gowthami r, neelam sharma & vartika srivastava · 2019-11-18 · pollen cryopreservation-an aid...

Pollen cryopreservation-an aid for

plant breeding

Eighth International Training Course on In Vitro and Cryopreservation Techniques for

Conservation of Plant Genetic Resources, ICAR-NBPGR, New Delhi, November 5 - 19 2019

Gowthami R, Neelam Sharma & Vartika Srivastava

04

03

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01 Pollen grains represent male gametophytes

Transmit nuclear genetic material

Abundantly produced

Provide phylogenetic evidence in plant systematics

Pollen grains may look like simple structures, but each pollen grain is an independent unit that underwent a precise developmental program

Tiny grains with greater important in plant lifecycle

POLLEN

Eighth International Training Course on ‘In vitro and Cryopreservation Approaches for Conservation of Plant Genetic Resources’, ICAR-NBPGR, New Delhi. Nov 5-19, 2019

1. Exine: The outer thick layer made up of sporopollenin (resistant to physical and biological decomposition) and provides protection

2. Intine: The intine is pecto-cellulose in nature, associated pollen tube formation

3. Vegetative cell: Contains abundant food reserve. Provides the medium for the movement of male gametes inside the pollen tube

4. Generative cell: the generative cell cytoplasm is highly reduced but it contains the usual cell organelles. It divides mitotically to produce two functional male gamete

STRUCTURE OF MATURE POLLEN GRAIN


Pollen – microscopic wonders of plant kingdom

Exine has the rare capacity to sustain heat upto 3,000 OC, could be located even in the Palaeozoic rocks of some 500 Ma old without being destroyed, where all other organic materials remain carbonized and distorted


POLLEN: BEAUTIFUL COLORS, FASCINATING FORMS

Pollen colour chart created by a beekeeping association in the UK

Pollen grains resembles seed in many respects

Pollen Seed Pollen develops in the anther Seeds develop in the ovary/fruit

Pollen is covered by exine, which contains sporopollenin (highly resistant to chemical degradation)

Seed (embryo) is protected by endosperm and hard integuments resistant to fungal attack

Two phases of growth • Development of the microspore up to the end of sporoderm formation • First haploid mitosis , subsequent development of male gametophyte

Two phases of growth • Development of the embryo up to heart-shaped stage • Storage of reserves in the embryo or in Separate endosperm tissue

Two cell types produced during development: •. Vegetative cell has limited function, then degenerates •. Generative cell gives rise to two male gametes

Zygote divides and gives rise to two cell types: •Suspensor has limited function, then degenerates •Terminal cell gives rise to embryo

Different degrees of dehydration before dispersal: orthodox (Desiccation resistant) and recalcitrant (desiccation Sensitive)

Rarely remains viable for few days Seeds may live for years

IS POLLEN AND SEED BEHAVES SAME?


Franchi et al., 2011

POLLEN AND SEED DEVELOPMENT IN ANGIOSPERMS


Why so many pollen grains?

200 2000 20000 200000

Vulpia myuros

Rostraria cristata

Lamarckia aurea

Brachypodium distachyon

Bromus lanceolatus

Bromus hordeaceus

Lindernia ciliata

Limnophila sessiliflora

Murdannia nudiflora

Stipa capensis

Holcus lanatus

Hordeum leporinum

Phalaris minor

Cyanotis cristata

Enydra fluctuans

Trisetaria panicea

Briza maxima

Hydrocotyle sibthropioides

Holcus setiglumis

Aegilops geniculata

Hyparrhenia hirta

Avena barbata

Cynodon dactylon

Vulpia geniculata

Aegilops triuncialis

Elymus repens

Sorghum halepense

Eichhornia crassipes

Festuca arundinacea

Lolium rigidum

Arrhenatherum album

Ludwigia octovalvis subsp. Sessiliflora

Ipomoea aquatica

Nymphaeapubescens

Nymphaea rubra

No. of pollen grains/flower

Pollen production is species-specific

Autogamous (self-fertilizing ) <

Entomophilous (insect-pollinated) <

Anemophilous (wind-dispersed)

Draba reptans produces an average of only four pollen grains per ovule, while wind-pollinated species (e.g. Betula pendula) produce up to 2 million pollen grains per ovule


C. Subba Reddi & N. S. Reddi (1986)

Family P/O ratio Family P/O ratio Family P/O ratio Asclepiadaceae 11 Oxalidaceae 1632 Rhamnaceae 6570 Onagraceae 34 Fumariaceae 1731 Betulaceae 6734 Pyrolaceae 82 Lamiaceae 1808 Poaceae 8131 Alismataceae 175 Asteraceae 1939 Apiaceae 10363 Solanaceae 184 Rubiaceae 1991 Plantaginaceae 13157 Malvaceae 226 Juncaceae 2747 Boraginaceae 18015 Caryophyllaceae 266 Araceae 2750 Chenopodiaceae 18069 Geraniaceae 328 Fabaceae 3312 Rosaceae 23788 Polemoniaceae 441 Polygonaceae 3589 Ulmaceae 28367 Lentibulariaceae 450 Adoxaceae 3902 Balsaminaceae 80033 Saxifragaceae 634 Tiliaceae 4350 Aceraceae 94078 Campanulaceae 647 Trilliaceae 4831 Euphorbiaceae 122190 Ericaceae 665 Brassicaceae 5166 Hippocastanaceae 451543

Primulaceae 690 Liliaceae 5314 Fagaceae 636594 Scrophulariaceae 709 Ranunculaceae 6226 Corylaceae 2119717

What happens to the remaining pollen

Pollen as phytopathological amplifier Pollen as feed for animals Human consumption


Why to store pollen


To serve plant breeders to attempt hybridization between plants cultivated/grown in different geographic regions, or showing non-synchronous flowering.

To provide a constant supply of short-lived (recalcitrant) pollen and for those crops that have recalcitrant seeds and exhibit poor response to tissue culture methods

To facilitates the supplementary pollinations in orchard crops

To ensures the availability of male gametes for hybridization program and eliminate the need to grow male lines continuously in breeding programs

To facilitate continuous source of pollen for studying pollen biology

To ensure the availability of pollen throughout the year

To conserve species where no other conservation methods are available (wild, rare and endangered)


TYPES OF POLLEN- STORAGE PERCPECTIVE

Mature pollen is of two types

BICELLULAR

TRICELLULAR


BICELLULAR AND TRICELLULAR POLLEN

Firon et al., 2012

Water status and pollen development Phase 1

Phase 3 & 4 Phase 2

Phase 5

Microsporogenesis Pollen maturation and Dehydration

Presentation and Dispersal

Pollen stigma interaction and

rehydration

Bicellular pollen Tricellular pollen

Desiccation-resistant, orthodox, or partially-dehydrated

Desiccation-sensitive, hydrated, partially-hydrated, or recalcitrant

70% of the species release pollen in a bicellular stage

About 30% of species release pollen in a tricellular stage

Pollen shed at a lower moisture content, survives desiccation (upto 10%)

Pollen shed at a high moisture content and survives only limited desiccation

All phylogenetically primitive taxa are bi- nucleate

Originated independently at many times during angiosperm evolution

Dispersed in a sexually immature, somewhat dehydrated, metabolically quiescent state

Dispersed in a sexually mature, well-hydrated, metabolically active state

Pollen less differentiated, long lived, often dormant at dispersal

Pollen terminally differentiated and dispersed in a nutrient poor conditions

Pollen longevity under natural conditions is long

Pollen longevity under natural conditions is short

Tolerant to desiccation & freezing Sensitive to desiccation and freezing



Bicellular Tricellular Both

Monocots Musaceae Zingiberaceae Amaryllidaceae Palmae Pandanaceae Dioscoreaceae

Poaceae Euphorbiaceae Convolvulaceae Rutaceae

Dicots Solanaceae Cucurbitacea Moraceae Annonaceae Piperaceae Malvaceae Vitaceae

Amaranthaceae Asteraceae Cruciferae Chenopodiaceae



Brewbaker (1967)

BICELLULAR AND TRI CELLULAR POLLEN

Pollen grains store water in vacuoles and the pollen water content is generally higher than 60% during development

During presentation and dispersal of mature pollen, water-containing vacuoles are absent and the water content generally decreases below 40%

In Tri-nucleate recalcitrant pollen, water loss to a minimum of 20-25 % moisture content causes a complete loss of pollen viability. Hence Desiccation sensitive pollen should be dried till no freezable water exist but above the level where desiccation injury is apparent


Bi-nucleate pollen, water loss to a minimum of <10 % moisture content causes a reduction in pollen viability. Hence Desiccation tolerant pollen should be be dried to a moisture level below which freezable water does not exist

Franchi et al., 2011, Pacini et al., 2011

POLLEN: EXPLANT FOR CRYOPRESERVATION

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Pollen grains are simple, haploid organisms and can be

easily collected and stored in viable condition for long

period

Pollen grains can be cultured on simple medium

Pollen germination and pollen tube growth are rapid, and provide results within a few hours

Maintenance of aseptic conditions in collection and storage is not mandatory

Larger number of alleles can be stored in small space

Handling and Retrieval is easy


In case of sticky pollen, they can be extracted using organic solvents such as cyclohexane or hexane for 2-3 min (to remove the sticky pollen coat substances)

POLLEN COLLECTION

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02

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Pollen should be harvested during peak flowering period and soon after anthesis, usually in the morning hours (8 – 10 am) on a bright sunny day

Pollen should not be collected from infected or insect pest-damaged flowers

Pollen should not be collected on a rainy day or if it had rained overnight

Pollen should not be forced from anther and pollen should be free of anther debris


POLLEN VIABILITY ASSESSMENT

Fertilizing ability

Staining

In vitro germination

In vivo pollen germination and pollen

tube growth

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2

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4

1. Tetrazolium (TTC) test 2. Fluorochromatic Reaction (FCR) or

Fluorescein Diacetate Test (FDA) 3. Acetocarmine test 4. Potassium Iodide staining

1. Hanging Drop Culture (Cavity slide method)

2. Sitting Drop Culture 3. Suspension Culture 4. Surface culture

Pollen –pisitl interaction studies

Seed set and fruit set percentage


POLLEN VIABILITY: TTC

2,3,5-Triphenyltetrazolium chloride Prepare TTC in sucrose solution of suitable concentration, to prevent bursting of pollen grains

TTC solution undergoes photo-oxidation, hence store in a brown bottle TTC test is based on the reduction of a colorless soluble tetrazolium salt to a reddish formazan, in the presence of dehydrogenases in the pollen cytoplasm

Place a drop of TTC solution on a microslide

Viable pollen grains turn red

due to accumulation of

formazan


Suspend a small amount of pollen in the TTC drop

Gently place a coverglass on pollen drop

Distribute pollen uniformly in the drop Transfer the

preparation to a humidity chamber

(>95% RH) for 5-10min

Incubate for 5-10min. at room temperature


POLLEN VIABILITY: FDA OR FCR

Place a drop of sucrose-FDA mixture solution on

a microslide

Suspend a small amount of pollen in

the FDA solution drop


Distribute pollen uniformly in the drop Transfer the preparation

to a humidity chamber (>95% RH) for 5-10min

Incubate for 5-10min. at room temperature

Pollen grains that fluoresce

brightly as viable

Stock solution of FDA is prepared in acetone (2 mg/ml). Add drops of stock solution of FDA to 2-5 ml of sucrose solution until the resulting mixture shows persistent turbidity

Observe under the fluorescence microscope

The mixture (FDA and Sucrose) should be used within ca. 30 min from preparation; otherwise most of the FDA would precipitate

The non-polar non-fluorescent FDA enters the pollen cytoplasm, Esterases of cytoplasm hydrolyze FDA and release fluorescein, which is polar and fluorescent. Viable grains and gives green or yellowish-green fluorescence


POLLEN VIABILITY: POTASSIUM IODIDE

Place a drop of sucrose-FDA mixture solution on

a microslide

Suspend a small amount of pollen in the

TTC drop


Distribute pollen uniformly in the drop

Incubate for 5-10 min at room temp

The dark blue-black colored grains are viable pollens

1% Potassium Iodide (KI): Dissolve 1 gm of iodine and 2 gm of potassium iodide in 100 ml of distilled water

Viable

Non-Viable

Rice

Black gram

Iodine broke up in a watery arrangement of potassium iodide the tri-iodide-anion groups with starch, creating blueblack color


POLLEN VIABILITY: ACETOCARMINE

Place a drop of sucrose-FDA mixture solution on a

microslide

Suspend a small amount of pollen in the TTC drop



Incubate in laboratory temperature for 5-10 min

Observe under the

microscope

The dark red or pinkish colored grains are counted as viable

The pollen nucleus is rich in chromatin material and viable pollen stains pink to deep red with aceto-carmine, while sterile (mostly shriveled) pollen does not take any stain and thus remains almost white and transparent

Acetocarmine (2%): Weigh 2 g of carmine powder, dissolve it in 95ml of 45% glacial acetic. Add distilled water to make a total of 100ml solution. Boil it for 5 min, cool and filtered and stored in a refrigerator

Commonly used pollen culture media Ingredients Brewbaker &

Kwack's Medium Roberts' Medium

Hodgkin & Lyon's Medium

Sucrose 10% 20% 20%

Boric acid 100 mg/1 10 mg/1 100 mg/1

Calcium nitrate 300 mg/1 - 400 mg/1

Calcium chloride - 362 mg/1 -

Magnesium sulfate 200 mg/1 - 200 mg/1

Potassium nitrate 100 mg/1 100 mg/1 100 mg/1

Tris - 60-130 mg/1 -

TAPS - - 4.86 g/l

POLLEN VIABILITY: In vitro germination

In vitro pollen germination is the most commonly used and reliable viability testing method

Place a drop of pollen germination medium

Suspend a small amount of pollen in the

TTC drop



Incubate for 5-10min. at room

temp.


01 03 04

02 Hanging Drop

Culture (Cavity slide method)

Sitting Drop Culture

Suspension Culture

Surface culture

Solid media slice

Petriplate with solid media

POLLEN VIABILITY: In vitro germination methods


Total number of well stained pollens Pollen viability percentage = ----------------------------------------------------------- x 100 Total number of stained and unstained pollens

A minimum of three replications A minimum of 10 microscopic fields to be counted (500 grains) per replication

POLLEN VIABILITY SCORING

Staining methods

In vitro germination


Scoring of pollen germination (Stanley and Linskens, 1974)

Viability Per cent germination

Excellent More than 80

Good 60-80

Moderate 40-60

Low 20-40

Poor Less than 20

Pollen germination (%) = No. of germinated pollen per field of view

˟ 100 Total number of pollen grains per field of view

DETERMINATION OF MOISTURE CONTENT

The moisture content of pollen is of critical importance for the maintenance of viability during long periods of storage

Moisture content should be reduced to ~10 % or more (bicellular) for storage

Regular monitoring of the sample should be done to ensure high viability and low moisture content. Excessive desiccation can lead to a loss in viability

DESICCATION OF POLLEN

AIR DESICCATION SILICA GEL DESICCATION

Charged silica contained in

airtight desiccators Room temperature


4o C

Few Hours

Your Text Here

-20o C to - 40o C

- 80o C

- 196o C

Several days

Several Months

Several Years (∞) Ce

ll ac

tivi

tie

s

Temp.

POLLEN STORAGE


All the metabolic activities arrested during long

term conservation

POLLEN CRYOBANK

POLLEN COLLECTION

AIR DESSICATION SILICA GEL DESSICATION

POLLEN VIABILITY AND MOISTURE CONTENT

PACK ALUMINUM FOIL POUCHES/ CRYOVIALS

STORAGE IN LIQUID NITROGEN


POLLEN CRYOPRESERVATION- APPLICATION

Recalcitrant seed spp, wild, rare and endangered

Germplasm conservation

Biochemical and physiological studies

Haploid production

Pollen mediated gene transfer

In vitro pollination and In vitro fertilization

Plants that flowers different times, different places

Germplasm exchange

POLLEN CRYOPRESERVATION: OKRA


Fully opened flower

Undehisced anther

Dehiscing anther

Fully opened inflorescence

Partially opened

inflorescence

Fully dehisced anther

Different stages of inflorescence and anthers

Place flowers under a table

lamp for dehiscing

Extract pollen using cyclohexane using a millipore filter and

collect the disc containing pollen

Place disc containing

pollen into a cryovial

Store in liquid nitrogen

POLLEN CRYOPRESERVATION: ALLIUMS


Botanical name Duration Percentage of germination Reference

Before

cryostorage

After

cryostorage

Allium sepa 1 year 45.13 46.90 Ganeshan (1986)

Carica papaya 5 yrs 65 52 Ganeshan (1986)

Abelmoschus esculentus 1 year 82.3 51.7 Reka chaudhury et al.,

2010 Brassica juncea 6 months 70 72 ()

Cucumis sativus 6 months 30 25

Crotalaria tetragona 1 year 88.5 68.32

Elaeis guineensis 8 years 60 54.0

Litchi chinensis (CHES-6) 4 years 69.9 72.2

Litchi chinensis (Chaina) 4 years 48.7 46.5

Litchi chinensis (Kasba) 4 years 58.7 60.0

Mangifera indica (Amrapali) 4 years 85.5 87.0

Mangifera indica

(Bombay Green)

4 years 55.0 56.8

Mangifera indica (Neelum) 4 years 75.0 70.6

Mangifera indica (Kishan Bhog) 4 years 82.5 80.0

Vitis vinifera L. 5 yrs 73 76 Ganeshan (1985)

Cryopreservation of pollen

POLLEN CRYOPRESERVATION- ICAR- NBPGR, NEW DELHI

Bitter Gourd (Momordica charantia), Cherry (Prunus nepalensis), Citrus (Citrus indica), Coconut (Cocos nucifera), Cowpea (Vigna unguiculata), Cucumber (Cucumis sativus X Hardwickii), Indian Mustard (Brassica juncea), Kokum (Garcinia indica), Litchi (Litchi chinensis), Long Melon (Cucumis melo var. utilissimus), Maize (Zea mays), Malabar Tamarind (Garcinia gummi gutta), Mango (Mangifera indica), Mangosteen (Garcinia mangostana), Mustard (Brassica sp.), Okra (Abelmoschus spp),

A total of 576 accs in the form of pollen grains

Onion (Allium spp.), Oil palm (Elaeis guineensis), Pumpkin (Cucurbita moschata), Radish (Raphanus sativus), Rapeseed (Brassica rapa var. brown sarson), Snapmelon (Cucumis melo var. momordica), Sweet gourd (Momordica cochinchinensis), Sesame (Sesamum indicum), Toria (Brassica rapa var. toria), Watermelon (Citrullus vulgaris), Wheat (Triticum durum), Wild vigna (Vigna trinervia var. bourneae), Yellow mustard Wild barley (Hordeum spontaneum) etc.


ICAR-NBPGR- SUCCESS STORIES

Prunus sp

NBPGR RS, SHIMA

Mango

Central Institute of Subtropical

Horticulture, Lucknow

and IARI- New Delhi

Okra

Wide hybridization

NBPGR

Allium sp

NBPGR


Let us join hands to Protect and Conserve Plant Biodiversity for our

future generations



But what the poet doesn't see is the

xylem and the phloem and the

pollen and the thousands of

generations of breeding and the

billions of years before that

A poet sees a flower , as how beautiful the colors are.

All of that is visible to the scientists George M. Church

gowthami r, neelam sharma & vartika srivastava · 2019-11-18 · pollen cryopreservation-an aid...

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