 hours

(v) t-butyl alcohol (300ml) for 24 hours

(vi) t-butyl alcohol + eosin = 300ml for 4 hours

(vii) t-butyl alcohol (150ml) + Paraffin (150ml) =300ml for 24 hours

(viii) Paraffin (melting point 48-500 C) = 300 ml for 24 hours

(ix) Paraffin (melting point 600 C) = 300 ml for 24 hour

3.2.2 .2 Embedded to paraffin

(i) A small plastic box (1 x 1 x 0.5cm) was taken and put on slide and it was

filled with the paraffin (Liquid paraffin).

Plastic Box

Slide

(ii) The hydrated ovule was then placed in liquid paraffin (about 6 ovules can

be adjusted in one box)

Ovule

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33

(iii) Paraffin were allowed to solidify

(iv) The solidified paraffin is then removed from the plastic box

Paraffin block

Plastic box

(v) Cut the paraffin into small blocks having one ovule embedded in each

Separated paraffin blocks

3.2.2 .3 Attaching paraffin to the wooden stock

(i) Attached the paraffin block to the wooden block with the help of melted

paraffin

Paraffin block

Wooden block

3.2.2 .4 Trimming the paraffin

Trimming of the paraffin block is done if needed

3.2.2 .5 Section cutting of the tissue

(i) Mark the slide with the slide marker

(ii) Section cutting was done by placing the wooden block in the microtome.

The paraffin sections along with the tissue were placed on the slides.

Hot plate at 450 C

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34

(iii) Pour 5-10 drops of water on paraffin sections lying on the slide. These

slides were then placed on the hot plate at temperature 450 C.

(iv) Heat the slide till the water dry up and fix the section on slides

3.2.2 .6 Staining of the tissue

(i) The slides were placed in a basket and staining was carried in the

following way:

a) Placed the slides in xylene for 15-17minutes, then in

b) 100% ethyl alcohol for 5 minutes, then in

c) 70% ethyl alcohol for 5 minutes, then in

d) 30% ethyl alcohol for 5 minutes

e) Washed with flowing water for 5 minutes

f) Placed the slides in double distilled water for 5 minute, then in

g) safranine for 10 minutes (safranine 2g in 2-methoxyethanol/200ml)

h) Washed with flowing water for 5 minutes

i) Placed the slides in double distilled water for 5 minutes, then shifted to

j) 50% ethyl alcohol for 5 minutes, then shifted to

k) 95% ethyl alcohol( 0.5% picric acid) for 1 sec, then in

l) 95% ethyl alcohol ( 4 drops of ammonia) for 2 sec, then in

m) 95% ethyl alcohol for 5 minutes, then in

n) 100% ethyl alcohol for 5 minutes, then in

o) absolute alcohol for 5 minutes, then in

p) Fast green for 4 sec (fast green 1g+absolute alcohol 100ml

+ 2- methoxyethanol 100g + clove oil), then in

q) clove oil for 10 seconds, then in

r) clove oil: absolute alcohol : xylene for 20 seconds, then in

s) xylene for 10 seconds

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35

(ii) Placed one or two drops of entellan and put a cover glass of 24 x 60 mm

on section and sealed the section

(iii) The slides were put to dry for about 7 days and after one week these were

ready to visualise under microscope.

3.2.3 Enhancement of doubled haploid production efficiency

Two promising genotypes of wheat DH- 100 (disease resistant) and DH-

40 (high yielding) were hybridized to produce sufficient F1 seeds during rabi

2007-08 and 2008-09, which were utilized for colchicine manipulation experiment

at in vivo and in vitro levels during rabi 2009-10. The materials were raised in

Experimental Field of the Department of Crop Improvement and the methodology

being depicted in Plate 3.

3.2.3.1 In vivo colchicine application

The haploids were produced following the protocol standardized by

Chaudhary et al. (2002 and 2005) with the exception in colchicine application

method. Colchicine manipulations in vivo comprised of ten doses of colchicine

treatment at different concentration ranging from 100-10,000 ppm at the intervals

of 100 and seven high doses of 1200, 1500, 2000, 3000, 5000, 7000 and 10,000

ppm in combination with and without 2,4-D at different intervals of 24, 48, 72, 96

and 120 hours after pollination. The plan for application of colchicine for each

dose was as per table 3.5.

3.2.3.2 In vitro colchicine application

In the in vitro colchicine manipulation experiment, the pseudo seeds

obtained by crossing the F1 and I. cylindrica and harvested after 18-20 days of

pollination following Chaudhary et al. 2005 were screened for haploid embryos.

These embryos were cultured on colchicine enriched medium at different time

intervals of 24, 48, 72, 96 and 120 hours and then shifted to the normal MS

medium. The regenerated plants were further transferred to the pots and given

complete growth condition as per protocol established by Chaudhary et al. (2002

and 2005). The detailed plan for in vitro application of colchicine was as per

table 3.6.

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i

Table 3.5 Plan for in vivo application of colchicine for each dose

Colchicine treatment

Hours after pollination with I. cylindrica

24 48 72 96 120

T1 D+C *D D - -

T2 D D+C D - -

T3 D D D+C - -

T4 D D D **C -

T5 D D D - C

T6 D+C D+C D - -

T7 D D+C D+C - -

T8 D D D+C C -

T9 D D D C C

T10 D+C D+C D+C - -

T11 D D+C D+C C -

T12 D D D+C C C

Control D D D - -

* D=2, 4-D **C=Colchicine

Table 3.6 Plan for in vitro application of colchicine

Colchicine Concentration

Duration on colchicine enriched medium

24hrs 48hrs 72hrs 96hrs 120hrs

100ppm T1 T2 T3 T4 T5

200ppm T1 T2 T3 T4 T5

300ppm T1 T2 T3 T4 T5

400ppm T1 T2 T3 T4 T5

500ppm T1 T2 T3 T4 T5

600ppm T1 T2 T3 T4 T5

700ppm T1 T2 T3 T4 T5

800ppm T1 T2 T3 T4 T5

900ppm T1 T2 T3 T4 T5

1000ppm T1 T2 T3 T4 T5

1500ppm T1 T2 T3 T4 T5

2000ppm T1 T2 T3 T4 T5

2500ppm T1 T2 T3 T4 T5

3000ppm T1 T2 T3 T4 T5

ii

Control No application of colchicine

3.2.4 Protocols for Cytology

The cytological studies consisted of three steps:

3.2.4 .1. Cold treatment

3.2.4 .2. Fixing of roots and

3.2.4 .3. Slide preparation

3.2.4.1 Cold Treatment

Roots (2-3cm) were excised from regenerated plants and transferred to

the 5 ml vial containing distilled water. These roots were kept in vial in crushed

ice in ice box and cold treatment was given by keeping the ice box at 40C for 18-

20 hrs.

3.2.4.2 Fixing of roots

The roots were fixed in freshly prepared solution of absolute ethanol and

glacial acetic acid (3:1). Fixed stocks were kept at room temperature and after 4-

5 days of fixation, the cytological investigation was initiated.

3.2.4.3 Slide Preparation

The roots were first given staining treatment by keeping it in 0.2%

acetocarmine solution for 15 minutes. The slide preparation was done by placing

the fixed root on the whatman filter paper and, the root cap was removed by

using the surgical blade. Immediately after this, the blunt side of the razor/scalpel

was used to squeeze the root so as to take out the meristematic cells. The

meristematic cells were placed on the clean slide and a drop of 45 % acetic acid

is poured on it. Taping with fine wooden stick is exercised by placing a cover slip

inclined on one side with a sterilized razor blade. Proper taping is a crucial step

and highly useful in separating and spreading the cells on the slide. The slide is

subjected to warm treatment on a spirit lamp and immediately squashed by

placing the slide in a folded filter paper and putting thumb pressure on the area of

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cover slip. The prepared slide was observed in the phase contrast microscope.

Chromosomes were counted from good spread metaphase cells. Slide with good

preparation was placed on the dry ice with the cover-slip facing upside for 15-30

minutes. After dry ice treatment, the cover slip was removed using blade and

immediately kept in the 45% glacial acetic acid for 15-30 minutes. Thereafter, the

slides were removed from 45% acetic acid and kept for drying in slide stand. The

slides were then placed in the silica gel containing desiccators for overnight. The

dried slides were kept in the slides box, sealed and preserved at -20oC deep

freezer.

3.3 Recording of observations

3.3.1 Haploid induction parameters

Observations were recorded with respect to haploid induction parameters

on per cent basis as follows:

Number of seeds formed Seed formation frequency = –––––––––––––––––––––––––––––– X 100 (%) Total number of florets pollinated Number of seeds carrying embryos Embryo formation frequency = ––––––––––––––––––––––––––––––– X 100 (%) Total number of seeds formed Number of haploid plantlets developed Regeneration frequency = ––––––––––––––––––––––––––––––– X 100 (%) Total number of embryos cultured

3.3.2 Chromosome doubling

The cytological investigation was done as per the protocols mentioned

above (3.2.4.3). The effective dose of colchicine applied at in vivo and in vitro

levels was decided on the basis of doubling of chromosomes observed in the

metaphase cells preparation made from the fixed roots of the regenerated

embryos.

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3.4 Statistical methods applied

The data in respect of the effect of the colchicine application (in vivo and

in vitro levels) was computed as per the standard student’s t-test.

Student’s t-test

To test whether the mean difference between treatments/concentrations of

colchicine is significant or not when compared with control, student’s t-test was

performed as :

Student’s t-test = )SE(X

X

d

d (at n – 1 d.f.)

where

dX = mean-difference between two sets of related samples

SE (Xd) = Standard error of mean difference

n = Number of related samples