Bth 301 plant and agricultural biotechnology

PROF. BALASUBRAMANIAN SATHYAMURTHY 2016 EDITION BTH - 301: PLANT AND AGRICULTURAL BIOTECHNOLOGY
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FOR MSC BIOTECHNOLOGY STUDENTS
2014 ONWARDS
Biochemistry scanner
THE IMPRINT
BTH - 301: PLANT AND AGRICULTURAL BIOTECHNOLOGY
As per Bangalore University (CBCS) Syllabus
2016 Edition
BY: Prof. Balasubramanian Sathyamurthy
Supported By:
Ayesha Siddiqui
Kiran K.S.
THE MATERIALS FROM “THE IMPRINT (BIOCHEMISTRY SCANNER)” ARE NOT
FOR COMMERCIAL OR BRAND BUILDING. HENCE ONLY ACADEMIC CONTENT
WILL BE PRESENT INSIDE. WE THANK ALL THE CONTRIBUTORS FOR
ENCOURAGING THIS.
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DEDICATDEDICATDEDICATDEDICATIONIONIONION
I dedicate this material to my spiritual guru Shri Raghavendra swamigal,I dedicate this material to my spiritual guru Shri Raghavendra swamigal,I dedicate this material to my spiritual guru Shri Raghavendra swamigal,I dedicate this material to my spiritual guru Shri Raghavendra swamigal,
parents, teachers, well wishers and students who always increase my moraleparents, teachers, well wishers and students who always increase my moraleparents, teachers, well wishers and students who always increase my moraleparents, teachers, well wishers and students who always increase my morale
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PREFACEPREFACEPREFACEPREFACE
Biochemistry scanner ‘THE IMPRINT’ consists of last ten years solved question
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CONTRIBUTORS:
CHETAN ABBUR ANJALI TIWARI
AASHITA SINHA ASHWINI BELLATTI
BHARATH K CHAITHRA
GADIPARTHI VAMSEEKRISHNA KALYAN BANERJEE
KAMALA KISHORE
KIRAN KIRAN H.R
KRUTHI PRABAKAR KRUPA S
LATHA M MAMATA
MADHU PRAKASHHA G D MANJUNATH .B.P
NAYAB RASOOL S NAVYA KUCHARLAPATI
NEHA SHARIFF DIVYA DUBEY
NOOR AYESHA M PAYAL BANERJEE
POONAM PANCHAL PRAVEEN
PRAKASH K J M PRADEEP.R
PURSHOTHAM PUPPALA DEEPTHI
RAGHUNATH REDDY V RAMYA S
RAVI RESHMA
RUBY SHA SALMA H.
SHWETHA B S SHILPI CHOUBEY
SOUMOUNDA DAS SURENDRA N
THUMMALA MANOJ UDAYASHRE. B
DEEPIKA SHARMA
EDITION : 2016
PRINT : Bangalore
CONTACT : theimprintbiochemistry@gmail.com or 9980494461

M. SC. BIOTECHNOLOGY – SECOND SEMESTER
BTH - 301: PLANT AND AGRICULTURAL BIOTECHNOLOGY
4 units (52 hrs)
UNIT: 1 PLANT TISSUE CULTURE 8 hrs
Scope and importance of plant tissue culture – Media composition and types, hormones
and growth regulators, explants for organogenesis, somaclonal variation and cell line
selection, production of haploid plants and homozygous cell lines. Micro propagation,
somatic embryogenesis, protoplast culture and somatic hybridization. Selection and
maitainance of cell lines, cryopreservation, germplasm collection and conservation,
plant tissue culture certification.
UNIT: 2 PLANT TRANSFORMATION TECHNIQUE 10hrs
Mechanism of DNA transfer – Agro bacterium mediated gene transfer, Ti and Ri plasmid
as vectors, role of virulence genes, design of expression vectors; 35S promoter, genetic
markers, reporter genes; viral vectors. Direct gene transfer methods-particles
bombardment, electroporation and microinjection. Binary vectors-p bluescript IIKs,
pBin19, pGreen vectors, transgene stabilility and gene silencing
UNIT: 3 METABOLIC ENGINEERING OF PLANTS 10hrs
Plant cell culture for for the production of useful chemicals and secondary metabolites
(Hairy root culture, Biotransformation, Elicitation) – pigments, flavonoids , alkaloids ;
mechanism and manipulation of shikimate pathway.
Production of Industrial enzymes, biodegradable plastics, therapeutic proteins, edible
vaccines and antibiotics using transgenic technology.
UNIT: 4 PLANT DEVELOPMENT 6hrs
Plant growth regulators, auxin, gibberlins, cytokines, abscicic acid acetylene. Biological
nitrogen fixation, importance and mechanism.
Biofertilizers – types, production, VAM, Rhizobium, Azotobacter, Mycorhiza, Actinorhiza
, Vermicomposting technology. Biopesticides.
UNIT: 5 GM TECHNOLOGY 10hrs
Crop improvement, productivity, performance and fortification of agricultural products –
Bt cotton, Bt brinjal. Herbicide resistance, viral resistance, bacterial resistance, fungal
resistance crops, Golden rice and transgenic sweet potato. Stratagies for engineering
stress tolerance , transgenic plants ; current status of transgenic plants in India and

other countries , Ethical issue associated with GM crops GM food ; labelling of GM
plants and products . Importance of integrated pest management and terminator gene
technology. Environmental impact of herbicide resistance crops and super weeds
UNIT: 6 POST-HARVEST TECHNOLOGY 8hrs
RNAi and antisense RNA technology for extending shelf life of fruits and flowers (ACC
synthase gene and polygalatonase) delay of softening and ripening of fleshy fruits
(tomato, banana watermelons) Post-harvest protection of cereals, millets and pulses.
References:
1. Chrispeels M.J.et al. Plants, Genes and Agriculture-Jones and Bartlett Publishers,
Boston.1994.
2. Gamborg O.L. and Philips G.C.Plant cell, tissue and organ culture (2nd Ed.) Narosa
Publishing House. New Delhi.1998
3. Hammound J, P McGravey & Yusibov.V. Plant Biotechnology, Springer verlag.2000
4. Heldt. Plant Biochemistry and Molecular Biology. Oxford and IBH Publishing
Co.Pvt.Ltd. Delhi. 1997
5. Lydiane Kyte and John Kleyn. Plants from test tubes. An introduction to
Micropropagation (3rd Ed.). Timber Press, Portland. 1996
6. Murray D.R. Advanced methods in plant breeding and biotechnology.Panima Publishing
Corporation.1996
7. Nickoloff J.A.Methods in molecular biology, Plant cell electroporation and electrofusion
protocols-Humana press incorp, USA. 1995.
8. Sawahel W.A. Plant genetic transformation technology. Daya Publishing House,
Delhi.1997
9. Gistou, P and Klu, H.Hand book of Plant Biotechnology (Vol. I & II).John
Publication.2004
10. Slatu A et al.The genetic manipulation of plant. Oxford University Press.2003
11. Kirakosyan A and Kaufman P.B.Recent Advances in Plant Biotechnology (1st
Ed.).Springer Publishers.2009
12. Halford N.G. Plant biotechnology: current and future applications of genetically
modified crops. John Wiely Publishers.2006

UNIT: 1 PLANT TISSUE CULTURE
Scope and importance of plant tissue culture – Media composition and types,
hormones and growth regulators, explants for organogenesis, somaclonal variation
and cell line selection, production of haploid plants and homozygous cell lines.
Micro propagation , somatic embryogenesis, protoplast culture and somatic
hybridization. Selection and maitainance of cell lines, cryopreservation,
germplasm collection and conservation, plant tissue culture certification.
SCOPE AND IMPORTANCE OF PLANT TISSUE CULTURE
The present status of plant biotechnology cannot be evaluated without appreciation of
the many possibilities and the potential of organ, tissue and cell suspension cultures.
Plants of wide taxonomic origin have been subjected to culture under strictly aseptic
conditions. Completely chemically defined media supplemented with growth regulators
and phytohormones are the basis for the exploitation of this technique.
Depending on the explants and the culture conditions cells either preserve their state of
biochemical and morphological differentiation or return to a status of embryogenic,
undifferentiated cells. The former situation can be used for organ cultures (e.g., pollen,
anthers, flower buds, roots), whereas the latter leads to many callus and suspension
types of cultures. For example, the cell culture technique has opened a facile route to
haploid cells and plants, and such systems are of great importance for genetic and
breeding studies.
Whenever a heterogeneous group of cells can be turned into a state of practically
uniform cells, this much less complicated cellular system can then be exploited to study
many problems. This has been performed with plant cell cultures for some 30 years
now. Growth of cells in medium-size and large volumes has opened interesting
applications for plant biotechnology.
Numerous physiological, biochemical, genetic, and morphological results and data on
cellular regulation stem from such investigations. Various primary and secondary
metabolic routes have been elucidated with the help of cell culture systems. The typical
sequence in pathway identification was first product and intermediate characterization,
then enzyme studies, and finally isolation of genes. Furthermore, application of gene
technology in the field of transgenic plants depends to some extent on the tissue and
cell culture techniques.

Plants are characterized by totipotency, which means that each cell possesses and can
express the total genetic potential to form a fully fertile and complete plant body. This
fact, highly remarkable from a cell biological point of view, is the genetic basis for
important and widely used applications of the cell culture technique. Differentiation of
single cells or small aggregates of cells into embryos, tissues, and even plants allows the
selection of interesting genotypes for several different fields of plant application.
The well-established procedures for mass regeneration of valuable specimens of
ornamental and crop plants constitute an important business section in agriculture and
gardening. Endangered plant species can be saved from extinction so that valuable gene
pools will not disappear. Remarkable progress has been achieved in mass regeneration
of trees from single plants or tissue pieces. This will undoubtedly be of further great
benefit for forestry because several problems in tree multiplication can thus be
circumvented.
Furthermore, it should be mentioned that plant cell suspension cultures possess a
great potential for biotransformation reactions in which exogenously applied substrates
are converted in sometimes high yields. Position and stereospecific hydroxylations,
oxidations, reductions, and especially interesting glucosylation of very different
substrates have been found. The plant cell culture technique has allowed the facile
isolation of mutants from many plant species. The overwhelming importance of mutants
for biochemical and genetic studies has been known for decades. Over the years,
mutations from all areas of cellular metabolism have been selected and characterized.
A good deal of our basic knowledge of the functioning and regulation of organisms and
cells and their organelles stems from work with mutants. The various techniques of
plated, suspended, or feeder cell-supported cell systems and even protoplasts have
found wide applications.
The normal rate of mutation and also increased levels of mutated cells induced by
physical (UV light, high-energy irradiation) or chemical mutagens (many such
compounds are known) have been used. The specific advantage of cell cultures for
mutant selection is the possible isolation of single cells from a mass of unmutated ones.
Heterotrophic, photomixotrophic, and photoautotrophic cells are available, and thus
different areas of cell metabolism can be screened for mutations.
In the cell culture field, regulatory mutants (i.e., excessive accumulation of products of
primary and secondary metabolism including visible pigments), uptake mutants (i.e.,

the normal cellular transport systems of nutrients into cells are invalidated), and
resistance mutants (i.e., pronounced cellular tolerance against toxic compounds such
as mycotoxins, pesticides, amino acid analogues, or salt) have especially been
characterized. Various auxotrophic mutants in the field of growth regulators have also
been of considerable value.
As an example, a series of studies using photoautotrophic cell suspension cultures and
the highly toxic herbicide metribuzin blocking electron transport in photosystem II will
be cited. A series of single, double, or even triple mutants of the Dl protein coded by the
chloroplast psbA gene were selected and thoroughly characterized. The various lines
allowed interesting insights into the mechanism of herbicide interference with the Dl
protein.
In a discussion of plant mutants resistant to herbicides, the impressive results on
herbicide-resistant crop plants require mention. Many of the mechanistic and metabolic
aspects of herbicide resistance were first elucidated with cell cultures. Modern plant
biotechnology has a wide choice of biochemical solutions for herbicide resistance by
inactivation and detoxification reactions. Several major crop plant species (i.e., soybean,
cotton, maize, rape) are presently cultivated to a large extent in the form of
appropriately manipulated genotypes. This development is on the one hand regarded as
a major advantage for agriculture but on the other hand as a subject of extensive and
often very critical public debate.
MEDIA COMPOSITION AND TYPES
MEDIA COMPOSITION:
Optimization of medium nutrients is important to increase the productivity of the
particular secondary metabolites. There are a number of reports describing the effects
of medium nutrients on secondary metabolism in plant cell cultures. Many of these
investigations seem to indicate a negative correlation between cell proliferation and
secondary metabolism. It might be possible that any manipulation for inhibiting cell
growth leads to an increase in the productivity of secondary metabolites, leading to
establishment of a two-stage culture system for production of phytochemicals where the
cells are first cultured in the medium appropriate for maximum biomass production
and then transferred to the growth-limiting medium for maximum productivity of
secondary metabolites as established for shikonin production in Lithospermum
erythrorhizon cell cultures.

One of the most important nutritional factors is the phosphate level in the medium.
Since Nettlership and Slator (19) first reported in 1974 that use of a phosphate-free
medium increased the alkaloid productivity of Peganum harmala cells, it has been
recognized that reducing the phosphate concentration results in growth limitation and a
concomitant increase in the level of secondary products.
Nitrogen is essential to support cell growth as a source of protein and nucleic acid
synthesis, thus affecting secondary metabolism. Generally, culture medium contains N
sources as NH3 and NO2, and both the concentration of total nitrogen and the ratio of
NH3 to NO2 regulate cell growth and secondary metabolism. In many cases, reducing
the total nitrogen concentration in the medium leads to lower cell growth and higher
product formation as typically reported for anthocyanin production by Vitis vinifera cell
cultures.
However, cell growth and production of betacyanin in Phytolacca americana cell
cultures increased with an elevated nitrogen supply. If used as a sole nitrogen source,
NH3 is often toxic to cell growth.
Shikonin production in L. erythrorhizon cell suspension cultures was completely
inhibited when the cells were cultured in NH3-containing medium. It is important to
find an optimum ratio of NH3 to NO2 for attaining maximum production of secondary
metabolites.
Sucrose is utilized most as a carbon source. In contrast to phosphate and nitrogen, an
increase in the initial sucrose concentration in culture medium leads to an increase in
secondary metabolite production. The enhancing effect of sucrose was most
impressively shown in the case of rosmarinic acid formation in Coleus blumei cell
suspension cultures, where the rosmarinic content increased sixfold in a medium
containing 5% sucrose compared with that in the control medium (2% sucrose),
reaching 12% of dry weight. This effect was not due to the higher osmotic pressure
because addition of mannitol to low-sucrose medium did not increase rosmarinic acid
production. In contrast, the stimulatory effect of sucrose on anthocyanin production in
Vitis vinifera cell cultures was shown to be due to osmotic stress. The carbonto-
nitrogen ratio is also an important factor in secondary metabolism as shown by
anthocyanin production in Vitis cell cultures.
Although less investigated compared with macronutrients, micronutrients are also
expected to affect secondary metabolism. In fact, the shikonin content in L.

erythrorhizon cell cultures increased drastically with an increasing Cu2+ level in the
medium.
Callus and Cell Suspension Cultures
Cell cultures are induced as callus tissues. Callus cultures are also usually the
materials from which cell suspension cultures are obtained. In addition, selection of
high-producing cell lines for a particular secondary metabolite is carried out using
callus tissues of either small-aggregate or single-cell origin. Suspension cultures are
established by transferring the callus tissue into liquid medium of the same
composition as used for callus tissues and agitating the culture on a rotatory or
reciprocal shaker. Suspension cultures generally comprise more homogeneous and less
differentiated cell populations and grow more rapidly than their parent callus cultures.
Furthermore, cell suspensions are suitable for continuous and/or chemostat culture
and easy to feed various chemical factors during the culture. These properties make
suspension cultures the material of choice for biochemical and molecular biological
investigation of plant secondary metabolism. Scaling up from flask to bioreactor for the
production of phytochemicals is always performed using suspension cultures.
Immobilized Cultures
Over the last two decades, immobilized culture systems have attracted much attention
for efficient production of plant secondary metabolites. Cultured cells from high-density
suspension cultures are trapped in an inert matrix such as calcium alginate gel beads,
stainless steel, and foam particles. The immobilized entities are cultured in shaken
flasks or aerated bioreactors.
Alternatively, the cell-encapsulated beads can be packed into a column, which is
percolated with nutrient medium. The major advantage of the immobilized culture
system is that cell growth and secondary metabolite production can be separated by the
precise manipulation of the chemical environment, allowing continuous or
semicontinuous operation. However, establishing the immobilized culture system for
large-scale production of phytochemicals is expensive. In addition, for efficient
operation of the immobilized system, permeation of the product from the cells to the
medium is necessary, which has not yet been fully achieved.
Organ Cultures
In spite of prolonged and concentrated efforts, many valuable phytochemicals such as
morphinan alkaloids of Papaver somniferum, tropane alkaloids of various solanaceous

species, and dimeric indole alkaloids of Catharanthus roseus cannot be produced by
callus and cell suspension cultures. Because most of these compounds start to
accumulate when the proper organs are regenerated from the cultured cells, production
of these compounds in cultured cells requires decoupling of biochemical differentiation
from morphological differentiation, which has so far been unsuccessful. This situation
makes organ cultures a favored option. One major disadvantage of organ cultures is
reduced productivity in bioreactors because the physical structure of shoots or roots
results in various difficulties including handling problems at inoculation and shear of
the organs during culture.
Shoot Cultures
Multiple shoots regenerated either from callus cultures or directly from explants
including apical buds are cultured in solid or liquid medium. Shoot cultures have been
considered appropriate when the target secondary metabolites are produced in aerial
parts of plants. Monoterpenoid essential oil flavors, which are not produced in
dedifferentiated callus or cell suspension cultures because of lack of oil-secretory
tissues, have been reported to accumulate in shoot cultures.
Production of a sesquiterpene lactone artemisinin that exhibits potent antimalarial
activity in shoot cultures of Artemisia annua has also been actively investigated.
A dimeric indole alkaloid anhydrovinblastine that is a direct precursor of antileukemic
indole alkaloids, vinblastine and vincristine, accumulated in shoot cultures of
Catharanthus roseus at a level similar to that in the leaves of intact plants. Vindoline
and catharanthine, precursors of the dimeric indole alkaloids, were also produced in
multiple shoot cultures of C. roseus.
In a few cases, multiple shoots transformed with Agrobacterium tumefaciens were used
to investigate secondary metabolite production. It is interesting to note that providing
the cultured shoots with environments similar to those of the intact plants sometimes
results in enhancement of the particular secondary metabolism. For example, menthol
production in Mentha arvensis shoot cultures increased with light illumination. Dimeric
indole alkaloid production in C. roseus shoot cultures was also stimulated by near-
ultraviolet light irradiation. Rooting was also reported to enhance artemisinin formation
in cultured shoots of A. annua.

Root Culture
There are two types of root culture, untransformed root culture and hairy root culture,
which are obtained by transformation with Agrobacterium rhizogenes.
Hairy roots generally show more vigorous growth than untransformed roots. However,
untransformed roots sometimes show vigorous growth to an extent similar to that of
transformed roots when cultured in auxin-containing me dium. Production of
hyoscyamine and scopolamine, pharmacologically active tropane alkaloids, was more
active in normal root cultures of Duboisia myoporoides than in the hairy roots. Scaling
up of transformed Atropa belladonna root cultures was attained without any reduction
in tropane alkaloid productivity by combining cutting treatment of seed roots with use
of a stirred bioreactor with a stainless steel net. It should also be pointed out that the
research on tropane alkaloid production in root cultures of solanaceous plants starting
in the mid-1980s has fruited as molecular biological characterization and genetic
engineering of tropane alkaloid biosynthesis.
Bioreactor Cultures
Irrespective of suspended cells, immobilized cells, or whole organs, it is necessary to
establish efficient large-scale bioreactor systems for commercial production of
secondary metabolites.
HORMONES AND GROWTH REGULATORS
Effects of plant growth regulators, especially auxin and cytokinin, on secondary
metabolism in cell cultures have been extensively investigated. It is well known that
auxin is essential and cytokinin is preferable to induce cell dedifferentiation and to
maintain cell proliferation in vitro. In has also been widely recognized that the
concentration and balance of auxin and cytokinin affect organ regeneration from
cultured cells. These growth regulators regulate secondary metabolism in in vitro-
cultured cells probably through controlling cell differentiation. However, the effects of
auxin and cytokinin are variable from species to species and from product to product,
and the mechanism by which the plant growth regulator up- or down-regulates the
particular secondary metabolism is not clear in most cases.
Gibberellin is usually not added to culture medium, and only a few reports describe its
effect on natural product biosynthesis. Production of berberine in Coptis japonica cell
cultures was increased by gibberellin. In contrast, gibberellin inhibited shikonin
biosynthesis in Lithospermum erythrorhizon cell cultures.

For practical application of plant cell cultures to secondary metabolite production, it is
desirable to culture the cells without phytohormones, especially when the product is
used as a crude extract, for example, in the case of anthocyanin, because
contamination of the phytohormones from the culture medium may influence human
health. In mammalian cell cultures, hybridomas produced by fusion of antibody-
producing cells having no proliferation activity with highly proliferative myeloma cells
are used to produce monoclonal antibodies. A similar approach could be possible in
plant cell cultures. Actually, protoplasts from petals of Petunia hybrida were fused with
protoplasts from cultured crown gall tumor cells, and microcalli thus produced grew
vigorously on hormone-free medium and formed anthocyanin characteristic of parent
petals.
EXPLANTS FOR ORGANOGENESIS
An ability to regenerate plants fromcultured cells and tissues at high frequency has
been considered as the corner stone of plant biotechnology, as it serves as an
alternative means of vegetative propagation and is a pre-requisite for the production of
genetically modified crops. In eggplant, plants have been regenerated from somatic cells
and tissues, or from anthers and microspores via organogenesis and/or embryogenesis.
Organogenesis
Cultivated andwild species of eggplant have been regenerated through organogenesis.
Organogenesis has been achieved using various explants, including those of hypocotyl,
leaf, cotyledon and root.Regeneration has also been reported in cell suspensions and
protoplast cultures. However, hypocotyl explants have been most commonly used for
organogenesis, comparedto cotyledonexplants.
Organogenic response varies considerably with genotypes and explants. In addition, the
variation has also been detected within a single explant that follows a basipetal pattern.
This variability in morphogenetic response correlates to changes in the spatial
distribution of polyamines. This finding is supported by the results of a study showing
that organogenesis in cotyledon explant correlates with the hormone-mediated
enhancement of biosynthesis and conjugation of polyamines.

Organogenesis in eggplant from leaf explant. A Multiple shoot bud formation from
callus. B Rooting of shoots in vitro. C Regenerated plants. Bar 1 cm
The formation of shoot buds can be induced from cultured explants grown in medium
containing cytokinins (Gleddie et al. 1983).Direct organogenesis in eggplant has been
reported in the presence of a combination of benzylaminopurine
(BAP) and indole-3-acetic acid (IAA), kinetin or thidiazuron (TDZ).
Indirect shoot regeneration, via the intervening callus stage, was
obtainedusingcombinations of IAA with BAP, kinetin or zeatin, α- aphthaleneacetic acid
(NAA) andBAP, or combinations of IAA, 2,4-dichlorophenoxyacetic acid (2,4-D) and
kinetin, followed by IAA and 2-isopentyladenine (2iP).
In contrast, cell suspension cultures grown in the presence of IAA and 2iP gave rise to a
nodule-like structure that, however, failed to develop into shoots. Shoot formulation
occurred after the culture was transferred to medium containing ascorbic acid or the
antiauxin, p-chlorophenoxyisobutyric acid.
In order to generate lines resistant or tolerant to diseases or abiotic stress, tissue and
cell cultures of eggplant have been used to regenerate plants on media supplemented
with pathogen toxins or abiotic stress agents. A similar approach has also been
employed in an attempt to understand gene function and activity under stress
conditions. Electrolyte release fromregenerating eggplant calli has been used in
screening for resistance and susceptibility to Verticillium dahliae. The induction of
laterals in eggplant root cultures in hormone-free liquid medium was reported by
Sharma and Rajam (1997) and this system was used to demonstrate the role of
polyamines in root growth and differentiation of lateral roots. These workers showed

that polyamines, particularly spermidine, were intimately involved in root growth and
differentiation of lateral roots.
SOMACLONAL VARIATION AND CELL LINE SELECTION
This technique has been described in detail by Larkin and Scowcroft (1981) and
specifically in fruit crops by Hammerschlag (1992). It arises when plant explants are
subjected to a tissue culture cycle. The cycle includes establishment of a
dedifferentiated cell or tissue culture under defined conditions and the subsequent
regeneration of plants. Variation at cellular level occurs either in cells before explant
excision or during the tissue culture cycle.
The degree of variation depends on many factors, including:
The origin of the explant used (organ, age, genotype)
The time that cells or tissues are maintained in vitro
The time and intensity of the mutagenic agents used
Reduction of somaclonal variation is achieved by using appropriate culture media and
by shortening subculture intervals. Somaclonal variation can be 10,000 times higher
than spontaneous mutation rates in whole plants. Many phenotypic variations reported
in the regenerated fruit crop plants were extensively reviewed by Hammerschlag (1992).
Important changes include growth rate and reproductive apparatus modification
(sterility, precocious flowering and flower abnormalities, internodal length), and leaf
(variegation, albino, chlorotic, etc.), thornlessness, isoenzymatic activity changes, and
increased salt resistance, fruit colour, etc.
An increased ploidy level has been reported in kiwi subcultures and in grape. Some
changes are not hereditable, since they have epigenetic origin. These changes include:
Cytokinin and auxin habituation
Chilling resistance
Changing susceptibility to fungal attack
Susceptibility to certain pathogens, due maybe to virus elimination during regeneration,
which can also alter plant habit.
PRODUCTION OF HAPLOID PLANTS AND HOMOZYGOUS CELL LINES
Selection of parental lines for rye breeding is more effective at the level of homozygous
inbred lines than at the heterozygous level, especially with respect to quantitative
characters. Homozygous inbred lines are also very useful in test crosses, particularly in
hybrid breeding. Conventional production of inbred lines in a heterozygous crop, such

as rye, is frequently complicated by self-incompatibility and requires at least five
generations of controlled selffertilization.
Several methods of haploid production have been investigated in cereals, including
microspore and/or anther culture (androgenesis), ovule culture (gynogenesis), Hordeum
bulbosum L. or maize pollination methods and an alien cytoplasm system. In contrast to
other methods, androgenesis has been adapted for haploid formation in rye. Following
the culture of anthers or isolatedmicrospores, embryo formation from immature pollen
and subsequent regeneration of embryos into plants can result in the formation of
haploid plants. The formation of double haploids occurs either spontaneously, or is
induced by colchicine treatment.
Early studies onthe productionof double haploids in rye, beguninthe 1970s,
concentrated on anther culture. The best results were obtained using progenies of
Secale cereal × vavilovii, a genotype which lacks agronomic importance.
Immonen and Anttila (1996) and Rakoczy-Trojanowska et al. (1997) identified
responsive true rye lines. Deimling et al. (1994) reported the successful regeneration of
plants from isolated cultured microspores of semi-wild rye SC35. The culture of isolated
microspores eliminates the risk of plants arising from diploid tissue (septum, anther
wall or tapetum). Guo and Pulli (2000) demonstrated relatively high callus induction
and green plant regeneration fromisolated culturedmicrospores of true rye (Secale
cereale L.). Compared to anther culture, cultured isolated microspores resulted in more
green double haploid plants per anther in four rye genotypes.
Most advances toward improving anther or microspore culture methods used specific
genotypes and “stress” treatments, like cold or osmotic pretreatment, to induce
androgenesis from the preprogrammed gametophytic to the sporophytic pathway.
Compared to barley andwheat, the development of an efficient androgenic cell culture
system for rye is less advanced. Problems associated with rye anther and microspore
culture include poor embryogenic callus induction, limited green plant regeneration, a
high proportion of albinos and severe genotype dependency.More effective methods are
needed for producing double haploids from a wide range of genotypes.
MICRO PROPAGATION AND SOMATIC EMBRYOGENESIS
Breeders of woody plants, including ornamentals, fruit trees, and forest trees, are
especially inclined to adopt micropropagation in their breeding programs.

Clearly, this technique has reached practical application and can in some cases
considerably shorten the tail end of a breeding program: the production of material
used in cultivation. In some of the most drastic cases, mutations found in natural
populations can be directly micropropagated for usage, e.g., in propagating ornamental
trees of special form or color.
In Finland this technique has found direct application in propagating various birch
clones (curly birch, laciniate forms, and high-yielding single pair matings) and hybrid
aspen clones. However, the advantages of micropropagation must be evaluated
economically in comparison with other cloning techniques, such as root and shoot
explants or direct rooting of cuttings. It appears in many cases that the more
conventional rooting of cuttings is simply less expensive and therefore applicable to
forest trees, where the magnitude of cloning is frequently in the million ramets range.
Economic comparisons of cloning techniques must be viewed in the light of final plant
prices. For example, plants produced for forestry plantations must sell for a maximum
$1 apiece as the landowner needs to plant stands of approximately 2000 plants per
hectare, whereas ornamental rhododendron plants may sell for $20 apiece as the
landowner considers only single plants and not stands. Thus, the extra price added
through micropropagation is essential in case of a forestry plant but futile in the case of
an expensive ornamental. The final method of cloning plants depends on the numbers
required and unit prices.
The dream of every plant and tree breeder is the application of somatic embryogenesis
to a breeding program. First, in this way one could avoid the tissue and plant aging
phenomenon, which causes many problems particularly in woody species, including
trees. It may cause plagiotrophic growth of tissues taken from mature trees as the
basically totipotent cells are genetically fixed in developmental stages.
Second, it may, in combination with cryopreservation, be used for keeping cell lines or
tissues embryogenic and ready for multiplication once the same material has been
through comparative field testing for yield or other special values for a number of years.
Third, a somatically induced embryonic cell line or tissue can eventually be handled for
the production of "artificial seed." This would be not only a seed but also a "clonal seed"
that could be multiplied in great numbers. If the somatic tissue in addition has a
desirable transgene, it would open great vistas for fast adoption of transgenic plants.
Fourth, somatic embryogenesis could be used for "fixing heterosis" in clones. In this

way one could circumvent the tedious construction of male sterility systems to produce
hybrid seed.
Basically, one would need only one heterotic plant for manufacturing large numbers of
hybrid clonal seeds. There are several examples of vigorous growth in interspecific
hybrids of trees. However, it has often been impossible in practice to utilize hybrid vigor
because of difficulties in producing hybrid seed. This is the case in larch hybrids, such
as Larix decidua X L.gmelini japonica (Fig. 1). To have practical importance, hybrid seed
must be produced in hundreds of kilograms. This has not been possible by the seed
orchard mode of breeding because of species differences in timing of flowering. Selection
of superior larch hybrids, already present as mature trees, and the conversion of their
somatic cells to an embryonic state would solve many present problems for practical
forestry. Alternatively, cell lines or tissues could be kept cryopreserved until field testing
indicates which hybrid genotypes to clone.
However, this section has to be ended with the statement that there is still a long way to
go before things work on a practical scale. What you can manipulate in the research
laboratory is a long way from practical application in a plant or tree breeding program.
Eventually, however, with the large concentration on basic research at the moment,
new useful applications will be adopted.
PROTOPLAST CULTURE
Protoplasts are plant cells from which the cell wall has been enzymatically or
mechanically removed. As with animal cells, the contents of the protoplast cytoplasm
are enclosed in a cell membrane, and the transformation of protoplasts
can be achieved using many of the procedures routinely used to transfect cultured
animal cells. Two procedures that are commonly used to introduce DNA into protoplasts
include the uptake of naked DNA mediated by polyethylene glycol and a divalent cation
(either Ca2+ or Mg2+) and electroporation, although other agents such as lipofectin
have also been used. Plant protoplasts can also be transformed using Agrobacterium.
Following transformation, protoplasts are placed on selective medium and allowed to
regenerate new cell walls. The cells then proliferate and form a callus from which
embryos or shoots and roots can be regenerated with appropriate hormonal treatments.
In principle, protoplasts from any plant species can be transformed, but the technology
is limited by the ability of protoplasts to regenerate into whole plants, which is not
possible for all species. Although economical and potentially a very powerful procedure,

direct transformation of protoplasts is disadvantageous because of the long culture
times involved. Not only does this mean that the transformation process itself is time
consuming, but cells cultured for extensive periods either fail to regenerate or
frequently regenerate plants that show full or partial sterility and other phenotypic
abnormalities (somaclonal variation). Protoplast preparation, maintenance, and
transformation is a skilled technique, which, compared with Agrobacterium mediated
transformation and particle bombardment procedures, requires a greater investment in
training. Advances in other transformation techniques are making protoplast
transformation obsolete.
SOMATIC HYBRIDIZATION
Pelletier introduces the use of somatic hybridization techniques in plant breeding as
follows: "Somatic hybridization needs the 'isolation' of intact protoplasts, the
interparental 'fusion' of these naked cells, the 'sustained divisions' of fusion products
before or after their 'selection' and the 'regeneration' of plants.'"
The first time this was successfully done was in 1972, when Carlson and his group
reported on parasexual plant hybridization in Solanaceae.
Since then, the technique has been greatly refined and many protocols are available for
producing fusion products. Basically, these protocols can be classified as chemical or
electrochemical.
The somatic hybridization techniques should be used where sexual means do not
function because of some form of incompatibility. Also, one must clearly define what
characters may be useful to manipulate by fusion and how such hybrids or cybrids can
be integrated in breeding programs. Generally, wide hybridization in plants is used in
order to integrate or incorporate valuable traits between species or in some cases even
between genera. It comes at the very beginning of a breeding program, generally to
widen the scope of genetic variability, later selected for useful and balanced gene
combinations. It can also be seen as a means of bridging between species, well
exemplified in the bridging for disease resistance in barley.
Such wide hybrids, whether sexual or parasexual, generally show distorted sexual
balance, growth, and adaptation. Thus, they have to undergo a considerable period of
"balancing" by either direct selection in populations or backcrossing to either parent or
in some cases to a third parent.

Therefore, such undertakings have recently been classified as "prebreeding" to
underline their time requirement and separation from typical breeding programs.
Looking at the economics of breeding programs, we have come across several cases in
which plant breeders doubt that the extra economic burden of interspecific
hybridization can be tolerated in plant breeding programs working in a competitive
environment. It therefore seems more plausible that such activities should be carried
out as direct extensions of national or international genetic resources units (gene
banks), whereby the extra economic burdens are carried by national or international
research centers, publicly funded.
Pelletier points at the future possibilities. Thus, it can be stated that the technology has
developed to a point at which plant regeneration from protoplasts is functioning in most
of the major crop species, including the cereals. In conjunction with polyploidy, a
number of new methods of transferring organelle genomes and combining cytoplasms
with nuclei of widely differing species seems possible.
One must keep in mind that from a plant breeder's point of view, the new techniques
are not shortcuts to superior cultivars but they extend classical breeding programs.
Still, these techniques must be used; they all widen the genetic variability that the plant
breeder considers imperative for combining new genes for better cultivated plants in a
future world that suffers from food, feed, fiber, and fuel shortages.
SELECTION AND MAINTENANCE OF CELL LINES
Anthocyanin constitutes a major flavonoid pigment. It is ubiquitous in the plant
kingdom and provides scarlet to blue colors in flowers, fruits, leaves, and storage
organs. Chemically, they are based on a single aromatic molecule, that of delphinidin
and all are derived from this pigment by hydroxylation, methylation, or glycosylation.

Interest in anthocyanins as food colorants has been increasing not only because they
are less toxic than synthetic red dyes but also because they exhibit significant
antioxidant activity, which might protect against cardiovascular diseases and certain
cancers.
There have been a number of publications describing anthocyanin production by plant
cell cultures; some of the more recent ones are summarized in Table.
Progress in sophisticated spectroscopic technology such as fast atom bombardment
mass spectrometry (FAB-MS) and two-dimensional nuclear magnetic resonance (2D-
NMR) together with high-performance liquid chromatography (HPLC) separation has led
to elucidation of complex structures of anthocyanin as reported in cell suspension
cultures of Perilla sp., Daucus carota, Ajuga reptans, and Ajuga pyramidalis.
Although it is suggested that polyacylated anthocyanins are stable in neutral aqueous
solution and thus suitable for application as food colorants, the occurrence of such
acylated anthocyanins in plant cell cultures has not been reported in many cases.
However, this does not necessarily mean that plant cell cultures lack the ability to
synthesize acylated anthocyanins but may mean that they have been overlooked
because of the lability of the acylated pigments in the extraction solvent containing
hydrochloric acid.
Because anthocyanin is relatively inexpensive, extensive optimization of anthocyanin
production in cultured plant cells is necessary to reduce the production costs for
commercial, including cell line selection, manipulation of the physical and chemical

environment, process management of largescale culture, and manipulation of the
genome using genetic engineering.
Cell Line Selection
Even when cell cultures accumulate anthocyanin, it is apparent that the cultures are
mostly heterogeneous and the population of the pigmented cells is usually low. For
example, only 10% of Cathcimnthus roseus cells in culture actively accumulated
anthocyanin. However, because anthocyanins are colored compounds, it is relatively
easy to perform visual cell aggregate selection by repeatedly subculturing red sectors of
the callus tissues. Such cell aggregate selection was described most typically and
systematically in Euphorbia milli callus, whose anthocyanin levels increased sevenfold,
reaching 13% of dry weight, and the capability was stable for 24 passages, and it has
been applied to various anthocyanin-producing plant cell cultures.
By small-aggregate cloning combined with HPLC analysis, a cell line of Vitis vinifera
accumulating malvidin-3-glucoside, a main anthocyanin in most red wines, at 63% of
total anthocyanins was established from an initial culture with a malvidin-3-glucoside
level of 13%.
CRYOPRESERVATION - GERMPLASM COLLECTION AND CONSERVATION
Cryopreservation is the technique of freezing cells and tissues at very low temperatures
at which the biological material remains genetically stable and metabolically inert, while
minimizing ice crystal formation. In general, when a tissue
is subjected to low temperatures, ice crystals will eventually form. These crystals may
disrupt the cell membrane leading to the death of the cell.
The goal of cryopreservation is to replace some of the water with other compounds that
will not form large crystals when frozen. The most commonly used replacements are
DMSO (dimethyl sulfoxide) and glycerol. These are mixed into a solution with media or
serum in which cells are suspended and placed in a liquid nitrogen freezer. As the
media begins to freeze, the salt concentration outside the cells will become greater than
that in the cells and water will leave the cells to be replaced by the cryopreservative.
Cryopreservation techniques
Most plant living cells have high quantities of water and they are extremely sensitive to
temperatures below 0ºC. Therefore, cells should be dehydrated to avoid ice crystal
formation (Mazur, 1984). However, extreme desiccation also produces damages (on cell
membrane, due to high concentration of internal solutes, protein

denaturation).Cryopreservation techniques have been developed to minimize both types
of damages.
Classical methods
Plant cryopreservation procedures were f irstly developed following the success of
animal cell cryopreservation (Grout and Morris, 1987). They were based on chemical
cryoprotection and slow cooling, followed by rapid immersion in liquid nitrogen. It is
named controlled freezing, slow freezing or two stepfreezing method. By decreasing
temperature at a relatively slow rate, ice crystals are formed in the extracellular solution
and water is removed from the intracellular one, leading to cellular dehydration and
therefore avoiding intracellular ice formation (Meryman and Williams, 1985). Such
«classical» cryopreservation procedures have been highly successful with callus and cell
cultures, consisting of rather uniform and small units (Schrijnemakers and Van Iren,
1995; Lynch, 2000). For example, sugarcane and banana embryonic calli were
successfully cryopreserved with this method and the field performance of the recovered
plants studied. In both cases, no differences between cryopreserved and control plants,
on agronomic traits and/or growth descriptors, were found after a period (two culture
cycles or 12 months) of growth in the field. This technique is usually not very effective
to cryopreserve larger structures which comprise different cell types, such as shoot
apices. However, there are some successful examples of two-step shoot-tip
cryopreservation, some of them with vegetatively propagated species such as potato or
chrysanthemum.
In this method, shoot apices are treated with cryoprotective substances such as
dimethyl sulfoxide (DMSO), ethylene glycol, polyethylene glycol, mannitol, sorbitol or
sucrose, either alone or in mixtures. Subsequently, they are slowly cooled, usually at
rates of 0.5-2ºC min-1, to –40ºC and then rapidly immersed in liquid nitrogen.
Rapid rewarming is usually required to avoid recrystallisation phenomena. Transfer to
fresh medium after one day in culture is recommended due to the toxic effect of
cryoprotective mixtures. Controlled cooling rate can be achieved with the use of
(expensive) programmable freezers or by more simple devices. A cooling rate of 0.4-
0.6ºC min-1 was obtained with an ethanol bath and a –40ºC freezer for cryopreservation
of sugarcane cultures.

New methods
For the last ten years, new plant cryopreservation procedures have been developed,
which are based on vitrif ication. Vitrification can be defined as «the solidification of a
liquid brought about not by crystallization but by an extreme elevation in viscosity
during cooling» (Fahy et al., 1984). During vitrification, the solution is said to become
an amorphous glassy solid, or glass. Vitrif ication of solutions is achieved by reducing
intra- and extracellular freezable water, either by exposure of plant tissues to highly
concentrated cryoprotective mixtures or by physical desiccation, and subsequent very
rapid cooling, generally by direct immersion in liquid nitrogen.
Cryopreservation techniques based on vitrification are usually simple to carry out and
applicable to complex structures such as embryos and shoot apices.
There are two main types of new cryopreservation techniques, although combinations of
them have also been used. The first one is actually termed vitrification (sensu stricto)
and the second one encapsulationdehydration.
In the vitrification technique, the plant material is exposed to highly concentrated
cryoprotectant solutions for short periods. Previously, to induce desiccation tolerance,
tissues are cultured on medium with high sucrose (e.g. 0.3 M) or sorbitol (e.g. 1.4 M)
concentration and subsequently transferred to a glycerol-sucrose solution, called
loading solution (e.g. 2 M glycerol + 0.4 M sucrose) (Sakai, 2000). A widely used
vitrification solution is that developed by Sakai et al. (1990) and named PVS2, which
consists of 30% (w v-1) glycerol, 15% (w /v) ethylene glycol and 15% (w/v) DMSO in
liquid medium with 0.4 M sucrose.
The droplet (see Table) technique can be considered a modification of the previous one.
It was developed for potato germplasm cryopreservation at FAL (Germany) and consists
on treating shoot apices in drops of a 10% DMSO solution placed on aluminium foil
strips, which are rapidly immersed in liquid nitrogen (Mix-Wagner et al., 2003).
The encapsulation-dehydration technique is based on the artificial seed technology.
This technique was developed by Fabre and Dereuddre (1990), and consists on the
inclusion of apices in alginate beads and their subsequent culture in a highly
concentrated (0.7-1.5 M) sucrose solution followed by physical dehydration, and direct
immersion in liquid nitrogen.
Culture of apices on sucrose enriched medium (0.3- 0.7 M), prior to encapsulation,
usually improves survival after desiccation and freezing.

Physical desiccation is carried out either with silica gel or in the air flow of the laminar
flow cabinet (Paulet et al., 1993). Water contents of around 20% (fresh weight basis)
have resulted appropriate for high survival after freezing of vegetative explants in
several species.
More recently, protocols combining the above techniques have been developed and
named encapsulation- vitrification techniques (Matsumoto et al., 1995a; Hirai et al.,
1998; Sakai et al., 2000). Apices are firstly encapsulated and then submitted to
vitrification, with no requirement for physical desiccation.
In many temperate species, incubation of the in vitro-shoots, nodal segments or apices
at low temperature (generally 4º to 10ºC), for periods ranging from days to weeks,
increases survival after freezing for both types of techniques, classical and new. During
cold acclimation cellular changes (numerous smaller vacuoles, more abundant
mitochondria and rough endoplasmic reticulum) and accumulation of certain proteins
occur, as it has been shown in cryopreservation studies of peach cell suspensions
(Arora and Wisniewski, 1995).
During shoot tip cryopreservation of non cold-hardy tropical species, preculture on
sucrose enriched medium (0.3-0.8 M) improved survival after cooling either by
encapsulationdehydration or by vitrification (Takagi, 2000).
In some cases, apices pregrowth on sucrose rich medium has proven sufficient for
survival after cooling in liquid nitrogen. That is the case of banana meristem clumps
cultured on 0.4 M sucrose for 2 weeks.
Uragami et al. (1990) obtained 63% survival, after cooling in liquid nitrogen, in
asparagus nodal explants that had been previously precultured for 2 days on 0.7 M
sucrose and subsequently desiccated to 20% water content with silica gel. These two
methods, named «pregrowth» and «pregrowth and desiccation», have been especially
successful to cryopreserve zygotic and somatic embryos of several species (Engelmann,
1997a).
List of vegetatively propagated plant species for which shoot-apex
cryopreservation has been developed using different Techniques

Conservation and recovery
Long-term conservation can take place in liquid nitrogen (–196ºC) or in the vapour
phase. However, storage temperature should not be higher than –130ºC to prevent
solution devitrification and large ice crystal formation (Towill, 1991). Those
temperatures can be also achieved by deep freezers running at –150ºC. At the Federal

Centre for Breeding Research on Cultivated Plants (BAZ, Germany) the viability of
potato apices after long-term storage in liquid nitrogen has been studied (Mix-Wagner et
al., 2003). The cryopreservation technique employed was the droplet method and
storage was carried out in liquid nitrogen.
Plant regeneration from long-term (3 to 8 years) versus short-term stored apices was
studied in 51 cultivars. Only in three of them recovery showed a significant decrease in
the long-term stored apices. For the recovery of apices after cryostorage, rapid
rewarming is usually required to avoid recrystallisation (Towill, 1991). Vials containing
the apices are usually immersed in a water bath at 35-40ºC. When apices are not
included in vials (e.g. the droplet technique, Mix- Wagner et al., 2003) rewarming
usually takes place in liquid medium at room temperature.
In many species, recovery of apices cryopreserved with the new techniques is direct,
without callus formation. By contrast with the classic techniques, the structural
integrity of most cells is well preserved (Engelmann, 1997a).
Some studies have shown the importance of the appropriate post-thawing culture
conditions to enhance organised growth. For example, potato cv. Desirée shoot tips
showed higher recovery when incubation during the first week after thawing was
performed under low light intensity (Benson et medium for apex recovery may be
necessary. Adjustment of growth regulator concentration (Withers et al., 1988) or even
medium salt formulation (Pennycooke and Towill, 2001; Decruse and Seeni, 2002)
could be required for the normal development of frozen shoot apices. Some media
supplements such as iron chelating agents or surfactants have been shown to improve
recovery (Benson et al., 1995; Pennycooke and Towill, 2001).
Application of cryopreservation techniques for plant genetic resources
conservation and management
Work is being carried out in several research institutes and universities to develop
appropriate cryopreservation techniques for vegetatively propagated species (Ashmore,
1997; Engelmann, 1997b). However, the number of plant genetic resources centers
where cryopreservation is used is still low and usually concerns few accessions only
(Ashmore, 1997; Reed, 2001; Reed, pers. com.). Potato is the crop in which the number
of accessions is the highest. At BAZ (Germany) a total of 519 varieties are stored under
cryopreservation (Mix- Wagner et al., 2003). Apices have been cryopreserved using the
droplet method. At the Centro Internacional de la Papa (International Potato Centre, CIP,

Perú) 197 accessions are cryopserved by the vitrification method (Reed, 2001). Lower
numbers, but still important collections, are those of cassava (95 accessions, CIAT,
Colombia) and pear (106 accessions, National Clonal Germplasm Repository, Corvallis,
USA) (Reed, 1990, 2001; Roca et al., 2000).
Certain aspects, for which there is still not too much experience, should be considered
before the establishment and management of a cryogenic germplasm bank. Probably,
the two first questions to answer are the technique to use and the number of replicates
required.
The new cryopreservation techniques do not require expensive equipment
(programmable freezer). In all cases, personnel should be properly trained in in vitro and
cryopreservation techniques, otherwise results may vary greatly (Mix-Wagner et al.,
2003). Some preliminary testing should be carried out with a few cultivars. Vitrification
usually requires less handling than encapsulation-dehydration, but solutions may be
toxic. Encapsulation-dehydration protocols are usually simple, but more handling of
alginate beads (although easier due to their size) is required and some species do not
tolerate the high sucrose concentrations employed. The advantages and disadvantages
of each technique should be considered. However, other factors such as personnel,
available facilities, and type of plant could influence the selection of the technique
(Reed, 2001).
Those first tests with few cultivars could give indications on how the protocol should be
improved to obtain high percentage recovery. But, which recovery percentage should be
aimed and in how many cultivars? For genebank management, recovery as normal apex
regrowth giving healthy in vitro plants should be the goal. Care should be taken when
interpreting some published cryopreservation works where survival (any kind of growth)
is reported.
In some germplasm conservation centers, 20% recovery is considered enough for long-
term preservation (Golmirzaie and Panta, 2000). Other authors consider that survival
should be higher than 40% (Reed et al., 2000; Reed, 2001). It is important that those
percentages be reproducible. These survival percentages may not seem high but we
should take into account that we are dealing with clonally propagated plants, where
homogeneity should be expected. The case is different when cryopreservation of
heterogeneous material is considered (e.g. zygotic embryos from species with
recalcitrant seeds). As genotypes usually respond differently to the same protocol

(Golmirzaie and Panta, 2000; Mix-Wagner et al., 2003), curators should make the
decision of: 1) either modify the protocol for each genotype or group of genotypes to
obtain high recovery in most of them, or 2) store more propagules (to compensate for
low recovery) with a protocol that may be not optimal for all genotypes.
More recently, Dussert et al. (2003) developed a probabilistic method to help curators to
take certain decisions, for example, on the number of cryopreserved propagules to be
tested in order to get a precise estimation of the expected recovery percentage, or on the
minimum number of propagules which should be rewarmed per accession to obtain a
fixed number of viable plants.
Three examples on the number of replicates conserved (number of vials and propagules
in each one) are those used for potato and Pyrus. Twelve potato apices are kept per
cryovial and around 300 apices per variety at BAZ (Mix-Wagner et al., 2003). At CIP,
250 apices are stored per genotype (five cryovials containing 50 shoot tips each)
(Golmirzaie and Panta 2000). In the National Seed Storage Laboratory (Fort
Collins, USA) 100 Pyrus meristems (25 per cryovial) of each genotype are stored as base
collection (Reed et al., 2000). The number of replicates conserved should take into
account the recovery percentage expected and the planned periodical monitoring.
Storage should be carried out under optimal conditions (no danger of temperature
rising and therefore of solution recrystallisation), and therefore periodical monitoring
would be necessary with very low frequency. It would be advisable that the container
used for long-term conservation is employed only for that purpose. Appropriate
handling procedure for inserting or removing accessions should be carefully eveloped
and carried out to avoid accidental temperature increase. It would be desirable that the
liquid nitrogen container or freezer (–150ºC) is in a different site to the field collection
(Reed, 2001). It is very important that liquid nitrogen or power supply are secured.
Automatic-filling liquid nitrogen containers are available. Alarms for temperature
decrease detection and constant personnel availability are required. For further
security, a duplicate of the cryopreserved collection could be established in a different
site. As in all types of ex situ conservation banks, information recording is very
important. A good recording method (accession numbering, data bases) should be
established to collect information about the mother plant (passport data; Anon., 1996),
the in vitro culture phase and the cryopreservation protocol used, including all phases:
apex excision, pretreatment, cryopreservation method, thawing method and recovery

medium and incubation conditions. The last two phases are very important to secure
adequate recovery and reproducibility in the future (probably after many years) and as
much detail as possible should be included (Reed, 2001). It is important to record as
well the recovery percentage after a short conservation period (days).
A major concern of curators is the genetic stability of conserved material. In vitro
culture has been reported to induce genetic changes (somaclonal variation) in some
cases (Scowcroft, 1984). Care should be taken to test that the in vitro culture phase
does not produce genetic instability (Harding, 1999). Although not many studies have
been performed yet, cryopreservation protocols seem to ensure genetic stability of the
plant material. Sugarcane plants derived from cryopreserved embryonic clumps did not
show field performance differences when compared with non-cryopreserved ones
(González-Arnao et al., 1999; Martínez-Montero et al., 2002). Genetic stability of
cryopreserved potato shoot tips has been studied through ploidy status or ribosomal
genes stability (Harding, 1991; Benson et al., 1996; Harding, 1997). Study of the DNA
variation through amplified fragment length polymorphism assays have been carried
out in strawberry, apple, grape, and kiwi (Hao et al., 2001, 2002; Zhai et al., 2003).
To date, there is no substantial evidence to suggest that plants regenerated from
cryopreserved shoot apices are genetically changed. In potato, DNA polymorphism
observed was not induced by cryopreservation but by the whole process employed,
including the tissue culture phase (Harding, 1997). In other works where variants were
regenerated from cryopreserved apices, those were due to their chimeric structure
(Fukai et al., 1994).
PLANT TISSUE CULTURE CERTIFICATION.
Introduction
Plant Tissue Culture Technology offers great promise for the production of quality
planting material on account of disease free and true to type plants produced through
micropropagation techniques. The need for a certification programme for the tissue
culture plants is imperative since inadvertent micropropagation of virus infected plants
will not only result in its poor performance, but also in undesirable spread of viruses
wherever such plants are grown. Also, failure to use prescribed standard protocols will
result in variations in the plants produced. The most deleterious variants in tissue
culture raised plants are those that effect yield, genetic fidelity and carry infection of

viruses, and other fastidious pathogens, which are difficult to diagnose. This is an area
of great concern, and requires a well-structured system be put in place to provide
support to the tissue culture industry for the commercialization of tested virus free and
high quality planting material.
Ministry of Agriculture has vide Gazette of India Notification dated 10th March 2006
notified that “In exercise of the powers conferred under section 8 of the seeds Act, 1966
(54 of 1966), the Central Government hereby authorizes Department of Biotechnology,
Ministry of Science and Technology, Government of India to act as Certification Agency for
the purpose for certification of the tissue culture-raised propagules up to laboratory level
and to regulate its genetic fidelity as prescribed by them”
Accordingly a National Certification System for Tissue Culture Plants (NCS-TCP) has
been developed for the first time, not only in the Country but also globally, where
currently no such organized structure exists for certification of Tissue Culture material.
Role and Responsibility
Tissue Culture Certification Agency (TCCA)

The Certification Agency (DBT) is responsible for implementing the National
Certification System for Tissue Culture Plants (NCS-TCP) in the Country. An
Accreditation Unit has been setup for undertaking Accreditation of Test Laboratories
for testing of Virus and Genetic Fidelity and also Certification of Tissue Culture
Production Facilities, based on the established guidelines and criteria. Referral
Laboratories have been identified for carrying out confirmatory tests, if required, and
also for developing standard protocols, maintenance of referral material, training etc.
The Certification Agency is overall responsible for developing standard tests, production
protocols/guidelines and manuals. It will also set-up a Tissue Culture Certification
Monitoring Unit which will be responsible for monitoring issue of certificates by
Accredited Test Laboratories.
Accreditation Unit (AU):
An Accreditation Unit (AU) has been established by the Certification Agency at Biotech
Consortium India Limited (BCIL), 5th Floor, Anuvrat Bhawan, 210 Deendayal
Upadhyaya Marg, New Delhi-110002. for undertaking Accreditation of laboratory
facilities for virus diagnosis and genetic fidelity testing of tissue culture raised plants
and Certification of Tissue Culture Production Facilities.
Accreditation Panel (AP):
The AU will maintain a panel of experts to undertake initial assessment of laboratory
facilities for virus diagnosis and genetic fidelity testing for accreditation/and or
periodical auditing of accredited facilities for granting renewal/revalidation/
reinstatement. The Accreditation Panel would also make an assessment of the tissue
culture production facility. The panel comprises experts specialized in plant tissue
culture/plant biotechnology/plant virology/plant bacteriology/ molecular biology and a
phytosanitary expert. The Accreditation Panel will submit its assessment report based
on established criteria/guidelines to the accreditation unit for grant of accreditation/
certification. The guidelines for Accreditation of laboratory facilities for Virus diagnosis
and genetic fidelity testing and for Certification of tissue culture production facilities are
at Annexure-I. The AU should ensure that the essential criteria are met with prior to
initial assessment of facility for accreditation/certification.
Referral Laboratory (RL):

The DBT has designated Referral laboratory (ies) for virus diagnosis/genetic fidelity
testing of tissue cultures plants.
Referral Center for Virus Diagnosis – Indian Agriculture Research Institute (IARI), New
Delhi referral Centers for Genetic Fidelity - Centre for DNA Fingerprinting and
Diagnostics (CDFD), Hyderabad.
The referral laboratory is responsible for carrying out confirmatory tests in the event of
dispute or nonconformity of test results. The referral laboratory usually will not involve
in routine virus diagnosis/genetic fidelity testing of tissue culture raised plants,
however as per the decision of the Tissue Culture Certification Monitoring Unit, the
Referral Laboratories will undertake random testing of samples at different Test
Laboratories.
Accredited Test Laboratories (ATL):
Test laboratories will be Accredited entities of the public sector, responsible for testing
the Tissue Culture material for virus diagnosis and genetic fidelity, for purpose of
certification. The Test laboratory will prepare a Test Report based on tests conducted in
conformity with the standards/protocols/guidelines. Based on the Test Report, each
Accredited Test Laboratories will be authorized to issue the Certificate of Quality for the
Tissue Culture Plant (CQ-TCP) on behalf of the Certification Agency. ATL will be
responsible for maintaining all diagnostic kits, primer, probes etc required for routine
testing. Each ATL would perform both tests-for virus diagnosis and true-to-type
Appellate Authority (AA):
An Appellate Authority under the Chairpersonship of Secretary, DBT established to
review the decision taken with regard to Accreditation of Test laboratories, certification
of Tissue Culture production facilities and also for Certification of tissue culture
material. The Nodal Officer designated by the Certification Agency will act as Member
Secretary. The members represented in the appellate panel will include: a. Officer in-
charge, Accreditation Unit
Head, Certification Monitoring Unit
Two Co-opted non-officio expert by Chairperson (Virology and Genetic fidelity Testing)
Modalities of Implementation
Accreditation of Laboratories and Certification of Production Facility:
The Applicant will register its application with the Accredited Unit (BCIL) (NCS-TCP
Form 1&2). Presently only public sector laboratories, universities, Government funded

institute, NGO’s would be considered for Accreditation as Test Laboratories. For
Certification of Tissue Culture Production Facility, all laboratories in the Public
sector/private sector/NGOs are eligible to apply. The requisite fee as indicated in the
Application Form (NCS-TCP Form 2) will need to be deposited. The Accreditation Unit
will examine the applications and refer it to the Accreditation Panel for further
processing. The criteria for Accreditation of Test Laboratories and Certification of tissue
culture production units are provided at Appendix-I and Appendix-II respectively. Based
on the report of the Accreditation Panel, the Certificate / Accreditation will be issued.
Accreditation and Certification would be for a period of Two years .Thereafter it would
be assessed for renewal
Certification of Tissue Culture Plants:
The Tissue Culture Production Facility will register its application for Certification of
Tissue Culture material with the nearest ATL (NCS-TCP Form 3). Only Certified
Production facilities will be eligible to register for certification of plant tissue culture
raised material. The ATL will examine the application and if found complete in all
respect it will intimate the applicant (in two working days), regarding requirements for
sending the samples. Guidelines for dispatch of material, sample size etc will be

provided. The ATL, will arrange for site inspection, receipt of samples, testing etc as per
prescribed format. The fees for certification will be deposited along with the sample at
the ATL. The ATL will generate the Test Report within the prescribed time frame and
based on the test report the Certificate of quality will be issued as per prescribed
norms. The fee structure charged by ATL will vary depending on the plant species,
sample size etc.
Standards for Production of Tissue Culture Material:
Standards/Guidelines for production of Tissue Culture material are currently being
prepared for different crops as per requirements, by DBT in consultation with
scientists/institutes, working in the area. The Accredited Test Laboratories will issue
the certificates only if Tissue Culture material is produced in conformity with these
notified guidelines. The Guidelines for potato have already been notified by Ministry of
Agriculture at Annexure II. Guidelines are being prepared for other crops like Banana,
Sugarcane, Vanilla, Black pepper, Bamboo etc. These will be notified and placed on the
website shortly.

UNIT: 2 PLANT TRANSFORMATION TECHNIQUES
Mechanism of DNA transfer – Agro bacterium mediated gene transfer, Ti and Ri
plasmid as vectors, role of virulence genes, design of expression vectors; 35S
promoter, genetic markers, reporter genes; viral vectors. Direct gene transfer
methods-particles bombardment, electroporation and microinjection. Binary
vectors-p bluescript IIKs, pBin19, pGreen vectors, transgene stabilility and gene
silencing
INTRODUCTION
In general, plant transformation systems are based on the introduction of DNA into
totipotent plant cells, followed by the regeneration of such cells into whole fertile plants.
Two essential requirements for plant transformation are therefore an efficient method
for introducing DNA into plant cells and the availability of cells or tissues that can
easily and reproducibly regenerate whole plants. DNA can be introduced into isolated
cells or protoplasts, explanted tissues, callus, or cell suspension cultures. However, the
process is characteristically inefficient and only a proportion of cells in a target
population will be transformed. These cells must be induced to proliferate at the
expense of nontransformed cells, and this can be achieved by introducing a selectable
marker gene and regenerating plants under the appropriate selective regime. Efficient
DNA delivery, competence for regeneration, and a suitable selection system are
therefore prerequisites for most plant transformation systems, although there has been
recent development in the application of in planta transformation strategies, which
circumvent the requirement for extensive tissue culture. Other criteria that define an
efficient transformation system are listed in Table.

MECHANISM OF DNA TRANSFER
DNA transfer to plants was first attempted in the 1960s, although the lack of selectable
markers and molecular tools to confirm transgene integration and expression made the
outcome of such experiments unclear. A breakthrough came in the late 1970s with the
elucidation of the mechanism of crown gall formation by Agrobacterium tumefaciens.
The discovery that virulent strains of A. tumefaciens carried a large plasmid that
conferred the ability to induce crown galls and that part of the plasmid (the T-DNA) was
transferred to the plant genome of crown gall cells provided a natural gene transfer
mechanism that could be exploited for plant transformation.
Tobacco plants carrying recombinant T-DNA sequences were first generated in 1981,
although the foreign genes were driven by their own promoters and were not expressed
in plant cells.
The first transgenic tobacco plants expressing recombinant genes in integrated T-DNA
sequences were reported in 1983. The technique of Agrobacterium-mediated
transformation has been developed and refined since then to become a widely used
strategy for gene transfer to plants.
Although it is convenient and versatile, a major limitation of Agrobacterium- mediated
transformation is its restricted host range, which until relatively recently excluded most
monocotyledonous plants. The development of strategies to extend the range of plants
susceptible to Agrobacterium infection is discussed in the following. A number of
alternative plant transformation methods were developed to facilitate gene transfer to
these recalcitrant species. These methods can be grouped under the term "direct DNA
transfer" and include the transformation of protoplasts using polyethylene glycol (PEG)
or electroporation, microinjection, the use of silicon carbide whiskers, and particle
bombardment. So far, only direct DNA transfer to protoplasts and particle
bombardment has gained widespread use.
The development and application of Agrobacterium-mediated transformation, particle
bombardment, protoplast transformation, and other transformation techniques is
discussed in more detail below.
AGRO BACTERIUM MEDIATED GENE TRANSFER
Agrobacterium tumefaciens is a Gram-negative soil bacterium responsible for crown gall
disease, a neoplastic disease of many dicotyledonous plants characterized by the
appearance of large tumors (galls) on the stems. Virulence is conferred by a large

tumor-inducing plasmid (Ti plasmid) containing genes encoding plant hormones (auxins
and cytokinins) and enzymes that catalyze the synthesis of amino acid derivatives
termed opines. The plant hormones are responsible for the deregulated cell proliferation
that accompanies crown gall growth, while the opines are secreted by the plant cells
and used by the bacteria as food. These genes are contained on a specific region of the
Ti plasmid, the T-DNA (transfer DNA), so called because it is transferred to the plant
nuclear genome under the control of vir (virulence) genes carried elsewhere on the Ti
plasmid. It is this natural gene transfer mechanism that is exploited for plant
transformation.
TI AND RI PLASMID AS VECTORS; ROLE OF VIRULENCE GENES
The earliest indication that T-DNA could be used as a plant transformation vector was
the demonstration that DNA from an Escherichia coli plasmid (the Tn7 transposon)
could be stably transferred to the plant genome by first incorporating it into the T-DNA.
However, transgenic plants could not be recovered from the transformed cells, either by
regeneration or by grafting onto normal plants, because the hormones encoded by the
T-DNA oncogenes caused unregulated and disorganized callus growth.
In rare cases, shoots were derived from such callus tissue, and analysis showed that
much of the T-DNA (including the oncogenes) had been deleted from the genome.
An important step in the development of T-DNA vectors was the realization that the only
requirements for T-DNA transfer to the plant genome were the vir genes and the 24-bp
direct repeat structures marking the left and right borders of the T-DNA. No genes
within the T-DNA were necessary for transformation, and any sequence could be
incorporated therein. This allowed the development of disarmed Ti plasmids lacking all
the oncogenes, facilitating T-DNA transfer to plant cells without causing neoplastic
growth.
Once suitable selectable markers had been incorporated into the TDNA (Table), Ti
plasmids became very powerful gene delivery vectors.

However, wild-type Ti plasmids were unsuitable for the task due to their large size,
which made them difficult to manipulate in vitro (large plasmids have a tendency to
fragment and they lack unique restriction sites for subcloning).
An early strategy to overcome this problem was the development of intermediate
vectors, where the T-DNA was subcloned into a standard E. coli plasmid vector, allowing
in vitro manipulation by normal procedures, and then integrated into the T-DNA
sequence of a disarmed Ti plasmid resident in A. tumefaciens by homologous
recombination. This system was simple to use but relied on a complex series of
conjugative interactions between E. coli and A. tumefaciens, requiring three different
bacterial strains (triparental matings). However, because the vir genes act in trans to
mobilize the T-DNA, it was soon discovered that the use of large natural Ti plasmids
was unnecessary. Intermediate vectors have been largely superseded by binary vectors,
in which the vir genes and the T-DNA are cloned on separate plasmids. These can be

introduced into A. tumefaciens by conjugation with an E. coli donor or by freeze-thaw
cycles or electroporation. Most contemporary Agmbacterium-mediated transformation
systems employ binary vectors.
General Protocol for Agrobacterium-Mediated Transformation
At first, Agrobacterium-mediated transformation was achieved by cocultivating a virulent
A. tumefaciens strain containing a recombinant Ti plasmid with plant protoplasts and
then obtaining callus from the protoplasts from which fertile plants were regenerated.
This strategy was widely replaced by a simpler method in which small discs were
punched from the leaves of recipient species and incubated in medium containing A.
tumefaciens prior to transfer to solid medium. Infection can be promoted under
conditions that induce virulence (presence of 10-200 /jM acetosyringone or a-
hydroxyacetosyringone, acidic pH, and room temperature), although these need to be
optimized for different species. After coculture for several days, the discs are transferred
to a medium containing selective agents to eliminate non transformed plant cells,
antibiotics to kill the bacteria, and hormones to induce shoot growth.
After a few weeks, shoots develop from transformed callus cells. These can be removed
and transferred to rooting medium or grafted onto seedling rootstock. Most current
protocols for the Agrobacterzwm- mediated transformation of solanaceous plants are
variations on the leaf disc theme, although different tissue explants are suitable
transformation targets in different species. Alternative methods are required for the
transformation of monocots. Rapidly dividing embryonic cells (e.g., immature embryos
or callus induced from scutellar tissue) are required for the transformation of rice and
other cereals. These are cocultured with Agrobacterium in the presence of
acetosyringone.
Recent Advances—Expanding the Agrobacterium Host Range
Although versatile and efficient for many plants, Agrobacterium-mediated
transformation was, until recently, limited to dicots and monocots of the orders Liliales
and Arales (which excludes most of the agronomically important cereals). The range of
plants amenable to genetic manipulation by direct DNA transfer is limited only by the
availability of cells competent for regeneration, and the range of plants amenable to
genetic manipulation by A. tumefaciens is further restricted to the species with cells
that are both competent for regeneration and susceptible to infection by A. tumefaciens.

Driven in part by the potential financial benefits of transgenic crop plants, much
research effort has been directed toward extending the Agrobacterium host range. In
some cases careful optimization of transformation conditions is required. For example,
the addition of a surfactant to the inoculation medium was responsible for the
successful transformation of wheat embryos and callus. Such optimization can also
dramatically increase the efficiency of transformation in susceptible plants. The
duration of cocultivation was shown to be important for the transformation of citrange.
The use of feeder cells can also significantly increase transformation efficiency; for
example, Niu et al.have shown that cocultivation on a tobacco cell feeder layer
significantly increases the efficiency of Agrobacterium infection of peppermint. Artificial
infiltration of the bacteria into plant tissues using a syringe or by vacuum infiltration
can improve transformation efficiencies in tobacco and is routinely used for the
transformation of Arabidopsis.
A further consideration is the fact that A. tumefaciens usually infects wounded cells in
its natural hosts, being attracted to phenolic compounds such as acetosyringone that
induce expression of the vir genes. Infection and T-DNA transfer are therefore
stimulated by wounding tissues (e.g., by crushing or cutting) or by treating explants
with acetosyringone. Several monocot species have been infected by pretreating the cells
with the exudates from wounded dicot plants, such as potato. The transformation of
recalcitrant species such as barley and sunflower has been achieved by prewounding
embryonic or meristematic tissue, respectively, with metal particles or glass beads,
while sonication has facilitated the uptake of Agrobacterium by soybean tissue. The
preinduction of virulence genes may circumvent the necessity to induce a wounding
response. Infection may be achieved by testing a range of different Agrobacterium
strains, and hypervirulent strains such as ALG1 in which the Ti plasmid contains vir
genes enhanced by mutation have proved especially useful.
Virulence can also be increased by the use of superbinary vector systems wherein the
vir component carries multiple copies of the virulence genes. Superbinary vector
systems such as pTOK have facilitated the transformation of important monocots,
including maize and sugarcane (Table).

Finally, some crops are amenable to Agrobacterium infection but resist gene transfer in
other ways. Comparative studies of T-DNA gene expression in tobacco and maize cells,
for example, have suggested that maize inhibits the integration of T-DNA by
suppressing gene expression. Other plants defend themselves against T-DNA
integration by forming necrotic boundaries. In some species (e.g., grape), such defenses
have been overcome by treatment with antioxidants.
Recent Advances—Increasing the Capacity of T-DNA
Another early problem with Agrobacterium-mediated transformation was the inability to
transfer large DNA fragments to the plant genome. Although a maximum size limit for
T-DNA gene transfer was not determined, the transfer of foreign DNAs larger than 30
kbp was not routinely achievable until about five years ago. This prevented the transfer
of large genes or T-DNA sequences containing multiple genes. The problem was
addressed by Hamilton and colleagues, who developed a novel binary vector system in
which the T-DNA vector was based on a bacterial artificial chromosome and the vir
component, expressed higher levels of virG and virE. This bibac (binary BAG) vector
facilitates the transfer of up to 150 kbp of DNA to the plant genome, allowing the
introduction of gene libraries into plants and the simultaneous introduction of many
genes on a cointegrate vector.
Conjugation Systems and Ri Plasmids
The mechanism of T-DNA transfer has many similarities with bacterial conjugation
systems, and indeed it has been shown that broad host range bacterial plasmids will

transfer from Agrobacterium to the plant genome under the control of their own
mobilization genes. Another system with close similarities to the A. tumefaciens Ti
plasmid is the Ri plasmid of Agrobacterium rhizogenes. This causes hairy root disease
though the integration of its own T-DNA, carrying genes that activate endogenous plant
hormones.
Ri plasmids have also been exploited as gene transfer vectors and have the advantage
that they do not induce cell proliferation in the host plant (i.e., they are naturally
disarmed. Agrobacterium strains containing both Ti and Ri plasmids often transfer both
T-DNAs to the host plant.
The Ri T-DNA induces hairy root disease and acts as a marker for transformation, while
the disarmed Ti plasmid carries the transgenes of interest. In this system, dominant
markers are not required and regeneration is not prevented by the Ri T-DNA.
Agrobacterium rhizogenes and Ri plasmids
Agrobacterium rhizogenes causes hairy-root disease in plants and this is induced by
root-inducing (Ri) plasmids, which are analogous to the Ti plasmids of A. tumefaciens.
The Ri T-DNA includes genes homologous to the iaaM (tryptophan 2-mono-oxygenase)
and iaaH (indoleacetamide hydrolase) genes of A.tumefaciens. Four other genes present
in the Ri T-DNA are named rol for root locus. Two of these, rolB and rolC, encode P-
glucosidases able to hydrolyse indole and cytokine-N-glucosides. A. rhizogenes therefore
appears to alter plant physiology by releasing free hormones from inactive or less active
conjugated forms.
Ri plasmids are of interest from the point of view of vector development, because opine-
producing root tissue induced by Ri plasmids in a variety of dicots can be regenerated
into whole plants by manipulation of phytohormones in the culture medium. Ri TDNA is
transmitted sexually by these plants and affects a variety of morphological and
physiological traits, but does not in general appear deleterious.
The Ri plasmids therefore appear to be already equivalent to disarmed Ti plasmids.
Transformed roots can also be maintained as hairyroot cultures, which have the
potential to produce certain valuable secondary metabolites at higher levels than
suspension cultures and are much more genetically stable.
The major limitation for the commercial use of hairy-root cultures is the difficulty
involved in scale-up, since each culture comprises a heterogeneous mass of

interconnected tissue, with highly uneven distribution. Many of the principles explained
in the context of disarmed Ti plasmids are applicable to Ri plasmids.
A cointegrate vector system has been developed and applied to the study of nodulation
in transgenic legumes. Van Sluys et al. (1987) have exploited the fact that
Agrobacterium containing both an Ri plasmid and a disarmed Ti plasmid can frequently
cotransfer both plasmids. The Ri plasmid induces hairy-root disease in recipient
Arabidopsis and carrot cells, serving as a transformation marker for the cotransferred
recombinant TDNA and allowing regeneration of intact plants. No drug-resistance
marker on the T-DNA is necessary with this plasmid combination.
DESIGN OF EXPRESSION VECTORS; 35S PROMOTER
Cauliflower Mosaic Virus (CaMV) is a member of the genus Caulimovirus, one of the
six genera in the Caulimoviridae family, which are pararetroviruses that infect plants.
Pararetroviruses replicate through reverse transcription just like retroviruses, but the
viral particles contain DNA instead of RNA
Structure
The CaMV particle is an icosahedron with a diameter of 52 nm built from 420 capsid
protein (CP) subunits arranged with a triangulation T = 7, which surrounds a solvent-
filled central cavity. It contains a circular double-stranded DNA molecule of about 8.0
kilobases, interrupted by site-specific discontinuities resulting from its replication by
reverse transcription. After entering the host, the single stranded nicks in the viral DNA
are repaired, forming a supercoiled molecule that binds to histones. This DNA is
transcribed into a full length, terminally redundant, 35S RNA and a subgenomic 19S
RNA.
Genome
The promoter of the 35S RNA is a very strong constitutive promoter responsible for the
transcription of the whole CaMV genome. It is well known for its use in plant
transformation. It causes 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. Interestingly, recent
study has indicated that the CaMV 35S promoter is also functional in some animal
cells, although the promoter elements used are different from those in plants. While this
promoter had low activity compared to canonical animal promoters, levels of reporter

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