TOPIC- DETERMINING HYBRIDITY OF SELF-POLLINATED

WHEAT CROP
Submitted by -Diksha
j18-biotech-210

INTRODUCTION:

Self-pollination-.   Transfer of pollen grains from the anther to the

stigma of the same flower is known as self-pollination. This
process can take place in the same flower or a different flower of
the same plant. It occurs in genetically identical flowers.  Flowers
that carry out self-pollination are  hermaphrodites. A hermaphrodite flower
is one in which both the stamen and the carpel are present. The stamen is
the reproductive organ of the flower that produces the pollen whereas the
carpel is that part of the flower producing the ovule. Self-pollination is
common in many plant species. It confers the advantage of having a rather
stable genotype and the benefit of not being dependent on pollinating
agents.

Hybridization-Hybridization can be defined as the process of crossing two

organisms that are genetically distant from each other. This can be an
artificial or natural process. It is important to note that hybridization does
not change the genetic composition of an individual; it creates variability by
producing a new combination of the allele.  The main goal of this process is
to induce heterozygosity and reduce homozygosity in the genotypes of the
population.
Hybrid varieties- are the first-generation offspring of a cross between
parents with contrasting genotypes. A hybrid variety differs from a variety
produced by hybridization. With the hybrid variety, the f1 generation is
grown and the hybrid genotype is reproduced faithfully in every plant if the
parents are homozygous. With hybridization in self-pollinated crops, a line
combining genes from the parents is selected in several generations of
inbreeding

PROCEDURE:-
To prepare master mix and to minimize the possibility of pipetting errors,
prepare a PCR mater mix by mixing water, buffer, dNTPs, and primer and Taq
DNA polymerase
 Prepare a sufficient master mix. Aliquot the master mix into individual
PCR tubes and then add template DNA
 The four samples which are used are
1) JAUW-584
2) HDR 77
3) HD 2385
4) Mixture of JAUW 584 and HS 365
 which are used along with this are – xgwn 219and xgwm 114
 Gently vortex and Primers briefly centrifuge all solutions after
thawing. Place in thin walled PCR tube and add the following
components for the PCR reaction:

1x
Taq buffer 1x 1.5 ul
dNTPs(10Mm) 0.2mm 0.3ul
Forward primer(10uM) 0.2uM 0.3ul
Reverse primer(10Um) 0.2uM 0.3ul
Taq polymerase(5U/ul) 0.75U 0.15ul
ddH20 10.45ul
DNA 2ul 2ul
13+2ulDNA

 Gently mix and spin down the samples

 Perform PCR using recommended thermal cycling conditions
STEPS TIME TEMPERATURE (°C)

Initial denaturation 3 min 94°C

Denaturation 1 min 94°C

Annealing 1 min 60°C

Extension 1 min 72 °C

Final extension 10 min 72 ° C

NUMBER OF CYCLES: 45

PREPARATION OF GEL:
1. If 100ml of gel has to be prepared, Weigh following amount of
electrophoresis grade agarose and melt it in electrophoresis buffer (1XTAE
or 0.5XTBE) by heating with continuous stirring till a clear solution is
obtained for 1% agarose gel ;
Melt 1g of agarose in 100ml (1xbuffer)
For 0.8% agarose gel, melt 0.8g agarose.
2. Place the cellophane tape around the casting tray.

3. Place the comb into the casting tray.

4. Ethidium bromide is added to the gel (final concentration

0.5%ug/ml.

5. Pour the molten agarose in gel casting tray/ platform with a comb
inserted.
 6. after the gel hardens, the comb is withdrawn, tape is removed
carefully.

7. Place the gel in the electrophoresis tank. The gel tank is filled with
sufficient volume of electrophoresis buffer in which the gel should be
submerged.

LOADING AND RUNNING THE GEL:

1. Mix the DNA sample with loading buffer and then pipette then into
simple wells.
2. The lid and power leads are placed in apparatus and current is applied.
Electrophoresis is carried out at a constant voltage of 100 volts.
3. DNA will migrate towards the positive electrode which is colored RED.
4. When adequate migration has occurred, DNA fragments are visualized
by staining with ETHIDIUM BROMIDE.

VISUALIZATION OF DNA IN AGAROSE GEL:

1. To visualize DNA keeps the agarose gel on the platform of UV

transilluminator and switch on uv light to visualize ETBr stained
DNA bands in gel.

2. Take photography of CCD camera attached gel documentation

system with a computer.

OBSERVATIONS;