Plant reproduction.

Haploid and anther culture (Lecture 9-10)

Normal pollen development
The plant life cycle has mitosis occurring in spores, produced by meiosis, that germinate into the
gametophyte phase.
Gametophyte size ranges from three cells (in pollen) to several million (in a "lower plant" such as
moss).
Alternation of generations occurs in plants, where the sporophyte phase is succeeded by the
gametophyte phase.
The sporophyte phase produces spores by meiosis within a sporangium.

Terminology
Sporangia - The structures in which spores are produced (sing.: sporangium).
spores - Impervious structures formed by some cells that encapsulate the cells and protect them
from the environment;
sporophyte - The diploid stage of a plant exhibiting alternation of generations. The diploid, spore
producing phase of the plant life cycle.

pollen grains - the containers for male gametophytes of seed plants produced in a
microsporangium by meiosis. Microspores produced by seed plants that contain the male
gametophyte.
pollen tube - structure produced by the tube nucleus in the pollen grain through which the sperm
nucleus (or nuclei in angiosperms) proceed to travel through to reach the egg.

pollination - the transfer of pollen from the anthers to the stigma by a pollinating agent such as
wind, insects, birds, bats, or in a few cases the opening of the flower itself.
Gametophyte - the haploid stage of a plant exhibiting alternation of generations, generates
gametes by the process of mitosis.

The gametophyte phase produces gametes by mitosis within an antheridium (producing sperm)
and/or archegonium (producing eggs).
Within the plant kingdom the dominance of phases varies.
Nonvascular plants, the mosses and liverworts, have the gametophyte phase dominant.
Vascular plants show a progression of increasing sporophyte dominance from the ferns and "fern
allies" to angiosperms.

Angiosperms
Flowering plants, the angiosperms, were the last of the seed plant groups to evolve, appearing over
100 million years ago during the middle of the Age of Dinosaurs (late Jurassic).
All flowering plants produce flowers and if they are sexually reproductive, they produce a diploid
zygote and triploid endosperm.
Clearly angiosperms are descended from some group of Mesozoic-aged gymnosperm seed
plant....but which one. The classical view of flowering plant evolution suggests early angiosperms
were evergreen trees that produced large Magnolia-like flowers.

Flowers
Flowers are collections of reproductive and sterile tissue arranged in a tight whorled array having
very short internodes.
Sterile parts of flowers are the sepals and petals.
When these are similar in size and shape, they are termed tepals.
Reproductive parts of the flower are the stamen (male, collectively termed the androecium) and
carpel (often the carpel is referred to as the pistil, the female parts collectively termed the
gynoecium).

Androecium
The individual units of the androecium are the stamens, which consist of a filament which
supports the anther. The anther contains four microsporangia within which microspores (pollen)
are produced by meiosis.
anther - The top of a stamen's filament; divided into pollen sacs in which the pollen grains form.
microsporangia - Structures of the sporophyte in which microspores are produced by meiosis. In
flowering plants the microsporangia are known as anther sacs.

Stamens are thought to represent modified sporophylls (leaves with sporangia on their upper
surface). Examinations by James E. Canright in the 1950s suggested an evolutionary series from
primitive angiosperms (like Austrobaileya ) which have leafish stamens to others with "normal"
stamens (Lilium).

Pollen
Pollen grains (from the greek palynos for dust or pollen) contain the male gametophyte
(microgametophyte) phase of the plant.
Pollen grains are produced by meiosis of microspore mother cells that are located along the inner
edge of the anther sacs (microsporangia).
The outer part of the pollen is the exine, which is composed of a complex polysaccharide,
sporopollenin. Inside the pollen are two (or, at most, three) cells that comprise the male
gametophyte.
The tube cell (also referred to as the tube nucleus) develops into the pollen tube.
The germ cell divides by mitosis to produce two sperm cells. Division of the germ cell can occur
before or after pollination.

Gynoecium
The gynoecium consists of the stigma, style, and ovary containing one or more ovules.
These three structures are often termed a pistil or carpel. In many plants, the pistils will fuse for all
or part of their length.

Like the stamen, the carpel is thought to be a modified leaf. Work by I.W. Bailey and his students
pointed to an evolutionary sequence from primitive angiosperms (like Drimys ) to "normal" carpels
like those of Lilium.

Hypothesized steps in the evolution of the carpel.

The Stigma and Style

The stigma functions as a receptive surface on which pollen lands and germinates its pollen tube.
Corn silk is part stigma, part style. The style serves to move the stigma some distance from the
ovary. This distance is species specific.
The Ovary
The ovary contains one or more ovules, which in turn contain one female gametophyte, also
referred to in angiosperms as the embryo sac. Some plants, such as cherry, have only a single
ovary which produces two ovules. Only one ovule will develop into a seed.

Anther/pollen culture
Pollen mother cells are in anther primordia.
First phase meiosis pollen mother cell
A tetrad forms from each PMC
Second phase microspores released from tetrads
Third phase microspores mature into pollen grains first pollen mitosis
Second pollen mitosis, maybe after germination
Generative and vegetative cells formed

Stage of development
Dicots first pollen mitosis
Between tetrad formation and exine formation
Monocots early uninucleate stage
Staging

Nutritional requirements
Sucrose essential
Mineral salts iron
Complex organics coconut milk
Activated charcoal

Influencing factors
HormonesSpecies requiring no hormones tobacco, petunia direct embryogenesis
Species requiring hormones callus phase monocots
Other factors
Cold or heat shock
Physiological status of donor plant
Albino plants
Isolated microspore culture

Anther/pollen culture
Method to produce haploid plants
Spontaneous occurrence in low frequency
Induction by physical and/or chemical treatment (nitrous oxide)
Chromosome elimination following interspecific hybridization
Anther culture 1966 pollen grains of Datura
Typically haploids can only be produced in polyploid plants wheat, tobacco, clover
Used in over 200 species

Haploid culture advantages

Technique is fairly simple

A large proportion of the anthers may respond
Haploids can be produced in large numbers very quickly

Haploid culture disadvantages

The majority of plants produced may not be haploid
May be albino or chimeric
Tedious

Haploids are useful because:

They carry only one allele of each gene.
Thus any recessive mutation or characteristic is apparent.
Plants with lethal genes are eliminated from the gene pool.
One can produce homozygous diploid or polyploid plants inbreeding
Haploids
is a naturally occurring process like Parthenogenesis
It is also possible to achieve via three processes including:
Chromosome elimination / Embryo Rescue
Anther CultureMicrospore Culture

Parthenogenesis
Parthenogenesis is a form of vegetative propagation in which an unfertilized egg develops into an
embryo. It is therefore maternal in origin.
Parthenogenesis occurs during meiosis and can result in a haploid or double haploid embryo. If
somatic doubling occurs (chromosomes in the egg double but do not divide) then the embryo will
be diploid, if not, then the resulting embryo will be haploid.
Embryos can be developed by parthenogenesis two different ways. The first is by apospory where
the embryo can be produced by a cell from the female parent.

The second is through diplospory, where an embryo can develop from a diploid spore from the
megaspore mother cell.
Parthenogenesis has been used in breeding peppers and corn. However anther culture has proven to
be more effective for these specie's. Parthenogenesis occurs in low frequencies because it cannot

be induced. The inefficiencies of screening for embryos, and the problems associated with
doubling chromosomes make it more worthwhile to study, anther or microspore culture.

Chromosome Elimination
Chromosome Elimination has been successful in barley. Barley haploids are obtained by crossing Hordeum
vulgare and Hordeum bulbosum. Since very few spikelets will develop from this pollenation, a hormone is
applied 1-2 days after crossing to increase seed set.

(a)

(b)

(a) Pollen is collected from plants of Hordeum bulbosum, a wild relative of cultivated barley (H. vulgare).

(b) The H. bulbosum pollen is brushed onto emasculated barley florets.

The Hordeum bulbosum chromosomes are eliminated during cell division in the hybrid zygotic
embryo resulting in a haploid Hordeum vulgare embryo. After several days the embryo is cultured
on nutrient agar, and a haploid plantlet develops.

The haploid plantlet is then placed in soil and later treated with colchicine.

Anther Culture
Anter culture is the process of using anthers to culture haploid plantlets.
The technique was discovered in 1964 by Guha and Maheshwari.
This technique can be used in over 200 species, including tomato, rice, tobacco, barley, and
geranium.
Some of the advantages which make this a valuable method for obtaining haploid plants are:
the technique is fairly simple
it is easy to induce cell division in the immature pollen cells in some species
a large proportion of the anthers used in culture respond (induction frequency is high)
haploids can be produced in large numbers very quickly.

In experiments using Datura innoxia, induction frequencies of almost 100% and a yield of more
than one thousand plantlets or calluses have occurred under optimal conditions from one anther.
Success can be determined within 24 hours as cells begin to divide.
Some disadvantages of using anther culture to obtain haploids are:
when working with some species, the majority of plants produced have been non-haploid
in cereals, very few green plants are obtained; many of the plants are albinos or green-albino
chimeras
it is tedious to remove the anthers without causing damage
sometimes a particular orientation is necessary to achieve a desired response

Microspore Culture

Microspores are immature pollen cells. Buds are surface sterilized and homogenized to release the
microspores.
In many microspore culture procedures, a heat treatment causes the microspores to undergo equal
mitosis and essentially behave like egg cells and develop into an embryo.
The microspore culture technique can be so efficient that thousands of plants are produced at a
time; plant breeders can hardly handle the numbers.
The procedure for culturing microspores is as follows:
Unopened flower buds are removed and surface sterilized

Buds are blended with B5 wash. The homogenate is filtered and centrifuged, and the cells in the pellet are
resuspended on culture medium
The number of microspore cells are counted using a microscope and hemacytometer (a grid on a slide for
counting microspores).
The microspores are plated at densities of around 100,000 cells per mL.
The culture cells are incubated for four weeks in the dark.

The haploid embryos that are formed are cultured further to obtain plantlets.
The haploid embryos must be germinated in vitro.

Endosperm
Most angiosperms form endosperm tissue
Consumed by the developing embryo or stored in seed
Triploid tissue
Homogeneous mass of parenchymatous tissue

Endosperm culture
To produce triploids
Seedless plants
Study of endosperm biosynthesis and metabolism

Production

of Doubled Haploid Plants from Anther Culture.

Doubled haploid plants have been used in a number of studies for several things.
1.They are used by plant breeders to get immediate, homozygous plants rather a
series of long selfings which will ultimately yield the same thing.
2.As we will see for mapping, they solve the problem of any residual
homozygosity which can cause problems for mapping.
3.They can be used for quantitative genetic studies for looking at linkage, etc. I.
I. In anther culture, immature pollen grains (n) are removed form the plant and
placed in culture.
II. The haploid anther cells are then induced to undergo differentiation into an
embryo where they undergo a distinct series of stages from torpedo to heart.
(Draw out).
III. The developing embryos are then placed on a media which will cause them to
form plantlets and roots. After some time on this media they can be transplanted
into pots and grown as normal plants.

IV. Doubling can either occur spontaneously, or be triggered by adding

cholchicine. The development of a haploid anther culture system required
adaptation of such treatments as cold or heat shock, hormonal regimes, etc.

Genotypic and Phenotypic Variability

Term somaclonal variation coined by Larkin and Scowcraft:

Larkin P. J. and Scowcroft W. R. (1981) Somaclonal variation-a novel source of
variability from cell cultures. Theor. App. Genet. 67:197-201
Ryan S.A., Larkin P.J. and Ellison F.W. (1987) Somaclonal variation in some
agronomic and quality parameters in wheat. Theor App Genet74:77-82
For the most part, other researchers did not find the vast amount of variability
reported by Shepard and few useful plants were obtained from somaclonal
variation.
Therefore, what variability that is induced in culture is typically negative in
nature. Several causes for this variability have been identified:
Genetic variability - mutations or other changes in the DNA of the tissue that
are heritable
Epigenetic variability non-heritable phenotypic variation
Somaclonal variation - Shepard and Secor. 1981. Variability of protoplastderived potato clones. Crop Sci. 21:102-105
Plants regenerated from potato protoplasts varied in a wide range of plant
characteristics, including tuper shape, yield, maturity date, photoperiod
requirement, plant morphology, resistance to pests, leaf color, shape, glossiness,
hairiness, and overall plant growth habit.

Mechanisms producing variability

Ploidy Changes
As plant cells mature (age), their ploidy level tends to increase. In tobacco:
5 cm below the apex - equal percentages of diploid (2n) and tetraploid (4n)
cells
20 cm below the apex - 70% tetraploid cells, only 9% diploid cells, 16%
octaploid (8n) cells, and 5% aneuploid cells

Changes in ploidy level in cultures may be due to:

the source of the explant material used
effects of the culture process itself (lengthy culture periods, growth regulators,
nutritional stress)
Three phenomena that occur during mitosis lead to most changes in ploidy:
endomitosis (sister chromatids separate within the nuclear membrane, but there
is no spindle formation nor cytoplasmic division)
endoreduplication (chromosomes at interphase undergo extra duplications)
spindle fusion (giving binucleate or multinucleate cells).
One of the triggers of polyploidy in vitro is growth regulators; both kinetin and
2,4-D have been implicated.
Other aspects of the culture medium may also affect the ploidy of the cultured
cells.
Medium that places cells under nutrient limitation will favor the development of
"abnormal" cells.
A large variation in ploidy in culture may not result in high percentages of
polyploid plants.
This is due to diplontic drift (also called diplontic selection), which is the
increased fitness of diploid cells over polyploid cells.
In mixed poplations of cells with different ploidys, diploid cells retain their
organogenic potential better than polyploid and aneuploid cells (probably due to
an enhanced ability to form meristems).
The existence of higher ploidy levels in explant tissues can also be useful.
In tobacco, for instance, culture of leaf midrib tissue, which has a high percentage
of cells with increased ploidy, followed by regeneration and rooting of shoots, is
the common method for producing doubled haploids from haploid plants and
tetraploids from diploid plants.
Chimeral rearrangement of tissue layers
Many horticultural plants are periclinal chimeras, that is, the genetic composition
of each concentric cell layer (LI, LII, LIII) in the tunica of the meristematic tissues
is different.
These layers can be rearranged during rapid cellular proliferation.
Therefore, regenerated plants may contain a different chimeral composition or
may no longer be chimeric at all.
Structural changes in the DNA sequence
Chromosomal rearrangements, point mutations, or transposition of transposable
elements can occur during culture. These changes can occur spontaneously or
can be induced with chemicals or radiation.

Gross structural rearrangements appear to be a major cause of somaclonal

variation.
These involve large segments of chromosomes and so may affect several genes
at a time.
Deletions
Inversions
Duplications
Translocations (whole chromosomal segments moved to a new location).
Point mutations (the change of a single DNA base), if they take place within a
coding region of a gene and result in the alteration of an amino acid, can lead to
somaclonal variation. Point mutations are often spontaneous and are more
difficult to detect. Note that they result in single gene changes.
Transposable elements are segments of DNA that are mobile and can insert into
coding regions of genes, typically resulting in a lack of expression of the gene.
The culture environment may make the transposable elements more likely to
excise and move.
Chromosomal alterations, like ploidy changes, increase with increased lengths of
culture.
Temporary (epigenetic) phenotypic alterations
Epigenetic changes can be temporary and are ultimately reversible. However,
they may also persist through the life of the regenerated plant.
One common alteration seen in plants produced through tissue culture is
rejuvenation, especially in woody species.
Rejuvenation may lead to changes in morphology, earlier flowering, improved
adventitious root formation, and/or increased vigor.
In other cases, morphology of a regenerated plant is altered either temporarily or
for the long term.
The causes of these nonheritable alterations are not known, but are undoubtedly
due to some alteration in gene expression, probably induced by the culture
environment.

Part of the material was adapted from

http://www.hos.ufl.edu/mooreweb/TissueCulture/tcclass.html
Images from Purves et al., Life: The Science of Biology

From
gopher://wiscinfo.wisc.edu:2070/I9/.image/.bot/.130/Angiosperm/Lilium/Flower_dissection/Flowe
rhttp://www.plant.uoguelph.ca/research/biotech/haploid/haploids.htm