VARIETAL IDENTIFICATION VARIETAL IDENTIFICATION USING LABORATORY TECHNIQUESUSING LABORATORY TECHNIQUES

Siddhartha Swarup JenaRAD/10-30

Ph.D. Mol. Bio & Biotech

Siddhartha Swarup JenaRAD/10-30

Ph.D. Mol. Bio & Biotech

Introduction

Morphological features

Special laboratory techniques

Rapid chemical tests Other laboratory techniques

Conclusion

Contents

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Intense crop improvement programme – development of large number of hybrids & varieties.

Due to widely grown seed industries – every chance of varietal mixture.

Varietal identification therefore attained critical importance to maintain genetic purity of seeds.

INTRODUCTIONINTRODUCTION

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Morphological features have been a major component of cultivar identification.

Thus Grow-out test has got importance under certification.

Limitations:Time consuming & expensive Require large areas of land & highly skilled personnel many morphological descriptors are multigenic, Quantitative characters altered by environment.

Morphological features

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1. Rapid chemical tests:

a) Phenol tests : rice, wheat, barley, oats

b) Peroxidase test : soybean, groundnut

c) Potassium hydroxide bleach test : sorghum, rice, sunflower

d) Ferrous sulphate test : oat, sunflower

e) Hydrochloric acid test : oat

f) Vanillin test : faba bean

g) Alkaloid test : lupine

h) Copper sulphate-ammonia test : sweet clover

i) Sodium hydroxide test : rice, sunflower. Wheat

j) Seedling growth response to GA3 : rice, maize

k) Seedling growth response to 2,4-D : rice, maize

Special laboratory techniques

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2. Fluorescence tests : oats, rye grass, peas, soybean

3. Seedling pigmentation : maize, sunflower, pearl millet

4. Herbicide injury test : sunflower, soybean

5. Machine vision image analysis: all crops

6. Seed radiography : all crops

7. Chromosome count : wheat, cotton

8. High performance liquid

chromatography (HPLC) : all crops

Special laboratory techniques cont.

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a) Phenol test:

Based on phenol oxidase activity

Reaction involves melanin formation by oxidizing phenol via orthoquinones and hydroxyquinones.

Reaction is controlled by tyrosinase in seed coat and is under genetic control.

The colour reaction is constant for a variety.

1. Rapid chemical tests 1. Rapid chemical tests

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Methods of phenol test:

Seeds presoaked in water for 24 hr at 25 °C

Transferred to whatman no. 1 filter paper moistened with 1% phenol solution in petridish.

The petridishes are covered and incubated at 30 °C

Colour reaction is noted after 24 hrs

Based on colour genotypes are classified into following reaction groups

No colour : no visible change in colour

Light brown : colour of lemma and palea turned light brown

Brown : colour of lemma and palea turned brown

Dark brown : colour of lemma and palea turned dark brown

Black : colour of lemma and palea turned blackS S Jena

b) Peroxidase test:

Seed coats of some soybean var contain a high or low activity peroxidase enzyme. This difference in enzyme activity is used to distinguish between varieties.

Remove seed coats and place in separate test tubes

Add 0.5-1.0 ml of 0.5% guaiacol & wait 10 min

Then add 0.1 ml of 0.1% H2O2 solution to each tube

If solution turns dark reddish brown → +ve reaction

If solution remains clear → -ve reactionS S Jena

c) Potassium hydroxide–bleach test:

The presence or absence of a darkly pigmented testa can be used to differentiate sorghum cultivars.

The dark pigment in the testa is tannic acid.

Prepare 1.5 (w/v) sol. of KOH & fresh bleach (5.25% NaOCl)

Put seeds in glass container & completely cover with KOH bleach sol.

Soak the seeds with brown seed coats for 10 min and seeds with white coat for 5 min.

Gently rinse with tap water, then air dried.

Record number of dark seeds & light seeds

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d) Ferrous sulphate test:

soak the oat seed in water for 5-6 hr at 20°C

Place in petridish lined with filter paper presoaked with 1% FeSO4 sol.

The colour developed is noted.

The varieties are classified into following FeSO4 colour reaction group Grey coloration of whole seed Grey spots Grey streaks Brown spots Brown streak

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e) Hydrochloric acid test:

Useful for testing treated seeds when the result of fluorescence are in doubt.

The fluorescent seeds stain tan and non-fluorescent seeds stain yellow.

Place oat seeds in glass beaker & cover with a sol. consisting of one part conc. HCl & four parts

distilled water

After 6 hrs of soaking remove the seeds & air-dry on filter paper

Classify the seeds as tan (fluorescent) or yellow (non-fluorescent)

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f) Vanillin test:

Presence or absence of tannins in varieties with colored seeds can be determined after about 6 months of storage, since the testa of seeds containing tannins darken with time.

Reagent: add 1% (w/v) vanillin in conc. HCl to an equal amount of 50% (v/v) ethanol.

A drop of acid vanillin reagent is placed on the inside surface of a piece of testa previously removed

from seed

After 1-2 min a dark pink color on the inner surface of testa indicates the presence of tannins. If no change in color occurs, tannins are absent.

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g) Alkaloid test:

A bitter poisonous alkaloid levels in varieties of Lupine sp. can be tested with lugol’s solution

This test can distinguish seeds with low alkaloid content called sweet lupine from seeds with high alkaloid content called bitter lupine.

Seeds are imbibed in water for 18-24 hrs & cut longitudinally to separate cotyledons.

Cotyledons are placed on a glass plate & a drop of Lugol’s sol. (0.3 g of iodine & 0.6 g of potassium iodide in 100

ml of water) is put on cut surface

Soak for 30 sec in a beaker & rinsed in water

Cotyledons with high alkaloid develop a reddish brown color & low alkaloid content don’t develop any color.

h) Sodium hydroxide test:

Seeds are soaked in 2% NaOH sol. for 1 hr and change in colour of the solution & seeds are observed.

Based on the intensity of color reaction the genotypes are classified into two groups as yellow & light yellow.

e.g. Rice, sunflower

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i) Seedling growth response to GA3:

One hundred seeds from each genotype are placed on a two layer of blotter paper moistened with GA3 @ 25 ppm in petriplates & incubated for 4 days at 30°C

The test is conducted in 4 replicates of 25 seeds each

The observation on coleoptile length is measured in cm and the percentage of increase in coleoptile length over control is worked out.

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Seedling growth response to GA3 cont.

Based on the growth response, the genotypes will be classified into following groups

Very slow response type : < 25% increase over control

Low response type : 26-50% increase over control

Medium response type : 51-75% increase over control

High response type : 76-100% increase over control

Very high response type : > 100% increase over control

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j) Seedling growth response to 2,4-D:

The methodology is same like GA3. Percentage reduction of coleoptile growth over control is worked out.

Genotypes will be classified into following groups based on growth response

High resistance type : < 30% reduction over control

Medium resistant type: 30-60% reduction over control

Low resistant type : > 60% reduction over control

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2. Fluorescence tests 2. Fluorescence tests

Under UV & near UV light, seeds, parts of seedlings or secretion from seedling reflect particular colour light, in others this phenomenon doesn’t occur.

Hellobo in 1933 reported fluorescence test as an aid to variety identification in oats, where lemma and palea of white, yellow, grey and black oats reacted differently with fluorescence reactions.

Funkur et.al. (1954) studied the inheritance of the fluorescence character of the lemma and postulated that it was governed by one or two major factors.

Fluorescence was found dominant to non-fluorescence.

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Examples of Fluorescence tests

Examples of Fluorescence tests

Oat: yellow oat absorbs UV light but seeds of white seeded var fluoresce clearly in UV light.

Pea: UV light is used to distinguish garden & fodder pea since fodder pea seeds don’t fluoresce

Vicia faba: the testa of some varieties clearly fluoresce in UV light

Allium: seedlings of A. cepa fluoresce yellow where as A. porum – bluish.

Beet: roots fluoresce in different varietiesS S Jena

3. Seedling pigmentation3. Seedling

pigmentationSeed samples grown for evaluation of anthocyanin or purple pigment development in hypocotyls, coleoptiles or shoots should be grown in inert sand.

Seeds of sunflower should be planted 1 cm deep & spaced 2 cm apart in rows 4 cm apart. Watered every day with nutrient free water. A photoperiod of 24hrs should be used. Samples should be grown at 25°C.

Seedlings can be evaluated for coleoptile and shoot color for up to 14 days after emergence.

Four possible shoot pigmentation patterns are- dark purple, intermediate purple, light purple & green.

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4. Herbicide injury4. Herbicide injury

The growth chamber & green house testing involves exposing seedlings and plants to chemicals such as herbicides (Metribuzin).

The degree of herbicide induced injury or chemically altered growth can be recorded.

Varieties can be classified as -

Most sensitive: plants show signs of injury after 9 days of application & are dead after 16 days of application

Moderately sensitive: seedlings show signs of injury after 13 days of application & are dead after 32 days of application.

Least sensitive: seedlings show signs of injury after 20 days of application & survive 38 days of application.

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5. Leaf epidermal morphology

5. Leaf epidermal morphology

Use of epidermal characters for identification of plants was proposed by Stoddard (1968).

He described a convenient technique for preparing cellulose acetate replicas of the pattern of epidermal cells

A thin film of about 1 cm2 of nail polish or quick fix applied over the upper surface of 30 day old plants, allowed to dry on

Leaf is removed after 10 min, mounted dry on a slide

Under magnification it would reveal the cellular impressions of the leaf surface like size, shape & arrangement of cells

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6. Machine vision image analysis

6. Machine vision image analysis

A computer based system used by Travis & Draper (1985) for image analysis of seeds.

Analyze the length, width, thickness etc. of the seed with precision and high speed at a very reasonable cost.

The image of the seed is captured using a video system linked to an analogue of a digital converter. Computer can be programmed to make a detailed analysis of all captured images.

A great advantage of machine vision is that it is possible to make detailed comparisons of sets of data i.e. to operate pattern recognition systems.

Attractive system for both variety description & identification.S S Jena

Advantages:

Allows rapid analysis to resolve cultivar differences in the shape, size of the seeds and other plant parts.

Quantification of different parts of the plants is also possible

High speed-up of operation; results are producible & very low running cost

Minute difference in cultivar can be worked out

Disadvantages:

o Involves high initial cost

o High skill is needed in processing & classification of analysis of data & programming.

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X-radiography was first reported by Lundstorm (1903) for assessing the seed quality.

In recent years it is widely used to assess the physical & physiological quality of seeds.

It is quick, easy, reliable and non-destructive

The soft x-rays that pass through the seed are differentially absorbed by different parts depending upon their thickness. Later they strike a photographic film & form the object called radiographs.

Depicts the internal structure of the seeds showing extent of seed filling, insect attack & presence of embryo.

7. Seed radiography7. Seed radiography

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Seed radiography Seed radiography

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In plants with different ploidy levels, counting the number of chromosomes in the root tips is the most conclusive way to differentiate between var with different ploidy levels

Counting the number of plastids in the stomata is another way to determine the ploidy level.

8. Chromosome count8. Chromosome count

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High performance liquid chromatography is a separation technique that can separate a mixture of compounds.

9. HPLC9. HPLC

It uses a pump that moves the mobile phase and analyte through the column and a detector that detects the characteristic retention time for the analyte.

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The recent & most advanced method of varietal identification is by the use of molecular markers.

Various markers commonly used for this are -

RAPDRFLPAFLPSSR, ISSRDArT

10. Molecular markers10. Molecular markers

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Laboratory techniques coupled with morphological methods for varietal identification gives most reliable data and perfection for the purpose.

In situation where morphological differences may be small, the characterization of var./seeds can be done through laboratory techniques.

Many of the techniques are simple & inexpensive and require short time.

CONCLUSION

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