This document discusses various methods and trends in plant taxonomy, including: 1. Analyzing gross morphology, anatomy, pollen morphology, chemical constituents, serology, paleontology, ontogeny, cytogenetics, and embryology. 2. Numerical taxonomy involves using mathematical methods to classify plants based on quantitative evaluations of their similarities. 3. Descriptions cover anatomical features, chemotaxonomy, serotaxonomy, paleontological evidence of plant evolution, relationships shown by ontogeny and floral development, the role of cytogenetics and biosystematics, and using embryology for phylogenetic studies.

1. GPB-504
Assignment
Seed viability testing.
Testing of pollen viability.
Tissue culture of crop plants.
Description of flowering plants in botanical terms
in relation to taxonomy.
Preparation of different agro-chemical doses in
field and pot applications.
Submitted to
Dr. Abhinav Dayal
Genetics and Plant Breeding
Submitted by
Muppala Tanuja
Genetics and Plant Breeding

2. CONTENTS
Seed viability testing.
Testing of pollen viability.
Tissue culture of crop plants.
Description of flowering plants in
botanical terms in relation to taxonomy.
Preparation of different agro-chemical
doses in field and pot applications.

3. Seed viability is the ability of the embryo to germinate
and is affected by a number of different conditions
(or)
Seed viability is the capability of plant structure (seed,
cuttings etc.) to show living properties like germination
and growth.
( or )
The degree to which the seed is alive (metabolically
active)

4. SEED VIABILITY
 Viability is highest at the point of physiological maturity
and then gradually declines
 Average life span of a seed is 10 to 15 years.
 Some are very short-lived. e.g. willow(< 1 week)
 Some are very long-lived. e.g. mimosa 221years
 Conditions are very important for longivity.
 Cold, dry, anaerobic conditions
 These are the conditions which are maintained in seed
banks

5.  Tetrazolium test ( Tz )
 Germination test
 Cut test
 X-ray analysis
 Spectral imaging .
 Ferric Chloride Test for Mechanical Damage .
 Indoxyl Acetate Test for Seed Coat Damage.
 Non invasive diagnosis of seed viability using
infrared thermograph.
SEED VIABILITY TESTS

6. TETRAZOLIUM TEST
 TETRAZOLIUM TEST (George Lakon in 1942)
 OBJECTIVE: Rapid assessment of viability
PRINCIPLE:
“A colorless tetrazolium solution is used as an indicator producing in
living cells a red, stable and non-diffusible substance, named
Formazan.
Thus, it’s possible to distinguish the red colored living tissues from the
colorless dead ones and the seeds are classified into viable and non
viable seed classes.”

7. PRECONDITIONING OF SEED BEFORE
TETRAZOLIUM (Tz) TEST
 Preconditioing of seeds before Tetrazolium (Tz) test no
moistening or preparation required (small seeded
legumes with soft coats).
 Seeds directly placed in Tz solution in case of peas and
beans bisect longitudinally before placing in Tz
solution.
 Eg: The seed coat may be removed e.g. cucurbits The
seed coat may be scratched above embryo e.g. lettuce

8. METHODOLOGY
SEED HYDRATION
 It is done by soaking seeds in water for a specific period
of time .
 This is done to active hydrolytic enzyme
(dehydrogenase) and stimulate respiration.
CUTTING OR PUNCTURING OF SEED
 This is done to allow the penetration of Tz solution in
to internal tissues.

9. STAINING OF SEEDS
Staining of seeds: It is done by soaking seeds in Tz
solution for a specific period of time to allow staining of
viable tissue in the seed.
Tz is used @ 0.1 or 1.0 % solution, at 30-35º C
temperature for 24-48 hours at pH of 6-8.

10. EVALUATION OF SEED
 Evaluation of seeds evaluated according to staining
pattern.

11. GERMINATION TEST
OBJECTIVE:
 To gain information about the field planting value of the seed lot.
GERMINATION :
Germination in a laboratory test refer to
the emergence from the seed embryo
of those essential structures which for the
kind or seed being tested; indicate its
ability to develop into a normal plant
under favourable conditions.

12. Testing Of Pollen Viability
•Pollen viability is critical for any studies on pollen biology. It is
imperative to know the extent of viability of the pollen sample to be
used for experimentation or pollination.
•With stored pollen also, its viability needs to be monitored under
different storage conditions. There is no universal viability test which is
quick, simple, and reliable.
•Treating the pollen grains with non vital stains such as acetocarmine,
iodine in potassium iodide, and aniline blue in lactophenol essentially
imparts colour to the contents of the pollen in fresh as well as
fixed/dead pollen.
•Staining with non vital stains may be useful to determine the degree
of pollen sterility in plants of hybrid origin or those grown under
unfavourable conditions (Alexander 1969, 1980), but it is not useful for
assessing pollen viability

14. In Vitro Germination Test
•This is the most commonly used test for pollen viability.
It is rapid and reasonably simple; in many species the test
shows correlation with fruit set and seed set (Visser 1955;
Akihama et al. 1978; Janssen and Hermsen 1980).
•A major limitation of this test is the difficulty in
achieving satisfactory germination in many species,
especially in three-celled pollen systems. Also, the
medium which elicits optimal germination of fresh pollen
may not be suitable for stored pollen (see Johri and Vasil
1961).

15. TetrazoliumTest
•The tetrazolium test is based on the reduction of a colorless soluble
tetrazolium salt to a reddish insoluble substance called formazan, in the
presence of dehydrogenases.
•Nitroblue tetrazolium and 2,3,5-triphenyl tetrazolium chloride are the
most commonly used tetrazolium salts (Hauser and Morrison 1964;
Stanley and Linskens 1974). With many species, the tetrazolium test has
proved satisfactory in assessing pollen viability. In many other systems,
however, the tetrazolium test tends to give false positive scores for pollen
viability, and does not show positive correlation with the in vitro
germination test.
• Also, pollen grains often show a gradation in color development (from
very light to deep red); therefore judging a cut-off point for color
intensity to determine pollen viability becomes subjective.

16. Special Requirements
•2,3,5-Triphenyltetrazolium chloride (TIC) or any other
suitable tetrazolium salt.
•Prepare 0.2-0.5% TIC in sucrose solution of suitable
concentration, to prevent bursting of pollen grains.
Addition of a small amount of triazine derivatives to TIC
solution in sucrose acts as an intermediate co-factor and
thus facilitates acceptance of hydrogen ions by the
tetrazolium salt and consequently improves the pollen
response.
•V (Precaution: TIC solution undergoes photo-
oxidation; hence store it in a brown bottle; TIC solution
can be stored in the refrigerator for a couple of weeks.)

17. Procedure
1. Take a drop of TIC solution on a microslide.
2. Suspend a small amount of pollen in the TIC drop and distribute it
uniformly in the drop.
3. Apply a coverglass. (Note: oxygen inhibits reduction of TIC, and
therefore hanging drop or sitting drop cultures are not suitable.)
4. Transfer the preparation to a humidity chamber (>95% RH).
5. Incubate the preparation in dark (keep the preparations in a closed
chamber such as table drawer) under laboratory temperature or at 30 ±
2°C for 30-60 min.
6. At the end of the chosen incubation period, observe the preparation
under microscope and score it for percentage of viable pollen grains,
i.e., pollen grains that have turned red due to accumulation of
formazan V (Precaution: score pollen grains from only the central
area in the preparation;
pollen grains lying near the margin of the coverglass show variable
degrees of red coloration due to higher oxygen availability.)

18. WHAT IS TISSUE CULTURE?
TISSUE CULTURE is the term
used for “the process of
growing cells artificially in
the laboratory.
PLANT TISSUE CULTURE-
Technique of growing plant
cells, tissues, and organs in
an artificially prepared
nutrient medium under
aseptic condition.
Plant cells are totipotent.
Origin : Early
2 0 t h century
with the work
of Gottleib
Haberlandt
(plants).

19. REQUIREMENT OF TISSUE CULTURE
Appropriate tissue (explant)
• A suitable growth medium (Some of the
common media are MS, LS, Gamborg B5,
White’s, tfellar for haploids etc)
• Growth regulators
• Aseptic (sterile) conditions
• Frequent subculturing :

20. TYPES OF PLANT TISSUE
TYPES OF PLANT TISSUE CULTURE
• SEED CULTURE
• EMBRYO CULTURE
• MERISTEM CULTURE
• BUD CULTURE
• CALLUS CULTURE
• CELL SUSPENSION CULTURE
• ANTHER CULTURE/OVARY CULTURE
• PROTOPLAST CULTURE

21. PROCESS OF PLANT TISSUE CULTURE
Selection of
explant from
mother plant
Parent plant Inoculation Light & war m t h
for shoot initiation
Shoots are
transferred to
rootingmedia(hig
her auxin conc)
Shoot
formation
Callus induction
Plants are kept
for hardening in
green house
,sold to a
nursery and

22. Tissue culture plants sold to
a nursery & then potted up
The rooted shoots
are potted up
(deflasked) and
‘hardened off’
This isnecessary as
many young tissue
culture plants have
no waxy cuticle to
prevent water loss.

23. Description of Flowering Plants in botanical
terms in relation to taxonomy
The following points highlight the ten modern trends in
taxonomy of Indian flowering plants.
The trends are:
1. Gross Morphological
2. Anatomical
3. Pollen Morphology or Palynological
4. Chemotaxonomy
5. Serotaxonomy
6. Palaeontological
7. Ontogenetical
8. Cytogenetics and Biosystematics, Embryology and Taxonomy
10. Numerical Taxonomy.

24. Taxonomy: Trend # 1. Gross Morphological:
•Turril says, the morphology, either of plants or animals, now days
includes a good deal more than the mere study of shape.
•The term ‘morphology’ is very wide and includes anatomy, and much of
cytology, ontogeny, embryology, palaeontology and genetics. He says,
“even within morphology in a very strict sense the taxonomist is
forced to consider matters which involve physiology.”
Taxonomy: Trend # 2. Anatomical:
•The study of morphology with a compound microscope is known as
anatomy (histology). According to some anatomists the anatomical
features are more conservative than those of- gross morphology and are,
therefore, of greater use in tracing phylogeny or organogenesis.
•Vesque has given much emphasis upon the value of anatomy in
taxonomic and phylogenetic research.

25. The following anatomical characters are of taxonomic
significance:
1.Epidermal appendages, trichomes and emergences of all
kinds.
2.Structure of stomata.
3.The wood anatomy including the elements of the
secondary xylem; this is very useful in modem taxonomic
studies, particularly in establishing the phylogenetic
relationships among taxa, and in many cases serves as an
aid in the identification of orders.
4.The nodal anatomy of the axis, the leaf trace nature.
5.Floral anatomy.

27. Taxonomy: Trend # 4. Chemotaxonomy:
•The science of chemical taxonomy (chemotaxonomy) is based on the
classification of plants on the basis of their chemical constituents which
are deeply concerned with the molecular characteristics.
• The method of chemical taxonomy is simple in principle and is based
on the investigation of the distribution of chemical compounds, or groups
of biosynthetically related compounds in series of related plants.
•Different plants sometimes contain substances which although belong to
different chemical compounds appear to be biosynthetically analogous.
•Such plants may contain similar enzyme system, and the compounds
produced by such enzymes infer that relationships exist between related
plants.
•However, the chemotaxonomic studies include the investigations of the
patterns of the compounds existing in plants, and in all the individual
parts of the plants, such as bark, wood, leaves, roots, etc.

28. Taxonomy: Trend # 5. Serotaxonomy:
•Serotaxonomic test and consequent phylogenetic
relationships between the taxa of angiosperms were
established in Germany by Professor K. C. Mez (1866-1944)
at the University of Koenigsberg in 1926, and this was
modified in 1936.
•He established that relationship between larger group of
angiospermic plants could be determined by serological test;
and the closely related taxa and plants could be arranged
accordingly.
•Serotaxonomy consists of the study and analysis of protein
reaction of plants of different families with the blood serum of
either rabbit or guineapig.

29. Taxonomy: Trend # 6. Palaeontological:
Darrah has proposed the following palaeontological evidences in support of the
phylogenetic evolution of the plants:
•The invasion of land by an undifferentiated thalloid plant took place not later than
Silurian.
•The simple upright undifferentiated and protostelic axis formed in Silurian.
•Enlargement of the plant body with specialization towards a division of labour-sterile
and fertile, took place apparently in late Silurian.
•Origin of the photosynthetic leaf took place in Devonian.
•Specialization in the service of support, with the resultant secondary body took place in
Devonian.
•Development of the strobilus also took place in Devonian.
•The development of heterospory took place in upper Devonian if not earlier.
•Retention of the gametophyte within megaspore, within the megasporangium, where
fertilization takes place with the ultimate attainment of the seed took place probably in
upper Devonian.
•Evolution of the pollen grain took place in Carboniferous if not earlier.
•Evolution of the bisporangiate flower took place in Triassic period.
•Attainment of the condition of angiospermy took place in Jurassic and Triassic periods.
•Evolution of the herbaceous habit of the angiosperms took place in Cretaceous (chiefly
Cenozoic).

30. Taxonomy: Trend # 7. Ontogenetical:
•According to Gunderson the evolution of the flowering plants is based on floral
structure and presumed organogenesis. He points out that the flowering tendencies are
widely accepted as progressive in organogenesis and agree with the findings in
development.
•Petals — from separate to united.
•Petals — from actinomorphy to zygomorphy. Sepals — from separate to united.
•Ovary — from superior to inferior.
•Carpels — from separate to united.
• Placentation — from parietal to axile.
Taxonomy: Trend # 8. Cytogenetics and Biosystematics:
•In modern days cytology has played an important role in taxonomy to achieve a
classification which represents mutual relationships and is useful in indexing plants.
•Modern taxonomy takes up cytological evidence based on chromosome numbers,
chromosome morphology, chromosome behaviour in meiosis and in aberrant forms of
reproduction.
•The branch of taxonomy principally based on cytology is known as ‘cytotaxonomy’, it
is a part of experimental taxonomy.
•It includes cytological aspects, study of cytogenetics and phenomeria together with
consideration of classical aspects of taxonomy.

31. Taxonomy: Trend # 9. Embryology and Taxonomy:
•The comparative or phylogenetic embryology deals with the embryological data,
which are used as a tool for ascertaining inter-relationships and taxonomic positions. In
a symposium on comparative Embryology of Angiosperms held at Delhi in 1967 the
systematic positions of various families were discussed in the light of embryological
data.
•This symposium enabled to bring together in a comprehensive manner the scattered
embryological literature on various families.
Taxonomy: Trend # 10. Numerical Taxonomy:
•Numerical plant taxonomy may be defined as the science in which for the purpose of
classifying the plants, mathematical methods are used. In other words, the application of
simple mathematical principles or techniques or methods in taxonomical studies of
plants may be defined as numerical taxonomy.
•Heywood (1967) defines numerical taxonomy as “the numerical evaluation of the
similarity between groups of organisms and the ordering of these groups into
higher-ranking taxa on the basis of these similarities”.
•According to Sneath and Sokal (1973) “numerical taxonomy aims to develop
methods that are objective, explicit and repeatable, both in evaluation of
taxonomic relationships and in the erection of taxa.”

32. Preparation of different
Agro-chemical doses for field
& Pot Application

33. Introduction
Agrochemical (or agrichemical), a contraction
of agricultural chemical, is a generic term for
the various chemical products used in
agriculture.
Many agrichemicals are toxic, and agrichemicals
in bulk storage may pose significant
environmental and/or health risks, particularly
in the event of accidental spills.
In many countries, use of agrichemicals is highly
regulated.

34. The broad categoryof
agrochemicals are:
AGROCHEMICALS
Pesticides
Insecticide
Acaricide
Nematicide
Molluscicide
Rodenticide
Fungicide
Bactericide
Herbicide/
Weedicide
Synthetic
Fertilizers
Growth
regulators

35. Synthetic Fertilizers
A compound made artificially by chemical
reactions. Not genuine or natural.
They are specifically designed to feed a
plant a certain amount of specific
nutrients.
An example of a synthetic fertilizer input
would be urea. This common nitrogen
source is famous for its quick release and
soluble nature.
Phosphoric acid and potash are also the
most common phosphorus and
potassium ingredients in synthetic
fertilizers

36. Nearly all nitrogen that plants useisin the form of NH3
or NO3 compounds. The usable phosphorus compounds
are usually in the form of phosphoric acid (H3PO4) and
the potassium (K) is typically in the form of potassium
chloride (KCl).
In commercial fertilizers the same required
compounds are available in easily dissolved
compounds that require no decay—they can be used
almost immediately after water isapplied.
Inorganic fertilizers are usually much more
concentrated with up to 64% (18-46-0) of their weight
being a given plant nutrient, compared to organic
fertilizers that only provide 0.4% or lessof their weight
asa given plant nutrient.

37. Compound fertilizers often combine N, P and K
fertilizers into easily dissolved pellets. The N:P:K
ratios quoted on fertilizers give the weight
percent of the fertilizer in nitrogen (N),
phosphate (P2O5) and potash (K2O equivalent)

38. Synthetic fertilizers are commonly used for growing all
crops, with application rates depending on the soil fertility,
usually as measured by a soil test and according to the
particular crop. Legumes, for example, fix nitrogen from the
atmosphere and generally do not require nitrogen fertilizer.
Studies have shown that application of nitrogen fertilizer on
off-season cover crops can increase the biomass (and
subsequent green manure value) of these crops, while
having a beneficial effect on soil nitrogen levels for the main
crop planted during the summer season
Application

39. Nutrients in soil can be thrown out of balance
with high concentrations of fertilizers.
Applying excessive amounts of fertilizer has
negative environmental effects.
To avoid over-application, the nutrient status
of crops should be assessed. Nutrient
deficiency can be detected by visually
assessing the physical symptoms of the crop.
Both soil tests and Plant Tissue Test are used
in agriculture to fine-tune nutrient
management to the crops needs.

40. DIFFERENT
DOSES FOR POT
APPLICATION

41. POT APPLICATION
Pesticide Technical Name Field
(1acre)
Potdose
(1litre)
Application
Fame Flubendamide
39.35 SG
20-40ml
For 200L
0.1 - 0.2ml/L Foliar insecticide(Corn,cotton,
fruits, grapes, nuts, orchard
crops, rice,
soybean, tree fruit,vegetables)
Proclaime Imamactin
Benzoiate 5SG
90g For
200L
0.45g/L Bollworm in cotton
Glamore Idaprole 40%+
Imidacloprid
40% UUG
50g For
200L
0.25g/L Aphids, canebeetles, sucking
insect, termites, soil insects
Tokain Danitoporain
20% SG
80g For
200L
0.40g/L
Polo Diafenthiuron
50%SC
240g For
200L
1.2g/L Insecticide or acaricide (aphids &
white fly in various fruits,
vegetables & cotton)

42. Fungicide Technical Name Field
(1 Acre)
Pot Dose
(1 Litre)
Application
Score Difenoconazole
2.5% EC
100ml Per 200l 0.5ml/L Foliar fungicide or seed
treatment (rice,wheat, burley,
potato, banana,grape)
Raxile Tebokonazole 2%
DS
40g Per 200l 0.2g/L Seed treatment
fungicide (Cereals)
Cabrio
Top
Metirame 55%+
Piraclostrobina5%
WG
600g Per 200l 3g/L Fungal diseases in carrot,
cole crops, onion, tomato,
leafy & tuber vegetables,
grapes
Poliram Metirame 70%WG 800g Per 200l 4g/L Early blight, late blight in
potatoes, applerust
Avatar Hexaconazole 4%+
Zineb 68%WP
400-500g Per
200l
2-2.5g/L Tea plants, sheath blight of
paddy, leafspots, red rot of
sugar cane

43. Bactericide Technical Name Field
(1 Acre)
Pot Dose
(1 Litre)
Application
Phytolan Copper
Oxychloride 50%
WP
600g Per
200L
3g/L Leaf spot, fruit rot, foot
rot, black rot, rust,
blight, brown leaf spot,
bud rot, early blight, red
rust, downy mildew
Coside Copper
Hydrochloride 77%
WP
600g Per
200L
3g/L Leaf spot, anthracnose,
tikka leaf spot, blister
blight, False smut.
Thiovit
Sulphex
Wettable Sulphur 600g Per
200L
3g/L All citrus fruits
Zed -78 Zenab 75% WP 400g Per
200L
2g/L Downy Mildew, Scab,
Early Blight. Forapple,
Grapes, Beans, Potato,
Tomato

44. Weedicide Technical
Name
Field
(1 Acre)
Pot Dose
(1 Litre)
Application
Millchlore,
Billchlore
Butachlore 1L/200L 5ml/L Rice field
Anilogaurd,
Anilthan
Anilophos 0.5L/200L 2.5ml/L Paddy, soya bean
Stamp,
Dhanutop
Pendimethali
n
1L/200L 5ml/L Wheat, corn, soybeans
potatoes, cabbage, peas,
carrots and asparagus
Weedmar,
Walkweed
2, 4 -
Dichlorophe
noxy Acetic
Acid
500g/200L 2.5g/L For perennial and broad
leaf weeds in rice &
wheat field
Leader,
Phatae,
korosan
Ethoxysulph
uran 75WP
60ml/200L 0.3ml/L Rice & wheat field

45. 1. Bhaduri,d., Gautam,p., 2013,optimization & Validation Of
Targeted Yield Equation –Based Fertilizer Doses Under INM
For Wheat In Tarai Region Of Uttarakhand, India, Indian
Journal Of Agricultural Research, Vol-47(1), PP 16-25.
2. https://www.slideshare.net/Supta2013/preparation-of-different-
agro-chemical-doses-for-field-pot application
3. https://www2.slideshare.net/KomalJalan/plant-tissue-cultureits-
methods
4. https://www2.slideshare.net/search/slideshow?searchfrom=he
ader&q=testing+of+pollen+viability
5. https://www2.slideshare.net/SandeepVunnam/seed-
viability?from_action=save
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