Totipotency and birth of tissue culture introduction important definitions birth if tissue culture

1. Lec. 1 b
Totipotency and the Birth of
Tissue Culture

2. The plant totipotency concept
• Totipotency is the ability of a single cell
to divide and produce all the
differentiated cells in an organism
• Totipotent cells have total potential
• Totipotent cells are formed during sexual
and asexual reproduction

3. The concept of totipotency
Many somatic plant cells provided they contain intact nuclear, plastid and
mitochondrial genomes, have the capacity to regenerate into whole plants.
This phenomenon is totipotency, an amazing developmental plasticity that
sets plant cells apart from most of their animal counterparts, first
demonstrated by Steward and Reinert in the 1950s
Often totipotency is revealed in tissue culture. A differentiated plant cell
that is selectively expressing its genetic information can instead initiate
expression of the program required for generation of an entire new plant
The first step in expression of regenerative totipotency is for mature cells
to re-enter the cell cycle and resume cell division — a process known as
dedifferentiation
Expression of totipotency depends on:
competence, by which we mean the ability of cells to be induced along a
particular developmental pathway, and
Determination, in which cells become irreversibly committed to a
particular pathway.

4. Three Fundamental Cellular Abilities of Plants
Totipotency
the potential or inherent capacity of a plant cell to develop into an
entire plant if suitably stimulated. It implies that all the information
necessary for growth and reproduction of the organism is
contained in the cell
Dedifferentiation
Capacity of mature cells to return to meristematic condition and
development of a new growing point, follow by re-differentiation
which is the ability to reorganize into new organ
Competency
the endogenous potential of a given cell or tissue to develop in a
particular way
Determination
in which cells become irreversibly committed to a particular
pathway (occurrence of non-regenerative calli in cereals)

5. Cells of adult plants remain totipotent:
cloning a carrot
Moore et al Figure 9.2
Wm C Brown Publishing
1 mm3 fragments (“explants”)
from adult root…
Culture explants in liquid culture medium…
Cells “dedifferentiate” and begin to divide,
forming “callus” tissue…
Induce with hormones to initiate shoot
and root formation…
Culture “embroid” in liquid culture,
then agar…
Move to soil…
Regenerated adult plant…

6. Cell potency
The process of specializing cells’ functions is called cell differentiation .
It is accompanied by morphogenesis, the change of the cells’ morphology.
Differentiation occurs by turning on certain genes and turning off some others
at a certain time.
Therefore, for a highly differentiated cell to grow into a full plant, the
differentiation process has to be reversed (called de-differentiation ) and
repeated again ( called re-differentiation).
Theoretically, all living cells can revert to an un differential status through this
process. However, the more differentiated a cell has been, the more difficult it
will be to induce its de-differentiation.
Practically, the younger or the less differentiated a cell is, the easier to culture
it into a full plant. The ease of fulfilling the cell totipotency also varies tissue by
tissue, genotype by genotype and species by species. Genotype dependency
is often the bottleneck in plant tissue culture and also in plant genetic
engineering.

7. Stem cells in plants are localized in “meristems”
MBoC (4) figure 21-111 and 112 © Garland Publishing
Shoot apical meristem
Root apical meristem
Lateral or axial
meristems
Shoot apical
meristem
Shoot apical
meristem

8. Cell Potenty
• – Totipotent (unlimited potential)
• – Pluripotent (slight limitation to potential)
• – Multipotent (limited potential)
• -Unipotent
The totipotency of plant cells is reflected by the variety of cell
types that can undergo embryogenesis and give rise to the fully
differentiated organism.

9. Loss of Totipotency
• Loss of totipotency is probably due to genetic
(physical changes to chromosomes, for example loss
of DNA, nucleotide sub-stitution, endopolyploidy) or
epigenetic blocks (changes in gene expression as a
consequence of development, for example DNA
methylation)

10. Basis for expression of
plasticity in Plant Tissue Culture
• Hormones Affect Plant Differentiation:
– Auxin: Stimulates Root Development
– Cytokinin: Stimulates Shoot Development
• Generally, the ratio of these two hormones can
determine plant development:
– Auxin ↓Cytokinin = Root Development
– Cytokinin ↓Auxin = Shoot Development
– Auxin = Cytokinin = Callus Development

11. Epigenetics is mostly the study of heritable changes that are
not caused by changes in the DNA sequence; to a lesser
extent, epigenetics also describes the study of stable, long-
term alterations in the transcriptional potential of a cell that are
not necessarily heritable.

12. PLANT CELL BIOTECHNOLOGY

13. BIOTECHNOLOGY
“The controlled exploitation of biological
agents (microorganisms or cellular
components) for beneficial use”
Microbio Biochem
Biology
Biotechnology

14. Biotechnology in general has become a very
broad based field of scientific research,
spanning the range of development for
genetic engineering, medical and the
cultivation of cells, tissues, and organs for
research. This has resulted in the field being
broken down into several subfields identified
by color, including green, blue, white, and
gray biotechnology.

15. Fields of Biotechnology
•Medicinal Biotech
•Industrial Biotech
•Plant Biotech
•Environmental Biotech

16.  Medicinal Biotechnology
a branch of biotechnology that deals specifically
with human health care and methods of
treatment through the development of
medicines such as antibiotics.
 Monoclonal antibodies
 Manipulation of genes
 Valuable drugs
 Parenting/criminals identification by DNA
Fingerprinting

18.  Industrial Biotechnology
 Production of useful compounds
 Production of antibiotics
 Transformation of less useful compounds to
valuable ones
 Fuel production from cheap sources
 Mineral extraction through leaching
 Production of immunotoxins

19.  Environmental Biotechnology
 Clean up environmental waste
 Prevent pollution
 Production of waste-based feed stocks
 Efficient utilization of land
Environmental Biotechnology Cooperative Research
Centr (EBCRC )Australia
Center for Environmental Biotechnology (CEB)USA

20.  Plant Biotechnology
 Rapid clonal multiplication
 Virus free plant production
 Embryo rescue of inviable hybrids
 Rapid isolation of homozygous lines
 Germplasm conservation
 Exploitation of somaclonal variation
 Development of transgenic plants
 Molecular markers and marker-assisted
breeding

21. Plant Biotechnology
“ Pertains to all such activities other than
conventional approaches that aim at either
improving the genetic make up, phenotypic
performance or multiplication rates of
economic plants or at exploiting plant cells
or cell constituents for generating useful
products”

22.  Increase crop production
 Improve nutritional quality
 Broaden crop tolerance for
drought, salinity and other
abiotic stresses
 Increase resistance to pests
and diseases
Potentials of Plant Biotechnology

23. PLANT BIOTECHNOLOGY

TISSUE CULTURE
GENETIC ENGINEERING

24. Whole plants can be regenerated via cell and
tissue culture

25. TRADITIONAL BIOTECHNOLOGY
TISSUE CULTURE
EMBRYO CULTURE
MERISTEM CULTURE
MICROPROPAGATION
PROTOPLAST CULTURE
CRYOPRESERVATION
ANTHER CULTURE

26. ADVANCED BIOTECHNOLOGY
Genetic Engineering
Genetic
Transformation
Somatic Hybridization

27.  EMBRYO CULTURE
The term “embryo rescue” refers to a number of in vitro
techniques to promote the development of an inherently weak,
immature or hybrid embryos into a viable plant.
Young embryos removed from developing seeds are cultured
in vitro to get seedlings
Applications
 Recovery of distant hybrids
 Propagation of orchids
 Shortening of breeding cycle
 Overcoming Dormancy

28.  MERISTEM CULTURE
Cultivation of axillary meristems
Applications
 Recovery of virus free stocks
 Germplasm exchange
 Germplasm conservation

29.  ANTHER CULTURE
In vitro culture of anthers for the
production of haploids
Applications
 Haploid plant production
 Homozygous diploid lines
 Gametoclonal variation

31.  MICROPROPAGATION
In vitro multiplication of stock plants
Applications
 Rapid, large-scale, year round production of
desired crop varieties
 Propagation of plant species that are difficult
to grow from seed
 Production of genetically uniform plant
material (clones)
 Development of plant culture systems that
can be used for genetic transformation
 Production of disease-free plant material

32.  PROTOPLAST CULTURE
In vitro plant regeneration from wall-less plant cells
(Protoplasts)

33. PLANT PROTOPLASTS

34.  Basic and applied research
 Plant cell metabolic studies; photosynthesis
 Cell wall synthesis and deposition
 Isolation of organelles; vacuoles and nuclei
 Application of flow cytometry
 Genetic manipulation studies; Transformation
& Somatic hybridization
APPLICATIONS OF PROTOPLAST TECHNOLOGY

35.  CRYOPRESERVATION
Storage of living cells at ultra low temperatures in a
way that viability is restored after thawing
Applications
 Ensures long-term, safe storage of plant germplasm
 Requirement of relatively very small space
 Germplasm storage free from diseases/insects etc
 Give clean source of nucleus seed
 Facilitates germplasm exchange

36. SOMATIC HYBRIDIZATION
Formation of hybrids by the fusion of
somatic cells (protoplasts) of two
different plant species

37. BENEFITS OF SOMATIC HYBRIDIZATION
 Elimination of crossing barriers
 No loss of genetic information during the formation
of gametes
 Specific addition of the genomes of two plants
 Combination of complex traits without loosing any
gene
 Unique combinations of nuclear and organellar
genomes generate novel germplasm
 No strict maternal inheritance of organelles

38. PROTOPLAST FUSION: Technique
A
B
SH
A+B

39. GENETIC TRANSFORMATION OF PLANTS
Transfer and stable integration of genes into the
genome of plant from other plants or other
organisms
 Methods for plant transformation
 Agrobacterium-mediated transformation
 Direct gene transfer methods
 Particle bombardment
 PEG-mediated transformation
 Electroporation
 Whiskers

40. Applications:
 Making crop plants resistant to herbicides
 Making crops more resistant to stress
 Improving the nutritional quality of crops
 Improving the storage properties of fruits
etc.
 Making crop plants which are resistant to
pests