Clonal Propagation: Introduction, Techniques, Factors, Applications and Disadvantages Multiplication of Apical or Axillary bud, Shoot tip or meristem culture Production of Disease free plants by Micropropagation techniques: their Advantages and Disadvantages

1. Clonal Propagation:
Techniques, Factors, Applications
and Disadvantages
DR. DIVYA SHARMA
ASSISTANT PROFESSOR
A Biodiction (A Unit of Dr. Divya Sharma)

2. Micropropagation
 Plant Tissue Culture technique:
Increasingly popular alternative means -- Plant vegetative propagation
 Plants can be propagated by sexual (through generation of seeds) or asexual
(through multiplication of vegetative parts).
 Plant tissue culture involves –
o Asexual method of propagation
o Primary goal -- crop improvement
 In-vitro selection, genetic manipulation techniques -- Higher plant

3. Clonal Propagation
Clonal propagation ------- In-vitro selection Micropropagation
Greek word --- Clone --- Twig ---- Identical copies
 The process of multiplication of genetically identical copies of individual plants by
asexual reproduction is called Clonal Propagation
 The term clone is used to represent a plant population derived from a single
individual by asexual reproduction.
Process -- Asexual reproduction --- Multiplication occur ----- Individual
plants genetically identical copies

4.  Multiplication of genetically, identically individual by asexual reproduction
 Apomixes (seed development without meiosis and fertilization) and vegetative
propagation (regeneration of new plants from vegetative parts)
 Short time
 Rapid process -- Commeralization of improvement plants.
Eg.: Apple, pear etc.
 Asexual reproduction through multiplication of vegetative parts is the only
method for the in-vivo propagation of certain plants, as they do not produce
viable seeds.
Eg.: Banana, Grape, Fig and Chrysanthemum.
 Clonal propagation has been successfully applied for the propagaton.
Eg.: Apple, Potato, Tuberous and several Ornamental plants

5. Advantages of Vegetative Propagation
 Faster multiplication --- Large number of plants can be produced from a single
individual in a short period.
 Produce genetically identical plants
 Sexually ---- Sterile hybrids can be propagated.
 Seed --- Raised plants pass through an undesirable juvenile phase which is
avoided in asexual propagation.
 Gene banks can be more easily established by clonally propagated plants

6. Micropropagation
 Aseptic method of clonal propagation is called as
Micropropagation
 Synonyms -- tissue culture
 Rapid multiplying stock plant material to produce
a large number of progeny plants, using modern
plant tissue culture methods
 Widely used for Orchids, Ferns, many interior
foliage plants etc.

7. Features of Micropropagation
 Relatively short time
 Easy to manipulate production cycles
 Disease free plants can be produced
 In-vitro production can be planned
 Recycled many times to produce an unlimited number of clones
 Micropropagated plants may acquire new desirable traits, like bushy habit of
ornamental plants and increased number of runners in strawberry
 Commercially viable method for horticultural crops
Eg.: Orchids, Ferns, many interior foliage plants etc.

8. In-vitro Clonal Propagation
In-vivo clonal propagation --- Tedious, expensive and frequently unsuccessful
In-vitro clonal propagation --- Through tissue culture --- Micropropagation
 From cells, tissue or organs
 Cultured aseptically on defined media
 Contained in culture vessels
 Maintained under controlled conditions of light and temperature

9. Commercialization of Micropropagation
Broad commercial applications ------ 1970s and 1980s
Murashige (1974)

10. In-vitro Clonal
Propagation
Use of tissue culture technique for
micropropagation was first started by
Morel (1960) for propagation of orchids,
and is now applied to several plants.
Micropropagation is a handy technique for
rapid multiplication of plants

11. Selection of Plant Material
 Part of plant
 Genotype
 Physiological condition
 Season
 Position on plant
 Size of explants

12. Explants used in Micropropagation
Different kinds of explants used in micropropagation:
Eg.: In case of orchids, shoot tips (Anacamptis pyramidalis, Aranthera,
Calanthe, Dendrobium); Axillary bud (Aranda, Brassocattleya, Cattleys Laelia);
Inflorescence segment (Aranda, Ascofinetia, Neostylis, Vascostylis); Lateral
bud (Cattleya, Rhynocostylis gigantean); Leaf base (Cattleya); Nodal segment
(Dendrobium; Flower stalk segment (Dendrobium, Phalaenopsis); and Root
tips (Neottis, Vanilla)

13. Stages of Micropropagation
Stage 0: Preparation of the mother plant
Stage 1: Initiation and establishment of cultures
Stage 2: Multiplication [shoot or rapid somatic embryo formation]
Stage 3: In-vitro germination of somatic embryo
and rooting of shoots
Stage 4: Transfer of plantlets to sterilized soil for Hardening
under greenhouse/ field conditions (transplantation)

14. Stages of
Micropropagation  Maintained light, temperature and
moisture regimes under which the
mother plants
 Loosely covering growing branches
with polythene
 Application of growth regulators
 Watering of plant from the base
Stage 0: Preparatory
stage
Involves the preparation of
mother plants to provide
quality explants for better
establishment of aseptic
cultures

15. Stages of
Micropropagation
 Explant selection
 Sterilization treatment
 Choice of growth medium
Stage 1: Initiation of
Cultures

16. Stage 2: Multiplication
Through Callusing
Initiation of culture on high Auxin media
Development of unorganized mass of cells
called callus
Transfer small piece of callus on
regeneration media
Shoot bud regeneration
Somatic embryo

17. Stage 2: Multiplication
Adventitious Bud Formation
 Transfer to proliferation media
 Shoots can be constantly divided

18.  Small bud present
 Apical dominance is removed
 Cytokinins
 Auxins
 Cluster formation
 Off types
Stage 2: Multiplication
Enhanced Axillary Branching

19.  Transfer of shoots to a medium for rapid development into shoots
 Sometimes, the shoots are directly planted in soil to develop roots
 In-vitro of shoots is preferred while simultaneously handling a large number of
species
Stage 3: Rooting of shoots
Rooting & Hardening

20.  Somatic embryos carry a preformed radical and may develop directly into
plantlet.
 Embryos sow very poor conversion into plantlets, especially under in-vitro
conditions
 Require an additional step of maturation to acquire the capability for normal
germination
 Adventitious and axillary shoots developed in cultures in the presence of a
cytokinins generally lack roots
 Obtain full plants the shoots must be transferred to a rooting medium which is
different from the shoot multiplication medium, particularly in its hormonal and
salt compositions
 For rooting, individual shoots measuring 2 cm in length are excised and
transferred to the rooting medium
Stage 3: Rooting of shoots
Rooting & Hardening

21. Stage 4: Transplantation & Accilimitization
Transferring the plantlets of stage 3 from the laboratory to the environment f
green house.
Some plant species, stage 3 skipped, and unrooted stage 2 shoots are planted in
pots or in suitable compost mixture
Tissue culture raised plants are characterized by:
 Low photosynthetic rates
 Non-functional stomata
 Low cuticular wax
 Abnormal leaf morphology
 Accilimitization
 Green house
 2 weeks
 Temperature (±10º)

22. Stages of Micropropagation

23. In-vitro regenerative protocol of V. reitzii
Nodule cluster culture formation and shoot regeneration in V. reitzii; a). Donor plant (inflorescence
of adult plant); b). Yellow nodular cultures induced in MS medium free of PGR; c). Nodule cluster
subcultureed in MS culture medium supplemented with GA3 resulted in high proliferation rate and
the subsequent development of adventitious microshoots; d). Elongation and growth of shoot in MS
culture medium free of PGR; e). Acclimatization of plantlets; f). Growth in green house

24. Barley Propagation

25. Potato Propagation

26. Methods of Clonal Micropropagation
 Multiplication by axillary buds/apical shoots
 Multiplication by adventitious shoots
Beside the above two approaches, the plant regeneration processes namely
Organogenesis and Somatic embryogenesis may also be treated as
micropropagation
 Organogenesis (Formation of individual organs such as shoots, roots, directly
from an explant or from callus and cell culture induced from the explant)
 Somatic embryogenesis (Regeneration of embryos from somatic cells, tissue or
organs)

27. Methods of Micropropagation
 Organogenesis
 Organogenesis via callus formation
 Direct adventitious organ formation
 Embryogenesis
 Direct embryogenesis
 Indirect embryogenesis
 Microcutting
 Meristem culture (Mericloning)
 Bud culture

28. 1. Multiplication by Axillary Buds and Apical
Shoots
 Quiescent or actively dividing meristems are present at the axillary and apical
shoots tips.
 The axillary buds located in the axils of leaves are capable of developing into
shoots.
 In in-vivo state, only limited number of axillary meristems can form shoots.
 Induced in-vitro multiplication in micropropagation, possible to develop plants
from meristems and shoot tip culture and form bud cultures

29. a. Meristem and Shoot Tip Cultures
 Apical meristem is a dome of tissue located at the extreme tip of a shoot. The
apical meristem along with the young leaf primordia constitutes the shoot apex.
For the development of disease-free plants, meristem tips should be cultured.
 Meristem or shoot tip is isolated from a stem by a V-shaped cut. The size
(frequently 0.2 to 0.5 mm) of the tip is critical for culture. In general, the larger the
explant (shoot tip), the better are the chances for culture survival. For good results
of micro propagation, explants should be taken from the actively growing shoot
tips, and the ideal timing is at the end of the plants dormancy period.

30. Shoot tip (or Meristem) Culture in Micropropagation
Stage 1, 2,and 3 represent in Micropropagation

31. a. Meristem and Shoot Tip Cultures
Stage 1: Establishment of meristem on culture. Addition of growth regulators
like Cytokinins (Kinetin, BAP) and Auxins (NAA or IBA)
Stage 2: Shoot development along with axillary shoot proliferation occurs.
High levels of cytokinins required
Stage 3: The root formation is facilitated by low cytokinins and high auxin
concentration. This is opposite to shoot formation since high level of
cytokinins is required (in stage II).
Consequently, stage II medium and stage III medium should be different in
composition. The optimal temperature for culture is in the range of 20-28°C
(for majority 24-26°C). Lower light intensity is more appropriate for good
micro propagation

32. b. Bud Cultures
 The plant buds possess quiescent or active meristems depending on the
physiological state of the plant.
 Two types of bud cultures involves:
o Single node culture
o Axillary bud culture

33. Single Node Cultures
 Natural method for vegetative propagation of plants both in-vivo and in-vitro
conditions.
 The bud found in the axil of leaf is comparable to the stem tip, for its ability in
micro propagation.
 A bud along with a piece of stem is isolated and cultured to develop into a
plantlet. Closed buds are used to reduce the chances of infections.
 In single node culture, no Cytokinins is added.

34. Single Node Technique for Micropropagation

35. Axillary Bud Cultures
 Isolated shoot tip along with axillary bud.
 The cultures are carried out with high cytokinins concentration.
 As a result of this, apical dominance stops and axillary buds develop.
Eg.: Rosette plant and an elongate plants
For a good axillary bud culture, the cytokinin/auxin ratio is around 10:1. However,
variable and depends on the nature of the plant species and the developmental stage
of the explant used. In general, juvenile explants require less cytokinin compared to
adult explants. Sometimes, the presence of apical meristem may interfere with axillary
shoot development. In such a case, it has to be removed.

36. Micropropagation of plants by axillary bud (or shoot tips) method:
(A) Rosetle plant; (B) Elongate plant

37. 2. Multiplication by Adventitious Shoots
 The stem and leaf structures that are naturally formed on plant tissues located in
sites other than the normal leaf axil regions are regarded as adventitious shoots.
 Many adventitious shoots which include stems, bulbs, tubers and rhizomes.
 The adventitious shoots are useful for in-vivo and in-vitro clonal propagation.
 The meristematic regions of adventitious shoots can be induced in a suitable
medium to regenerate to plants.

38. 3. Organogenesis
 Organogenesis is the process of morphogenesis involving the formation of plant
organs like shoots, roots, flowers, buds from explant or cultured plant tissues.
 Two types:
o Direct organogenesis
o Indirect organogenesis

39. a. Direct Organogenesis
 Tissues from leaves, stems, roots and inflorescences can be directly cultured to
produce plant organs. In direct organogenesis, the tissue undergoes
morphogenesis without going through a callus or suspension cell culture stage.
The term direct adventitious organ formation is also used for direct organogenesis.
 Induction of adventitious shoot formation directly on roots, leaves and various
other organs of intact plants is a widely used method for plant propagation. This
approach is particularly useful for herbaceous species.
 For appropriate organogenesis in culture system, exogenous addition of growth
regulators (Auxin and Cytokinin) is required.
 The concentration of the growth promoting substance depends on the age and
nature of the explant, besides the growth conditions.

40. b. Indirect Organogenesis
 When the organogenesis occurs through callus or suspension cell culture
formation, it is regarded as indirect organogenesis.
 Callus growth can be established from many explants (leaves, roots, cotyledons,
stems, flower petals etc.) for subsequent organogenesis.
 The explants for good organogenesis should be mitotically active immature tissues.
In general, the bigger the explant the better the chances for obtaining viable
callus/cell suspension cultures.
 It is advantageous to select meristematic tissues (shoot tip, leaf, and petiole) for
efficient indirect organogenesis. This is because their growth rate and survival rate
are much better.
 For indirect organogenesis, the cultures may be grown in liquid medium or solid
medium. Many culture media (MS, B5 White’s etc.) can be used in organogenesis.
The concentration of growth regulators in the medium is critical for organogenesis.

41. Micropropagation of plants by organogenesis:
(A) Direct organogenesis; (B) Indirect organogenesis through
callus; (C) Indirect organogenesis though cell culture

42. By varying the concentrations of Auxin and Cytokinin, In-
vitro organogenesis can be manipulated
 Low Auxin and low Cytokinin concentration will induce callus formation.
 Low Auxin and high Cytokinin concentration will promote shoot organogenesis
from callus.
 High Auxin and low Cytokinin concentration will induce root formation.

43. 4. Somatic Embryogenesis
 Somatic embryogenesis may result in non-zygotic embryos or somatic embryos (directly
formed from somatic organs), parthogenetic embryos (formed from unfertilized egg)
and androgenic embryos (formed from male gametophyte).
 In a general usage, when the term somatic embryo is used it implies that it is formed
from somatic tissues under in vitro conditions. Somatic embryos are structurally similar
to zygotic (sexually formed) embryos, and they can be excised from the parent tissues
and induced to germinate in tissue culture media.
 Development of somatic embryos can be done in plant cultures using somatic cells,
particularly epidermis, parenchymatous cells of petioles or secondary root phloem.
Somatic embryos arise from single cells located within the clusters of meristematic cells
in the callus or cell suspension. Firstly, proembryo is formed which then develops into
an embryo, and finally a plant.
 Two routes of somatic embryogenesis — Direct and Indirect Embryogenesis

44. a. Direct Embryogenesis
 When the somatic embryos develop directly on the excised plant (explant) without
undergoing callus formation, it is referred to as direct somatic embryogenesis.
 Due to the presence of pre-embryonic determined cells (PEDQ found in certain
tissues of plants. The characteristic features of direct somatic embryogenesis is
avoiding the possibility of introducing somaclonal variations in the propagated
plants.

45. b. Indirect Embryogenesis
 In indirect embryogenesis, the cells from explant (excised plant tissues) are made to
proliferate and form callus, from which cell suspension cultures can be raised.
Certain cells referred to as induced embryo genic determined cells (IEDC) from the
cell suspension can form somatic embryos. Embryogenesis is made possible by the
presence of growth regulators (in appropriate concentration) and under suitable
environmental conditions.
 Somatic embryogenesis (direct or indirect) can be carried on a wide range of media
(e.g. MS, White’s). The addition of the amino acid L-glutamine promotes
embryogenesis. The presence of auxin such as 2, 4-dichlorophenoxy acetic acid is
essential for embryo initiation. On a low auxin or no auxin medium, the embryo
genic clumps develop into mature embryos.
 Indirect somatic embryogenesis is commercially very attractive since a large
number of embryos can be generated in a small volume of culture medium. The
somatic embryos so formed are synchronous and with good regeneration
capability.

46. Artificial Seeds from Somatic Embryo
 Artificial seeds can be made by encapsulation of somatic embryos.
 The embryos, coated with sodium alginate and nutrient solution, are dipped in
calcium chloride solution.
 The calcium ions induce rapid cross-linking of sodium alginate to produce small
gel beads, each containing an encapsulated embryo.
 These artificial seeds (encapsulated embryos) can be maintained in a viable state
till they are planted

47. Micropropagation of plants by embryogenesis:
(A) Direct embryogenesis; (B) Indirect embryogenesis

48. Factors Affecting Micropropagation
 Genotype of the plant
 Physiological status of the explants
 Culture media
 Culture environment (Light, Temperature, Composition of gas phase )

49. Factors Affecting in-vitro Rooting
 For efficient in vitro rooting during micropropagation, low concentration of
salts (reduction to half to one quarter from the original) is advantageous.
 Induction of roots is also promoted by the presence of suitable Auxin (NAA or
IBA).

50. Applications of Clonal Propagation
 Production of disease and virus free plantlets
 Production of seeds in some crops
 High rate of plant propagation
 Cost effective process
 Automated micropropagation

51. Advantages of Clonal Propagation
 From one to many propagules rapidly
 Multiplication in controlled lab conditions
 Continuous propagation year round
 Potential for disease free propagules
 Inexpensive per plant once establishment

52. Disadvantages of Clonal Propagation
 Contamination of cultures
 Brewing of medium
 Genetic cultures
 Vetrification
 Cost factor

53. Horticultural uses for plant tissue culture
 Clonal mass propagation
 Difficult or slow to propagate plants
 Introduction of new cultivars
 Vegetative propagation of sterile hybrids
 Pathology – eliminate viruses, bacteria, fungi etc
 Storage of germplasm

54. Production of Disease-free Plants
 Many plant species are infected with pathogens — viruses, bacteria, fungi,
mycoplasma and nematodes that cause systemic diseases. Although these diseases do
not always result in the death of plants, they reduce the quality and yield of plants. The
plants infected with bacteria and fungi frequently respond to chemical treatment by
bactericides and fungicides.
 However, it is very difficult to cure the virus-infected plants. Further, viral disease are
easily transferred in seed- propagated as well as vegetatively propagated plant
species.
 Plant breeders are always interested to develop disease-free plants, particularly viral
disease-free plants. This have become a reality through tissue cultures.

55. Apical Meristems with Low Concentration of Viruses:
 Absence of vascular tissue in the meristems through which viruses readily move in
the plant body.
 Rapidly dividing meristematic cells with high metabolic activity do not allow viruses
to multiply.
 Virus replication is inhibited by a high concentration of endogenous auxin in shoot
apices. Tissue culture techniques employing meristem-tips are successfully used for
the production of disease-free plants, caused by several pathogens — viruses,
bacteria, fungi, mycoplasmas.

56. List of the plants with virus elimination by meristem culture

57. Merits and Demerits of Disease-Free
Plant Production
 Among the culture techniques, meristem-tip culture is the most reliable method for
virus and other pathogen elimination. However, requires good knowledge of plant
pathology and tissue culture.
 Virus-free plants exhibit increased growth and vigor of plants, higher yield (E.g.,
Potato), increased flower size (E.g., Chrysanthemum), and improved rooting of stem
cuttings (E.g., Pelargonium)
 Limitation: Virus-free plants are more susceptible to the same virus when exposed
again. Reinfection of disease-free plants can be minimized with good knowledge of
greenhouse maintenance.

58. Thank you
A Biodiction (A Unit of Dr. Divya Sharma)