Description of plant propagation methods

1. Plant Propagation

2. Plant propagation...
• Sexual methods - seed propagation
• Asexual methods
– rooting cuttings
– layering; air layering
– grafting
– specialized structures
– tissue culture; micropropagation
“the art and science of multiplying
plants by sexual or asexual means.”

3. Plant Propagation Types
• Sexual Propagation
• Asexual Propagation/Vegetative propogation

4. Sexual Propagation
• Sexual propagation involves contribution of
both female & male sexes for creation of new
plants. It is a natural process in which a parent
species create offspring that are genetically
different from them.
• The process starts with flowering followed by
pollination, fertilization and ultimately seed
formation. These seeds when sown, results in
formation of new plants.

5. Advantages of Sexual Propagation
• Simplest, easiest and the most economical process among
various types of plant propagation.
• Some plants, trees, vegetables or fruits species can propagate only
through sexual propagation.
• This type of propagation leads to better crop species that are
stronger, disease-resistant and have longer life-span.
• Viral transmission can be prevented in this type of propagation.
• Sexual propagation is responsible for production of large number of
crops and that too with different varieties.
• It is the only propagation process in which resultant offspring have
genetic variation and exhibit diversity of characters from parent
crops.
• This genetic variation is responsible for continuous evolution that
keeps on producing better & better offspring.
• Easy storage and transportation of seeds.

6. Disadvantages of Sexual Propagation:
• Seeds take a long time to turn into mature
plants i.e. time interval between sowing and
flowering is longer.
• Seedlings propagated through sexual
propagation are unlikely to have same genetic
characteristics as that of parent plants.
• Some plant species do not produce viable
seeds through sexual propagation and hence
are unsuitable to propagate for the same.
• Plants that do not have seeds can’t be
propagated through this process.

7. Vegetative propagations

9. Asexual propagation...
“vegetative reproduction, i.e.,
multiplication that does not
involve the seed cycle - clonal
propagation.”

10. Clone...
A genetically identical assemblage of
individuals produced from a plant
entirely by vegetative means.
Hartmann and Kester

11. Vegetative propagation
• Vegetative propagation, this process involves
production of species through vegetative parts of
the plants such as roots, leaves, stems, bulbs,
tubers etc.
• In this process, no exchange of genetic
information takes place as the offspring is formed
through material of a single parent. Thus the
resultant plants formed are identical to the
parent plant (also known as clones).

12. Different Propagation methods
• Cuttings
• Layering
• Grafting
• Budding

14. Benefits of clonal propagation...
• Some plants produce few (if any) viable seeds.
• Clonal progeny are highly uniform in all characters.
• Outcrossing plants produce highly variable progeny.
• Plants may have extended juvenile period.
• Cloning allows for combining genotypes in one plant.
• Seeds may have lengthy and complex dormancies.

15. Cutting propagation...
• Cutting capable of regenerating roots (or shoots) from adventitious meristem.
• Cuttings defined by size and location on parent plant:
– stem tip
– nodal
– root
– leaf
• Cuttings also defined by condition of growth:
– softwood, semihardwood, hardwood

16. Conditions required for rooting cuttings...
• Hardwood and root cuttings require well drained medium, but little moisture control.
• Leafy cuttings require high humidity to prevent excessive dehydration:
– intermittent mist systems.
– poly tents or cold frames.
– fog.
• Medium into which cuttings are stuck must be disease free, well drained, and light.
• Auxin application now standard treatment.

17. Examples of cutting propagation...
Species Cutting Type
African violet, begonia leaf cuttings
Many woody shrubs stem tip cuttings (softwood)
brambles root cuttings
Willow, poplars dormant hardwood cuttings

18. Grafted and budded plants...
Species Graft/bud Type
apples chip, T-bud, cleft
conifers side veneer
pecans patch
roses T-bud (shield)
grapes modified wedge

19. Layering...
• Air layering - interrupt cambium and cover wound with moistened medium. Ficus elastica, Magnolia
• Simple layering - low hanging branch covered with soil (with or without wounding) - many shrubs
• Tip layering - tips of plants (brambles) at certain times of year (rat-tail condition) develop roots where they touch the soil Blackberries,
raspberries
• Mound layering - soil mounded to cover base of specially pruned young tree (also referred to as stool layering) Apple rootstocks
“Layering involves inducing roots on an intact (or
nearly so) plant .”

20. Air Layering

21. Specialized
Structures
Modified Stems
bulb, corm, tuber, rhizome
pseudobulb, runner
Modified Roots
tuberous root

23. Apomixis...
“the development of an embryo within a seed or
flowering structure from a source other than the egg,
resulting in the formation of an embryo (sometimes in
addition to the sexual embryo) that is a clone of the
maternal parent. Examples include polyembryony in
citrus and crabapples, and the formation of bulbils in
garlic.”
“an exception to the rule of nonclonal embryony.”

24. Micropropagation...
• Micropropagation is rapid, continuous, and efficient.
• Specialized equipment, facilities, and technically trained personnel are
required.
• Steps can be taken to obtain and maintain certified pest-free plants.
• Cost effective if large numbers of a given clone are produced.
• Widely used for orchids, ferns, many interior foliage plants,
rootstocks, etc.

25. Plants amenable to micropropagation...
Flowering pot plants - Begonia, African violets, orchids
Interior foliage plants - ferns, Syngonium, Ficus,
Diffenbachia
Woody ornamentals - red maples, Rhododendrons,
Nandina
Forest trees - Poplar, birch, loblolly pine
Fruit trees - apple, cherry, pear (many rootstocks)
Vegetable crops - potato, celery, tomato, onion (male
sterile)
Plantation crops - banana, date palm, coffee

26. Types of development in vitro...
Proliferation of axillary buds from shoot tip cultures.
Differentiation of adventitious shoots from leaves,
stems, or roots.
Formation and proliferation of somatic embryos.
Seed germination - orchids.
Development of haploid plants from anthers or ovules.
Protoplast fusion and somatic hybrid development.

27. Propagation by seeds...
• Seeds are widely available, inexpensive, and easy to handle.
• Hybrid seeds are more expensive, but may have production benefits that offset the cost.
• Large scale agriculture (including vegetable crop production) is dependent upon seed propagation.
• Seeds (especially seeds of woody plants) may have complex dormancies that impede germination.
“Propagation by seeds is the major method by
which plants reproduce in nature and one of the
most efficient and widely used propagation
methods for cultivated crops.”
Hartmann and Kester

29. What is plant tissue culture ??
“Plant tissue culture is a collection of
techniques used to maintain or grow plant
cells, tissues or organs under sterile
conditions on a nutrient culture medium
of known composition”

30. Different techniques in plant tissue culture may offer
certain advantages over traditional methods of propagation,
including:
 The production of exact copies of plants that produce particularly good
flowers, fruits, or have other desirable traits.
 To quickly produce mature plants.
 The production of multiples of plants in the absence of seeds or
necessary pollinators to produce seeds.
 The regeneration of whole plants from plant cells that have been
genetically modified.
 The production of plants from seeds that otherwise have very low
chances of germinating and growing.

31. Organogenesis
Definition
“The formation of roots, shoots or flower buds from the cells in
culture in manner similar to adventitious root or shoot formation in
cuttings is called organogenesis’’
 Organogenesis starts in the callus in response to the stimulation given by the
chemicals in the medium.
 Organogenesis takes place in two stages, namely caulogenesis or shoot initiation
and rhizogenesis or root initiation.
 Both types of organogenesis are controlled by the hormones present in the
medium. generally a high auxin:cytokinin ratio induce shoot formation.
 Organogenesis starts with the development of a group of meristematic cells
called meristemoids, which initiate the formation of a primordium.
 Depending on the factors within the system, this primordium develops into shoot,
root or embryoid.

34. Two types of organoginasis
1. Direct regeneration
2. Indirect organogenesis
 This two types depend on hormonal combination of the
culture media.
Intermediate ratios around 1:1 favor callus growth.
Indirect organogenesis
Auxin/cytokinin 10:1-100:1 induces roots Direct regeneratio
Auxin/cytokinin 1:10-1:100 induces shoots.
Rule of thumb:

35. Direct organogenesis
Explant → Meristemoid → Primordium
 In many plants, subculturing of callus results in undesired
variations of clones (somaclonal variations).
 To avoid this, direct regeneration of the explants
into plantlets can be tried.
 This has been achieved in many plant species by altering the
hormonal combination of the culture media.

37. Indirect organogenesis
Explant → Callus → Meristemoid → Primordium
• In indirect organogenesis, callus is first produced from
the explant. Organs can then be produced from the callus
tissue or from a cell suspension produced from that callus.

38. The major factors affecting the process of regeneration are:
1. Source of Explant
 The organ that is to be served as tissue source
 The physiological and ontogenic age of the organ
 The season in which the explant is obtained
 The size of the explant
 The overall quality of the plant from which explants are
taken.
2. Culture Environment
 Physical form of the medium i.e. presence or absence of agar
 The pH of the medium3. Light quality and quantity
 Temperature
 Relative humidity
 The gaseous atmosphere within the vessel.
3. Nutrient Media and Constituents

39. Embryogenesis
• Somatic Embryogenesis
“The process of a single cell or a group of cells
initiating the developmental pathway that leads to
reproducible regeneration of non-zygotic embryos
capable of germinating to form complete plants”
• Under natural conditions, this pathway is not
normally followed, but from tissue cultures
somatic embryogenesis occurs most frequently
and as an alternative to organogenesis for
regeneration of whole plants.

40. How Somatic Embryos produced?
• In somatic embryogenesis, embryo-like
structures, which can develop into whole plants
in a way analogous to zygotic embryos, are
formed from somatic tissues.
• These somatic embryos (SE) can be produced
either directly or indirectly.
• Two ways of somatic embryogenesis:

41. 1. Direct embryogenesis
 In direct somatic embryogenesis, the embryo is formed
directly from a cell or small group of cells without the
production of an intervening callus.
 Direct somatic embryogenesis is generally rare in
comparison with indirect somatic embryogenesis.
2. Indirect embryogenesis
 In indirect somatic embryogenesis, callus is first
produced from the explant.
 Embryos can then be produced from the callus tissue or
from a cell suspension produced from that callus.

43. Somatic embryogenesis - Synthetic
seeds

44. Importance
 In poly embroyonic crops like citrus, zygotic as well as nucellar
embryonic plants are obtained separately.
 Embryos of big and heavy fruits like coconut can be taken out of the
fruits and preserve in tube in sterile distilled water for about two
months and then cultured in media.
 In this process easy international exchange of germplasm is possible.
 In many interspecific and intergeneric crosses the hybrid embryos fail
to develop to maturity. In such cases before the embryo gets damaged
can be taken out of the ovule and cultured in artificial media, which
gives rise to complete plantlets.
 One major path of regeneration
 Mass multiplication
 Production of artificial seeds

45. Micrografting (shoot tip grafting)
• Micrografting consists of the placement in aseptic conditions of a
maintained scion onto an in vitro grown rootstock.
• The results of in vitro micrografting and the plant material derived
from it can be further multiplied in tissue culture conditions or
acclimatized to outdoor conditions.
• Micrografting is a technique that potentially can combine the
advantages of rapid in vitro multiplication with increased
productivity that results from grafting, superior rootstock and scion
combinations.
• Among various methods of micrografting, slit or wedge grafting has
been found most suitable in case of fruit crops.
• In vitro shoot tips are better as compared to in vivo shoot tips for
carrying out micrografting resulting in higher graft success, less
contamination, lower shoot tip necrosis and good vigour of
micrografts.

46. This comprises the following steps
• Aseptic condition was maintained throughout all stages
of micrografting process.
• Seedlings were removed carefully from the agar medium
with sterilized forceps.
• For use as rootstock, seedlings were cut back to 1 cm
above the cotyledonary nodes and leaves were excised
using a microscalpel.
• Terminal shoots to be used as scions were excised from
the other seedlings at 2 cm below the apex.
• Lower leaves removed from the basal 1 cm of scion.
• Excised scion and rootstocks were stored in sterile de-
ionized water until used in the grafting process.

47. • The shoot tip was inserted at the top of
decapitated rootstock by making an incision.
• The cortex was exposed by the horizontal cut of
the incision.
• Grafted plants were cultured in a liquid nutrient
medium containing plant cell culture salt solution
of MS, modified White's vitamins and 75 g/1
sucrose.
• When at least two expanded leaves were formed
micrografted plants were transferred to pots
containing a steam sterilized soil mixture suitable

50. Importance
• Overcoming graft incompatibility
• Rapid mass propagation of elite scions by graftin
g onto rootstocks that have desirable traits like
resistance to soil borne pathogens and diseases
• To allow survival of difficult to root /shoots
• Development of virus free plant

51. Meristem culture
Cultivation of axillary or apical shoot
meristems, particularly of shoot apical
meristem, is known as meristem culture.
Meristem culture involves the development
of an already existing shoot meristem and
subsequently, the regeneration of
adventitious roots from the developed
shoots.

52.  Shoot apical meristem lies in the ‘shoot tip’ beyond the
youngest leaf or the first leaf primordium; it measures upto
about 100 µm in diameter and 250 µm in length.
 Thus a shoot tip of 100- 500 µm would contain 1-3 leaf
primordia in addition to the apical meristem.
 Shoot tip culture is widely used for rapid clonal propagation
for which much larger, e.g., 5-10 mm, explants are used.
 Therefore, most cases of meristem culture are essentially
shoot-tip cultures. Nodal explants of various sizes are also
commonly employed for rapid clonal propagation.

55. • Procedure
 Collect rapidly growing apex of a shoot
 Remove all the leaves except the smallest leaves, wash
thoroughly under running water with one drop of Tween
20.
 Disinfect the working area of the laminar flow cabinet
with 70% alcohol or rectified spirit.
 Dip washed explants in 0.1% mercuric chloride solution
for 5-7 min for surface sterilization, and wash with
sterile distilled water thoroughly for 3-4 times.

56.  Place the shoot tips on sterile filter paper.
 Hold the stem firmly with a forcep and remove the young
leaves with a needle.
 Remove the underlying leaf primordia gradually.
 Remove up to the 3rd and 4th leaf primordia and keep intact
the 1st and 2nd leaf
primordia carefully without damaging the fragile dome shaped
apical meristem.
 With a surgical scalpel remove the apical dome (0.1-0.3 mm).
 Transfer the dome to the culture tubes containing medium.
 The culture tubes maintain at 25 ± 2° C for 12 h light (3000
lux)/12 h dark cycle.

57. Growth and development:
 Within 2-3 weeks of inoculation apical meristem grows
and forms shoots.
 Each single shoot undergo proliferation, these shoots are
separated and cultured in rooting media.
 Root initiation occurs within 18-21 days of inoculation.
 Once the plantlet has two to three leaves and strong root,
they are transferred to liquid basal media with the help of
Paper Bridge
 After about 8 weeks the plantlets are soaked in 0.2%
Bavisiin a fungicide for 10 min and are potted in
vermiculite mixture, till they grow.

58. • Importance:
Production of virus free germplasm.
Mass production of desirable genotypes.
Facilitation of exchange between locations
(production of clean material).
Cryopreservation (cold storage) or in vitro
conservation of germplasm.
can produce virus-free plantlets for micropropagation
and international exchange of germplasm.
Viruses persist in the vegetative plant parts, which
are used for propagation.

59. • References:
• Methods in plant tissue culture by U. Kumar
• http://www.whatisthebiotechnology.com/blog/wp-
content/uploads/2013/10/ORGANOGENESIS-IN-
PLANTS.pdf
• https://www.scribd.com/doc/35044234/Importance-of-Plant-
Tissue-Culture
• http://www.biologydiscussion.com/essay/plant-breeding-
essay/essay-on-plant-tissue-culture-history-methods-and-
application/17639
• https://en.wikipedia.org/wiki/Plant_tissue_culture