B. Sc. (Hons.

) Agriculture, 6th semester, 3rd Year

GPB- 322 : Principles of Seed Technology
Unit III
Biochemical Tests for Varietal Identification

Lecture No. 16
Dr. Jai P. Gupta

Assistant Professor, School of Agriculture

Outlines

Introduction
Procedure
Biochemical Tests
Assignment/exercise on topic
References
Introduction

Varietal Identification is the processes in which confirm the genetic

purity of any seed lot of prescribe variety.

Genetic purity of a seed lot is determined on the basis of distinct

morphological characters Of the variety expressed at seed, seedling and
plant level by comparing its submitted sample with authentic sample under
identical environmental situation.
Procedure

Grow-Out Test for determination of genetic purity on the basis of plant

morphological characters.

Determination of genetic purity on the basis of expression of

distinguishing traits at seed level.
 Biochemical Tests
Objective
Verification of genetic purity of cultivars based on variation in
expression in response to biochemical tests at seed level.
1)Fluorescent test
2)Phenol colour test
3)KOH bleach test
4)KOH test
5)Peroxidase activity test
6)Copper sulphate ammonia test
7)Coleoptile : Anthocyanin colouration
8)Reaction of seedling root to UV light
9)Ammonia fluorescent test
Fluorescent test
Seeds of oat are exposed to UV light at 360 – 380 nm wave length on a
dark background.

Varieties are classified in two groups on the basis of fluorescent effect

i.e., fluorescent or non- fluorescent (Anon, 1985).
Phenol colour test
For cereals particulary wheat, rice and sorghum through involvement of
enzyme tyrosinase using phenol as a substance (Walls, 1965).

The test is approved for DUS testing of Wheat varieties.

Seed rinsed with methanol and placed in beaker and immersed in distilled
water.

Two sheets of filter paper arranged in petriplate and seed arranged on

filter paper.

1% solution of carbolic acid is applied in each petriplate till ¾ part of

seed is covered.

Seed are observed after prescribed period for development of colour and
its intensity.
KOH bleach test
Genetic purity of sorghum varieties can be verified on the basis of  dark
pigmentation present on the tegmen of the seed coat of some  varieties due
to tannic acid. This character can be confirmed by KOH bleach test (Payne
and Koszykowski, 1980). 

5.25% solution of bleach is prepared in water.  

1: 5 (weight/ volume) solution of KOH is prepared with 5.25%  bleach

solution.  

Seeds are soaked in KOH - bleach solution for 10 minutes and Seeds are
gently swirled in the solution.  

Seeds are placed on filter paper for air drying.  

Varieties are verified based on dark or light colour developed on the

seed. 
KOH test
KOH  test is recommended to verify presence of objectionable weed seed
in  the seed lot of rice (Rosta, 1975).  

Seed suspected as wild rice are sorted out from the sample.  

Seed sorted out as of objectionable weed are placed in a test tube.  Two
drops of 2% KOH solution is added in each test tube.  

Colour of the KOH solution is observed after 10 minutes.  

Development of red color in the solution confirms presence of wild rice

Seed. Whereas, no change in the color of KOH solution confirms that the
seed is of normal rice.
Peroxidase activity test
Presence or absence of peroxidase enzyme in seeds of crops from  family
Leguminoceae is under genetic control therefore, variety may  be verified
based on variation in the expression with the reaction of  hydrogen
peroxidase (Buttery and Buzzell, 1968). The test is  approved for DUS
testing of soybean.
Coat of 20 seed sorted as genetic impure 13 removed with the help of a
sharp razor. No part of embryo or  cotyledon should remain attached with
the separated seed coat.
Seed coat is placed in test tube in such a way that one test tude contains
coat of only one seed. Guaicol solution of 0.5% concentration is prepared
in water, in each test tube containing seed coat, 10 drops of 0.5% guaicol
solution is added. 
After 10 minutes, one drop of 0.1% hydrogen peroxide (H202) is added
in each test tube. The change in the colour of the solution is observed
immediately after addition of  hydrogen peroxide.  
Copper sulphate ammonia test
Seed of Melilotus alba is of white colour whereas, M. officinalis is of
 yellow colour, but the differentiation in a seed lot on the basis of seed  coat
color and expression of other morphological traits of the seed is difficult.
Seed of both the species have different expression for copper  sulphate
ammonia test (Maxon and Hurst, 1983).  

A solution of tetra amine copper  sulphate is prepared by dissolving 3 g

CuSO4 in 30 ml NH4OH2  (4.8%) in a Stopper bottle. In the event of
precipitation in the  solution more quantity of CuS04 is added.  

Seeds are soaked in the solution for 20 minutes. Both the species have
different expression,  seeds of M. alba exhibits olive or yellow  
green, while M. officinalis shows dark  brown to black colour.  
Coleoptile : Anthocyanin colouration

The test is approved for DUS testing of wheat.

20 seeds of wheat are placed on a moistened filter paper in  Petriplate

for germination.  

The germinated seed with 1 cm coleoptile is shifted to artificial  light

of 15,000 lux at 150 to 200C. 

After one week, when leaf is within coleoptile (stage 9-10) the colour
of coleoptile is observed for presence or absence of anthocynin
pigmentation by naked eye.  
Reaction of seedling root to UV light

Seeds of rye grass are arranged on a moist filter paper  eliminating the
chances of root entanglement with each other. 

This is kept for germination in dark.  

5-10 days old seedlings are examined under UV light at 300-400  nm

wavelength for fluorescent effect on root.  

Varieties are verified as of positive response (annual rye grass)  or

negative response (perennial rye grass).  

Similar test has been recommended for verification of soybean 

varieties (Grabe, 1957).  
Ammonia fluorescent test
Three pieces of filter paper are moistened and placed in a  Petriplate.

Seeds are arranged on the Petriplate in such a way that the  roots are
not entangled with each other during germination. 

The Petriplate is placed in a germinator at alternate tempera ture (10-

30°C or 15-25°C) in weak light (1000 lux) for 14 days. 

On 14th day 0.5% solution of ammonium hydroxide is sprayed on  the

seedlings.  

After spray seedlings are observed under UV light.  

Root of F. rubra shows fluorescent effect of yellow green, while

F. ovina exhibits blue green colour. 
Assignment/Exercise

1. Write a assignment on method of cultivar identification based on

biochemical tests at seed level.
References

1. Agarwal, P.K. 1994. Principles of Seed technology. ICAR, New

Delhi.

2. Dhirendra Khare and Mohan S. Bhale. 2007. Seed Technology.

Scientific Publishers (India), Jodhpur.

3. Seed Technology- Har Pal Singh Tomar , Aman pub. House