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 (C 6 H 6 Cl 2 O 3 ) 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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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,
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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(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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(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
(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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(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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(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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(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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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
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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