SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE,

TECHNOLOGY & SCIENCES,

PRYAGRAJ -211007 (U.P.)

TOPIC: PROJECT SYNOPSIS ON SEED HEALTH

COURSE CODE: SST - 722

COURSE TITLE: SEED PROCESSING AND STORAGE

Submitted to:

DR. Bineeta

Dept. of Genetics and Plant Breeding

SHUATS

Submitted by:

M.SAI YASWANTH

19MSSDCI065

M.Sc.Ag. (SEED SCIENCE AND TECHNOLOGY)

Content

1. Title of the project

2. Introduction

3. Priority area

4. Proposed duration of the project

5. Objectives of the project

6. Methodology

7. Financial outlay of the project

8. Expect outcome

9. Useful internets resources

1. Title : Project Synopsis on Seed health

2. Introduction

Seed health : Seed health refers to presence or absence of disease causing organisms like fungi,
bacteria and viruses, animal pests such as eelworms and insects, as well physiological conditions
such as trace element deficiency (ISTA, 2003).

The seed-borne pathogens may result in

(i) reduced germination/crop establishment

(ii) discoloration and shriveling of seeds

(iii) development of plant diseases leading to epidemics

(iv) loss of yield and quality

(v) distribution of pathogens to new areas

(vi) introduction of new strains or physiologic races of the pathogen along with new germplasm
from other countries

(vii) toxin production in infected seed.

3. Priority Area

Development of diagnostic tools for different seed borne diseases and storage pests

The seed health testing methods have become very critical and target oriented.The methods
employed for the detection of seed borne viruses and bacteria are very sensitive and accurate,
however needs fairly good knowledge of microbiology, plant biotechnology and plant pathology
considering the present set up of seed analysis. Tremendous scope exists for the application of
these methods. Seed health consciousness is gaining importance. Seed health awareness
isincreasing. Academically more and more methods are being evolved which areaccurate and
less time consuming but general adoption is far away due to the involvement of scientific
infrastructure, manpower and expertise in our conditions.

4. Proposed duration of the project

6 months period is required to install and run the testing

Seed health test Duration

Wash test 1 hr

Blotter test 7 days

Plate culture test 8 days

ELISA test 1 - 3 hours

PCR test 1 hr

5. Objectives of the project

1. To satisfy quarantine requirement of a country

2. To know storage quality of seed lot

3. To know the efficacy of seed treating chemicals

4. For checking advisability of seed treatment

5. To know the planting value of a seed lot in order to forecast the field emergence and predict
the health of mature crop

6. Methodology

Tests for a wide variety of organisms on many species of seed:

 Wash test (nematodes, cysts, spores)

 Blotter test (fungi, bacteria)
 Plate culture tests (fungi, bacteria)
 ELISA test (viruses, bacteria)
 PCR test (bacteria)

Wash test
The sample is suspended in required amount of water to cover the seeds (10-100 ml)
shaken thoroughly and spun in centrifuge at 1500-3000 rpm for 5 minutes. The
suspension is mounted in glycerol and observed under the compound binocular
microscope (40 x). The method is suitable to detect oospores or other fungal conidia
present as contaminants.

standard blotter method test

Four hundred seeds are subjected to the standard blotter paper method (SBM) by
following the rules of ISTA (2003). Ten seeds per plate are plated on three layered
moistened blotter discs in sterilized perspex plastic plates. These plates are incubated at
25 +_ 2°C under alternating cycles of 12/12h of near ultra violet (NUV) light and
darkness for seven to thirteen days depending on the crop. The plated seeds are examined
under a stereoscopic binocular microscope for fungal colonies. Subcultures are made on
fresh PDA from fungi emerging on blotter and agar plates for proper identification and
pathogenicity tests. Relative Percent Occurrence (RPO or Infection Incidence) is
reported.

RPO is calculated using the formula:

Agar plate method
Fungal contamination was also determined by agar plating method (Lacey, 1988). Two hundred
seeds in four replicates of 50 seeds each are surface sterilized in 0.35 % sodium hypochlorite
solution for 2 minutes and plated at 5 seeds per plate on PDA in an aseptic condition. The plates
are incubated for 5 days at 25 +_ 2 °C under alternative cycles of 12/12 h of NUV and darkness.
They were observed using stereo-binocular microscope and the percent incidence of fungal
contamination are recorded.

ELISA ( Enzyme Linked Immuno Sorbent Assay)

Basic principles of ELISA

 Enzyme is used to detect the binding of Antibody - Antigen

 Enzyme converts colorless substrate into colored product, indicating the presence of
Antibody - Antigen complex

 ELISA can be used to detect either presence of Antibodies or Antigens

Procedure

Step 1 : Add sample

Step 2 : Add Antibodies

Step 3 : washing

Step 4 : Add substrate

PCR test

Polymerase Chain Reaction (PCR) is a high-tech method to extract, increase, detect and identify
DNA from biotech traits (if present). It is highly sensitive and especially good in determining
absence of biotech traits. PCR can give qualitative or quantitative results. Attention to detail and
in depth knowledge of molecular biology and available biotech traits are critical to success in
using PCR.

WHEN TO TEST
Seed health testing should be done when pathogens are suspected for crop or seed damage. It
should also be conducted when local, regional, national or international rules apply to seed and
crop shipping.

Seed health standards and tests differ from country to country. Ensure your crops meet the
destination market requirements with testing services.

7. Financial outlay of project

Other facilities - Incubation room

Specifications: Air-conditioned room that could be maintained at 20-25°C. Shelves with two
near ultraviolet light tubes hung at 40 cm height from the bench and 20 cm apart, light cycle
controlling device

Other facilities

Screen house Rs. ₹1,500,000

Climate room Rs. ₹1,500,000

Molecular detection of seed borne pathogens

Thermo cycler Rs. ₹600,000

Ultra freezer (-80°C) Preservation of reference culture Rs. ₹450,000

Gel documentation unit Scan and store gel data Rs. ₹600,000

List of equipment needed to set up a seed health-testing laboratory

Equipment Unit Approx. Price (rs)

Low speed centrifuge 1 7500

Stereo binocular microscope 4 300,000

Compound binocular microscope 2 150,000

Laminar flow 1 150,000

Hot air oven 1 30,000

Micro plate reader (ELISA reader) 1 300,000 - 600,000

Deep freezer (- 20°C) 1 112,000

Autoclaves 1 75,000

UV lamp 1 15,000

8. Expect outcome :

 Earliest possible establishment of the infection in seedlings is prevented

 Prevention of inoculating uninfested soil
 use of healthy seed is a key factor to boost agricultural production and quality of produce

9. Useful Internet Resources

1. https://www.sgsgroup.in/en-gb/agriculture-food/seed-and-crop/seed-services/seed-health-
testing
2. https://www.seedtest.org/en/ista-seed-health-testing-methods-_content---1--1132--
234.html

3. https://seedhealth.org/seed-health-testing-methods/#fdf55cc8b36a02338

4. https://www.biologydiscussion.com/plants/plant-diseases/3-techniques-for-detection-of-
fungal-pathogens-in-plants/43184#:~:text=The%20seeds%20are%20plated
%20as,24%20hours%20under%20usual%20conditions.