NEHRU ARTS AND SCIENCE COLLEGE
DEPARTMENT OF BIOTECHNOLOGY
E-LEARNING
CLASS
SUBJECT
: II MSC BIOTECHNOLOGY
: PLANT BIOTECHNOLOGY
UNIT I
Conventional plant breeding methods- Selection, hybridization, mutation and
polyploidy, Cell and tissue culture in plants: Tissues culture media (Composition
and preparation), Micropropagation: Callus and suspension culture, somaclonal
variation, somatic embryogenesis: Embryo culture, protoplast isolation and
somatic hybridization; cybrids; Haploid plants, Artificial seeds and hardening.
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Part – A
1. Who is called as the father of plant tissue culture?
Gottlieb Haberlandt
2. Emasculation
Removal of stamens or anthers to prevent self-pollination
3. Ovule
The part of the ovary of seed plants that contains the female germ cell and
after fertilization becomes the seed
4. Mass selection
As practised in plant and animal breeding, the choosing of individuals for
reproduction from the entire population on the basis of individual
phenotypes
5. Pollen tube
A pollen tube of pollen grain grows from the stigma, through the style, and
into the ovary where it enters an ovule. The sperm nuclei then pass through
the pollen tube into the ovary where fertilization takes place.
6. Plant Growth Regulator
Plant hormones (also known as phytohormones) are chemicals that regulate
plant growth, which, in the UK, are termed 'plant growth substances'. Plant
hormones are signal molecules produced within the plant, and occur in
extremely low concentrations
7. Agar agar
Agar or agar agar is a gelatinous substance derived from seaweed.
Historically and in a modern context, it is chiefly used as an ingredient in
desserts throughout Japan, but in the past century has found extensive use as
a solid substrate to contain culture medium.
8. Pure line selection
Pure line is a self pollinated descendent of a self pollinated plant. Pure-line
selection generally involves three more or less distinct steps: (1) numerous
superior appearing plants are selected from a genetically variable population;
(2) progenies of the individual plant selections are grown and evaluated by
simple observation, frequently over a period of several years; and (3) when
selection can no longer be made on the basis of observation alone, extensive
trials are undertaken, involving careful measurements to determine whether
the remaining selections are superior in yielding ability and other aspects of
performance. Any progeny superior to an existing variety is then released as a
new “pure-line” variety
9. Cross pollination
Pollination is the process by which pollen is transferred in plants, thereby
enabling fertilization and sexual reproduction
10. Meiosis
Meiosis is a special type of cell division necessary for sexual reproduction. In
animals, meiosis produces gametes like sperm and egg cells, while in other
organisms like fungi it generates spores. Meiosis begins with one diploid cell
containing two copies of each chromosome-one from the organism's mother
and one from its father and produces four haploid cells containing one copy
of each chromosome. Each of the resulting chromosomes in the gamete cells is
a unique mixture of maternal and paternal DNA, ensuring that offspring are
genetically distinct from either parent.
Part – B
1. Describe protoplast fusion.
Somatic fusion, also called protoplast fusion, is a type of genetic modification
in plants by which two distinct species of plants are fused together to form a
new hybrid plant with the characteristics of both, a somatic hybrid. Hybrids
have been produced either between the different varieties of the same species
(e.g. between non-flowering potato plants and flowering potato plants) or
between two different species (e.g. between wheat triticum and rye secale to
produce Triticale).
Uses of somatic fusion include making potato plants resistant to potato leaf
roll disease. Through somatic fusion, the crop potato plant Solanum
tuberosum – the yield of which is severely reduced by a viral disease
transmitted on by the aphid vector – is fused with the wild, non-tuber-bearing
potato Solanum brevidens, which is resistant to the disease. The resulting
hybrid has the chromosomes of both plants and is thus similar to polyploid
plants.
Fused protoplast (left) with chloroplasts (from a leaf cell) and coloured
vacuole (from a petal).
The somatic fusion process occurs in four steps:
1. The removal of the cell wall of one cell of each type of plant using cellulase
enzyme to produce a somatic cell called a protoplast
2. The cells are then fused using electric shock (electrofusion) or chemical
treatment to join the cells and fuse togethor the nuclei. The resulting fused
nucleus is called heterokaryon.
3. The somatic hybrid cell then has its cell wall induced to form using
hormones
4. The cells are then grown into calluses which then are further grown to
plantlets and finally to a full plant, known as a somatic hybrid.
Different from the procedure for seed plants describe above, fusion of
moss protoplasts can be initiated without electric shock but by the use of
polyethylene glycol (PEG). Further, moss protoplasts do not need
phytohormones for regeneration, and they do not form a callus. Instead,
regenerating moss protoplasts behave like germinating moss spores . Of
further note sodium nitrate and calcium ion at high pH can be used, although
results are variable depending on the organism
2. Describe the characteristics of Somatic Hybridization and Cybridization
1. Somatic cell fusion appears to be the only means through which two
different parental genomes can be recombined among plants that cannot
reproduce sexually (asexual or sterile).
2. Protoplasts of sexually sterile (haploid, triploid, and aneuploid) plants can
be fused to produce fertile diploids and polyploids.
3. Somatic cell fusion overcomes sexual incompatibility barriers. In some cases
somatic hybrids between two incompatible plants have also found application
in industry or agriculture.
4. Somatic cell fusion is useful in the study of cytoplasmic genes and their
activities and this information can be applied in plant breeding experiments.
Inter-specific and inter-generic fusion achievements
Cross
Oat
Brassica sinensis
Torrentia fourneri
Brassica oleracea
Datura innoxia
Nicotiana tabacum
Datura innoxia
Arabidopsis thaliana
Petunia hybrida
Crossed with
Maize
B. oleracea
T. bailloni
B. campestris
Atropa belladonna
N. glutinosa
D. candida
Brassica campestris
Vicia faba
3. Describe the synthetic seed preparation and its uses
Synthetic seeds or artificial seeds are the living-seed like structure derived
from somatic embryoids (or, somatic embryos) in-vitro culture after
encapsulation by a hydrogel. The preserved embryoids are called synthetic
seeds.
Synthetic seeds or artificial seeds are the living-seed like structure derived
from somatic embryoids (or, somatic embryos) in vitro culture after
encapsulation by a hydrogel. The preserved embryoids are called synthetic
seeds.
Father of synthetic seeds in India is Bapat who worked on sandalwood and
Morus alba in1980.
Advantages
It could be preserved for long time at lower temperature
Rooting, hardening and conversion steps are waved off as these seeds can
directly be sowed in the fields like natural seeds
Potential Uses
For the development of hybrid plants which have unstable genotypes or
show seed sterility. In case that the seeds become sterile, the immature
embryo from the seed can be rescued (called as embryo rescue) and then can
be encapsulated artificially with appropriate growth medium to allow its
maturation and desiccation for its germination.
Germplasm conservation
For various research and analysis purposes like studying the role of
endosperm, etc
To produce large number of the clones of elite species at the cheaper cost
To supply adjuvant like plant growth regulators, pesticides, etc
Preparation of synthetic seeds:
For preparation seeds need to be encapsulated. This can be done using
various water soluble hydrogels like alginate, gel rite, carboxy methyl
cellulose, locust bean gum, sodium alginate with gelatin. However, alginate
based encapsulation was found to be best.
Encapsulation can be done in two ways:
1. Dropping method
In this method, somatic embryos are mixed with 2% sodium alginate
solution prepared in MS medium. However if the sodium alginate is
purchased from an Indian based company the add 4% sodium alginate.
Then these encapsulated beads are then added as drops into a calcium
nitrate solution (100mM) and then they were kept for around 20-30 minutes
after which embryos are completely gelled by calcium alginate.. Then these
gelled embryos are rinsed with water and then can be used as synthetic seeds.
This method has been used Redenbaugh et al. (1986) for alfalfa somatic
embryos encapsulation.
However, in place of calcium nitrate, calcium chloride can also be used
with by product of encapsulation as sodium chloride.
Gelling with sodium alginate is not done since sodium is toxic for cells
2. Molding
In this, embryos are mixed with temperature dependent gel like gel rite
where cells get gelled as the temperature is lowered.
However, the synthetic seeds obtained by above methods cannot be used
directly into the fields because:
They are very fragile
Their nutrients will be attacked by the micro flora present in the soil which
will starve the embryo to death
So, to prevent above problems, one or two more coatings of alginate are
given and also, certain antibiotics, insecticides, pesticides, etc are added to
prevent the microbial attack on the nutrient medium.
4. Explain somaclonal variation with its advantages and disadvantages
Somaclonal variation It is the term used to describe the variation seen in
plants that have been produced by plant tissue culture. Chromosomal
rearrangements are an important source of this variation.
Somaclonal variation is not restricted to, but is particularly common in
plants regenerated from callus. The variations can be genotypic or
phenotypic, which in the latter case can be either genetic or epigenetic in
origin. Typical genetic alterations are: changes in chromosome numbers
(polyploidy and aneuploidy), chromosome structure (translocations,
deletions, insertions and duplications) and DNA sequence (base mutations).
Typical epigenetic related events are: gene amplification and gene
methylation.
If no visual, morphogenic changes are apparent, other plant screening
procedures must be applied. There are both benefits and disadvantages to
somaclonal variation. The phenomenon of high variability in individuals from
plant cell cultures or adventitious shoots has been named somaclonal
variation.
Benefits:
The major likely benefit of somaclonal variation in plant is improvement.
Somaclonal variation leads to the creation of additional genetic variability.
Characteristics for which somaclonal mutants can be enriched during in vitro
culture includes resistance to disease pathotoxins, herbicides and tolerance to
environmental or chemical stress, as well as for increased production of
secondary metabolites. Micropropagation can be carried out throughout the
year independent of the seasons.
Disadvantages:
A serious disadvantage of somaclonal variation occurs in operations
which require clonal uniformity, as in the horticulture and forestry industries
where tissue culture is employed for rapid propagation of elite genotypes.
Ways of reducing somaclonal variation: Different steps can be used. It is well
known that increasing numbers of subculture increases the likelihood of
somaclonal variation, so the number of subcultures in micropropagation
protocols should be kept to a minimum. Regular reinitiation of clones from
new explants might reduce variability over time. Another way of reducing
somaclonal variation is to avoid 2,4-D in the culture medium, as this hormone
is known to introduce variation.Vitrification[hyperhydracity] may be a
problem in some species. In case of forest trees, mature elite trees can be
identified and rapidly cloned by this technique.
High production cost has limited the application of this technique to more
valuable ornamental crops and some fruit trees.
5. Explain the principle and significance of embryo rescue techniques
Embryo Rescue is one of the earliest and successful forms of in-vitro culture
techniques that is used to assist in the development of plant embryos that
might not survive to become viable plants. Embryo rescue plays an important
role in modern plant breeding, allowing the development of many
interspecific and intergeneric food and ornamental plant crop hybrids. This
technique nurtures the immature or weak embryo, thus allowing it the chance
to survive. Plant embryos are multicellular structures that have the potential
to develop into a new plant. The most widely used embryo rescue procedure
is referred to as embryo culture, and involves excising plant embryos and
placing them onto media culture. Embryo rescue is most often used to create
interspecific and intergeneric crosses that would normally produce seeds
which are aborted. Interspecific incompatibility in plants can occur for many
reasons, but most often embryo abortion occurs. In plant breeding, wide
hybridization crosses can result in small shrunken seeds which indicate that
fertilization has occurred, however the seed fails to develop. Many times,
remote hybridizations will fail to undergo normal sexual reproduction, thus
embryo rescue can assist in circumventing this problem.
Embryo rescue was first documented in the 18th century when Charles
Bonnet excised Phaseolus and Fagopyrum embryos, planted them in soil and
the cross resulted in dwarf plants. Soon after this, scientists began placing the
embryos in various nutrient media. During the period of 1890 to 1904,
systems for embryo rescue became systematic by applying nutrient solutions
that contained salts and sugars and applying aseptic technique. The first
successful in vitro embryo culture was performed by Hanning in 1904, he
however described problems with precocious embryos that resulted in small,
weak, and often inviable plantlets.
Applications
Breeding of incompatible interspecific and intergeneric species
Overcoming seed dormancy
Determination of seed viability
Recovery of maternal haploids that develop as a result of chromosome
elimination following interspecific hybridization
Used in studies on the physiology of seed germination and development
Techniques
Depending on the organ cultured, it may be referred to as either embryo,
ovule, or ovary culture. Ovule culture or in ovolo embryo culture is a
modified technique of embryo rescue whereby embryos are cultured while
still inside their ovules to prevent damaging them during the excision
process. Ovary or pod culture, on the other hand employs the use of an entire
ovary into culture. It becomes necessary to excise the entire small embryo to
prevent early embryo abortion. However, it is technically difficult to isolate
the tiny intact embryos, so often ovaries with young embryos, or entire
fertilized ovules will be used.
Factors to consider
Embryos are manually excised and placed immediately onto a culture
media that provides the proper nutrients to support survival and growth
(Miyajima 2006). While the disinfestation and explant excision processes
differ for these three techniques, many of the factors that contribute to the
successful recovery of viable plants are similar. The main factors that
influence success are; the time of culture, the composition of the medium, and
temperature and light. Timing mainly refers to the maturation stage of the
embryo before excision. The optimal time especially for the rescue of embryos
involving incompatible crosses would be just prior to embryo abortion.
Nevertheless, due to difficulties involved with the rearing of young embryos
compared to those that have reached the autotrophic phase of development,
embryos are normally allowed to develop in vivo as long as possible. While in
general, two main types of basal media are the most commonly used for
embryo rescue studies, i.e Murashige and Skoog medium (MS) and
Gamborg’s B-5 media (Bridgen, 1994), the composition of the media will vary
in terms of the concentrations of media supplements required. This will
generally depend on the stage of development of the embryo. For instance,
young embryos would require a complex medium with high sucrose
concentrations, while more mature embryos can usually develop on a simple
medium with low levels of sucrose. The temperature and light requirement is
generally species specific and thus its usually regulated to be the within the
same temperature requirement as that of its parent with embryos of coolseason crops requiring lower temperatures than those of warm-season crops.
Part – C
1. Describe various stages involved in micro propagation techniques.
Micropropagation is the practice of rapidly multiplying stock plant
material to produce a large number of progeny plants, using modern plant
tissue culture methods.
Micropropagation is used to multiply novel plants, such as those that have
been genetically modified or bred through conventional plant breeding
methods. It is also used to provide a sufficient number of plantlets for
planting from a stock plant which does not produce seeds, or does not
respond well to vegetative reproduction.
Micropropagation begins with the selection of plant material to be
propagated. Clean stock materials that are free of viruses and fungi are
important in the production of the healthiest plants. Once the plant material is
chosen for culture, the collection of explant(s) begins and is dependent on the
type of tissue to be used; including stem tips, anthers, petals, pollen and
others plant tissues. The explant material is then surface sterilized, usually in
multiple courses of bleach and alcohol washes and finally rinsed in sterilized
water. This small portion of plant tissue, sometimes only a single cell, is
placed on a growth medium, typically containing sucrose as an energy source
and one or more plant growth regulators (plant hormones). Usually the
medium is thickened with agar to create a gel which supports the explant
during growth. Some plants are easily grown on simple media but others
require more complicated media for successful growth; The plant tissue
grows and differentiates into new tissues depending on the medium. For
example, media containing cytokinins are used to create branched shoots
from plant buds. and it happens in a vegetative form
Multiplication
Multiplication is the taking of tissue samples produced during the first
stage and increasing their number. Following the successful introduction and
growth of plant tissue, the establishment stage is followed by multiplication.
Through repeated cycles of this process, a single explant sample may be
increased from one to hundreds or thousands of plants. Depending on the
type of tissue grown, multiplication can involve different methods and media.
If the plant material grown is callus tissue, it can be placed in a blender and
cut into smaller pieces and recultured on the same type of culture medium to
grow more callus tissue. If the tissue is grown as small plants called plantlets,
hormones are often added that cause the plantlets to produce many small
offshoots that can be removed and recultured.
Pretransplant
This stage involves treating the plantlets/shoots produced to encourage
root growth and "hardening." It is performed in vitro, or in a sterile "test tube"
environment.
"Hardening" refers to the preparation of the plants for a natural growth
environment. Until this stage, the plantlets have been grown in "ideal"
conditions, designed to encourage rapid growth. Due to lack of necessity, the
plants are likely to be highly susceptible to disease and often do not have fully
functional dermal coverings and will be inefficient in their use of water and
energy. In vitro conditions are high in humidity and plants grown under
these condition do not form a working cuticle and stomata that keep the plant
from drying out, when taken out of culture the plantlets need time to adjust to
more natural environmental conditions. Hardening typically involves slowly
weaning the plantlets from a high-humidity, low light, warm environment to
what would be considered a normal growth environment for the species in
question. This is done by moving the plants to a location high in humidity,
Transfer from culture
In the final stage of plant micropropagation, the plantlets are removed
from the plant media and transferred to soil or (more commonly) potting
compost for continued growth by conventional methods.
This stage is often combined with the "pretransplant" stage.
2. Write an essay on conventional breeding techniques
Plant breeding is defined as identifying and selecting desirable traits in plants
and combining these into one individual plant. Since 1900, Mendel's laws of
genetics provided the scientific basis for plant breeding. As all traits of a plant
are controlled by genes located on chromosomes, conventional plant breeding
can be considered as the manipulation of the combination of chromosomes. In
general, there are three main procedures to manipulate plant chromosome
combination. First, plants of a given population which show desired traits can
be selected and used for further breeding and cultivation, a process called
(pure line-) selection. Second, desired traits found in different plant lines can
be combined together to obtain plants which exhibit both traits
simultaneously, a method termed hybridization. Heterosis, a phenomenon of
increased vigor, is obtained by hybridization of inbred lines. Third,
polyploidy (increased number of chromosome sets) can contribute to crop
improvement. Finally, new genetic variability can be introduced through
spontaneous or artificially induced mutations.
Selection
Selection is the most ancient and basic procedure in plant breeding. It
generally involves three distinct steps. First, a large number of selections are
made from the genetically variable original population. Second, progeny rows
are grown from the individual plant selections for observational purposes.
After obvious elimination, the selections are grown over several years to
permit observations of performance under different environmental conditions
for making further eliminations. Finally, the selected and inbred lines are
compared to existing commercial varieties in their yielding performance and
other aspects of agronomic importance.
Hybridization
The most frequently employed plant breeding technique is hybridization.
The aim of hybridization is to bring together desired traits found in different
plant lines into one plant line via cross- pollination. The first step is to
generate homozygous inbred lines. This is normally done by using selfpollinating plants where pollen from male flowers pollinates female flowers
from the same plants. Once a pure line is generated, it is outcrossed, i. e.
combined with another inbred line. Then the resulting progeny is selected for
combination of the desired traits. If a trait from a wild relative of a crop
species, e.g. resistance against a disease, is to be brought into the genome of
the crop, a large quantity of undesired traits (like low yield, bad taste, low
nutritional value) are transferred to the crop as well. These unfavorable traits
must be removed by time-consuming back-crossing, i. e. repeated crossing
with the crop parent. There are two types of hybrid plants: interspecific and
intergeneric hybrids. Beyond this biological boundary, hybridization cannot
be accomplished due to sexual incompatibility, which limits the possibilities
of introducing desired traits into crop Plants.
Heterosis is an effect which is achieved by crossing highly inbred lines of
crop plants. Inbreeding of most crops leads to a strong reduction of vigor and
size in the first generations. After six or seven generations, no further
reduction in vigor or size is found. When such highly inbred plants are
crossed with other inbred varieties, very vigorous, large sized, large-fruited
plants may result. The term "heterosis" is used to describe the phenomenon of
hybrid vigor. The most notable and successful hybrid plant ever produced is
the hybrid maize. By 1919, the first commercial hybrid maize was available in
the United States. Two decades later, nearly all maize was hybrid, as it is
today, although the farmers must buy new hybrid seed every year, because
the heterosis effect is lost in the first generation after hybridization of the
inbred parental lines.
Polyploidy
Most plants are diploid. Plants with three or more complete sets of
chromosomes are common and are referred to as polyploids. The increase of
chromosomes sets per cell can be artificially induced by applying the chemical
colchicine, which leads to a doubling of the chromosome number. Generally,
the main effect of polyploidy is increase in size and genetic variability. On the
other hand, polyploid plants often have a lower fertility and grow more
slowly.
Induced mutation
Instead of relying only on the introduction of genetic variability from the
wild species gene pool or from other cultivars, an alternative is the
introduction of mutations induced by chemicals or radiation. The mutants
obtained are tested and further selected for desired traits. The site of the
mutation cannot be controlled when chemicals or radiation are used as agents
of mutagenesis. Because the great majority of mutants carry undesirable
traits, this method has not been widely used in breeding programs.
Success in using conventional plant breeding principles and agricultural
techniques reached its peak when high-yielding wheat and rice lines were
cultivated in the 1960s. The doubling and tripling of productivity of these
important crops in Asia signaled a agricultural revolution in the developing
countries. This breakthrough in food production was termed the "Green
Revolution" to describe the social, economic, and nutritional impact of the
new high-yielding wheat and rice strains. Norman Borlaug was awarded the
Noble Price in 1970 for his contribution in breeding new high-yielding strains
of cereals. However, these strains were highly dependent on fertilizers,
irrigation and agrochemicals and required energy-intensive investments. This
led to degradation and loss of soils and other severe environmental problems
all over the world. For these reasons, the "Green Revolution" has been both
praised and damned.
3. Write an essay on Biotechnological Methods of plant breeding
Biotechnology is the discipline which deals with the use of living
organisms or their products. In this wide sense, also traditional agriculture
may be seen as a form of biotechnology. The European Federation of
Biotechnology defines biotechnology as "the integrated use of biochemistry,
microbiology and engineering sciences in order to achieve technological
(industrial) application of the capability of microorganisms, cultured tissue
cells and parts thereof". In recent years, biotechnology has developed rapidly
as a practical means for accelerating success in plant breeding and improving
economically important crops. The most important methods used to achieve
these goals are described below. The techniques of genetic engineering, which
are a part of biotechnology, will be discussed in more detail in the next
chapter.
In Vitro Cultivation of Plant Cells and Regeneration of Plants from
Cultured Cells Certain isolated somatic plant cells can be cultured in vitro (in
the test tube) and are capable of proliferation and organization into tissues
and eventually into complete plants. The process of regenerating whole plants
out of plant cells is called in vitro regeneration. The three factors affecting
plant regeneration are genotype, explant source, and culture conditions,
including culture medium and environment. Different mixtures of plant
hormones and other compounds in varying concentrations are used to
achieve regeneration of plants from cultured cells and tissues. As the plant
hormonal mechanisms are not yet understood completely, the development
of in vitro cultivation and regeneration systems is still largely based on
empirically testing variations of the three above mentioned factors.
In Vitro Selection and Somaclonal Variation : Plants regenerated from cell
cultures may exhibit phenotypes differing from their parent plants,
sometimes at quite high frequencies. If these are heritable and affecting
desirable agronomic traits, such "somaclonal variation" can be incorporated
into breeding programs. However, finding of specific valuable traits by this
method is largely left to chance and hence inefficient. Rather than relying on
this undirected process, selection in vitro targets specific traits by subjecting
large populations of cultured cells to the action of a selective agent in the Petri
dish. For purposes of disease resistance, this selection can be provided by
pathogens, or isolated pathotoxins that are known to have a role in
pathogenesis. The selection will only allow those cells to survive and
proliferate that are resistant to the challenge. Selection of cells also plays an
important role in genetic engineering, where special marker genes are used to
select for transgenic cells.
Somatic Hybrid Plants : Somatic hybrid plants are plants derived from the
fusion of somatic cells. Cell fusion was developed after the successful culture
of a large number of plant cells stripped of their cell walls. The resulting cells
without walls are referred to as protoplasts. Since also protoplasts from
phylogenetically unrelated species can be fused, attempts have been made to
overcome sexual incompatibility using protoplast fusion. In most cases, these
attempts failed because growth and division of the fused cells did not take
place when only distantly related cells were fused. Successful fusions between
sexually incompatible petunia species and between potatoes and tomatoes
did not lead to economically interesting products, but important contributions
to the understanding of cell wall regeneration and other mechanisms were
achieved
Genetic engineering is a term used for the directed manipulation of genes, i. e.
the transfer of genes between organisms or changes in the sequence of a gene.
Closely related to this field are methods which use genes or specific sequences
for the identification of traits and other analytical purposes. In plant breeding,
the most important and already widely used method of this kind is
Restriction Fragment Length Polymorphism (RFLP). The basics of this
technique are described below. The principles of plant genetic engineering
will be described in the next chapter. For those who are not familiar with the
principles of genetics, the glossary at the end of this report explains the most
important terms.
4. List down various applications of in vitro propagation techniques
Micropropagation /Clonal Propagation
Clonal propagation refers to the process of asexual reproduction by
multiplication of genetically identical copies of individual plants. The
vegetative propagation of plants is labour-intensive, low in productivity and
seasonal. The tissue culture methods of plant propagation, known as
'micropropagation' utilizes the culture of apical shoots, axillary buds and
meristems on suitable nutrient medium.The regeneration of plantlets in
cultured tissue was described by Murashige in 1974. Fossard (1987) gave a
detailed account of stages of micropropagation.
The micropropagation is rapid and has been adopted for
commercialization of important plants such as banana, apple, pears,
strawberry, cardamom, many ornamentals (e.g. Orchids) and other plants.The
micropropagation techniques are preferred over the conventional asexual
propagation methods because of the following reasons: (a) In the
micropropagation method, only a small amount of tissue is required to
regenerate millions of clonal plants in a year., (b) micropropagation is also
used as a method to develop resistance in many species., (c) in vitro stock can
be quickly proliferated as it is season independent,. (d) long term storage of
valuable germplasm possible.
The steps in micropropagation method are: a) Initiation of culture - from
an explant like shoot tip on a suitable nutrient medium, b) multiple shoots
formation from the cultured explant, c) rooting of in vitro developed shoots
and, d) transplantation - transplantation to the field following acclimatization.
The factors that affect micropropagation are: (a) genotype and the
physiological status of the plant e.g. plants with vigorous germination are
more suitable for micropropagation., (b) the culture medium and the culture
environment like light, temperature etc. For example an illumination of 16
hours a day and 8 hours night is satisfactory for shoot proliferation and a
temperature of 250C is optimal for the growth.
The benefits of micropropagation this method are:
a) rapid multiplication of superior clones can be carried out through out the year,
irrespective of seasonal variations.
b) multiplication of disease free plants e.g. virus free plants of sweet potato
(Ipomea batatus), cassava (Manihot esculenta)
c) multiplication of sexually derived sterile hybrids
d) It is a cost effective process as it requires minimum growing space.
Somaclonal variation
The genetic variations found in the in vitro cultured cells are collectively
referred to as somaclonal variation and the plants derived from such cells are
called as ‘somaclones’. It has been observed that the long-term callus and cell
suspension culture and plants regenerated from such cultures are often
associated with chromosomal variations. It is this property of cultured cells
that finds potential application in the crop improvement and in the
production of mutants and variants (e.g. disease resistance in potato).
Larkin and Scowcroft (1981) working at the division of Plant Industry,
C.S.I.R.O., Australia gave the term 'somaclones' for plant variants obtained
from tissue cultures of somatic tissues. Similarly, if the tissue from which the
variants have been obtained is having gametophytic origin such as pollen or
egg cell, it is known as 'gametoclonal' variation.They explained that it may be
due to: (a) reflection of heterogeneity between the cells and explant tissue, (b)
a simple representation of spontaneous mutation rate, and (c) activation by
culture environment of transposition of genetic materials.
Shepard et al. (1980) also contributed by screening about 100 somaclones
produced from leaf protoplasts of Russet Burbank. They found that there was
a significant amount of stable variation in compactness of growth habit,
maturity, date, tuber uniformity, tuber skin colour and photoperiodic
requirements.
Somaclonal Variations has been used in plant breeding programmes where
the genetic variations with desired or improved characters are introduced into
the plants and new varieties are created that can exhibit disease resistance,
improved quality and yield in plants like cereals, legumes, oil seeds tuber
crops etc. Somaclonal variation is applicable for seed
Applications of Somaclonal Variations
a) Methodology of introducing somaclonal variations is simpler and easier as
compared to recombinant DNA technology.
b) Development and production of plants with disease resistance e.g. rice, wheat,
apple, tomato etc.
c) Develop biochemical mutants with abiotic stress resistance e.g. aluminium
tolerance in carrot, salt tolerance in tobacco and maize.
d) Development of somaclonal variants with herbicide resistance e.g. tobacco
resistant to sulfonylurea
e) Development of seeds with improved quality e.g. a new variety of Lathyrus
sativa seeds (Lathyrus Bio L 212) with low content of neurotoxin.
f) Bio-13 – A somaclonal variant of Citronella java (with 37% more oil and 39%
more citronellon), a medicinal plant has been released as Bio-13 for
commercial cultivation by Central Institute for Medicinal and Aromatic
Plants (CIMAP), Lucknow, India.
g) Supertomatoes- Heinz Co. and DNA plant Technology Laboratories (USA)
developed Supertomatoes with high solid component by screening somaclones
which helped in reducing the shipping and processing costs.
Production of virus free plants
The viral diseases in plants transfer easily and lower the quality and yield of
the plants. It is very difficult to treat and cure the virus infected plants
therefore te plant breeders are always interested in developing and growing
virus free plants.
In some crops like ornamental plants, it has become possible to
produce virus free plants through tissue culture at the commercial level. This
is done by regenerating plants from cultured tissues derived from a) virus
free plants, b) meristems which are generally free of infection - In the
elimination of the virus, the size of the meristem used in cultures play a very
critical role because most of the viruses exist by establishing a gradient in
plant tissues. The regeneration of virus-free plants through cultures is
inversely proportional to the size of the meristem used., c) meristems treated
with heat shock (34-360C) to inactivate the virus, d) callus, which is usually
virus free like meristems.e) chemical treatment of the media- attempts have
been made to eradicate the viruses from infected plants by treating the culture
medium with chemicals e.g. addition of cytokinins suppressed the
multiplication of certain viruses.
Among the culture techniques, meristem-tip culture is the most reliable
method for virus and other pathogen elimination.
Viruses have been eliminated from a number of economically
important plant species which has resulted in a significant increase in the
yield and production e.g. potato virus X from potato, mosaic virus from
cassava etc. These virus free plants are not disease resistant so there is a need
to maintain stock plants to multiply virus free plants whenever required.
Production of synthetic seeds
In synthetic seeds, the somatic embryos are encapsulated in a suitable
matrix (e.g. sodium alginate), along with substances like mycorrhizae,
insecticides, fungicides and herbicides. These artificial seeds can be utilized
for the rapid and mass propagation of desired plant species as well as hybrid
varieties.
The major benefits of synthetic seeds are:
a) They can be stored up to a year with out loss of viability
b) Easy to handle and useful as units of delivery
c) Can be directly sown in the soil like natural seeds and do not need
acclimatization in green house.
Mutant selection
An important use of cell cultures is in mutant selection in relation to
crop improvement. The frequency of mutations can be increased several fold
through mutagenic treatments and millions of cells can be screened. A large
number of reports are available where mutants have been selected at cellular
level. The cells are often selected directly by adding the toxic substance
against which resistance is sought in the mutant cells. Using this method, cell
lines resistant to amino acid analogues, antibiotics, herbicides, fungal toxins
etc have actually been isolated.
Production of secondary metabolites
The most important chemicals produced using cell culture are
secondary metabolites, which are defined as’ those cell constituents which are
not essential for survival’. These secondary metabolites include alkaloids,
glycosides (steroids and phenolics), terpenoids, latex, tannins etc. It has been
observed that as the cells undergo morphological differentiation and
maturation during plant growth, some of the cells specialize to produce
secondary metabolites. The in vitro production of secondary metabolites is
much higher from differentiated tissues when compared to non-differentiated
tissues.
The cell cultures contribute in several ways to the production of
natural products. These are: (a) a new route of synthesis to establish products
e.g. codeine, quinine, pyrethroids, (b) a route of synthesis to a novel product
from plants difficult to grow or establish e.g. thebain from Papaver
bracteatum, (c) a source of novel chemicals in their own right e.g. rutacultin
from culture of Ruta, (d) as biotransformation systems either on their own or
as part of a larger chemical process e.g. digoxin synthesis.
The advantages of in vitro production of secondary metabolites
a) The cell cultures and cell growth are easily controlled in order to facilitate
improved product formation.
b) The recovery of the product is easy.
c) As the cell culture systems are independent of environmental factors, seasonal
variations, pest and microbial diseases, geographical location constraints, it is
easy to increase the production of the required metabolite.
d) Mutant cell lines can be developed for the production of novel and
commercially useful compounds.
e) Compounds are produced under controlled conditions as per the market
demands.
f) The production time is less and cost effective due to minimal labour involved.
Production of Somatic hybrids and cybrids
The Somatic cell hybridization/ parasexual hybridization or Protoplast
fusion offers an alternative method for obtaining distant hybrids with
desirable traits significantly between species or genera, which can not be
made to cross by conventional method of sexual hybridization.
Somatic hybridization
Somatic hybridization broadly involves in vitro fusion of isolated
protoplasts to form a hybrid cell and its subsequent development to form a
hybrid plant. The process involves: a) fusion of protoplasts, (b) Selection of
hybrid cells, (c) identification of hybrid plants.
During the last two decades, a variety of treatments have been used to bring
about the fusion of plant protoplasts. Protoplast fusion can be achieved by
spontaneous, mechanical, or induced fusion methods.. These treatments
include the use of fusogens like NaNO3, high pH with high Ca2++ ion
concentration, use of polyethylene glycol (PEG), and electrofusion. These
inducing agents used in protoplast fusion are called ‘fusogen’.
PEG treatment is the most widely used method for protoplast fusion as it has
certain advantages over others. These are : (a) it results in a reproducible
high-frequency of heterokaryon formation., (b) The PEG fusion is non specific
and therefore can be used for a wide range of plants., (c) It has low toxicity to
the cell and (d) The formation of binucleate heterokaryons is low.
Mechanism of fusion : The fusion of protoplasts takes place in three phasesagglutination, plasma membrane fusion and formation of heterokaryons.
When the two protoplasts come in close contact with each other, they adhere
to each other. This agglutination can be induced by PEG, high pH and high
Ca2+. The protoplast membranes get fused at localized sites at the point of
adhesion. This leads to the formation of cytoplasmic bridges between
protoplasts. High pH and high Ca2+ ions neutralize the surface charges on
the protoplasts which allows closer contact and membrane fusion between
agglutinated protoplasts. The fused protoplasts become round as a result of
cytoplasmic bridges which leads to the formation of spherical homokaryon or
heterokaryon.
Selection of hybrid cells : The methods used for the selection of hybrid cells
are biochemical, visual and cytometric methods using fluorescent dyes. The
biochemical methods for selection of hybrid cells are based on the use of
biochemical compounds in the medium. The drug sensitivity method is useful
for the selection hybrids of two plants species, if one of them is sensitive to a
drug. Another method, auxotrophic mutant selection method involves the
auxotrophs which are mutants that cannot grow on a minimal medium.
Therefore specific compounds are added in the medium. The selection of
auxotropic mutants is possible only if the hybrid cells can grow on a minimal
medium. The visual method involves the identification of heterokaryons
under the light microscope. In some of the somatic hybridizations, the
chloroplast deficient protoplast of one plant species is fused with the green
protoplast of another plant species. The heterokaryons obtained are bigger
and green in colour while the parental protoplasts are either small or
colourless. The cytometric method uses flow cytometry and flourescentactivated cell sorting techniques for the analysis of plant protoplasts.
Applications of Somatic hybridization
a) Creation of hybrids with disease resistance - Many disease resistance genes (e.g.
tobacco mosaic virus, potato virus X, club rot disease) could be successfully
transferred from one species to another. E.g resistance has been introduced in
tomato against diseases such as TMV, spotted wilt virus and insect pests.
b) Environmental tolerance - using somatic hybridization the genes conferring
tolerance for cold, frost and salt were introduced in e.g. in tomato.
c) Cytoplasmic male sterility - using cybridization method, it was possible to
transfer cytoplasmic male sterility.
d) Quality characters - somatic hybrids with selective characteristics have been
developed e.g. the production of high nicotine content.
Chromosome number in somatic hybrids : The chromosome number in the
somatic hybrids is generally more than the total number of both of the
parental protoplasts. If the chromosome number in the hybrid is the sum of
the chromosomes of the two parental protoplasts, the hybrid is said to be
symmetric hybrid. Asymmetric hybrids have abnormal or wide variations in
the chromosome number than the exact total of two species.
In 1972, Carlson and his associates produced the first inter-specific somatic
hybrid between Nicotiana glauca and N. langsdorffii. In 1978, Melchers and
his co-workers developed the first inter-genetic somatic hybrids between
Solanum tuberosum (potato) and Lycopersicon esculentum (tomato). The
hybrids are known as ‘Pomatoes or Topatoes’.
Limitations of Somatic Hybridization :
a) Somatic hybridization does not always produce plants that give fertile and
visible seeds.
b) There is genetic instability associated with protoplast culture.
c) There are limitations in the selection methods of hybrids, as many of them
are not efficient.
d) Somatic hybridization between two diploids results in the formation of an
amphidiploid which is not favourable therefore haploid protoplasts are
recommended in somatic hybridization.
e) It is not certain that a specific character will get expressed in somatic
hybridization.
f) Regenerated plants obtained from somatic hybridization are often variable
due to somaclonal variations, chromosomal elimination, organelle
segregation etc.
g) Protoplast fusion between different species/genus is easy, but the
production of viable somatic hybrids is not always possible.
Cybrids
The cytoplasmic hybrids where the nucleus is derived from only one
parent and the cytoplasm is derived from both the parents are referred to as
cybrids. The process of formation of cybrids is called cybridization. During
the process of cybridization and heterokaryon formation, the nuclei are
stimulated to segregate so that one protoplast contributes to the cytoplasm
while the other contributes nucleus alone. The irradiation with gamma rays
and X-rays and use of metabolic inhibitors makes the protoplasts inactive and
non-dividing. Some of the genetic traits in certain plants are cytoplasmically
controlled. This includes certain types of male sterility, resistance to certain
antibiotics and herbicides. Therefore cybrids are important for the transfer of
cytoplasmic male sterility (CMS), antibiotic and herbicide resistance in
agriculturally useful plants. Cybrids of Brassica raphanus that contain
nucleus of B. napus, chloroplasts of atrazinc resistant B. capestris and male
sterility from Raphanus sativas have been developed.
In vitro plant germplasm conservation
Germplasm refers to the sum total of all the genes present in a crop and
its related species.
The conservation of germplasm involves the preservation of the
genetic diversity of a particular plant or genetic stock for it’s use at any time
in future. It is important to conserve the endangered plants or else some of the
valuable genetic traits present in the existing and primitive plants will be lost.
A global organization- International Board of Plant Genetic Resources
(IBPGR) has been established for germplasm conservation and provides
necessary support for collection, conservation and utilization of plant geneic
resources through out the world. The germplasm is preserved by the
following two ways:
(a) In-situ conservation- The germplasm is conserved in natural environment
by establishing biosphere reserves such as national parks, sanctuaries. This is
used in the preservation of land plants in a near natural habitat along with
several wild types.
(b) Ex-situ conservation- This method is used for the preservation of
germplasm obtained from cultivated and wild plant materials. The genetic
material in the form of seeds or in vitro cultures are preserved and stored as
gene banks for long term use.
In vivo gene banks have been made to preserve the genetic resources by
conventional methods e.g. seeds, vegetative propagules, etc. In vitro gene
banks have been made to preserve the genetic resources by non - conventional
methods such as cell and tissue culture methods. This will ensure the
availability of valuable germplasm to breeder to develop new and improved
varieties.
The methods involved in the in vitro conservation of germplasm are:
(a) Cryopreservation- In cryopreservation (Greek-krayos-frost), the cells are
preserved in the frozen state. The germplasm is stored at a very low
temperature using solid carbon dioxide (at -790C), using low temperature
deep freezers (at -800C), using vapour nitrogen (at- 1500C) and liquid
nitrogen (at-1960C). The cells stay in completely inactive state and thus can be
conserved for long periods. Any tissue from a plant can be used for
cryopreservation e.g. meristems, embryos, endosperms, ovules, seeds,
cultured plant cells, protoplasts, calluses. Certain compounds like- DMSO
(dimethyl sulfoxide), glycerol, ethylene, propylene, sucrose, mannose,
glucose, praline, acetamide etc are added during the cryopreservation. These
are called cryoprotectants and prevent the damage caused to cells (by freezing
or thawing) by reducing the freezing point and super cooling point of water.
(b) Cold Storage- Cold storage is a slow growth germplasm conservation
method and conserves the germplasm at a low and non-freezing temperature
(1-90C). The growth of the plant material is slowed down in cold storage in
contrast to complete stoppage in cryopreservation and thus prevents
cryogenic injuries. Long term cold storage is simple, cost effective and yields
germplasm with good survival rate. Virus free strawberry plants could be
preserved at 100C for about 6 years. Several grape plants have been stored for
over 15 years by using a cold storage at temperature around 90C and
transferring them in the fresh medium every year.
(c) Low pressure and low oxygen storage- In low- pressure storage, the
atmospheric pressure surrounding the plant material is reduced and in the
low oxygen storage, the oxygen concentration is reduced. The lowered partial
pressure reduces the in vitro growth of plants. In the low-oxygen storage, the
oxygen concentration is reduced and the partial pressure of oxygen below 50
mmHg reduces plant tissue growth. Due to the reduced availability of O2,
and reduced production of CO2, the photosynthetic activity is reduced which
inhibits the plant tissue growth and dimension. This method has also helped
in increasing the shelf life of many fruits, vegetables and flowers.
The germplasm conservation through the conventional
limitations such as short-lived seeds, seed dormancy,
and high inputs of cost and labour. The techniques
(freezing cells and tissues at -1960c) and using cold
overcome these problems.
methods has several
seed-borne diseases,
of cryo-preservation
storages help us to
5. Explain the principles of isolation and culture of protoplasts
Isolation of protoplasts
Protoplasts (cell minus cell wall) is the biologically active and most significant
material of cells. When cell wall is mechanically or enzymatically removed the
isolated protoplast is known as "naked plant cell" on which most of recent
researches are based.
Plant cell wall acts as physical barrier and protects cytoplasm from microbial
invasion and environmental stress. It consists of a complex mixture of
cellulose, hemicellulose, pectin, lignin, lipids, protein, etc. For dissolution of
different components of the cell wall it is essential to have the respective
enzymes.
Until suitable methods were developed, protoplasts were isolated by cutting
the plasmolysed plant tissues and releasing protoplast through deplasmolysis
of cells. Cooking (1960) for the first time isolated the protoplasts of plant
tissues by using cell wall degrading enzymes viz., cellulase, hemicellulase,
pectinase, and protease extracted from a saprophytic fungus Trichoderma
viride. Later on protoplasts were cultured in vitro.
Microorganisms are well equipped with a system to produce substrate
specific extracellular enzymes, the extent of which depends on the genetic
variability of the specific species and strains. A detailed account of enzyme
preparation and their uses in isolation of protoplast has been given by
Cooking (1972 ); Bajaj (1977b) and Patnaik et al (1981). However, the basic
techniques of isolation and culture of protoplast are given in Figs. 8.5. and 8.6
with a brief description.
Surface Sterilization of Leaf Samples : Mature leaves are collected from
healthy plants which are washed in tap water to remove adhering soil
particles and sterilized with sodium hypochlorite solution.
Rinsing in Suitable Osmoticum : After 10 min, sample is properly washed
with sterile distilled water or MS medium adjusted to a suitable pH and
buffer to maintain osmotic pressure. Washing should be done for about 6
times to remove the traces of sodium hypochlorite.
Plasmolysis of Cells : The lower epidermis covered by thin wax cuticle is
removed with a forcep. Stripping should be done from midrib to margin of
lamina. The stripped surface of leaf is kept in mannitol solution (13% W/V)
for 3 hours to allow plasmolysis of cells.
Peeling of Lower Epidermis : Thereafter, about 1 gm leaves are peeled off and
transferred into enzyme mixture already sterilized through a Seitz filter (0.45
mm). This facilitates the penetration of enzyme into tissue within 12-18 hours
at 25°C.
Isolation and Purification of Portoplasts : Leaf debris are removed with
forcep, and enzyme solution containing protoplasts are filtered with a nylon
mesh (45mm). Filtrate is centrifuged at 75 X g for 5 min and supernatant is
decanted. Again a fresh MS medium plus 13% mannitol is added to
centrifuge. Repeated washing with nutrient medium, centrifugation and
decantation are done for about three time. Finally specific concentration of
protoplast suspension is prepared.
Protoplast culture and regeneration
From the protoplast solution of known density (about 105 protoplast/ml)
about 1 ml suspension is poured on sterile and cooled down nutrient medium
in Petri dishes. The plates are incubated at 25°C in a dim white light.
The protoplasts regenerate a cell wall, undergo cell division and form
callus. The callus can also be subcultured. Embryogenesis begins from callus
when it is placed on nutrient medium lacking mannitol and auxin. The
embryo develops into seedlings and finally mature plants
6. Explain somatic embryogenesis with suitable diagrams
Somatic embryos are mainly produced in vitro and for laboratory purposes,
using either solid or liquid nutrient media which contain plant growth
regulators (PGR’s). The main PGRs used are auxins but can contain cytokinin
in a smaller amount. Somatic embryogenesis is a process where a plant or
embryo is derived from a single somatic cell or group of somatic cells. This is
in contrast to zygotic embryogenesis, where ,in diploid species, two haploid
cells combine to form one diploid cell. Somatic embryos are produced when
somatic cells are restructured through a series of morphological and
biochemical changes in the embryogenic pathway. Development of somatic
embryos is not that different from zygotic embryos. Three examples of
somatic embryogenesis from nature are ovules in Paeonia and on the leaves of
Asplenium and Kalanchoe. Shoots and roots are monopolar while somatic
embryos are bipolar, allowing them to form a whole plant without culturing
on multiple media types. Somatic embryogenesis has served as a model to
understand the physiological and biochemical events that occur plant
developmental processes as well as a component to biotechnological
advancement. The first documentation of somatic embryogenesis was by
Steward et al in 1958 and Reinert in 1959 with carrot cell suspension cultures.
7. Explain haploid culture techniques and describe their advantages
Anther and Pollen Culture (Production of Haploid Plants) :
Anther, a male reproductive organ, is diploid in chromosome numbers. As a
result of microsporogenesis, tetrads of microspores are formed from a single spore
mother cell. They are known as pollen grains after release from tetrads (Bhojwani
and Bhatnagar, 1974). The aim of anther and pollen culture is to get haploid plants by
induction of embryogenesis. Haploid plants have single complete set of
chromosomes that in turn may be useful for the improvement of many crop plants
(Sunderland, 1979). Moreover, chromosome set of these haploids can be doubled by
mutagenic chemicals (e.g. colchicine) or regeneration technique to obtain fertile
homozygous diploi4Su(Vasil and Nitsch, 1975).
Tulecke (1951) cultured pollen grains of Ginkgo biloba (gymnosperm) and succeeded
to induce the development of haploid callus. Guha and Maheswari (1964) made a
remarkable discovery by culturing pollen grains of an angiospermic plant, Datura
innoxia on the nutrient agar medium and also developed torpedo- shaped embryoids
that metamorphosed into plantlets through the process.
Sunderland (1979) has described that the anthers to be cultured should be one of the
three categories i.e. premitotic, mitotic and postmitotic. In premitotic anthers, where
the microspores have completed meiosis but not started first pollen division, the best
response is achieved e.g. Hordeum vulgare. In mitotic anthers where first pollen
division has started the optimum responses are achieved e.g. N. tabacum and D.
innoxia. In post mitotic anthers, the early bicellular stage of pollen development is
the best time to culture e.g. Atropa belladona.
Haploid plants are very useful in (i) direct screening of recessive mutation because in
diploid or polyploid screening of recessive mutation is not possible, and (ii)
development of homozygous diploid plants following chromosome doubling of
haploid plant cells.
In China, the most widely grown wheat is a doubled haploid produced through
homozygous diploid lines. Anther culture of rice is also successfully grown. Haploid
plants have been produced in tobacco, wheat and rice through pollen culture. These
are used for the development of disease resistant and superior diploid lines.
At present, more than 247 plant species and hybrids belonging to 38 genera and 34
families of dicots and monocots have been regenerated using anther culture
technique. They include economically important crops and trees such as rice, wheat
maize, coconut, rubber trees, etc, (Maheswari et al.1983).
8. Describe plant growth regulators with their characteristic feature
Plant hormones (also known as phytohormones) are chemicals that regulate
plant growth, which, in the UK, are termed 'plant growth substances'. Plant
hormones are signal molecules produced within the plant, and occur in
extremely low concentrations. Hormones regulate cellular processes in
targeted cells locally and when moved to other locations, in other locations of
the plant. Hormones also determine the formation of flowers, stems, leaves,
the shedding of leaves, and the development and ripening of fruit. Plants,
unlike animals, lack glands that produce and secrete hormones, instead each
cell is capable of producing hormones. Plant hormones shape the plant,
affecting seed growth, time of flowering, the sex of flowers, senescence of
leaves and fruits. They affect which tissues grow upward and which grow
downward, leaf formation and stem growth, fruit development and ripening,
plant longevity, and even plant death. Hormones are vital to plant growth
and lacking them, plants would be mostly a mass of undifferentiated cells.
Characteristics
The word hormone is derived from Greek, meaning 'set in motion.' Plant
hormones affect gene expression and transcription levels, cellular division,
and growth. They are naturally produced within plants, though very similar
chemicals are produced by fungi and bacteria that can also affect plant
growth. A large number of related chemical compounds are synthesized by
humans, they are used to regulate the growth of cultivated plants, weeds, and
in vitro-grown plants and plant cells; these manmade compounds are called
Plant Growth Regulators or PGRs for short. Early in the study of plant
hormones, "phytohormone" was the commonly used term, but its use is less
widely applied now.
Plant hormones are not nutrients, but chemicals that in small amounts
promote and influence the growth, development, and differentiation of cells
and tissues. The biosynthesis of plant hormones within plant tissues is often
diffuse and not always localized. Plants lack glands to produce and store
hormones, because, unlike animals, which have two circulatory systems
(lymphatic and cardiovascular) powered by a heart that moves fluids around
the body, plants use more passive means to move chemicals around the plant.
Plants utilize simple chemicals as hormones, which move more easily through
the plant's tissues. They are often produced and used on a local basis within
the plant body, plant cells even produce hormones that affect different
regions of the cell producing the hormone.
Hormones are transported within the plant by utilizing four types of
movements. For localized movement, cytoplasmic streaming within cells and
slow diffusion of ions and molecules between cells are utilized. Vascular
tissues are used to move hormones from one part of the plant to another;
these include sieve tubes that move sugars from the leaves to the roots and
flowers, and xylem that moves water and mineral solutes from the roots to
the foliage.
Not all plant cells respond to hormones, but those cells that do are
programmed to respond at specific points in their growth cycle. The greatest
effects occur at specific stages during the cell's life, with diminished effects
occurring before or after this period. Plants need hormones at very specific
times during plant growth and at specific locations. They also need to
disengage the effects that hormones have when they are no longer needed.
The production of hormones occurs very often at sites of active growth within
the meristems, before cells have fully differentiated. After production they are
sometimes moved to other parts of the plant where they cause an immediate
effect or they can be stored in cells to be released later. Plants use different
pathways to regulate internal hormone quantities and moderate their effects;
they can regulate the amount of chemicals used to biosynthesize hormones.
They can store them in cells, inactivate them, or cannibalise already-formed
hormones by conjugating them with carbohydrates, amino acids or peptides.
Plants can also break down hormones chemically, effectively destroying
them. Plant hormones frequently regulate the concentrations of other plant
hormones. Plants also move hormones around the plant diluting their
concentrations.
The concentration of hormones required for plant responses are very low
(10−6 to 10−5 mol/L). Because of these low concentrations, it has been very
difficult to study plant hormones, and only since the late 1970s have scientists
been able to start piecing together their effects and relationships to plant
physiology. Much of the early work on plant hormones involved studying
plants that were genetically deficient in one or involved the use of tissuecultured plants grown in vitro that were subjected to differing ratios of
hormones, and the resultant growth compared. The earliest scientific
observation and study dates to the 1880s; the determination and observation
of plant hormones and their identification was spread-out over the next 70
years.
Classes of plant hormonesIn general, it is accepted that there are five major
classes of plant hormones, some of which are made up of many different
chemicals that can vary in structure from one plant to the next. The chemicals
are each grouped together into one of these classes based on their structural
similarities and on their effects on plant physiology. Other plant hormones
and growth regulators are not easily grouped into these classes; they exist
naturally or are synthesized by humans or other organisms, including
chemicals that inhibit plant growth or interrupt the physiological processes
within plants. Each class has positive as well as inhibitory functions, and most
often work in tandem with each other, with varying ratios of one or more
interplaying to affect growth regulation.
The five major classes are:
Abscisic acid
Abscisic acid also called ABA, was discovered and researched under two
different names before its chemical properties were fully known, it was called
dormin and abscicin II. Once it was determined that the two latter
compounds were the same; it was named abscisic acid. The name "abscisic
acid" was given because it was found in high concentrations in newly
abscissed or freshly fallen leaves.
This class of PGR is composed of one chemical compound normally
produced in the leaves of plants, originating from chloroplasts, especially
when plants are under stress. In general, it acts as an inhibitory chemical
compound that affects bud growth, seed and bud dormancy. It mediates
changes within the apical meristem causing bud dormancy and the alteration
of the last set of leaves into protective bud covers. Since it was found in
freshly abscissed leaves, it was thought to play a role in the processes of
natural leaf drop but further research has disproven this. In plant species
from temperate parts of the world it plays a role in leaf and seed dormancy by
inhibiting growth, but, as it is dissipated from seeds or buds, growth begins.
In other plants, as ABA levels decrease, growth then commences as
gibberellin levels increase. Without ABA, buds and seeds would start to grow
during warm periods in winter and be killed when it froze again. Since ABA
dissipates slowly from the tissues and its effects take time to be offset by other
plant hormones, there is a delay in physiological pathways that provide some
protection from premature growth. It accumulates within seeds during fruit
maturation, preventing seed germination within the fruit, or seed germination
before winter. Abscisic acid's effects are degraded within plant tissues during
cold temperatures or by its removal by water washing in out of the tissues,
releasing the seeds and buds from dormancy.
In plants under water stress, ABA plays a role in closing the stomata. Soon
after plants are water-stressed and the roots are deficient in water, a signal
moves up to the leaves, causing the formation of ABA precursors there, which
then move to the roots. The roots then release ABA, which is translocated to
the foliage through the vascular system and modulates the potassium and
sodium uptake within the guard cells, which then lose turgidity, closing the
stomata. ABA exists in all parts of the plant and its concentration within any
tissue seems to mediate its effects and function as a hormone; its degradation,
or more properly catabolism, within the plant affects metabolic reactions and
cellular growth and production of other hormones. Plants start life as a seed
with high ABA levels, just before the seed germinates ABA levels decrease;
during germination and early growth of the seedling, ABA levels decrease
even more. As plants begin to produce shoots with fully functional leaves ABA levels begin to increase, slowing down cellular growth in more "mature"
areas of the plant. Stress from water or predation affects ABA production and
catabolism rates, mediating another cascade of effects that trigger specific
responses from targeted cells. Scientists are still piecing together the complex
interactions and effects of this and other phytohormones.
Auxins
The auxin indoleacetic acidAuxins are compounds that positively
influence cell enlargement, bud formation and root initiation. They also
promote the production of other hormones and in conjunction with
cytokinins, they control the growth of stems, roots, and fruits, and convert
stems into flowers.[11] Auxins were the first class of growth regulators
discovered.[12] They affect cell elongation by altering cell wall plasticity.
Auxins decrease in light and increase where it is dark. They stimulate
cambium, a subtype of meristem cells, to divide and in stems cause secondary
xylem to differentiate. Auxins act to inhibit the growth of buds lower down
the stems (apical dominance), and also to promote lateral and adventitious
root development and growth. Leaf abscission is initiated by the growing
point of a plant ceasing to produce auxins. Auxins in seeds regulate specific
protein synthesis,[13] as they develop within the flower after pollination,
causing the flower to develop a fruit to contain the developing seeds. Auxins
are toxic to plants in large concentrations; they are most toxic to dicots and
less so to monocots. Because of this property, synthetic auxin herbicides
including 2,4-D and 2,4,5-T have been developed and used for weed control.
Auxins, especially 1-Naphthaleneacetic acid (NAA) and Indole-3-butyric acid
(IBA), are also commonly applied to stimulate root growth when taking
cuttings of plants. The most common auxin found in plants is indoleacetic
acid or IAA. The correlation of auxins and cytokinins in the plants is a
constant (A/C = const.).
Cytokinins
The cytokinin zeatin , Zea, in which it was first discovered in immature
kernels.Cytokinins or CKs are a group of chemicals that influence cell division
and shoot formation. They were called kinins in the past when the first
cytokinins were isolated from yeast cells. They also help delay senescence or
the aging of tissues, are responsible for mediating auxin transport throughout
the plant, and affect internodal length and leaf growth. They have a highly
synergistic effect in concert with auxins and the ratios of these two groups of
plant hormones affect most major growth periods during a plant's lifetime.
Cytokinins counter the apical dominance induced by auxins; they in
conjunction with ethylene promote abscission of leaves, flower parts and
fruits.[14] The correlation of auxins and cytokinins in the plants is a constant
(A/C = const.).
Ethylene
EthyleneEthylene is a gas that forms through the Yang Cycle from the
breakdown of methionine, which is in all cells. Ethylene has very limited
solubility in water and does not accumulate within the cell but diffuses out of
the cell and escapes out of the plant. Its effectiveness as a plant hormone is
dependent on its rate of production versus its rate of escaping into the
atmosphere. Ethylene is produced at a faster rate in rapidly growing and
dividing cells, especially in darkness. New growth and newly germinated
seedlings produce more ethylene than can escape the plant, which leads to
elevated amounts of ethylene, inhibiting leaf expansion (see Hyponastic
response). As the new shoot is exposed to light, reactions by phytochrome in
the plant's cells produce a signal for ethylene production to decrease,
allowing leaf expansion. Ethylene affects cell growth and cell shape; when a
growing shoot hits an obstacle while underground, ethylene production
greatly increases, preventing cell elongation and causing the stem to swell.
The resulting thicker stem can exert more pressure against the object
impeding its path to the surface. If the shoot does not reach the surface and
the ethylene stimulus becomes prolonged, it affects the stems natural
geotropic response, which is to grow upright, allowing it to grow around an
object. Studies seem to indicate that ethylene affects stem diameter and
height: When stems of trees are subjected to wind, causing lateral stress,
greater ethylene production occurs, resulting in thicker, more sturdy tree
trunks and branches. Ethylene affects fruit-ripening: Normally, when the
seeds are mature, ethylene production increases and builds-up within the
fruit, resulting in a climacteric event just before seed dispersal. The nuclear
protein Ethylene Insensitive2 (EIN2) is regulated by ethylene production, and,
in turn, regulates other hormones including ABA and stress hormones.
Gibberellins
Gibberellin A1 Gibberellins, or GAs, include a large range of chemicals that
are produced naturally within plants and by fungi. They were first discovered
when Japanese researchers, including Eiichi Kurosawa, noticed a chemical
produced by a fungus called Gibberella fujikuroi that produced abnormal
growth in rice plants. Gibberellins are important in seed germination,
affecting enzyme production that mobilizes food production used for growth
of new cells. This is done by modulating chromosomal transcription. In grain
(rice, wheat, corn, etc.) seeds, a layer of cells called the aleurone layer wraps
around the endosperm tissue. Absoption of water by the seed causes
production of GA. The GA is transported to the aleurone layer, which
responds by producing enzymes that break down stored food reserves within
the endosperm, which are utilized by the growing seedling. GAs produce
bolting of rosette-forming plants, increasing internodal length. They promote
flowering, cellular division, and in seeds growth after germination.
Gibberellins also reverse the inhibition of shoot growth and dormancy
induced by ABA.
Unit – II
Genome organization in plants: Nuclear genome, chloroplast genome,
mitochondrial genome, CMS, Protein targeting to chloroplast and mitochondria,
Heat shock proteins, seed storage proteins.
________________________________________________________________________
Part – A
1. maintainer line
A maintainer line is similar to a CMS line except that it has viable pollen
grains and normal seed setting. •The maintainer line is used as a pollinator
for maintaining a CMS line. The maintainer is also called the B line. The B line
cannot restore fertility to the F1 generation when it is crossed with a CMS line.
2. CMS
Cytoplasmic male sterility is the total or partial male sterility associated with
plant biology as the result of specific nuclear and mitochondrial
interactions.[1] Male sterility is the failure of plants to produce functional
anthers, pollen, or male gametes
3. HSP
Heat shock proteins (HSP) are a class of functionally related proteins whose
expression is increased when cells are exposed to elevated temperatures or
other stress.[1] This increase in expression is transcriptionally regulated. The
dramatic upregulation of the heat shock proteins is a key part of the heat
shock response and is induced primarily by heat shock factor (HSF).
Part – B
1. Explain chloroplast genome organization in plants.
All angiosperms and land plants have cpDNAs which range in size from 120160 kb; three expceptions are: N. accuminati (171 kb), Duckweed (180 kb),
Geranium (217 kb).
All cpDNA molecules are circular and spinach is used as the basis for all
comparisons. Very few repeat elements are found other than short sequences
of less than 100 bp. The notable exception is a large (10-76 kb) inverted repeat
section, which when present, always contains the rRNA genes. (Legumes
such as pea do not contain this repeat.) For the majority of species, this repeat
region is 22-26 kb in size. Finally,the genetic order of the ribosomal unit is
conserved in all species:
16S - tRNAile - tRNAala - 23S - 5S
Recent research has also described two other features of chloroplast DNA.
First it was shown to that it can exist in in two orientations This implies that
the molecule can undergo an isomerization event. Second is has been shown
that spinach, corn, tomato and pea can all exist as multimers (PNAS 86:4156,
June 1989).
Multimer
Monomer
Dimer
Trimer
Tetramer
Relative Abundance
1
1/3
1/9
1/27
Percent
67.5
22.5
7.5
2.5
Because photosysnthesis is the primary function of the chloroplast it is not
surprising that the chlroplast genome contains genes which encode for
proteins that are involved in that process.
Reaction
Function
Dark Reactions rbcS (nuclear encoded)
rbcL (chloroplast encoded)
Light Reactions apoproteins for PSI andPSII, cytochrome b6, cytochrome f
6 of 9 ATPase subunits, cab, LHC proteins (nuclear encoded)
plastocyanin (nuclear encoded), ferredoxin (nuclear encoded)
Other
19/60 ribosome binding proteins, translation factors
RNA polymerase subunits, tRNA and rRNA genes
Atrazine resistance is apparantley mediated through the psbA gene
sequences of the 32 kd protein which is encoded by cpDNA. DNA sequence
analysis revealed the following amino acid changes that are thought to be
important.
Species
Blue green algae
Chlamydomonas
Solanum nigrum
Amaranthus
AA#
264
264
264
228
Susceptible
Ser (TCG)
Ser (TCT)
Ser (AGT)
Ser (AGT)
Resistant
Ala (GCG)
Ala (GCT)
Gly (GGT)
Gly (GGT)
Evolutionary Changes of cpDNA
The majority of changes are small insertions and deletions of 1-106bp;
significantly, a few length mutations of 50-1200 bp are clusted in "hot spots".
The largest deletion occured in pea where an entire rRNA cluster is lost.
The most common evolutionary change is in gene order. Small changes in
the gene order occur, especially in the algae, but inversions have generated
large scale order changes:
legumes
- about 50 kb inversion brought rbcL closer to psbA
wheat
- about 25 kb inversion brought atpA closer to rbcL
2. Explain mitochondrial genome organization in plants
Mitochondrial DNA (mtDNA) is the DNA located in organelles called
mitochondria, structures within eukaryotic cells that convert the chemical
energy from food into a form that cells can use, adenosine triphosphate
(ATP). Most other DNA present in eukaryotic organisms is found in the cell
nucleus.
Origin
Nuclear and mitochondrial DNA are thought to be of separate evolutionary
origin, with the mtDNA being derived from the circular genomes of the
bacteria that were engulfed by the early ancestors of today's eukaryotic cells.
This theory is called the endosymbiotic theory. Each mitochondrion is
estimated to contain 2-10 mtDNA copies. In the cells of extant organisms, the
vast majority of the proteins present in the mitochondria (numbering
approximately 1500 different types in mammals) are coded for by nuclear
DNA, but the genes for some of them, if not most, are thought to have
originally been of bacterial origin, having since been transferred to the
eukaryotic nucleus during evolution.
Mitochondrial inheritance
In most multicellular organisms, mtDNA is inherited from the mother
(maternally inherited). Mechanisms for this include simple dilution (an egg
contains 100,000 to 1,000,000 mtDNA molecules, whereas a sperm contains
only 100 to 1000), degradation of sperm mtDNA in the fertilized egg, and, at
least in a few organisms, failure of sperm mtDNA to enter the egg. Whatever
the mechanism, this single parent (uniparental) pattern of mtDNA inheritance
is found in most animals, most plants and in fungi as well.
Female inheritance
In sexual reproduction, mitochondria are normally inherited exclusively from
the mother. The mitochondria in mammalian sperm are usually destroyed by
the egg cell after fertilization. Also, most mitochondria are present at the base
of the sperm's tail, which is used for propelling the sperm cells. Sometimes
the tail is lost during fertilization. In 1999 it was reported that paternal sperm
mitochondria (containing mtDNA) are marked with ubiquitin to select them
for later destruction inside the embryo. Some in vitro fertilization techniques,
particularly injecting a sperm into an oocyte, may interfere with this.
The fact that mitochondrial DNA is maternally inherited enables researchers
to trace maternal lineage far back in time. ( Y-chromosomal DNA, paternally
inherited, is used in an analogous way to trace the agnate lineage.) This is
accomplished in humans by sequencing one or more of the hypervariable
control regions (HVR1 or HVR2) of the mitochondrial DNA, as with a
genealogical DNA test. HVR1 consists of about 440 base pairs. These 440 base
pairs are then compared to the control regions of other individuals (either
specific people or subjects in a database) to determine maternal lineage. Most
often, the comparison is made to the revised Cambridge Reference Sequence.
Vilà et al. have published studies tracing the matrilineal descent of domestic
dogs to wolves. The concept of the Mitochondrial Eve is based on the same
type of analysis, attempting to discover the origin of humanity by tracking the
lineage back in time.
Because mtDNA is not highly conserved and has a rapid mutation rate, it is
useful for studying the evolutionary relationships - phylogeny - of organisms.
Biologists can determine and then compare mtDNA sequences among
different species and use the comparisons to build an evolutionary tree for the
species examined.
Because mtDNA is transmitted from mother to child (both male and female),
it can be a useful tool in genealogical research into a person's maternal line.
Male inheritance :
It has been reported that mitochondria can occasionally be inherited from the
father in some species such as mussels. Paternally inherited mitochondria
have additionally been reported in some insects such as fruit flies, honeybees,
and periodical cicadas.
Evidence supports rare instances of male mitochondrial inheritance in some
mammals as well. Specifically, documented occurrences exist for mice, where
the male-inherited mitochondria was subsequently rejected. It has also been
found in sheep, and in cloned cattle. It has been found in a single case in a
human male.
While many of these cases involve cloned embryos or subsequent rejection of
the paternal mitochondria, others document in vivo inheritance and
persistence under lab conditions.
Structure
In humans (and probably in metazoans in general), 100-10,000 separate
copies of mtDNA are usually present per cell (egg and sperm cells are
exceptions). In mammals, each double-stranded circular mtDNA molecule
consists of 15,000-17,000 base pairs. The two strands of mtDNA are
differentiated by their nucleotide content with the guanine rich strand
referred to as the heavy strand, and the cytosine rich strand referred to as the
light strand. The heavy strand encodes 28 genes, and the light strand encodes
9 genes for a total of 37 genes. Of the 37 genes, 13 are for proteins
(polypeptides), 22 are for transfer RNA (tRNA) and two are for the small and
large subunits of ribosomal RNA (rRNA). This pattern is also seen among
most metazoans, although in some cases one or more of the 37 genes is absent
and the mtDNA size range is greater. Even greater variation in mtDNA gene
content and size exists among fungi and plants, although there appears to be
a core subset of genes that are present in all eukaryotes (except for the few
that have no mitochondria at all). Some plant species have enormous
mtDNAs (as many as 2,500,000 base pairs per mtDNA molecule) but,
surprisingly, even those huge mtDNAs contain the same number and kinds of
genes as related plants with much smaller mtDNAs.
3. Explain CMS and its role in hybrid breeding.
Cytoplasmic male sterility is the total or partial male sterility associated with
plant biology as the result of specific nuclear and mitochondrial interactions.
Male sterility is the failure of plants to produce functional anthers, pollen, or
male gametes. As the name indicates, is under extra-nuclear genetic control
(under the control of the mitochondrial or plastid genomes). They show nonMendelian inheritance and are under the regulation of cytoplasmic factors. In
this type, male sterility is inherited maternally. In general there are two types
of cytoplasm: N (normal) and the aberrant S (sterile) cytoplasms. These types
exhibit reciprocal differences.
The first documentation of male sterility came in Joseph Gottlieb Kölreuter
observed anther abortion within species and specific hybrids. Cytoplasmic
male sterility has now been identified in over 150 plant species.[2] It is more
prevalent than female sterility, either because the male sporophyte and
gametophyte are less protected from the environment than the ovule and
embryo sac, or because it results from natural selection on mitochondrial
genes which are maternally inherited and are thus not concerned with pollen
production. Male sterility is easy to detect because a large number of pollen
grains are produced and are easily studied. Male sterility is assayed through
staining techniques (carmine, lactophenol or iodine); while detection of
female sterility is detectable by the absence of seeds. Male sterility has
propagation potential in nature since it can still set seed and is important for
crop breeding, while female sterility does not. Male sterility can be aroused
spontaneously via mutations in nuclear and/or cytoplasmic genes.
Male sterility can be either cytoplasmic or cytoplasmic-genetic. Cytoplasmic
male sterility (CMS) is caused by the extranuclear genome (mitochondria or
chloroplast) and shows maternal inheritance. Manifestation of male sterility in
CMS may be either entirely controlled by cytoplasmic factors or by the
interaction between cytoplasmic and nuclear factors.
Cytoplasmic-genetic male sterility
While CMS is controlled by an extranuclear genome often times nuclear
genes can have the capability to restore fertility. When nuclear restorations of
fertility genes (“Rf”) are available for CMS system in any crop, it is
cytoplasmic-genetic male sterility; the sterility is manifested by the influence
of both nuclear (Mendelian inheritance) and cytoplasmic (maternally
inherited) genes. There are also restorers of fertility (Rf) genes, which are
distinct from genetic male sterility genes. The Rf genes do not have any
expression of their own unless the sterile cytoplasm is present. Rf genes are
required to restore fertility in S cytoplasm which causes sterility. Thus N
cytoplasm is always fertile and S cytoplasm with genotype Rf- produces
fertiles; while S cytoplasm with rfrf produces only male steriles. Another
feature of these systems is that Rf mutations (i.e., mutations to rf or no fertility
restoration) are frequent, so N cytoplasm with Rfrf is best for stable fertility.
Cytoplasmic-genetic male sterility systems are widely exploited in crop
plants for hybrid breeding due to the convenience to control the sterility
expression by manipulating the gene–cytoplasm combinations in any selected
genotype. Incorporation of these systems for male sterility evades the need for
emasculation in cross-pollinated species, thus encouraging cross breeding
producing only hybrid seeds under natural conditions.
Cytoplasmic male sterility in hybrid breeding
Hybrid production requires a female plant in which no viable male
gametes are borne. Emasculation is done to make a plant devoid of pollen so
that it is made female. Another simple way to establish a female line for
hybrid seed production is to identify or create a line that is unable to produce
viable pollen. This male sterile line is therefore unable to self-pollinate, and
seed formation is dependent upon pollen from the male line.
Cytoplasmic male sterility is used in hybrid seed production. In this case,
the sterility is transmitted only through the female and all progeny will be
sterile. This is not a problem for crops such as onions or carrots where the
commodity harvested from the F1 generation is produced during vegetative
growth. These CMS lines must be maintained by repeated crossing to a sister
line (known as the maintainer line) that is genetically identical except that it
possesses normal cytoplasm and is therefore male fertile. In cytoplasmicgenetic male sterility restoration of fertility is done using restorer lines
carrying nuclear restorer genes in crops. The male sterile line is maintained by
crossing with a maintainer line which has the same genome as that of the MS
line but carrying normal fertile cytoplasm.
Cytoplasmic male sterility in hybrid maize breeding
Cytoplasmic male sterility is an important part of hybrid maize
production. The first commercial cytoplasmic male sterile, discovered in
Texas, is known as CMS-T. The use of CMS-T, starting in the 1950s,
eliminated the need for detasseling. In the early 1970’s plants containing
CMS-T genetics were susceptible to southern corn leaf blight and suffered
from widespread loss of yield. Since then CMS types C and S are used
instead.[3] Unfortunately these types are prone to environmentally induced
fertility restoration and must be carefully monitored in the field.
Environmentally induced restoration is when certain environmental stimuli
signal the plant to bypass sterility restrictions and produce pollen anyway.
Environmentally induced restoration differs from genetic restoration in that it
is rooted in external signals rather than genetic DNA.
The systematic sequencing of new plant species in recent years has
uncovered the existence of several novel RF genes and their encoded proteins.
A unified nomenclature for the RF extended protein families across all plant
species, fundamental in the context of comparative functional genomics. This
unified nomenclature accommodates functional RF genes and pseudogenes,
and offers the flexibility needed to incorporate additional RFs as they become
available in future.
4. Write briefly about the role of HSP in plant kingdom.
Heat shock proteins (HSP) are a class of functionally related proteins
whose expression is increased when cells are exposed to elevated
temperatures or other stress. This increase in expression is transcriptionally
regulated. The dramatic upregulation of the heat shock proteins is a key part
of the heat shock response and is induced primarily by heat shock factor
(HSF). HSPs are found in virtually all living organisms, from bacteria to
humans.
Heat-shock proteins are named according to their molecular weight. For
example, Hsp60, Hsp70 and Hsp90 (the most widely-studied HSPs) refer to
families of heat shock proteins on the order of 60, 70 and 90 kilodaltons in
size, respectively. The small 8 kilodalton protein ubiquitin, which marks
proteins for degradation, also has features of a heat shock protein
Discovery
It is known that rapid heat hardening can be elicited by a brief exposure of
cells to sub-lethal high temperature, which in turn provides protection from
subsequent and more severe temperature. In 1962, Ritossa reported that heat
and the metabolic uncoupler dinitrophenol induced a characteristic pattern of
puffing in the chromosomes of Drosophila.[5][6] This discovery eventually
led to the identification of the heat-shock proteins (HSP) or stress proteins
whose expression these puffs represented. Increased synthesis of selected
proteins in Drosophila cells following stresses such as heat shock was first
reported in 1974.
Beginning in the mid-1980s, investigators recognized that many HSPs
function as molecular chaperones and thus play a critical role in protein
folding, intracellular trafficking of proteins, and coping with proteins
denatured by heat and other stresses. Accordingly, the study of stress
proteins has undergone explosive growth.
Functions
Upregulation in stressProduction of high levels of heat shock proteins can
also be triggered by exposure to different kinds of environmental stress
conditions, such as infection, inflammation, exercise, exposure of the cell to
toxins (ethanol, arsenic, trace metals and ultraviolet light, among many
others), starvation, hypoxia (oxygen deprivation), nitrogen deficiency (in
plants), or water deprivation. Consequently, the heat shock proteins are also
referred to as stress proteins and their upregulation is sometimes described
more generally as part of the stress response.
The mechanism by which heat-shock (or other environmental stressors)
activates the heat shock factor has not been determined. However, some
studies suggest that an increase in damaged or abnormal proteins brings
HSPs into action.
Some bacterial heat shock proteins are upregulated via a mechanism
involving RNA thermometers such as the FourU thermometer, ROSE element
and the Hsp90 cis-regulatory element.
Role as chaperone : Heat shock proteins function as intra-cellular
chaperones for other proteins. They play an important role in protein-protein
interactions such as folding and assisting in the establishment of proper
protein conformation (shape) and prevention of unwanted protein
aggregation. By helping to stabilize partially unfolded proteins, HSPs aid in
transporting proteins across membranes within the cell.
Some members of the HSP family are expressed at low to moderate levels
in all organisms because of their essential role in protein maintenance.
Housekeeping : Heat-shock proteins also occur under non-stressful
conditions, simply "monitoring" the cell's proteins. Some examples of their
role as "monitors" are that they carry old proteins to the cell's "recycling bin"
(proteasome) and they help newly synthesised proteins fold properly.
These activities are part of a cell's own repair system, called the "cellular
stress response" or the "heat-shock response".
Cardiovascular : Heat shock proteins appear to serve a significant
cardiovascular role. Hsp90, hsp84, hsp70, hsp27, hsp20, and alpha B crystallin
all have been reported as having roles in the cardiovasculature.
Hsp90 binds both endothelial nitric oxide synthase and soluble guanylate
cyclase which in turn are involved in vascular relaxation.
A downstream kinase of the nitric oxide cell signalling pathway, protein
kinase G, phosphorylates a small heat shock protein, hsp20. Hsp20
phosphorylation correlates well with smooth muscle relaxation and is one
significant phosphoprotein involved in the process. Hsp20 appears significant
in development of the smooth muscle phenotype during development. Hsp20
also serves a significant role in preventing platelet aggregation, cardiac
myocyte function and prevention of apoptosis after ischemic injury, and
skeletal muscle function and muscle insulin response.
Hsp27 is a major phosphoprotein during muscle contraction. Hsp27
functions in smooth muscle migration and appears to serve an integral role.
Immunity: Extracellular and membrane bound heat-shock proteins,
especially Hsp70 are involved in binding antigens and presenting them to the
immune system.
Applications
Cancer vaccine adjuvant : Given their role in antigen presentation, HSPs
are useful as immunologic adjuvants in boosting the response to a vaccine.
Furthermore, some researchers speculate that HSPs may be involved in
binding protein fragments from dead malignant cells and presenting them to
the immune system. Therefore HSPs may be useful for increasing the
effectiveness of cancer vaccines.
Anticancer therapeutics : Intracellular heat shock proteins are highly
expressed in cancerous cells and are essential to the survival of these cell
types. Hence small molecule inhibitors of Hsps, especially Hsp90 show
promise as anticancer agents. The potent Hsp90 inhibitor 17-AAG is currently
in clinical trials for the treatment of several types of cancer.
Agricultural : Researchers are also investigating the role of HSPs in
conferring stress tolerance to hybridized plants, hoping to address drought
and poor soil conditions for farming
Part – C
1. Explain nuclear genome organization in plants.
A genome is the complete set of chromosomes found in each nucleus of a
given species which contains the entire genetic material. The nuclear genome
is the largest in the plant cell, both in terms of picograms of DNA and in
number of genes encoded (complexity). Eukaryotic nuclear genomes can be
distinguished from organelle and prokaryotic genomes by size and
complexity. A typical higher plant genome, for example, contains about 5 x
109 base pairs of DNA per haploid set of chromosomes. This is about 30,000
times as much DNA as in a single chloroplast genome and some 10,000 times
as much as in a moderately sized plant mitochondrial genome. It is also 1000
times more than that of bacterial DNA present in Escherichia coli.
The typical plant genome with 5 x 109 base pairs of DNA would be about
three metres long if the entire DNA were to be laid out in a straight line.
Chromosomes are composed of two types of large organic molecules
(macromolecules) called proteins and nucleic acids.
Nucleic acids are of two types: deoxyribonucleic acids. (DNA) and ribonucleic
acids (RNA).For many years scientists disputed which of these three
macromolecules (proteins, RNA and DNA) carries genetic information.
During the 1940s and early 1950s several elegant experiments were carried
out which clearly established that genetic information resides in the nucleic
acids and not in the proteins. More specifically, these experiments showed
that genetic information resides in DNA. (In a few simple viruses, however,
RNA carries the genetic information; these particular viruses contain no
DNA.) Nuclear DNA is packaged into chromosomes along with histones and
nonhistone proteins, all of which play important roles in gene expression.
These various components are held together to form chromatin by both
hydrophobic and electrostatic forces. While the DNA encodes the genetic
information, the proteins are involved in controlling packaging of DNA and
in regulating its availability for transcription. Although the structure of
eukaryotic chromatin has been fairly well characterized, the roles of various
individual proteins in chromatin structure and gene regulation have yet to be
elucidated.
Genetically, a plant genome is the most complex one found in living systems.
It comprises three interacting genome. Aside from the nuclear genome,
complete genetic systems are located in the plastids and the mitochondria.
These organelles are semiautonomous bodies; they have their own
organizational and functional properties but do not synthesize all their own
proteins. The nuclear genome plays an important role in organelle biogenesis.
Techniques in molecular cloning and deoxyribonucleic acid (DNA)
sequencing have provided the tools for studying the structure of genes at the
nucleotide level. Hence our knowledge of the structure, organization and
expression of a plant genome has come largely through, the use of
recombinant DNA techniques. This technology allows isolation and
characterization of specific pieces of DNA by cloning the DNA sequences into
bacterial cells in which they can be replicated and large quantities obtained
for analysis.
Some organisms have multiple copies of chromosomes, diploid, triploid,
tetraploid and so on. In classical genetics, in a sexually reproducing organism
(typically eukarya) the gamete has half the number of chromosomes of the
somatic cell and the genome is a full set of chromosomes in a gamete. In
haploid organisms, including cells of bacteria, archaea, and in organelles
including mitochondria and chloroplasts, or viruses, that similarly contain
genes, the single or set of circular and/or linear chains of DNA (or RNA for
some viruses), likewise constitute the genome. The term genome can be
applied specifically to mean that stored on a complete set of nuclear DNA
(i.e., the "nuclear genome") but can also be applied to that stored within
organelles that contain their own DNA, as with the "mitochondrial genome"
or the "chloroplast genome". Additionally, the genome can comprise
nonchromosomal genetic elements such as viruses, plasmids, and
transposable elements
When people say that the genome of a sexually reproducing species has been
"sequenced", typically they are referring to a determination of the sequences
of one set of autosomes and one of each type of sex chromosome, which
together represent both of the possible sexes. Even in species that exist in only
one sex, what is described as "a genome sequence" may be a composite read
from the chromosomes of various individuals. In general use, the phrase
"genetic makeup" is sometimes used conversationally to mean the genome of
a particular individual or organism. The study of the global properties of
genomes of related organisms is usually referred to as genomics, which
distinguishes it from genetics which generally studies the properties of single
genes or groups of genes.
Both the number of base pairs and the number of genes vary widely from one
species to another, and there is only a rough correlation between the two (an
observation known as the C-value paradox). At present, the highest known
number of genes is around 60,000, for the protozoan causing trichomoniasis
(see List of sequenced eukaryotic genomes), almost three times as many as in
the human genome.
Comparison of different genome sizes
Organism
Arabidopsis thaliana
Genlisea margaretae
Genome size
(base pairs)
Note
First plant genome sequenced,
December 2000.
Smallest recorded flowering plant
63,400,000
genome, 2006.
157,000,000
Fritillaria assyrica
Populus trichocarpa
130,000,000,000
480,000,000
First tree genome sequenced,
September 2006
Pieris japonica
150,000,000,000 Largest plant genome known
(Japanese-native, pale-petal)
Unit – III
Molecular markers aided breeding, Rflp Maps, RAPD markers, STS,
microsatellites SCAR (sequence characterized amplified Regions), SSCT (Single
strand conformational Polumorphism), AFLP, molecular marker assisted
selection. Arid and semi – arid technology, green house and green – home
technology
________________________________________________________________________
Part – A
1. RAPD
RAPD (pronounced "rapid") stands for Random Amplification of
Polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that
are amplified are random. The scientist performing RAPD creates several
arbitrary, short primers (8-12 nucleotides), then proceeds with the PCR using
a large template of genomic DNA, hoping that fragments will amplify. By
resolving the resulting patterns, a semi-unique profile can be gleaned from a
RAPD reaction
2. RFLP
In molecular biology, restriction fragment length polymorphism, or RFLP
(commonly pronounced “rif-lip”), is a technique that exploits variations in
homologous DNA sequences. It refers to a difference between samples of
homologous DNA molecules that come from differing locations of restriction
enzyme sites, and to a related laboratory technique by which these segments
can be illustrated. In RFLP analysis, the DNA sample is broken into pieces
(digested) by restriction enzymes and the resulting restriction fragments are
separated according to their lengths by gel electrophoresis. Although now
largely obsolete due to the rise of inexpensive DNA sequencing technologies,
RFLP analysis was the first DNA profiling technique inexpensive enough to
see widespread application. In addition to genetic fingerprinting, RFLP was
an important tool in genome mapping, localization of genes for genetic
disorders, determination of risk for disease, and paternity testing.
3. STS
A sequence-tagged site (or STS) is a short (200 to 500 base pair) DNA
sequence that has a single occurrence in the genome and whose location and
base sequence are known. STSs can be easily detected by the polymerase
chain reaction (PCR) using specific primers. For this reason they are useful for
constructing genetic and physical maps from sequence data reported from
many different laboratories. They serve as landmarks on the developing
physical map of a genome.
4. Microsatellite
Microsatellites, also known as Simple Sequence Repeats (SSRs) or short
tandem repeats (STRs), are repeating sequences of 1-6 base pairs of DNA.
Microsatellites are typically neutral and co-dominant. They are used as
molecular markers in genetics, for kinship, population and other studies.
They can also be used to study gene duplication or deletion.
5. Probe
In molecular biology, a hybridization probe is a fragment of DNA or RNA of
variable length (usually 100-1000 bases long), which is used in DNA or RNA
samples to detect the presence of nucleotide sequences (the DNA target) that
are complementary to the sequence in the probe. The probe thereby
hybridizes to single-stranded nucleic acid (DNA or RNA) whose base
sequence allows probe-target base pairing due to complementarity between
the probe and target. The labeled probe is first denatured (by heating or
under alkaline conditions such as exposure to sodium hydroxide) into single
stranded DNA (ssDNA) and then hybridized to the target ssDNA (Southern
blotting) or RNA (northern blotting) immobilized on a membrane or in situ.
6. SCAR
Sequence characterised amplified region (SCAR). A locus representing a
single RAPD fragment which has been sequenced. Primers specific to the
locus can be designed and used in PCR amplification.
7. Annealing
Nucleic acid thermodynamics is the study of the thermodynamics of nucleic
acid molecules, or how temperature affects nucleic acid structure. For
multiple copies of DNA molecules, the melting temperature (Tm) is defined
as the temperature at which half of the DNA strands are in the double-helical
state and half are in the random coil states.[1] The melting temperature
depends on both the length of the molecule, and the specific nucleotide
sequence composition of that molecule
8. Southern hybridization
A Southern blot is a method routinely used in molecular biology for detection
of a specific DNA sequence in DNA samples. Southern blotting combines
transfer of electrophoresis-separated DNA fragments to a filter membrane
and subsequent fragment detection by probe hybridization. The method is
named after its inventor, the British biologist Edwin Southern. Other blotting
methods (i.e., Western blot, Northern blot, Eastern blot, Southwestern blot)
that employ similar principles, but using RNA or protein, have later been
named in reference to Edwin Southern's name.
9. AFLP
Amplified Fragment Length Polymorphism PCR (or AFLP-PCR or just AFLP)
is a PCR-based tool used in genetics research, DNA fingerprinting, and in the
practice of genetic engineering. Developed in the early 1990s by Keygene,
AFLP uses restriction enzymes to digest genomic DNA, followed by ligation
of adaptors to the sticky ends of the restriction fragments. A subset of the
restriction fragments is then selected to be amplified. This selection is
achieved by using primers complementary to the adaptor sequence, the
restriction site sequence and a few nucleotides inside the restriction site
fragments (as described in detail below). The amplified fragments are
visualized
on
denaturing
polyacrylamide
gels
either
through
autoradiography or fluorescence methodologies.
Part – B
1. Explain the methodology of Restriction Fragment Length Polymorphism
The basic technique for detecting RFLPs involves fragmenting a sample of
DNA by a restriction enzyme, which can recognize and cut DNA wherever a
specific short sequence occurs, in a process known as a restriction digest. The
resulting DNA fragments are then separated by length through a process
known as agarose gel electrophoresis, and transferred to a membrane via the
Southern blot procedure. Hybridization of the membrane to a labeled DNA
probe then determines the length of the fragments which are complementary
to the probe. A RFLP occurs when the length of a detected fragment varies
between individuals. Each fragment length is considered an allele, and can be
used in genetic analysis.
RFLP analysis may be subdivided into single- (SLP) and multi-locus probe
(MLP) paradigms. Usually, the SLP method is preferred over MLP because it
is more sensitive, easier to interpret and capable of analyzing mixed-DNA
samples.[citation needed] Moreover data can be generated even when the
DNA is degraded (e.g. when it is found in bone remains.)
Schematic for RFLP by cleavage site loss.
Analysis and inheritance of allelic RFLP fragments (NIH).
Schematic for RFLP by VNTR length variation.
Examples
There are two common mechanisms by which the size of a particular
restriction fragment can vary. In the first schematic, a small segment of the
genome is being detected by a DNA probe (thicker line). In allele "A", the
genome is cleaved by a restriction enzyme at three nearby sites (triangles), but
only the rightmost fragment will be detected by the probe. In allele "a",
restriction site 2 has been lost by a mutation, so the probe now detects the
larger fused fragment running from sites 1 to 3. The second diagram shows
how this fragment size variation would look on a Southern blot, and how
each allele (two per individual) might be inherited in members of a family.
In the third schematic, the probe and restriction enzyme are chosen to detect a
region of the genome that includes a variable VNTR segment (boxes). In allele
"c" there are five repeats in the VNTR, and the probe detects a longer
fragment between the two restriction sites. In allele "d" there are only two
repeats in the VNTR, so the probe detects a shorter fragment between the
same two restriction sites. Other genetic processes, such as insertions,
deletions, translocations, and inversions, can also lead to RFLPs.
2. Define SSCP with suitable diagram
Single-strand conformation polymorphism (SSCP), or single-strand chain
polymorphism, is defined as conformational difference of single-stranded
nucleotide sequences of identical length as induced by differences in the
sequences under certain experimental conditions. This property allows to
distinguish the sequences by means of gel electrophoresis, which separates
the different conformations.
Physical background
A single nucleotide change in a particular sequence, as seen in a doublestranded DNA, cannot be distinguished by electrophoresis, because the
physical properties of the double strands are almost identical for both alleles.
After denaturation, single-stranded DNA undergoes a 3-dimensional folding
and may assume a unique conformational state based on its DNA sequence.
The difference in shape between two single-stranded DNA strands with
different sequences can cause them to migrate differently on an
electrophoresis gel, even though the number of nucleotides is the same, which
is, in fact, an application of SSCP.
Applications in molecular biology
SSCP used to be a way to discover new DNA polymorphisms apart from
DNA sequencing, but is now being supplanted by sequencing techniques on
account of efficiency and accuracy. These days, SSCP is most applicable as a
diagnostic tool in molecular biology. It can be used in genotyping to detect
homozygous individuals of different allelic states, as well as heterozygous
individuals that should each demonstrate distinct patterns in an
electrophoresis experiment.
SSCP is also widely used in virology to detect variations in different strains of
a virus, the idea being that a particular virus particle present in both strains
will have undergone changes due to mutation, and that these changes will
cause the two particles to assume different conformations and, thus, be
differentiable on an SSCP gel
3. Describe arid and semi arid technology
Salinity and drought still remain as major abiotic stresses that pose a threat to
agricultural production in many parts of the world (Altmann, 1999). Arable
lands are lost annually due to desertification and salination, toxication, and
mismanagement of the natural resource base for agriculture (Evans, 1998).
Water is becoming a scarce resource that requires careful economic and
environmental management (Barghouti, 1999a). Expansion of irrigation does
not seem feasible in many countries in Asia, the Middle
5
East, and North Africa, where most of the available and easily accessible
water resources have been already developed. Furthermore, public irrigation
systems need substantial investments for rehabilitation, modernisation,
operation and maintenance. Likewise, irrigated soils are affected by salting,
although the yield loss estimates vary widely. Desertification may be
aggravated by both over exploitation by native populations and regional
climatic changes. Hence, breeding programs must rank high the development
of crops with tolerance to both drought and salinity stress. The genetically
complex control of these stresses in the plant genome may be facilitated
through the manipulation of specific genes governing the component
characteristics needed to achieve tolerance to salt or drought in plant crops.
Genetic enhancement for crops of the semi-arid tropics
The genetic resources available in the crops grown in the semi-arid tropics has
provided the source of genes for their betterment. A few genes from wild
species have been also transferred to the cultivated gene pool by interspecific
hybridisation followed by embryo culture (Mallikarjuna, 1999), which
overcomes the cross incompatibility that often occurs after pollination in the
so-called "wide-crosses" between species. In some interspecific incompatible
crosses, fertilization occurs but the embryo aborts a few days later. Hybrid
plants may be obtained by preventing pod abscission and saving aborting
hybrid embryos with "rescue" techniques.
Biotechnology and conventional cross-breeding
The new tools of molecular genetics enable researchers to understand better
and faster the full potential of the genetic resources endowment of crops, to
preserve this genetic heritage, and to develop improved plant materials. The
identification, isolation and cloning of new genes controlling specific
characteristics will also facilitate the development of a more stable, diversified
germplasm with improved resistance to diseases and pests, stress tolerance,
better food quality, and higher productivity. For example genes allowing a
reduced crop cycle or modified plant structure will provide pathways for new
cropping systems. Nonetheless, conventional cross-breeding will be still
required for an appropriate testing and further transfer of these genes to the
advanced breeding pools of the crop. Furthermore, seed delivery systems of
improved genotypes should be in place to promote the utilisation of new
cultivars, which will enhance and stabilise the agricultural production, farm
income, and farm-family welfare. In brief, the new tools of biotechnology
alone cannot provide the answer to genetic improvement, but they are
facilitating and accelerating the pace in the development of new cultivars.
The role of biotechnology in crop improvement
The management of pests and diseases affecting crops can be achieved by
exploiting host plant resistance, biological control or improved crop
husbandry. Genetic enhancement appears as one of the best options because it
offers an easy, cheap and sustainable technology to farmers, i.e., improved
cultivars. However, there are some pests and diseases for which resistance
genes are lacking in the primary gene pool of the host plant for an easy gene
transfer through conventional cross-breeding. Hence, genetic transformation
provides a complementary means to crop breeding, especially for traits that
are rare or not available in the investigated gene pool. Some transformation
systems, especially for legumes have been developed in recent years. These
protocols are now being adapted or improved for some of the legume crops of
the semi-arid tropics (Sharma and Ortiz, 2000).
4. Describe RAPD technique.
RAPD (pronounced "rapid") stands for Random Amplification of
Polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that
are amplified are random. The scientist performing RAPD creates several
arbitrary, short primers (8-12 nucleotides), then proceeds with the PCR using
a large template of genomic DNA, hoping that fragments will amplify. By
resolving the resulting patterns, a semi-unique profile can be gleaned from a
RAPD reaction.
No knowledge of the DNA sequence for the targeted gene is required, as
the primers will bind somewhere in the sequence, but it is not certain exactly
where. This makes the method popular for comparing the DNA of biological
systems that have not had the attention of the scientific community, or in a
system in which relatively few DNA sequences are compared (it is not
suitable for forming a DNA databank). Because it relies on a large, intact
DNA template sequence, it has some limitations in the use of degraded DNA
samples. Its resolving power is much lower than targeted, species specific
DNA comparison methods, such as short tandem repeats. In recent years,
RAPD has been used to characterize, and trace, the phylogeny of diverse
plant and animal species.
RAPD markers are decamer (10 nucleotide length) DNA fragments from
PCR amplification of random segments of genomic DNA with single primer
of arbitrary nucleotide sequence and which are able to differentiate between
genetically distinct individuals, although not necessarily in a reproducible
way. It is used to analyse the genetic diversity of an individual by using
random primers. Due to problems in experiment reproducibility, many
scientific journals do not accept experiments merely based on RAPDs
anymore.
Unlike traditional PCR analysis, RAPD does not require any specific
knowledge of the DNA sequence of the target organism: the identical 10-mer
primers will or will not amplify a segment of DNA, depending on positions
that are complementary to the primers' sequence. For example, no fragment is
produced if primers annealed too far apart or 3' ends of the primers are not
facing each other. Therefore, if a mutation has occurred in the template DNA
at the site that was previously complementary to the primer, a PCR product
will not be produced, resulting in a different pattern of amplified DNA
segments on the gel.
Example
RAPD is an inexpensive yet powerful typing method for many bacterial
species. RAPD profiles, example Silver-stained polyacrylamide gel showing
three distinct RAPD profiles generated by primer OPE15 for Haemophilus
ducreyi isolates from Tanzania, Senegal, Thailand, Europe, and North
America.
Selecting the right sequence for the primer is very important because different
sequences will produce different band patterns and possibly allow for a more
specific recognition of individual strains.
Limitations of RAPD
Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish
whether a DNA segment is amplified from a locus that is heterozygous (1
copy) or homozygous (2 copies). Co-dominant RAPD markers, observed as
different-sized DNA segments amplified from the same locus, are detected
only rarely.
PCR is an enzymatic reaction, therefore the quality and concentration of
template DNA, concentrations of PCR components, and the PCR cycling
conditions may greatly influence the outcome. Thus, the RAPD technique is
notoriously laboratory dependent and needs carefully developed laboratory
protocols to be reproducible.
Mismatches between the primer and the template may result in the total
absence of PCR product as well as in a merely decreased amount of the
product. Thus, the RAPD results can be difficult to interpret.
Developing Locus-specific, Co-Dominant Markers from RAPDs
The polymorphic RAPD marker band is isolated from the gel.
It is amplified in the PCR reaction.
The PCR product is cloned and sequenced.
New longer and specific primers are designed for the DNA sequence,
which is called the Sequenced Characterized Amplified Region Marker
(SCAR).
5. Describe microsatellite technique
Microsatellites, also known as Simple Sequence Repeats (SSRs) or short
tandem repeats (STRs), are repeating sequences of 1-6 base pairs of DNA.
Microsatellites are typically neutral and co-dominant. They are used as
molecular markers in genetics, for kinship, population and other studies.
They can also be used to study gene duplication or deletion.
One common example of a microsatellite is a (CA)n repeat, where n varies
between alleles. These markers often present high levels of inter- and intraspecific polymorphism, particularly when the number of repetitions is 10 or
greater. The repeated sequence is often simple, consisting of two, three or four
nucleotides (di-, tri-, and tetranucleotide repeats respectively), and can be
repeated 3 to 100 times, with the longer loci generally having more alleles due
to the greater potential for slippage (see below). CA nucleotide repeats are
very frequent in human and other genomes, and are present every few
thousand base pairs. As there are often many alleles present at a microsatellite
locus, genotypes within pedigrees are often fully informative, in that the
progenitor of a particular allele can often be identified. In this way,
microsatellites are ideal for determining paternity, population genetic studies
and recombination mapping. It is also the only molecular marker to provide
clues about which alleles are more closely related. Microsatellites are also
predictors of SNP density and human–chimpanzee divergence differing from
the genome-wide average in regions extending thousands of nucleotides.
The variability of microsatellites is due to a higher rate of mutation compared
to other neutral regions of DNA. These high rates of mutation can be
explained most frequently by slipped strand mispairing (slippage) during
DNA replication on a single DNA strand. Mutation may also occur during
recombination during meiosis. Some errors in slippage are rectified by
proofreading mechanisms within the nucleus, but some mutations can escape
repair. The size of the repeat unit, the number of repeats and the presence of
variant repeats are all factors, as well as the frequency of transcription in the
area of the DNA repeat. Interruption of microsatellites, perhaps due to
mutation, can result in reduced polymorphism. However, this same
mechanism can occasionally lead to incorrect amplification of microsatellites;
if slippage occurs early on during PCR, microsatellites of incorrect lengths can
be amplified.
Amplification
Microsatellites can be amplified for identification by the polymerase chain
reaction (PCR) process, using the unique sequences of flanking regions as
primers. DNA is repeatedly denatured at a high temperature to separate the
double strand, then cooled to allow annealing of primers and the extension of
nucleotide sequences through the microsatellite. This process results in
production of enough DNA to be visible on agarose or polyacrylamide gels;
only small amounts of DNA are needed for amplification because in this way
thermocycling creates an exponential increase in the replicated segment.[7]
With the abundance of PCR technology, primers that flank microsatellite loci
are simple and quick to use, but the development of correctly functioning
primers is often a tedious and costly process.
A number of DNA samples from specimens of Littorina plena amplified using polymerase
chain reaction with primers targeting a variable simple sequence repeat (SSR, a.k.a.
microsatellite) locus. Samples have been run on a 5% polyacrylamide gel and visualized
using silver staining
Creation of microsatellite primers
If searching for microsatellite markers in specific regions of a genome, for
example within a particular exon of a gene, primers can be designed
manually. This involves searching the genomic DNA sequence for
microsatellite repeats, which can be done by eye or by using automated tools
such as repeat masker. Once the potentially useful microsatellites are
determined (removing non-useful ones such as those with random inserts
within the repeat region), the flanking sequences can be used to design
oligonucleotide primers which will amplify the specific microsatellite repeat
in a PCR reaction.
Random microsatellite primers can be developed by cloning random
segments of DNA from the focal species. These random segments are inserted
into a plasmid or bacteriophage vector, which is in turn implanted into
Escherichia coli bacteria. Colonies are then developed, and screened with
fluorescently–labelled oligonucleotide sequences that will hybridize to a
microsatellite repeat, if present on the DNA segment. If positive clones can be
obtained from this procedure, the DNA is sequenced and PCR primers are
chosen from sequences flanking such regions to determine a specific locus.
This process involves significant trial and error on the part of researchers, as
microsatellite repeat sequences must be predicted and primers that are
randomly isolated may not display significant polymorphism.[2][8]
Microsatellite loci are widely distributed throughout the genome and can be
isolated from semi-degraded DNA of older specimens, as all that is needed is
a suitable substrate for amplification through PCR.
More recent techniques involve using oligonucleotide sequences consisting of
repeats complementary to repeats in the microsatellite to "enrich" the DNA
extracted. The oligonucleotide probe hybridizes with the repeat in the
microsatellite, and the probe/microsatellite complex is then pulled out of
solution. The enriched DNA is then cloned as normal, but the proportion of
successes will now be much higher, drastically reducing the time required to
develop the regions for use. However, which probes to use can be a trial and
error process in itself.
Part – C
1. Write a detailed essay on AFLP technique with its advantages and disadvantages
Amplified Fragment Length Polymorphism PCR (or AFLP-PCR or just AFLP) is a
PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of
genetic engineering. Developed in the early 1990s by Keygene[1], AFLP uses
restriction enzymes to digest genomic DNA, followed by ligation of adaptors to the
sticky ends of the restriction fragments. A subset of the restriction fragments is then
selected to be amplified. This selection is achieved by using primers complementary
to the adaptor sequence, the restriction site sequence and a few nucleotides inside the
restriction site fragments (as described in detail below). The amplified fragments are
visualized on denaturing polyacrylamide gels either through autoradiography or
fluorescence methodologies.
AFLP-PCR is a highly sensitive method for detecting polymorphisms in DNA. The
technique was originally described by Vos and Zabeau in 1993[2][3]. In detail, the
procedure of this technique is divided into three steps: [1]
1. Digestion of total cellular DNA with one or more restriction enzymes and ligation
of restriction half-site specific adaptors to all restriction fragments.
2. Selective amplification of some of these fragments with two PCR primers that have
corresponding adaptor and restriction site specific sequences.
3. Electrophoretic separation of amplicons on a gel matrix, followed by visualisation
of the band pattern.
A variation on AFLP is cDNA-AFLP, which is used to quantify differences in gene
expression levels.
Another variation on AFLP is TE Display, used to detect transposable element
mobility.
Applications
The AFLP technology has the capability to detect various polymorphisms in different
genomic regions simultaneously. It is also highly sensitive and reproducible. As a
result, AFLP has become widely used for the identification of genetic variation in
strains or closely related species of plants, fungi, animals, and bacteria. The AFLP
technology has been used in criminal and paternity tests, also to determine slight
differences within populations, and in linkage studies to generate maps for
quantitative trait locus (QTL) analysis.
There are many advantages to AFLP when compared to other marker technologies
including randomly amplified polymorphic DNA (RAPD), restriction fragment
length polymorphism (RFLP), and microsatellites. AFLP not only has higher
reproducibility, resolution, and sensitivity at the whole genome level compared to
other techniques, but it also has the capability to amplify between 50 and 100
fragments at one time. In addition, no prior sequence information is needed for
amplification (Meudt & Clarke 2007). As a result, AFLP has become extremely
beneficial in the study of taxa including bacteria, fungi, and plants, where much is
still unknown about the genomic makeup of various organisms.
The AFLP technology is covered by patents and patent applications of Keygene N.V.
AFLP is a registered trademark of Keygene N.V.
2. Describe the principles of PCR
The polymerase chain reaction (PCR) is a scientific technique in molecular
biology to amplify a single or a few copies of a piece of DNA across several
orders of magnitude, generating thousands to millions of copies of a
particular DNA sequence.
Developed in 1983 by Kary Mullis, PCR is now a common and often
indispensable technique used in medical and biological research labs for a
variety of applications. These include DNA cloning for sequencing, DNAbased phylogeny, or functional analysis of genes; the diagnosis of hereditary
diseases; the identification of genetic fingerprints (used in forensic sciences
and paternity testing); and the detection and diagnosis of infectious diseases.
In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael
Smith for his work on PCR.
The method relies on thermal cycling, consisting of cycles of repeated heating
and cooling of the reaction for DNA melting and enzymatic replication of the
DNA. Primers (short DNA fragments) containing sequences complementary
to the target region along with a DNA polymerase (after which the method is
named) are key components to enable selective and repeated amplification.
As PCR progresses, the DNA generated is itself used as a template for
replication, setting in motion a chain reaction in which the DNA template is
exponentially amplified. PCR can be extensively modified to perform a wide
array of genetic manipulations.
Almost all PCR applications employ a heat-stable DNA polymerase, such as
Taq polymerase, an enzyme originally isolated from the bacterium Thermus
aquaticus. This DNA polymerase enzymatically assembles a new DNA strand
from DNA building-blocks, the nucleotides, by using single-stranded DNA as
a template and DNA oligonucleotides (also called DNA primers), which are
required for initiation of DNA synthesis. The vast majority of PCR methods
use thermal cycling, i.e., alternately heating and cooling the PCR sample to a
defined series of temperature steps. These thermal cycling steps are necessary
first to physically separate the two strands in a DNA double helix at a high
temperature in a process called DNA melting. At a lower temperature, each
strand is then used as the template in DNA synthesis by the DNA polymerase
to selectively amplify the target DNA. The selectivity of PCR results from the
use of primers that are complementary to the DNA region targeted for
amplification under specific thermal cycling conditions.
PCR principles
PCR is used to amplify a specific region of a DNA strand (the DNA
target). Most PCR methods typically amplify DNA fragments of up to ~10
kilo base pairs (kb), although some techniques allow for amplification of
fragments up to 40 kb in size.
A basic PCR set up requires several components and reagents. These
components include:
1. DNA template that contains the DNA region (target) to be amplified.
2. Two primers that are complementary to the 3' (three prime) ends of each
of the sense and anti-sense strand of the DNA target.
3. Taq polymerase or another DNA polymerase with a temperature optimum
at around 70 °C.
4. Deoxynucleoside triphosphates (dNTPs;
nucleotides containing
triphosphate groups), the building-blocks from which the DNA
polymerase synthesizes a new DNA strand.
5. Buffer solution, providing a suitable chemical environment for optimum
activity and stability of the DNA polymerase.
6. Divalent cations, magnesium or manganese ions; generally Mg2+ is used,
but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher
Mn2+ concentration increases the error rate during DNA synthesis[7]
7. Monovalent cation potassium ions.
The PCR is commonly carried out in a reaction volume of 10–200 μl in
small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal
cycler heats and cools the reaction tubes to achieve the temperatures required
at each step of the reaction (see below). Many modern thermal cyclers make
use of the Peltier effect, which permits both heating and cooling of the block
holding the PCR tubes simply by reversing the electric current. Thin-walled
reaction tubes permit favorable thermal conductivity to allow for rapid
thermal equilibration. Most thermal cyclers have heated lids to prevent
condensation at the top of the reaction tube. Older thermocyclers lacking a
heated lid require a layer of oil on top of the reaction mixture or a ball of wax
inside the tube.
PCR procedure
Typically, PCR consists of a series of 20-40 repeated temperature changes,
called cycles, with each cycle commonly consisting of 2-3 discrete temperature
steps, usually three (Fig. 2). The cycling is often preceded by a single
temperature step (called hold) at a high temperature (>90°C), and followed by
one hold at the end for final product extension or brief storage. The
temperatures used and the length of time they are applied in each cycle
depend on a variety of parameters. These include the enzyme used for DNA
synthesis, the concentration of divalent ions and dNTPs in the reaction, and
the melting temperature (Tm) of the primers.
Initialization step: This step consists of heating the reaction to a temperature of
94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is
held for 1–9 minutes. It is only required for DNA polymerases that require
heat activation by hot-start PCR.[9]
Denaturation step: This step is the first regular cycling event and consists of
heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of
the DNA template by disrupting the hydrogen bonds between
complementary bases, yielding single-stranded DNA molecules.
Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40
seconds allowing annealing of the primers to the single-stranded DNA
template. Typically the annealing temperature is about 3-5 degrees Celsius
below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are
only formed when the primer sequence very closely matches the template
sequence. The polymerase binds to the primer-template hybrid and begins
DNA synthesis.
Extension/elongation step: The temperature at this step depends on the DNA
polymerase used; Taq polymerase has its optimum activity temperature at 75–
80 °C, and commonly a temperature of 72 °C is used with this enzyme. At this
step the DNA polymerase synthesizes a new DNA strand complementary to
the DNA template strand by adding dNTPs that are complementary to the
template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs
with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand.
The extension time depends both on the DNA polymerase used and on the
length of the DNA fragment to be amplified. As a rule-of-thumb, at its
optimum temperature, the DNA polymerase will polymerize a thousand
bases per minute. Under optimum conditions, i.e., if there are no limitations
due to limiting substrates or reagents, at each extension step, the amount of
DNA target is doubled, leading to exponential (geometric) amplification of
the specific DNA fragment.
Final elongation: This single step is occasionally performed at a temperature of
70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any
remaining single-stranded DNA is fully extended.
Final hold: This step at 4–15 °C for an indefinite time may be employed for
short-term storage of the reaction.
Ethidium
bromide-stained
PCR
products after gel electrophoresis. Two
sets of primers were used to amplify a
target sequence from three different
tissue samples. No amplification is
present in sample #1; DNA bands in
sample #2 and #3 indicate successful
amplification of the target sequence.
The gel also shows a positive control,
and a DNA ladder containing DNA
fragments of defined length for sizing
the bands in the experimental PCRs.
Schematic drawing of the PCR cycle.
(1) Denaturing at 94–96 °C. (2)
Annealing at ~65 °C (3) Elongation at
72 °C. Four cycles are shown here. The
blue lines represent the DNA template
to which primers (red arrows) anneal
that are extended by the DNA
polymerase (light green circles), to give
shorter DNA products (green lines),
which themselves are used as
templates as PCR progresses.
______________________________
Electrophoresis
of
PCR-amplified
DNA fragments. (1) Father. (2) Child.
(3) Mother. The child has inherited
some, but not all of the fingerprint of
each of its parents, giving it a new,
unique fingerprint
To check whether the PCR generated the anticipated DNA fragment (also
sometimes referred to as the amplimer or amplicon), agarose gel
electrophoresis is employed for size separation of the PCR products. The
size(s) of PCR products is determined by comparison with a DNA ladder (a
molecular weight marker), which contains DNA fragments of known size, run
on the gel alongside the PCR products (see Fig. 3).
Unit – IV
Plant transformation technology: features of Ti and Ri plasmids, uses of Ti and Ri
as vectors, binary vectors, use of 35S and other promoters, genetic markers, use of
reporter genes, methods of nuclear transformation, virtual vectors and their
applications. Transgenic biology; Role of virulence genes. Gene transfer methods
in plants: multiple gene transfers, vector – less or direct DNA transfer
________________________________________________________________________
Part – A
1. Selection marker
A selectable marker is a gene introduced into a cell, especially a bacterium or
to cells in culture, that confers a trait suitable for artificial selection. They are a
type of reporter gene used in laboratory microbiology, molecular biology, and
genetic engineering to indicate the success of a transfection or other
procedure meant to introduce foreign DNA into a cell. Selectable markers are
often antibiotic resistance genes; bacteria that have been subjected to a
procedure to introduce foreign DNA are grown on a medium containing an
antibiotic, and those bacterial colonies that can grow have successfully taken
up and expressed the introduced genetic material.
2. GUS
The GUS reporter system (GUS: beta-glucuronidase) is a reporter gene
system, particularly useful in plant molecular biology. [1] Several kinds of
GUS reporter gene assay are actually available, depending on the substrate
used. The term GUS staining refers to the most common of these, a
histochemical technique
3. Reporter gene
In molecular biology, a reporter gene (often simply reporter) is a gene that
researchers attach to a regulatory sequence of another gene of interest in cell
culture, animals or plants. Certain genes are chosen as reporters because the
characteristics they confer on organisms expressing them are easily identified
and measured, or because they are selectable markers. Reporter genes are
often used as an indication of whether a certain gene has been taken up by or
expressed in the cell or organism population.
4. Binary vector
Used to generate transgenic plants, binary vectors are cloning vectors
which are able to replicate in both E. coli and Agrobacterium tumefaciens,
which are bacteria that are often used in biotechnology.
Systems in which T-DNA and vir genes are located on separate replicons
are called T-DNA binary systems. T-DNA is located on the binary vector (the
non-T-DNA region of this vector containing origin(s) of replication that could
function both in E. coli and in Agrobacterium tumefaciens, and antibioticresistance genes used to select for the presence of the binary vector in bacteria,
became known as vector backbone sequences). The replicon containing the vir
genes became known as the vir helper. Strains harboring this replicon and a
T-DNA are considered disarmed if they do not contain oncogenes that could
be transferred to a plant.
There are several binary vector systems that differ mainly in the plasmid
region that facilitates replication in Agrobacterium. Commonly used binary
vectors include: pBIN19, pPVP, pGreen.
5. Acetosyringone
Acetosyringone is a volatile chemical compound related to acetophenone. It is
a natural product that can be found in Posidonia oceanica. The compound is
also produced by the male leaffooted bug (Leptoglossus phyllopus) and used
in its communication system.
This substance is involved in plant-pathogen recognition. The VirA gene of Ti
plasmid, part of the genetic equipment that Agrobacterium tumefaciens and
Agrobacterium rhizogenes soil bacteria use to infect plants, codes for a
receptor to acetosyringone leaking from plant wounds. This compound
allows higher transformation efficiency in plants
6. Crown gall tumour
Protein involved in crown gall tumor formation, a plant tumor caused by the
bacterium Agrobacterium tumefaciens
7. Genetic colonization
Natural introduction of genetic material into the deoxyribonucleic acid of a
host cell; for example, transmission of a tumor-inducing plasmid into a plant
cell by the bacterium Agrobacterium tumefaciens.
8. Liposome
Liposomes are artificially prepared vesicles made of lipid bilayer. Liposomes
can be filled with drugs, and used to deliver drugs for cancer and other
diseases.[2] Liposomes can be prepared by disrupting biological membranes,
for example by sonication.
Liposomes can be composed of naturally-derived phospholipids with mixed
lipid chains (like egg phosphatidylethanolamine) or other surfactants.
Liposomes should not be confused with micelles and reverse micelles
composed of monolayers
Part – B
1. Explain the role of vir genes in Ti plasmid.
The T-DNA transfer is mediated by products encoded by the 30-40 kb vir
region of the Ti plasmid. This region is composed by at least six essential
operons (vir A, vir B, vir C, vir D, vir E, virG ) and two non-essential (virF,
virH). The number of genes per operon differs, virA, virG and virF have only
one gene; virE, virC, virH have two genes while virD and virB have four and
eleven genes respectively. The only constitutive expressed operons are virA
and virG, coding for a two-component (VirA-VirG) system activating the
transcription of the other vir genes. The VirA-VirG two component system
has structural and functional similarities to other already described for other
cellular mechanisms (Nixon, 1986, Iuchi, 1993).
VirA is a transmembrane dimeric sensor protein that detects signal molecules,
mainly small phenolic compounds, released from wounded plants (Pan et al.,
1993). The signals for VirA activation include acidic pH, phenolic compounds,
such as acetosyringone (Winans et al., 1992), and certain class of
monosaccharides which acts sinergistically with phenolic compounds
(Ankenbauer et al., 1990; Cangelosi et al., 1990; Shimoda et al., 1990; Doty et
al., 1996). VirA protein can be structurally defined into three domains: the
periplasmic or input domain and two transmembrane domains (TM1 and
TM2). The TM1 and TM2 domains act as a transmitter (signaling) and receiver
(sensor) (Parkinson, 1993). The periplasmic domain is important for
monosaccharide detection (Chang and Winans, 1992). Within the periplasmic
domain, adjacent to the TM2 domain is an amphipatic helix, with strong
hydrophilic and hydrophobic regions (Heath et al., 1995). This structure is
characteristic for other transmembrane sensor proteins and folds the protein
to be simultaneously aligned with the inner membrane and anchored in the
membrane (Seligman and Manoil, 1994). The TM2 is the kinase domain and
plays a crucial role in the activation of VirA, phosphorylating itself on a
conserved His-474 residue (Huang et al., 1990; Jin et al. 1990ª, 1990b) in
response to signaling molecules from wounded plant sites. Monosaccharide
detection by VirA is an important amplification system and responds to low
levels of phenolic compounds. The induction of this system is only possible
through the periplasmic sugar (glucose/galactose) binding protein ChvE
(Ankenbauer and Nester, 1990; Cangelosi et al., 1990), which interacts with
VirA (Shimoda et al., 1990, 1993; Turk et al., 1993; Chang and Winans, 1992).
Recent studies for determination of VirA regions, important for its sensing
activity suggested the position, which may be involved on TM1-TM2
interaction. This interaction causes the exposure of the amphipathic helix to
small phenolic compounds and suggests a putative model for the VirA-ChvE
interaction (Doty et al., 1996).
Activated VirA has the capacity to transfer its phosphate to a conserved
aspartate residue of the cytoplasmic DNA binding protein VirG (Jin et al.
1990a, 1990b; Pan et al., 1993). VirG functions as a transcriptional factor
regulating the expression of vir genes when it is phosphorylated by VirA (Jin
et al., 1990a, 1990b). The C-terminal region is responsible for the DNA binding
activity, while the N-terminal is the phosphorylation domain and shows
homology with the VirA receiver (sensor) domain.
The activation of vir system also depends on external factors like temperature
and pH. At temperatures greater than 32°C, the vir genes are not expressed
because of a conformational change in the folding of VirA induce the
inactivation of its properties (Jin et al., 1993). The effect of temperature on
VirA is suppressed by a mutant form of VirG (VirGc), which activates the
constitutive expression of the vir genes. However, this mutant cannot confers
the virulence capacity at that temperature to Agrobacterium, probably
because the folding of other proteins that actively participate in the T-DNA
transfer process are also affected at high temperature (Fullner and Nester,
1996).
2. Explain: Direct gene transfer.
In the direct gene transfer methods, the foreign gene of interest is delivered
into the host plant cell without the help of a vector. The methods used for
direct gene transfer in plants are:
Chemical mediated gene transfer e.g. chemicals like polyethylene glycol
(PEG) and dextran sulphate induce DNA uptake into plant
protoplasts.Calcium phosphate is also used to transfer DNA into cultured
cells.
Microinjection where the DNA is directly injected into plant protoplasts or
cells (specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0
micrometerdiameter) glass needle or micropipette. This method of gene
transfer is used to introduce DNA into large cells such as oocytes, eggs, and
the cells of early embryo.
Electroporation involves a pulse of high voltage applied to protoplasts/cells/
tissues to make transient (temporary) pores in the plasma membrane which
facilitates the uptake of foreign DNA.
The cells are placed in a solution containing DNA and subjected to electrical
shocks to cause holes in the membranes. The foreign DNA fragments enter
through the holes into the cytoplasm and then to nucleus.
Particle gun/Particle bombardment - In this method, the foreign DNA
containing the genes to be transferred is coated onto the surface of minute
gold or tungsten particles (1-3 micrometers) and bombarded onto the target
tissue or cells using a particle gun (also called as gene gun/shot
gun/microprojectile gun).The microprojectile bombardment method was
initially named as biolistics by its inventor Sanford (1988). Two types of plant
tissue are commonly used for particle bombardment- Primary explants and
the proliferating embryonic tissues.
Transformation - This method is used for introducing foreign DNA into
bacterial cells e.g. E. Coli. The transformation frequency (the fraction of cell
population that can be transferred) is very good in this method. E.g. the
uptake of plasmid DNA by E. coli is carried out in ice cold CaCl2 (0-50C)
followed by heat shock treatment at 37-450C for about 90 sec. The
transformation efficiency refers to the number of transformants per
microgram of added DNA. The CaCl2 breaks the cell wall at certain regions
and binds the DNA to the cell surface.
Conjuction - It is a natural microbial recombination process and is used as a
method for gene transfer. In conjuction, two live bacteria come together and
the single stranded DNA is transferred via cytoplasmic bridges from the
donor bacteria to the recipient bacteria.
Liposome mediated gene transfer or Lipofection - Liposomes are circular lipid
molecules with an aqueous interior that can carry nucleic acids. Liposomes
encapsulate the DNA fragments and then adher to the cell membranes and
fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and
then to the nucleus. Lipofection is a very efficient technique used to transfer
genes in bacterial, animal and plant cells.
Selection of transformed cells from untransformed cells
The selection of transformed plant cells from untransformed cells is an
important step in the plant genetic engineering. For this, a marker gene (e.g.
for antibiotic resistance) is introduced into the plant along with the transgene
followed by the selection of an appropriate selection medium (containing the
antibiotic). The segregation and stability of the transgene integration and
expression in the subsequent generations can be studied by genetic and
molecular analyses (Northern, Southern, Western blot, PCR).
3. Describe the features of Ti and Ri plasmids
Among Higher plants, Ti plasmid of Agrobacterium tumefaciens or Ri
plasmid of rhizogenes is the best known vector. T- DNA, from Ti or Ri
plasmid Agrobacterium, is considered to be a very potential vector for cloning
experiments with higher plants. This will involve the following steps
(i)foreign DNA has to be first cloned into T-DNA of Ti or Ri plasmid
(ii) modified hybrid T-DNA can be transferred to the genome of plan cells by
Agrobacterium infection. Recombinant Ti plasmids can also b obtained by
cloning of a large fragment of Ti plasmid in pBR322. The recombinant Ti
plasmids can then be used for transformation of higher plants.
This system can be widely used, since Agrobacterium infects nearly all
dicotyledonous plants. Such cloning in plants has proved to be of immense
use to modify agricultural plants to increase the productivity.
Structure and Functions of Ti and Ri Plasmids - The most commonly used
vectors for gene transfer in higher plants are based on tumour inducing
mechanism of the soil bacterium Agrobacterium tumefaciens, which is the
causal organism for crown gall disease, A closely related species A.
rhizogenes causes hairy root disease. An understanding of the molecular basis
of these diseases led to the utilization of these bacteria for developing gene
transfer systems. It has been shown that the disease is caused due to the
transfer of a DNA segment from the bacterium to the plant nuclear genome.
The DNA segment, which is transferred is called T - DNA and is part of a
large Ti (tumour inducing) plasmid found in virulent strains of
Agrobacterium tumefaciens. Similarly Ri (root inducing) megaplasmids are
found in the virulent strains of A. rhizogenes. The Ti and Ri plasmids,
inducing crown gall disease and hairy root disease respectively have been
studied in great detail during the last decade. However, we will discuss only
those aspects of these plasmids which are relevant to the design of vectors for
gene transfer in higher plants.
Most Ti plasmids have four regions in common,
(i) Region A, comprising T-DNA is responsible for tumour induction, so that
mutations in this region lead to the production of tumours with altered
morphology (shooty or rooty mutant galls). Sequences homologous to
this region are always transferred to plant nuclear genome, so that the
region is described as T-DNA (transferred DNA).
(ii) Region B is responsible for replication.
(iii) Region C is responsible for conjugation.
(iv) Region D is responsible for virulence, so that mutation in this region
abolishes virulence. This region is therefore called virulene
(v) region and plays a crucial role in the transfer of T-DNA into the plant
nuclear genome. The components of this Ti plasmid have been used for
developing efficient plant transformation vectors.
The T-DNA consists of the following regions:
(i) An one region consisting of three genes (two genes tms and tms2
representing 'shooty locus' and one gene tmr representing 'rooty locus')
responsible for the biosynthesis of two phytohormones, namely indole acetic
acid or lAA (an auxin) and isopentyladenosine 5'-monophosphate (a
cytokinin). These genes encode the enzymes responsible for the synthesis of
these phytohormones, so that the incorporation of these genes in plant
nuclear genome leads to the synthesis of these phytohormones in the host
plant. The phytohormones in their turn alter the developmental programme,
leading to the formation of crown gall
(ii) An os region responsible for the synthesis of unusual amino acid or sugar
derivatives, which are collectively given the name opines. Opines are derived
from a variety of compounds (e.g. arginine + pyruvate), that are found in
plant cells. Two most common opines are octopine and nopaline. For the
synthesis of octopine and nopaline, the corresponding enzymes octopine
synthase and nopaline synthase are coded by T- DNA.
Depending upon whether the Ti plasmid encodes octopine or nopaline, it is
described as octopine-type Ti plasmid or nopalinetype Ti plasmid. Many
organisms including higher plants are incapable of utilizing opines, which can
be effectively utilized by Agrobacterium. Outside the T-DNA region, Ti
plasmid carries genes that, catabolize the opines, which are utilized as a
source of carbon and nitrogen.
The T-DNA regions on all Ti and Ri plasmids are flanked by almost perfect
25bp direct repeat sequences, which are essential for T-DNA transfer, acting
only in cis orientation. It has also been shown that any DNA sequence,
flanked by these 25bp repeat sequences in the correct orientation, can be
transferred to plant cells, an attribute that has been successfully utilized for
Agrobacterium mediated gene transfer in higher plants leading to the
production of transgenic plants.
Besides 25bp flanking border sequences (with T DNA), vir region is also
essential for T-DNA transfer. While border sequences function in cis
orientation with respect to T -DNA, vir region is capable of functioning even
in trans orientation. Consequently physical separation of T-DNA and vir
region onto two different plasmids does not affect T-DNA transfer, provided
both the plasmids are present in the same Agrobacterium cell. This property
played an important role in designing the vectors for gene transfer in higher
plants, as will be discussed later.
The vir region (approx 35 kbp) is organized into six operons, namely vir A, vir
B, vir C, vir D, vir E, and vir G, of which four operons (except vir A and vir G)
are polycistronic. Genes vir A, B, D, and G are absolutely required for
virulence; the remaining two genes vir C and E are required for tumour
formation. The vir A locus is expressed constitutively under all conditions.
The vir G locus is expressed at low levels in vegetative cells, but is rapidly
induced to higher expression levels by exudates from wounded plant tissue.
The vir A and vir G gene products regulate the expression of other vir loci.
The vir A product (Vir A) is located on the inner membrane of Agrobacterium
cells and is probably a chemoreceptor, which senses the presence of phenolic
compounds (found in exudates of wounded plant tissue), such as
acetosyringone and β-hydroxyaceto syringone.
Signal transduction proceeds via activation (possibly phosphorylation) of Vir
G (product of gene vir G), which in its turn induces expression of other vir
genes.
4. What are 35S promoters? Explain with diagram.
Analysis on the CaMV 35 promoter is divided into a discussion of:
•the promoter itself
•sequences identified in patents as "35S enhancer regions"
•the "minimal" promoter
At the beginning of the 1980s, Chua and collaborators at the Rockefeller
University isolated the promoter responsible for the transcription of the
whole genome of a Cauliflower mosaic virus (CaMV) infecting turnips. The
promoter was named CaMV 35S promoter ("35S promoter") because the
coefficient of sedimentation of the viral transcript whose expression is
naturally driven by this promoter is 35S. It is one of the most widely used,
general-purpose constitutive promoters.
The 35S promoter is a very strong constitutive promoter, causing high levels
of gene expression in dicot plants. However, it is less effective in monocots,
especially in cereals. The differences in behavior are probably due to
differences in quality and/or quantity of regulatory factors. The promoter
responsible for the transcription of another part of the genome of CaMV, the
CaMV 19S promoter, is also used as a constitutive promoter, but is not as
widely used as the 35S promoter.
Part – C
1. Elaborate on antisense RNA technology and emphasis its role in fruit
ripening.
Antisense RNA is a single-stranded RNA that is complementary to a
messenger RNA (mRNA) strand transcribed within a cell. Antisense RNA
may be introduced into a cell to inhibit translation of a complementary
mRNA by base pairing to it and physically obstructing the translation
machinery. This effect is therefore stoichiometric. An example of naturally
occurring mRNA antisense mechanism is the hok/sok system of the E.coli R1
plasmid. Antisense RNA has long been thought of as a promising technique
for disease therapy; the only such case to have reached the market is the drug
fomivirsen. One commentator has characterized antisense RNA as one of
"dozens of technologies that are gorgeous in concept, but exasperating in
[commercialization]". Generally, antisense RNA still lack effective design,
biological activity, and efficient route of administration.
Historically, the effects of antisense RNA have often been confused with the
effects of RNA interference (RNAi), a related process in which doublestranded RNA fragments called small interfering RNAs trigger catalytically
mediated gene silencing, most typically by targeting the RNA-induced
silencing complex (RISC) to bind to and degrade the mRNA. Attempts to
genetically engineer transgenic plants to express antisense RNA instead
activate the RNAi pathway, although the processes result in differing
magnitudes of the same downstream effect, gene silencing. Well-known
examples include the Flavr Savr tomato and two cultivars of ringspotresistant papaya.
Transcription of longer cis-antisense transcripts is a common phenomenon in
the mammalian transcriptome. Although the function of some cases have
been described, such as the Zeb2/Sip1 antisense RNA, no general function
has been elucidated. In the case of Zeb2/Sip1, the antisense noncoding RNA
is opposite the 5' splice site of an intron in the 5'UTR of the Zeb2 mRNA.
Expression of the antisense ncRNA prevents splicing of an intron that
contains a ribosome entry site necessary for efficient expression of the Zeb2
protein. Transcription of long antisense ncRNAs is often concordant with the
associated protein-coding gene, but more detailed studies have revealed that
the relative expression patterns of the mRNA and antisense ncRNA are
complex
2. Narrate the process of T-DNA transfer during agrobacterium mediated
transformation
The transferring vehicle to the plant nucleus is a ssT-DNA-protein
complex. Is must be translocated to the plant nucleus passing through three
membranes, the plant cell wall and cellular spaces. According to the most
accepted model, the ssT-DNA-VirD2 complex is coated by the 69 kDa VirE2
protein, a single strand DNA binding protein. This cooperative association
prevents the attack of nucleases and, in addition, extends the ssT-DNA strand
reducing the complex diameter to approximately 2 nm, making the
translocation through membrane channels easier. However, that association
does not stabilizes T-DNA complex inside Agrobacterium (Zupan et al., 1996).
VirE2 contains two plant nuclear location signals (NLS) and VirD2 one (Bravo
Angel et al, 1998). This fact indicates that both proteins presumably play
important roles once the complex is in the plant cell mediating the complex
uptake to the nucleus (Herrera-Estrella et al., 1990; Shurvinton et al., 1992;
Rossi et al., 1993, Tinland et al., 1995, Zupan et al., 1996). The deletion of NLS
in one of these proteins reduces, but does not totally inhibit, the ssT-DNA
transfer and its integration into plant genome, evidencing the other partner
can at least partially assume the function of the absent protein.
It is known that VirE1 is essential for the export of VirE2 to the plant cell,
although other specific functions are still uncharacterized (Binns et al., 1995).
Bacterial strains mutated in virE1, cannot export VirE2 which is accumulated
inside the bacterium. Such mutants can be complemented if coinfected with a
strain that can export VirE2, indicating that this protein can be exported
independently and that the transfer of VirE2 as part of the ss-T-DNA complex
is not necessary for the transmission event (Sundberg et al., 1996) being
possible to transfer naked T-DNA to the plant cell (Binns et al., 1995;
Sundberg et al., 1996).
From these experimental evidences, an alternative model was brought to light
for ssT-DNA complex transfer. This model proposes that the transfer complex
is a single-strand DNA covalently bound at its 5'-end with VirD2, but
uncoated by VirE2. The independent export of VirE2 to plant cell is presented
as a natural process, and once the naked ssT-DNA-VirD2 complex is inside
the plant cell, it is coated by VirE2 (Binns et al., 1995; Lessl et al., 1994). It is
also possible that the process can be performed by one of the proposed
alternatives ways according to the infection conditions.
Previous researches described the role of 9.5 kb virB operon in the
generation of a suitable cell surface structure for the ssT-DNA complex
transfer from bacterium to plant (Finberg et al., 1995; Stephens et al., 1995;
Zhou and Christie, 1997; Dang and Christie, 1997; Rashkova et al., 1997,
Fernandez et al., 1996; Beaupré et al., 1997). The VirD4 protein is also required
for the ss-T-DNA transport. The function of VirD4 is the ATP-dependent
linkage of protein complex necessary for T-DNA translocation (Firth et al.,
1996).
VirB are proteins that presents hydrophathy characteristics similar to other
membrane-associated proteins (Kuldau et al., 1990; Shirasu et al., 1990, 1994;
Thompson et al., 1988; Ward et al., 1988). VirD4 is a transmembrane protein
but predominantly located at the cytoplasmatic side of the cytoplasmic
membrane (Okamoto et al., 1991). Comparative studies showed a high degree
of homology between the virB operon and transfer regions of broad host
range (BHR) plasmids in genetic organization, nucleotide sequence and
protein function (Pohlman et al., 1994; Lessl et al., 1992). Both systems deliver
non-self transmissible DNA-protein complex to recipient host cell. In
addition, they have the capacity to DNA interkingdom delivery (Heinemann
and Sprague, 1989; Bundock et al., 1995; Piers et al., 1996) suggesting that the
T-DNA transfer apparatus and conjugation systems are related and probably
evolved from a common ancestral (Christie et al., 1997; Oger et al., 1998).
The majority of VirB proteins are assembled as a membrane-spanning
protein channel involving both membranes (Shirasu and Kado, 1993a, 1993b;
Shirasu et al., 1994; Stephens et al., 1995; Das and Xie, 1998). Except for
VirB11, they have multiple periplasmic domains (Christie, 1997). VirB1 is the
only member of VirB proteins found in the extracellular milieu (Baron et al.,
1997) although it is possible that some of the other VirB proteins may be
redistributed during the process of biogenesis and functioning of the
transcellular conjugal channel (Christie, 1997). That could be the case of the
VirB2, a protein with deduced extracellular functions. Vir B2 is translated as a
12 kDa proprotein, which is later processed by proteolysis to its mature 7 kDa
functional form (Jones et al., 1996).
VirB4 and VirB11 are hydrophilic ATPases necessary for active DNA
transfer. Vir B11 lacks continuous sequence of hydrophobic residues, formiing
periplasmic domains. Despite these structural characteristics, less than one
third of VirB11 constitutes its soluble fraction, while the rest of the protein
remains associated with the cytoplasmic membrane (Rashkova et al., 1997).
These characteristics are atypical for this type of protein and evidence the
possible dynamic co-existence of different conformational forms in vivo .
VirB4 tightly associates with the cytoplasmic membrane (Dang and Christie,
1997). It contains two putative extracellular domains conferring
transmembrane topology to this protein, which presumably allows the ATPdependent conformational change in the conjugation channel. Probably, the
functional forms of VirB4 and VirB11 are homo- and heterodimers (Dang and
Christie, 1997). The VirB4 synthesis is well correlated with the accumulation
and distribution of VirB3. Other protein, VirB7, seems to be crucial for the
conformation of the transfer apparatus. VirB7 interacts with VirB9 forming
heterodimers and probably higher-order multimeric complexes. The synthesis
of VirB9 and its stable accumulation depends of heterodimer conformation,
indicating that VirB9 alone may be unstable and requires the association with
VirB7 (Anderson et al., 1996). In this intermolecular conformation the
monomeric subunits are joined by disulfide bridges. The VirB7-VirB9
heterodimer is assumed to stabilize other Vir proteins during assembly of
functional transmembrane channels (Fernandez et al., 1996; Spudich et al.,
1996).
Some of the initial steps of biogenesis of ssT-DNA complex apparatus. have
been recently identified. Firstly, VirB7 and VirB9 monomers are exported to
the membrane and processed. They interact each other to form covalently
cross-linked homo- and heterodimers. Although the role of both types of
dimers in the biogenesis of the transfer apparatus is widely accepted, it is
likely that only heterodimers are essential (Fernández et al., 1996; Spudich et
al., 1996). Subsequently the VirB7-VirB9 heterodimer is sorted to the outer
membrane. The sorting mechanism has not been elucidated (Christie, 1997)
the next step implies the interaction with the other Vir proteins for assembling
the transfer channel with the contribution of the transglycosidase VirB1.
It is known that VirB2 through VirB11 are essential for DNA transfer,
suggesting that these proteins are fundamental component of the transfer
apparatus (Berger and Christie, 1993, 1994) while VirB1 has a lesser
contribution to this process.
Two accessory vir operons, present in the octopine Ti-plasmid, are virF and
virH. The virF operon encodes for a 23 kDa protein that functions once the TDNA complex is inside the plant cells via the conjugal channel or
independently, as it was assumed for VirE2 export. The role of VirF seems to
be related with the nuclear targeting of the ssT-DNA complex but its
contribution is less important than in the case of VirF (Hooykass and
Schilperoort, 1992). The virH operon consists of two genes that code for VirH1
and VirH2 proteins. These Vir proteins are not essential but could enhance the
transfer efficiency, detoxifying certain plant compounds that can affect the
bacterial growth (Kanemoto et al., 1989). If that is the function of VirH
proteins, they play a role in the host range specificity of bacterial strain for
different plant species.
Integration of T-DNA into plant genome
Inside the plant cell, the ssT-DNA complex is targeted to the nucleus
crossing the nuclear membrane. Two Vir proteins have been found to be
important in this step: VirD2 and VirE2, which are the most important, and
probably VirF, which has a minor contribution to this process (Hooykaas and
Schilperoort, 1992). The nuclear location signals (NLS) of VirD2 and VirE2
play an important role in nuclear targeting of the delivered ss-T-DNA
complex, as early described. VirD2 has one functional NLS. The ssT-DNA
complex is a large (up to 20 kb) nucleoprotein complex containing only one
5'end covalently attached VirD2 protein per complex. But the complex is
coated by a large number of VirE2 molecules (approximately 600 per a 20 kb
T-DNA), and each of them has two NLS. The two NLS of VirE2 have been
considered important for the continuos nuclear import of ss-T-DNA complex,
probably by keeping both sides of nuclear pores simultaneously open. The
nuclear import is probably mediated also by specific NLS-binding proteins,
which are present in plant cytoplasm.
The final step of T-DNA transfer is its integration into the plant genome.
The mechanism involved in the T-DNA integration has not been
characterized. It is considered that the integration occurs by illegitimate
recombination (Gheysen et al., 1991, Lehman et al., 1994; Puchta, 1998).
According to this model, paring of a few bases, known as micro-homologies,
are required for a pre-annealing step between T-DNA strand coupled with
VirD2 and plant DNA. These homologies are very low and provide jus a
minimum specificity for the recombination process by positioning VirD2 for
the ligation. The 3´-end or adjacent sequences of T-DNA find some low
homologies with plant DNA resulting in the first contact (synapses) between
the T-strand and plant DNA and forming a gap in 3'-5' strand of plant DNA.
Displaced plant DNA is subsequently cut at the 3'-end position of the gap by
endonucleases, and the first nucleotide of the 5' attaches to VirD2 pairs with a
nucleotide in the top (5'-3') plant DNA strand. The 3' overhanging part of TDNA together with displaced plant DNA are digested away, either by
endonucleases or by 3'-5' exonucleases. Then, the 5' attached to VirD2 end and
other 3'-end of T-strand (paired with plant DNA during since the first step of
integration process) joins the nicks in the bottom plant DNA strand. Once the
introduction of T-strand in the 3'-5' strand of the plant DNA is completed, a
torsion followed by a nick into the opposite plant DNA strand is produced.
This situation activates the repair mechanism of the plant cell and the
complementary strand is synthesized using the early inserted T-DNA strand
as a template (Tinland et al., 1995).
VirD2 has an active role in the precise integration on T-strand in the plant
chromosome. The release of VirD2 protein may provide the energy containing
in its phosphodiester bond, at the Tyr29 residue, with the first nucleotide of Tstrand, providing the 5'-end of the T-strand for ligation to the plant DNA.
This phosphodiester bond can serve as electrophilic substrate for nucleophilic
3'-OH from nicked plant DNA. (Jayaram, 1994). When the mutant VirD2
protein is transferred attached to the T-strand, the integration process take
place with the loss of nucleotides at the 5'-end of the T-strand (Tinland et al.,
1995).
3. What are the methods of gene transfer in plants?
In conventional breeding, the pool of available genes and the traits they code
for is limited due to sexual incompatibility to other lines of the crop in
question and to their wild relatives. This restriction can be overcome by using
the methods of genetic engineering, which in principle allow introducing
valuable traits coded for by specific genes of any organism (other plants,
bacteria, fungi, animals, viruses) into the genome of any plant. The first gene
transfer experiments with plants took place in the early 1980s. Normally,
transgenes are inserted into the nuclear genome of a plant cell. Recently it has
become possible to introduce genes into the genome of chloroplasts and other
plastids (small organelles of plant cells which possess a separate genome).
Transgenic plants have been obtained using Agrobacterium-mediated DNAtransfer and direct DNA-transfer, the latter including methods such as
particle
bombardment,
electroporation
and
polyethylenglycol
permeabilisation. The majority of plants have been transformed using
Agrobacterium mediated transformation.
Agrobacterium-mediated Gene Transfer :
The Agrobacterium-mediated technique involves the natural gene
transfer system resident in the bacterial plant pathogens of the genus
Agrobacterium. In nature, Agrobacterium tumefaciens and Agrobacterium
rhizogenes are the causative agents of the crown gall and the hairy root
diseases, respectively. The utility of Agrobacterium as a gene transfer system
was first recognized when it was demonstrated that these plant diseases were
actually produced as a result of the transfer and integration of genes from the
bacteria into the genome of the plant. Both Agrobacterium species carry a
large plasmid (small circular DNA molecule) called Ti in A. tumefaciens and
Ri in A. rhizogenes. A segment of this plasmid, designated T-(for transfer)
DNA, is transmitted by this organism into individual plant cells, usually
within wounded tissue (see figure 2.1). The T-DNA segment penetrates the
plant cell nucleus and integrates randomly into the genome where it is stably
incorporated and inherited like any other plant gene in a predictable,
dominant Mendelian fashion. Expression of the natural genes on the T-DNA
results in the synthesis of gene products that direct the observed
morphological changes, i. e. tumor or hairy root formation.
In genetic engineering, the tumor inducing genes within the T-DNA which
cause the plant disease are removed and replaced by foreign genes. These
genes are then stably integrated into the genome of the plant after infection
with the altered strain of Agrobacterium, just like the natural T-DNA. Because
all tumor-inducing genes are removed, the gene transfer does not induce any
disease symptoms. This reliable method of gene transfer is well suited for
plants which are susceptible to infection by Agrobacterium. Unfortunately,
many species, especially economically important legumes and
monocotyledons such as cereals, do not respond positively to Agrobacteriummediated transformation. For these plants, the following methods of direct
DNA uptake must be applied.
Particle Bombardment :
This method, also referred to as biolistic transformation (from
biological ballistics) involves coating biologically active DNA onto small
tungsten or gold particles (1-5 ?m in diameter) and accelerating them into
plant tissue at high velocity. The particles penetrate the plant cell wall and
lodge themselves within the cell where the DNA is liberated resulting in
transformation of the individual plant cell in an explant. This technique is
generally less efficient than Agrobacterium-mediated transformation, but has
nevertheless been particularly useful in several plant species, most notably in
cereal crops. The introduction of DNA into organized, morphogenic tissues
such as seeds, embryos or meristems has enabled the successful
transformation and regeneration of rice, wheat, soybean and maize, thus
demonstrating the enormous potential of this method .
Electroporation and Direct DNA Entry into Protoplasts
Electroporation is a process whereby very short pulses of electricity are
used to reversibly permeabilize lipid bilayers of plant cell membranes. The
electrical discharge enables the diffusion of macromolecules such as DNA
through an otherwise impermable plasma membrane. Because the plant cell
wall will not allow the efficient diffusion of many transgene constructs,
protoplasts (cells without cell walls) must be prepared. This requirement
presents a major obstacle for many applications as protocols making possible
the regeneration of protoplasts into complete plants do not exist for many
species.
DNA uptake by plant protoplasts can also be stimulated by phosphate
or calcium/polyethylene glycol (PEG) coprecipitation. However, these
methods all suffer from the drawback that they use protoplasts as the
recipient host which often cannot be regenerated into whole plants.
Transgene Expression
In most cases, the introduction of a gene into the plant genome will
only have an effect on the plant if the transgene is expressed, i. e. transcribed
into mRNA and translated into a protein. A promoter is a sequence of nucleic
acids where the RNA polymerase (a complex enzyme synthesizing the mRNA
transcript) attaches to the DNA template. The nature of the promoter defines
(together with other expression-regulating elements), under which conditions
and with which intensity a gene will be transcribed. The promoter of the 35S
gene of cauliflower mosaic virus is used very frequently in plant genetic
engineering. This promoter confers high-level expression of exogenous genes
in most cell types from virtually all species tested. As it is often advantageous
to express a transgene only in certain tissues or quantities or at certain times, a
number of other promoters are available, e.g. promoters inducing gene
expression after wounding or during fruit ripening only.
Methods of gene transfer currently employed result in the random
integration of foreign DNA throughout the genome of recipient cells. The site
of insertion may have a strong influence on the expression levels of the
exogenous gene, resulting in different expression levels of an introduced
gene, even if the same promoter/gene construct was used. The exact
mechanism of this phenomenon are not yet fully understood.
Selection and Plant Regeneration
In a transformation experiment, the proportion of transformed cells is
usually small compared to the number of cells which remain unaltered. In
order to select only cells which have actually incorporated the new genes, the
genes coding for the desired trait are fused to a gene which allows selection of
transformed cells, so-called marker genes. The expression of the marker gene
enables the transgenic cells to grow in presence of a selective agent, usually an
antibiotic or a herbicide, while cells without the marker gene die. One of the
most commonly used marker genes is the bacterial aminoglycoside-3'
phosphotransferase gene (APH(3')II), also referred to as neomycin
phosphotransferase 11 (NPTII). This gene codes for an enzyme which
inactivates the antibiotics kanamycin, neomycin and G418 through
phosphorylation. In addition to NPTII, a number of other antibiotic resistance
genes have been used as selective markers, e.g. hygromycin
phosphotransferase gene conferring resistance to hygromycin.
Another group of selective markers are herbicide tolerance genes.
Herbicide tolerance has been obtained through the incorporation and
expression of a gene which either detoxifies the herbicide in a similar manner
as the antibiotic resistance gene products or a gene that expresses a product
which acts like the herbicide target but is not affected by the herbicide.
Herbicide tolerance may not only serve as a trait useful for selection in the
development of transgenic plants, but also has some commercial interest.
Herbicide tolerance transgenic plants are therefore among the first crops
approaching market introduction.
Transformation of plant protoplasts, cells and tissues is usually only
useful if the they can be regenerated into whole plants. The rates of
regeneration vary greatly not only among different species, but also between
cultivars of the same species. As mentioned in capter 2.2, in many cases
regeneration of whole plants from cells is not possible or very difficult.
Besides the ability to introduce a gene into the genome of a plant species,
regeneration of intact, fertile plants out of transformed cells or tissues is the
most limiting step in developing transgenic plants.
Unit – V
Application of plant transformations for productivity and performance:
Engineering plants for herbicide resistance, insect resistance, virus resistance,
disease resistance, antifungal proteins, PR Proteins, nematode resistance, abiotic
stress, long shelf life of fruits and flowers
________________________________________________________________________
Part – A
1. Reserpine is isolated from the plant snakeroot plant ( Rauwolfia serpentina)
2. What are PR proteins? Give an example
Protein induced in several plant species when they are infected by viruses,
viroids, fungi or bacteria. The occurrence of these proteins is not pathogenspecific, but determined by the type of reaction of the host plant. They form a
protective barrier against pathogens by collecting at infection sites and act to
decrease susceptibility of plants. They may have anti-fungal or anti-bacterial
activity
Example :
Family Type member
Properties
PR-1
tobacco PR-1a
antifungal?, 14-17kD
PR-2
tobacco PR-2
class I, II, and III endo-beta-1,3-glucanases,
PR-3
tobacco P, Q
class I, II, IV, V, VI, and VII endochitinases,
PR-4
tobacco R
antifungal, win-like proteins, endochitinase
PR-5
tobacco S
antifungal, thaumatin-like proteins,
PR-6
tomato
inhibitor I protease inhibitors, 6-13kD
PR-7
tomato P69
endoproteases
PR-8
cucumber chitinase class III chitinases, chitinase/lysozyme
PR-9
lignin-forming
peroxidase, peroxidase-like proteins
PR-10
parsley PR-1
ribonucleases, Bet v 1-related proteins
PR-11
tobacco
class V chitinase endochitinase activity
PR-12
radish Ps-AFP3
plant defensins
PR-13
Arabidopsis THI2.1 thionins
PR-14
barley LTP4
nonspecific lipid transfer proteins (ns-LTPs)
PR-15
barley OxOa
(germin) oxalate oxidase
PR-16
barley OxOLP
oxalate-oxidase-like proteins
PR-17
tobacco PRp27
unknown
3. Luciferase
Luciferase is a generic term for the class of oxidative enzymes used in
bioluminescence and is distinct from a photoprotein. One famous example is
the firefly luciferase (EC 1.13.12.7) from the firefly Photinus pyralis. "Firefly
luciferase" as a laboratory reagent usually refers to P. pyralis luciferase
although recombinant luciferases from several other species of fireflies are
also commercially available. The name is derived from Lucifer, the root of
which means 'light-bearer' (lucem ferre).
4. PHB in transgenic technology.
Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA), a polymer
belonging to the polyesters class that was first isolated and characterized in
1925 by French microbiologist Maurice Lemoigne. PHB is produced by microorganisms (like Ralstonia eutrophus or Bacillus megaterium) apparently in
response to conditions of physiological stress. The polymer is primarily a
product of carbon assimilation (from glucose or starch) and is employed by
micro-organisms as a form of energy storage molecule to be metabolized
when other common energy sources are not available. Microbial biosynthesis
of PHB starts with the condensation of two molecules of acetyl-CoA to give
acetoacetyl-CoA which is subsequently reduced to hydroxybutyryl-CoA. This
latter compound is then used as a monomer to polymerize PHB.[1]
Studies showed that transgenic plants can produce PHB 20-40 % of their dry
mass. It is known that bioplastic synthesis occurs in plant cytoplasm, plastid
or peroxisome. Researches found that transgenic plants give up starch
production when they start to produce PHB. They use all their energy to
produce polymer.
PHB genes isolated from Alcaligenes eutrophus translated to corn, potato,
and some other plants. These plants could produce polymer and Imperial
Chemical Company obtained 50 tons PHB-HV co-polymer.
Researches still search to produce PHA, which is degradable plastic, from
sunflower and soybean. They reported that corn must be harvested at early
stage to obtain plastic. PHA gain from modified corn is very hard and
difficult process but carbon dioxide and sun-shine is used as a carbon and
energy source and there is not more to do than fermentation process.
Studies show that cheaper PHB can be obtained from transgenic plants, big
probably “plastic potatoes“ will be grown in the future.
5. Atrazine
Atrazine, 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine, an organic
compound consisting of an s-triazine-ring is a widely used herbicide. Its use is
controversial due to widespread contamination in drinking water and its
associations with birth defects, menstrual problems, and cancer when
consumed by humans at concentrations below government standards.
Although it has been excluded from a re-registration process in the European
Union, it is still one of the most widely used herbicides in the world
6. Glyphosate
Glyphosate (N-(phosphonomethyl)glycine) is a broad-spectrum systemic
herbicide used to kill weeds, especially annual broadleaf weeds and grasses
known to compete with crops grown widely across the Midwest of the United
States. Initially patented and sold by Monsanto Company in the 1970s under
the tradename Roundup, its U.S. patent expired in 2000. Glyphosate is the
most used herbicide in the USA, where every year, 5–8 million pounds (2,300–
3,600 tonnes) are used on lawns and yards and another 85–90 million pounds
(39,000–41,000 t) are used in agriculture.
7. Polygalacturonase
Polygalacturonase (PG) is an enzyme produced in plants which is involved in
the ripening process, and by some bacteria and fungi which is involved in the
rotting process. PGs degrades polygalacturonan present in the cell walls of
plants by hydrolysis of the glycosidic bonds that link galacturonic acid
residues. Polygalacturonan is a significant carbohydrate component of the
pectin network that comprises plant cell walls.[1] The activity of the
endogenous plant PGs work to soften and sweeten fruit during the ripening
process. Similarly, phytopathogens use PGs as a means to weaken the pectin
network, so that a host of digestive enzymes can be excreted into the plant
host to acquire nutrients
Part – B
1. What is the role of glyphosphate in herbicide resistance?
Glyphosate kills plants by interfering with the synthesis of the amino acids
phenylalanine, tyrosine and tryptophan. It does this by inhibiting the enzyme
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the
reaction of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form 5enolpyruvyl-shikimate-3-phosphate
(ESP).
ESP
is
subsequently
dephosphorylated to chorismate, an essential precursor in plants for the
aromatic amino acids: phenylalanine, tyrosine and tryptophan.[12][13] These
amino acids are used as building blocks in peptides, and to produce
secondary metabolites such as folates, ubiquinones and naphthoquinone. Xray crystallographic studies of glyphosate and EPSPS show that glyphosate
functions by occupying the binding site of the phosphoenolpyruvate,
mimicking an intermediate state of the ternary enzyme substrates
complex.[14] The shikimate pathway is not present in animals, which instead
obtain aromatic amino acids from their diet. Glyphosate has also been shown
to inhibit other plant enzymes,[15][16] and also has been found to affect
animal enzymes
2. Explain: Edible vaccines.
In the past few years, agricultural biotechnology has progressed in leaps and
bounds. It started with the commercial success of “Golden Rice”- a genetically
modified crop, aimed to provide better “Vitamin-A” nutrition to those
populations that suffer from Vitamin- A deficiency. After Golden Rice came
the Indian invention- Bt. Cotton. This cotton crop could reduce the damage
caused by Bollworm, by causing an adverse effect on the insects that feed on
it. It therefore helped in increasing yield by decreasing the damage. The other
commercial GM crops were namely, “Flavr Savr” Tomatoes, Bt. Brinjal and
many more, each aiming at either increasing yield or reducing spoilage.
Biotechnologists in recent years have come up with a new concept, enhancing
the idea behind “Golden Rice”. This new concept is about Food Vaccines. The
difference here lies, that crops like “golden rice” provided extra nutrition that
naturally didn’t occur in it. But food vaccines are GM crops that would
provide extra added “immunity” from certain diseases.
Food Vaccines or Edible Vaccines have many potential advantages. These GM
plants could be grown locally, and cheaply, using the standard growing
methods of a given region. Homegrown vaccines would also avoid the
logistical and economic problems posed by having to transport traditional
preparations over long distances. And, being edible, the vaccines would
require no syringes.
But with advancement, come many hurdles and problems. One of the key
goals of the edible-vaccine pioneers is to reduce immunization costs. It is
postulated that edible vaccines would be far cheaper than current injectable
vaccines since they would not have to undergo the expensive purification and
refrigeration of traditional vaccines, and transportation costs would be much
reduced. Even if edible vaccines are cheaper, it is not assured that this will
lead to increased vaccination coverage, since in many cases the cost of the
vaccine is a small part of the whole package. According to the WHO, to
immunize a child, the cost is no more than $1 for six big vaccines, but $14 is
the cost for the total programme which includes- laboratories, transport, cold
chain, personnel and research.
In special cases, for the newer, more expensive vaccines, such as hepatitis B
and AIDS, the cost of the vaccine plays a more significant role, but the nature
of the vehicle (apples or syringe) will still only represent a small part of the
total cost.
Research into edible vaccines is still at a very early stage and scientists have a
long way to go in proving their efficacy. Getting plants to express adequate
amounts of the vaccine, is proving challenging enough, let alone translating
that into an appropriate immunological response in people. Producing stable
and reliable amounts of vaccines in plants is complicated by the fact that
tomatoes and bananas don’t come in standard sizes! There may also be sideeffects due to the interaction between the vaccine and the vehicle. People
could ingest too much of the vaccine, which could be toxic, or too little, which
could lead to disease outbreaks among populations believed to be immune.
Despite the un-surety of these vaccines, they can prove to be bliss for the
underprivileged, if they are a success as they will be easily reachable to a wide
population. Even “Golden Rice” faced a lot of problems till its final approval
and now it has proved as a major success to the underprivileged population
of Africa
3. Cry gene
Bacillus thuringiensis (Bt), is a gram-positive soil bacterium, with a genome
size of 2.4 to 5.7 million basepairs. The prevalence of this strain is not
restricted and has been isolated worldwide from many habitats, including
soil, stored-product dusts, insects, deciduous and coniferous leaves. Bacillus
thuringiensis forms parasporal crystals during the stationary phase of its
growth cycle. These crystals are specifically toxic to certain orders and species
of insects, like Lepidoptera, Diptera, and Coleoptera. Many different strains of
Bt have been shown to produce these inclusions of insecticidal crystal protein
(ICP). Bt also produces antibiotic compounds that have antifungal activity.
During sporulation, it synthesizes a cytoplasmic inclusion containing one
or more proteins that are toxic to insect larvae. Upon completion of
sporulation the parent bacterium lyses to release the spore and the inclusion.
In these inclusions, the toxins exist as inactive protoxins. When the inclusions
are ingested by insect larvae, the alkaline pH solubilizes the crystal. The
protoxin is then converted in to an active toxin after processing by the host
proteases present in the midgut.
It has been indicated that the activated toxin binds to insect-specific receptors
exposed on the surface of the plasma membrane of midgut epithelial cells and
then inserts into the membrane to create transmembrane pores that cause cell
swelling and lysis and eventually death of the insect.
Due to their high specificity for these unique receptors on the membrane of
the gut epithelial cells, these toxins (delta-endotoxins) are harmless to nontarget insects and the end-user and are compatible with integrated pest
management programs. The fact that they are proteins ensures that they are
readily biodegraded.
The Cry Gene Family:
These toxins can be categorized under the d-endotoxins, which is highly
specific to only certain insects. The family of genes coding for this toxin is the
Cry gene family. A common characteristic of the cry genes is their expression
during the stationary phase. Their products generally accumulate in the
mother cell compartment to form a crystal inclusion that can account for 2030% of the dry weight of the sporulated cells. The high level of synthesis and
coordination with the stationary phase are controlled by a variety of
mechanisms occurring at the transcriptional, posttranscriptional and
posttranslational levels.
At the transcriptional level, the development of sporulation is controlled
by the successive activation of sigma factors.
At the posttranscriptional level, the stability of the mRNA is enhanced by the
formation of a stemloop structure during termination of transcription. This
protects the mRNA from the activity of exonucleases present in the cell from
degrading the mRNA. The 5’ end of the mRNA is protected due to the
presence of the perfect Shine-Dalgarno sequence designated as STAB-SD. The
interaction with the 16s rRNA of the 30s subunit of the ribosome confers
stability to the mRNA.
At the posttranslational level, these proteins form crystalline inclusions in
the mother cell compartment. Depending on the protoxin composition, the
crystals have various forms. This ability to crystallize helps in protecting the
protein itself from premature proteolytic degradation.
Structure-Function Interpretation of the Cry Proteins:
The Cry toxin has three domains which are, from N to C terminus, a seven
helix bundle, (Domain I), a triple anti-parallel beta sheet domain (Domain II)
and a beta-sheet sandwich (Domain III). (1)
The core of the molecule is built from five sequence blocks, which are a highly
conserved feature of all the Bt toxins indicating that all the proteins in this Cry
family will adopt the same general fold.
The long, hydrophobic and amphipathic alpha helices of Domain I is
equipped for transmembrane pore formation. The seven alpha helix domain I
structure resembles the pore forming domain of Colicin A and is important
for the membrane insertion step.
Pore formation is initiated by insertion of a helical hairpin (alpha4/alpha5)
from domain I with subsequent association of alpha4/alpha5 hairpins from
several molecules to form an oligomeric helical bundle pore with a radius of
5-10 Angstroms.
Before one or more of these Cry helices can insert into the membrane to
initiate oligomerization and pore formation, a major conformational change
must occur, since in the water soluble pre-insertion form all the hydrophobic
faces of the Cry Domain I helical bundle face inwards.
Membrane penetration occurs in two steps: binding to a specific receptor
exposed on the membrane surface, followed by insertion of the deltaendotoxin protein into the membrane leading to pore formation.
The three beta sheet structure (beta prism) of domain II is involved in receptor
binding and specificity determination. This is further supported by reports
that domain II shared the same structural fold with three carbohydrate
binding proteins: the vitelline membrane outer layer protein I from hen's
eggs, the plant lectin jacalin and the Maclura pomifera agglutinin.
Domain III of the Bt toxin (see below) may also be a determinant of insect
specificity/receptor binding.
The striking similarity between the structure of domain II of the Bt toxins and
the three dimensional structures of two known lectins suggests that
insecticidal specificity might be determined by the carbohydrate affinity of
the domain II lectin fold. A recent discovery that domain III is also a lectinlike domain suggests that the insecticidal specificity of these toxins could be
determined by two lectin-like domains acting in concert or independently.
To fully realize the potential of Bacillus thuringiensis d-endotoxins as
biopesticides, progress is required in several areas. First, we must increase the
yield or efficiency of toxin protein production. Second, we must gain a
sufficient understanding of the mechanism of toxicity to allow engineering of
the toxins for maximum activity. Third, we must continue to isolate new
strains with novel toxin structures and activities either on known B.
thuringiensis targets or on pests thought to be insensitive to B. thuringiensis.
And fourth, we must gain a better understanding of the mechanism, and
management, of insect resistance to B. thuringiensis toxins. All the
requirement can be easily met by the process of DNA shuffling of the Cry
genes.
4. How abiotic stress is playing a role in plants?
Abiotic stress is defined as the negative impact of non-living factors on the
living organisms in a specific environment. The non-living variable must
influence the environment beyond its normal range of variation to adversely
affect the population performance or individual physiology of the organism
in a significant way. Whereas a biotic stress would include such living
disturbances as fungi or harmful insects, abiotic stress factors, or stressors, are
naturally occurring, often intangible, factors such as intense sunlight or wind
that may cause harm to the plants and animals in the area affected. Abiotic
stress is essentially unavoidable. Abiotic stress affects animals, but plants are
especially dependent on environmental factors, so it is particularly
constraining. Abiotic stress is the most harmful factor concerning the growth
and productivity of crops worldwide. Research has also shown that abiotic
stressors are at their most harmful when they occur together, in combinations
of abiotic stress factors
Abiotic stress comes in many forms. The most common of the stressors are the
easiest for people to identify, but there are many other, less recognizable
abiotic stress factors which affect environments constantly. The most basic
stressors include: high winds, extreme temperatures, drought, flood, and
other natural disasters, such as tornados and wildfires. The lesser-known
stressors generally occur on a smaller scale and so are less noticeable, but they
include: poor edaphic conditions like rock content and pH, high radiation,
compaction, contamination, and other, highly specific conditions like rapid
rehydration during seed germination
A plant’s first line of defense against abiotic stress is in its roots. If the soil
holding the plant is healthy and biologically diverse, the plant will have a
higher chance of surviving stressful conditions. Facilitation, or the positive
interactions between different species of plants, is an intricate web of
association in a natural environment. It is how plants work together. In areas
of high stress, the level of facilitation is especially high as well. This could
possibly be because the plants need a stronger network to survive in a harsher
environment, so their interactions between species, such as cross-pollination
or mutualistic actions, become more common to cope with the severity of
their habitat. This facilitation will not go so far as to protect an entire species,
however. For example, cold weather crops like rye, oats, wheat, and apples
are expected to decline by about 15% in the next fifty years and strawberries
will drop as much as 32% simply because of projected climate changes of a
few degrees. Plants are extremely sensitive to such changes, and do not
generally adapt quickly. Plants also adapt very differently from one another,
even from a plant living in the same area. When a group of different plant
species was prompted by a variety of different stress signals, such as drought
or cold, each plant responded uniquely. Hardly any of the responses were
similar, even though the plants had become accustomed to exactly the same
home environment
5. What is a herbicide resistance and how will you achieve it using gene transfer
technology.
Excessive weed growth forces crops to compete for sunlight and nutrients,
often leading to significant losses. Because herbicides cannot differentiate
between plants that are crops and plants that are weeds, conventional
agricultural systems can only use 'selective' herbicides. Such herbicides do not
harm the crop, but are not effective at removing all types of weeds. If farmers
use herbicide resistant crops, 'non-selective' herbicides can be used to remove
all weeds in a single, quick application. This means less spraying, less traffic
on the field, and lower operating costs.
Herbicides for controlling weeds in sugar beet fields (left: field treated with
herbicides; right: without herbicides)
'Non-selective' herbicides: Not always useful
'Broad-spectrum', or non-selective herbicides are effective at killing a wide
range of weeds. The problem is, they can also kill valuable crops. Therefore,
broad-spectrum herbicides are only useful before seedlings emerge or in
special cases like fruit orchards, vineyards, and tree nurseries.
Herbicide resistant crops are changing weed managment
Several crops have been genetically modified to be resistant to non-selective
herbicides. These transgenic crops contain genes that enable them to degrade
the active ingredient in an herbicide, rendering it harmless. Farmers can
thereby easily control weeds during the entire growing season and have more
flexibility in choosing times for spraying.
Herbicide resistant crops also facilitate low or no tillage cultural practices,
which many consider to be more sustainable. Another advantage is that
farmers can manage weeds without turning to some of the more
environmentally suspect types of herbicides.
Critics claim that in some cases, the use of herbicide resistant crops can lead to
an increase in herbicide use, promote the development of herbicide resistant
weeds, and damage biodiversity on the farm. Extensive ecological impact
assessments have been addressing these issues.
Among the field trials conducted on herbicide resistant crops, studies in the
United Kingdom have shown that different herbicides and different herbicide
application practices can affect the amount of wild plants on the farm. In
comparison with conventional cropping systems, weed and animal
populations were negatively affected by herbicide tolerant sugar beet and
rapeseed, but biodiversity was increased with the use of herbicide tolerant
maize.
Currently, two herbicide resistant cropping systems are common for soybean,
maize, rapeseed, and cotton
Part – C
1. Explain Shikimate pathway and its role in the production of secondary
metabolites.
Shikimic acid, more commonly known as its anionic form shikimate, is an
important biochemical metabolite in plants and microorganisms. Its name
comes from the Japanese flower shikimi (Illicium anisatum), from which it
was first isolated.
Shikimic acid is a precursor for: the aromatic amino acids phenylalanine and
tyrosine, indole, indole derivatives and aromatic amino acid tryptophan,
many alkaloids and other aromatic metabolites, tannins, flavonoids, and
lignin.
In the pharmaceutical industry, shikimic acid from the Chinese star anise
is used as a base material for production of oseltamivir (Tamiflu). Although
shikimic acid is present in most autotrophic organisms, it is a biosynthetic
intermediate and generally found in very low concentrations. The low
isolation yield of shikimic acid from the Chinese star anise is blamed for the
2005 shortage of oseltamivir. Shikimic acid can also be extracted from the
seeds of the sweetgum fruit, which is abundant in North America, in yields of
around 1.5%. 4 kg of sweetgum seeds is needed for fourteen packages of
Tamiflu. By comparison, star anise has been reported to yield 3 to 7% shikimic
acid. Recently biosynthetic pathways in E. coli have been enhanced to allow
the organism to accumulate enough material to be used commercially.
2. Explain biochemistry and molecular pathogenecity related with nif genes
Nif genes have both positive and negative regulators. Some of nif genes are:
Nif A, D, L,K, F,H S,U,Y,W,Z
The nif genes are genes encoding enzymes involved in the fixation of
atmospheric nitrogen. The primary enzyme encoded by the nif genes is the
nitrogenase complex which is in charge of converting atmospheric nitrogenN2 To other nitrogen forms such as ammonia which the plant can use for
various purposes. Besides the nitrogenase enzyme, the nif genes also encode a
number of regulatory proteins involved in nitrogen fixation. The nif genes are
found in both free living nitrogen fixing bacteria and in symbiotic bacteria in
various plants. The expression of the nif genes is induced as a response to low
concentrations of fixed nitrogen and oxygen concentrations (the low oxygen
concentrations are actively maintained in the root environment).
The expression and regulation of nif genes while sharing common features in
all or most of the nitrogen fixing organisms in nature, have distinct characters
and qualities differed from one diazotroph to another. Examples of nif genes
structure and regulation in different diazotrophs:
Klebsiella pneumoniae – a free living anaerobic nitrogen fixing bacteria. It
contains a total of 20 nif genes located on the bacteria's chromosome in a 24kb
region. nifH, nifK and nifD encode the nitrogenase's subunits, while nifE,
nifN, nifU, nifS, nifV, nifW, nifX, nifB and nifQ encode proteins involved the
assembly and incorporation of the Fe and Mo into the nitrogenase's subunits.
nifF and nifJ encode proteins related to electron transfer taking place in the
reduction process and nifA is a regulatory protein in-charge of regulating the
nif genes expression. Regulation of the nif genes expression is done through
nifA and nifL in the same way described in the previous chapter of the nif
genes structure and regulation. (3,5) Nif genes structure in K. pneumoniae (5):
Rhodospirillum rubrum – a free living anaerobic photosynthetic bacteria
which in addition to the transcriptional control described before, regulates
expression of the nif genes also in a metabolic way through a reversible ADPribosylation of a specific arginine residue in the nitrogenase complex. The
ribosylation takes place when reduced nitrogen is present and it causes a
barrier in the electron transfer flow and by so, inactivates nitrogenase's
activity. The enzymes catalyzing the ribosylation are called DRAG and DRAT.
(5,9)
Rhodobacter capsulatus - free living anaerobic phototroph containing a
transcriptional nif genes regulatory system. R. capsulatus regulates nif genes
expression through nifA in the same manner described before, but it uses a
different nifA activator called factor which initiates the RcNtrC. RcNtrC
activates a different Expression of nifA and the- nif genes. (5,9).
Rhizobium spp. - Gram negative, symbiotic nitrogen fixing bacteria. It usually
forms symbiotic relationship with Legum species. In some Rhizobia, the nif
genes are located on plasmids called the sym plasmids (sym = symbiosis)
which contain genes related to nitrogen fixing and some housekeeping genes,
while the chromosomes contain most of the housekeeping genes of the
bacteria. Regulation of the nif genes expression is done at the transcription
level inside the symbiotic plant
3. Explain the importance of plant secondary metabolites to human society. Add
a note on the contribution of biotechnology to this area.
Secondary metabolites are organic compounds that are not directly involved
in the normal growth, development, or reproduction of an organism.[1]
Unlike primary metabolites, absence of secondary metabolities does not result
in immediate death, but rather in long-term impairment of the organism's
survivability, fecundity, or aesthetics, or perhaps in no significant change at
all. Secondary metabolites are often restricted to a narrow set of species
within a phylogenetic group.[2] Secondary metabolites often play an
important role in plant defense against herbivory[3] and other interspecies
defenses. Humans use secondary metabolites as medicines, flavorings, and
recreational drugs.
Most of the secondary metabolites of interest to humankind fit into categories
which classify secondary metabolites based on their biosynthetic origin. Since
secondary metabolites are often created by modified primary metabolite
synthases, or "borrow" substrates of primary metabolite origin, these
categories should not be interpreted as saying that all molecules in the
category are secondary metabolites (for example the steroid category), but
rather that there are secondary metabolites in these categories.
Small "small molecules"





Alkaloids (usually a small, heavily derivatized amino acid):
o Hyoscyamine, present in Datura stramonium
o Atropine, present in Atropa belladonna, Deadly nightshade
o Cocaine, present in Erythroxylon coca the Coca plant
o Scopolamine, present in the Solanaceae (nightshade) plant family
o Codeine and Morphine, present in Papaver somniferum, the opium
poppy
o Tetrodotoxin, a microbial product in Fugu and some salamanders
o Vincristine & Vinblastine, mitotic inhibitors found in the Rosy
Periwinkle
Terpenoids (come from semiterpene oligomerization):
o Azadirachtin, (Neem tree)
o Artemisinin, present in Artemisia annua Chinese wormwood
o tetrahydrocannabinol, present in cannabis
o Steroids (Terpenes with a particular ring structure)
 Saponins (plant steroids, often glycosylated)
Glycosides (heavily modified sugar molecules):
o Nojirimycin
o Glucosinolates
Natural phenols:
o Resveratrol
Phenazines:
o Pyocyanin
o Phenazine-1-carboxylic acid (and derivatives)
Big "small molecules", produced by large, modular, "molecular
factories"



Polyketides:
o Erythromycin
o Discodermolide
Fatty acid synthase products :
o FR-900848
o U-106305
o phloroglucinols
Nonribosomal peptides:
o Vancomycin
o Thiostrepton
o Ramoplanin
o Teicoplanin
o Gramicidin
Bacitracin
Hybrids of the above three:
o Epothilone
Polyphenols
o


Non-"small molecules" - DNA, RNA, ribosome, or polysaccharide
"classical" biopolymers

Ribosomal peptides:
o Microcin-J25
4. Explain certain applications of transgenic technology in terms of
manipulation of plants.
Progress is being made on several fronts to introduce new traits into plants
using recombinant DNA technology. The genetic manipulation of plants has
been going on since the dawn of agriculture, but until recently this has
required the slow and tedious process of cross-breeding varieties. Genetic
engineering promises to speed the process and broaden the scope of what can
be done.
There are several methods for introducing genes into plants, including
• infecting plant cells with plasmids as vectors carrying the desired gene;
• shooting microscopic pellets containing the gene directly into the cell.
In contrast to animals, there is no real distinction between somatic cells and
germline cells. Somatic tissues of plants, e.g., root cells grown in culture
[View],
• can be transformed in the laboratory with the desired gene;
• grown into mature plants with flowers.
If all goes well, the transgene will be incorporated into the pollen and eggs
and passed on to the next generation.
In this respect, it is easier to produce transgenic plants than transgenic
animals.
Some Achievements
1. Improved Nutritional Quality
Milled rice is the staple food for a large fraction of the world's human
population. Milling rice removes the husk and any beta-carotene it contained.
Beta-carotene is a precursor to vitamin A, so it is not surprising that vitamin A
deficiency is widespread, especially in the countries of Southeast Asia. The
synthesis of beta-carotene requires a number of enzyme-catalyzed steps. In
January 2000, a group of European researchers reported that they had
succeeded in incorporating three transgenes into rice that enabled the plants
to manufacture beta-carotene in their endosperm.
2. Insect Resistance.
Bacillus thuringiensis is a bacterium that is pathogenic for a number of insect
pests. Its lethal effect is mediated by a protein toxin it produces. Through
recombinant DNA methods, the toxin gene can be introduced directly into the
genome of the plant where it is expressed and provides protection against
insect pests of the plant. Link to illustrated discussion of Bacillus
thuringiensis.
3. Disease Resistance.
Genes that provide resistance against plant viruses have been successfully
introduced into such crop plants as tobacco, tomatoes, and potatoes.
Tomato plants infected with tobacco mosaic virus (which attacks tomato
plants as well as tobacco). The plants in the back row carry an introduced
gene conferring resistance to the virus. The resistant plants produced three
times as much fruit as the sensitive plants (front row) and the same as control
plants. (Courtesy Monsanto Company.)
4. Herbicide Resistance.
Questions have been raised about the safety — both to humans and to the
environment — of some of the broad-leaved weed killers like 2,4-D.
Alternatives are available, but they may damage the crop as well as the weeds
growing in it. However, genes for resistance to some of the newer herbicides
have been introduced into some crop plants and enable them to thrive even
when exposed to the weed killer. Effect of the herbicide bromoxynil on
tobacco plants transformed with a bacterial gene whose product breaks down
bromoxynil (top row) and control plants (bottom row). "Spray blank" plants
were treated with the same spray mixture as the others except the bromoxynil
was left out. (Courtesy of Calgene, Davis, CA.)
5. Salt Tolerance
A large fraction of the world's irrigated crop land is so laden with salt that it
cannot be used to grow most important crops. However, researchers at the
University of California Davis campus have created transgenic tomatoes that
grow well in saline soils. The transgene was a highly-expressed
sodium/proton antiport pump that sequestered excess sodium in the vacuole
of leaf cells. There was no sodium buildup in the fruit.
6. "Terminator" Genes
This term is used (by opponents of the practice) for transgenes introduced
into crop plants to make them produce sterile seeds (and thus force the farmer
to buy fresh seeds for the following season rather than saving seeds from the
current crop).
The process involves introducing three transgenes into the plant:
• A gene encoding a toxin which is lethal to developing seeds but not to
mature seeds or the plant. This gene is normally inactive because of a
stretch of DNA inserted between it and its promoter.
• A gene encoding a recombinase — an enzyme that can remove the spacer
in the toxin gene thus allowing to be expressed.
• A repressor gene whose protein product binds to the promoter of the
recombinase thus keeping it inactive.
How they work
When the seeds are soaked (before their sale) in a solution of tetracycline
• Synthesis of the repressor is blocked.
• The recombinase gene becomes active.
• The spacer is removed from the toxin gene and it can now be turned on.
Because the toxin does not harm the growing plant — only its developing
seeds — the crop can be grown normally except that its seeds are sterile.
The use of terminator genes has created much controversy:
• Farmers — especially those in developing countries — want to be able to
save some seed from their crop to plant the next season.
• Seed companies want to be able to keep selling seed.
7. Transgenes Encoding Antisense RNA.
Messenger RNA (mRNA) is single-stranded. Its sequence of nucleotides is called
"sense" because it results in a gene product (protein). Normally, its unpaired
nucleotides are "read" by transfer RNA anticodons as the ribosome proceeds to
translate the message. (See mechanism of translation.)
However, RNA can form duplexes just as DNA does. All that is needed is a second
strand of RNA whose sequence of bases is complementary to the first strand; e.g.,
5´ C A U G 3´
3´ G U A C 5´
mRNA
Antisense RNA
The second strand is called the antisense strand because its sequence of nucleotides
is the complement of message sense. When mRNA forms a duplex with a
complementary antisense RNA sequence, translation is blocked. This may occur
because


the ribosome cannot gain access to the
nucleotides in the mRNA or
duplex RNA is quickly degraded by
ribonucleases in the cell (see RNAi
below).
With recombinant DNA methods, synthetic
genes (DNA) encoding antisense RNA
molecules can be introduced into the
organism.
8. Biopharmaceuticals
The genes for proteins to be used in human (and animal) medicine can be
inserted into plants and expressed by them.
Advantages:
• Glycoproteins can be made (bacteria like E. coli cannot do this).
• Virtually unlimited amounts can be grown in the field rather than in
expensive fermentation tanks.
• It avoids the danger from using mammalian cells and tissue culture
medium that might be contaminated with infectious agents.
• Purification is often easier.
Corn is the most popular plant for these purposes, but tobacco, tomatoes,
potatoes, and rice are also being used.
Some of the proteins that are being produced by transgenic crop plants:
• human growth hormone with the gene inserted into the chloroplast DNA
of tobacco plants.
• humanized antibodies against such infectious agents as
◦HIV
◦ respiratory syncytial virus (RSV)
◦ sperm (a possible contraceptive)
◦ herpes simplex virus, HSV, the cause of "cold sores"
• protein antigens to be used in vaccines
◦ An example: patient-specific antilymphoma (a cancer) vaccines. B-cell
lymphomas are clones of malignant B cells expressing on their surface a
unique antibody molecule. Making tobacco plants transgenic for the
RNA of the variable (unique) regions of this antibody enables them to
produce the corresponding protein. This can then be incorporated into a
vaccine in the hopes (early trials look promising) of boosting the
patient's immune system — especially the cell-mediated branch — to
combat the cancer.
• other useful proteins like lysozyme and trypsin
5. Explain the methods of virus resistance in plants.
There have been many reports about the development of transgenic plants
which have become resistant to virus infections. These plants showed
resis-tance to virus infections when they were transformed with sequences
re-lated to several gene functions.
Some of the areas where successes have been seen related to sequences
concerning viral capsid protein, viral move-ment protein, antisense RNA,
antibody-mediated resistance, interferon-related genes and host genes
involved in plant protection.
Viruses are biochemical complexes consisting of a RNA or a DNA genome
packaged into a protein capsid which mayor may not be sur-rounded by a
membrane envelope. The protein coat 'covered genome is referred to as the
nucleocapsid. The proteins on the surface of the capsid and envelope
determine the interaction of the virus with the host and elicit the protective
immune response against the virus. Some virus particles also contain
enzymes required to facilitate the replication of the virus.
The tobacco mosaic virus (TMV) is an example of a virus with helical
symmetry whose capsomeres (many protein subunits of the capsid) appear as
projections that are assembled on the RNA genome into rods extending to the
length of the genome.
In other viruses the capsomere arrangement is cubical or icosahedral
enclosing its nucleic acid component. Engineering resistance in plants
involves either countering the capsid properties or dis-rupting the virus
replicating mechanisms in the hot.
6. What are the methods of insect resistance in plants.
Insect attack is a serious agricultural problem leading to yield losses and
reduced product quality. Insects can cause damage both in the field and
during storage in silos. Each year, insects destroy about 25 percent of food
crops worldwide. The larvae of Ostrinia nubilalis, the European corn borer,
can destroy up to 20 percent of a maize crop.
European corn borer: A major pest in southern and central Europe. Insect resistant Bt
maize is already being grown in Spain, France, Germany, Portugal and the Czech
Republic.
Western corn rootworm beetles feeding on a maize cob. Certain cultivars of Bt maize are
resistant to this serious pest. GM rootworm resistant crops are not approved for cultivation
in the European Union but are now being grown in the US.
The “Bt concept” – pest resistant transgenic plants
Bacillus thuringiensis, or Bt, is a bacterium that has attracted much attention
for its use in pest control. The soil bacterium produces a protein that is toxic to
various herbivorous insects. The protein, known asBt toxin, is produced in an
inactive, crystalline form.
When consumed by insects, the protein is converted to its active, toxic form
(delta endotoxin), which in turn destroys the gut of the insect. Bt preparations
are commonly used in organic agriculture to control insects, as Bt toxin occurs
naturally and is completely safe for humans.
More than 100 different variations of Bt toxin have been identified in diverse
strains of Bacillus thuringiensis. The different variations have different target
insect specificity. For example, the toxins classified under Cry1a group target
Lepidoptera (butterflies), while toxins in the Cry3 group are effective against
beetles.
Researchers have used genetic engineering to take the bacterial genes needed
to produce Bt toxins and introduce them into plants. If plants produce Bt
toxin on their own, they can defend themselves against specific types of
insects. This means farmers no longer have to use chemical insecticides to
control certain insect problems.
Critics claim that in some cases the use of insect resistant crops can harm
beneficial insects and other non-target organisms. Extensive ecological impact
assessments have been addressing these issues. In the field, no significant
adverse effects on non-target wildlife nor long term effects of higher Bt
concentrations in soil have yet been observed.
New concepts on the way
Bt crops have been planted commercially for more than eight years. Other
naturally occuring insecticidal compounds are now becoming available as
alternatives to the Bt approach. Among these are chitinase, lectins, alphaamylase inhibitors, proteinase inhibitors, and cystatin. Plants genetically
modified to express these defense proteins are still in early stages of
development.