TISSUE CULTURE IN PLANTS

Project Report Submitted to

University of Lucknow

For the Degree of

MASTER OF SCIENCE
IN
BOTANY
BY
SWATI SRIVASTAVA
UNDER THE SUPERVISION OF

DR. ASHOK KUMAR SINGH

ASSOCIATE PROFESSOR

DEPARTMENT OF BOTANY

RAJAT P.G. COLLEGE, KAMTA, LUCKNOW

 ACKNOWLEDGEMENT

On the very outset of this report, I would like to extend my sincere and heartfelt
obligation towards all the personages who have helped me in this endeavour,
without their active guidance, help cooperation and encouragement, I wouldn’t
have made headway in this project.

I am grateful to Dr. Ashok Kumar Singh, (Ph. D.), Assistant Professor, Department
of Botany, Rajat P.G. College, Lucknow for providing me an opportunity to do this
project work under his guidance and giving support that helped me in completing
this project duly.

I am extremely thankful to principal Dr.Shyam Kishore (Head, Department of

Botany), Dr. Deepak Kumar Agrahari (Assistant Professor), Dr. Naheed
Lohani(Assistant Professor).

Also, I want to thank my parents and members of family for continuously

supporting and encouraging me both morally and economically.

At last, I would like to thanks my friends who directly or indirectly helped me

completing this project report.

Swati Srivstava

M. Sc. 4th semester

Roll no. 190631210008

Rajat P.G. College

 CONTENT

1. Introduction

2. Media Composition

3. Types of Culture

i) Explant Culture

ii) Callus Culture

iii) Root Culture

iv) Shoot Culture

v) Cell Suspension Culture

vi) Protoplast Culture

4. Protoplast fusion and Somatic Hybridisation

5. Somaclonal Variation

6. Applications

7. Reference
 INTRODUCTION
Tissue culture is the in vitro aseptic culture of cells, tissues, organs or
whole plant under controlled nutritional and environmental conditions
often to produce the clones of plants. The resultant clones are true-to
type of the selected genotype. The controlled conditions provide the
culture an environment conducive for their growth and multiplication.
These conditions include proper supply of nutrients, pH medium,
adequate temperature and proper gaseous and liquid environment.

Plant tissue culture technology is being widely used for large scale plant
multiplication. Apart from their use as a tool of research, plant tissue
culture techniques have in recent years, become of major industrial
importance in the area of plant propagation, disease elimination, plant
improvement and production of secondary metabolites.Small pieces of
tissue (named explants) can be used to produce hundreds and thousands
of plants in a continuous process. A single explant can be multiplied into
several thousand plants in relatively short time period and space under
controlled conditions, irrespective of the season and weather on a year
round basis. Endangered, threatened and rare species have successfully
been grown and conserved by micropropagation because of high
coefficient of multiplication and small demands on number of initial
plants and space.
In addition, plant tissue culture is considered to be the most efficient
technology for crop improvement by the production of somaclonal and
gametoclonal variants. The micropropagation technology has a vast
potential to produce plants of superior quality, isolation of useful
variants in well-adapted high yielding genotypes with better disease
resistance and stress tolerance capacities. Certain type of callus cultures
give rise to clones that have inheritable characteristics different from
those of parent plants due to the possibility of occurrence of somaclonal
variability, which leads to the development of commercially important
improved varieties. Commercial production of plants through
micropropagation techniques has several advantages over the traditional
methods of propagation through seed, cutting, grafting and air-layering
etc. It is rapid propagation processes that can lead to the production of
plants virus free. Coryodalisyanhusuo, an important medicinal plant was
propagated by somatic embryogenesis from tuber-derived callus to
produce disease free tubers . Meristem tip culture of banana plants
devoid from banana bunchy top virus (BBTV) and brome mosaic virus
(BMV) were produced. Higher yields have been obtained by culturing
pathogen free germplasmin vitro. Increase in yield up to 150% of virus-
free potatoes was obtained in controlled conditions. The main objective
of writing this chapter is to describe the tissue culture techniques,
various developments, present and future trends and its application in
various fields.
 MEDIA COMPOSITION

Product Description :Murashige and Skoog Medium (MS) was

originally formulated by Murashige and Skoog in 1962 to optimize
tobacco callus bioassay system for facilitating the study of cytokinins.
Since then, it is widely used for micro propagation, organ culture, callus
culture and suspension culture. The formulation is a nutrient blend of
inorganic salts, vitamins and amino acid. Murashige and Skoog Medium
(MS) provides all the essential macroelements and microelements.
Potassium dihydrogen phosphate serves as a source of phosphate.
Microelements like Boron, Manganese, Molybdenum, Copper and Zinc
play vital role in plant metabolism. Boron plays a key role in
carbohydrate metabolism. Thiamine, nicotinic acid, pyridoxine, inositol
act as enzymatic cofactors in universal pathways including glycolysis
and TCA cycle along with primary and secondary metabolism in the
plants. Glycine serves as a source of amino acid. The product is plant
tissue culture tested but it is the sole responsibility of the user to ensure
the suitability of the medium for individual species.
Composition :
 TYPES OF CULTURE
EXPLANT CULTURE -

In biology, explant culture is a technique to organotypically culture cells

from a piece or pieces of tissue or organ removed from a plant or animal.
The term explant can be applied to samples obtained from any part of
the organism. The extraction process is extensively sterilized, and the
culture can be typically used for two to three weeks.

The major advantage of explant culture is the maintenance of near in

vivo environment in the laboratory for a short duration of time. This
experimental setup allows investigators to perform experiments and
easily visualize the impact of tests.

This ex vivo model requires a highly maintained environment in order to

recreate original cellular conditions. The composition of extracellular
matrix, for example, must be precisely similar to that of in
vivo conditions in order to induce naturally observed behaviors of cells.
The growth medium also must be considered, as different solutions may
be needed for different experiments.

The tissue must be placed and harvested in an aseptic environment such

as sterile laminar flow tissue culture hood. The samples are often
minced, and the pieces are placed in a cell culture dish
containing growth media. Over time, progenitor cells migrate out of the
tissue onto the surface of the dish. These primary cells can then be
further expanded and transferred into fresh dishes
through micropropagation.

Explant culture can also refer to the culturing of the tissue pieces

themselves, where cells are left in their surrounding extracellular
matrix to more accurately mimic the in vivo environment
e.g. cartilage explant culture,or blastocyst implant culture.

Historically, explant culture has been used in several areas of biological

research. Organogenesis and morphogenesis in fetus have been studied
with explant cultures. Since the explant culture is grown in the lab, the
area or cells of interest can be labeled with fluorescent markers. These
transgenic labels can help researchers observe growth of specific cells.
For example, neural tissue development and central nervous system
regeneration have been studied with organotypic explant culture. The
role of a specific gene, gene expression, and the mechanism of action all
can be studied with explant culture as well. Certain factors that control
or contribute to growth could be identified during different stages of
embryogenesis. Looking at the expression pattern would allow tracking
of where the gene transcripts have been. How much gene has been
expressed could be quantified too. Coupling with stem cell research,
researchers have successfully grown simple organs derived from
autologous human pluripotent stem cells. So far bladder and trachea
have been developed. This method attempts to address tissue rejection,
and there are already cases of successful transplantation. A research
team from Wake Forest Institute for Regenerative Medicine in Winston-
Salem, North Carolina, successfully transplanted stem cell-engineered
bladders to seven pediatric patients suffering from malfunctioning
bladders. Another case was from a team at University College, London,
UK, which transplanted a wind pipe derived from the patient’s own stem
cells. Even with all the advantages to explant culture, there still are
several caveats. The downside of explant culture is that it does not
provide sufficient time to study chronic diseases. Although two to three
weeks may be enough time to study acute changes, it is not fit for
experiments requiring long-term observations.

CALLUS CULTURE-
Plant callus (plural calluses or calli) is a growing mass of unorganized
plant parenchyma cells. In living plants, callus cells are those cells that
cover a plant wound. In biological research and biotechnology callus
formation is induced from plant tissue samples (explants) after surface
sterilization and plating onto tissue culture medium in vitro (in a closed
culture vessel such as a Petri dish). The culture medium is supplemented
with plant growth regulators, such as auxin, cytokinin, and gibberellin,
to initiate callus formation or somatic embryogenesis. Callus initiation
has been described for all major groups of land plants.
Plant species representing all major land plant groups have been shown
to be capable of producing callus in tissue culture. A callus cell culture
is usually sustained on gel medium. Callus induction medium consists of
agar and a mixture of macronutrients and micronutrients for the given
cell type. There are several types of basal salt mixtures used in plant
tissue culture, but most notably modified Murashige and Skoog medium,
White's medium, and woody plant medium. Vitamins are also provided
to enhance growth such as Gamborg B5 vitamins. For plant cells,
enrichment with nitrogen, phosphorus, and potassium is especially
important. Plant callus is usually derived from somatic tissues. The
tissues used to initiate callus formation depends on plant species and
which tissues are available for explant culture. The cells that give rise to
callus and somatic embryos usually undergo rapid division or are
partially undifferentiated such as meristematic tissue. In
alfalfa, Medicago truncatula, however callus and somatic embryos are
derived from mesophyll cells that undergo dedifferentiation.[17] Plant
hormones are used to initiate callus growth.
Callus Culture is the culture of dedifferentiated plant cells induced on
media usually containing relatively high auxin concentrations or a
combination of auxin and cytokinin under in vitro conditions. In plants,
where sought after metabolites are present in leaves, establishing in
vitro cultures from leaves and using them for the extraction of
compounds would be an ideal alternative. Callus cultures containing the
bioactive substances are collected at a specific stage (usually during the
stationary phase of their growth cycle, since secondary metabolite
production is greater during the stationary phase), dried, extracted, and
the extract then taken for identification and quantification of the desired
medicinal compound using HPLC, LC-MS, etc. The further scale-up and
yield enhancement studies of the compound are performed by raising the
callus in suspension, first in a shake-flask culture, and then in a suitably
designed bioreactor, to maximize its production.

ROOT CULTURE-
Root culture can be defined as the culture of excised radical tips of
aseptically germinated seeds in a liquid medium where they are induced
to grow independently under controlled conditions.Intact in vivo plants
are not suitable for the isolation of intact root tips because the roots of
‘the plant are buried deeply in the soil. Again, root tips from young
seedlings are very sensitive to toxic sterilants. So it is better to avoid the
surface sterilization of young root tips for the establishment of root
cultures. Root cultures can be successfully initiated from the excised
radicle tips of aseptically germinated seeds.Root tip cultures are
generally maintained in moving liquid medium. In culture, root tips are
induced to grow like that of root system of an intact plant. A clone of
excised roots can also be established from a single root culture by
repeatedly cutting and transferring of the main root tips or of lateral tips
into fresh medium in every subculture at the interval of definite period.
Growth of excised roots can be expressed in terms of fresh and dry
weight, increase in length of the main axis, number of emergent laterals
and total length of laterals per culture.
SHOOT CULTURE-
The term shoot culture is now preferred for cultures started from
explants bearing an intact shoot meristem, whose purpose is shoot
multiplication by the repeated formation of axillary branches. In this
technique, newly formed shoots or shoot bases serve as explants for
repeated proliferation; severed shoots, explant size, shoot cultures are
conventionally started from the apices of lateral or main shoots, up to 20
mm in length, dissected from actively-growing shoots or dormant buds.
Larger explants are also sometimes used with advantage: they may
consist of a larger part of the shoot apex or be stemming segments
bearing one or more lateral buds; sometimes shoots from other in vitro
cultures are employed. When apical or lateral buds were used almost
exclusively as explants, the name shoot tip culture came to be widely
used for cultures of this kind. In shoot culture, apical meristem (the
region of shoot apex laying distal to leaf primordium) is cultured. It
clearly differs from shoot apex by having shoot apex and a few leaf
primordia. Because of culture of relatively large tips (5-10 mm long
sections), this technique is also known as meristem culture,
meristemming and mericlones (Murashige, 1974). Shoot tip culture is
extensively applied in horticulture, agriculture and forestry. Morel
(1960) pioneered the work of shoot apex culture of
orchid, Cymbidium, for the clonal multiplication. Among the workers
Murashige significantly contributed for establishing micropropagation
technique and its further biotechnological application. Because of
minute size of the propagules in culture, the in vitro propagation
technique is known as micropopagation. Murashige (1974) has described
the procedure of development of micropropagation into three different
developmental stages:
Stage I, establishment of explant aseptically;
stage II, multiplication of propagules by repeated subcultures on a
specific nutrient medium;
stage III, rooting and hardening of plantlets and planting into soil.
CELL SUSPENSION CULTURE-
Suspension culture is a type of culture in which single cells or small
aggregates of cells multiply while suspended in agitated liquid medium.
It is also referred to as cell culture or cell suspension culture.Callus
proliferates as an unorganised mass of cells. So it is very difficult to
follow many cellular events during its growth and developmental
phases. To overcome such limitations of callus culture, the cultivation of
free cells as well as small cell aggregates in a chemically defined liquid
medium as a suspension was initiated to study the morphological and
biochemical changes during their growth and developmental phases.To
achieve an ideal cell suspension, most commonly a friable callus is
transferred to agitated liquid medium where it breaks up and readily
disperses. After eliminating the large callus pieces, only single cells and
small cell aggregates are again transferred to fresh medium and after two
or three weeks a suspension of actively growing cells is produced.This
suspension can then be propagated by regular sub-culture of an aliquot
to fresh medium. Ideally suspension culture should consist of only single
cells which are physiologically and biochemically uniform. Although
this ideal culture has yet to be achieved, but it can be achieved if it is
possible to synchronize the process of cell division, enlargement and
differentiation within the cell population.The culture of single cells and
cell aggregates in moving liquid medium can be handled as the culture
of microbes. The suspension culture eliminates many of the
disadvantages ascribed to the callus culture on agar medium. Movement
of the cells in relation to nutrient medium facilitates gaseous exchange,
removes any polarity of the cells due to gravity and eliminates the
nutrient gradients within the medium and at the surface of the cells.

PROTOPLAST CULTURE-

Protoplasts have been isolated from both intact plants and cell cultures
of many plant species. Leaves are an especially suitable cell source
because mesophyll cells have relatively thin walls which are readily
degraded. It may be necessary to remove the epidermis because enzymes
have difficulty in penetrating the cuticle. Pretreatment with α-
glucuronidase or pectinglucosidase can partially substitute for epidermis
stripping. Other plant parts such as root tips and embryos may also be
used but generally have cells with more resistant walls. Rapidly growing
cell cultures are a most suitable source of protoplasts. Old cells or cells
from slowly growing cultures often have walls which are highly resistant
to enzyme degradation. The medium should be slightly hypertonic
because it appears that plasmolysis hastens the wall degradation, perhaps
by allowing enzyme attack from the inner surface of the wall.
Protoplasts appear to be more stable than the cells in the incubation
mixtures. Generally, all cells not converted to protoplasts are dead after
18 hours incubation with the enzyme mixture.

The basic principle of protoplast culture is the aseptic isolation of large

number of intact living protoplasts removing their cell wall and cultures
them on a suitable nutrient medium for their requisite growth and
development. Protoplast can be isolated from varieties of plant tissues.
Convenient and suitable materials are leaf mesophyll and cells from
liquid suspension culture. Protoplast yield and viability are greatly
influenced by the growing condition of the plant as well as the cells.
The essential step of the isolation of protoplast is the removal of the cell
wall without damaging the cell or protoplasts. The plant cell is an
osmotic system. The cell wall exerts the inward pressure upon the
enclosed protoplasts. Likewise, the protoplast also puts equal and oppo-
site pressure upon the cell wall. Thus, both the pressures are balanced.

Now if the cell wall is removed, the balanced pressures will be

disturbed. As a result, the outward pressure of protoplast will be greater
and at the same time in absence of cell wall, irresistible expansion of
protoplast takes place due to huge inflow of water from the external
medium. Greater outward pressure and the expansion of protoplast cause
it to burst.

So, the isolated protoplast is an osmotically fragile structure at its

nascent stage. Therefore, if the cell wall is to be removed to isolate
protoplast, the cell or tissue must be placed in a hypertonic solution of a
metabolically inert sugar such as

mannitol at higher concentration (13%) to plasmolysis the cell away

from the cell wall Mannitol, an alcoholic sugar, is easily transported
across the plasmodesmata, provides a stable osmotic environment for the
protoplasts and prevents the usual expansion and bursting of protoplast
even after loss of cell wall. That is why, this hypertonic solution is
known as osmotic stabilizer or plasmolyticum or osmolyticum.

Once the cells are stabilized in such a manner by plasmolysis the

protoplasts are released from the containing cell wall either
mechanically or enzymatically. Mechanical isolation (Fig 12.3) involves
breaking open each cell compartment to liberate the protoplast. This
operation can be done carefully on small pieces of tissue under a
microscope using a micro-scalpel.

But very few protoplasts are obtained for a lot of time and effort. Large-
scale attempts at mechanical isolation involves the disrupting tissue with
fine stainless steel-bristled brush. This process may liberate more
protoplasts with less efforts, but the percentage of yield of intact
protoplasts is still very low.
A considerably more efficient way of liberating the protoplasts is to
digest the cell walls away around them, using cell wall degrading
enzymes such as cellulase, hemicellulose, pectinase or macerozyme etc.
These enzymes are isolated from fungi and available commercially
Period of treatment and concentration of enzymes are the critical factors
and both factors should be standardized for particular plant tissue. Intact
tissue can be incubated with a pectinase or macerozyme solution which
will dissolve the middle lamella between the cells and so separate them.

Subsequent treatment with cellulase will digest away the cellulosic layer
of the cell wall. This process is known as sequential enzyme treatment or
two step method as opposed to a mixed enzyme treatment (one step
method) in which both cellulase and pectinase or macerozyme are mixed
so that the entire wall is broken down in a single operation (Fig. 12.4).
The isolated protoplasts can be cultured either static liquid or agarified
medium. The protoplast media consist of mineral salts, vitamins, carbon
sources and plant growth hormones as well as osmotic stabilizers and
possibly organic nitrogen sources, coconut milk and organic acids.

In culture protoplast can reform a new cell wall around them. Once the
wall is formed, the protoplast becomes a cell. The cells from protoplasts
subsequently enter cell division which is followed by the formation of
callus and cell cultures. Such callus also retain the capacity for
morphogenesis and plant regeneration. A brief list of plant regeneration
from plant protoplast culture is given below .
 PROTOPLAST FUSION

AND SOMATIC HYBRIDISATION-

Protoplast fusion followed by regeneration by SE is a technique that can

be used in plant breeding when sexual crosses are impossible. Somatic
hybridization, which brings together two different nuclear genomes that
are not genetically close, may not produce fertile plants. However, this
approach has been used to produce rootstocks in citrus species, where
fertility is unnecessary. Transfer of more limited amounts of DNA in
asymmetric or highly asymmetric somatic hybridization still has
potential as fertility is possible. Organellar genomes can also be
transferred by protoplast fusion, with the capacity to by-pass maternal
inheritance. This has utility in transferring male sterility; mitochondrial
fusion can also produce recombinant mitochondrial genomes.

Protoplast fusion is a novel tool for transferring genes for a desired

quality and quantity of production. In this technology, two different
genetically originated protoplasts from different somatic cells are fused
in order to obtain parasexual hybrid protoplasts. Genes representing
useful characteristics such as higher bioproduct productivity, improved
protein quality, heat and cold resistance can be transferred from one
species to another. For instance, intra-strain protoplast fusion was
carried out in T. reesei strain PTr2 using lysing enzymes, 0.6 M KCl as
osmotic stabilizer, 40% polyethylene glycol with STC (sorbitol, Tris–
HCl, CaCl2) buffer. The majority of the fusants showed faster mycelia
growth, abundant sporulation, and high levels of extracellular
carboxymethyl cellulase (80% of the fusants showed higher enzyme
activity and two fusants, SFTr2 and SFTr3 were found with twofold
increase in enzyme activity) than the nonfusants and wild strains .

Protoplast fusion is the fruit of an original idea. If you can load and fuse
liposomes with DNA, why should you go to the trouble of
manufacturing these, because bacteria are really nothing more than large
liposomes filled with plasmid DNA.
The bacteria are treated with chloramphenicol to increase the proportion
of plasmid DNA, and the lysozyme digests the cell wall. In this way,
protoplasts can be fused to the cells like liposomes.
The well-versed cell culturologist develops gray hair merely from the
idea that bacteria should voluntarily be added in large numbers to the
cells without there being any emergency. The problem is solved through
the application of large quantities of antibiotics. The simultaneous
transfer of plasmid DNA and bacterial chromosomes is not entirely
problem free, and the method has not prevailed.
Currently, protoplast fusion is perhaps the most important and
significant new tool in plant breeding because of its potential to create
‘cybrids’, a new method of hybridizing divergent taxa that were isolated
by genetic barriers. Cybrids contain organelles from both parents, and
thus increase genetic variability. Somatic hybridization also permits the
combination of desirable cytoplasmic traits not normally obtainable by
conventional breeding. Cytoplasmically encoded traits are usually
inherited through the maternal parent.
Schenck and Robbelen were the first to synthesize B. napus through
protoplast fusion of B. campestris and B. oleracea (including cabbage).
This work was repeated by other researchers. Taguchi and
Kameya fused protoplast from leaf mesophyll cells of red cabbage line
ER-159 and the Chinese cabbage cultivar ‘Chihili 70’, the first example
of a somatic hybrid between heading type vegetables. Morphologically,
the regenerated plants exhibited intermediate characteristics between the
two parental species. Cytological analysis showed that the hybrid plants
had 2n = 38 chromosome (18 + 20).
Yamashita et al. obtained asymmetric somatic hybrids through
protoplast fusion between B. campestris and cabbage. Some of the
hybrids contained the complete genome of cabbage plus a part of the B.
campestris genome.
Intergeneric hybrid plants were obtained through protoplast fusion
between red cabbage and Moricandia arvensis (L.) DC. and between red
cabbage and radish. Hybrid plants of red cabbage and radish were male
sterile but female fertile. Leaf morphology, chromosome number,
isozyme patterns and Smart cleavage patterns (restriction enzyme
analysis) of chloroplast DNA showed that the regenerated plants were
truly intergeneric hybrids, having the nucleus of cabbage and the
chloroplasts of radish.
Kagami et al. suggested that a more efficient method of producing
cytoplasmic male sterile somatic hybrids was by fusion
between Brassica oleracea recipient protoplast and Raphanus CMS
(Ogura) donor protoplast.
Cell selection and protoplast fusion are discussed before evaluating the
potential benefits of transgenic plant species. Cell selection involves
actively selecting cell lines with an altered phenotype. The new
phenotype could arise from either genetic or non-genetic events and so
the selected cell lines are referred to as variants. The term mutant is
retained for those cell lines where a genetic event has taken place. In
brief, the variant must be screened and then selected. This involves
imposing a selection pressure on the cell population so that only the
individuals with the altered phenotype can survive or grow. Genetic
variations in regenerated plants have a chromosomal bias and may
involve complete chromosomal sets (polyploidy), loss or gain of
individual chromosomal sets (aneuploidy), or structural rearrangements
within individual chromosomes (deletions, duplications, inversions and
translocations). The modified cell line with the stable phenotype can
then be isolated from the cell culture. The altered phenotype may
include increased resistance to stress such as drought, freezing and heat,
resistance to herbicides, fungicides, heavy metals, aluminum, salinity,
pathogens and viruses. Unfortunately, the molecular basis for most
agriculturally important phenotypes are so poorly understood that it is
not really possible to design effective selection protocols and define
potential selection parameters.
Protoplast fusion permits the production of the somatic hybrids. One cell
can be induced to fuse with another cell to form the heterokaryon which
consists of cytoplasms containing chloroplasts, mitochondria and nuclei
from both parent cells. Generation of the protoplast from the plant cell
can be performed simply by incubating the tissue in a cocktail of
enzymes that include cellulases, hemicellulases and pectinases. After the
cell wall has been removed from the cell, a hypo-osmotic external milieu
will lyse the protoplast unless a suitable osmoticum such
as mannitol and sorbitol is present in the incubation medium.
Protoplast fusion may be induced by an electric field or chemically, by
addition of 20–40% polyethylene glycol which results
in protoplasts aggregation and dilution of polyethylene glycol which
causes protoplast fusion. The somatic hybrid must be selected after
fusion of the protoplasts. The selection procedure can for example, be
based on biochemical marker analysis or a differential effect of a
metabolic inhibitor. One protoplast could be irreversibly inhibited by
one inhibitor and another protoplast could be irreversibly inhibited by
another inhibitor. However, the somatic hybrid will survive and grow in
the presence of the inhibitors because both parent cells will contribute
enzymes that remain unaffected by each inhibitor. At present, it has
proved difficult to regenerate crop plants from protoplasts.
Transformation of plants with foreign DNA has proved difficult in the
past. One of the most common ways of introducing genes into plants is
based on the natural gene transfer ability of the Gram-negative soil
bacterium Agrobacterium tumefaciens. This bacterium infects
wounded plant tissue of many dicotyledonous and
some monocotyledonous plants and causes crown gall disease. The
crown gall is a plant tumor, a lump of undifferentiated tissue that
represents true oncogenic transformation. This bacterium contains large
plasmids and the virulence trait is known to be plasmid-borne and they
are therefore referred to as Ti (tumour-inducing) plasmids. Complete Ti-
plasmid DNA is not found in plant tumor cells, but a small segment of
DNA (approximately 20 kb) is found integrated in the plant genome.
This DNA segment is called T-DNA. The plant cells express the T-DNA
genes that encode enzymes responsible for
phytohormone biosynthesis (and consequently tumor growth) and opine
production.
The Ti-plasmid is, therefore, an excellent natural vector for genetically
engineering plant cells because it can transfer its T-DNA from the
bacterium into the plant genome. It is possible to insert foreign DNA
into T-DNA which would then incorporate into the plant genome and the
foreign gene(s) would then be expressed by the plant cell. It is important
to note that the wild type Ti-plasmids are too large to act as vectors.
Smaller plasmid vectors containing the T-DNA have been constructed
that permit considerably simpler transfer of foreign genes to plant cells.
This small plasmid known as mini-Ti is transferred conjugatively to A.
tumefaciens and can induce opine tumors if the bacterium contains a
plasmid that carries the virulence genes but not the T-DNA. Thus
the vir functions can be donated by a separate plasmid and confer
complete tumorigenicity. The plant cell can be regenerated from the
transformed cells and the foreign DNA can then be transmitted normally
by sexual reproduction to subsequent generations. One of the problems
with the transfer of foreign DNA by the T-DNA is that transfer is often
closely associated with transformation of the cells to tumors. It is
sometimes difficult to induce regeneration into normal plantlets or into
normal tissue that can be grafted on to healthy plants. Some researchers
have overcome this problem by deleting one or more of the tumor-
inducing genes.
In addition, the Ti-plasmid approach cannot often be applied to cereal
plants because these plants are insensitive to crown gall disease. One
way to overcome the restrictions imposed by the host range
of Agrobacterium tumefaciens is to introduce DNA directly into cells
using a physical, rather than a biological, process such
as electroporation. A high concentration of plasmid DNA containing the
cloned gene is added to a suspension of protoplasts and the mixture is
applied to an electric field of 200–600 V cm−1. After electroporation, the
cells are grown in tissue culture for 1–2 weeks and then the cells that
have taken up the DNA can be selected. Maize and rice protoplasts have
been successfully transformed with 0.1–1% efficiency. However,
electroporation still requires the use of protoplasts with the comcomitant
difficulties associated with regenerating whole plants from protoplasts
and problems of somaclonal variation resulting from prolonged periods
of tissue culture.
Genes have been transferred into plant cells with intact cell walls by
shooting extremely small metal beads of 1 μm in diameter coated with
DNA at velocities of about 430 ms −1. This procedure has been used
with suspension cultures of embryonic cells plated on filters and intact
leaves. Embryonic maize cells have been transformed by this procedure
with reporter genes and the bar gene that encodes the
enzyme phosphinothricin acetyltransferase which inactivates
phosphinothricin, a component of herbicides. Whole plants regenerated
from these transformed cells were found to be resistant to a herbicide
applied directly to leaves. The E. coli gene for the enzyme β-
glucuronidase (GUS) is particularly important as a reporter gene in
plants. Plants have virtually undetectable levels of GUS. When plant
cells expressing β-glucuronidase are incubated with a
chromogenic glucuronide substrate, a blue color is produced which can
be observed histochemically or if a different substrate is used, GUS can
be determined quantitatively with a fluorimeter. Alternatively, the living
cells expressing a cloned gene can be detected by using
the luciferase gene as a potential reporter gene. A particular problem
with the use of reporter genes is that they give no indication of
either mRNA stability or post-translational modification of the co-
transformed protein.
These powerful molecular biological tools have and are anticipated to
make a substantial impact on biotechnology. Recombinant
DNA technology can enhance our understanding of the molecular basis
of physiological processes. Traditional plant physiology has for too long
been poorly defined at the molecular level. Today, there are many
techniques that can be employed to investigate the role of proteins
involved in stress, photosynthesis and defense.
Plants activate a large array of inducible defense responses during
microbial infection. These include accumulation of phytoalexins,
production of hydrolytic enzymes (e.g. invertases, chitinases), enhanced
activity of peroxidases, deposition of callose and enhanced
insolubilization of hydroxyproline-rich glycoproteins in the cell wall. An
important enzyme in flavanoid (phytoalexin) biosynthesis is
chalcone synthase. The gene encoding this protein has been isolated.
A recombinant gene consisting of the chalcone synthase promoter
from Antirrhinum majus, the neomycin phosphotransferase III reporter
gene and the termination region of the chalcone synthase gene from
parsley (Petroselinum hortense) have been inserted into tobacco. The
promoter functioned normally in tobacco and was induced by UV-
B irradiation. Two regulatory regions were identified by deletion
analysis of the promoter sequence. One region participated in UV-B
light responsivity and the other region was essential for optimal
expression of the gene.
Genetically manipulating plants can have significant importance in
agriculture. Plants can be genetically altered so that they express a
bacterial toxin which is toxic to larvae of a number of insects. This could
have desirable effects on reducing the use of chemical pesticides. Plants
can be used to express heterologous proteins because they are cheap and
a large quantity of protein can be obtained from a single field. Some
heterologous proteins such as enkephalin, a
human neuropeptide and human serum albumin have been expressed in
plants. Perhaps potato tubers could be engineered to synthesize large
amounts of therapeutically important proteins.
Protoplast fusion, in which living cells with cell walls removed are fused
to form new living entities, has also been used successfully to overcome
pre- and postfertilization barriers. Protoplast fusions
between Brassica and Arabidopsis were reported to yield stable
genotypes that incorporated chromosomes or fragments from each
species (Hoffman and Adachi, 1981). Gene exchange was also reported
in intergeneric somatic hybrids from protoplast fusion
between B. napus and Diplotaxis harra (Klimaszewska and Keller, 1988)
as well as between Sinapis turgida Del. and Brassica (Toriyama et al.,
1987).
Protoplast fusion is also the only means of combining two
cytoplasmically inherited characteristics in a single genotype. In
addition, fusion can result in a reassortment of nuclear and cytoplasmic
elements. Thus, it is possible in B. napus to combine in one cell the
nucleus of one parent with foreign chloroplasts and mitochondria
genomes, or a mixed foreign and domestic chloroplast–mitochondrion
combination, or a choice of chloroplast genomes with a
recombined mitochondrial genome. For example, CMS B. napus plants,
containing radish (R. sativus) cytoplasm, exhibited chlorosis, especially
at low temperatures, and contained low chlorophyll levels at high
temperatures (Bannerot et al., 1977; Rousselle, 1982; McCollum, 1981).
In addition, these plants had poorly developed nectaries and reduced
nectar flow (Mesquida and Renard, 1978). The chloroplast problem was
solved through protoplast fusion by replacing the radish chloroplast
genome with B. napus chloroplasts but retaining the radish mitochondria
controlling the CMS trait (Pelletier et al., 1983). In the resulting hybrids
variability of flower morphology was also observed, with some plants
having their nectar production restored to 81% of the male fertile
control. These observations indicate that a recombination of the
mitochondrial genome resulted in a restoration of nectaries while
retaining the male sterility trait (Pelletier, 1990).
In B. napus plants, protoplast fusion and plant regeneration have also
made possible the combination of the B. rapa chloroplasts that impart
tolerance to the triazine family of herbicides with the mitochondria that
control male sterility in the Ogura (Pelletier et al., 1983; Kao et al.,
1991), Polima (Barsby et al, 1987; Chuong et al, 1988),
and nap (Yarrow et al, 1986) CMS systems. Thus two cytoplasmically
inherited characteristics were combined in a single genotype.
Setting up the Experimental Conditions for Protoplast Fusion, Culture
and Selection of Heterokaryons, and Plant Regeneration from Selected
Calli
Protoplast fusion can be induced by various treatments. These include
(1) divalent cations (Ca2+) in alkaline solutions, (2) water-soluble
polymers, and (3) electric field pulses. Plant protoplasts are negatively
charged, and therefore spontaneous fusion rarely occurs. The
neutralization of this negative charge by divalent cations such as Ca 2+ in
a solution of high pH can promote protoplast aggregation. In the areas
where the protoplasts are in close contact, the plasma membranes of the
naked cells coalesce, inducing cytoplasmic continuity, which broadens
to the whole surface of membrane adhesion. The same effect of
protoplast agglutination and membrane coalescence is obtained
with polyethylene glycol (PEG), dextran, and polyvinyl alcohol (PVA).
Many laboratories use PEG in combination with high pH and high
Ca2+ concentration in the fusion buffer. However, PEG preparations are
often toxic for plant cells. This problem can be overcome by deionizing
PEG solutions or by reducing their concentration. In the electric field–
mediated method, protoplasts are first brought in close contact by the
application of a nonuniform alternating electric field (AC), followed by
the application of electric field pulses (DC) of high intensity for a short
time, which leads to the breakdown of membranes at the points of
adhesion, and later, to cell fusion. Figure 1(a)–1(d) shows the different
phases of electrofusion of two protoplasts. This method has proved to be
very efficient in terms of hybrid yield because it avoids the
nonphysiological conditions imposed on protoplasts by PEG and/or high
pH. Furthermore, by proper adjustment of the fusion parameters, it is
possible to maximize the number of binucleate fusion products.
Figure 1. (a–d) The different phases of electrofusion of two protoplasts. (a) The protoplasts

are aligned by the application of the AC field. (b and c) The DC pulses induce the

breakdown of membranes and (d) the heterokaryon is formed, but the cytoplasms are not
 mixed yet. (e) Electrofusion products: h – heterokaryon from mesophyll and callus

protoplasts; c and s – homokaryons of callus and mesophyll protoplasts, respectively.

(f) Mesophyll protoplasts observed under ultraviolet (UV) light. (g) The arrowhead

indicates a heterokaryon derived form mesophyll and callus protoplasts, observed under UV

light. (h) Callus protoplasts treated with fluorescein isothiocyanate and observed under UV

light. (i) Somatic hybrid plants (middle) between Medicago arborea (left) and Medicago

sativa (right)

Morphological differences such as the presence or absence of

chloroplasts in mesophyll and cultured cell protoplasts, respectively, can
be used to identify fusion products by light microscopy. The physical
isolation of heterokaryons with a micropipette is feasible, but tedious
and labor consuming. Moreover, the hybrids must be recovered within a
few hours of fusion and before the mixing of parental cytoplasms,
otherwise the hybrid cells become undistinguishable from parental cells.
The addition of fluorescent dyes such as fluorescein
isothiocyanate (FITC) or fluorescein diacetate (FDA) to the colorless
protoplasts makes them fluoresce green-yellow under ultraviolet light
(UV) (Figure 1(h)), while mesophyll protoplasts appeared red because
of chlorophyll fluorescence (Figure 1(f)); the hybrid cells appear with
red spots (Figure 1(g)). This treatment extends the time limit for hybrid
identification by another 3–4 days; it reduces the risks of
misinterpretations and allows the use of equipment such as FACS for the
automatic screening and isolation of differentially stained protoplasts.
Since neither antibiotic resistant nor auxotrophic mutants were available
in Medicago, hybrid cells were selected on the basis of their color using
the FITC staining procedure. Fusion products were embedded in
semisolid culture medium 2–3 days after fusion, and heterokaryon
development followed visually until the stage of 50- to 80-cell clusters,
which could be picked up manually with the needle of a syringe.
Prolonged culture of heterokaryons in close proximity to rapidly
dividing parental protoplasts can be critical as it results in a higher
percentage of dividing heterokaryons. Similarly, the surrounding of
newly isolated hybrid calli with actively dividing nurse calli greatly
enhanced their growth.
The culture of hybrid cells does not differ substantially from that of
normal plant cells, and a culture medium with a high ratio of
auxin/cytokinin growth regulators is initially required to enhance mitotic
division of hybrid cells. When a callus is formed as a result of many
divisions, the regeneration process is triggered by reducing the auxin
to cytokinin ratio. In general, the regeneration of hybrid plants is more
difficult than that of the corresponding parental genotypes. This is
probably due to the number of stresses to which the hybrid cells are
subjected (enzyme treatment for protoplast isolation, exposure to
fusogenic agents, selective pressures with drugs of various nature). In
addition, either polyploidization or the genetic distance of the parents
contributes to prolong the regeneration phase of hybrid calli.
Even when the sources of parental protoplasts were correctly selected,
regeneration from hybrid calli in the genus Medicago was not easy. In
fact, although M. sativa + M. coerulea somatic hybrids (S + C) followed
the regeneration pathway of the M. sativa parental line, the other two
somatic hybrid plants, M. sativa + M. arborea (S + A)
and M. sativa + M. falcata (S + F) regenerated with extreme difficulty
only after more than 1 year of culture.
Even when the hybrid cells were subjected to selective pressure or
physically isolated, the hybrid nature of the regenerated plants should be
confirmed because escapes from the selective agent and
misclassification of hybrid cells cannot be excluded a
priori. Isozymes that are different forms of the same enzymatic
activity are often polymorphic between parental lines of somatic
hybrids and are visualized as bands of different molecular weights in an
electrophoretic pattern. Each band corresponds to a gene that can be
taken as a marker of the presence of a determinate genome or a fraction
of it in the hybrid genome. The general rule is that polymorphic bands
must be simultaneously present in the hybrids. However, isozyme
analysis provides only circumstantial evidence of hybridity; chimerism,
i.e., the simultaneous presence of both parental cells in the hybrid plants
that have not originated from a real fusion event, cannot be ruled out. If
the parental lines have different isozymes and they are simultaneously
present in the hybrids, the analysis of the number, size, and shape of the
chromosomes of the hybrid cells can confirm hybridity and exclude
chimerism.
With the advent of the DNA-based technologies, a range of genetic
(molecular) markers are available allowing the contribution of each
parental genome in the hybrid to be quantified at both the cytoplasmic
and nuclear levels. This is particularly useful for those hybrids in which
one parent contributes poorly to the constitution of the hybrid genome
(asymmetric somatic hybrids). The most commonly used markers are
restriction fragment length polymorphisms (RFLPs), random amplified
polymorphic DNA (RAPD), amplified fragment length polymorphisms,
and simple sequence repeats (SSRs). They are all based on variation
in DNA sequences and therefore are able to reveal a great number of
parent-specific alleles that allow the nuclear contribution of each parent
to the hybrid genome to be quantified. Similarly, the DNA of
chloroplasts and mitochondria can be analyzed with these markers to
investigate the cytoplasmic genetic constitutions of somatic hybrids.
Somatic hybridization and cybridization have great potential in plant
improvement. Somatic hybridization through protoplast fusion provides
the ability to combine parent genes in higher plants to overcome sexual
incompatibility among plant species or genera. Protoplast fusion enables
transfer of desirable qualities, for example, resistance to diseases
(bacterial, fungal, viral), pests, herbicides, and other stress factors.
Hundreds of reports on somatic hybridization have been published
during the past four decades. A recent review of somatic hybrids was
provided by Liu et al. (2005a). Grosser and Gmitter (2005) and Singh
and Rajam (2009) reviewed applications of somatic hybridization and
cybridization in citrus improvement. Wang et al. (2009) reviewed
progress in somatic hybridization in banana (Musa spp.). Application to
genetics and breeding in somatic hybrids of potato (Solanum tuberosum)
was reviewed by Orczyk et al. (2003). Somatic hybridization in the
family Brassicaceae was reviewed by Navratilova (2004). Reddy et al.
(2008) reviewed developments in seaweed protoplast research and their
potential in genetic improvement. This section primarily focuses on
literature published in the past decade, with particular emphasis on types
of somatic hybrids, protoplast fusion methods, selection and
identification of somatic hybrids, and the recent efforts in somatic
hybridization are reviewed.
Somatic hybridization is a technique that has been used to tap the
potential of related or distant species/genera of crops and is especially
useful for creating novel combinations of nuclear and/or cytoplasmic
genomes. Numerous intergeneric, and intra- and interspecific somatic
hybrids have been reported and genes for quality improvement,
resistance against bacterial, fungal, viral and nematode diseases, as well
as abiotic stress tolerance have been transferred (Liu et al., 2005; Nagata
and Bajaj, 2001). Several new commercial varieties of potato and
oilseed rape have also been produced through this technology (Murphy,
2007).
Drawbacks of somatic hybridization include genetic instability and low
fertility of the hybrids, and over the past few years, this technology has
been replaced to a large extent by transgenesis. Developing protocols
for somatic hybridization is often a long and cumbersome process, and
poor plantlet regeneration efficiency in vitro is a limitation in many
species. However, somatic hybridization has considerable potential for
the future since, unlike transgenesis, it is more efficient for transferring
polygenic traits, it does not require the same regulatory approval, and
advances in tissue culture and molecular marker techniques have
increased the success rate in regenerating genetically stable progeny 
Selection of Heterokaryons and Hybrid Somatic Tissues
The most difficult aspect of somatic hybridization is the selection
of heterokaryons, cells, or tissues derived from heterokaryons or hybrid
plants. In a number of studies, the protoplasts subjected to fusion
treatments were cultured, and the regenerated plants were selected based
on their traits. This procedure is laborious and takes up a large amount
of space.
When two populations of protoplasts are easily identified, individual
heterokaryons can be separated using micropipettes. This method works
well for leaf protoplast fusions, which contain chlorophyll, and
discolored protoplasts can be isolated from cell suspensions. Great
advances have been made in this field with the development of the
current system of fluorescent labeling in heterokaryons. The protoplasts
are stained green through treatment with fluorescein diacetate (1–
20 mg L−1) and are fused with protoplasts emitting red fluorescence,
derived from autofluorescing chlorophyll and exogenously
applied rhodamine isocyanate, respectively.
A number of procedures inhibit the growth of homokaryons of at least
one of the parental populations. Hormone autotrophic, auxotrophic, and
antibiotic resistant mutants, amino acid analogs, and fungal toxins have
been used in various combinations. Albino mutants have also been used
successfully, and hybrid tissues have been identified through the
production of chlorophyll following exposure of the cultures to light.
A second outcome or byproduct from the widely used citrus somatic
hybridization model, where protoplasts isolated from embryogenic
callus/cell suspension cultures are fused with the protoplasts derived
from nonembryogenic leaf mesophyll cells of the second parent, is the
production of cybrid plants (reviewed by Guo et al., 2004a). Somatic
hybrids contain a hybrid tetraploid nucleus, whereas cybrids retain the
diploid nucleus from the nonembryogenic parent (Grosser et al.,
2000; Guo et al., 2013). Citrus cybrid plants regenerated from protoplast
fusion events almost always possess the mitochondrial DNA (mtDNA)
of the embryogenic parent, indicating a critical role of the mitochondrial
genome in the regeneration of cybrids (Moreira-Dias et al.,
2000; Yamamoto et al., 1995; Moriguchi et al., 1996; Saito et al.,
1993; Cabasson et al., 2001). Chloroplast regeneration in citrus cybrid
plants is generally random from either parent, with mixtures not
observed. Cybridization in citrus provides another unique source
of genetic variation that can be exploited for cultivar development by the
creation of novel alloplasmic combinations of the nuclear and organelle
genomes that are present in fusion parents.
 SOMACLONAL VARIATION

Somaclonal variation is defined as genetic or epigenetic changes that

arise in vitro between clonal regenerants and their corresponding donor
plants. The genetic changes are cytogenetic abnormalities and alterations
to specific sequences of DNA; epigenetic changes are alterations of gene
expression without changes to DNA sequences. Somaclonal variation,
independent from the mechanisms involved, has been reported for a
number of plant species. The occurrence of somaclonal variation in
tissue culture has a negative effect on the rapid production of clonal
plants of elite cultivars, but may promote the production of
novel horticultural crop genotypes. An improved understanding of the
mechanisms of variation in tissue culture, specifically epigenetic
variation, may be a potential tool in producing cultivars adapted to meet
the growing demand for food. Somaclonal variation generated through
tissue culture can form the basis of selection, either in vitro or among
regenerated plants, for oat genotypes with altered grain quality profiles.
Selection among regenerated oat lines for altered levels of grain quality
factors (e.g., β-glucan, avenanthramides, protein, fat, etc.) may provide a
means to improve grain quality of oat varieties that have high agronomic
performance but low grain quality. Additionally, selection for resistance
traits in vitro (such as mycotoxin or herbicide resistance) may indirectly
improve oat quality through enhanced agronomic performance. Finally,
genetic engineering approaches to studying and altering oat grain quality
currently rely on successful culture and regeneration of oat plants.
Somaclonal variation and in vitro mutation induction have been used in
sugarcane, banana and potato, with the objective of improving specific
characters, mainly the resistance to diseases and other defects that limit
the use of some important commercial varieties.
Sugarcane was the first crop and the one where the most important
results have been obtained. A complete selection scheme was
established and it was demonstrated that, only after three vegetative
multiplications in field, a selection of true mutants for the main
quantitative characters could be carried out. Sixteen somaclones were
obtained and 5 of them were approved for the extension and introduction
in the production as commercial varietes. The selection process lasted
from 5 to 7 years in total, these results demonstrated the efficiency of in
vitro mutation in comparison with the conventional improvement for
specific characters. This advantage has been outlined by several
investigators, but not with practical results to date (Larkin and
Scowcroft, 1981, Micke and Donini, 1994).
In potato, mutation induction and in vitro selection have been used in
order to improve the resistance against Alternaría solani, in the variety
Desiree that occupies around 80% of the cultivated area in the country.
Four resistant somaclones were selected which are under field testing
for agronomic traits. Bananas and plantains have two main
phytopathologic problems: Fusarium and Black Sigatoka.
For Fusarium resistance 17 000 somaclones from the variety Gross
Michel were studied and after 5 years of selection in greehouses and
field, 5 resistant somaclones have been selected. They are evaluated
under production conditions. For Black Sigatoka, 50 000 somaclones
from Grand Naine have been studied and no resistant individuals were
selected. Although, with the development of an efficient regeneration
system via somatic embriogenesis combined with in vitro selection
better results are expected.
The results obtained with the use of somaclonal variation, mutation
induction and in vitro selection have created the methodological bases
for the handling and selection in plant populations obtained from tissue
culture. The main problems for selection in such populations are
associated with epigenetic and juvenile effects induced by in
vitro conditions. These problems are also present in transformed plants.
This program not only has given knowledge, but it has also generated
materials of great value for molecular biology and transformation
projects. For instance, in sugarcane 3 rust (Puccinia melanocephala)
resistant somaclones were obtained, as well as a clone selected from
classical mutation in buds. These clones are used as resistant progenitors
for hybridization and they also were crossed with the original variety to
determine the inheritance of the resistance that was obtained by mutation
induction. The final goal is to clone and characterize the gene(s)
involved in the resistance mechanism to be used in genetic
transformation.
Somaclonal variation is 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 is called somaclonal
variation . Therefore, it can be defined as the variation that occurs
because of genetic mutation caused by in vitro conditions or by chimeral
separation. Somaclonal variation is usually undesirable. In some cases,
somaclonal variation can lead to new cultivars (e.g., disease resistance,
new leave pattern) that may have desirable ornamental characteristics or
increased pest resistance.
The occurrence of somaclonal variation can be reduced by:

 Avoiding long-term cultures.

 Using axillary shoot induction systems where possible.
 Propagating chimeras by other clonal systems.

 It is well known that increasing numbers of subcultures

increase the likelihood of somaclonal variation, so the
number of subcultures in micropropagation protocols should
be kept to a minimum.
  Regularly reinitiating clones from new explants, which might
reduce variability over time.
 Avoiding 2,4-D in the culture medium, as this hormone is
known to introduce variation.

Considering the sources of somaclonal variation, it is unpractical the

management of the preexisting variability into the sugarcane modem
genotypes. Both the choice of the starting material for transformation
and the cell targeted for transformations are random events.
However, somaclonal variation induced by tissue culture could be
management.
Genomic variability induced or evidenced by tissue culture has been
demonstrated. Results of polymorphism indexes by studying non-
regenerable calli and albino plants could be considered high (manuscript
in preparation) and, point that these polymorphisms could be present, at
least partially, within the transgenic plants. It is well know that
callogenesis is a source of additional variability in plant tissue cultures.
All available transformation protocol for sugarcane used the callogenesis
as way for propagates the transformed cells and it further plant
regeneration (Arencibia et al., 1992; 1995; 1998; Bower and Birch,
1992; Enriquez et al., 1998). For that reason, our recent work has been
based on the hypothesis that direct organogenesis must give shoots
carrying and evidencing only the pre-existing variability, and not
inducing a new variability.
As result, we developed a novel approach for sugarcane transformation
based on the A. tumefaciens infection of the basal zone of in vitro plants.
Repetitive selective-micropropagation steps eliminated probability for
the recovery of mosaic plants. GUS staining was uniform in all parts of
the hygromicin-resistant plants. Optimized transformation conditions
were plant size between 1.1-3 cm, co-cultivation time ranging 72-96
hours in the dark and bacterial concentration of DO 620nm= 1. Frequency
for hygromicin-resistant (30 mg/L)-plant recovery was 1,87 % and
2,5 % using EHA105 (pCambia1301) and LBA4404 (pTOK233),
respectively.
Advantages of this methodology regarding with the presence of
somaclonal variants into the transgenic plants will be studies based on
high-volume molecular tools for genome exploration as AFLP.
Somaclonal variation can disturb both basic and applied studies
on transgenic plants. The use of transgenic plants in order to assign a
role to cloned genes of unknown function may be impaired by
concomitant variant traits due to somaclonal variation. For example, in
the agronomic and industrial exploitation of transgenic plants, transgenic
elite cultivars might show undesirable changes compared with the
original plant. For instance, when insect-resistant, transgenic sugarcane
plants were produced, they were found to be morphologically identical,
but the agronomic analysis of selected plants showed changes in
some agronomic traits. Rare DNA changes were also observed
by AFLP analysis.
Nevertheless, a large array of commercially exploitable transgenic plants
has been produced. In fact, several different approaches can be applied
to overcome the problem of somaclonal variation in transgenic plants.
One may be that of using recurrent backcrossings to restore the original
genotype while retaining the foreign gene. A second approach may be to
utilize the best-performing transgenic plants, regardless of genomic
changes. The former approach is particularly suitable for annual crops,
such as rice, while the latter is suitable for plants that are commercially
reproduced by cuttings, such as sugarcane or poplar. But these
approaches are not appropriate for all cases. For instance, in the
hypothetical case of transgenic olive trees, it would not be practical to
wait years before verifying that somaclonal variation had not affected
flavor, yield, ripening, or other industrial traits in selected trees.
The term somaclonal variation is defined as genetic variation that is
present in plants regenerated from tissue cultures, either uncovered or
induced by a tissue culture process (Larkin and Scowcroft, 1981). The
first reports of somaclonal variation were primarily from solanaceous
or cereal crops, affecting a wide range of traits including plant height,
growth habit, flower, fruit and leaf morphology, juvenility, maturity
date, disease resistance, yield, and various biochemical characteristics.
Sweet orange and other polyembryonic citrus cultivars are well suited
for studies of somaclonal variation due to their biology and good
performance in tissue culture. The sweet orange is polyembryonic and
forms adventive somatic embryos from nucellar tissue, including
embryogenic callus and suspension cultures (Kochba et al., 1972). The
sweet orange was also shown early on to be amenable
to protoplast regeneration via somatic embryogenesis (Vardi et al., 1982)
and to plant regeneration via organogenesis through adventitious
shoot induction (Grinblat, 1972), which is a key to its
efficient Agrobacterium-mediated transformation (Moore et al., 1992).
The UF/CREC citrus improvement program has produced and evaluated
> 2000 sweet orange somaclones regenerated from Hamlin, Valencia,
Vernia, and a noncommercial selection designated as OLL (Orie Lee
Late) for fresh market and processing potential (Grosser et al., 1997).
Cultivars released for commercial production from this work are
provided in Improved attributes include earlier maturity, seedlessness,
and improved fruit/juice quality, all achieved while maintaining cultivar
integrity. Somaclonal variation has proven to be a valuable yet
underexploited source of genetic variation for citrus improvement,
especially in cultivars that are difficult to improve by conventional
breeding. Somaclonal variation could also be exploited in
monoembryonic cultivars such as Clementines, depending on the ability
of each selection to respond efficiently on shoot induction
(organogenesis) media.
When the in vitro cultured cells have gone through genetic variation it is
then called somaclonal variation and the plants derived from such cells
are called “somaclones.” Somaclonal variation is always associated with
chromosomal variations, which have been generally found in long-term
callus, cell suspension culture, and plants regenerated from such
cultures. This type of genetic variation generates various potential
applications such as crop improvement, in the production of mutants and
variants (e.g., disease resistance in potato). Larkin and Scowcroft coined
the term “somaclones” for plant variants obtained from tissue cultures of
somatic tissues. Similarly, if the somatic tissue-derived variants have a
gametophytic origin such as pollen or egg cell, then it is known as
“gametoclonal” variation . Several causes of this type of variation are
heterogeneity between the cells and explant tissue, spontaneous mutation
and activation of culture environment of transposition of genetic
materials. In 1980, Shepard and his coworkers screened 100 somaclones
produced from leaf protoplasts of large white potato (Russet Burbank) .
They established that there was a significant amount of stable variation
in compactness of growth habit, maturity, date, tuber uniformity, tuber
skin color, and photoperiodic requirements. Among the various
applications, plant breeding is the major application of somaclonal
variations where the new traits with desired or improved characters are
introduced into the plants.
 APPLICATIONS

 The commercial production of plants used as potting,

landscape, and florist subjects, which uses meristem and
shoot culture to produce large numbers of identical
individuals.
 To conserve rare or endangered plant species.
 A plant breeder may use tissue culture to screen cells rather
than plants for advantageous
characters,e.g. herbicide resistance/tolerance.
 Large-scale growth of plant cells in liquid culture
in bioreactors for production of valuable compounds,
like plant-derived secondary metabolites and recombinant
proteins used as biopharmaceuticals.
 To cross distantly related species by protoplast fusion and
regeneration of the novel hybrid.
 To rapidly study the molecular basis for physiological,
biochemical, and reproductive mechanisms in plants, for
example in vitro selection for stress tolerant plants.
 To cross-pollinate distantly related species and then tissue
culture the resulting embryo which would otherwise
normally die (Embryo Rescue).
 For chromosome doubling and induction of polyploidy, for
example doubled haploids, tetraploids, and other forms
of polyploids. This is usually achieved by application
of antimitotic agents such as colchicine or oryzalin.
 As a tissue for transformation, followed by either short-term
testing of genetic constructs or regeneration
of transgenic plants.
 Certain techniques such as meristem tip culture can be used
to produce clean plant material from virused stock, such as
sugarcane, potatoes and many species of soft fruit.
 Production of identical sterile hybrid species can be
obtained.
 Large scale production of artificial seeds through somatic
embryogenesis
 CONCLUSION
In reality, there are numerous methods used for tissue culture
given that there are different types of tissues that require specific
conditions for the culture process yield desired results. Both
plant and animal tissue can be used for tissue culture purposes
for a wide range of purposes. For instance, animal tissue culture
may serve such purposes as preservation of an organ/tissue,
studying the tutors or given tissues or for diagnosis purposes. On
the other hand, plant tissue culture may be used for cloning
purposes, genetic modification of a given plant or simply to
accelerate or increase yield of the plant of interest. Tissue
culture is therefore of great significance in biological studies due
to its wide range of applications. The processes involved in
tissue culture may be complex, requiring a lot of care to avoid
such effects as contamination. Because of the complexities that
may be involved in some of the steps, this may not be an
experiment for everyone.

Plant tissue culture as an important tool for the continuous

production of active compounds including secondary
metabolites and engineered molecules. Novel methods (gene
editing, abiotic stress) can improve the technique. Humans have
a long history of reliance on plants for a supply of food, shelter
and, most importantly, medicine. Current-day pharmaceuticals
are typically based on plant-derived metabolites, with new
products being discovered constantly. Nevertheless, the
consistent and uniform supply of plant pharmaceuticals has
often been compromised. One alternative for the production of
important plant active compounds is in vitro plant tissue culture,
as it assures independence from geographical conditions by
eliminating the need to rely on wild plants. Plant transformation
also allows the further use of plants for the production of
engineered compounds, such as vaccines and multiple
pharmaceuticals. This review summarizes the important
bioactive compounds currently produced by plant tissue culture
and the fundamental methods and plants employed for their
production.
 REFERENCES
1. "Explant Culture for Developmental Studies Protocol". www.jove.com.
Retrieved 2017-05-25.
2. ^  Jump up to:a b
   "Explant Culture of Neural Tissue". JoVE Science Education
Database.
3. ^ Orth MW et al. (2000) Cartilage turnover in embryonic chick tibial
explant cultures. Poult Sci 79:990-993.
4. ^ Ivan Bedzhov, Magdalena Zernicka-Goetz.(2014). Self-Organizing
Properties of Mouse Pluripotent Cells Initiate Morphogenesis upon
Implantation. Cell,; DOI: 10.1016/j.cell.2014.01.023
5. ^ Hunter, Philip (2017-05-25). "One organ at a time: Research has been
making much progress to create in vitro human tissues for transplantation
but laboratory-grown complex organs still remain decades away". EMBO
Reports. 15 (3): 227–230. doi:10.1002/embr.201438528. ISSN 1469-
221X. PMC 3989688. PMID 24554301.
6. ^ Garg, Aakriti; Yang, Jin; Lee, Winston; Tsang, Stephen H. (2017-02-
02). "Stem Cell Therapies in Retinal Disorders
7. Takeda, Reiji; Katoh, Kenji. "Growth and sesquiterpenoid production
by Calypogeia granulata inoue cells in suspension
culture". Planta. 151 (6): 525–
530. doi:10.1007/BF00387429. PMID 24302203.
8. ^ Peterson, M (2003). "Cinnamic acid 4-hydroxylase from cell cultures of
the hornwort Anthoceros agrestis". Planta. 217 (1): 96–
101. doi:10.1007/s00425-002-0960-9. PMID 12721853.
9. ^ Beutelmann, P.; Bauer, L. (1 January 1977). "Purification and
identification of a cytokinin from moss callus cells". Planta. 133 (3): 215–
217. doi:10.1007/BF00380679. PMID 24425252.
10. ^ Atmane, N. "Histological analysis of indirect somatic embryogenesis in
the Marsh clubmoss Lycopodiella inundata (L.) Holub
(Pteridophytes)". Plant Science. 156 (2): 159–167. doi:10.1016/S0168-
9452(00)00244-2.
11. ^ Yang, Xuexi; Chen, Hui; Xu, Wenzhong; He, Zhenyan; Ma, Mi.
"Hyperaccumulation of arsenic by callus, sporophytes and gametophytes
of Pteris vittata cultured in vitro". Plant Cell Reports. 26 (10): 1889–
1897. doi:10.1007/s00299-007-0388-6.
12. ^ Chavez, V. M.; Litz, R. E.; Monroy, M.; Moon, P. A.; Vovides, A. M.
"Regeneration of Ceratozamia euryphyllidia (Cycadales, Gymnospermae)
plants from embryogenic leaf cultures derived from mature-phase
trees". Plant Cell Reports. 17 (8): 612–616. doi:10.1007/s002990050452.
13. ^ Jeon, MeeHee; Sung, SangHyun; Huh, Hoon; Kim, YoungChoong.
"Ginkgolide B production in cultured cells derived from Ginkgo biloba L.
leaves". Plant Cell Reports. 14 (8). doi:10.1007/BF00232783.
14. ^ Finer, John J.; Kriebel, Howard B.; Becwar, Michael R. (1 January
1989). "Initiation of embryogenic callus and suspension cultures of eastern
white pine (Pinus strobus L.)". Plant Cell Reports. 8 (4): 203–
206. doi:10.1007/BF00778532.
15. ^ O'Dowd, Niamh A.; McCauley, Patrick G.; Richardson, David H. S.;
Wilson, Graham. "Callus production, suspension culture and in
vitro alkaloid yields of Ephedra". Plant Cell, Tissue and Organ
Culture. 34 (2): 149–155. doi:10.1007/BF00036095.
16. ^ Chen, Ying-Chun; Chang, Chen; Chang, Wei-chin. "A reliable protocol
for plant regeneration from callus culture of Phalaenopsis". In Vitro
Cellular & Developmental Biology – Plant. 36 (5): 420–
423. doi:10.1007/s11627-000-0076-5.
17. ^ Burris, Jason N.; Mann, David G. J.; Joyce, Blake L.; Stewart, C. Neal
(10 October 2009). "An Improved Tissue Culture System for Embryogenic
Callus Production and Plant Regeneration in Switchgrass (Panicum
virgatum L.)". BioEnergy Research. 2 (4): 267–274. doi:10.1007/s12155-
009-9048-8.
18. ^ Murashige, Toshio; F. Skoog (July 1962). "A Revised Medium for Rapid
Growth and Bio Assays with Tobacco Tissue Cultures". Physiologia
Plantarum. 15 (3): 473–497. doi:10.1111/j.1399-3054.1962.tb08052.x.
19. ^  Jump up to:a b
   White, P. R. (Feb 1939). "Potentially unlimited growth of
excised plant callus in an artificial nutrient". American Journal of
Botany. 26 (2): 59–4. doi:10.2307/2436709. JSTOR 2436709.
20. ^ Lloyd, G; B McCown (1981). "Commercially-feasible micropropagation
of mountain laurel, Kalmia latifolia, by use of shoot-tip culture". Combined
Proceedings, International Plant Propagators' Society. 30: 421–427.
21. ^ Gamborg, OL; RA Miller; K Ojima (April 1968). "Nutrient requirements
of suspension cultures of soybean root cells". Experimental Cell
Research. 50 (1): 151–158. doi:10.1016/0014-4827(68)90403-
5. PMID 5650857.
22. ^ Wang, X.-D.; Nolan, K. E.; Irwanto, R. R.; Sheahan, M. B.; Rose, R. J. (10
January 2011). "Ontogeny of embryogenic callus in Medicago truncatula:
the fate of the pluripotent and totipotent stem cells". Annals of
Botany. 107 (4): 599–
609. doi:10.1093/aob/mcq269. PMC 3064535. PMID 21224270.
23. ^ He, Yang; Guo, Xiulian; Lu, Ran; Niu, Bei; Pasapula, Vijaya; Hou, Pei;
Cai, Feng; Xu, Ying; Chen, Fang. "Changes in morphology and biochemical
indices in browning callus derived from Jatropha curcas hypocotyls". Plant
Cell, Tissue and Organ Culture. 98 (1): 11–17. doi:10.1007/s11240-009-
9533-y.
24. ^ Dan, Yinghui; Armstrong, Charles L.; Dong, Jimmy; Feng, Xiaorong;
Fry, Joyce E.; Keithly, Greg E.; Martinell, Brian J.; Roberts, Gail A.; Smith,
Lori A.; Tan, Lalaine J.; Duncan, David R. "Lipoic acid—an unique plant
transformation enhancer". In Vitro Cellular & Developmental Biology -
Plant. 45 (6): 630–638. doi:10.1007/s11627-009-9227-5.
25. ^ Pazuki, Arman & Sohani, Mehdi (2013). "Phenotypic evaluation of
scutellum-derived calluses in 'Indica' rice cultivars" (PDF). Acta
Agriculturae Slovenica. 101 (2): 239–247. doi:10.2478/acas-2013-0020.
Retrieved February 2, 2014.
26. ^ Razdan, M. K. (2003). Introduction to plant tissue culture (2. ed.).
Enfield, NH [u.a.]: oxford Publishers. ISBN 1-57808-237-4.
27. ^ Gautheret, Roger J. (1 December 1983). "Plant tissue culture: A
history". The Botanical Magazine Tokyo. 96 (4): 393–
410. doi:10.1007/BF02488184.
28. ^ Chawla, H.S. (2002). Introduction to plant biotechnology (2nd ed.).
Enfield, N.H.: Science Publishers. ISBN 1-57808-228-5.  Sathyanarayana,
B.N. (2007). Plant Tissue Culture: Practices and New Experimental
Protocols. I. K. International. pp. 106–. ISBN 978-81-89866-11-2.
29. ^ Bhojwani, S. S.; Razdan, M. K. (1996). Plant tissue culture: theory and
practice (Revised ed.). Elsevier. ISBN 978-0-444-81623-8.
30. ^ Vasil, I.K.; Vasil, V. (1972). "Totipotency and embryogenesis in plant cell
and tissue cultures". In Vitro. 8 (3): 117–
125. doi:10.1007/BF02619487. PMID 4568172. S2CID 20181898.
31. ^ Brian James Atwell; Colin G. N. Turnbull; Paul E. Kriedemann
(1999). Plants in Action: Adaptation in Nature, Performance in
Cultivation (1st ed.). Archived from the original on March 27, 2018.
Retrieved May 7, 2020.
32. ^ Indra K. Vasil; Trevor A. Thorpe (1994). Plant Cell and Tissue Culture.
Springer. pp. 4–. ISBN 978-0-7923-2493-5.
33. ^  Jump up to:a  b Pazuki, Arman & Sohani, Mehdi (2013). "Phenotypic evaluation
of scutellum-derived calluses in 'Indica' rice cultivars" (PDF). Acta
Agriculturae Slovenica. 101 (2): 239–247. doi:10.2478/acas-2013-0020.
34. ^ Mukund R. Shukla; A. Maxwell P. Jones; J. Alan Sullivan; Chunzhao Liu;
Susan Gosling; Praveen K. Saxena (April 2012). "In vitro conservation of
American elm (Ulmus americana): potential role of auxin metabolism in
sustained plant proliferation". Canadian Journal of Forest
Research. 42 (4): 686–697. doi:10.1139/x2012-022.
35. ^ Georgiev, Milen I.; Weber, Jost; MacIuk, Alexandre (2009).
"Bioprocessing of plant cell cultures for mass production of targeted
compounds". Applied Microbiology and Biotechnology. 83 (5): 809–
23. doi:10.1007/s00253-009-2049-x. PMID 19488748. S2CID 30677496.
36. ^ Manoj K. Rai; Rajwant K. Kalia; Rohtas Singh; Manu P. Gangola; A.K.
Dhawan (April 2011). "Developing stress tolerant plants through in vitro
selection—An overview of the recent progress". Environmental and
Experimental Botany. 71 (1): 89–98. doi:10.1016/j.envexpbot.2010.10.021.
37. ^ Aina, O; Quesenberry, K.; Gallo, M (2012). "In vitro induction of
tetraploids in Arachis paraguariensis". Plant Cell, Tissue and Organ
 Culture. 111 (2): 231–238. doi:10.1007/s11240-012-0191-
0. S2CID 9211804.
38. ^ Pawar, K. R., Waghmare, S. G., Tabe, R., Patil, A. and Ambavane, A. R.
2017. In vitro regeneration of Saccharum officinarum var. Co 92005 using
shoot tip explant. International Journal of Science and Nature 8(1): 154-
157.
39. ^ Waghmare, S. G., Pawar, K. R., and Tabe, R. 2017. Somatic
embryogenesis in Strawberry (Fragaria ananassa) var. Camarosa. Global
Journal of Bioscience and Biotechnology 6(2): 309 - 313.

40.Abbasi, B. H., Stiles, A. R., Saxena, P. K., Liu, C. Z. (2012). Gibberellic acid
increases secondary metabolite production in Echinacea purpurea hairy
roots. Appl. Biochem. Biotechnol. 168, 2057–2066. doi: 10.1007/s12010

41. "Some landmarks in the development of tissue and cell culture".

Retrieved 2006-04-19.
42. ^ "Cell Culture". Retrieved 2006-04-19.
43. ^ "Whonamedit - Ringer's solution". whonamedit.com. Retrieved 2014-06-
09.
44. ^ "Animals and alternatives in testing". Archived from the original on
2006-02-25. Retrieved 2006-04-19.
45. ^ Schiff J (February 2002). "An unsung hero of medical research". Yale
Alumni Magazine. Retrieved 2006-04-19.
46. ^ Voigt N, Pearman CM, Dobrev D, Dibb KM (September 2015). "Methods
for isolating atrial cells from large mammals and humans". Journal of
Molecular and Cellular Cardiology. 86: 187–
98. doi:10.1016/j.yjmcc.2015.07.006. PMID 26186893.
47. ^ Louch WE, Sheehan KA, Wolska BM (September 2011). "Methods in
cardiomyocyte isolation, culture, and gene transfer". Journal of Molecular
and Cellular Cardiology. 51 (3): 288–
98. doi:10.1016/j.yjmcc.2011.06.012. PMC 3164875. PMID 21723873.
48. ^ Hemeda, H., Giebel, B., Wagner, W. (16Feb2014) Evaluation of human
platelet lysate versus fetal bovine serum for culture of mesenchymal stromal
cells Cytotherapy p170-180 issue 2 doi.10.1016
49. ^ "Post - Blog | Boval BioSolutions, LLC". bovalco.com. Retrieved 2014-
12-02.
50. ^ "LipiMAX purified lipoprotein solution from bovine serum". Selborne
Biological Services. 2006. Archived from the original on 2012-06-08.
Retrieved 2010-02-02.
51. ^ Portela VM, Zamberlam G, Price CA (April 2010). "Cell plating density
alters the ratio of estrogenic to progestagenic enzyme gene expression in
cultured granulosa cells". Fertility and Sterility. 93 (6): 2050–
5. doi:10.1016/j.fertnstert.2009.01.151. PMID 19324349.
52. ^ Humpel C (October 2015). "Organotypic brain slice cultures: A
review". Neuroscience. 305: 86–
98. doi:10.1016/j.neuroscience.2015.07.086. PMC 4699268. PMID 26254
240.
53. ^ Neimark J (February 2015). "Line of attack". Science. 347 (6225): 938–
40. Bibcode:2015Sci...347..938N. doi:10.1126/science.347.6225.938. PMI
D 25722392.
54. ^ Drexler HG, Dirks WG, MacLeod RA (October 1999). "False human
hematopoietic cell lines: cross-contaminations and
misinterpretations". Leukemia. 13 (10): 1601–
7. doi:10.1038/sj.leu.2401510. PMID 10516762.
55. ^ Drexler HG, MacLeod RA, Dirks WG (December 2001). "Cross-
contamination: HS-Sultan is not a myeloma but a Burkitt lymphoma cell
line". Blood. 98 (12): 3495–
6. doi:10.1182/blood.V98.12.3495. PMID 11732505.
56. ^ Cabrera CM, Cobo F, Nieto A, Cortés JL, Montes RM, Catalina P,
Concha A (June 2006). "Identity tests: determination of cell line cross-
contamination". Cytotechnology. 51 (2): 45–50. doi:10.1007/s10616-006-
9013-8. PMC 3449683. PMID 19002894.
57. ^  Jump up to:a  b Chatterjee R (February 2007). "Cell biology. Cases of mistaken
identity". Science. 315 (5814): 928–
31. doi:10.1126/science.315.5814.928. PMID 17303729. S2CID 13255156
.
58. ^ Liscovitch M, Ravid D (January 2007). "A case study in misidentification
of cancer cell lines: MCF-7/AdrR cells (re-designated NCI/ADR-RES) are
derived from OVCAR-8 human ovarian carcinoma cells". Cancer
Letters. 245 (1–2): 350–
2. doi:10.1016/j.canlet.2006.01.013. PMID 16504380.
59. ^ MacLeod RA, Dirks WG, Matsuo Y, Kaufmann M, Milch H, Drexler HG
(November 1999). "Widespread intraspecies cross-contamination of human
tumor cell lines arising at source". International Journal of Cancer. 83 (4):
555–63. doi:10.1002/(SICI)1097-0215(19991112)83:4<555::AID-
IJC19>3.0.CO;2-2. PMID 10508494.
60. ^ Masters JR (April 2002). "HeLa cells 50 years on: the good, the bad and
the ugly". Nature Reviews. Cancer. 2 (4): 315–
9. doi:10.1038/nrc775. PMID 12001993. S2CID 991019.
61. ^  Jump up to:a b
   Dunham J, Guthmiller P (2008). "Doing good science:
Authenticating cell line identity" (PDF). Cell Notes. 22: 15–17. Archived
from the original (PDF) on 2008-10-28. Retrieved 2008-10-28.
62. ^ Nguyen HT, Geens M, Spits C (2012). "Genetic and epigenetic instability
in human pluripotent stem cells". Human Reproduction Update. 19 (2):
187–205. doi:10.1093/humupd/dms048. PMID 23223511.
63. ^  Jump up to:a b
   Lagziel S, GottliebE, Shlomi T (2020). "Mind your
media". Nature Metabolism. 2 (12): 1369–1372. doi:10.1038/s42255-020-
00299-y. PMID 33046912.
64. ^ Lagziel S, Lee WD, Shlomi T (2019). "Inferring cancer dependencies on
metabolic genes from large-scale genetic screens". BMC Biol. 17 (1):
37. doi:10.1186/s12915-019-0654-4. PMC 6489231. PMID 31039782.
65. ^ Vande Voorde J, Ackermann T, Pfetzer N, Sumpton D, Mackay G, Kalna
G; et al. (2019). "Improving the metabolic fidelity of cancer models with a
physiological cell culture medium". Sci Adv. 5 (1):
eaau7314. Bibcode:2019SciA....5.7314V. doi:10.1126/sciadv.aau7314. PM
C 6314821. PMID 30613774.
66. ^ Cantor JR, Abu-Remaileh M, Kanarek N, Freinkman E, Gao X, Louissaint
A; et al. (2017). "Physiologic Medium Rewires Cellular Metabolism and
Reveals Uric Acid as an Endogenous Inhibitor of UMP
Synthase". Cell. 169 (2): 258–
272.e17. doi:10.1016/j.cell.2017.03.023. PMC 5421364. PMID 28388410.
67. ^ "Moore v. Regents of University of California (1990) 51 C3d 120".
Online.ceb.com. Retrieved 2012-01-27.
68. ^ Hayflick L (September 1998). "A brief history of the mortality and
immortality of cultured cells". The Keio Journal of Medicine. 3. 47 (3):
174–82. doi:10.2302/kjm.47.174. PMID 9785764.
69. ^ "Worthington tissue guide". Retrieved 2013-04-30.
70. ^ Qian L, Saltzman WM (2004). "Improving the expansion and neuronal
differentiation of mesenchymal stem cells through culture surface
modification". Biomaterials. 25 (7–8): 1331–
7. doi:10.1016/j.biomaterials.2003.08.013. PMID 14643607.
71. ^ Maguire G (May 2016). "Therapeutics from Adult Stem Cells and the
Hype Curve". ACS Medicinal Chemistry Letters. 7 (5): 441–
3. doi:10.1021/acsmedchemlett.6b00125. PMC 4867479. PMID 27190588.
72. ^  Jump up to:a b
   Prieto D, Aparicio G, Sotelo-Silveira JR (November
2017). "Cell migration analysis: A low-cost laboratory experiment for cell
and developmental biology courses using keratocytes from fish
scales". Biochemistry and Molecular Biology Education. 45 (6): 475–
482. doi:10.1002/bmb.21071. PMID 28627731.
73. ^ Discher DE, Janmey P, Wang YL (November 2005). "Tissue cells feel and
respond to the stiffness of their substrate". Science. 310 (5751): 1139–
43. Bibcode:2005Sci...310.1139D. CiteSeerX 10.1.1.318.690. doi:10.1126/s
cience.1116995. PMID 16293750. S2CID 9036803.
74. ^ Gilbert PM, Havenstrite KL, Magnusson KE, Sacco A, Leonardi NA, Kraft
P, et al. (August 2010). "Substrate elasticity regulates skeletal muscle stem
cell self-renewal in culture". Science. 329 (5995): 1078–
81. Bibcode:2010Sci...329.1078G. doi:10.1126/science.1191035. PMC 292
9271. PMID 20647425.
75. ^ Chowdhury F, Li Y, Poh YC, Yokohama-Tamaki T, Wang N, Tanaka TS
(December 2010). Zhou Z (ed.). "Soft substrates promote homogeneous self-
renewal of embryonic stem cells via downregulating cell-matrix
tractions". PLOS ONE. 5 (12):
 e15655. Bibcode:2010PLoSO...515655C. doi:10.1371/journal.pone.001565
5. PMC 3001487. PMID 21179449.
76. ^ Engler AJ, Sen S, Sweeney HL, Discher DE (August 2006). "Matrix
elasticity directs stem cell lineage specification". Cell. 126 (4): 677–
89. doi:10.1016/j.cell.2006.06.044. PMID 16923388.
77. ^ Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, Gefen A,
et al. (September 2005). "Tensional homeostasis and the malignant
phenotype". Cancer Cell. 8 (3): 241–
54. doi:10.1016/j.ccr.2005.08.010. PMID 16169468.
78. ^ Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, et al.
(November 2009). "Matrix crosslinking forces tumor progression by
enhancing integrin signaling". Cell. 139 (5): 891–
906. doi:10.1016/j.cell.2009.10.027. PMC 2788004. PMID 19931152.
79. ^ Tilghman RW, Cowan CR, Mih JD, Koryakina Y, Gioeli D, Slack-Davis
JK, et al. (September 2010). Hotchin NA (ed.). "Matrix rigidity regulates
cancer cell growth and cellular phenotype". PLOS ONE. 5 (9):
e12905. Bibcode:2010PLoSO...512905T. doi:10.1371/journal.pone.001290
5. PMC 2944843. PMID 20886123.
80. ^ Liu F, Mih JD, Shea BS, Kho AT, Sharif AS, Tager AM, Tschumperlin DJ
(August 2010). "Feedback amplification of fibrosis through matrix stiffening
and COX-2 suppression". The Journal of Cell Biology. 190(4): 693–
706. doi:10.1083/jcb.201004082. PMC 2928007. PMID 20733059.
81. ^ Wipff PJ, Rifkin DB, Meister JJ, Hinz B (December 2007). "Myofibroblast
contraction activates latent TGF-beta1 from the extracellular matrix". The
Journal of Cell Biology. 179 (6): 1311–
23. doi:10.1083/jcb.200704042. PMC 2140013. PMID 18086923.
82. ^ Georges PC, Hui JJ, Gombos Z, McCormick ME, Wang AY, Uemura M,
et al. (December 2007). "Increased stiffness of the rat liver precedes matrix
deposition: implications for fibrosis". American Journal of Physiology.
Gastrointestinal and Liver Physiology. 293 (6): G1147–
54. doi:10.1152/ajpgi.00032.2007. PMID 17932231. S2CID 201357.
83. ^ Li L, Sharma N, Chippada U, Jiang X, Schloss R, Yarmush ML, Langrana
NA (May 2008). "Functional modulation of ES-derived hepatocyte lineage
cells via substrate compliance alteration". Annals of Biomedical
Engineering. 36 (5): 865–76. doi:10.1007/s10439-008-9458-
3. PMID 18266108. S2CID 21773886.
84. ^ Semler EJ, Lancin PA, Dasgupta A, Moghe PV (February 2005).
"Engineering hepatocellular morphogenesis and function via ligand-
presenting hydrogels with graded mechanical compliance". Biotechnology
and Bioengineering. 89 (3): 296–
307. doi:10.1002/bit.20328. PMID 15744840.
85. ^ Friedland JC, Lee MH, Boettiger D (January 2009). "Mechanically
activated integrin switch controls alpha5beta1
function". Science. 323(5914): 642–
4. Bibcode:2009Sci...323..642F. doi:10.1126/science.1168441. PMID 1917
9533. S2CID 206517419.
86. ^ Chan CE, Odde DJ (December 2008). "Traction dynamics of filopodia on
compliant substrates". Science. 322 (5908): 1687–
91. Bibcode:2008Sci...322.1687C. doi:10.1126/science.1163595. PMID 19
074349. S2CID 28568350.
87. ^ Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, et al.
(June 2011). "Role of YAP/TAZ in
mechanotransduction". Nature. 474(7350): 179–
 83. doi:10.1038/nature10137. hdl:11380/673649. PMID 21654799. S2CID 
205225137.
88. ^ "drug discovery@nature.com". Nature.com. Retrieved 2013-03-26.
89. ^ Duell BL, Cripps AW, Schembri MA, Ulett GC (2011). "Epithelial cell
coculture models for studying infectious diseases: benefits and
limitations". Journal of Biomedicine & Biotechnology. 2011:
852419. doi:10.1155/2011/852419. PMC 3189631. PMID 22007147.
90. ^ Barrila J, Radtke AL, Crabbé A, Sarker SF, Herbst-Kralovetz MM, Ott
CM, Nickerson CA (November 2010). "Organotypic 3D cell culture models:
using the rotating wall vessel to study host-pathogen interactions". Nature
Reviews. Microbiology. 8 (11): 791–
801. doi:10.1038/nrmicro2423. PMID 20948552. S2CID 6925183.
91. ^ Mapanao, Ana Katrina; Voliani, Valerio (June 2020). "Three-dimensional
tumor models: Promoting breakthroughs in nanotheranostics translational
research". Applied Materials Today. 19:
100552. doi:10.1016/j.apmt.2019.100552.
92. ^ Cassano, Domenico; Santi, Melissa; D’Autilia, Francesca; Mapanao, Ana
Katrina; Luin, Stefano; Voliani, Valerio (2019). "Photothermal effect by
NIR-responsive excretable ultrasmall-in-nano architectures". Materials
Horizons. 6 (3): 531–537. doi:10.1039/C9MH00096H. ISSN 2051-6347.
93. ^ Edmondson R, Broglie JJ, Adcock AF, Yang L (May 2014). "Three-
dimensional cell culture systems and their applications in drug discovery
and cell-based biosensors". Assay and Drug Development
Technologies. 12 (4): 207–
18. doi:10.1089/adt.2014.573. PMC 4026212. PMID 24831787.
94. ^ Bhattacharya M, Malinen MM, Lauren P, Lou YR, Kuisma SW, Kanninen
L, et al. (December 2012). "Nanofibrillar cellulose hydrogel promotes
 three-dimensional liver cell culture". Journal of Controlled
Release. 164 (3): 291–
8. doi:10.1016/j.jconrel.2012.06.039. PMID 22776290.
95. ^ DeRosa MC, Monreal C, Schnitzer M, Walsh R, Sultan Y (February
2010). "Nanotechnology in fertilizers". Nature Nanotechnology. 5 (2):
91. Bibcode:2010NatNa...5...91D. doi:10.1038/nnano.2010.2. PMID 20130
583.
96. ^ Hsiao AY, Tung YC, Qu X, Patel LR, Pienta KJ, Takayama S (May
2012). "384 hanging drop arrays give excellent Z-factors and allow
versatile formation of co-culture spheroids". Biotechnology and
Bioengineering. 109 (5): 1293–
304. doi:10.1002/bit.24399. PMC 3306496. PMID 22161651.
97. ^ Mapanao AK, Santi M, Faraci P, Cappello V, Cassano D, Voliani V
(September 2018). "Endogenously Triggerable Ultrasmall-in-Nano
Architectures: Targeting Assessment on 3D Pancreatic Carcinoma
Spheroids". ACS Omega. 3 (9): 11796–
11801. doi:10.1021/acsomega.8b01719. PMC 6173554. PMID 30320273.
98. ^ Simon EM (1988). "NIH Phase I Final Report: Fibrous Substrates for
Cell Culture (R3RR03544A) (PDF Download Available)". ResearchGate.
Retrieved 2017-05-22.
99. ^ Tibbitt MW, Anseth KS (July 2009). "Hydrogels as extracellular matrix
mimics for 3D cell culture". Biotechnology and Bioengineering. 103 (4):
655–63. doi:10.1002/bit.22361. PMC 2997742. PMID 19472329.
100. ^ "Quickie Bird Flu Vaccine Created". Wired. Wired.com. Reuters.
2006-01-26. Retrieved 2010-01-31.
101. ^ Gao W, Soloff AC, Lu X, Montecalvo A, Nguyen DC, Matsuoka Y,
et al. (February 2006). "Protection of mice and poultry from lethal H5N1
 avian influenza virus through adenovirus-based immunization". Journal of
Virology. 80 (4): 1959–64. doi:10.1128/JVI.80.4.1959-
1964.2006. PMC 1367171. PMID 16439551.
102. ^ "NIAID Taps Chiron to Develop Vaccine Against H9N2 Avian
Influenza". National Institute of Allergy and Infectious Diseases (NIAID).
2004-08-17. Retrieved 2010-01-31.
103. ^ Rapanan JL, Cooper KE, Leyva KJ, Hull EE (August 2014).
"Collective cell migration of primary zebrafish keratocytes". Experimental
Cell Research. 326 (1): 155–
65. doi:10.1016/j.yexcr.2014.06.011. PMID 24973510.
104. ^ Lee J, Jacobson K (November 1997). "The composition and
dynamics of cell-substratum adhesions in locomoting fish
keratocytes". Journal of Cell Science. 110 ( Pt 22) (22): 2833–
44. doi:10.1242/jcs.110.22.2833. PMID 9427291.
105. ^ Hunt P, Robertson D, Weiss D, Rennick D, Lee F, Witte ON (March
1987). "A single bone marrow-derived stromal cell type supports the in vitro
growth of early lymphoid and myeloid cells". Cell. 48 (6): 997–
1007. doi:10.1016/0092-8674(87)90708-2. PMID 2435412. S2CID 314996
11.
106. ^ van den Berg-Bakker CA, Hagemeijer A, Franken-Postma EM, Smit
VT, Kuppen PJ, van Ravenswaay Claasen HH, et al. (February 1993).
"Establishment and characterization of 7 ovarian carcinoma cell lines and
one granulosa tumor cell line: growth features and
cytogenetics". International Journal of Cancer. 53 (4): 613–
20. doi:10.1002/ijc.2910530415. PMID 8436435. S2CID 6182244.
107. ^ Lee YG, Korenchuk S, Lehr J, Whitney S, Vessela R, Pienta KJ
(2001). "Establishment and characterization of a new human prostatic
cancer cell line: DuCaP". In Vivo. 15 (2): 157–62. PMID 11317521.
108. ^ Ou D, Mitchell LA, Décarie D, Tingle AJ, Nepom GT (March
1998). "Promiscuous T-cell recognition of a rubella capsid protein epitope
restricted by DRB1*0403 and DRB1*0901 molecules sharing an HLA DR
supertype". Human Immunology. 59 (3): 149–57. doi:10.1016/S0198-
8859(98)00006-8. PMID 9548074.
109. Hanstein, J (1880). Das Protoplasma. Heidelberg.
110. ^ Sharp, LW (1921). Introduction To Cytology. New York: McGraw
Hill, p. 24.
111. ^  Jump up to:a b c
     Davey MR, Anthony P, Power JB, Lowe KC (2005).
"Plant protoplasts: status and biotechnological
perspectives". Biotechnology Advances. 23 (2): 131–
71. doi:10.1016/j.biotechadv.2004.09.008. PMID 15694124.
112. ^  Jump up to:a b c d
       Cushnie, TP; O’Driscoll, NH; Lamb, AJ
(2016). "Morphological and ultrastructural changes in bacterial cells as an
indicator of antibacterial mechanism of action". Cellular and Molecular
Life Sciences. 73 (23): 4471–4492. doi:10.1007/s00018-016-2302-
2. hdl:10059/2129. PMID 27392605. S2CID 2065821.
113. ^  Jump up to:a b c d
       "Protoplasts and
spheroplasts". www.encyclopedia.com. Encyclopedia.com. 2016.
Retrieved July 21, 2019.
114. ^  Jump up to:a b
   Dahiya, N; Tewari, R; Hoondal, GS (2006).
"Biotechnological aspects of chitinolytic enzymes: a review". Applied
Microbiology and Biotechnology. 71 (6): 773–782. doi:10.1007/s00253-
005-0183-7. PMID 16249876. S2CID 852042.
115. ^ "Definition of spheroplast". www.merriam-webster.com. Merriam-
Webster. 2019. Retrieved July 21, 2019.
116. ^ Thorpe TA (2007). "History of plant tissue culture". Molecular
Biotechnology. 37 (2): 169–80. doi:10.1007/s12033-007-0031-
3. PMID 17914178. S2CID 25641573.
117. ^ Bhatla SC, Kiessling J, Reski R (2002): Observation of polarity
induction by cytochemical localization of phenylalkylamine-binding
receptors in regenerating protoplasts of the moss Physcomitrella
patens. Protoplasma 219, 99–105.
118. ^ Hain R, Czernilofsky AP, et al. (1985). "Uptake, integration,
expression and genetic transmission of a selectable chimaeric gene by plant
protoplasts". Molecular and General Genetics 199:161–168.