Plant regeneration is possible through organogenesis or somatic embryogenesis where differentiated plant cells can become totipotent using hormones. The process involves taking an explant from a plant and culturing it in a nutrient medium under sterile conditions to regenerate a whole new plant. Somaclonal variation may occur during this process, introducing genetic changes into the regenerated plants.

1. PLANT REGENERATION

2. PLANT REGENERATION
 The process of growing an entire plant from a single
cell or group of cells.
 Regeneration is possible because plant cells can be
made totipotent using hormones.
 Differentiated tissue: stems, leaves, roots, etc.
 Undifferentiated (embryonic) cells are totipotent: can
become a whole new plant by differentiating into a
whole new plant.

3. THE PLANTS CAN BE REGENERATED BY
ORGANOGENESIS
It refers to the formation of organs from the cultured
explants. The shoot buds or monopolar structures are
formed by manipulating the ratio of cytokinins to auxins
in the cultures.
 SOMATIC EMBRYOGENESIS
In Somatic embryogenesis, the totipotent cells may
undergo embryogenic pathway to form somatic embryos
which are grown to regenerate into complete plants.

4. PLANT MATERIAL—THE EXPLANT
Any part of a plant taken out and grown in test tube
under sterile conditions in special nutrient media is
called explant.

6. HORMONES USED IN PLANT TISSUE
CULTURE
1. Auxins
2. Cytokinins
3. Gibberellins
4. Abscisic Acid
5. Polyamines

7. ENVIRONMENTAL CONDITIONS:
(i) NUTRIENT MEDIUM
inorganic salts , a carbon source ,vitamins ,amino acids and
growth regulators
(ii) ASEPTIC CONDITIONS
The microbes compete with growing tissue and finally kill it. It is
essential to maintain aseptic conditions of tissue culture. Thus
sterilization means complete destruction or killing of.

8. microorganisms so that complete aseptic conditions are
created for in vitro culturing.
(iii) AERATION OF THE TISSUE
Proper aeration of the cultured tissue is also an important
aspect of culture technique. It is achieved by occasionally
stirring the medium by sterring or by automatic shaker.

9. REGENERATION OF PLANTLETS
1. Preparation of Suitable Nutrient Medium:
Suitable nutrient medium as per objective of culture is prepared and
transferred into suitable containers.
2. Selection of Explants:
Selection of explants such as shoot tip should be done.
3. Sterilization of Explants:
Surface steriization of the explants by disinfectants and then washing
the explants with sterile distilled water is essential.
4. Inoculation:
Inoculation (transfer) of the explants into the suitable nutrient medium
(which is sterilized by filter-sterilized to avoid microbial
contamination) in culture vessels under sterile conditions is done.

10. 5. Incubation:
Growing the culture in the growth chamber or plant tissue culture room,
having the appropriate physical condition (i.e., artificial light; 16 hours of
photoperiod), temperature (-26°C) and relative humidity (50-60%) is required.
6. Regeneration:
Regeneration of plants from cultured plant tissues is carried out.
7. Hardening:
Hardening is gradual exposure of plantlets to an environmental conditions.
8. Plantlet Transfer:
After hardening plantlets transferred to the green house or field conditions
following acclimatization (hardening) of regenerated plants

12. PLANT PROPAGATION

13. INTRO:
 Propagation of plant is defined as production of new
individuals from a selected plant having all the characters
of the original one.
 New plants are created from a variety of
sources: seeds, cuttings, bulbs, tissue culture etc.
 Plant propagation in done sexually and asexually as well
as on artificial platform using TISSUE CULTURE
TECHNEQUIES.

14. PRINCIPLE:
The basis of plant propagation is totipotency, the
capability of cells to regenerate missing parts and,
subsequently, an entire organism.

16. TYPES:
 Sexual propagation is with the use of seed or spore
that is separated from the parent plant. This method
is so termed "sexual" because there is the
involvement of the sexes, referring to the
contribution of both the male and female gametes in
the production of new plants.
 Pollen is transferred from anther to stigma.
 Fertilization occurs and seeds are produce.

18.  Asexual propagation, also called vegetative
propagation, is with the use of planting materials
which are vegetative parts of any plant rather than
seeds or spores which are reproductive parts.
 E.g. cutting, layering, grafting etc.

19.  Tissue culture, also called in vitro culture
andmicropropagation, is the technique of
propagating plants indoor under aseptic or sterile
conditions in artificial growth media. The growth
medium, also called nutrient solution, is a mixture
of essential elements. The initial propagule in tissue
culture is called explant

20. MICROPROPAGATION
• Micro-propagation is the propagation of plants
through tissue culture
• It is a proven means of producing millions of
identical plants under a controlled and aseptic
condition independent of seasonal constraints
• Small pieces of plant tissues ( ex plants )use for
regeneration ( shoot system rooting and
growing into full size plant)

21. STARTING MATERIAL
 Tip bud
 Leaf
 Axillary bud
 Internode
 Root
 Seed

22. STEPS OF MICROPROPAGATION
1. Selection and preparation of mother plant
2. Initiation of culture
3. Multiplication
4. Rooting
5. Transfer to soil

23. 1.ESTABLISHMENT:
 The objective of this stage is to get clean cultures
that can begin the process of shoot multiplication
 The explant is usually sterilized with a combination
of detergent and bleach. In difficult situations alcohol
or a fungicide may be used.
2. INITIATION:
Explant placed into growth media.

24. 3. MULTIPLICATION:
•Explant transferred to shoot media where shoots
can be constantly divided.
•The objective of the shoot multiplication stage is to
increase the number of shoots produced by the
original explant.

25.  The culture or a portion of the culture is removed
from the jar and placed on a sterile paper towel.
 A scalpel and forceps are used to cut and separate
the larger culture into smaller pieces for transfer to a
new jar to complete the subculturing procedure.
 A high cytokinin to auxin ratio is used during the
multiplication stage to induce axillary or adventitious
shoot formation

26.  This ratio is decided upon by preliminaryresearch.
 Too high a concentration of cytokinin will result in a
high number of adventitious shoots that do not
elongate.
 Common cytokinins used in culture are
benzyladenine and kinetin.

27. 4. ROOTING:
 Shoots multiplied in culture must be rooted in order
to create a new plantlet.
 In the rooting stage, microcuttings are induced to
form roots - usually by application of auxin.
 Micrcuttings can either be rooted in vitro or ex vitro .
 In general, microcuttings rooted ex vitro have a more
normal root system and acclimatize to a normal
growing environment better than cuttings rooted in
vitro .
 However, the propagator has more control over the
rooting environment in vitro and this method may fit
their production scheme better.

29.  Microcuttings are inserted directly into the rooting
substrate often using forceps to handle the small
cuttings.
5. TRANSFER TO SOIL:
 Finally, after roots have become well established on
the microcutting, plantlets must be acclimatized to a
normal growing environment
 This involves gradually moving to open-air conditions
where the humidity is reduced and the light levels
increased

30. APPLICATIONS:
 Rapid multiplication
 Seasonal independent production
 Use to conserve rare or endangered species
 Greater output

31. SOMACLONAL VARIATION

32. SOMACLONAL VARIATIONS:
 The term somaclonal variation was first used by
Larkin and Scowcroft in 1981.
 Somaclonal variation is the variation seen in plants
that have been produced by plant tissue culture.
 The term somaclonal variation is used for genetic
variability present among all kinds of cells or
obtained from cells cultured in vitro.
 Plants derived from such cells or progeny of such
plant is called somaclones.
 These variations can be detected as genetic or
phenotypic traits.

33. BASIC FEATURES:
 Variations in number and structure of chromosomes
are commonly observed.
 Regenerated plants with altered chromosomal
changes often show changes in leaf shape and
colour, growth rate and habit, and sexual fertility.
 It is generally heritable mutations and persist in plant
population even after plantation into the field.

35. CAUSES OF S.C VARIATIONS:
The causes may be;
Physiological
Genetic
Biochemical

36. 1. GENETIC
 Pre-existing variations in the somatic cells of explant
that are Caused by mutations and other DNA
changes.
 Typical genetic alterations are;
1. changes in chromosome numbers
(polyploidy and aneuploidy),
2. Change in chromosome structure (translocations,
deletions, insertions and duplications).
3. DNA sequence (base mutations).
 Occur at high frequency

37. 2. Physiological
 Exposure of culture to plant growth regulators.
 Culture conditions
3. BIOCHEMICAL
 Lack of photosynthetic ability due to alteration in
carbon metabolism
 Antibiotic resistance

38. ADVANTAGES:
 • Rather stable and high frequencies.
 New alleles or even novel mutations may be isolated
which were not available in the germplasm.
 Can be performed in all types of plants.
 Reduce the time required for the release of new
variety by two years as compared to mutation
breeding.
 A very effective selection method.

39. DISADVANTAGES:
 Applicable only to those species cell cultures which
regenerate into complete plants.
 Selected cell lines often show reduced or no
regeneration potential.
 Show undesirable features such as reduced fertility,
growth rate and even overall performance of plant.
 Selected clones are unstable not always stable and
heritable.