This document discusses molecular and biochemical methods for varietal identification of seed. It describes electrophoresis, which separates proteins and enzymes extracted from seeds based on banding patterns that can differentiate varieties. PCR (polymerase chain reaction) amplifies specific DNA fragments, allowing detection and identification of genes through size and charge visualization via gel electrophoresis. Molecular markers like RFLPs and VNTRs also detect contamination and segregation by obtaining unique patterns for each variety. Biochemical methods like electrophoresis analysis of proteins and isozymes provide efficient, cost-effective cultivar identification and purity testing as seed storage proteins are independent of environmental fluctuations.

1. Molecular and biochemical for
varietal identification of seed
By
Srijan lama
School of agriculture science
2nd year(4th sem)

2. Veritial identification
• To determine the genetic purity status of a
given seed lot of the notified cultivar / hybrid
and the extent to which the sample in
question conforms to the prescribed
standards

3. Molecular methods
1. Electrophoresis
2. PCR
3. Molecular marker
1. Electrohoresis- It is the latest method of cultivar identification
based on protein banding and isoenzyme activity. Here single
seeds are defatted and extracted for protein and esterases. The
extracted protein are separated by polyacrylamide gel
electrophoresis. Based on the banding pattern of protein and
esterase's the varieties can be differentiated and identified.

4. Electrophoresis for proteins and
enzymes
Seeds, seedlings or mature leaves etc. of a crop plant have a
specific mix of proteins which are not only crop specific but also
variety specific (genotype specific). The electrophoresis in a
suitable medium separates the mixture of proteins extracted
from seeds, seedlings or mature leaves into distinct bands. Each
variety (or genotype) thus has a specific "banding pattern" on
the basis of which admixtures of other varieties, differing in
"banding pattern" could be detected. This is done by comparing
the banding pattern of analysis sample with the standard
banding pattern of that variety. The electrophoresis is now being
increasingly used for determining the genetic purity of seed
samples.

5. 2.PCR(POLYMARIES CHAIN
REACTION)
PCR is a very sensitive technique that allows rapid amplification of a
specific segment of DNA. PCR makes billions of copies of a specific
DNA fragment or gene, which allows detection and identification of
gene sequences using visual techniques based on size and charge.
The key ingredients of a PCR reaction are polymerase, primers,
template DNA, and nucleotides. The ingredients are assembed in a
tube along with cofactors needed by enzyme are put through
repeated cycles of heating and cooling that allow DNA synthesized.
The result of PCR reaction usually visualized using gel
electrophoresis.

6. The basic steps are
1.Denaturation[96]
2.Annealing[55-65]
3.Extension[72]

7. 3.MOLICULAR MARKER
In genetic purity assessment, molecular markers detect the
degree of contamination due to selfing and outcrossing in hybrid
seed lot, segregation of genotypes in DUS testing and trait
confirmation in transgenics. The goal for variety identification is
to obtain a specific/unique pattern for each variety.
Types of molicular markers
1.RELFs- Restriction fragment length polymorhphosis
2.VNTRs- variable number of tandem repeats

8. General methodology for molicular
markers
• DNA extraction
• Denaturation
• Annealing
• Extension
• Final extension
• Repeated cycle

9. • Advantage of molecular method
1. Very fast method
2. Time saving
3. Give quick result
Limitation of molecular method
1.Very costly
2.Need expert staff

10. Biochemical method
• Biochemical is term associated to biochemistry. It refers to chemical
substance present in a living organism or chemical processes
occuring in a living organism. for Exmples - protein, enzymes,
carbohydrate, etc. The biochemical are generally present in distinct
forms in the various varieties in differenrt forms.
• Electrophoresis analysis of proteins and isoenzyme offers an
efficient and cost effective method towards cultivar identification
and variety purity tests in seed lots.
• Simple and extensively used biochemical techniques for analysis of
cultivar identification. As seed storage proteins are largely
independent of environment fluctuation .

11. Thank you ☺️☺️☺️