Lectures 2

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In vitro production of haploids

The term haploid refers to those plants which

possess a gametophytic number of chromosomes

(single set) in their sporophytes (2n)

Potential in plant breeding

Detection of mutations

Low frequency (0.001-0.01%) in nature

spontaneously through apomixis or parthenogenesis

Anther cultures (ponsiviljelmt): immature pollen

haploid plants

Haploids

1. Monoploids (monohaploids)

half of the number of chromosomes from a diploidspecies, e.g. maize, barley

2. Polyhaploids half of the number of chromosomes (gametophytic set)

from a polyploid species, e.g. wheat, potato

Androgenesis = haploid production through antherculture

Gynogenesis = haploid production through ovary orovule culture

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An overview of angiosperm reproduction

Filament

AntherStamen

Anther attip of stamen

Pollen tube

Germinated pollen grain

(n) (male gametophyte)on stigma of carpel

Ovary (base of carpel)

Ovule

Embryo sac (n)(female gametophyte)

FERTILIZATIONEgg (n)

Sperm (n)

Petal

Receptacle

Sepal

Stigma

Style

Ovary

Key

Haploid (n)

Diploid (2n)

(a) An idealized flower.

(a) Simplified angiosperm life cycle.See Figure 30.10 for a more detailed

version of the life cycle, including meiosis.

Mature sporophyte

plant (2n) with

flowers

Seed

(develops

from ovule)

Zygote(2n)

Embryo (2n)

(sporophyte)Simple fruit(develops from ovary)

Germinating

seed

Seed

Carpel

The development of angiosperm gametophytes (pollen

grains and embryo sacs)

Diploid (2n) 100

m

3

1

2

Three mitotic divisionsof the megaspore formthe embryo sac, amulticellular femalegametophyte. Theovule now consists ofthe embryo sac alongwith the surroundinginteguments (protectivetissue).

Development of a male gametophyte(pollen grain). Pollen grains developwithin the microsporangia (pollensacs) of anthers at the tips of thestamens.

(a)

Each one of themicrosporangiacontains diploidmicrosporocytes(microsporemother cells).

Each microsporo-cytedivides bymeiosis to producefour haploidmicrospores,each of whichdevelops intoa pollen grain.

A pollen grain becomes amature male gametophytewhen its generative nucleusdivides and forms two sperm.This usually occurs after apollen grain lands on the stigmaof a carpel and the pollentube begins to grow. (SeeFigure 38.2b.)

Pollen sac(microsporangium)

Micro-sporocyte

Micro-Spores (4)

Each of 4microspores

Generativecell (wellform 2sperm)

75 m

MaleGametophyte(pollen grain)

Nucleusof tube cell

RagweedPollengrain

KeyTo labels

MITOSIS

MEIOSIS

Ovule

Ovule

Integuments

Haploid (2n)

Embryosac

Mega-sporangium

Mega-sporocyte

Integuments

Micropyle

Survivingmegaspore

AntipodelCells (3)

PolarNuclei (2)

Egg (1)

Synergids(2)

Development of a female gametophyte(embryo sac). The embryo sac developswithin an ovule, itself enclosed by theovary at the base of a carpel.

(b)

20 m

Within the ovulesmegasporangiumis a large diploidcell called themegasporocyte(megasporemother cell).

The megasporo-cytedivides bymeiosis and givesrise to four haploidcells, but in mostspecies only oneof these survivesas the megaspore.

1

2

3

Female gametophyte(embryo sac)

Diploid (2n) 100m

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Androgenic methods (andro = male)

From the male gametophyte of an angiosperm plant,i.e. microspore (immature pollen)

The underlying principle is to stop the developmentof pollen cell direct development in a plant (nogamete phasis)

Anther techniques are simple,quick and efficient:

immature anthers sterilized acetocarmine test for pollen development

solid media, pollen callus shoots

disadvantages: plants may originate from various parts ofthe anther plants with various ploidy levels

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Microspore culture

Haploid plants can also be produced through cultureof male gametophytic cells i.e. microspores orimmature pollen embryo (directly) or via callus

Advantages: uncontrolled effects of the anther wall and other

associated tissue are eliminated

the sequence of androgenesis can be observed startingfrom a single cell

microspores are ideal for mutagenic and transformationstudies

high yield of plants per anther can be obtained

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Diploidization

Haploids can be diploidized to produce

homozygeous plants:

colchicine treatment (spindle inhibitor

chromosome duplication)

endomitosis: haploid cells are unstable in culture

and have a tendency to undergo endomitosis (=

chromosome duplication without nuclear

division), can be enhanced in callus by auxin-

cytokinin medium

Significances and uses of haploids

1. Development of pure homozygous lines

reduction in time from 6-8 years to a few monthsor a year

2. Hybrid development

3. Induction of mutations

majority of induced mutations are recessive andtherefore not expressed in the diploid cells

resistance to antibiotics, herbicides, toxins, UV-light, gamma radiation, extreme temperaturesetc.

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Protoplasts

Naked plant cells, all components of a plant

cell excluding the cell wall

Cell wall degrading enzymes (cellulase)

Used in cell fusion studies, and to take up

foreign DNA, cell organelles, bacteria and

virus particles

Protoplast isolation

1. Mechanical method

suitable for large and highly vacuolated cells,such as onion bulb scales

cells are plasmolysed, tissue is dissected anddeplasmolyzed

2. Enzymatic method

commercial mixture of cell wall degradingenzymes in solution containing osmoticstabilizers

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Enzymes

Pectinase mainly degrades the middle lamella

Cellulase and hemicellulase are required for other

main component

Helicase, colonase, cellulysin, glusulase,

zymolyase, pectolyase etc.

1. Two-step or sequential method

pectinase (or macerozyme) cellulase

2. One-step or simultaneous method

one mixture of enzymes

Osmoticum

In isolating protoplasts, the wall pressure that is

mechanically supported by cell wall must be

replaced with an appropriate osmotic pressure (also

later in the culture medium) osmotic stress has harmful effects on cell metabolism and

growth: condensation of DNA in cell nuclei and

decreased protein synthesis

Lower osmotic potentials by addition of various

ionic and non-ionic solutes: mannitol, sorbitol,

glucose, fructose, galactose, NaCl, CaCl etc.

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Protoplast viability and density

FDA (fluorescein diacetate) staining

accumulates inside the plasmalemma of

viable protoplasts

Phenosafranine staining, Calcofluor White

(CFW), oxygen uptake, photosynthesis

Maximum and minimum plating densities forgrowth

Protoplast culture techniques

Semisolid agar media

protoplasts remain in a fixed position (no clumping)

Liquid culture

easy dilution, transfer of protoplasts and adjustment ofosmotic pressure

Liquid dropled method

Hanging droplet methods

Feeder layer

Co-culturing slow and fast growing protoplasts

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Protoplast development

Regeneration of cell wall starts within a few

hours after isolation

cell expansion

cell division (within 2-7 days)

cell colonies

callus in an osmotic free medium

organogenesis/embryogenic differentation

Somatic hybridization

Sexual hybridization is limited in most cases tocultivars within a species or a few wild speciesclosely related to a cultivated crop due to incompatibility

Plant propoplasts offer exciting possibilities in thesomatic cell genetics and crop improvement

Somatic hybridization = fusion of isolated somaticprotoplasts and development of their product(heterokaryon) to a hybrid plant

The nucleus and cytoplasm of both parents are fused(except in cybrids)

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Protoplast fusion

Mixing of protoplasts of two different genomes

Spontaneous fusion: from callus tissue, do notregenerate in to whole plants

Induced fusion by

polyethylene glycol method (PEG): high yield

NaNO3 (sodium nitrate)

Ca2+

at high pH electrofusion

3 main phases: agglutination or adhesion, plasmamembrane fusion at localized sites, fused protoplasts

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Selection: use of visual characteristics

The most efficient but the most tedious method is tovisually identify hybrid cells and mechanicallyisolate them

Morphologically distinct cells: green chloroplasts +colorless cells (containing starch granules)

Leaf protoplasts + petal chloroplasts

Fluorescent labeling: isothiocyanate, rhodamineisothiocyanate

The growth pattern of hybrid callus is different fromeither parental line (more vigorous)

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Verification and characterization of somatichybrids

Morphological features (both vegetative and floral)are usually intermediate between the two parents not observed in dominant single gene traits such as

anthocyanin pigment, flower pigment, leaf size

Isoenzyme analysis unique banding patterns e.g. amylase, malate and lactate

dehydrogenase, esterase aspartate aminotransferase

Chromosomal constitution the chromosome number of the somatic hybrids should be

the sum of chromosome number of two parentalprotoplasts

however, variation is often observed (chromosomenumber frequently higher)

Verification and characterization of somatic

hybrids

Molecular techniques

genetic constitution can be studied e.g. using

specific restriction patterns of chloroplast and

mitochondrial DNA, and molecular markers such

as

AFLP (Amplified Fragment Length Polymorphism)

RFLP (Restriction Fragment Length Polymorphism)

RAPD (Random Amplified Polymorphic DNA)

microsatellites

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Chromosome number in somatic hybrids

Variation in chromosome number (aneuploidy) isgenerally observed in hybrids but frequentlychromosome number is more than the total numberof both the parental protoplasts due to

multiple fusion

fusion of more than two protoplasts

actively dividing tissue of one parent and quiescent tissue

of the other parent unequal rates of DNA replication in two fusing partners

somaclonal variation in cultured cells used for protoplastisolation

Cybrids

Nucleus derived from one parent and cytoplasm

from both parents, producing cytoplasmic hybrids =

cybrids

alloplasmic cell lines

approaches: lethal doses of X or gamma irradiation to one

parental protoplast population; iodoacetate metabolic

inactivation; fusion of normal protoplasts with enucleated

protoplasts/nuclear division-suppressed protoplasts

applications: directed transfer of cytoplasmic male

sterility (CMS) or herbicide resistance from a donor to a

recipient crop plant species

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Potential of somatic hybridization

Hybridization becomes possible between plants that

are still in juvenile phase

Studies on cytoplasmic genes and their activities

The production of unique nuclear-cytoplasmic

combinations

Production of heterozygous lines within a single

species that normally could only be propagated by

vegetative means, e.g. potato and other tubers and

root crops

Problems and limitations

Efficient plant regeneration is needed, limited to a few plantspecies

The lack of an efficient selection method for fused product

The end-product is often unbalanced, not viable

Somatic hybridization of two diploids amphidiploid(genetically unfavorable)

It is never certain that a particular trait will be expressed aftersomatic hybridization

To achieve successful integration into a breeding program,somatic hybrids must be capable of sexual reproduction (

backgrossing to the cultivated crop to develop new varieties)

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Somaclonal variation

Genetic variation is essential for any crop

breeding programs

Plant cell cultures are potential sources of useful geneticvariation

Somaclonal variation = variability generated by the use of atissue culture cycle (explant callus plant regeneration)

Although particular genotypes are cloned in tissue culture,

phenotypic variants are observed amongst regenerated plants Caused by changes in chromosome number and structure:

the expression of chromosomal mosaicism or genetic disorders inexplant cells

new irregularities brought about by culture conditions throughspontaneous mutations

Schemes for obtaining somaclonal

variation

1. Without in vitro selection:

explant explant derived callus shootregeneration plant transfer to the field

screening for desirable traits agronomic trials both dominant and homozygous recessive traits

can be directly selected

progenies of heterozygous regenerants used forselection of recessive traits

time consuming, not effective

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Schemes for obtaining somaclonalvariation

2. With in vitro selection:

protoplasts, cell suspensions and callus cultures

can be searched for biochemical mutants

selection for resistance: inhibitors such as

antibiotics, amino acid analogs, herbicides,

pathotoxins

these compounds are put in the medium at a

concentration such that some cell populations survives

and can be further grown on a selective medium

Factors influencing somaclonal variation

Genotype: frequency of regeneration andsomaclones

Explant source

Duration of cell culture: most long-termcultures are chromosomally variable

Culture conditions: 2,4-D, NAA, BAP

In vitro selection from suspensionculture/protoplasts/calli

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Factors important during in vitro selectionof somaclonal variants

Selective agent: a dose response curve with respectto growth inhibition must be performed for eachselection agent of interest

Selection technique in vitro selection of cell lines against an inhibitor is best

carried out at concentration that kill 70-90% of wild typecells

Regeneration of plants

In vivo testing

Agronomic analysis: testing agronomic value in thefirst generation and also in the second generation forstability

Application of somaclonal variation

Novel variants:

improved scented Geranium variety Velvet Rose

tornless blackberries (Rubus) Lincoln Logan

Disease resistance eye spot disease (Helminthosporium sacchari), downy

mildew (Sclerospora sacchari), Fiji virus disease insugarcane

Abiotic stress resistance

freezing tolerance in Norstar winter wheat (prolineaccumulation)

salt and drought tolerance in alfalfa, maize, rice, tobacco

aluminium tolerance: tomato, alfalfa, tobacco, carrot

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Application of somaclonal variation

Herbicide resistance

tobacco, soybean wheat, maize

resistance to glyphosate, sulfonylurea,

imidazolinones etc.

Insect resistance

Russian wheat aphid (kirva) in wheat

Seed quality

Lathyrus sativa in central India

Alien gene introgression

Basis of somaclonal variation

Karyotype changes

polyploidy, commonly associated with reduced fertility

Changes in chromosome structure

translocations, deletions, duplications, inversions, breaks,

multinucleate cells, abnormal anaphase etc.

Single-gene mutations

Cytoplasmic genetic changes

variations in mitochondrial and chloroplastic DNA

Mitotic crossing over (MCO)

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Basis of somaclonal variation

Gene amplification and nuclear changes

nuclear genes are affected by tissue culture stress

increased amount of total DNA and an increasedproportion of highly repeated sequences (AT- andGC-rich fractions in tobacco)

ribosomal DNA is known to alter directly in

response to environmental and cultural pressures:methylation, structural rearrangements

Transposable elements

Disadvatages of somaclonal variation

Uncontrolled and unpredictable, most

variations are of no apparent use

The variation is cultivar-dependent The variation obtained is not always stable

and heritable

Not all the changes obtained are novel

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Germplasm storage and cryopreservation

The aim is to ensure the availability of usefulgermplasm at any time

Breeding programs rely heavily on locally adaptedancient plant varieties and their wild relatives assources of germplasm

Continuing search for high yielding varieties of cropplants with resistance to pathogens and pests

In species which are propagated through seeds, it iseconomical to preserve the seeds dried to a water content 5-8%, stored at < -18oC , low

humidity

Germplasm storage and cryopreservation

Sometimes it is not feasible to store seeds

because:

some plants do not produce fertile seeds

some seeds remain viable only for a limited

duration

some seeds are very heterozygous and therefore,

not suitable for maintaining true genotypes

seeds of certain species deteriorate rapidly due to

seed borne pathogens

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Germplasm storage in vitro

The in vitro system is extremely suitable forstorage of plant material

can be stored on a small scale, disease free andgrowth-limiting conditions (large land resourcesnot needed)

Advantages overin vivo conservation:

plants species that are in danger of being extinct great savings in storage space and time

sterile plants

growth can be reduced efficiently

Cryopreservation (kryopreservaatio,

syvjdytys) = preservation in the frozen state

storage at very low temperatures: solid carbon dioxide (-79oC), low temperature deep freezers (-80oC), in vapor

phase of nitrogen (-150oC) or in liquid nitrogen (-196oC)

Metabolic processes and biological deterioration isslowing down or halting

Susceptibility or degree of freeze tolerance displayedby a given genotype to reduced temperatures andformation of ice crystals within the cells must betaken into account

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Cryopreservation

The morphological and physiological conditions ofthe plant material influence the ability of an explantto survive

In general, small, richly cytoplasmic, meristematiccells survive better than the larger, highly vacuolatedcells

Cell suspensions have been successfully frozed (latelag phase or exponential phase)

Callus tissues are rather resistant to freezing damage(rapidly growing phase)

Shoot apices, embryos or young plantlets(meristems) are preferred in many species

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Addition of cryoprotectants andpretreatment

The freeze tolerance can be improved by timing the harvest ofcells or by hardening process (growing plants as +4oC for 3-7days before taking explant)

Two potential sources of cell damage: formation of large ice crystals inside the cells

intracellular concentration of solutes increases to toxic levels as aresult of dehydration

Addition of cryoprotectants controls the appearance of ice

crystals and protects the cells from the toxic solution effect DMSO, glyserol, glycols, acetamides, sucrose, mannose, glucose,

proline, PVP

5-10% DMSO: low molecular weight, easily dissolved, non-toxic atlow concentrations, easily permeable and washable

Freezing

Rapid freezing in liquid N

the quicker the freezing is done, the smaller the

intracellular ice crystals are

Slow freezing 0.1-10 oC/min from 0 oC to -100oC liquid N

slow cooling permits the flow of water from the cells to

the outside (extracellular ice formation)

Stepwise freezing

combines the advantages of both rapid and slow freezing

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Storage and thawing

Long-term storage is best done at -196oC

Thawing in warm water bath

The freshly thawed cell is strongly prone to further

damage and requires appropriate nursing

Determination of survival/viability of cells

cell viability tests

Regrowth of plants from stored tissues or cells is the

only realistic test of survival of plant materials

Slow growth method

Cells or tissues can be stored at non-freezingconditions in a slow growth state

comparable to Japanese Bonsai-technique

reduced growing processes by temperature (1-4oC), nutrition medium, hormones (ABA), lowoxygen pressure or osmotic inhibitors (mannitol,sucrose)

grape plants have been stored for 15 years,strawberries for 6 years at 4oC

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Applications of cryopreservation

Conservation of genetic material

Freeze storage of cell culture

suppression of cell division and reducing the needfor periodical subculturing in cell lines to bemaintained

Maintenance of disease free stocks

Cold acclimation and frost resistance

material for selection of cold resistant mutant celllines, which could later differentiate into frostresistant plants