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The cloning is much more widely spread in plant organisms. At
present, somatic embryogenesis is one of the main methods of
regenerating whole plants from tissue and cell cultures cultivated in vitro.
As phenomenon, the somatic embryogenesis is observed first in a
suspension (Steward et al., 1958 a, b) and callus (Reinert, 1959) of carrots.
Now it is established in more than 100 plant species and became a
promising method for intensive developing the production of numerous
important agricultural crops. Some authors divide it into direct and indirect.
The direct somatic embryogenesis is realized without callusogenesis. In
contrast to it, in indirect embryogenesis the cells should be dedifferentiated
to form callus and then to be induced and form embryoids.
Independently whether it is direct or indirect, the beginning of somatic
embryogenesis is preceded by cell division, leading to the formation of
spheric structures, called globules, from which the new explants are
formed. An illustration of this process is given in Figure 4–5. A regeneration
of plants can be also obtained from protoplast cultures (cell population with
disintegrated cell walls), as well as by using other in vitro techniques.

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Figure 4–5. Regeneration of a whole plant from a somatic cell of 
tobacco (Nicotiana tabacum) on medium for direct organogenesis, at a 
definite proportion of growth regulators — auxins and cytokinins 
(Courtesy of S. B. Slavov, Institute of Genetic Engineering, 
Kostinbrod). 
a — leaf explants; b — beginning of regeneration; c — shaped 
regenerated plant; d — in vitro rooting of a shaped plant.

The reproduction of living organisms through sexual cells is widely
spread in animal and plant kingdoms. Lately, widely discussed is the
problem about so-called cloning, i.e. obtaining viable progeny from single
vegetative (somatic) cells of definite cell lines obtained through genetic
methods and manipulations. It is outlined that cloning has not only scientific
and practical, but also social importance if it is applied to the man.
For the present, in animal organisms cloning is realized through
transplanting nuclei of somatic embryonic cells in enucleated unfertilized
eggs. The development of this problem begins after the 1950s, when the
role of nucleus for determining characteristic features of individual cell or
organism was proven. The difficulties in this direction met by the
investigators are connected mainly with the specificity of organism in
dependence of its position in evolutionary hierarchy and the development
stage of embryonic cells used as donor of nuclei. The earlier stage of
embryonic development is, the better the results are.
After the encouraging success of experiments with the unicellular
green alga Acetabularia and different amphibia (mainly frog tadpoles) the
investigators direct their attention to higher animal organisms — mice,
sheep, cows, etc. Willadsen (1986) obtained a lamb through cloning, by
using as donor cells nuclei of embryo in early morula stage (8 cells). Wilmut
and co-authors (1997) extended the age limits. They proved the possibility
to obtain a viable lamb not only from embryonic cells in early stage, but
also from adult animal cells (Fig. 4–4). The cloned lambs possesses the
same genotype as the donor-cells and differ from that of recipient.
According to the authors “the fact that a lamb was derived from an adult cell
confirms that differentiation of that cell did not involve the irreversible
modification of genetic material required for development”.

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Figure 4–4. Lamb number 6LL3 derived from the mammary gland of a 
Finn Dorset ewe with the Scottish Blackface ewe which was the 
recipient. The lamb possesses the donor-cell genotype differing from 
that of recipient (After Wilmut et al., 1997).

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Figure 4–3. Fresh-water hydra (Hydra vulgaris): a — general 
appearance; b — budding ; c — sexually mature hydra with eggs (From 
Life of Animals, vol. 1, 1968).

However, from genetical point of view one cannot pass over a very
important question related to the development of this process: to what
extent is the autonomy of the cells, building multicellular organisms,
preserved and is there a clearly expressed dividing line between the
constituent cells and the organisms as a whole. It may well be that many
processes, phenomena, troubles originate from the condition and activity of
a cell or group of cells. The elucidation of this question is forthcoming.
In the initial stages of evolution the formed multicellular organisms
reproduce asexually by forming new coenobia and colonies, or by budding.
Such capabilities can be traced in volvox algae, sea fungi, hydra, etc. In the
course of complicating their organization, along with the asexual
reproduction, the sexual process arises. In higher organisms, including
man, evolution differentiates the two types of cells — vegetative (somatic)
and sexual (reproductive).
As it was already mentioned, in multicellular organisms the vegetative or
somatic cells normally posses a diploid set of chromosomes (2n), and the sexual
ones — haploid (1n). To develop a new organism, it is necessary two sexual
cells (male and female) to fuse and form a zygote in which the genomes of the
initial parent organisms are combined. This is the biological sense of the sexual
reproduction irrespectively of its variety regarding ways and forms of realization.

primitive multicellular animals. Their body is built of uneven, porous mass of
cells connected with channels of different direction. These mostly
immovable sea animals live on the sea and ocean bottom attached to
different subaquatic objects. According to the shape and colour they are
extremely varied. As to the size, they from range millimeters to a meter and
more. On their surface there are a lot of pores from where their Latin name
Porifera, i.e. porous animals.
The reproduction of sea fungi is realized by both asexual and sexual
way. They posses large possibilities to regenerate from single cells. If a
part of their body is ground to individual cells and then filtered, the cells
preserve their viability, amoeba-like motion, gather together in small
groups, form shapeless associations of cells which after several days
become new small fungi.
The hydra (Hydra vulgaris) — see Fig. 4–3, is a typical fresh-water
animal, which place is at the beginning of the evolution of animal
multicellular organisms. Firstly it is examined under the microscope by van
Leeuwenhoek. It is a small beast of prey feeding with infusoria, plankton
crabs, small fishes, small worms, etc. It is known with its high ability to
regenerate as from separate parts, as well as from single cells. Dissected
in half, it rapidly regenerates the missing part. The astonishing thing in this
case is that on the anterior end of the piece always a “head” with tentacles
grows, and on the posterior one — pedicel. The cells in the middle of the
body reproduce more intensively, from where they move in opposite
directions. From single cells daughter hydras are developed by budding.
Except asexually (by budding) hydra also reproduces sexually, developing
male and female sexual cells (gonads). Here, there is a well-expressed
specialization and differentiation of the cells.
The brief survey of the organization of some of the lower multicellular
plant and animal organisms gives good reason to accept that their
formation is a result of integrating of individual cells in common biosystems.
As a result of the cell interactions, covering layers and membranes (like
those of independently existed unicellular organisms) arise. They protect
the cells from outer influences, take or secrete outwards different chemical
elements and substances, thus creating the necessary favourable
conditions for a normal realization of life processes in them. The
consolidation of cells in a united organism gives rise to the necessity of
specializing their functions. There appear different types of cells, tissues
and organs, and in the course of evolution not only their morphological
diversity increases, but also their strict functional specificity. Studying this
“evolutionary heritage” is a subject of separate branches of biology —
anatomy, histology, etc.

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Figure 4–2. Thread-like structures of blue-green algae: 1 — most simple 
structure of the fibre with diffusion growth in Oscillatoria; 2 — fibre with 
differentiated basis in Endonema; 3 — upper part of a fibre in Rivularia, 
extended like a hair; 4 — intercalated growth in Gloeotrichia; 5 — apical 
growth at the ends of branches in Scytonoma; 6 — basal growth in 
Fischirella; 7 — complex branchy fibrious structure in Fischirella (From 
Life of Plants, vol. 3, 1977). 

Sea fungi of Porifera (or Spongia) type also are a community of cells
organized in a different way. They are peculiar multicellular organisms.
Because of their appearance and structure, for a long time taxonomists
have been hesitated where is to assign them — to the plants or to the
animals. After intensive research, now they are considered to be the most

Figure 4–1. Coenobial and colonial forms in representatives of four 
genera of Volvocaceae family: 1 — Gonium pectorale; 2 — Eudorina
elegans; 3 — Pandorina morum; 4 — Volvox aureus (From Life of 
Plants, vol. 3, 1977).

A higher stage of organization is demonstrated by the colonial forms of
Volvox genus. Their colonies are mucous, spherical, with a diameter up to
2 mm. In their peripheral layer there are a lot of chlamydomonada-like cells
joined together through their mucous side walls. The motion of flagella is
also coordinated. In this case a better expressed specialization of the cells
in the colony is observed. Largest is the share of vegetative cells, which do
not participate in reproduction. Among them there are groups of cells
(connected by cytoplasmic fibres) from which new colonies are formed in
asexual way. At the end of vegetation sexual cells (oogonia and antheridia)
appear. As a result of their fusion new initial colonies arise. The cells are
mutually dependent and incapable of independently existence. Breacking
down the colony leads to their death.
Some of the lower multicellular blue-green algae (Cyanophyta) also
are a community of cells, arranged like fibres of different size. The fibres
can be single or branchy (Fig. 4–2). The cells posses pores, channels and
realize cytoplasmic bridges crossing through the transverse cell walls.
Separating the fibres into sections (segments) or individual cells is one
usual mode of vegetative reproduction. In evolutionary respect the thread-
like structure is interesting as a starting point for the formation of
determinate line (fila) of multicellular organisms.

FROM SINGLE CELLS TO MULTICELULAR ORGANISMS.
CLONING

CHAPTER 4

The cell theory postulates the general principle of biology, that all plant
and animal organisms are built up of cells. As it was already mentioned in
Chapter 2 (Section 2. 2), immediately after its appearance a lot of
questions arise. One of them, deserving greatest attention, is the
following: are the multicellular organisms a mechanical sum of
autonomous cells or they represent a specific community?
Different opinions are expressed on this question. Some authors
defend the idea that multicellular organisms are a sum of cells with
completely independent functions. Ardent adherents of this view are:
R. Virchow — in his book “Cellular Pathology”, M. Verworn — “General
Physiology”, E. Haeckel — with his formulation “state of cells, in which
they are independent citizens”, etc. In their opinion the general
biological problems have their roots in the individual cells. The concept
that multicellular organisms are a sum of constituents (cells) is also
included in the formulation of Th. Schwann substantiating the cell
theory. Other authors, like W. Ritter, E. Russell and Z. Katznelson
resolutely oppose to that concept and consider it a mechanistic
approach.
The above-mentioned gives some notion about the two extreme
concepts on the question posed. As early as in 1864 in his book
“Principles of Biology” the English philosopher H. Spencer expresses the
idea, that “in the course of emerging the multicellularity not only a simple
summing but integration of cells occurs” (cited by Katznelson, 1963).
By now nobody chalenge that multicellular organisms are built of cells.
As most logical, it is accepted that they have been formed through
integration of individual cells in multicellular systems. Diagrammaticaly this
process is presented with Volvox green algae in Fig. 4–1.
Volvocaceae family includes coenobial and colonial forms, more
typical are the representatives of Gonium and Eudorina genera. The
cells included in them form 16- or 32-cell coenobia and are located in a
single loose layer in the form of a packet with a common mucous
membrane. In contrast to them, in Pandorina genus the cells are more
compact. The motions of outer flagella are coordinate which enables the
cell formation to move in the same direction. They reproduce asexually
by forming a new coenobia from each cell, but toward the end of
vegetation, i.e. when conditions become adverse, sexual process is
realized.

unicellular organisms. These two processes are represented in Figure 3–30.

Reasonably, one arrives at the conclusion that the key to the biological
aging and the neoplasms lies in the division spindle which, as it was
already mentioned, is a demarcation line between eukaryotic and
prokaryotic cells. Revealing the causes and mechanisms that determine
and realize the starting or stopping of the division spindle will serve as a
basis for creating a substantial theory of these two processes. That is an
exeptoinaly difficult task, but it should be solved in the interest of
irreversible needs of the human race.

1. Mutations in somatic cells, which lead to disturbances in the normal
mechanisms controlling the division of cells.
2. Inducing under the action of physical and chemical mutagenic
factors. To the physical factors different ionizing irradiations can be reffered
— X-ray, γ-ray, UV-light, α- and β-particles, fast neutrons, etc., and to the
chemical ones — some analogues of purine and pyrimidine bases,
alkylating agents, acridines, etc.
3. Transformations with the participation of oncogenic viruses. The
virus theory gained currency since 1912 after the discovery of the first
oncogenic virus of Rous, causing sarcomas in the connective tissue in
birds, the virus SV-40 known for the formation of polyomes in monkeys,
etc. But this theory was not proved because of the unclear mechanisms
of integrating DNA and RNA-containing oncoviruses into the cell genome
and the numerous observed cases of virus presence in them without
causing tumour formations, as well as the unreceptiveness of many cells
to them.
Without ignoring the influence of the factors of surroundings, there is
reason to consider the transforming normal cells of higher organisms
into their neoplasmic analogues to be a process of returning from limited
to unlimited division, which is a norm in unicellular organisms. Thus, the
cells in multicellular organisms “remind” of their phylogenesis, which can
be accepted as a proof in favour of the concept about the origin of the
higher multicellular organisms from unicellular ancestors (Nicolov, 1997,
b).
Noteworthy is the assumption of Bungo Wada (1979) that there exist
genes controlling the appearance and retention of division spindle. For
convenience sake he has marked them by SFI (the spindle formation
inducer gene) and SFR (the spindle repressor gene). Under the
influence of SFI-gene the cells form a division spindle and divide up to
the termination of its activity. If this gene is defective or inhibited under
the influence of SFR-gene, then the cells cannot form a division spindle
and as a result they will not divide, and under certain conditions can turn
into differentiated state. Though hypothetical, the expressed opinion
deserves attention, since it directs the investigations to the discussed
problems of the division spindle, which ultimately determine the
behaviour of cell, i.e. whether to start dividing, to remain undivided and
to die, or to prolong its existence in differentiated state.
On the basis of all stated in this Section, the biological aging and
the neoplasms can be considered to be natural phenomena approved in
the course of the evolution of multicellular organisms. These are two
mutually connected and at the same time opposite processes. The one
of them has emerged as a result of cell specialization and differentiation
that have limited the number of divisions, and the other — returning to
unlimited division, which is a norm in the independently existing

Neoplasms

The neoplasms (called also tumours, cancer or carcinogenic formations)
are observed usually in animals, but sometimes are also met in plants.
They became a scourge for the human race. The struggle against them is a
foremost task of science and medical practice. For that reason the methods
and remedies for their treatment are permanently improved. Such type of
cells is HeLa-line (Fig. 3–29).

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Figure 3–29. Scanning electron micrograph of malignant HeLa-cells 
(Courtesy of K. Porter; From Fawcett, 1981).

An essential peculiarity of neoplasmic cells is their capability to divide
continiously (non-stop) without “obeying” the mechanisms of division
acquired in the process of cell differentiation, which are characteristic of the
other cells forming the different tissues and organs of a certain organism. In
this way conflicting interrelations arise between them, the effect in most
cases being lethal as a result.
According the prevailing concept, transforming the normal cells into
neoplasm (tumour) cells is a multistage process, which depends on many
factors realizing the interaction between the genome and surroundings.
Concerning this aspect, different opinions and hypotheses are expressed
about the causes for transformation resulting in neoplasmic formations.
They can be reduced to three main groups.